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Sample records for nuclear progesterone-binding protein

  1. Dermatophyte-hormone relationships: characterization of progesterone-binding specificity and growth inhibition in the genera Trichophyton and Microsporum.

    PubMed Central

    Clemons, K V; Schär, G; Stover, E P; Feldman, D; Stevens, D A

    1988-01-01

    We reported previously that Trichophyton mentagrophytes contains a cytoplasmic macromolecule which specifically binds progesterone. Progesterone is also an effective inhibitor of growth of the fungus. We report here studies which characterize more fully the specific binding properties and the functional responses of T. mentagrophytes and taxonomically related fungi to a series of mammalian steroid hormones. Scatchard analysis of [3H]progesterone binding in both the + and - mating types of Arthroderma benhamiae and in Microsporum canis revealed a single class of binding sites with approximately the same affinity as that in T. mentagrophytes (Kd, 1 X 10(-7) to 2 X 10(-7) M). Trichophyton rubrum had a protein with a higher binding affinity (Kd, 1.6 X 10(-8) M). Characterization of the [3H]progesterone-binding sites in T. mentagrophytes showed the binder to be a protein which was destroyed by trypsin and heating to 56 degrees C. Previous examination of the steroid-binding specificity in T. mentagrophytes had demonstrated that deoxycorticosterone (DOC) and dihydrotestosterone (DHT) were effective competitors for [3H]progesterone binding. Expansion of this study to include other competitors revealed that R5020 (a synthetic progestin), androstenedione, and dehydroepiandosterone possessed relative binding affinities which were 20, 11, and 9% of that of progesterone, respectively. Other ligands tested were less effective. Competition studies for the binder in M. canis resulted in similar findings: DOC and DHT were effective competitors for [3H]progesterone binding. The growth of A. benhamiae + and -, M. canis, and T. rubrum were all inhibited by progesterone in a dose-responsive manner, with 50% inhibition achieved at concentrations of 9.8 x 10(-6), 1.2 x 10(-5), 1.5 x 10(-5), and 2.7 x 10(-6) M. respectively,. PMID:3182998

  2. A membrane-associated progesterone-binding protein, 25-Dx, is regulated by progesterone

    E-print Network

    Jarvis, Erich D.

    on the perineum (1). This model system underscores the behavior's dependence on E, P, and tactile stimulation (2 of the neural circuitry responsible for lordosis. One brain structure important for integrating the endocrine.g., Hsc73) (5) and others that may serve specific functions in intracellular trafficking in endocrine

  3. Nuclear Pore Proteins and Cancer

    PubMed Central

    Xu, Songli; Powers, Maureen A.

    2009-01-01

    Nucleocytoplasmic trafficking of macromolecules, a highly specific and tightly regulated process, occurs exclusively through the Nuclear Pore Complex. This immense structure is assembled from approximately 30 proteins, termed nucleoporins. Here we discuss the four nucleoporins that have been linked to cancers, either through elevated expression in tumors (Nup88) or through involvement in chromosomal translocations that encode chimeric fusion proteins (Tpr, Nup98, Nup214). In each case we consider the normal function of the nucleoporin and its translocation partners, as well as what is known about their mechanistic contributions to carcinogenesis, particularly in leukemias. Studies of nucleoporin-linked cancers have revealed novel mechanisms of oncogenesis and. in the future, should continue to expand our understanding of cancer biology. PMID:19577736

  4. Nuclear protein in the liver in protein–calorie deficiency

    PubMed Central

    Hryniewiecki, L.

    1965-01-01

    1. The effect of protein–calorie deficiency on nuclear proteins was studied in growing and adult rats by using chemical and electrophoretic methods of fractionation. 2. After 7 days on protein-deficient diets, the amount of neutral-soluble proteins increased, and their electrophoretic pattern changed, with the appearance of a new component. 3. The histone fraction of the liver nuclei in rapidly growing rats was lower than that in either deficient or adult animals. 4. The possible role of nuclear proteins in relation to synthesis of the proteins and nucleic acids of the other parts of the cells is discussed. ImagesFig. 1. PMID:14333563

  5. Properties of proteins binding plasma progesterone in pregnant Cape porcupines (Hystrix africaeaustralis)

    E-print Network

    Pretoria, University of

    as that of guinea-pigs. Concentrations of progesterone-binding proteins in plasma increased during preg- nancy) are similar in composition to those in guinea-pigs (New World hystricomorph). Keywords: Cape porcupine; plasma¬ trations of progesterone are similar to those in pregnant New World hystricomorph rodents, such as guinea-pigs

  6. Dynamic Nuclear Polarization of Deuterated Proteins

    E-print Network

    Akbey, Umit

    For D's a jolly good fellow: Deuteration of proteins significantly increases the signal enhancements observed in dynamic nuclear polarization (DNP) magic-angle spinning (MAS) NMR experiments. In 13C?CP-MAS spectra an ...

  7. Direct Visualization of a Protein Nuclear Architecture

    PubMed Central

    Hendzel, Michael J.; Boisvert, F.-Michel; Bazett-Jones, David P.

    1999-01-01

    Whether the cell nucleus is organized by an underlying architecture analagous to the cytoskeleton has been a highly contentious issue since the original isolation of a nuclease and salt-resistant nuclear matrix. Despite electron microscopy studies that show that a nuclear architecture can be visualized after fractionation, the necessity to elute chromatin to visualize this structure has hindered general acceptance of a karyoskeleton. Using an analytical electron microscopy method capable of quantitative elemental analysis, electron spectroscopic imaging, we show that the majority of the fine structure within interchromatin regions of the cell nucleus in fixed whole cells is not nucleoprotein. Rather, this fine structure is compositionally similar to known protein-based cellular structures of the cytoplasm. This study is the first demonstration of a protein network in unfractionated and uninfected cells and provides a method for the ultrastructural characterization of the interaction of this protein architecture with chromatin and ribonucleoprotein elements of the cell nucleus. PMID:10359614

  8. Compartmentalization and Functionality of Nuclear Disorder: Intrinsic Disorder and Protein-Protein Interactions in Intra-Nuclear Compartments.

    PubMed

    Meng, Fanchi; Na, Insung; Kurgan, Lukasz; Uversky, Vladimir N

    2015-01-01

    The cell nucleus contains a number of membrane-less organelles or intra-nuclear compartments. These compartments are dynamic structures representing liquid-droplet phases which are only slightly denser than the bulk intra-nuclear fluid. They possess different functions, have diverse morphologies, and are typically composed of RNA (or, in some cases, DNA) and proteins. We analyzed 3005 mouse proteins localized in specific intra-nuclear organelles, such as nucleolus, chromatin, Cajal bodies, nuclear speckles, promyelocytic leukemia (PML) nuclear bodies, nuclear lamina, nuclear pores, and perinuclear compartment and compared them with ~29,863 non-nuclear proteins from mouse proteome. Our analysis revealed that intrinsic disorder is enriched in the majority of intra-nuclear compartments, except for the nuclear pore and lamina. These compartments are depleted in proteins that lack disordered domains and enriched in proteins that have multiple disordered domains. Moonlighting proteins found in multiple intra-nuclear compartments are more likely to have multiple disordered domains. Protein-protein interaction networks in the intra-nuclear compartments are denser and include more hubs compared to the non-nuclear proteins. Hubs in the intra-nuclear compartments (except for the nuclear pore) are enriched in disorder compared with non-nuclear hubs and non-nuclear proteins. Therefore, our work provides support to the idea of the functional importance of intrinsic disorder in the cell nucleus and shows that many proteins associated with sub-nuclear organelles in nuclei of mouse cells are enriched in disorder. This high level of disorder in the mouse nuclear proteins defines their ability to serve as very promiscuous binders, possessing both large quantities of potential disorder-based interaction sites and the ability of a single such site to be involved in a large number of interactions. PMID:26712748

  9. Inner nuclear membrane proteins: targeting and influence on genome organization 

    E-print Network

    Zuleger, Nikolaj

    2012-06-22

    The nuclear envelope is a complex double membrane system that separates the activities of the nuclear and cytoplasmic compartments. A recent explosion in the number of proteins associated with this subnuclear organelle together ...

  10. Nuclear G protein signaling: new tricks for old dogs.

    PubMed

    Campden, Rhiannon; Audet, Nicolas; Hébert, Terence E

    2015-02-01

    According to the standard model of G protein-coupled receptor (GPCR) signaling, GPCRs are localized to the cell membrane where they respond to extracellular signals. Stimulation of GPCRs leads to the activation of heterotrimeric G proteins and their intracellular signaling pathways. However, this model fails to accommodate GPCRs, G proteins, and their downstream effectors that are found on the nuclear membrane or in the nucleus. Evidence from isolated nuclei indicates the presence of GPCRs on the nuclear membrane that can activate similar G protein-dependent signaling pathways in the nucleus as at the cell surface. These pathways also include activation of cyclic adenosine monophosphate, calcium and nitric oxide synthase signaling in cardiomyocytes. In addition, a number of distinct heterotrimeric and monomeric G proteins have been found in the nucleus of various cell types. This review will focus on understanding the function of nuclear G proteins with a focus on cardiac signaling where applicable. PMID:25590750

  11. Nuclear protein import is reduced in cells expressing nuclear envelopathy-causing lamin A mutants

    SciTech Connect

    Busch, Albert; Kiel, Tilman; Heupel, Wolfgang-M.; Wehnert, Manfred; Huebner, Stefan

    2009-08-15

    Lamins, which form the nuclear lamina, not only constitute an important determinant of nuclear architecture, but additionally play essential roles in many nuclear functions. Mutations in A-type lamins cause a wide range of human genetic disorders (laminopathies). The importance of lamin A (LaA) in the spatial arrangement of nuclear pore complexes (NPCs) prompted us to study the role of LaA mutants in nuclear protein transport. Two mutants, causing prenatal skin disease restrictive dermopathy (RD) and the premature aging disease Hutchinson Gilford progeria syndrome, were used for expression in HeLa cells to investigate their impact on the subcellular localization of NPC-associated proteins and nuclear protein import. Furthermore, dynamics of the LaA mutants within the nuclear lamina were studied. We observed affected localization of NPC-associated proteins, diminished lamina dynamics for both LaA mutants and reduced nuclear import of representative cargo molecules. Intriguingly, both LaA mutants displayed similar effects on nuclear morphology and functions, despite their differences in disease severity. Reduced nuclear protein import was also seen in RD fibroblasts and impaired lamina dynamics for the nucleoporin Nup153. Our data thus represent the first study of a direct link between LaA mutant expression and reduced nuclear protein import.

  12. Nuclear protein import is reduced in cells expressing nuclear envelopathy-causing lamin A mutants.

    PubMed

    Busch, Albert; Kiel, Tilman; Heupel, Wolfgang-M; Wehnert, Manfred; Hübner, Stefan

    2009-08-15

    Lamins, which form the nuclear lamina, not only constitute an important determinant of nuclear architecture, but additionally play essential roles in many nuclear functions. Mutations in A-type lamins cause a wide range of human genetic disorders (laminopathies). The importance of lamin A (LaA) in the spatial arrangement of nuclear pore complexes (NPCs) prompted us to study the role of LaA mutants in nuclear protein transport. Two mutants, causing prenatal skin disease restrictive dermopathy (RD) and the premature aging disease Hutchinson Gilford progeria syndrome, were used for expression in HeLa cells to investigate their impact on the subcellular localization of NPC-associated proteins and nuclear protein import. Furthermore, dynamics of the LaA mutants within the nuclear lamina were studied. We observed affected localization of NPC-associated proteins, diminished lamina dynamics for both LaA mutants and reduced nuclear import of representative cargo molecules. Intriguingly, both LaA mutants displayed similar effects on nuclear morphology and functions, despite their differences in disease severity. Reduced nuclear protein import was also seen in RD fibroblasts and impaired lamina dynamics for the nucleoporin Nup153. Our data thus represent the first study of a direct link between LaA mutant expression and reduced nuclear protein import. PMID:19442658

  13. Sun2 is a novel mammalian inner nuclear membrane protein.

    PubMed

    Hodzic, Didier M; Yeater, David B; Bengtsson, Luiza; Otto, Henning; Stahl, Philip D

    2004-06-11

    Sun protein (Sun1 and Sun2) cDNAs were previously cloned based on the homology of their C-terminal regions (SUN (Sad1 and UNC) domain) with the Caenorhabditis elegans protein UNC-84 whose mutation disrupts nuclear migration/positioning. In this study, we raised an anti-Sun2 serum and identified Sun2 in mammalian cells. In HeLa cells, Sun2 displays a nuclear rim-like pattern typical for a nuclear envelope protein. The Sun2 antibody signal co-localizes with nuclear pore and INM markers signals. The rim-like pattern was also observed with the recombinant full-length Sun2 protein fused to either EGFP or V5 epitopes. In addition, we found that a recombinant truncated form of Sun2, extending from amino acids 26 to 339, is sufficient to specify the nuclear envelope localization. Biochemical analyses show that Sun2 is an 85-kDa protein that is partially insoluble in detergent with high salt concentration and in chaotropic agents. Furthermore, Sun2 is enriched in purified HeLa cell nuclei. Electron microscopy analysis shows that Sun2 localizes in the nuclear envelope with a sub-population present in small clusters. Additionally, we show that the SUN domain of Sun2 is localized to the periplasmic space between the inner and the outer nuclear membranes. From our data, we conclude that Sun2 is a new mammalian inner nuclear membrane protein. Because the SUN domain is conserved from fission yeast to mammals, we suggest that Sun2 belongs to a new class of nuclear envelope proteins with potential relevance to nuclear membrane function in the context of the involvement of its components in an increasing spectrum of human diseases. PMID:15082709

  14. TTRAP is a novel PML nuclear bodies-associated protein

    SciTech Connect

    Xu Guanlan; Pan Yukun; Wang Bingyin; Huang Lu; Tian Ling; Xue Jinglun; Chen Jinzhong Jia, William

    2008-10-24

    PML nuclear body (PML NB) is an important macromolecular nuclear structure that is involved in many essential aspects of cellular function. Tens of proteins have been found in PML NBs, and promyelocytic leukemia protein (PML) has been proven to be essential for the formation of this structure. Here, we showed that TRAF and TNF receptor-associated protein (TTRAP) was a novel PML NBs-associated protein. TTRAP colocalized with three important PML NBs-associated proteins, PML, DAXX and Sp100 in the typical fashion of PML NBs. By yeast mating assay, TTRAP was identified to interact with these PML NBs-associated proteins. The transcription and expression of TTRAP could be induced by IFN-{gamma}, representing another common feature of PML NBs-associated proteins. These results would not only be important for understanding PML NBs but also be helpful in studying the TTRAP function in the future.

  15. Protein quality control at the inner nuclear membrane

    PubMed Central

    Khmelinskii, Anton; Blaszczak, Ewa; Pantazopoulou, Marina; Fischer, Bernd; Omnus, Deike J.; Le Dez, Gaëlle; Brossard, Audrey; Gunnarsson, Alexander; Barry, Joseph D.; Meurer, Matthias; Kirrmaier, Daniel; Boone, Charles; Huber, Wolfgang; Rabut, Gwenaël; Ljungdahl, Per O.; Knop, Michael

    2015-01-01

    The nuclear envelope is a double membrane that separates the nucleus from the cytoplasm. The inner nuclear membrane (INM) functions in essential nuclear processes including chromatin organization and regulation of gene expression1. The outer nuclear membrane is continuous with the endoplasmic reticulum (ER) and is the site of membrane protein synthesis. Protein homeostasis in this compartment is ensured by ER-associated protein degradation (ERAD) pathways that in yeast involve the integral membrane E3 ubiquitin ligases Hrd1 and Doa10 operating with the E2 ubiquitin-conjugating enzymes Ubc6 and Ubc72,3. However, little is known regarding protein quality control at the INM. Here we describe a protein degradation pathway at the INM mediated by the Asi complex consisting of the RING domain proteins Asi1 and Asi34. We report that the As complex functions together with the ubiquitin conjugating enzymes Ubc6andUbc7to degrade soluble and integral membrane proteins. Genetic evidence suggest that the Asi ubiquitin ligase defines a pathway distinct from but complementary to ERAD. Using unbiased screening with a novel genome-wide yeast library based on a tandem fluorescent protein timer (tFT)5, we identify more than 50 substrates of the Asi, Hrd1 and Doa10 E3 ubiquity ligases. We show that the Asi ubiquitin ligase is involved in degradation of mislocalised integral membrane proteins, thus acting to maintain and safeguard the identity of the INM. PMID:25519137

  16. The Novel Nuclear Envelope Protein KAKU4 Modulates Nuclear Morphology in Arabidopsis[W

    PubMed Central

    Goto, Chieko; Tamura, Kentaro; Fukao, Yoichiro; Shimada, Tomoo; Hara-Nishimura, Ikuko

    2014-01-01

    In animals, the nuclear lamina is a fibrillar meshwork on the inner surface of the nuclear envelope, composed of coiled-coil lamin proteins and lamin binding membrane proteins. Plants also have a meshwork on the inner surface of the nuclear envelope, but little is known about its composition other than the presence of members of the CROWDED NUCLEI (CRWN) protein family, possible plant lamin analogs. Here, we describe a candidate lamina component, based on two Arabidopsis thaliana mutants (kaku2 and kaku4) with aberrant nuclear morphology. The responsible gene in kaku2 encodes CRWN1, and the responsible gene in kaku4 encodes a plant-specific protein of unknown function (KAKU4) that physically interacts with CRWN1 and its homolog CRWN4. Immunogold labeling revealed that KAKU4 localizes at the inner nuclear membrane. KAKU4 deforms the nuclear envelope in a dose-dependent manner, in association with nuclear membrane invagination and stack formation. The KAKU4-dependent nuclear envelope deformation was enhanced by overaccumulation of CRWN1, although KAKU4 can deform the nuclear envelope even in the absence of CRWN1 and/or CRWN4. Together, these results suggest that plants have evolved a unique lamina-like structure to modulate nuclear shape and size. PMID:24824484

  17. Lamins: nuclear intermediate filament proteins with fundamental functions in nuclear mechanics and genome regulation.

    PubMed

    Gruenbaum, Yosef; Foisner, Roland

    2015-01-01

    Lamins are intermediate filament proteins that form a scaffold, termed nuclear lamina, at the nuclear periphery. A small fraction of lamins also localize throughout the nucleoplasm. Lamins bind to a growing number of nuclear protein complexes and are implicated in both nuclear and cytoskeletal organization, mechanical stability, chromatin organization, gene regulation, genome stability, differentiation, and tissue-specific functions. The lamin-based complexes and their specific functions also provide insights into possible disease mechanisms for human laminopathies, ranging from muscular dystrophy to accelerated aging, as observed in Hutchinson-Gilford progeria and atypical Werner syndromes. PMID:25747401

  18. Nuclear envelope transmembrane proteins in differentiation systems 

    E-print Network

    Batrakou, Dzmitry G.

    2012-11-30

    Historically, our perception of the nuclear envelope has evolved from a simple barrier isolating the genome from the rest of a cell to a complex system that regulates functions including transcription, splicing, DNA ...

  19. Importin-? facilitates nuclear import of human GW proteins and balances cytoplasmic gene silencing protein levels.

    PubMed

    Schraivogel, Daniel; Schindler, Susann G; Danner, Johannes; Kremmer, Elisabeth; Pfaff, Janina; Hannus, Stefan; Depping, Reinhard; Meister, Gunter

    2015-09-01

    MicroRNAs (miRNAs) guide Argonaute (Ago) proteins to distinct target mRNAs leading to translational repression and mRNA decay. Ago proteins interact with a member of the GW protein family, referred to as TNRC6A-C in mammals, which coordinate downstream gene-silencing processes. The cytoplasmic functions of TNRC6 and Ago proteins are reasonably well established. Both protein families are found in the nucleus as well. Their detailed nuclear functions, however, remain elusive. Furthermore, it is not clear which import routes Ago and TNRC6 proteins take into the nucleus. Using different nuclear transport assays, we find that Ago as well as TNRC6 proteins shuttle between the cytoplasm and the nucleus. While import receptors might function redundantly to transport Ago2, we demonstrate that TNRC6 proteins are imported by the Importin-? pathway. Finally, we show that nuclear localization of both Ago2 and TNRC6 proteins can depend on each other suggesting actively balanced cytoplasmic Ago - TNRC6 levels. PMID:26170235

  20. Importin-? facilitates nuclear import of human GW proteins and balances cytoplasmic gene silencing protein levels

    PubMed Central

    Schraivogel, Daniel; Schindler, Susann G.; Danner, Johannes; Kremmer, Elisabeth; Pfaff, Janina; Hannus, Stefan; Depping, Reinhard; Meister, Gunter

    2015-01-01

    MicroRNAs (miRNAs) guide Argonaute (Ago) proteins to distinct target mRNAs leading to translational repression and mRNA decay. Ago proteins interact with a member of the GW protein family, referred to as TNRC6A-C in mammals, which coordinate downstream gene-silencing processes. The cytoplasmic functions of TNRC6 and Ago proteins are reasonably well established. Both protein families are found in the nucleus as well. Their detailed nuclear functions, however, remain elusive. Furthermore, it is not clear which import routes Ago and TNRC6 proteins take into the nucleus. Using different nuclear transport assays, we find that Ago as well as TNRC6 proteins shuttle between the cytoplasm and the nucleus. While import receptors might function redundantly to transport Ago2, we demonstrate that TNRC6 proteins are imported by the Importin-? pathway. Finally, we show that nuclear localization of both Ago2 and TNRC6 proteins can depend on each other suggesting actively balanced cytoplasmic Ago – TNRC6 levels. PMID:26170235

  1. Nuclear matrix proteins and their potential applications to diagnostic pathology.

    PubMed

    Hughes, J H; Cohen, M B

    1999-02-01

    The nuclear matrix is the nonchromatin scaffolding of the cell nucleus that confers nuclear shape, organizes the nuclear chromatin, and regulates many important intranuclear biochemical events. Although our understanding of the nuclear matrix and its proteins is still evolving, it is clear that nuclear matrix proteins (NMPs) hold considerable promise as diagnostic tools for pathologists. Early evidence suggests that NMPs may be useful biomarkers of neoplastic disease in serum, body fluids, and tissues. NMPs are also potential candidates for use as tumor prognostic factors and targets of anticancer drugs. Moreover, NMPs may hold the key to understanding important cellular events, such as neoplastic transformation, steroid hormone binding, and apoptosis. Despite impressive gains made by cellular biologists and biochemists toward understanding the structure and function of the nuclear matrix, many of the potential applications of NMPs to diagnostic pathology are largely unexplored. Thus, NMPs should prove an exciting and fruitful area of investigation for experimental and clinical pathologists who are interested in developing diagnostic tests for detecting, quantitating, and characterizing these proteins in human tissues and body fluids and translating these applications into the clinical pathology laboratory. PMID:9930151

  2. Nuclear export dynamics of RNA-protein complexes.

    PubMed

    Grünwald, David; Singer, Robert H; Rout, Michael

    2011-07-21

    The central dogma of molecular biology - DNA makes RNA makes proteins - is a flow of information that in eukaryotes encounters a physical barrier: the nuclear envelope, which encapsulates, organizes and protects the genome. Nuclear-pore complexes, embedded in the nuclear envelope, regulate the passage of molecules to and from the nucleus, including the poorly understood process of the export of RNAs from the nucleus. Recent imaging approaches focusing on single molecules have provided unexpected insight into this crucial step in the information flow. This review addresses the latest studies of RNA export and presents some models for how this complex process may work. PMID:21776079

  3. Nuclear pore proteins and the control of genome functions

    PubMed Central

    Ibarra, Arkaitz

    2015-01-01

    Nuclear pore complexes (NPCs) are composed of several copies of ?30 different proteins called nucleoporins (Nups). NPCs penetrate the nuclear envelope (NE) and regulate the nucleocytoplasmic trafficking of macromolecules. Beyond this vital role, NPC components influence genome functions in a transport-independent manner. Nups play an evolutionarily conserved role in gene expression regulation that, in metazoans, extends into the nuclear interior. Additionally, in proliferative cells, Nups play a crucial role in genome integrity maintenance and mitotic progression. Here we discuss genome-related functions of Nups and their impact on essential DNA metabolism processes such as transcription, chromosome duplication, and segregation. PMID:25691464

  4. Cellular stress induces Bax-regulated nuclear bubble budding and rupture followed by nuclear protein release

    PubMed Central

    Lindenboim, Liora; Sasson, Tiki; Worman, Howard J; Borner, Christoph; Stein, Reuven

    2014-01-01

    Cellular stress triggers many pathways including nuclear protein redistribution. We previously discovered that this process is regulated by Bax but the underlying mechanism has not yet been studied. Here we define this mechanism by showing that apoptotic stimuli cause Bax-regulated disturbances in lamin A/C and nuclear envelope (NE)-associated proteins which results in the generation and subsequent rupture of nuclear protein-containing bubbles. The bubbles do not contain DNA and are encapsulated by impaired nuclear pore-depleted NE. Stress-induced generation and rupture of nuclear bubbles ultimately leads to the discharge of nuclear proteins into the cytoplasm. This process precedes morphological changes of apoptosis and occurs independently of caspases. Rescue experiments revealed that this Bax effect is non-canonical, i.e. it requires the BH3 domain and ?-helices 5 and 6 but it is not inhibited by Bcl-xL. Targeting Bax to the NE by the Klarsicht/ANC-1/Syne-1 homology (KASH) domain effectively triggers the generation and rupture of nuclear bubbles. Overall, our findings provide evidence for a novel stress-response, which is regulated by a non-canonical action of Bax on the NE. PMID:25482068

  5. Molecular chaperone-mediated nuclear protein dynamics.

    PubMed

    Echtenkamp, Frank J; Freeman, Brian C

    2014-05-01

    Homeostasis requires effective action of numerous biological pathways including those working along a genome. The variety of processes functioning in the nucleus is considerable, yet the number of employed factors eclipses this total. Ideally, individual components assemble into distinct complexes and serially operate along a pathway to perform work. Adding to the complexity is a multitude of fluctuating internal and external signals that must be monitored to initiate, continue or halt individual activities. While cooperative interactions between proteins of the same process provide a mechanism for rapid and precise assembly, the inherent stability of such organized structures interferes with the proper timing of biological events. Further prolonging the longevity of biological complexes are crowding effects resulting from the high concentration of intracellular macromolecules. Hence, accessory proteins are required to destabilize the various assemblies to efficiently transition between structures, avoid off-pathway competitive interactions, and to terminate pathway activity. We suggest that molecular chaperones have evolved, in part, to manage these challenges by fostering a general and continuous dynamic protein environment within the nucleus. PMID:24694369

  6. Identification of nuclear retention domains in the RBM20 protein.

    PubMed

    Filippello, Agnese; Lorenzi, Pamela; Bergamo, Elisa; Romanelli, Maria Grazia

    2013-09-17

    RBM20 is a nuclear protein which regulates alternative splicing of expressed genes that have a key role in cardiac function. By cloning the human and mouse RBM20 cDNA, producing expressing vectors for truncated proteins, and comparing their sub-cellular distribution in transfected cells, we have identified the sequences necessary for RBM20 full nuclear retention. The region overlaps an RNA binding motif and a serine-arginine domain. The sequence is conserved in many species but belongs only to RBM20 orthologs. The RMB20 tissue specificity, together with the properties of its nuclear localization determinant, demonstrates a specific evolutionary selection of post-transcriptional regulation factors. PMID:23886709

  7. Nuclear actin and protein 4.1: Essential interactions during nuclear assembly in vitro

    SciTech Connect

    Krauss, Sharon Wald; Chen, Cynthia; Penman, Sheldon; Heald, Rebecca

    2003-06-11

    Structural protein 4.1, which has crucial interactions within the spectin-actin lattice of the human red cell membrane skeleton, also is widely distributed at diverse intracellular sites in nucleated cells. We previously showed that 4.1 is essential for assembly of functional nuclei in vitro and that the capacity of 4.1 to bind actin is required. Here we report that 4.1 and actin colocalize in mammalian cell nuclei using fluorescence microscopy and, by higher resolution cell whole mount electron microscopy, are associated on nuclear filaments. We also devised a cell-free assay using Xenopus egg extract containing fluorescent actin to follow actin during nuclear assembly. By directly imaging actin under non-perturbing conditions, the total nuclear actin population is retained and is visualized in situ relative to intact chromatin. We detected actin initially when chromatin and nuclear pores began assembling. As the nuclear lamina assembled, but preceding DNA synthesis, a discrete actin network formed throughout the nucleus. Protein 4.1 epitopes also were detected when actin began to accumulate in nuclei, producing a diffuse coincident pattern. As nuclei matured, actin was detected both coincident with and also independent of 4.1 epitopes. To test whether acquisition of nuclear actin is required for nuclear assembly, the actin inhibitor latrunculin A was added to Xenopus egg extracts during nuclear assembly. Latrunculin A strongly perturbed nuclear assembly and produced distorted nuclear structures containing neither actin nor protein 4.1. Our results suggest that actin as well as 4.1 is necessary for nuclear assembly and that 4.1-actin interactions may be critical.

  8. Chromatin condensation modulates access and binding of nuclear proteins.

    PubMed

    Martin, Robert M; Cardoso, M Cristina

    2010-04-01

    The condensation level of chromatin is controlled by epigenetic modifications and associated regulatory factors and changes throughout differentiation and cell cycle progression. To test whether changes of chromatin condensation levels per se affect access and binding of proteins, we used a hypertonic cell treatment. This shift to hyperosmolar medium increased nuclear calcium concentrations and induced a reversible chromatin condensation comparable to the levels in mitosis. However, this condensation was independent of mitotic histone H3 serine 10 phosphorylation. Photobleaching and photoactivation experiments with chromatin proteins-histone H2B-GFP and GFP-HP1alpha-before and after induced chromatin condensation demonstrated that hypercondensation reduced their dissociation rate and stabilized their chromatin binding. Finally, measuring the distribution of nucleoplasmic proteins in the size range from 30 to 230 kDa, we found that even relatively small proteins like GFP were excluded from highly condensed chromatin in living cells. These results suggest that structural changes in condensed chromatin by themselves affect chromatin access and binding of chromatin proteins independent of regulatory histone modifications.-Martin, R. M., Cardoso, M. C. Chromatin condensation modulates access and binding of nuclear proteins. PMID:19897663

  9. Nuclear polyglutamine-containing protein aggregates as active proteolytic centers

    PubMed Central

    Chen, Min; Singer, Lena; Scharf, Andrea; von Mikecz, Anna

    2008-01-01

    Protein aggregates and nuclear inclusions (NIs) containing components of the ubiquitin–proteasome system (UPS), expanded polyglutamine (polyQ) proteins, and transcriptional coactivators characterize cellular responses to stress and are hallmarks of neurodegenerative diseases. The biological function of polyQ-containing aggregates is unknown. To analyze proteasomal activity within such aggregates, we present a nanoparticle (NP)-based method that enables controlled induction of sodium dodecyl sulfate–resistant inclusions of endogenous nuclear proteins while normal regulatory mechanisms remain in place. Consistent with the idea that the UPS maintains quality control, inhibition of proteasomal proteolysis promotes extra large protein aggregates (1.4–2 ?m), whereas formation of NP-induced NIs is found to be inversely correlated to proteasome activation. We show that global proteasomal proteolysis increases in NP-treated nuclei and, on the local level, a subpopulation of NIs overlaps with focal domains of proteasome-dependent protein degradation. These results suggest that inclusions in the nucleus constitute active proteolysis modules that may serve to concentrate and decompose damaged, malfolded, or misplaced proteins. PMID:18283109

  10. DPF2 regulates OCT4 protein level and nuclear distribution.

    PubMed

    Liu, Chao; Zhang, Dijuan; Shen, Yuxian; Tao, Xiaofang; Liu, Lihua; Zhong, Yongwang; Fang, Shengyun

    2015-12-01

    The amount of transcription factor OCT4 is strictly regulated. A tight regulation of OCT4 levels is crucial for mammalian embryonic development and oncogenesis. However, the mechanisms underlying regulation of OCT4 protein expression and nuclear distribution are largely unknown. Here, we report that DPF2, a plant homeodomain (PHD) finger protein, is upregulated during H9 cell differentiation induced by retinoic acid. Endogenous interaction between DPF2 and OCT4 in P19 cells was revealed by an immunoprecipitation assay. GST-pull down assay proved that OCT4 protein in H9 cells and recombinant OCT4 can precipitate with DPF2 in vitro. In vitro ubiquitination assay demonstrated DPF2 might serve as an E3 ligase. Knock down of dpf2 using siRNA increased OCT4 protein level and stability in P19 cells. DPF2 siRNAs also up-regulates OCT4 but not NANOG in H9 cells. However, RA fails to downregulates OCT4 protein level in cells infected by lenitviruses containing DPF2 siRNA. Moreover, overexpression of both DPF2 and OCT4 in 293 cells proved the DPF2-OCT4 interaction. DPF2 but not PHD2 mutant DPF2 enhanced ubiquitination and degradation of OCT4 in 293 cells co-expressed DPF2 and OCT4. Both wild type DPF2 and PHD2 mutant DPF2 redistributes nuclear OCT4 without affecting DPF2-OCT4 interaction. Further analysis indicated that DPF2 decreases monomeric and mono-ubiquitinated OCT4, assembles poly-ubiquitin chains on OCT4 mainly through Ub-K48 linkage. These findings contribute to an understanding of how OCT4 protein level and nuclear distribution is regulated by its associated protein. PMID:26417682

  11. Regulation of Nuclear Localization of Signaling Proteins by Cytokinin

    SciTech Connect

    Kieber, J.J.

    2010-05-01

    Cytokinins are a class of mitogenic plant hormones that play an important role in most aspects of plant development, including shoot and root growth, vascular and photomorphogenic development and leaf senescence. A model for cytokinin perception and signaling has emerged that is similar to bacterial two-component phosphorelays. In this model, binding of cytokinin to the extracellular domain of the Arabidopsis histidine kinase (AHKs) receptors induces autophosphorylation within the intracellular histidine-kinase domain. The phosphoryl group is subsequently transferred to cytosolic Arabidopsis histidine phosphotransfer proteins (AHPs), which have been suggested to translocate to the nucleus in response to cytokinin treatment, where they then transfer the phosphoryl group to nuclear-localized response regulators (Type-A and Type-B ARRs). We examined the effects of cytokinin on AHP subcellular localization in Arabidopsis and, contrary to expectations, the AHPs maintained a constant nuclear/cytosolic distribution following cytokinin treatment. Furthermore, mutation of the conserved phosphoacceptor histidine residue of the AHP, as well as disruption of multiple cytokinin signaling elements, did not affect the subcellular localization of the AHP proteins. Finally, we present data indicating that AHPs maintain a nuclear/cytosolic distribution by balancing active transport into and out of the nucleus. Our findings suggest that the current models indicating relocalization of AHP protein into the nucleus in response to cytokinin are incorrect. Rather, AHPs actively maintain a consistent nuclear/cytosolic distribution regardless of the status of the cytokinin response pathway.

  12. Proteomic Approach to Identify Nuclear Proteins in Wheat Grain.

    PubMed

    Bancel, Emmanuelle; Bonnot, Titouan; Davanture, Marlène; Branlard, Gérard; Zivy, Michel; Martre, Pierre

    2015-10-01

    The nuclear proteome of the grain of the two cultivated wheat species Triticum aestivum (hexaploid wheat; genomes A, B, and D) and T. monococcum (diploid wheat; genome A) was analyzed in two early stages of development using shotgun-based proteomics. A procedure was optimized to purify nuclei, and an improved protein sample preparation was developed to efficiently remove nonprotein substances (starch and nucleic acids). A total of 797 proteins corresponding to 528 unique proteins were identified, 36% of which were classified in functional groups related to DNA and RNA metabolism. A large number (107 proteins) of unknown functions and hypothetical proteins were also found. Some identified proteins may be multifunctional and may present multiple localizations. On the basis of the MS/MS analysis, 368 proteins were present in the two species, and in two stages of development, some qualitative differences between species and stages of development were also found. All of these data illustrate the dynamic function of the grain nucleus in the early stages of development. PMID:26228564

  13. Cellular distribution of ACT domain repeat protein 9, a nuclear localizing protein, in rice (Oryza sativa).

    PubMed

    Kudo, Toru; Kawai, Akiko; Yamaya, Tomoyuki; Hayakawa, Toshihiko

    2008-06-01

    Regulatory ACT domains serve as amino acid-binding sites in certain amino acid metabolic enzymes and transcriptional regulators in bacteria. The ACT domain repeat protein (ACR) family in plants is primarily composed of four copies of the domain homologous to those of the bacteria Gln sensor GLND. In the current study, to evaluate the possible involvement of the protein OsACR9 in the Gln-sensing system related to nitrogen (N) metabolism in rice (Oryza sativa L.), subcellular localization of OsACR9 and its accumulation and cellular distribution in various rice organs were examined by transient expression analysis and immunological methods using a monospecific antibody, respectively. Transient expression analysis of OsACR9 fused with a synthetic green fluorescent protein in cultured rice cells suggested nuclear localization of OsACR9. In rice roots, OsACR9 protein was distributed in epidermis, exodermis, sclerenchyma and vascular parenchyma cells, and its accumulation markedly increased after supply of NH(+)(4). In rice leaf samples, OsACR9 protein was abundant in the vascular parenchyma and mestome-sheath cells of young leaf blades at the early stage of development and in the vascular parenchyma and phloem-companion cells of mature leaf sheaths. OsACR9 protein also showed a high level of accumulation in vascular parenchyma cells of dorsal vascular bundles and aleurone cells in young rice grains at the early stage of ripening. The possibility of the nuclear protein OsACR9 acting as a Gln sensor in rice is subsequently discussed through comparison of its spatiotemporal expression with that of Gln-responsive N-assimilatory genes. PMID:18282189

  14. Dynamics of Protein Kinases: Insights from Nuclear Magnetic Resonance

    PubMed Central

    Xiao, Yao; Liddle, Jennifer C.; Pardi, Arthur; Ahn, Natalie G.

    2015-01-01

    CONSPECTUS Protein kinases are ubiquitous enzymes with critical roles in cellular processes and pathology. As a result, researchers have studied their activity and regulatory mechanisms extensively. Thousands of X-ray structures give snapshots of the architectures of protein kinases in various states of activation and ligand binding. However, the extent of and manner by which protein motions and conformational dynamics underlie the function and regulation of these important enzymes is not well understood. Nuclear magnetic resonance (NMR) methods provide complementary information about protein conformation and dynamics in solution. However, until recently, the large size of these enzymes prevented researchers from using these methods with kinases. Developments in transverse relaxation-optimized spectroscopy (TROSY)-based techniques and more efficient isotope labeling strategies are now allowing researchers to carry out NMR studies on full-length protein kinases. In this Account, we describe recent insights into the role of dynamics in protein kinase regulation and catalysis that have been gained from NMR measurements of chemical shift changes and line broadening, residual dipolar couplings, and relaxation. These findings show strong associations between protein motion and events that control kinase activity. Dynamic and conformational changes occurring at ligand binding sites and other regulatory domains of these proteins propagate to conserved kinase core regions that mediate catalytic function. NMR measurements of slow time scale (microsecond to millisecond) motions also reveal that kinases carry out global exchange processes that synchronize multiple residues and allosteric interconversion between conformational states. Activating covalent modifications or ligand binding to form the Michaelis complex can induce these global processes. Inhibitors can also exploit the exchange properties of kinases by using conformational selection to form dynamically quenched states. These investigations have revealed that kinases are highly dynamic enzymes, whose regulation by interdomain interactions, ligand binding, and covalent modifications involve changes in motion and conformational equilibrium in a manner that can be correlated with function. Thus, NMR provides a unique window into the role of protein dynamics in kinase regulation and catalysis with important implications for drug design. PMID:25803188

  15. Characterization of nuclear targeting signal of hepatitis delta antigen: nuclear transport as a protein complex.

    PubMed Central

    Xia, Y P; Yeh, C T; Ou, J H; Lai, M M

    1992-01-01

    Hepatitis delta antigen (HDAg) is the only protein encoded by hepatitis delta virus (HDV). HDAg has been demonstrated in the nuclei of HDV-infected hepatocytes, and its nuclear transport may be important for the replication of HDV RNA. In this report, we investigated the mechanism of nuclear transport of HDAg. By expressing fusion proteins consisting of the different portions of HDAg and alpha-globin, we have identified a nuclear localization signal (NLS) within the N-terminal one-third of HDAg. It consists of two stretches of basic amino acid domains separated by a short run of nonbasic amino acids. Both of the basic domains are necessary for the efficient nuclear transport of HDAg. The nonbasic spacer amino acids could be removed without affecting the nuclear targeting of HDAg significantly. Thus, the HDAg NLS belongs to a newly identified class of NLS which consists of two discontiguous stretches of basic amino acids. This NLS is separated from a stretch of steroid receptor NLS-like sequence, which is also present but not functioning as an NLS, in HDAg. Furthermore, we have shown that subfragments of HDAg which do not contain the NLS can be passively transported into the nucleus by a trans-acting full-length HDAg, provided that these subfragments contain the region with a leucine zipper sequence. Thus, our results indicate that HDAg forms aggregates in the cytoplasm and that the HDAg oligomerization is probably mediated by the leucine zipper sequence. Therefore, HDAg is likely transported into the nucleus as a protein complex. Images PMID:1731113

  16. Nuclear targeting of the maize R protein requires two nuclear localization sequences

    SciTech Connect

    Shieh, M.W.; Raikhel, N.V. ); Wessler, S.R. )

    1993-02-01

    Previous genetic and structural evidence indicates that the maize R gene encodes a nuclear transcriptional activating factor. In-frame carboxyl- and amino-terminal fusions of the R gene to the reporter gene encoding [beta]-glucuronidase (GUS) were sufficient to direct GUS to the nucleus of the transiently transformed onion (Allium cepa) epidermal cells. Further analysis of chimeric constructs containing regions of the R gene fused to the GUS cDNA revealed three specific nuclear localization sequences (NLSs) that were capable of redirecting the GUS protein to the nucleus. Amino-terminal NLS-A (amino acids 100-109, GDRRAAPARP) contained several arginine residues; a similar localization signal is found in only a few viral proteins. The medial NLS-M (amino acids 419-428, MSERKRREKL) is a simian virus 40 large T antigen-type NLS, and the carboxyl-terminal NLS-C (amino acids 598-610, MISESLRKAIGKR) is a mating type [alpha]2 type. NLSs M and C are independently sufficient to direct the GUS protein to the nucleus when it is fused at the amino terminus of GUS, whereas NLS-A fused to GUS partitioned between the nucleus and cytoplasm. Similar partitioning was observed when localization signals NLS-A and NLS-C were independently fused to the carboxy-terminal portion of GUS. A sequential deletion of the localization signals indicated that the amino-terminal and carboxyl-terminal fusions of R and GUS were redirected to the nucleus only when both NLS-A and -M, or NLS-C and -M, were present. These results indicate that multiple localization signals are necessary for nuclear targeting of this protein. The conservation of the localization signals within the alleles of R and similar proteins from other organisms is also discussed. 45 refs., 6 figs.

  17. An analysis of the protein interactions involved in classical nuclear import 

    E-print Network

    Reedy, Brian Michael Thomas

    1999-01-01

    Every aspect of eukaryotic cellular life involves the trafficking of proteins and other biomolecules through the nuclear membrane for the purposes of bringing feedback to or information from the nucleus. Many proteins ...

  18. Nanobody-targeted E3-ubiquitin ligase complex degrades nuclear proteins

    PubMed Central

    Ju Shin, Yeong; Kyun Park, Seung; Jung Jung, Yoo; Na Kim, Ye; Sung Kim, Ki; Kyu Park, Ok; Kwon, Seung-Hae; Ho Jeon, Sung; Trinh, Le A.; Fraser, Scott E.; Kee, Yun; Joon Hwang, Byung

    2015-01-01

    Targeted protein degradation is a powerful tool in determining the function of specific proteins or protein complexes. We fused nanobodies to SPOP, an adaptor protein of the Cullin-RING E3 ubiquitin ligase complex, resulting in rapid ubiquitination and subsequent proteasome-dependent degradation of specific nuclear proteins in mammalian cells and zebrafish embryos. This approach is easily modifiable, as substrate specificity is conferred by an antibody domain that can be adapted to target virtually any protein. PMID:26373678

  19. Interaction between a plasma membrane-localized ankyrin-repeat protein ITN1 and a nuclear protein RTV1

    SciTech Connect

    Sakamoto, Hikaru; Sakata, Keiko; Kusumi, Kensuke; Kojima, Mikiko; Sakakibara, Hitoshi; Iba, Koh

    2012-06-29

    Highlights: Black-Right-Pointing-Pointer ITN1, a plasma membrane ankyrin protein, interacts with a nuclear DNA-binding protein RTV1. Black-Right-Pointing-Pointer The nuclear transport of RTV1 is partially inhibited by interaction with ITN1. Black-Right-Pointing-Pointer RTV1 can promote the nuclear localization of ITN1. Black-Right-Pointing-Pointer Both overexpression of RTV1 and the lack of ITN1 increase salicylic acids sensitivity in plants. -- Abstract: The increased tolerance to NaCl 1 (ITN1) protein is a plasma membrane (PM)-localized protein involved in responses to NaCl stress in Arabidopsis. The predicted structure of ITN1 is composed of multiple transmembrane regions and an ankyrin-repeat domain that is known to mediate protein-protein interactions. To elucidate the molecular functions of ITN1, we searched for interacting partners using a yeast two-hybrid assay, and a nuclear-localized DNA-binding protein, RTV1, was identified as a candidate. Bimolecular fluorescence complementation analysis revealed that RTV1 interacted with ITN1 at the PM and nuclei in vivo. RTV1 tagged with red fluorescent protein localized to nuclei and ITN1 tagged with green fluorescent protein localized to PM; however, both proteins localized to both nuclei and the PM when co-expressed. These findings suggest that RTV1 and ITN1 regulate the subcellular localization of each other.

  20. Protein targeting to the nuclear pore. What can we learn from plants. [Nuclear pore complex

    SciTech Connect

    Smith, H.M.S.; Raikhel, N.V. . DOE Plant Research Lab.)

    1999-04-01

    Characteristic of eukaryotic cells are the numerous types of membrane-bound organelles or compartments found in the cytoplasm, with each type carrying out an essential function for the cell. The spatial separation of proteins and biochemical pathways typical of the various types of organelles requires selective targeting apparatuses. Because each type of organelle contains its own targeting apparatus, proteins destined for a particular organelle must contain the proper targeting signal(s) for entry. These signal-dependent targeting pathways ensure that proteins are targeted to the proper organelle. Understanding how proteins are targeted to the different types of organelles is an important goal in the field of cell biology. In plants recent studies have highlighted a number of unusual features, and as the understanding of import in plants increases, the authors have gained new insights, such as a model for the targeting of proteins from the cytoplasm to the NPC. These advances will contribute to further expansion of the knowledge of nuclear import in eukaryotes.

  1. Radioprotective Thiolamines WR-1065 and WR-33278 Selectively Denature Nonhistone Nuclear Proteins

    NASA Technical Reports Server (NTRS)

    Booth, Valerie K.; Roberts, Jeanette C.; Warters, Raymond L.; Wilmore, Britta H.; Lepock, James R.

    2000-01-01

    Differential scanning calorimetry was used to study the interactions of nuclei isolated from Chinese hamster V79 cells with the radioprotector WR-1065, other thiol compounds, and polyamines. Differential scanning calorimetry monitors denaturation of macromolecules and resolves the major nuclear components (e.g. constrained and relaxed DNA, nucleosome core, and nuclear matrix) of intact nuclei on the basis of thermal stability. WR-1065 treatment (0.5-10 mM) of isolated nuclei led to the irreversible denaturation of nuclear proteins, a fraction of which are nuclear matrix proteins. Denaturation of 50% of the total nonhistone nuclear protein content of isolated nuclei occurred after exposure to 4.7 mM WR-1065 for 20 min at 23 C. In addition, a 22% increase in the insoluble protein content of nuclei isolated from V79 cells that had been treated with 4 mM WR-1065 for 30 min at 37 C was observed, indicating that WR-1065-induced protein denaturation occurs not only in isolated nuclei but also in the nuclei of intact cells. From the extent of the increase in insoluble protein in the nucleus, protein denaturation by WR-1065 is expected to contribute to drug toxicity at concentrations greater than approximately 4 mM. WR-33278, the disulfide form of WR1065, was approximately twice as effective as the free thiol at denaturing nuclear proteins. The proposed mechanism for nucleoprotein denaturation is through direct interactions with protein cysteine groups with the formation of destabilizing protein-WR-1065 disulfides. In comparison to its effect on nuclear proteins in isolated nuclei, WR-1065 had only a very small effect on non-nuclear proteins of whole cells, isolated nuclear matrix, or the thiol-rich Ca (2+) ATPase of sarcoplasmic reticulum, indicating that WR-1065 can effectively denature protein only inside an intact nucleus, probably due to the increased concentration of the positively charged drug in the vicinity of DNA.

  2. Examining weak protein-protein interactions in start codon recognition via nuclear magnetic resonance spectroscopy

    PubMed Central

    Luna, Rafael E.; Akabayov, Sabine R.; Ziarek, Joshua J.; Wagner, Gerhard

    2014-01-01

    Weak protein-protein interactions are critical in numerous biological processes. Unfortunately, they are difficult to characterize due to the high concentrations required for the production and detection of the complex population. The inherent sensitivity of nuclear magnetic resonance (NMR) spectroscopy to the chemical environment makes it an excellent tool to tackle this problem. NMR permits the exploration of interactions over a range of affinities, yielding essential insights into dynamic biological processes. The conversion of mRNA to protein is one such process that requires the coordinated association of many low affinity proteins. During start codon recognition, eukaryotic initiation factors assemble into high-order complexes that bind mRNA and bring it to the ribosome for decoding. Many of the structures of the eukaryotic initiation factors have been determined; however, only little is known regarding the weak binary complexes formed and their structure-function mechanisms. Herein, we use start codon recognition as a model system to review the relevant NMR methods for the characterization of weak interactions and the development of small molecule inhibitors. PMID:24393460

  3. Probing Protein Quinary Interactions by In-Cell Nuclear Magnetic Resonance Spectroscopy

    PubMed Central

    Majumder, Subhabrata; Xue, Jing; DeMott, Christopher M.; Reverdatto, Sergey; Burz, David S.; Shekhtman, Alexander

    2015-01-01

    Historically introduced by McConkey to explain the slow mutation rate of highly abundant proteins, weak protein (quinary) interactions are an emergent property of living cells. The protein complexes that result from quinary interactions are transient and thus difficult to study biochemically in vitro. Cross-correlated relaxation-induced polarization transfer-based in-cell nuclear magnetic resonance allows the characterization of protein quinary interactions with atomic resolution inside live prokaryotic and eukaryotic cells. We show that RNAs are an important component of protein quinary interactions. Protein quinary interactions are unique to the target protein and may affect physicochemical properties, protein activity, and interactions with drugs. PMID:25894651

  4. Tandem affinity purification to identify cytosolic and nuclear g??-interacting proteins.

    PubMed

    Campden, Rhiannon; Pétrin, Darlaine; Robitaille, Mélanie; Audet, Nicolas; Gora, Sarah; Angers, Stéphane; Hébert, Terence E

    2015-01-01

    It has become clear in recent years that the G?? subunits of heterotrimeric proteins serve broad roles in the regulation of cellular activity and interact with many proteins in different subcellular locations including the nucleus. Protein affinity purification is a common method to identify and confirm protein interactions. When used in conjugation with mass spectrometry it can be used to identify novel protein interactions with a given bait protein. The tandem affinity purification (TAP) technique identifies partner proteins bound to tagged protein bait. Combined with protocols to enrich the nuclear fraction of whole cell lysate through sucrose cushions, TAP allows for purification of interacting proteins found specifically in the nucleus. Here we describe the use of the TAP technique on cytosolic and nuclear lysates to identify candidate proteins, through mass spectrometry, that bind to G?1 subunits. PMID:25304356

  5. The nuclear translocation assay for intracellular protein-protein interactions and its application to the Bcr coiled-coil domain

    PubMed Central

    Dixon, Andrew S.; Lim, Carol S.

    2010-01-01

    Protein interactions are critical for normal biological processes and molecular pathogenesis. While it is important to study these interactions, there are limited assays that are performed inside the cell, in the native cell environment, where the majority of protein-protein interactions take place. Here we present a method of studying protein interactions intracellularly using one protein of interest fused to a localization-controllable enhanced GFP (EGFP) construct and the other protein of interest fused to the red fluorescent protein, DsRed. Nuclear translocation of the EGFP construct is induced by addition of a ligand, and the difference in nuclear localization between the induced and noninduced states of the DsRed construct provides an indication of the interaction between the two proteins. This assay, the nuclear translocation assay (NTA), is introduced here as broadly applicable for studying protein interactions in the native environment inside cells and is demonstrated using forms of the coiled-coil domain from the breakpoint cluster region (Bcr) protein. PMID:20615205

  6. Regulation of Greatwall kinase by protein stabilization and nuclear localization

    PubMed Central

    Yamamoto, Tomomi M; Wang, Ling; Fisher, Laura A; Eckerdt, Frank D; Peng, Aimin

    2014-01-01

    Greatwall (Gwl) functions as an essential mitotic kinase by antagonizing protein phosphatase 2A. In this study we identified Hsp90, Cdc37 and members of the importin ? and ? families as the major binding partners of Gwl. Both Hsp90/Cdc37 chaperone and importin complexes associated with the N-terminal kinase domain of Gwl, whereas an intact glycine-rich loop at the N-terminus of Gwl was essential for binding of Hsp90/Cdc37 but not importins. We found that Hsp90 inhibition led to destabilization of Gwl, a mechanism that may partially contribute to the emerging role of Hsp90 in cell cycle progression and the anti-proliferative potential of Hsp90 inhibition. Moreover, in agreement with its importin association, Gwl exhibited nuclear localization in interphase Xenopus S3 cells, and dynamic nucleocytoplasmic distribution during mitosis. We identified KR456/457 as the locus of importin binding and the functional NLS of Gwl. Mutation of this site resulted in exclusion of Gwl from the nucleus. Finally, we showed that the Gwl nuclear localization is indispensable for the biochemical function of Gwl in promoting mitotic entry. PMID:25483093

  7. TATA-binding protein and nuclear differentiation in Tetrahymena thermophila.

    PubMed Central

    Stargell, L A; Gorovsky, M A

    1994-01-01

    Unambiguous TATA boxes have not been identified in upstream sequences of Tetrahymena thermophila genes analyzed to date. To begin a characterization of the promoter requirements for RNA polymerase II, the gene encoding TATA-binding protein (TBP) was cloned from this species. The derived amino acid sequence for the conserved C-terminal domain of Tetrahymena TBP is one of the most divergent described and includes a unique 20-amino-acid C-terminal extension. Polyclonal antibodies generated against a fragment of Tetrahymena TBP recognize a 36-kDa protein in macronuclear preparations and also cross-react with yeast and human TBPs. Immunocytochemistry was used to examine the nuclear localization of TBP during growth, starvation, and conjugation (the sexual phase of the life cycle). The transcriptionally active macronuclei stained at all stages of the life cycle. The transcriptionally inert micronuclei did not stain during growth or starvation but surprisingly stained with anti-TBP throughout early stages of conjugation. Anti-TBP staining disappeared from developing micronuclei late in conjugation, corresponding to the onset of transcription in developing macronuclei. Since micronuclei do not enlarge or divide at this time, loss of TBP appears to be an active process. Thus, the transcriptional differences between macro- and micronuclei that arise during conjugation are associated with the loss of a major component of the basal transcription apparatus from developing micronuclei rather than its appearance in developing macronuclei. Images PMID:8264641

  8. Nuclear substructure reorganization during late stageerythropoiesis is selective and does not involve caspase cleavage ofmajor nuclear substructural proteins

    SciTech Connect

    Krauss, Sharon Wald; Lo, Annie J.; Short, Sarah A.; Koury, MarkJ.; Mohandas, Narla; Chasis, Joel Anne

    2005-04-06

    Enucleation, a rare feature of mammalian differentiation, occurs in three cell types: erythroblasts, lens epithelium and keratinocytes. Previous investigations suggest that caspase activation functions in lens epithelial and keratinocyte enucleation, as well as in early erythropoiesis encompassing BFU-E differentiation to proerythroblast. To determine whether caspase activation contributes to later erythropoiesis and whether nuclear substructures other than chromatin reorganize, we analyzed distributions of nuclear subcompartment proteins and assayed for caspase-induced cleavage of subcompartmental target proteins in mouse erythroblasts. We found that patterns of lamin B in the filamentous network interacting with both the nuclear envelope and DNA, nuclear matrix protein NuMA, and splicing factors Sm and SC35 persisted during nuclear condensation, consistent with effective transcription of genes expressed late in differentiation. Thus nuclear reorganization prior to enucleation is selective, allowing maintenance of critical transcriptional processes independent of extensive chromosomal reorganization. Consistent with these data, we found no evidence for caspase-induced cleavage of major nuclear subcompartment proteins during late erythropoiesis, in contrast to what has been observed in early erythropoiesis and in lens epithelial and keratinocyte differentiation. These findings imply that nuclear condensation and extrusion during terminal erythroid differentiation involve novel mechanisms that do not entail major activation of apoptotic machinery.

  9. Evolving Regular Expression-based Sequence Classifiers for Protein Nuclear Localisation

    E-print Network

    Fernandez, Thomas

    if they are needed there. Most are actively transported in an energy-consuming process through the so-called nuclearEvolving Regular Expression-based Sequence Classifiers for Protein Nuclear Localisation Amine discovered by researchers in the laboratory. For exam- ple, patterns representing nuclear localisation

  10. A Bayesian Network Model of Proteins' Association with Promyelocytic Leukemia (PML) Nuclear Bodies

    E-print Network

    Dellaire, Graham

    A Bayesian Network Model of Proteins' Association with Promyelocytic Leukemia (PML) Nuclear Bodies. Promyelocytic leukemia nuclear bodies are implicated in important regulatory processes. To understand leukemia nuclear bodies accurately when interaction data is available. At a false positive rate of 10

  11. Yeast-Plant Coupled Vector System for Identification of Nuclear Proteins1[OA

    E-print Network

    Citovsky, Vitaly

    Yeast-Plant Coupled Vector System for Identification of Nuclear Proteins1[OA] Adi Zaltsman, Bu and visualization of their nuclear accumulation in vivo. To this end, we developed a system of vectors that allows characterization would be facilitated by a convenient experimental system that allows identification of nuclear

  12. ER Membrane Protein Complex Required for Nuclear Fusion Davis T.W. Ng and Peter Walter

    E-print Network

    Walter, Peter

    ER Membrane Protein Complex Required for Nuclear Fusion Davis T.W. Ng and Peter Walter Department is localized to the luminal (i.e., noncytoplasmic) face of the ER mem- brane, yet nuclear fusion must initiate of Sec63p, Sec71p, and Sec72p plays a central role in mediating nuclear mem- brane fusion and requires ER

  13. Identification of a nuclear localization signal in the retinitis pigmentosa-mutated RP26 protein, ceramide kinase-like protein

    SciTech Connect

    Inagaki, Yuichi; Mitsutake, Susumu; Igarashi, Yasuyuki . E-mail: yigarash@pharm.hokudai.ac.jp

    2006-05-12

    Retinitis pigmentosa (RP) is a genetically heterogeneous disease characterized by degeneration of the retina. A mutation in a new ceramide kinase (CERK) homologous gene, named CERK-like protein (CERKL), was found to cause autosomal recessive retinitis pigmentosa (RP26). Here, we show a point mutation of one of two putative nuclear localization signal (NLS) sequences inhibited the nuclear localization of the protein. Furthermore, the tetra-GFP-tagged NLS, which cannot passively enter the nucleus, was observed not only in the nucleus but also in the nucleolus. Our results provide First evidence of the active nuclear import of CERKL and suggest that the identified NLS might be responsible for nucleolar retention of the protein. As recent studies have shown other RP-related proteins are localized in the nucleus or the nucleolus, our identification of NLS in CERKL suggests that CERKL likely plays important roles for retinal functions in the nucleus and the nucleolus.

  14. Light-induced nuclear import of phytochrome-A:GFP fusion proteins is differentially regulated in transgenic tobacco

    E-print Network

    Schäfer, Eberhard

    Light-induced nuclear import of phytochrome-A:GFP fusion proteins is differentially regulated of the fusion protein was observed. By contrast, light-dependent nuclear import of the same fusion protein have recently shown that nuclear import of rice phyA:GFP is regulated by VLFR in transgenic tobacco

  15. Insights into the origin of the nuclear localization signals in conserved ribosomal proteins

    PubMed Central

    Melnikov, Sergey; Ben-Shem, Adam; Yusupova, Gulnara; Yusupov, Marat

    2015-01-01

    Eukaryotic ribosomal proteins, unlike their bacterial homologues, possess nuclear localization signals (NLSs) to enter the cell nucleus during ribosome assembly. Here we provide a comprehensive comparison of bacterial and eukaryotic ribosomes to show that NLSs appear in conserved ribosomal proteins via remodelling of their RNA-binding domains. This finding enabled us to identify previously unknown NLSs in ribosomal proteins from humans, and suggests that, apart from promoting protein transport, NLSs may facilitate folding of ribosomal RNA. PMID:26066547

  16. Overlapping signals for protein degradation and nuclear localization define a role for intrinsic RAG-2 nuclear uptake in dividing cells.

    PubMed

    Ross, Ashley E; Vuica, Milena; Desiderio, Stephen

    2003-08-01

    Expression of the recombinase proteins RAG-1 and RAG-2 is discordant: while RAG-1 is relatively long lived, RAG-2 is degraded periodically at the G(1)-S transition. Destruction of RAG-2 is mediated by a conserved interval in the recombination-dispensable region. The need for RAG-2 to reaccumulate in the nucleus at each cell division suggested the existence of an intrinsic RAG-2 nuclear localization signal (NLS). RAG-1 or RAG-2, expressed individually, is a nuclear protein. A screen for proteins that bind the recombination-dispensable region of RAG-2 identified the nuclear transport protein Importin 5. Mutation of residues 499 to 508 in RAG-2 abolished Importin 5 binding, nuclear accumulation, and periodic degradation of RAG-2. The Importin 5 binding site overlaps an NLS, defined by mutagenesis. RAG-1 rescued the localization of degradation-defective, RAG-2 NLS mutants; this required an intact RAG-1 NLS. Mutations in RAG-2 that abolish intrinsic nuclear accumulation but spare periodic degradation impaired recombination in cycling cells; induction of quiescence restored recombination to wild-type levels. Recombination defects were correlated with a cell cycle-dependent defect in the ability of RAG-1 to rescue localization of the RAG-2 mutants. These results suggest that the intrinsic RAG-2 NLS functions in the nuclear uptake of RAG-2 following its reexpression in cycling cells. PMID:12861017

  17. cGMP-dependent protein kinase I gamma encodes a nuclear localization signal that regulates nuclear compartmentation and function

    PubMed Central

    Chen, Jingsi; Roberts, Jesse D.

    2014-01-01

    cGMP-dependent protein kinase I (PKGI) plays an important role in regulating how cGMP specifies vascular smooth muscle cell (SMC) phenotype. Although studies indicate that PKGI nuclear localization controls how cGMP regulates gene expression in SMC, information about the mechanisms that regulate PKGI nuclear compartmentation and its role in directly regulating cell phenotype is limited. Here we characterize a nuclear localization signal sequence (NLS) in PKGI?, a proteolytically cleaved PKGI kinase fragment that translocates to the nucleus of SMC. Immuno-localization studies using cells expressing native and NLS-mutant PKGI?, and treated with a small molecule nuclear transport inhibitor, indicated that PKGI? encodes a constitutively active NLS that requires importin ? and ? for regulation of its compartmentation. Moreover, studies utilizing a genetically encoded nuclear phospho-CREB biosensor probe and fluorescence lifetime imaging microscopy demonstrated that this NLS controls PKGI? nuclear function. In addition, although cytosolic PKGI?-activity was observed to stimulate MAPK/ERK-mediated nuclear CREB signalling in SMC, NLS-mediated PKGI? nuclear activity alone was determined to increase the expression of differentiation marker proteins in these cells. These results indicate that NLS-mediated nuclear PKGI? localization plays an important role in how PKGI regulates vascular SMC phenotype. PMID:25172423

  18. Identification of an unconventional nuclear localization signal in human ribosomal protein S2

    SciTech Connect

    Antoine, M.; Reimers, K.; Wirz, W.; Gressner, A.M.; Mueller, R.; Kiefer, P. . E-Mail: pkiefer@ukaachen.de

    2005-09-16

    Ribosomal proteins must be imported into the nucleus after being synthesized in the cytoplasm. Since the rpS2 amino acid sequence does not contain a typical nuclear localization signal, we used deletion mutant analysis and rpS2-{beta}-galactosidase chimeric proteins to identify the nuclear targeting domains in rpS2. Nuclear rpS2 is strictly localized in the nucleoplasm and is not targeted to the nucleoli. Subcellular localization analysis of deletion mutants of rpS2-{beta}-galactosidase chimeras identified a central domain comprising 72 amino acids which is necessary and sufficient to target the chimeric {beta}-galactosidase to the nucleus. The nuclear targeting domain shares no significant similarity to already characterized nuclear localization signals in ribosomal proteins or other nuclear proteins. Although a Nup153 fragment containing the importin{beta} binding site fused to VP22 blocks nuclear import of rpS2-{beta}-galactosidase fusion proteins, nuclear uptake of rpS2 could be mediated by several import receptors since it binds to importin{alpha}/{beta} and transportin.

  19. Efficient plant male fertility depends on vegetative nuclear movement mediated by two families of plant outer nuclear membrane proteins

    PubMed Central

    Zhou, Xiao; Meier, Iris

    2014-01-01

    Increasing evidence suggests that nuclear migration is important for eukaryotic development. Although nuclear migration is conserved in plants, its importance for plant development has not yet been established. The most extraordinary plant nuclear migration events involve plant fertilization, which is starkly different from that of animals. Instead of evolving self-propelled sperm cells (SCs), plants use pollen tubes to deliver SCs, in which the pollen vegetative nucleus (VN) and the SCs migrate as a unit toward the ovules, a fundamental but barely understood process. Here, we report that WPP domain-interacting proteins (WIPs) and their binding partners the WPP domain-interacting tail-anchored proteins (WITs) are essential for pollen nuclear migration. Loss-of-function mutations in WIT and/or WIP gene families resulted in impaired VN movement, inefficient SC delivery, and defects in pollen tube reception. WIPs are Klarsicht/ANC-1/Syne-1 Homology (KASH) analogs in plants. KASH proteins are key players in animal nuclear migration. Thus, this study not only reveals an important nuclear migration mechanism in plant fertilization but also, suggests that similar nuclear migration machinery is conserved between plants and animals. PMID:25074908

  20. Heterogeneity in the kinetics of nuclear proteins and trajectories of substructures associated with heterochromatin

    PubMed Central

    2011-01-01

    Background Protein exchange kinetics correlate with the level of chromatin condensation and, in many cases, with the level of transcription. We used fluorescence recovery after photobleaching (FRAP) to analyse the kinetics of 18 proteins and determine the relationships between nuclear arrangement, protein molecular weight, global transcription level, and recovery kinetics. In particular, we studied heterochromatin-specific heterochromatin protein 1? (HP1?) B lymphoma Mo-MLV insertion region 1 (BMI1), and telomeric-repeat binding factor 1 (TRF1) proteins, and nucleolus-related proteins, upstream binding factor (UBF) and RNA polymerase I large subunit (RPA194). We considered whether the trajectories and kinetics of particular proteins change in response to histone hyperacetylation by histone deacetylase (HDAC) inhibitors or after suppression of transcription by actinomycin D. Results We show that protein dynamics are influenced by many factors and events, including nuclear pattern and transcription activity. A slower recovery after photobleaching was found when proteins, such as HP1?, BMI1, TRF1, and others accumulated at specific foci. In identical cells, proteins that were evenly dispersed throughout the nucleoplasm recovered more rapidly. Distinct trajectories for HP1?, BMI1, and TRF1 were observed after hyperacetylation or suppression of transcription. The relationship between protein trajectory and transcription level was confirmed for telomeric protein TRF1, but not for HP1? or BMI1 proteins. Moreover, heterogeneity of foci movement was especially observed when we made distinctions between centrally and peripherally positioned foci. Conclusion Based on our results, we propose that protein kinetics are likely influenced by several factors, including chromatin condensation, differentiation, local protein density, protein binding efficiency, and nuclear pattern. These factors and events likely cooperate to dictate the mobility of particular proteins. PMID:21418567

  1. Nuclear localization and cell cycle regulation of a murine protein tyrosine phosphatase.

    PubMed Central

    Tillmann, U; Wagner, J; Boerboom, D; Westphal, H; Tremblay, M L

    1994-01-01

    MPTP is a murine homolog of the human T-cell protein tyrosine phosphatase (PTPase) and the rat PTP-S enzyme. Enzymatic activity of this ubiquitously expressed protein was demonstrated in immunoprecipitates from NIH 3T3 cells and in recombinant protein overexpressed in bacteria. Expression of beta-galactosidase-MPTP MPTP chimeric proteins in COS1 cells identified a nuclear localization signal at the carboxyl terminus of the MPTP that was sufficient to direct beta-galactosidase as well as a tagged version of the MPTP to the nucleus. Deletion analysis of amino acids within the nuclear targeting signal showed that this sequence does not conform to the bipartite type of nuclear localization signals. Furthermore, it was shown that the steady-state levels of MPTP RNA fluctuate in a cell cycle-specific manner. On the basis of these experiments, we discuss the possible function of MPTP in the cell cycle and other nuclear processes. Images PMID:8164659

  2. Identification of Protein-Bound Dinitrosyl Iron Complexes by Nuclear Resonance Vibrational Spectroscopy

    E-print Network

    Wang, Hongxin

    We have applied [superscript 57]Fe nuclear resonance vibrational spectroscopy (NRVS) to identify protein-bound dinitrosyl iron complexes. Intense NRVS peaks due to vibrations of the N?Fe?N unit can be observed between 500 ...

  3. Nuclear envelope localization of Ran-binding protein 2 and Ran-GTPase-activating protein 1 in psoriatic epidermal keratinocytes.

    PubMed

    Yasuda, Kayo; Sugiura, Kazumitsu; Takeichi, Takuya; Ogawa, Yasushi; Muro, Yoshinao; Akiyama, Masashi

    2014-02-01

    The nuclear localization signal (NLS)-containing proteins LEDGF and STAT3 localize to the nucleus in both the spinous and basal layers of the epidermis in psoriatic skin, where they function as transcription factors or co-factors to activate epidermal keratinocytes (KCs). However, the mechanism underlying the localization of these proteins remains to be elucidated. We investigated the differential nucleocytoplasmic transport of NLS-containing proteins as a potential pathogenic mechanism for psoriasis vulgaris. Nucleoporins play an important role in the Ran-GTP-dependent nucleocytoplasmic transport of NLS-containing proteins. We showed, using immunohistochemical staining, that the nucleoporins Ran-binding protein 2 (RanBP2) and Ran-GTPase-activating protein 1 (RanGAP1) have greater expression on the nuclear envelope in psoriatic epidermal KCs than in KCs from healthy controls. We then studied the signalling pathways involved in the regulation of these proteins in HaCaT cells. The two major downstream pathways of epidermal growth factor receptor (EGFR) signalling activated in psoriatic KCs are the MAPK/Erk/1/2 and the phosphatidylinositol-3-kinase/Akt pathways. Therefore, we treated HaCaT cells with inhibitors to disrupt the MAP kinase kinase 1 (MEK1), PI3-kinase, or mTOR pathways. RanBP2 and RanGAP1 protein expression levels were significantly greater in the nuclear envelope of HaCaT cells that were not treated with inhibitors than in cells treated with a combination of PI3-kinase and MEK1 inhibitors or mTOR and MEK1 inhibitors. These results suggest that adequate nuclear envelope expression of RanBP2 and RanGAP1 could be a prerequisite for nucleocytoplasmic transport in KCs in psoriatic epidermis. PMID:24438026

  4. Organellar proteomics reveals hundreds of novel nuclear proteins in the malaria parasite Plasmodium falciparum

    PubMed Central

    2012-01-01

    Background The post-genomic era of malaria research provided unprecedented insights into the biology of Plasmodium parasites. Due to the large evolutionary distance to model eukaryotes, however, we lack a profound understanding of many processes in Plasmodium biology. One example is the cell nucleus, which controls the parasite genome in a development- and cell cycle-specific manner through mostly unknown mechanisms. To study this important organelle in detail, we conducted an integrative analysis of the P. falciparum nuclear proteome. Results We combined high accuracy mass spectrometry and bioinformatic approaches to present for the first time an experimentally determined core nuclear proteome for P. falciparum. Besides a large number of factors implicated in known nuclear processes, one-third of all detected proteins carry no functional annotation, including many phylum- or genus-specific factors. Importantly, extensive experimental validation using 30 transgenic cell lines confirmed the high specificity of this inventory, and revealed distinct nuclear localization patterns of hitherto uncharacterized proteins. Further, our detailed analysis identified novel protein domains potentially implicated in gene transcription pathways, and sheds important new light on nuclear compartments and processes including regulatory complexes, the nucleolus, nuclear pores, and nuclear import pathways. Conclusion Our study provides comprehensive new insight into the biology of the Plasmodium nucleus and will serve as an important platform for dissecting general and parasite-specific nuclear processes in malaria parasites. Moreover, as the first nuclear proteome characterized in any protist organism, it will provide an important resource for studying evolutionary aspects of nuclear biology. PMID:23181666

  5. Isolation of nuclear proteins from flax (Linum usitatissimum L.) seed coats for gene expression regulation studies

    PubMed Central

    2012-01-01

    Background While seed biology is well characterized and numerous studies have focused on this subject over the past years, the regulation of seed coat development and metabolism is for the most part still non-elucidated. It is well known that the seed coat has an essential role in seed development and its features are associated with important agronomical traits. It also constitutes a rich source of valuable compounds such as pharmaceuticals. Most of the cell genetic material is contained in the nucleus; therefore nuclear proteins constitute a major actor for gene expression regulation. Isolation of nuclear proteins responsible for specific seed coat expression is an important prerequisite for understanding seed coat metabolism and development. The extraction of nuclear proteins may be problematic due to the presence of specific components that can interfere with the extraction process. The seed coat is a rich source of mucilage and phenolics, which are good examples of these hindering compounds. Findings In the present study, we propose an optimized nuclear protein extraction protocol able to provide nuclear proteins from flax seed coat without contaminants and sufficient yield and quality for their use in transcriptional gene expression regulation by gel shift experiments. Conclusions Routinely, around 250 ?g of nuclear proteins per gram of fresh weight were extracted from immature flax seed coats. The isolation protocol described hereafter may serve as an effective tool for gene expression regulation and seed coat-focused proteomics studies. PMID:22230709

  6. Identification of NTF2, a cytosolic factor for nuclear import that interacts with nuclear pore complex protein p62

    PubMed Central

    1995-01-01

    Protein import into the nucleus is a multistep process that requires the activities of several cytosolic factors. In this study we have purified a cytosolic factor that interacts with the nuclear pore complex glycoprotein p62. Isolation involved biochemical complementation of cytosol depleted of this activity by preadsorption with recombinant p62 and the use of a novel flow cytometry-based assay for quantitation of nuclear import. The purified activity (NTF2) is an apparent dimer of approximately 14-kD subunits and is present at approximately 10(6) copies per cell. We obtained a cDNA encoding NTF2 and showed that the recombinant protein restores transport activity to p62-pretreated cytosol. Our data suggest that NTF2 acts at a relatively late stage of nuclear protein import, subsequent to the initial docking of nuclear import ligand at the nuclear envelope. NTF2 interacts with at least one additional cytosolic transport activity, indicating that it could be part of a multicomponent system of cytosolic factors that assemble at the pore complex during nuclear import. PMID:7744965

  7. C. elegans Nuclear Envelope Proteins Emerin, MAN1, Lamin, and Nucleoporins Reveal Unique Timing of Nuclear Envelope Breakdown during Mitosis

    PubMed Central

    Lee, Kenneth K.; Gruenbaum, Yosef; Spann, Perah; Liu, Jun; Wilson, Katherine L.

    2000-01-01

    Emerin, MAN1, and LAP2 are integral membrane proteins of the vertebrate nuclear envelope. They share a 43-residue N-terminal motif termed the LEM domain. We found three putative LEM domain genes in Caenorhabditis elegans, designated emr-1, lem-2, and lem-3. We analyzed emr-l, which encodes Ce-emerin, and lem-2, which encodes Ce-MAN1. Ce-emerin and Ce-MAN1 migrate on SDS-PAGE as 17- and 52-kDa proteins, respectively. Based on their biochemical extraction properties and immunolocalization, both Ce-emerin and Ce-MAN1 are integral membrane proteins localized at the nuclear envelope. We used antibodies against Ce-MAN1, Ce-emerin, nucleoporins, and Ce-lamin to determine the timing of nuclear envelope breakdown during mitosis in C. elegans. The C. elegans nuclear envelope disassembles very late compared with vertebrates and Drosophila. The nuclear membranes remained intact everywhere except near spindle poles during metaphase and early anaphase, fully disassembling only during mid-late anaphase. Disassembly of pore complexes, and to a lesser extent the lamina, depended on embryo age: pore complexes were absent during metaphase in >30-cell embryos but existed until anaphase in 2- to 24-cell embryos. Intranuclear mRNA splicing factors disassembled after prophase. The timing of nuclear disassembly in C. elegans is novel and may reflect its evolutionary position between unicellular and more complex eukaryotes. PMID:10982402

  8. Evidence for Ubiquitin-Regulated Nuclear and Subnuclear Trafficking among Paramyxovirinae Matrix Proteins

    PubMed Central

    Pentecost, Mickey; Vashisht, Ajay A.; Beaty, Shannon M.; Park, Arnold; Wang, Yao E.; Yun, Tatyana E; Freiberg, Alexander N.; Wohlschlegel, James A.; Lee, Benhur

    2015-01-01

    The paramyxovirus matrix (M) protein is a molecular scaffold required for viral morphogenesis and budding at the plasma membrane. Transient nuclear residence of some M proteins hints at non-structural roles. However, little is known regarding the mechanisms that regulate the nuclear sojourn. Previously, we found that the nuclear-cytoplasmic trafficking of Nipah virus M (NiV-M) is a prerequisite for budding, and is regulated by a bipartite nuclear localization signal (NLSbp), a leucine-rich nuclear export signal (NES), and monoubiquitination of the K258 residue within the NLSbp itself (NLSbp-lysine). To define whether the sequence determinants of nuclear trafficking identified in NiV-M are common among other Paramyxovirinae M proteins, we generated the homologous NES and NLSbp-lysine mutations in M proteins from the five major Paramyxovirinae genera. Using quantitative 3D confocal microscopy, we determined that the NES and NLSbp-lysine are required for the efficient nuclear export of the M proteins of Nipah virus, Hendra virus, Sendai virus, and Mumps virus. Pharmacological depletion of free ubiquitin or mutation of the conserved NLSbp-lysine to an arginine, which inhibits M ubiquitination, also results in nuclear and nucleolar retention of these M proteins. Recombinant Sendai virus (rSeV-eGFP) bearing the NES or NLSbp-lysine M mutants rescued at similar efficiencies to wild type. However, foci of cells expressing the M mutants displayed marked fusogenicity in contrast to wild type, and infection did not spread. Recombinant Mumps virus (rMuV-eGFP) bearing the homologous mutations showed similar defects in viral morphogenesis. Finally, shotgun proteomics experiments indicated that the interactomes of Paramyxovirinae M proteins are significantly enriched for components of the nuclear pore complex, nuclear transport receptors, and nucleolar proteins. We then synthesize our functional and proteomics data to propose a working model for the ubiquitin-regulated nuclear-cytoplasmic trafficking of cognate paramyxovirus M proteins that show a consistent nuclear trafficking phenotype. PMID:25782006

  9. The farnesylated nuclear proteins KUGELKERN and LAMIN B promote aging-like phenotypes in Drosophila flies.

    PubMed

    Brandt, Annely; Krohne, Georg; Grosshans, Jörg

    2008-08-01

    The nuclear lamina consists of a meshwork of lamins and lamina-associated proteins, which provide mechanical support, control size and shape of the nucleus, and mediate the attachment of chromatin to the nuclear envelope. Abnormal nuclear shapes are observed in aging cells of humans and nematode worms. The expression of laminDelta50, a constitutively active lamin A splicing variant in Hutchinson-Gilford progeria syndrome patients, leads to the lobulation of the nuclear envelope accompanied by DNA damage, and loss of heterochromatin. So far, it has been unclear whether these age-related changes are laminDelta50 specific or whether proteins that affect nuclear shape such as KUGELKERN or LAMIN B in general play a causative role in senescence. Here we show that in adult Drosophila flies, the size of the nuclei increases with age and the nuclei assume an aberrant shape. Moreover, induced expression of the farnesylated lamina proteins Lamin B and Kugelkern cause aberrant nuclear shapes and reduce the lifespan of adult flies. The shorter lifespan correlates with an early decline in age-dependent locomotor behaviour. Expression of kugelkern or lamin B in mammalian cells induces a nuclear lobulation phenotype in conjunction with DNA damage, and changes in histone modification similar to that found in cells expressing laminDelta50 or in cells from aged individuals. We conclude that lobulation of the nuclear membrane induced by the insertion of farnesylated lamina-proteins can lead to aging-like phenotypes. PMID:18494863

  10. Structure Determination of Membrane Proteins by Nuclear Magnetic Resonance Spectroscopy

    NASA Astrophysics Data System (ADS)

    Opella, Stanley J.

    2013-06-01

    Many biological membranes consist of 50% or more (by weight) membrane proteins, which constitute approximately one-third of all proteins expressed in biological organisms. Helical membrane proteins function as receptors, enzymes, and transporters, among other unique cellular roles. Additionally, most drugs have membrane proteins as their receptors, notably the superfamily of G protein-coupled receptors with seven transmembrane helices. Determining the structures of membrane proteins is a daunting task because of the effects of the membrane environment; specifically, it has been difficult to combine biologically compatible environments with the requirements for the established methods of structure determination. There is strong motivation to determine the structures in their native phospholipid bilayer environment so that perturbations from nonnatural lipids and phases do not have to be taken into account. At present, the only method that can work with proteins in liquid crystalline phospholipid bilayers is solid-state NMR spectroscopy.

  11. Nuclear localization and in vivo dynamics of a plant-specic serine/arginine-rich protein

    E-print Network

    Reddy, A.S.N

    Nuclear localization and in vivo dynamics of a plant-speci®c serine/arginine-rich protein Gul Shad@colostate.edu). y These authors have contributed equally to this work. Summary Serine/arginine-rich (SR) proteins; Schuler, 1998; Zhou et al., 2002). Among the splicing factors, the serine/arginine-rich (SR) family

  12. Benzo[a]pyrene treatment leads to changes in nuclear protein expression and alternative splicing.

    PubMed

    Yan, Chunlan; Wu, Wei; Li, Haiyan; Zhang, Guanglin; Duerksen-Hughes, Penelope J; Zhu, Xinqiang; Yang, Jun

    2010-04-01

    Benzo[a]pyrene (BaP) is a potent pro-carcinogen generated from the combustion of fossil fuel and cigarette smoke. Previously, using a proteomic approach, we have shown that BaP can induce changes in the expression of many cellular proteins, including transcription regulators. In the present study, using a similar approach, we examined the nuclear protein response to BaP in HeLa cells and found that BaP treatment caused expression changes in many nuclear proteins. Twenty-four of these proteins were successfully identified, several of which are involved in the alternative splicing of mRNA, DNA replication, recombination, and repair. The changed expression levels were further confirmed by immunoblot analysis using specific antibodies for two proteins, Lamin A and mitotic checkpoint protein Bub3. The nuclear localization of these two proteins was also confirmed by confocal microscopy. To determine whether alternative splicing was activated following BaP treatment, we examined Fas and CD44, two genes previously shown to be targets of alternative splicing in respond to DNA damage. While no significant activation of alternative splicing was observed for Fas, CD44 splicing variants were found after BaP treatment. Together, these data show that DNA damage induces dramatic changes in nuclear protein expression, and that alternative splicing might be involved in the cellular response to DNA damage. PMID:20097212

  13. Dynamical deductions from nuclear magnetic resonance relaxation measurements at the water-protein interface.

    PubMed Central

    Bryant, R G; Shirley, W M

    1980-01-01

    Nuclear magnetic resonance (NMR) measurements provide both structural and dynamical information about the molecules in which nuclear resonances are observed. This manuscript addresses NMR relaxation of water protons in protein powder systems. Inclusion of magnetic communication between the water proton spins and protein proton spins leads to a clearer view of water molecule dynamics at the protein surface than has been previously available. We conclude that water molecule motion at the protein surface is somewhat slower than in the solute free solvent, but it is orders of magnitude faster than motions in a rigid ice lattice even in samples hydrated to levels well below what is generally thought to be the full hydration complement of the protein. The NMR relaxation data on lysozyme powders support a model that leaves adsorbed water very fluid at the protein surface with reorientational correlation times for the water shorter than nanoseconds. PMID:7248450

  14. Nuclear Cytoplasmic Trafficking of Proteins is a Major Response of Human Fibroblasts to Oxidative Stress

    PubMed Central

    Baqader, Noor O.; Radulovic, Marko; Crawford, Mark; Stoeber, Kai; Godovac-Zimmermann, Jasminka

    2014-01-01

    We have used a subcellular spatial razor approach based on LC–MS/MS-based proteomics with SILAC isotope labeling to determine changes in protein abundances in the nuclear and cytoplasmic compartments of human IMR90 fibroblasts subjected to mild oxidative stress. We show that response to mild tert-butyl hydrogen peroxide treatment includes redistribution between the nucleus and cytoplasm of numerous proteins not previously associated with oxidative stress. The 121 proteins with the most significant changes encompass proteins with known functions in a wide variety of subcellular locations and of cellular functional processes (transcription, signal transduction, autophagy, iron metabolism, TCA cycle, ATP synthesis) and are consistent with functional networks that are spatially dispersed across the cell. Both nuclear respiratory factor 2 and the proline regulatory axis appear to contribute to the cellular metabolic response. Proteins involved in iron metabolism or with iron/heme as a cofactor as well as mitochondrial proteins are prominent in the response. Evidence suggesting that nuclear import/export and vesicle-mediated protein transport contribute to the cellular response was obtained. We suggest that measurements of global changes in total cellular protein abundances need to be complemented with measurements of the dynamic subcellular spatial redistribution of proteins to obtain comprehensive pictures of cellular function. PMID:25133973

  15. A network of nuclear envelope membrane proteins linking centromeres to microtubules.

    PubMed

    King, Megan C; Drivas, Theodore G; Blobel, Günter

    2008-08-01

    In the fission yeast S. pombe, nuclei are actively positioned at the cell center by microtubules. Here, we show that cytoplasmic microtubules are mechanically coupled to the nuclear heterochromatin through proteins embedded in the nuclear envelope. This includes an integral outer nuclear membrane protein of the KASH family (Kms2) and two integral inner nuclear membrane proteins, the SUN-domain protein Sad1 and the previously uncharacterized protein Ima1. Ima1 specifically binds to heterochromatic regions and promotes the tethering of centromeric DNA to the SUN-KASH complex. In the absence of Ima1, or in cells harboring mutations in the centromeric Ndc80 complex, inefficient coupling of centromeric heterochromatin to Sad1 leads to striking defects in the ability of the nucleus to tolerate microtubule-dependent forces, leading to changes in nuclear shape, loss of spindle pole body components from the nuclear envelope, and partial dissociation of SUN-KASH complexes. This work highlights a framework for communication between cytoplasmic microtubules and chromatin. PMID:18692466

  16. A functional nuclear localization sequence in the VP1 capsid protein of coxsackievirus B3

    SciTech Connect

    Wang, Tianying; Yu, Bohai; Lin, Lexun; Zhai, Xia; Han, Yelu; Qin, Ying; Guo, Zhiwei; Wu, Shuo; Zhong, Xiaoyan; Wang, Yan; Tong, Lei; Zhang, Fengmin; Si, Xiaoning; Zhao, Wenran; Zhong, Zhaohua

    2012-11-25

    The capsid proteins of some RNA viruses can translocate to the nucleus and interfere with cellular phenotypes. In this study we found that the VP1 capsid protein of coxsackievirus B3 (CVB3) was dominantly localized in the nucleus of the cells transfected with VP1-expressing plasmid. The VP1 nuclear localization also occurred in the cells infected with CVB3. Truncation analysis indicated that the VP1 nuclear localization sequence located near the C-terminal. The substitution of His220 with threonine completely abolished its translocation. The VP1 proteins of other CVB types might have the nuclear localization potential because this region was highly conserved. Moreover, the VP1 nuclear localization induced cell cycle deregulation, including a prolonged S phase and shortened G2-M phase. Besides these findings, we also found a domain between Ala72 and Phe106 that caused the VP1 truncates dotted distributed in the cytoplasm. Our results suggest a new pathogenic mechanism of CVB. - Highlights: Black-Right-Pointing-Pointer The VP1 protein of coxsackievirus B3 can specifically localize in the nucleus. Black-Right-Pointing-Pointer The nuclear localization signal of coxsackievirus B3 VP1 protein locates near its C-terminal. Black-Right-Pointing-Pointer The VP1 nuclear localization of coxsackievirus B3 can deregulate cell cycle. Black-Right-Pointing-Pointer There is a domain in the VP1 that determines it dotted distributed in the cytoplasm.

  17. Nuclear-cytoplasmatic shuttling of proteins in control of cellular oxygen sensing.

    PubMed

    Depping, Reinhard; Jelkmann, Wolfgang; Kosyna, Friederike Katharina

    2015-06-01

    In order to pass through the nuclear pore complex, proteins larger than ?40 kDa require specific nuclear transport receptors. Defects in nuclear-cytoplasmatic transport affect fundamental processes such as development, inflammation and oxygen sensing. The transcriptional response to O2 deficiency is controlled by hypoxia-inducible factors (HIFs). These are heterodimeric transcription factors of each ?100-120 kDa proteins, consisting of one out of three different O2-labile ? subunits (primarily HIF-1?) and a more constitutive 1? subunit. In the presence of O2, the ? subunits are hydroxylated by specific prolyl-4-hydroxylase domain proteins (PHD1, PHD2, and PHD3) and an asparaginyl hydroxylase (factor inhibiting HIF-1, FIH-1). The prolyl hydroxylation causes recognition by von Hippel-Lindau tumor suppressor protein (pVHL), ubiquitination, and proteasomal degradation. The activity of the oxygen sensing machinery depends on dynamic intracellular trafficking. Nuclear import of HIF-1? and HIF-1? is mainly mediated by importins ? and ? (?/?). HIF-1? can shuttle between nucleus and cytoplasm, while HIF-1? is permanently inside the nucleus. pVHL is localized to both compartments. Nuclear import of PHD1 relies on a nuclear localization signal (NLS) and uses the classical import pathway involving importin ?/? receptors. PHD2 shows an atypical NLS, and its nuclear import does not occur via the classical pathway. PHD2-mediated hydroxylation of HIF-1? occurs predominantly in the cell nucleus. Nuclear export of PHD2 involves a nuclear export signal (NES) in the N-terminus and depends on the export receptor chromosome region maintenance 1 (CRM1). Nuclear import of PHD3 is mediated by importin ?/? receptors and depends on a non-classical NLS. Specific modification of the nuclear translocation of the three PHD isoforms could provide a promising strategy for the development of new therapeutic substances to tackle major diseases. PMID:25809665

  18. Nuclear envelope-associated endosomes deliver surface proteins to the nucleus

    PubMed Central

    Chaumet, Alexandre; Wright, Graham D.; Seet, Sze Hwee; Tham, Keit Min; Gounko, Natalia V.; Bard, Frederic

    2015-01-01

    Endocytosis directs molecular cargo along three main routes: recycling to the cell surface, transport to the Golgi apparatus or degradation in endolysosomes. Pseudomonas exotoxin A (PE) is a bacterial protein that typically traffics to the Golgi and then the endoplasmic reticulum before translocating to the cytosol. Here we show that a substantial fraction of internalized PE is also located in nuclear envelope-associated endosomes (NAE), which display limited mobility, exhibit a propensity to undergo fusion and readily discharge their contents into the nuclear envelope. Electron microscopy and protein trapping in the nucleus indicate that NAE mediate PE transfer into the nucleoplasm. RNAi screening further revealed that NAE-mediated transfer depends on the nuclear envelope proteins SUN1 and SUN2, as well as the Sec61 translocon complex. These data reveal a novel endosomal route from the cell surface to the nucleoplasm that facilitates the accumulation of extracellular and cell surface proteins in the nucleus. PMID:26356418

  19. Nuclear envelope-associated endosomes deliver surface proteins to the nucleus.

    PubMed

    Chaumet, Alexandre; Wright, Graham D; Seet, Sze Hwee; Tham, Keit Min; Gounko, Natalia V; Bard, Frederic

    2015-01-01

    Endocytosis directs molecular cargo along three main routes: recycling to the cell surface, transport to the Golgi apparatus or degradation in endolysosomes. Pseudomonas exotoxin A (PE) is a bacterial protein that typically traffics to the Golgi and then the endoplasmic reticulum before translocating to the cytosol. Here we show that a substantial fraction of internalized PE is also located in nuclear envelope-associated endosomes (NAE), which display limited mobility, exhibit a propensity to undergo fusion and readily discharge their contents into the nuclear envelope. Electron microscopy and protein trapping in the nucleus indicate that NAE mediate PE transfer into the nucleoplasm. RNAi screening further revealed that NAE-mediated transfer depends on the nuclear envelope proteins SUN1 and SUN2, as well as the Sec61 translocon complex. These data reveal a novel endosomal route from the cell surface to the nucleoplasm that facilitates the accumulation of extracellular and cell surface proteins in the nucleus. PMID:26356418

  20. Inner nuclear membrane protein Ima1 is dispensable for intranuclear positioning of centromeres.

    PubMed

    Hiraoka, Yasushi; Maekawa, Hiromi; Asakawa, Haruhiko; Chikashige, Yuji; Kojidani, Tomoko; Osakada, Hiroko; Matsuda, Atsushi; Haraguchi, Tokuko

    2011-10-01

    Inner nuclear membrane (INM) proteins play a role in spatial organization of chromosomes within the nucleus. In the fission yeast Schizosaccharomyces pombe, Sad1, an INM protein of the conserved SUN-domain family, plays an active role in moving chromosomes along the nuclear membranes during meiotic prophase. Ima1 is another conserved INM protein recently identified. A previous study claimed that Ima1 is essential for mitotic cell growth, linking centromeric heterochromatin to the spindle-pole body. However, we obtained results contradictory to the previously proposed role for Ima1: Ima1 was dispensable for mitotic cell growth or centromere positioning. This discrepancy was attributed to incorrect ima1 deletion mutants used in the previous study. Our results show that Ima1 collaborates with two other conserved INM proteins of the LEM-domain family that are homologous to human Man1 and Lem2. Loss of any one of three INM proteins has no effect on mitotic cell growth; however, loss of all these proteins causes severe defects in mitotic cell growth and nuclear membrane morphology. Considering that all three INM proteins interact with Sad1, these results suggest that Ima1, Lem2 and Man1 play at least partially redundant roles for nuclear membrane organization. PMID:21880100

  1. Identification of amino acid sequences in the polyomavirus capsid proteins that serve as nuclear localization signals

    NASA Technical Reports Server (NTRS)

    Chang, D.; Haynes, J. I. Jr; Brady, J. N.; Consigli, R. A.; Spooner, B. S. (Principal Investigator)

    1993-01-01

    The molecular mechanism participating in the transport of newly synthesized proteins from the cytoplasm to the nucleus in mammalian cells is poorly understood. Recently, the nuclear localization signal sequences (NLS) of many nuclear proteins have been identified, and most have been found to be composed of a highly basic amino acid stretch. A genetic "subtractive" and a biochemical "additive" approach were used in our studies to identify the NLS's of the polyomavirus structural capsid proteins. An NLS was identified at the N-terminus (Ala1-Pro-Lys-Arg-Lys-Ser-Gly-Val-Ser-Lys-Cys11) of the major capsid protein VP1 and at the C-terminus (Glu307 -Glu-Asp-Gly-Pro-Glu-Lys-Lys-Lys-Arg-Arg-Leu318) of the VP2/VP3 minor capsid proteins.

  2. Identification of two functional nuclear localization signals in the capsid protein of duck circovirus

    SciTech Connect

    Xiang, Qi-Wang; Zou, Jin-Feng; Wang, Xin; Shandong Provincial Key Laboratory of Animal Biotechnology and Disease Control and Prevention, Shandong, Taian 271018 ; Sun, Ya-Ni; Gao, Ji-Ming; Xie, Zhi-Jing; Shandong Provincial Key Laboratory of Animal Biotechnology and Disease Control and Prevention, Shandong, Taian 271018 ; Wang, Yu; Zhu, Yan-Li; Shandong Provincial Key Laboratory of Animal Biotechnology and Disease Control and Prevention, Shandong, Taian 271018 ; Jiang, Shi-Jin; Shandong Provincial Key Laboratory of Animal Biotechnology and Disease Control and Prevention, Shandong, Taian 271018

    2013-02-05

    The capsid protein (CP) of duck circovirus (DuCV) is the major immunogenic protein and has a high proportion of arginine residues concentrated at the N terminus of the protein, which inhibits efficient mRNA translation in prokaryotic expression systems. In this study, we investigated the subcellular distribution of DuCV CP expressed via recombinant baculoviruses in Sf9 cells and the DNA binding activities of the truncated recombinant DuCV CPs. The results showed that two independent bipartite nuclear localization signals (NLSs) situated at N-terminal 1-17 and 18-36 amino acid residue of the CP. Moreover, two expression level regulatory signals (ELRSs) and two DNA binding signals (DBSs) were also mapped to the N terminus of the protein and overlapped with the two NLSs. The ability of CP to bind DNA, coupled with the karyophilic nature of this protein, strongly suggests that it may be responsible for nuclear targeting of the viral genome.

  3. Characterization of the nuclear localization signal of the mouse TET3 protein

    SciTech Connect

    Xiao, Peng; Zhou, Xiao-long; Zhang, Hong-xiao; Xiong, Kai; Teng, Yun; Huang, Xian-ju; Cao, Rui; Wang, Yi; Liu, Hong-lin

    2013-09-27

    Highlights: •Amino acid sequence KKRK is responsible for nuclear localization of TET3. •Amino acid sequence KKRK are capable of targeting the cytoplasmic proteins to the nucleus. •Amino acid sequence KKRK are conserved in TET3 orthologs. -- Abstract: DNA demethylation is associated with gene activation and is mediated by a family of ten-eleven translocation (TET) dioxygenase. The TET3 protein is a 1668-amino-acid DNA demethylase that is predicted to possess five nuclear localization signals (NLSs). In this paper, we used a series of green fluorescent protein-tagged and mutation constructs to identify a conserved NLS (KKRK) embedded between amino acid 1615 and 1618 of mouse TET3. The KKRK sequence facilitates the cytoplasmic protein’s translocation into the nucleus. Additionally TET3 may be imported into the nucleus by importin-? and importin-?.

  4. Active Degradation Explains the Distribution of Nuclear Proteins during Cellular Senescence

    PubMed Central

    Giampieri, Enrico; De Cecco, Marco; Remondini, Daniel; Sedivy, John; Castellani, Gastone

    2015-01-01

    The amount of cellular proteins is a crucial parameter that is known to vary between cells as a function of the replicative passages, and can be important during physiological aging. The process of protein degradation is known to be performed by a series of enzymatic reactions, ranging from an initial step of protein ubiquitination to their final fragmentation by the proteasome. In this paper we propose a stochastic dynamical model of nuclear proteins concentration resulting from a balance between a constant production of proteins and their degradation by a cooperative enzymatic reaction. The predictions of this model are compared with experimental data obtained by fluorescence measurements of the amount of nuclear proteins in murine tail fibroblast (MTF) undergoing cellular senescence. Our model provides a three-parameter stationary distribution that is in good agreement with the experimental data even during the transition to the senescent state, where the nuclear protein concentration changes abruptly. The estimation of three parameters (cooperativity, saturation threshold, and maximal velocity of the reaction), and their evolution during replicative passages shows that only the maximal velocity varies significantly. Based on our modeling we speculate the reduction of functionality of the protein degradation mechanism as a possible competitive inhibition of the proteasome. PMID:26115222

  5. The mammalian heterochromatin protein 1 binds diverse nuclear proteins through a common motif that targets the chromoshadow domain

    SciTech Connect

    Lechner, Mark S. . E-mail: msl27@drexel.edu; Schultz, David C.; Negorev, Dmitri; Maul, Gerd G.; Rauscher, Frank J.

    2005-06-17

    The HP1 proteins regulate epigenetic gene silencing by promoting and maintaining chromatin condensation. The HP1 chromodomain binds to methylated histone H3. More enigmatic is the chromoshadow domain (CSD), which mediates dimerization, transcription repression, and interaction with multiple nuclear proteins. Here we show that KAP-1, CAF-1 p150, and NIPBL carry a canonical amino acid motif, PxVxL, which binds directly to the CSD with high affinity. We also define a new class of variant PxVxL CSD-binding motifs in Sp100A, LBR, and ATRX. Both canonical and variant motifs recognize a similar surface of the CSD dimer as demonstrated by a panel of CSD mutants. These in vitro binding results were confirmed by the analysis of polypeptides found associated with nuclear HP1 complexes and we provide the first evidence of the NIPBL/delangin protein in human cells, a protein recently implicated in the developmental disorder, Cornelia de Lange syndrome. NIPBL is related to Nipped-B, a factor participating in gene activation by remote enhancers in Drosophila melanogaster. Thus, this spectrum of direct binding partners suggests an expanded role for HP1 as factor participating in promoter-enhancer communication, chromatin remodeling/assembly, and sub-nuclear compartmentalization.

  6. Physical and Functional Interaction between Heterochromatin Protein 1? and the RNA-binding Protein Heterogeneous Nuclear Ribonucleoprotein U*

    PubMed Central

    Ameyar-Zazoua, Maya; Souidi, Mouloud; Fritsch, Lauriane; Robin, Philippe; Thomas, Audrey; Hamiche, Ali; Percipalle, Piergiorgio; Ait-Si-Ali, Slimane; Harel-Bellan, Annick

    2009-01-01

    By combining biochemical purification and mass spectrometry, we identified proteins associated with human heterochromatin protein 1? (HP1?) both in the nucleoplasm and in chromatin. Some of these are RNA-binding proteins, and among them is the protein heterogeneous nuclear ribonucleoprotein U (hnRNP U)/SAF-A, which is linked to chromatin organization and transcriptional regulation. Here, we demonstrate that hnRNP U is a bona fide HP1?-interacting molecule. More importantly, hnRNP U depletion reduces HP1?-dependent gene silencing and disturbs HP1? subcellular localization. Thus, our data demonstrate that hnRNP U is involved in HP1? function, shedding new light on the mode of action of HP1? and on the function of hnRNP U. PMID:19617346

  7. Characterization of nuclear localizing sequences derived from yeast ribosomal protein L29.

    PubMed Central

    Underwood, M R; Fried, H M

    1990-01-01

    Two particular seven-amino-acid segments from yeast ribosomal protein L29 caused a non-nuclear reporter protein to associate almost exclusively with the yeast nucleus. The two L29-derived nuclear localizing sequences were identical in five of the seven residues, many of which were basic amino acids. Generally, localization of the reporter protein was most impaired by replacement of the basic residues. A particular Arg residue was unique; substitution by any amino acid including Lys diminished nuclear localization of the reporter protein. In L29 the corresponding Arg 25----Lys substitution within the nuclear localizing sequence distal to the N-terminus was without effect, as evidence by normal rates of ribosome assembly and cell growth. However, the analogous Arg 8----Lys substitution within the localizing sequence proximal to the N-terminus led to greatly reduced rates of ribosome assembly and cell growth. Finally, when both localizing sequences contained the Arg----Lys substitution a still greater decrease in ribosome assembly and cell growth was observed. These results were as expected if the two short peptide sequences functioned in nuclear localization and/or assembly of yeast ribosomal protein L29. Images Fig. 1. Fig. 3. Fig. 5. PMID:2104804

  8. Scapinin, a putative protein phosphatase-1 regulatory subunit associated with the nuclear nonchromatin structure.

    PubMed

    Sagara, Junji; Higuchi, Tsukasa; Hattori, Yukiko; Moriya, Mie; Sarvotham, Haritha; Shima, Hiroshi; Shirato, Haruki; Kikuchi, Kunimi; Taniguchi, Shunichiro

    2003-11-14

    It is thought that the nuclear nonchromatin structures, such as the nuclear matrix and lamina, play regulatory roles in gene expression. In this study, we identified an insoluble protein that was associated with the chromatin-depleted nuclear structure of proliferating human leukemia HL-60 cells. Preparation of the chromatin-depleted nuclear structure, referred to as the nuclear matrix-intermediate filament scaffold (Fey, E., Krochmalnic, G., and Penman, S. (1986) J. Cell. Biol. 102, 1654-1665), involved cell extraction using a series of buffers containing Triton X-100, DNase I, and 2 M NaCl. A yeast two-hybrid assay revealed that this protein bound to the catalytic subunit of protein phosphatase-1 (PP1). Furthermore, it inhibited PP1 activity in vitro. We therefore named it scapinin (scaffold-associated PP1 inhibiting protein). cDNA cloning revealed that scapinin had two splicing variants of 448 amino acids (scapinin-S) and 518 amino acids (scapinin-L). Scapinin was down-regulated by differentiation in HL-60 cells. These results suggest that scapinin is a putative regulatory subunit of PP1 and is involved in transformed or immature phenotypes of HL-60 cells. We also describe the presence of scapinin family proteins from worm to human. PMID:12925532

  9. Protein kinase C isoforms and lipid second messengers: a critical nuclear partnership?

    PubMed

    Neri, L M; Borgatti, P; Capitani, S; Martelli, A M

    2002-10-01

    A growing body of evidence, accumulated over the past 15 years, has highlighted that the protein kinase C family of isozymes is capable of translocating to the nucleus or is resident within the nucleus. The comprehension of protein kinase C isoform regulation within this organelle is under development. At present, it is emerging that lipid second messengers may play at least two roles in the control of nuclear protein kinase C: on one side they serve as chemical attractants, on the other they directly modulate the activity of specific isoforms. One of the best characterized lipid second messenger that could be involved in the regulation of nuclear PKC activity is DAG. The existence of two separate pools of nuclear DAG suggests that this lipid second messenger might be involved in distinct pathways that lead to different cell responses. Nuclear phosphatidylglycerol, D-3 phosphorylated inositol lipids and nuclear fatty acids are involved in a striking variety of critical biological functions which may act by specific PKC activation. The fine tuning of PKC regulation in cells subjected to proliferating or differentiating stimuli, might prove to be of great interest also for cancer therapy, given the fact that PKC-dependent signaling pathways are increasingly being seen as possible pharmacological target in some forms of neoplastic diseases. In this article, we review the current knowledge about lipid second messengers that are involved in regulating the translocation and/or the activity of different protein kinase C isoforms identified at the nuclear level. PMID:12371155

  10. Lamin-dependent Localization of UNC-84, A Protein Required for Nuclear Migration in Caenorhabditis elegans

    PubMed Central

    Lee, Kenneth K.; Starr, Daniel; Cohen, Merav; Liu, Jun; Han, Min; Wilson, Katherine L.; Gruenbaum, Yosef

    2002-01-01

    Mutations in the Caenorhabditis elegans unc-84 gene cause defects in nuclear migration and anchoring. We show that endogenous UNC-84 protein colocalizes with Ce-lamin at the nuclear envelope and that the envelope localization of UNC-84 requires Ce-lamin. We also show that during mitosis, UNC-84 remains at the nuclear periphery until late anaphase, similar to known inner nuclear membrane proteins. UNC-84 protein is first detected at the 26-cell stage and thereafter is present in most cells during development and in adults. UNC-84 is properly expressed in unc-83 and anc-1 lines, which have phenotypes similar to unc-84, suggesting that neither the expression nor nuclear envelope localization of UNC-84 depends on UNC-83 or ANC-1 proteins. The envelope localization of Ce-lamin, Ce-emerin, Ce-MAN1, and nucleoporins are unaffected by the loss of UNC-84. UNC-84 is not required for centrosome attachment to the nucleus because centrosomes are localized normally in unc-84 hyp7 cells despite a nuclear migration defect. Models for UNC-84 localization are discussed. PMID:11907270

  11. Screen of Nuclear Localized Effecters Proteins in Coxiella burnetii 

    E-print Network

    Galvan, Gloria

    2013-02-04

    In some pathogens such as Ehrlichia chaffeensis, Anaplasma phagocytophilum, Chlamydia, and Shigella, secreted effector proteins have been found to localize to the nucleus and alter the host’s immune response by modulating chromatin or binding...

  12. CHARACTERIZING THE NUCLEAR LOCALIZATION OF ADENOMATOUS POLYPOSIS COLI PROTEIN

    E-print Network

    Cunningham, Jamie Renee Zerbe

    2008-08-18

    Mutation of tumor suppressor protein Adenomatous Polyposis Coli (APC) has been shown to be an early, if not rate limiting step in the progression of both hereditary and sporadic colorectal cancers. The observation that APC ...

  13. Nuclear Import and Export of Venezuelan Equine Encephalitis Virus Nonstructural Protein 2?

    PubMed Central

    Montgomery, Stephanie A.; Johnston, Robert E.

    2007-01-01

    Many RNA viruses, which replicate predominantly in the cytoplasm, have nuclear components that contribute to their life cycle or pathogenesis. We investigated the intracellular localization of the multifunctional nonstructural protein 2 (nsP2) in mammalian cells infected with Venezuelan equine encephalitis virus (VEE), an important, naturally emerging zoonotic alphavirus. VEE nsP2 localizes to both the cytoplasm and the nucleus of mammalian cells in the context of infection and also when expressed alone. Through the analysis of a series of enhanced green fluorescent protein fusions, a segment of nsP2 that completely localizes to the nucleus of mammalian cells was identified. Within this region, mutation of the putative nuclear localization signal (NLS) PGKMV diminished, but did not obliterate, the ability of the protein to localize to the nucleus, suggesting that this sequence contributes to the nuclear localization of VEE nsP2. Furthermore, VEE nsP2 specifically interacted with the nuclear import protein karyopherin-?1 but not with karyopherin-?2, -3, or -4, suggesting that karyopherin-?1 transports nsP2 to the nucleus during infection. Additionally, a novel nuclear export signal (NES) was identified, which included residues L526 and L528 of VEE nsP2. Leptomycin B treatment resulted in nuclear accumulation of nsP2, demonstrating that nuclear export of nsP2 is mediated via the CRM1 nuclear export pathway. Disruption of either the NLS or the NES in nsP2 compromised essential viral functions. Taken together, these results establish the bidirectional transport of nsP2 across the nuclear membrane, suggesting that a critical function of nsP2 during infection involves its shuttling between the cytoplasm and the nucleus. PMID:17652399

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

    PubMed

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

    2012-01-01

    Plant cells have a well organized nucleus and nuclear matrix, but lack orthologues of the main structural components of the metazoan nuclear matrix. Although data is limited, most plant nuclear structural proteins are coiled-coil proteins, such as the NIFs (nuclear intermediate filaments) in Pisum sativum that cross-react with anti-intermediate filament and anti-lamin antibodies, form filaments 6-12 nm in diameter in vitro, and may play the role of lamins. We have investigated the conservation and features of NIFs in a monocot species, Allium cepa, and compared them with onion lamin-like proteins. Polyclonal antisera against the pea 65 kDa NIF were used in 1D and 2D Western blots, ICM (imunofluorescence confocal microscopy) and IEM (immunoelectron microscopy). Their presence in the nuclear matrix was analysed by differential extraction of nuclei, and their association with structural spectrin-like proteins by co-immunoprecipitation and co-localization in ICM. NIF is a conserved structural component of the nucleus and its matrix in monocots with Mr and pI values similar to those of pea 65 kDa NIF, which localized to the nuclear envelope, perichromatin domains and foci, and to the nuclear matrix, interacting directly with structural nuclear spectrin-like proteins. Its similarities with some of the proteins described as onion lamin-like proteins suggest that they are highly related or perhaps the same proteins. PMID:22950797

  15. In vivo mobility of proteins involved in nuclear transport studied by fluorescence correlation spectroscopy

    NASA Astrophysics Data System (ADS)

    Fradin, Cecile; Elbaum, Michael

    2003-07-01

    Using fluorescence correlation spectroscopy we measured the apparent mobility of a nuclear transport cargo (a streptavidin labeled with a nuclear localization signal) both in the cytoplasm and the nucleus of living cells, and we compared it to the mobility of a streptavidin labeled with mutations of the nuclear localization signal known not to support nuclear import, and with the mobility of a set of inert molecules (dextrans) of different sizes. In the cytoplasm, the mobility of the transport cargo is found to be significantly reduced compared to its mobility in the nucleus, or to the mobility of the streptavidins labeled with a mutant nuclear localization signal. This can be partly explained by the fact that the transport cargo forms a complex with two nuclear import mediator proteins (importin ? and importin ?) in the cytoplasm, but could also be partly due to specific interactions of this cargo with the cell cytoskeleton.

  16. Identification of unique SUN-interacting nuclear envelope proteins with diverse functions in plants

    PubMed Central

    Zhou, Xiao; Graumann, Katja; Wirthmueller, Lennart; Jones, Jonathan D.G.

    2014-01-01

    Although a plethora of nuclear envelope (NE) transmembrane proteins (NETs) have been identified in opisthokonts, plant NETs are largely unknown. The only known NET homologues in plants are Sad1/UNC-84 (SUN) proteins, which bind Klarsicht/ANC-1/Syne-1 homology (KASH) proteins. Therefore, de novo identification of plant NETs is necessary. Based on similarities between opisthokont KASH proteins and the only known plant KASH proteins, WPP domain–interacting proteins, we used a computational method to identify the KASH subset of plant NETs. Ten potential plant KASH protein families were identified, and five candidates from four of these families were verified for their NE localization, depending on SUN domain interaction. Of those, Arabidopsis thaliana SINE1 is involved in actin-dependent nuclear positioning in guard cells, whereas its paralogue SINE2 contributes to innate immunity against an oomycete pathogen. This study dramatically expands our knowledge of plant KASH proteins and suggests that plants and opisthokonts have recruited different KASH proteins to perform NE regulatory functions. PMID:24891605

  17. Red Light-Regulated Reversible Nuclear Localization of Proteins in Mammalian Cells and Zebrafish.

    PubMed

    Beyer, Hannes M; Juillot, Samuel; Herbst, Kathrin; Samodelov, Sophia L; Müller, Konrad; Schamel, Wolfgang W; Römer, Winfried; Schäfer, Eberhard; Nagy, Ferenc; Strähle, Uwe; Weber, Wilfried; Zurbriggen, Matias D

    2015-09-18

    Protein trafficking in and out of the nucleus represents a key step in controlling cell fate and function. Here we report the development of a red light-inducible and far-red light-reversible synthetic system for controlling nuclear localization of proteins in mammalian cells and zebrafish. First, we synthetically reconstructed and validated the red light-dependent Arabidopsis phytochrome B nuclear import mediated by phytochrome-interacting factor 3 in a nonplant environment and support current hypotheses on the import mechanism in planta. On the basis of this principle we next regulated nuclear import and activity of target proteins by the spatiotemporal projection of light patterns. A synthetic transcription factor was translocated into the nucleus of mammalian cells and zebrafish to drive transgene expression. These data demonstrate the first in vivo application of a plant phytochrome-based optogenetic tool in vertebrates and expand the repertoire of available light-regulated molecular devices. PMID:25803699

  18. Rho, nuclear actin, and actin-binding proteins in the regulation of transcription and gene expression

    PubMed Central

    Rajakylä, Eeva Kaisa; Vartiainen, Maria K

    2014-01-01

    Actin cytoskeleton is one of the main targets of Rho GTPases, which act as molecular switches on many signaling pathways. During the past decade, actin has emerged as an important regulator of gene expression. Nuclear actin plays a key role in transcription, chromatin remodeling, and pre-mRNA processing. In addition, the “status” of the actin cytoskeleton is used as a signaling intermediate by at least the MKL1-SRF and Hippo-pathways, which culminate in the transcriptional regulation of cytoskeletal and growth-promoting genes, respectively. Rho GTPases may therefore regulate gene expression by controlling either cytoplasmic or nuclear actin dynamics. Although the regulation of nuclear actin polymerization is still poorly understood, many actin-binding proteins, which are downstream effectors of Rho, are found in the nuclear compartment. In this review, we discuss the possible mechanisms and key proteins that may mediate the transcriptional regulation by Rho GTPases through actin. PMID:24603113

  19. Identification and characterisation of a nuclear localisation signal in the SMN associated protein, Gemin4

    SciTech Connect

    Lorson, Monique A.; Dickson, Alexa M.; Shaw, Debra J.; Todd, Adrian G.; Young, Elizabeth C.; Morse, Robert; Wolstencroft, Catherine; Lorson, Christian L.; Young, Philip J.

    2008-10-10

    Gemin4 is a ubiquitously expressed multifunctional protein that is involved in U snRNP assembly, apoptosis, nuclear/cytoplasmic transportation, transcription, and RNAi pathways. Gemin4 is one of the core components of the Gemin-complex, which also contains survival motor neuron (SMN), the seven Gemin proteins (Gemin2-8), and Unrip. Mutations in the SMN1 gene cause the autosomal recessive disorder spinal muscular atrophy (SMA). Although the functions assigned to Gemin4 predominantly occur in the nucleus, the mechanisms that mediate the nuclear import of Gemin4 remain unclear. Here, using a novel panel of Gemin4 constructs we identify a canonical nuclear import sequence (NLS) in the N-terminus of Gemin4. The Gemin4 NLS is necessary and independently sufficient to mediate nuclear import of Gemin4. This is the first functional NLS identified within the SMN-Gemin complex.

  20. Multidimensional profiling of cell surface proteins and nuclear markers

    SciTech Connect

    Han, Ju; Chang, Hang; Andarawewa, Kumari; Yaswen, Paul; Helen Barcellos-Hoff, Mary; Parvin, Bahram

    2009-01-30

    Cell membrane proteins play an important role in tissue architecture and cell-cell communication. We hypothesize that segmentation and multidimensional characterization of the distribution of cell membrane proteins, on a cell-by-cell basis, enable improved classification of treatment groups and identify important characteristics that can otherwise be hidden. We have developed a series of computational steps to (i) delineate cell membrane protein signals and associate them with a specific nucleus; (ii) compute a coupled representation of the multiplexed DNA content with membrane proteins; (iii) rank computed features associated with such a multidimensional representation; (iv) visualize selected features for comparative evaluation through heatmaps; and (v) discriminate between treatment groups in an optimal fashion. The novelty of our method is in the segmentation of the membrane signal and the multidimensional representation of phenotypic signature on a cell-by-cell basis. To test the utility of this method, the proposed computational steps were applied to images of cells that have been irradiated with different radiation qualities in the presence and absence of other small molecules. These samples are labeled for their DNA content and E-cadherin membrane proteins. We demonstrate that multidimensional representations of cell-by-cell phenotypes improve predictive and visualization capabilities among different treatment groups, and identify hidden variables.

  1. CGGBP1 is a nuclear and midbody protein regulating abscission

    SciTech Connect

    Singh, Umashankar Westermark, Bengt

    2011-01-15

    Abscission marks the completion of cell division and its failure is associated with delayed cytokinesis and even tetraploidization. Aberrant abscission and consequential ploidy changes can underlie various diseases including cancer. Midbody, a transient structure formed in the intercellular bridge during telophase, contains several proteins including Aurora kinase B (AURKB), which participate in abscission. We report here an unexpected expression pattern and function of the transcription repressor protein CGG triplet repeat-binding protein 1 (CGGBP1), in normal human fibroblasts. We show that CGGBP1, a chromatin-associated protein, trans-localizes to spindle midzone and midbodies in a manner similar to that of AURKB. CGGBP1 depletion resulted in a cell cycle block at G2, characterized by failure of cells to undergo mitosis and also reduced entry into S phase. Consistent with its presence in the midbodies, live microscopy showed that CGGBP1 deficiency caused mitotic failure at abscission resulting in tetraploidy, which could be rescued by CGGBP1 overexpression. These results show that CGGBP1 is a bona fide midbody protein required for normal abscission and mitosis in general.

  2. The Inner Nuclear Membrane Protein Nemp1 Is a New Type of RanGTP-Binding Protein in Eukaryotes

    PubMed Central

    Shibano, Takashi; Mamada, Hiroshi; Hakuno, Fumihiko; Takahashi, Shin-Ichiro; Taira, Masanori

    2015-01-01

    The inner nuclear membrane (INM) protein Nemp1/TMEM194A has previously been suggested to be involved in eye development in Xenopus, and contains two evolutionarily conserved sequences in the transmembrane domains (TMs) and the C-terminal region, named region A and region B, respectively. To elucidate the molecular nature of Nemp1, we analyzed its interacting proteins through those conserved regions. First, we found that Nemp1 interacts with itself and lamin through the TMs and region A, respectively. Colocalization of Nemp1 and lamin at the INM suggests that the interaction with lamin participates in the INM localization of Nemp1. Secondly, through yeast two-hybrid screening using region B as bait, we identified the small GTPase Ran as a probable Nemp1-binding partner. GST pulldown and co-immunoprecipitation assays using region B and Ran mutants revealed that region B binds directly to the GTP-bound Ran through its effector domain. Immunostaining experiments using transfected COS-7 cells revealed that full-length Nemp1 recruits Ran near the nuclear envelope, suggesting a role for Nemp1 in the accumulation of RanGTP at the nuclear periphery. At the neurula-to-tailbud stages of Xenopus embryos, nemp1 expression overlapped with ran in several regions including the eye vesicles. Co-knockdown using antisense morpholino oligos for nemp1 and ran caused reduction of cell densities and severe eye defects more strongly than either single knockdown alone, suggesting their functional interaction. Finally we show that Arabidopsis thaliana Nemp1-orthologous proteins interact with A. thaliana Ran, suggesting their evolutionally conserved physical and functional interactions possibly in basic cellular functions including nuclear transportation. Taken together, we conclude that Nemp1 represents a new type of RanGTP-binding protein. PMID:25946333

  3. The Inner Nuclear Membrane Protein Nemp1 Is a New Type of RanGTP-Binding Protein in Eukaryotes.

    PubMed

    Shibano, Takashi; Mamada, Hiroshi; Hakuno, Fumihiko; Takahashi, Shin-Ichiro; Taira, Masanori

    2015-01-01

    The inner nuclear membrane (INM) protein Nemp1/TMEM194A has previously been suggested to be involved in eye development in Xenopus, and contains two evolutionarily conserved sequences in the transmembrane domains (TMs) and the C-terminal region, named region A and region B, respectively. To elucidate the molecular nature of Nemp1, we analyzed its interacting proteins through those conserved regions. First, we found that Nemp1 interacts with itself and lamin through the TMs and region A, respectively. Colocalization of Nemp1 and lamin at the INM suggests that the interaction with lamin participates in the INM localization of Nemp1. Secondly, through yeast two-hybrid screening using region B as bait, we identified the small GTPase Ran as a probable Nemp1-binding partner. GST pulldown and co-immunoprecipitation assays using region B and Ran mutants revealed that region B binds directly to the GTP-bound Ran through its effector domain. Immunostaining experiments using transfected COS-7 cells revealed that full-length Nemp1 recruits Ran near the nuclear envelope, suggesting a role for Nemp1 in the accumulation of RanGTP at the nuclear periphery. At the neurula-to-tailbud stages of Xenopus embryos, nemp1 expression overlapped with ran in several regions including the eye vesicles. Co-knockdown using antisense morpholino oligos for nemp1 and ran caused reduction of cell densities and severe eye defects more strongly than either single knockdown alone, suggesting their functional interaction. Finally we show that Arabidopsis thaliana Nemp1-orthologous proteins interact with A. thaliana Ran, suggesting their evolutionally conserved physical and functional interactions possibly in basic cellular functions including nuclear transportation. Taken together, we conclude that Nemp1 represents a new type of RanGTP-binding protein. PMID:25946333

  4. Review of methods to assign the nuclear magnetic resonance peaks of reductively methylated proteins.

    PubMed

    Roberson, Kevin J; Macnaughtan, Megan A

    2014-12-01

    Reductive methylation of lysyl side-chain amines has been a successful tool in the advancement of high-resolution structural biology. The utility of this method has continuously gained ground as a protein chemical modification, first as a tool to aid protein crystallization and later as a probe in protein nuclear magnetic resonance (NMR) spectroscopy. As an isotope-labeling strategy for NMR studies, reductive methylation has contributed to the study of protein-protein interactions and global conformational changes. Although more detailed structural studies using this labeling strategy are possible, the hurdle of assigning the NMR peaks to the corresponding reductively methylated amine hinders its use. In this review, we discuss and compare strategies used to assign the NMR peaks of reductively methylated protein amines. PMID:25175010

  5. The protein composition of Friend cell nuclear matrix stabilized by various treatments. Different recovery of nucleolar proteins B23 and C23 and nuclear lamins.

    PubMed

    Martelli, A M; Manzoli, L; Rubbini, S; Billi, A M; Bareggi, R; Cocco, L

    1995-01-01

    Using two-dimensional polyacrylamide gels stained with Coomassie blue we have studied the protein composition of the nuclear matrix obtained from mouse erythroleukemic nuclei kept at 0 degrees C throughout the isolation procedure to prepare the high ionic strength resistant fraction (control matrix) or stabilized in vitro or in vivo by different procedures prior to subfractionation (ie 37 degrees C incubation of isolated nuclei; sodium tetrathionate exposure of purified nuclei; heat shock of intact cells). When the matrix obtained from 37 degrees C incubated nuclei was compared with the control matrix, striking differences in the polypeptide pattern were seen if the protein was obtained in both cases from an equivalent number of nuclei. On the other hand, if the same amount of protein for both the samples was applied to the gels the differences were less evident. Sodium tetrathionate stabilization of isolated nuclei and heat shock of intact cells produced a matrix protein pattern that was very similar and differed from that of the in vitro heat-exposed matrix. Using specific polyclonal antisera, we demonstrate that nucleolar proteins B23/numatrin and C23/nucleolin were very abundant in the matrix obtained from chemically-treated nuclei or in vivo heat-stabilized nuclei but were recovered in very small amounts (B23) or completely absent (C23) in the matrix prepared from nuclei heated to 37 degrees C in vitro. Differences were seen also in the recovery of nuclear lamins, and especially lamin B, that was poorly represented in the sodium tetrathionate-stabilized matrix. The results demonstrate that in mouse erythroleukemia cells the increased recovery of nuclear matrix protein that is seen after in vitro heating of isolated nuclei is predominantly due to an additional recovery of the same types of polypeptides that are detected also in the absence of such a treatment. The data also indicate that in vivo heat shock of intact cells produces a nuclear matrix protein pattern that is more similar to the pattern seen after stabilization of purified nuclei with sodium tetrathionate and differs significantly from that obtained by exposing nuclei to 37 degrees C in vitro, unlike to that what previous reports have indicated. PMID:7647704

  6. Blocking protein farnesyltransferase improves nuclear shape in fibroblasts from humans

    E-print Network

    Gelb, Michael

    dermopathy (RD). Hutchinson­ Gilford progeria syndrome (HGPS) is caused by a mutant prelamin A that cannot dermopathy (RD) and Hutchinson­Gilford progeria syndrome (HGPS), are caused by defective biogenesis of lamin to the accumulation of prelamin A at the nuclear envelope, cause misshapen nuclei, and result in progeroid syndromes

  7. RNF38 encodes a nuclear ubiquitin protein ligase that modifies p53

    SciTech Connect

    Sheren, Jamie E.; Kassenbrock, C. Kenneth

    2013-11-01

    Highlights: •RNF38 is shown to be a nuclear protein with a bipartite nuclear localization signal. •RNF38 protein is purified and shown to have ubiquitin protein ligase (E3) activity. •We show that RNF38 binds p53 and can ubiquitinate p53 in vitro. •Overexpression of RNF38 increases p53 ubiquitination in HEK293T cells. •Overexpression of RNF38 in HEK293T cells alters p53 localization. -- Abstract: The RNF38 gene encodes a RING finger protein of unknown function. Here we demonstrate that RNF38 is a functional ubiquitin protein ligase (E3). We show that RNF38 isoform 1 is localized to the nucleus by a bipartite nuclear localization sequence (NLS). We confirm that RNF38 is a binding partner of p53 and demonstrate that RNF38 can ubiquitinate p53 in vitro and in vivo. Finally, we show that overexpression of RNF38 in HEK293T cells results in relocalization of p53 to discrete foci associated with PML nuclear bodies. These results suggest RNF38 is an E3 ubiquitin ligase that may play a role in regulating p53.

  8. Control of Protein Activity and Cell Fate Specification via Light-Mediated Nuclear Translocation

    PubMed Central

    Zimmerman, Seth P.; Bear, James E.; Goldstein, Bob; Hahn, Klaus; Kuhlman, Brian

    2015-01-01

    Light-activatable proteins allow precise spatial and temporal control of biological processes in living cells and animals. Several approaches have been developed for controlling protein localization with light, including the conditional inhibition of a nuclear localization signal (NLS) with the Light Oxygen Voltage (AsLOV2) domain of phototropin 1 from Avena sativa. In the dark, the switch adopts a closed conformation that sterically blocks the NLS motif. Upon activation with blue light the C-terminus of the protein unfolds, freeing the NLS to direct the protein to the nucleus. A previous study showed that this approach can be used to control the localization and activity of proteins in mammalian tissue culture cells. Here, we extend this result by characterizing the binding properties of a LOV/NLS switch and demonstrating that it can be used to control gene transcription in yeast. Additionally, we show that the switch, referred to as LANS (light-activated nuclear shuttle), functions in the C. elegans embryo and allows for control of nuclear localization in individual cells. By inserting LANS into the C. elegans lin-1 locus using Cas9-triggered homologous recombination, we demonstrated control of cell fate via light-dependent manipulation of a native transcription factor. We conclude that LANS can be a valuable experimental method for spatial and temporal control of nuclear localization in vivo. PMID:26083500

  9. Impaired nuclear import of mammalian Dlx4 proteins as a consequence of rapid sequence divergence

    SciTech Connect

    Coubrough, Melissa L.; Bendall, Andrew J. . E-mail: abendall@uoguelph.ca

    2006-11-15

    Dlx genes encode a developmentally important family of transcription factors with a variety of functions and sites of action during vertebrate embryogenesis. The murine Dlx4 gene is an enigmatic member of the family; little is known about the normal developmental function(s) of Dlx4. Here, we show that Dlx4 is expressed in the murine placenta and in a trophoblast cell line where the protein localizes to both the nucleus and cytoplasm. Despite the presence of several leucine/valine-rich motifs that match known nuclear export sequences, cytoplasmic Dlx4 is not due to CRM-1-mediated nuclear export. Rather, nuclear import of Dlx4 is compromised by specific residues that flank the nuclear localization signal. One of these residues represents a novel conserved feature of the Dlx4 protein in placental mammals, and the second represents novel variation within mouse Dlx4 isoforms. Comparison of orthologous protein sequences reveals a particularly high rate of non-synonymous change in the coding regions of mammalian Dlx4 genes. Since impaired nuclear localization is unlikely to enhance the function of a nuclear transcription factor, these data point to reduced selection pressure as the basis for the rapid divergence of the Dlx4 gene within the mammalian clade.

  10. Characterization of nuclear polyadenylated RNA-binding proteins in Saccharomyces cerevisiae

    PubMed Central

    1994-01-01

    To study the functions of heterogeneous nuclear ribonucleoproteins (hnRNPs), we have characterized nuclear polyadenylated RNA-binding (Nab) proteins from Saccharomyces cerevisiae. Nab1p, Nab2p, and Nab3p were isolated by a method which uses UV light to cross-link proteins directly bound to poly(A)+ RNA in vivo. We have previously characterized Nab2p, and demonstrated that it is structurally related to human hnRNPs. Here we report that Nab1p is identical to the Np13p/Nop3p protein recently implicated in both nucleocytoplasmic protein shuttling and pre-rRNA processing, and characterize a new nuclear polyadenylated RNA-binding protein, Nab3p. The intranuclear distributions of the Nab proteins were analyzed by three-dimensional immunofluorescence optical microscopy. All three Nab proteins are predominantly localized within the nucleoplasm in a pattern similar to the distribution of hnRNPs in human cells. The NAB3 gene is essential for cell viability and encodes an acidic ribonucleoprotein. Loss of Nab3p by growth of a GAL::nab3 mutant strain in glucose results in a decrease in the amount of mature ACT1, CYH2, and TPI1 mRNAs, a concomitant accumulation of unspliced ACT1 pre-mRNA, and an increase in the ratio of unspliced CYH2 pre-mRNA to mRNA. These results suggest that the Nab proteins may be required for packaging pre-mRNAs into ribonucleoprotein structures amenable to efficient nuclear RNA processing. PMID:7962083

  11. Nuclear protein kinase CLK1 uses a non-traditional docking mechanism to select physiological substrates.

    PubMed

    Keshwani, Malik M; Hailey, Kendra L; Aubol, Brandon E; Fattet, Laurent; McGlone, Maria L; Jennings, Patricia A; Adams, Joseph A

    2015-12-15

    Phosphorylation-dependent cell communication requires enzymes that specifically recognize key proteins in a sea of similar, competing substrates. The protein kinases achieve this goal by utilizing docking grooves in the kinase domain or heterologous protein adaptors to reduce 'off pathway' targeting. We now provide evidence that the nuclear protein kinase CLK1 (cell division cycle2-like kinase 1) important for splicing regulation departs from these classic paradigms by using a novel self-association mechanism. The disordered N-terminus of CLK1 induces oligomerization, a necessary event for targeting its physiological substrates the SR protein (splicing factor containing a C-terminal RS domain) family of splicing factors. Increasing the CLK1 concentration enhances phosphorylation of the splicing regulator SRSF1 (SR protein splicing factor 1) compared with the general substrate myelin basic protein (MBP). In contrast, removal of the N-terminus or dilution of CLK1 induces monomer formation and reverses this specificity. CLK1 self-association also occurs in the nucleus, is induced by the N-terminus and is important for localization of the kinase in sub-nuclear compartments known as speckles. These findings present a new picture of substrate recognition for a protein kinase in which an intrinsically disordered domain is used to capture physiological targets with similar disordered domains in a large oligomeric complex while discriminating against non-physiological targets. PMID:26443864

  12. Nuf2, a spindle pole body-associated protein required for nuclear division in yeast

    PubMed Central

    1994-01-01

    The NUF2 gene of the yeast Saccharomyces cerevisiae encodes an essential 53-kd protein with a high content of potential coiled-coil structure similar to myosin. Nuf2 is associated with the spindle pole body (SPB) as determined by coimmunofluorescence with known SPB proteins. Nuf2 appears to be localized to the intranuclear region and is a candidate for a protein involved in SPB separation. The nuclear association of Nuf2 can be disrupted, in part, by 1 M salt but not by the detergent Triton X-100. All Nuf2 can be removed from nuclei by 8 M urea extraction. In this regard, Nuf2 is similar to other SPB- associated proteins including Nufl/SPC110, also a coiled-coil protein. Temperature-sensitive alleles of NUF2 were generated within the coiled- coil region of Nuf2 and such NUF2 mutant cells rapidly arrest after temperature shift with a single undivided or partially divided nucleus in the bud neck, a shortened mitotic spindle and their DNA fully replicated. In sum, Nuf2 is a protein associated with the SPB that is critical for nuclear division. Anti-Nuf2 antibodies also recognize a mammalian 73-kd protein and display centrosome staining of mammalian tissue culture cells suggesting the presence of a protein with similar function. PMID:8188751

  13. Vaccinia Virus G8R Protein: A Structural Ortholog of Proliferating Cell Nuclear Antigen (PCNA)

    PubMed Central

    Da Silva, Melissa; Upton, Chris

    2009-01-01

    Background Eukaryotic DNA replication involves the synthesis of both a DNA leading and lagging strand, the latter requiring several additional proteins including flap endonuclease (FEN-1) and proliferating cell nuclear antigen (PCNA) in order to remove RNA primers used in the synthesis of Okazaki fragments. Poxviruses are complex viruses (dsDNA genomes) that infect eukaryotes, but surprisingly little is known about the process of DNA replication. Given our previous results that the vaccinia virus (VACV) G5R protein may be structurally similar to a FEN-1-like protein and a recent finding that poxviruses encode a primase function, we undertook a series of in silico analyses to identify whether VACV also encodes a PCNA-like protein. Results An InterProScan of all VACV proteins using the JIPS software package was used to identify any PCNA-like proteins. The VACV G8R protein was identified as the only vaccinia protein that contained a PCNA-like sliding clamp motif. The VACV G8R protein plays a role in poxvirus late transcription and is known to interact with several other poxvirus proteins including itself. The secondary and tertiary structure of the VACV G8R protein was predicted and compared to the secondary and tertiary structure of both human and yeast PCNA proteins, and a high degree of similarity between all three proteins was noted. Conclusions The structure of the VACV G8R protein is predicted to closely resemble the eukaryotic PCNA protein; it possesses several other features including a conserved ubiquitylation and SUMOylation site that suggest that, like its counterpart in T4 bacteriophage (gp45), it may function as a sliding clamp ushering transcription factors to RNA polymerase during late transcription. PMID:19421403

  14. V-myc- and c-myc-encoded proteins are associated with the nuclear matrix.

    PubMed Central

    Eisenman, R N; Tachibana, C Y; Abrams, H D; Hann, S R

    1985-01-01

    A series of extraction procedures were applied to avian nuclei which allowed us to define three types of association of v-myc- and c-myc-encoded proteins with nuclei: (i) a major fraction (60 to 90%) which is retained in DNA- and RNA-depleted nuclei after low- and high-salt extraction, (ii) a small fraction (1%) released during nuclease digestion of DNA in intact nuclei in the presence of low-salt buffer, and (iii) a fraction of myc protein (less than 10%) extractable with salt or detergents and found to have affinity for both single- and double-stranded DNA. Immunofluorescence analysis with anti-myc peptide sera on cells extracted sequentially with nucleases and salts confirmed the idea that myc proteins were associated with a complex residual nuclear structure (matrix-lamin fraction) which also contained avian nuclear lamin protein. Dispersal of myc proteins into the cytoplasm was found to occur during mitosis. Both c-myc and v-myc proteins were associated with the matrix-lamin, suggesting that the function of myc may relate to nuclear structural organization. Images PMID:3872410

  15. Changes in the nuclear protein kinase activities in the regenerating liver of partially irradiated rat

    SciTech Connect

    Asami, K.; Kobayashi, H.; Fujiwara, A.; Yasumasu, I. )

    1989-09-01

    X rays (4.8 Gy) inhibit both DNA synthesis and phosphorylation of histone H1 in the regenerating liver of the rat. To determine the cause of the inhibition of histone H1 phosphorylation, changes in the nuclear protein kinase activities during the prereplicative phase of regeneration were measured. The cAMP-dependent protein kinase activity was low during regeneration, and the changes in the activity were not statistically significant. The cAMP-independent protein kinase activity increased at 15 h, decreased at 18 h, and increased again at 24 h after partial hepatectomy. X irradiation prior to partial hepatectomy did not inhibit the increase at 15 h, but it did inhibit the increase at 24 h. The activity was not inhibited by isoquinolinesulfonamide inhibitors such as H-7, and it was activated by a commercial preparation of an inhibitor protein of the cAMP-dependent kinase. It was also inhibited by quercetin. The possibility that the radiation-sensitive nuclear protein kinase is a nuclear cAMP-independent protein kinase specific for histone H1 is considered.

  16. Recruitment of phosphorylated small heat shock protein Hsp27 to nuclear speckles without stress

    SciTech Connect

    Bryantsev, A.L.; Chechenova, M.B.; Shelden, E.A. . E-mail: eshelden@wsu.edu

    2007-01-01

    During stress, the mammalian small heat shock protein Hsp27 enters cell nuclei. The present study examines the requirements for entry of Hsp27 into nuclei of normal rat kidney (NRK) renal epithelial cells, and for its interactions with specific nuclear structures. We find that phosphorylation of Hsp27 is necessary for the efficient entry into nuclei during heat shock but not sufficient for efficient nuclear entry under control conditions. We further report that Hsp27 is recruited to an RNAse sensitive fraction of SC35 positive nuclear speckles, but not other intranuclear structures, in response to heat shock. Intriguingly, Hsp27 phosphorylation, in the absence of stress, is sufficient for recruitment to speckles found in post-anaphase stage mitotic cells. Additionally, pseudophosphorylated Hsp27 fused to a nuclear localization peptide (NLS) is recruited to nuclear speckles in unstressed interphase cells, but wildtype and nonphosphorylatable Hsp27 NLS fusion proteins are not. The expression of NLS-Hsp27 mutants does not enhance colony forming abilities of cells subjected to severe heat shock, but does regulate nuclear speckle morphology. These data demonstrate that phosphorylation, but not stress, mediates Hsp27 recruitment to an RNAse soluble fraction of nuclear speckles and support a site-specific role for Hsp27 within the nucleus.

  17. Specific interaction with the nuclear transporter importin ?2 can modulate paraspeckle protein 1 delivery to nuclear paraspeckles.

    PubMed

    Major, Andrew T; Hogarth, Cathryn A; Miyamoto, Yoichi; Sarraj, Mai A; Smith, Catherine L; Koopman, Peter; Kurihara, Yasuyuki; Jans, David A; Loveland, Kate L

    2015-04-15

    Importin (IMP) superfamily members mediate regulated nucleocytoplasmic transport, which is central to key cellular processes. Although individual IMP? proteins exhibit dynamic synthesis and subcellular localization during cellular differentiation, including during spermatogenesis, little is known of how this affects cell fate. To investigate how IMP?s control cellular development, we conducted a yeast two-hybrid screen for IMP?2 cargoes in embryonic day 12.5 mouse testis, a site of peak IMP?2 expression coincident with germ-line masculization. We identified paraspeckle protein 1 (PSPC1), the original defining component of nuclear paraspeckles, as an IMP?2-binding partner. PSPC1-IMP?2 binding in testis was confirmed in immunoprecipitations and pull downs, and an enzyme-linked immunosorbent assay-based assay demonstrated direct, high-affinity PSPC1 binding to either IMP?2/IMP?1 or IMP?6/IMP?1. Coexpression of full-length PSPC1 and IMP?2 in HeLa cells yielded increased PSPC1 localization in nuclear paraspeckles. High-throughput image analysis of >3500 cells indicated IMP?2 levels can directly determine PSPC1-positive nuclear speckle numbers and size; a transport-deficient IMP?2 isoform or small interfering RNA knockdown of IMP?2 each reduced endogenous PSPC1 accumulation in speckles. This first validation of an IMP?2 nuclear import cargo in fetal testis provides novel evidence that PSPC1 delivery to paraspeckles, and consequently paraspeckle function, may be controlled by modulated synthesis of specific IMPs. PMID:25694451

  18. Specific interaction with the nuclear transporter importin ?2 can modulate paraspeckle protein 1 delivery to nuclear paraspeckles

    PubMed Central

    Major, Andrew T.; Hogarth, Cathryn A.; Miyamoto, Yoichi; Sarraj, Mai A.; Smith, Catherine L.; Koopman, Peter; Kurihara, Yasuyuki; Jans, David A.; Loveland, Kate L.

    2015-01-01

    Importin (IMP) superfamily members mediate regulated nucleocytoplasmic transport, which is central to key cellular processes. Although individual IMP? proteins exhibit dynamic synthesis and subcellular localization during cellular differentiation, including during spermatogenesis, little is known of how this affects cell fate. To investigate how IMP?s control cellular development, we conducted a yeast two-hybrid screen for IMP?2 cargoes in embryonic day 12.5 mouse testis, a site of peak IMP?2 expression coincident with germ-line masculization. We identified paraspeckle protein 1 (PSPC1), the original defining component of nuclear paraspeckles, as an IMP?2-binding partner. PSPC1-IMP?2 binding in testis was confirmed in immunoprecipitations and pull downs, and an enzyme-linked immunosorbent assay–based assay demonstrated direct, high-affinity PSPC1 binding to either IMP?2/IMP?1 or IMP?6/IMP?1. Coexpression of full-length PSPC1 and IMP?2 in HeLa cells yielded increased PSPC1 localization in nuclear paraspeckles. High-throughput image analysis of >3500 cells indicated IMP?2 levels can directly determine PSPC1-positive nuclear speckle numbers and size; a transport-deficient IMP?2 isoform or small interfering RNA knockdown of IMP?2 each reduced endogenous PSPC1 accumulation in speckles. This first validation of an IMP?2 nuclear import cargo in fetal testis provides novel evidence that PSPC1 delivery to paraspeckles, and consequently paraspeckle function, may be controlled by modulated synthesis of specific IMPs. PMID:25694451

  19. Nuclear magnetic resonance studies of the hydration of proteins and DNA.

    PubMed

    Belton, P S

    2000-07-01

    The behaviour of water in the presence of proteins and DNA as elucidated by nuclear magnetic resonance is reviewed. The picture that emerges is that in dilute solution only those water molecules in the interior of the biopolymers or in clefts have their motions substantially affected. In concentrated systems the situation is more complicated because many more motions have to be considered, but there is no evidence of special effects due to the biopolymers being present. The case of nonfreezing water in protein solutions is considered, and it is suggested that this is not evidence for 'bound' water but simply due to the effects of the inhibition of protein precipitation. PMID:10961339

  20. Molecular characterization of three PRORP proteins in the moss Physcomitrella patens: nuclear PRORP protein is not essential for moss viability.

    PubMed

    Sugita, Chieko; Komura, Yoshihiro; Tanaka, Korechika; Kometani, Kazuki; Satoh, Hiroyuki; Sugita, Mamoru

    2014-01-01

    RNase P is a ubiquitous endonuclease that removes the 5' leader sequence from pre-tRNAs in all organisms. In Arabidopsis thaliana, RNA-free proteinaceous RNase Ps (PRORPs) seem to be enzyme(s) for pre-tRNA 5'-end processing in organelles and the nucleus and are thought to have replaced the ribonucleoprotein RNase P variant. However, the evolution and function of plant PRORPs are not fully understood. Here, we identified and characterized three PRORP-like proteins, PpPPR_63, 67, and 104, in the basal land plant, the moss Physcomitrella patens. PpPPR_63 localizes to the nucleus, while PpPPR_67 and PpPPR_104 are found in both the mitochondria and chloroplasts. The three proteins displayed pre-tRNA 5'-end processing activity in vitro. Mutants with knockout (KO) of the PpPPR_63 gene displayed growth retardation of protonemal colonies, indicating that, unlike Arabidopsis nuclear RPORPs, the moss nuclear PpPPR_63 is not essential for viability. In the KO mutant, nuclear-encoded tRNAAsp (GUC) levels were slightly decreased, whereas most nuclear-encoded tRNA levels were not altered. This indicated that most of the cytosolic mature tRNAs were produced normally without proteinaceous RNase P-like PpPPR_63. Single PpPPR_67 or 104 gene KO mutants displayed different phenotypes of protonemal growth and chloroplast tRNA(Arg) (ACG) accumulation. However, the levels of all other tRNAs were not altered in the KO mutants. In addition, in vitro RNase P assays showed that PpPPR_67 and PpPPR_104 efficiently cleaved chloroplast pre-tRNA(Arg) (CCG) and pre-tRNA(Arg) (UCU) but they cleaved pre-tRNA(Arg) (ACG) with different efficiency. This suggests that the two proteins have overlapping function but their substrate specificity is not identical. PMID:25272157

  1. The Nuclear Protein p34SEI-1 Regulates the Kinase Activity of Cyclin-Dependent

    E-print Network

    Tsai, Ming-Daw

    and tumorigenesis. p34SEI-1 , a nuclear protein originally cloned through a yeast two-hybrid approach using human p-14) are inhibitors of CDK4 and 6. Moreover, the oncoprotein Tax from human T-cell leukemia virus 1 (HTLV-1 gankyrin affects CDK4 by coun- teracting against the inhibition of p16 and p18 (17). The intricate

  2. Pygopus, a nuclear PHD-finger protein required for Wingless signaling in Drosophila.

    PubMed

    Parker, David S; Jemison, Jemileh; Cadigan, Kenneth M

    2002-06-01

    The secreted glycoprotein Wingless (Wg) acts through a conserved signaling pathway to regulate target gene expression. Wg signaling causes nuclear translocation of Armadillo, the fly beta-catenin, which then complexes with the DNA-binding protein TCF, enabling it to activate transcription. Though many nuclear factors have been implicated in modulating TCF/Armadillo activity, their importance remains poorly understood. This work describes a ubiquitously expressed protein, called Pygopus, which is required for Wg signaling throughout Drosophila development. Pygopus contains a PHD finger at its C terminus, a motif often found in chromatin remodeling factors. Overexpression of pygopus also blocks the pathway, consistent with the protein acting in a complex. The pygopus mutant phenotype is highly, though not exclusively, specific for Wg signaling. Epistasis experiments indicate that Pygopus acts downstream of Armadillo nuclear import, consistent with the nuclear location of heterologously expressed protein. Our data argue strongly that Pygopus is a new core component of the Wg signaling pathway that acts downstream or at the level of TCF. PMID:12015286

  3. TMEM120A and B: Nuclear Envelope Transmembrane Proteins Important for Adipocyte Differentiation

    PubMed Central

    Batrakou, Dzmitry G.; de las Heras, Jose I.; Czapiewski, Rafal; Mouras, Rabah; Schirmer, Eric C.

    2015-01-01

    Recent work indicates that the nuclear envelope is a major signaling node for the cell that can influence tissue differentiation processes. Here we present two nuclear envelope trans-membrane proteins TMEM120A and TMEM120B that are paralogs encoded by the Tmem120A and Tmem120B genes. The TMEM120 proteins are expressed preferentially in fat and both are induced during 3T3-L1 adipocyte differentiation. Knockdown of one or the other protein altered expression of several genes required for adipocyte differentiation, Gata3, Fasn, Glut4, while knockdown of both together additionally affected Pparg and Adipoq. The double knockdown also increased the strength of effects, reducing for example Glut4 levels by 95% compared to control 3T3-L1 cells upon pharmacologically induced differentiation. Accordingly, TMEM120A and B knockdown individually and together impacted on adipocyte differentiation/metabolism as measured by lipid accumulation through binding of Oil Red O and coherent anti-Stokes Raman scattering microscopy (CARS). The nuclear envelope is linked to several lipodystrophies through mutations in lamin A; however, lamin A is widely expressed. Thus it is possible that the TMEM120A and B fat-specific nuclear envelope transmembrane proteins may play a contributory role in the tissue-specific pathology of this disorder or in the wider problem of obesity. PMID:26024229

  4. The Caenorhabditis elegans Gene mfap-1 Encodes a Nuclear Protein That Affects Alternative Splicing

    E-print Network

    Horvitz, H. Robert

    The Caenorhabditis elegans Gene mfap-1 Encodes a Nuclear Protein That Affects Alternative Splicing regulatory mechanism for controlling eukaryotic gene expression. By generating various splice isoforms from used the endogenous gene tos-1 as a reporter for splicing and detected increased intron 1 retention

  5. Nuclear Matrix Protein Matrin3 regulates alternative splicing and forms overlapping regulatory networks with PTB

    E-print Network

    Coelho, Miguel B.; Attig, Jan; Bellora, Nicolás; König, Julian; Hallegger, Martina; Kayikci, Melis; Eyras, Eduardo; Ule, Jernej; Smith, Christopher W. J.

    2015-01-19

    Matrin3 is an RNA- and DNA-binding nuclear matrix protein found to be associated with neural and muscular degenerative diseases. A number of possible functions of Matrin3 have been suggested, but no widespread role in RNA metabolism has yet been...

  6. Characterization of the nuclear localization signal of high risk HPV16 E2 protein

    SciTech Connect

    Klucevsek, Kristin; Wertz, Mary; Lucchi, John; Leszczynski, Anna; Moroianu, Junona . E-mail: moroianu@bc.edu

    2007-03-30

    The E2 protein of high risk human papillomavirus type 16 (HPV16) contains an amino-terminal (N) domain, a hinge (H) region and a carboxyl-terminal (C) DNA-binding domain. Using enhanced green fluorescent protein (EGFP) fusions with full length E2 and E2 domains in transfection assays in HeLa cells, we found that the C domain is responsible for the nuclear localization of E2 in vivo, whereas the N and H domains do not contain additional nuclear localization signals (NLSs). Deletion analysis of EGFP-E2 and EGFP-cE2 determined that the C domain contains an {alpha} helix cNLS that overlaps with the DNA-binding region. Mutational analysis revealed that the arginine and lysine residues in this cNLS are essential for nuclear localization of HPV16 E2. Interestingly, these basic amino acid residues are well conserved among the E2 proteins of BPV-1 and some high risk HPV types but not in the low risk HPV types, suggesting that there are differences between the NLSs and corresponding nuclear import pathways between these E2 proteins.

  7. Differentiation- and stress-dependent nuclear cytoplasmic redistribution of myopodin, a novel actin-bundling protein

    PubMed Central

    Weins, Astrid; Schwarz, Karin; Faul, Christian; Barisoni, Laura; Linke, Wolfgang A.; Mundel, Peter

    2001-01-01

    We report the cloning and functional characterization of myopodin, the second member of the synaptopodin gene family. Myopodin shows no significant homology to any known protein except synaptopodin. Northern blot analysis resulted in a 3.6-kb transcript for mouse skeletal and heart muscle. Western blots showed an 80-kD signal for skeletal and a 95-kD signal for heart muscle. Myopodin contains one PPXY motif and multiple PXXP motifs. Myopodin colocalizes with ?-actinin and is found at the Z-disc as shown by immunogold electron microscopy. In myoblasts, myopodin shows preferential nuclear localization. During myotube differentiation, myopodin binds to stress fibers in a punctuated pattern before incorporation into the Z-disc. Myopodin can directly bind to actin and contains a novel actin binding site in the center of the protein. Myopodin has actin-bundling activity as shown by formation of latrunculin-A–sensitive cytosolic actin bundles and nuclear actin loops in transfected cells expressing green fluorescent protein–myopodin. Under stress conditions, myopodin accumulates in the nucleus and is depleted from the cytoplasm. Nuclear export of myopodin is sensitive to leptomycin B, despite the absence of a classical nuclear export sequence. We propose a dual role for myopodin as a structural protein also participating in signaling pathways between the Z-disc and the nucleus. PMID:11673475

  8. Beta-like importins mediate the nuclear translocation of mitogen-activated protein kinases.

    PubMed

    Zehorai, Eldar; Seger, Rony

    2014-01-01

    The rapid nuclear translocation of signaling proteins upon stimulation is important for the regulation of de novo gene expression. We have studied the stimulated nuclear shuttling of c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinases (MAPKs) and found that they translocate into the nucleus in a Ran-dependent, but NLS- or NTS-independent, manner, unrelated to their catalytic activity. We show that this translocation involves three ?-like importins, importins 3, 7, and 9 (Imp3/7/9). Knockdown of these importins inhibits the nuclear translocation of the MAPKs and, thereby, activation of their transcription factor targets. We further demonstrate that the translocation requires the stimulated formation of heterotrimers composed of Imp3/Imp7/MAPK or Imp3/Imp9/MAPK. JNK1/2 and p38?/? bind to either Imp7 or Imp9 upon stimulated posttranslational modification of the two Imps, while Imp3 joins the complex after its stimulation-induced phosphorylation. Once formed, these heterotrimers move to the nuclear envelope, where importin 3 remains, while importins 7 and 9 escort the MAPKs into the nucleus. These results suggest that ?-like importins are central mediators of stimulated nuclear translocation of signaling proteins and therefore add a central level of regulation to stimulated transcription. PMID:24216760

  9. The tight junction protein Z O-2 has several functional nuclear export signals

    SciTech Connect

    Gonzalez-Mariscal, Lorenza . E-mail: lorenza@fisio.cinvestav.mx; Ponce, Arturo; Alarcon, Lourdes; Jaramillo, Blanca Estela

    2006-10-15

    The tight junction (TJ) protein ZO-2 changes its subcellular distribution according to the state of confluency of the culture. Thus in confluent monolayers, it localizes at the TJ region whereas in sparse cultures it concentrates at the nucleus. The canine sequence of ZO-2 displays four putative nuclear export signals (NES), two at the second PDZ domain (NES-0 and NES-1) and the rest at the GK region (NES-2 and NES-3). The functionality of NES-0 and NES-3 was unknown, hence here we have explored it with a nuclear export assay, injecting into the nucleus of MDCK cells peptides corresponding to the ZO-2 NES sequences chemically coupled to ovalbumin. We show that both NES-0 and NES-3 are functional and sensitive to leptomycin B. We also demonstrate that NES-1, previously characterized as a non functional NES, is rendered capable of nuclear export upon the acquisition of a negative charge at its Ser369 residue. Experiments performed injecting at the nucleus WT and mutated ZO-2-GST fusion proteins revealed the need of both NES-0 and NES-1, and NES-2 and NES-3 for attaining an efficient nuclear exit of the respective amino and middle segments of ZO-2. Moreover, the transfection of MDCK cells with full-length ZO-2 revealed that the mutation of any of the NES present in the molecule was sufficient to induce nuclear accumulation of the protein.

  10. Ubiquitin-dependent Protein Degradation at the Yeast Endoplasmic Reticulum and Nuclear Envelope

    PubMed Central

    Zattas, Dimitrios; Hochstrasser, Mark

    2014-01-01

    The endoplasmic reticulum (ER) is the primary organelle in eukaryotic cells where membrane and secreted proteins are inserted into or across cell membranes. Its membrane bilayer and luminal compartments provide a favorable environment for the folding and assembly of thousands of newly synthesized proteins. However, protein folding is intrinsically error-prone, and various stress conditions can further increase levels of protein misfolding and damage, particularly in the ER, which can lead to cellular dysfunction and disease. The ubiquitin-proteasome system (UPS) is responsible for the selective destruction of a vast array of protein substrates, either for protein quality control or to allow rapid changes in the levels of specific regulatory proteins. In this review, we will focus on the components and mechanisms of ER-associated protein degradation (ERAD), an important branch of the UPS. ER membranes extend from subcortical regions of the cell to the nuclear envelope, with its continuous outer and inner membranes; the nuclear envelope is a specialized subdomain of the ER. ERAD presents additional challenges to the UPS beyond those faced with soluble substrates of the cytoplasm and nucleus. These include recognition of sugar modifications that occur in the ER, retrotranslocation of proteins across the membrane bilayer, and transfer of substrates from the ER extraction machinery to the proteasome. Here we review characteristics of ERAD substrate degradation signals (degrons), mechanisms underlying substrate recognition and processing by the ERAD machinery, and ideas on the still unresolved problem of how substrate proteins are moved across and extracted from the ER membrane. PMID:25231236

  11. Characterization of a nuclear deformed epidermal autoregulatory factor-1 (DEAF-1)-related (NUDR) transcriptional regulator protein.

    PubMed

    Huggenvik, J I; Michelson, R J; Collard, M W; Ziemba, A J; Gurley, P; Mowen, K A

    1998-10-01

    A monkey kidney cDNA that encodes a nuclear regulatory factor was identified by expression and affinity binding to a synthetic retinoic acid response element (RARE) and was used to isolate human placental and rat germ cell cDNAs by hybridization. The cDNAs encode a 59-kDa protein [nuclear DEAF-1-related (NUDR)] which shows sequence similarity to the Drosophila Deformed epidermal autoregulatory factor-1 (DEAF-1), a nonhomeodomain cofactor of embryonic Deformed gene expression. Similarities to other proteins indicate five functional domains in NUDR including an alanine-rich region prevalent in developmental transcription factors, a domain found in the promyelocytic leukemia-associated SP100 proteins, and a zinc finger homology domain associated with the AML1/MTG8 oncoprotein. Although NUDR mRNA displayed a wide tissue distribution in rats, elevated levels of protein were only observed in testicular germ cells, developing fetus, and transformed cell lines. Nuclear localization of NUDR was demonstrated by immunocytochemistry and by a green fluorescent protein-NUDR fusion protein. Site-directed mutagenesis of a nuclear localization signal resulted in cytoplasmic localization of the protein and eliminated NUDR-dependent transcriptional activation. Recombinant NUDR protein showed affinity for the RARE in mobility shifts; however it was efficiently displaced by retinoic acid receptor (RAR)/retinoid X receptor (RXR) complexes. In transient transfections, NUDR produced up to 26-fold inductions of a human proenkephalin promoter-reporter plasmid, with minimal effects on the promoters for prodynorphin or thymidine kinase. Placement of a RARE on the proenkephalin promoter increased NUDR-dependent activation to 41-fold, but this RARE-dependent increase was not transferable to a thymidine kinase promoter. Recombinant NUDR protein showed minimal binding affinity for proenkephalin promoter sequences, but was able to select DNA sequences from a random oligonucleotide library that had similar core-binding motifs (TTCG) as those recognized by DEAF-1. This motif is also present between the half-sites of several endogenous RAREs. The derived consensus- binding motif recognized by NUDR (TTCGGGNNTTTCCGG) was confirmed by mobility shift and deoxyribonuclease I (DNase I) protection assays; however, the consensus sequence was also unable to confer NUDR-dependent transcriptional activation to the thymidine kinase promoter. Our data suggests that NUDR may activate transcription independently of promoter binding, perhaps through protein-protein interaction with basal transcription factors, or by activation of secondary factors. The sequence and functional similarities between NUDR and DEAF-1 suggest that NUDR may also act as a cofactor to regulate the transcription of genes during fetal development or differentiation of testicular cells. PMID:9773984

  12. Nuclear translocation of doublecortin-like protein kinase and phosphorylation of a transcription factor JDP2

    SciTech Connect

    Nagamine, Tadashi; Nomada, Shohgo; Onouchi, Takashi; Kameshita, Isamu; Sueyoshi, Noriyuki

    2014-03-28

    Highlights: • Doublecortin-like protein kinase (DCLK) is a microtubule-associated protein kinase. • In living cells, DCLK was cleaved into two functional fragments. • zDCLK(kinase) was translocated into the nucleus by osmotic stresses. • Jun dimerization protein 2 (JDP2) was identified as zDCLK(kinase)-binding protein. • JDP2 was efficiently phosphorylated by zDCLK(kinase) only when histone was present. - Abstract: Doublecortin-like protein kinase (DCLK) is a microtubule-associated protein kinase predominantly expressed in brain. In a previous paper, we reported that zebrafish DCLK2 (zDCLK) was cleaved into two functional fragments; the N-terminal zDCLK(DC + SP) with microtubule-binding activity and the C-terminal zDCLK(kinase) with a Ser/Thr protein kinase activity. In this study, we demonstrated that zDCLK(kinase) was widely distributed in the cytoplasm and translocated into the nucleus when the cells were treated under hyperosmotic conditions with NaCl or mannitol. By two-hybrid screening using the C-terminal domain of DCLK, Jun dimerization protein 2 (JDP2), a nuclear transcription factor, was identified as zDCLK(kinase)-binding protein. Furthermore, JDP2 served as an efficient substrate for zDCLK(kinase) only when histone was present. These results suggest that the kinase fragment of DCLK is translocated into the nucleus upon hyperosmotic stresses and that the kinase efficiently phosphorylates JDP2, a possible target in the nucleus, with the aid of histones.

  13. Characterization of a nuclear compartment shared by nuclear bodies applying ectopic protein expression and correlative light and electron microscopy

    SciTech Connect

    Richter, Karsten; Reichenzeller, Michaela; Goerisch, Sabine M.; Schmidt, Ute; Scheuermann, Markus O.; Herrmann, Harald; Lichter, Peter . E-mail: m.macleod@dkfz.de

    2005-02-01

    To investigate the accessibility of interphase nuclei for nuclear body-sized particles, we analyzed in cultured cells from human origin by correlative fluorescence and electron microscopy (EM) the bundle-formation of Xenopus-vimentin targeted to the nucleus via a nuclear localization signal (NLS). Moreover, we investigated the spatial relationship of speckles, Cajal bodies, and crystalline particles formed by Mx1 fused to yellow fluorescent protein (YFP), with respect to these bundle arrays. At 37 deg C, the nucleus-targeted, temperature-sensitive Xenopus vimentin was deposited in focal accumulations. Upon shift to 28 deg C, polymerization was induced and filament arrays became visible. Within 2 h after temperature shift, arrays were found to be composed of filaments loosely embedded in the nucleoplasm. The filaments were restricted to limited areas of the nucleus between focal accumulations. Upon incubation at 28 deg C for several hours, NLS vimentin filaments formed bundles looping throughout the nuclei. Speckles and Cajal bodies frequently localized in direct neighborhood to vimentin bundles. Similarly, small crystalline particles formed by YFP-tagged Mx1 also located next to vimentin bundles. Taking into account that nuclear targeted vimentin locates in the interchromosomal domain (ICD), we conclude that nuclear body-sized particles share a common nuclear space which is controlled by higher order chromatin organization.

  14. Reduction of a 4q35-encoded nuclear envelope protein in muscle differentiation

    SciTech Connect

    Ostlund, Cecilia; Department of Pathology and Cell Biology, College of Physicians and Surgeons, Columbia University, New York, NY 10032 ; Guan, Tinglu; Figlewicz, Denise A.; Hays, Arthur P.; Worman, Howard J.; Department of Pathology and Cell Biology, College of Physicians and Surgeons, Columbia University, New York, NY 10032 ; Gerace, Larry; Schirmer, Eric C.

    2009-11-13

    Muscular dystrophy and peripheral neuropathy have been linked to mutations in genes encoding nuclear envelope proteins; however, the molecular mechanisms underlying these disorders remain unresolved. Nuclear envelope protein p19A is a protein of unknown function encoded by a gene at chromosome 4q35. p19A levels are significantly reduced in human muscle as cells differentiate from myoblasts to myotubes; however, its levels are not similarly reduced in all differentiation systems tested. Because 4q35 has been linked to facioscapulohumeral muscular dystrophy (FSHD) and some adjacent genes are reportedly misregulated in the disorder, levels of p19A were analyzed in muscle samples from patients with FSHD. Although p19A was increased in most cases, an absolute correlation was not observed. Nonetheless, p19A downregulation in normal muscle differentiation suggests that in the cases where its gene is inappropriately re-activated it could affect muscle differentiation and contribute to disease pathology.

  15. Deciphering the Nuclear Import Pathway for the Cytoskeletal Red Cell Protein 4.1R

    PubMed Central

    Gascard, Philippe; Nunomura, Wataru; Lee, Gloria; Walensky, Loren D.; Krauss, Sharon Wald; Takakuwa, Yuichi; Chasis, Joel A.; Mohandas, Narla; Conboy, John G.

    1999-01-01

    The erythroid membrane cytoskeletal protein 4.1 is the prototypical member of a genetically and topologically complex family that is generated by combinatorial alternative splicing pathways and is localized at diverse intracellular sites including the nucleus. To explore the molecular determinants for nuclear localization, we transfected COS-7 cells with epitope-tagged versions of natural red cell protein 4.1 (4.1R) isoforms as well as mutagenized and truncated derivatives. Two distant topological sorting signals were required for efficient nuclear import of the 4.1R80 isoform: a basic peptide, KKKRER, encoded by alternative exon 16 and acting as a weak core nuclear localization signal (4.1R NLS), and an acidic peptide, EED, encoded by alternative exon 5. 4.1R80 isoforms lacking either of these two exons showed decreased nuclear import. Fusion of various 4.1R80 constructs to the cytoplasmic reporter protein pyruvate kinase confirmed a requirement for both motifs for full NLS function. 4.1R80 was efficiently imported in the nuclei of digitonin-permeabilized COS-7 cells in the presence of recombinant Rch1 (human importin ?2), importin ?, and GTPase Ran. Quantitative analysis of protein–protein interactions using a resonant mirror detection technique showed that 4.1R80 bound to Rch1 in vitro with high affinity (KD = 30 nM). The affinity decreased at least 7- and 20-fold, respectively, if the EED motif in exon 5 or if 4.1R NLS in exon 16 was lacking or mutated, confirming that both motifs were required for efficient importin-mediated nuclear import of 4.1R80. PMID:10359596

  16. Inner nuclear envelope protein SUN1 plays a prominent role in mammalian mRNA export.

    PubMed

    Li, Ping; Noegel, Angelika A

    2015-11-16

    Nuclear export of messenger ribonucleoproteins (mRNPs) through the nuclear pore complex (NPC) can be roughly classified into two forms: bulk and specific export, involving an nuclear RNA export factor 1 (NXF1)-dependent pathway and chromosome region maintenance 1 (CRM1)-dependent pathway, respectively. SUN proteins constitute the inner nuclear envelope component of the l I: nker of N: ucleoskeleton and C: ytoskeleton (LINC) complex. Here, we show that mammalian cells require SUN1 for efficient nuclear mRNP export. The results indicate that both SUN1 and SUN2 interact with heterogeneous nuclear ribonucleoprotein (hnRNP) F/H and hnRNP K/J. SUN1 depletion inhibits the mRNP export, with accumulations of both hnRNPs and poly(A)+RNA in the nucleus. Leptomycin B treatment indicates that SUN1 functions in mammalian mRNA export involving the NXF1-dependent pathway. SUN1 mediates mRNA export through its association with mRNP complexes via a direct interaction with NXF1. Additionally, SUN1 associates with the NPC through a direct interaction with Nup153, a nuclear pore component involved in mRNA export. Taken together, our results reveal that the inner nuclear envelope protein SUN1 has additional functions aside from being a central component of the LINC complex and that it is an integral component of the mammalian mRNA export pathway suggesting a model whereby SUN1 recruits NXF1-containing mRNP onto the nuclear envelope and hands it over to Nup153. PMID:26476453

  17. Antibody-based analysis reveals “filamentous vs. non-filamentous” and “cytoplasmic vs. nuclear” crosstalk of cytoskeletal proteins

    SciTech Connect

    Kumeta, Masahiro; Hirai, Yuya; Yoshimura, Shige H.; Horigome, Tsuneyoshi; Takeyasu, Kunio

    2013-12-10

    To uncover the molecular composition and dynamics of the functional scaffold for the nucleus, three fractions of biochemically-stable nuclear protein complexes were extracted and used as immunogens to produce a variety of monoclonal antibodies. Many helix-based cytoskeletal proteins were identified as antigens, suggesting their dynamic contribution to nuclear architecture and function. Interestingly, sets of antibodies distinguished distinct subcellular localization of a single isoform of certain cytoskeletal proteins; distinct molecular forms of keratin and actinin were found in the nucleus. Their nuclear shuttling properties were verified by the apparent nuclear accumulations under inhibition of CRM1-dependent nuclear export. Nuclear keratins do not take an obvious filamentous structure, as was revealed by non-filamentous cytoplasmic keratin-specific monoclonal antibody. These results suggest the distinct roles of the helix-based cytoskeletal proteins in the nucleus. - Highlights: • A set of monoclonal antibodies were raised against nuclear scaffold proteins. • Helix-based cytoskeletal proteins were involved in nuclear scaffold. • Many cytoskeletal components shuttle into the nucleus in a CRM1-dependent manner. • Sets of antibodies distinguished distinct subcellular localization of a single isoform. • Nuclear keratin is soluble and does not form an obvious filamentous structure.

  18. The Karyopherin Kap142p/Msn5p Mediates Nuclear Import and Nuclear Export of Different Cargo Proteins

    PubMed Central

    Yoshida, Kimihisa; Blobel, Günter

    2001-01-01

    We have identified a novel pathway for protein import into the nucleus. Although the product of Saccharomyces cerevisiae gene MSN5 was previously shown to function as a karyopherin (Kap) for nuclear export of various proteins, we discovered a nuclear import pathway mediated by Msn5p (also referred to as Kap142p). We have purified from yeast cytosol a complex containing Kap142p and the trimeric replication protein A (RPA), which is required for multiple aspects of DNA metabolism, including DNA replication, DNA repair, and recombination. In wild-type cells, RPA was localized primarily to the nucleus but, in a KAP142 deletion strain, RPA was mislocalized to the cytoplasm and the strain was highly sensitive to bleomycin (BLM). BLM causes DNA double-strand breaks and, in S. cerevisiae, the DNA damage is repaired predominantly by RPA-dependent homologous recombination. Therefore, our results indicate that in wild-type cells a critical portion of RPA was imported into the nucleus by Kap142p. Like several other import-related Kap–substrate complexes, the endogenous RPA–Kap142p complex was dissociated by RanGTP, but not by RanGDP. All three RPA genes are essential for viability, whereas KAP142 is not. Perhaps explaining this disparity, we observed an interaction between RPA and Kap95p in a strain lacking Kap142p. This interaction could provide a mechanism for import of RPA into the nucleus and cell viability in the absence of Kap142p. Together with published results (Kaffman, A., N.M. Rank, E.M. O'Neill, L.S. Huang, and E.K. O'Shea. 1998. Nature. 396:482–486; Blondel, M., P.M. Alepuz, L.S. Huang, S. Shaham, G. Ammerer, and M. Peter. 1999. Genes Dev. 13:2284–2300; DeVit, M.J., and M. Johnston. 1999. Curr. Biol. 9:1231–1241; Mahanty, S.K., Y. Wang, F.W. Farley, and E.A. Elion. 1999. Cell. 98:501–512) our data indicate that the karyopherin Kap142p is able to mediate nuclear import of one set of proteins and nuclear export of a different set of proteins. PMID:11266464

  19. Primary structure of a human arginine-rich nuclear protein that colocalizes with spliceosome components

    SciTech Connect

    Chaudhary, N.; McMahon, C.; Blobel, G. )

    1991-09-15

    The cDNA for a 54-kDa nuclear protein (p54) has been cloned from a human hepatoma expression library. Contained within p54 is an arginine/serine-rich region similar to segments of several proteins that participate in pre-mRNA splicing including the 70-kDa component of U1 small nuclear ribonucleoprotein particle (snRNP) and the Drosophila transformer and suppressor-of-white-apricot proteins. The arginine/serine-rich region is dominated by a series of 8-amino acid imperfect repetitive motifs (consensus sequence, Arg-Arg-Ser-Arg-Ser-Arg-Ser-Arg). Antibodies raised against synthetic peptides of p54 react with an {approximately}70-kDa protein on immunoblots of HeLa cell and rat liver nuclear proteins. This apparent discrepancy in mass is also observed when p54 mRNA is translated in vitro. Indirect immunofluorescence studies in HeLa cells show that p54 is distributed throughout the nucleus in a speckled pattern, with an additional diffuse labeling of the nucleus excluding the nucleoli. Double immunofluorescence experiments indicate that these punctate regions are coincident with the speckles seen in cells stained with antibodies against several constituents of the pre-mRNA splicing machinery. Sedimentation analysis of HeLa cell extracts on sucrose gradients showed that p54 migrates at 4-6 S, indicating that the protein is not a tightly associated component of snRNPs. Although the function of p54 is not yet known, the structure and immunolocalization data suggest that this protein may have a role in pre-mRNA processing.

  20. TM4SF3 and AR: A Nuclear Complex that Stabilizes Both Proteins.

    PubMed

    Bhansali, Meenakshi; Zhou, Jun; Shemshedini, Lirim

    2016-01-01

    Transmembrane 4 superfamily 3 (TM4SF3) was identified as a novel androgen-regulated gene in prostate cancer (PCa) cells. Our data demonstrate that TM4SF3 exhibits androgen-induced repression of the mRNA but up-regulation of the protein. The androgen positive effect on the TM4SF3 protein is of significant interest in view of the procancer functions of both androgens and tetraspanin proteins. Androgen positively regulates TM4SF3 protein stability by inhibiting its proteasome-dependent degradation. This androgen stabilization of TM4SF3 is involved in promoting PCa cell invasion and migration of both androgen-dependent and androgen-independent PCa cells. Although confirming androgen up-regulation of the TM4SF3 protein, we observed that TM4SF3 is localized not only to the membrane, but also, surprisingly, the nuclei of PCa cells. This novel nuclear localization of TM4SF3 depends on androgen-induced nuclear localization of androgen receptor (AR) in both androgen-dependent and androgen-independent PCa cell lines. TM4SF3 interacts with AR both in PCa cell types and in vitro, strongly suggesting a direct interaction. This direct interaction is required for the stabilization of not only TM4SF3, but also remarkably AR, because down-regulation of TM4SF3 resulted in reduced AR protein levels. As expected of an important AR regulator, TM4SF3 regulates androgen-dependent gene expression in and proliferation of PCa cells. Importantly, a direct correlation between AR and TM4SF3 protein levels and nuclear colocalization were also observed in prostate tumors, strongly suggesting that the mutual stabilization resulting from the AR-TM4SF3 interaction is found in tumors and that this interaction is important in PCa biology. PMID:26649804

  1. A Crowdsourced nucleus: Understanding nuclear organization in terms of dynamically networked protein function

    PubMed Central

    Wood, Ashley M.; Garza-Gongora, Arturo G.; Kosak, Steven T.

    2014-01-01

    The spatial organization of the nucleus results in a compartmentalized structure that affects all aspects of nuclear function. This compartmentalization involves genome organization as well as the formation of nuclear bodies and plays a role in many functions, including gene regulation, genome stability, replication, and RNA processing. Here we review the recent findings associated with the spatial organization of the nucleus and reveal that a common theme for nuclear proteins is their ability to participate in a variety of functions and pathways. We consider this multiplicity of function in terms of Crowdsourcing, a recent phenomenon in the world of information technology, and suggest that this model provides a novel way to synthesize the many intersections between nuclear organization and function. PMID:24412853

  2. Nuclear factor erythroid-2 related factor 2 (Nrf2)-mediated protein quality control in cardiomyocytes.

    PubMed

    Cui, Taixing; Lai, Yimu; Janicki, Jospeh S; Wang, Xuejun

    2016-01-01

    Protein quality control (PQC) acts to minimize the level and toxicity of malfolded proteins in the cell. It is performed by an elaborate network of molecular chaperones and targeted protein degradation pathways. PQC monitors and maintains protein homeostasis or proteostasis in the cells. Whilst chaperones may actively promote refolding of malfolded proteins, the malfolded proteins which cannot be correctly refolded are degraded by the ubiquitin proteasome system (UPS) and the autophagic-lysosome pathway (ALP). The UPS degrades individual misfolded protein molecules, whereas the ALP removes large and less soluble protein aggregates and organelles. Emerging evidence indicates that dysregulated and inadequate PQC play an important role in the pathogenesis of not only classic conformational disease but more common forms of cardiac pathology such as cardiac pathological hypertrophy and heart failure. Nuclear factor erythroid 2-related factor 2 (Nrf2), a master transcription factor of cellular defense, appears to regulate the USP and the ALP by directly controlling the expression of UPS- and ALP- related genes. This article highlights an emerging role of Nrf2 in the regulation of intracellular PQC as well as its potential involvement in cardiac pathology. PMID:26709769

  3. Cloning and characterisation of a nuclear, site specific ssDNA binding protein.

    PubMed

    Smidt, M P; Russchen, B; Snippe, L; Wijnholds, J; Ab, G

    1995-07-11

    Estradiol inducible, liver-specific expression of the apoVLDL II gene is mediated through the estrogen receptor and a variety of other DNA-binding proteins. In the present study we report the cloning and characterisation of a single-strand DNA binding protein that interacts with the lower strand of a complex regulatory site, which includes the major estrogen responsive element and a site that resembles the rat albumin site D (apoVLDL II site D). Based on its binding specificity determined with electro-mobility shift assays, the protein is named single-strand D-box binding factor (ssDBF). Analysis of the deduced 302 amino acid sequence revealed that the protein belongs to the heteronuclear ribonucleoprotein A/B family (hnRNP A/B) and resembles other known eukaryotic single-strand DNA binding proteins. Transient transfection experiments in a chicken liver cell-line showed that the protein represses estrogen-induced transcription. A protein with similar binding characteristics is present in liver nuclear extract. The relevance of the occurrence of this protein to the expression of the apoVLDL II gene is discussed. PMID:7630716

  4. Cloning and characterisation of a nuclear, site specific ssDNA binding protein.

    PubMed Central

    Smidt, M P; Russchen, B; Snippe, L; Wijnholds, J; Ab, G

    1995-01-01

    Estradiol inducible, liver-specific expression of the apoVLDL II gene is mediated through the estrogen receptor and a variety of other DNA-binding proteins. In the present study we report the cloning and characterisation of a single-strand DNA binding protein that interacts with the lower strand of a complex regulatory site, which includes the major estrogen responsive element and a site that resembles the rat albumin site D (apoVLDL II site D). Based on its binding specificity determined with electro-mobility shift assays, the protein is named single-strand D-box binding factor (ssDBF). Analysis of the deduced 302 amino acid sequence revealed that the protein belongs to the heteronuclear ribonucleoprotein A/B family (hnRNP A/B) and resembles other known eukaryotic single-strand DNA binding proteins. Transient transfection experiments in a chicken liver cell-line showed that the protein represses estrogen-induced transcription. A protein with similar binding characteristics is present in liver nuclear extract. The relevance of the occurrence of this protein to the expression of the apoVLDL II gene is discussed. Images PMID:7630716

  5. Development of an ELISA detecting Tumor Protein 53-Induced Nuclear Protein 1 in serum of prostate cancer patients.

    PubMed

    Saadi, Houda; Seillier, Marion; Sandi, Maria José; Peuget, Sylvain; Kellenberger, Christine; Gravis, Gwenaëlle; Dusetti, Nelson J; Iovanna, Juan L; Rocchi, Palma; Amri, Mohamed; Carrier, Alice

    2013-01-01

    Tumor Protein 53-Induced Nuclear Protein 1 (TP53INP1) plays an important role during cell stress response in synergy with the potent "genome-keeper" p53. In human, the gene encoding TP53INP1 is expressed at very high level in some pathological situations, such as inflammation and prostate cancer (PC). TP53INP1 overexpression in PC seems to be a worse prognostic factor, particularly predictive of biological cancer relapse, making TP53INP1 a relevant specific target for molecular therapy of Castration Resistant (CR) PC. In that context, detection of TP53INP1 in patient biological fluids is a promising diagnostic avenue. We report here successful development of a new Enzyme-Linked Immunosorbent Assay (ELISA) detecting TP53INP1, taking advantage of molecular tools (monoclonal antibodies (mAbs) and recombinant proteins) generated in the laboratory during the course of basic functional investigations devoted to TP53INP1. The ELISA principle is based on a sandwich immunoenzymatic system, TP53INP1 protein being trapped by a first specific mAb coated on microplate then recognized by a second specific mAb. This new assay allows specific detection of TP53INP1 in serum of several PC patients. This breakthrough paves the way towards investigation of a large cohort of patients and assessment of clinical applications of TP53INP1 dosage. PMID:24600558

  6. Heterogeneous nuclear ribonucleoprotein B1 protein impairs DNA repair mediated through the inhibition of DNA-dependent protein kinase activity

    SciTech Connect

    Iwanaga, Kentaro; Sueoka, Naoko; Sato, Akemi; Hayashi, Shinichiro; Sueoka, Eisaburo . E-mail: sueokae@post.saga-med.ac.jp

    2005-08-05

    Heterogeneous nuclear ribonucleoprotein B1, an RNA binding protein, is overexpressed from the early stage of lung cancers; it is evident even in bronchial dysplasia, a premalignant lesion. We evaluated the proteins bound with hnRNP B1 and found that hnRNP B1 interacted with DNA-dependent protein kinase (DNA-PK) complex, and recombinant hnRNP B1 protein dose-dependently inhibited DNA-PK activity in vitro. To test the effect of hnRNP B1 on DNA repair, we performed comet assay after irradiation, using normal human bronchial epithelial (HBE) cells treated with siRNA for hnRNP A2/B1: reduction of hnRNP B1 treated with siRNA for hnRNP A2/B1 induced faster DNA repair in normal HBE cells. Considering these results, we assume that overexpression of hnRNP B1 occurring in the early stage of carcinogenesis inhibits DNA-PK activity, resulting in subsequent accumulation of erroneous rejoining of DNA double-strand breaks, causing tumor progression.

  7. Nuclear Import of the Yeast Hexokinase 2 Protein Requires ?/?-Importin-dependent Pathway*

    PubMed Central

    Peláez, Rafael; Fernández-García, Paula; Herrero, Pilar; Moreno, Fernando

    2012-01-01

    Hexokinase 2 (Hxk2) from Saccharomyces cerevisiae was one of the first metabolic enzymes described as a multifunctional protein. Hxk2 has a double subcellular localization and role, it functions as a glycolytic enzyme in the cytoplasm and as a regulator of gene transcription of several Mig1-regulated genes in the nucleus. However, the mechanism by which Hxk2 enters in the nucleus was unknown until now. Here, we report that the Hxk2 protein is an import substrate of the carriers ?-importin (Kap60 in yeast) and ?-importin (Kap95 in yeast). We also show that the Hxk2 nuclear import and the binding of Hxk2 with Kap60 are glucose-dependent and involve one lysine-rich nuclear localization sequence (NLS), located between lysine 6 and lysine 12. Moreover, Kap95 facilitates the recognition of the Hxk2 NLS1 motif by Kap60 and both importins are essential for Hxk2 nuclear import. It is also demonstrated that Hxk2 nuclear import and its binding to Kap95 and Kap60 depend on the Gsp1-GTP/GDP protein levels. Thus, our study uncovers Hxk2 as a new cargo for the ?/?-importin pathway of S. cerevisiae. PMID:22157003

  8. Mutational analyses of fs(1)Ya, an essential, developmentally regulated, nuclear envelope protein in Drosophila

    SciTech Connect

    Liu, Jun; Song, Kiwon; Wolfner, M.F.

    1995-12-01

    The fs(1)Ya protein (YA) is an essential, maternally encoded, nuclear lamina protein that is under both developmental and cell cycle control. A strong Ya mutation results in early arrest of embryos. To define the function of YA in the nuclear envelope during early embryonic development, we characterized the phenotypes of four Ya mutant alleles and determined their molecular lesions. Ya mutant embryos arrest with abnormal nuclear envelopes prior to the first mitotic division; a proportion of embryos from two leaky Ya mutants proceed beyond this but arrest after several abnormal divisions. Ya unfertilized eggs contain nuclei of different sizes and condensation states, apparently due to abnormal fusion of the meiotic products immediately after meiosis. Lamin is localized at the periphery of the uncondensed nuclei in these eggs. These results suggest that Ya function is required during and after egg maturation to facilitate proper chromatin condensation, rather than to allow a lamin-containing nuclear envelope to form. Two leaky Ya alleles that partially complement have lesions at opposite ends of the YA protein, suggesting that the N- and C-termini are important for YA function might interact with itself either directly or indirectly. 27 refs., 6 figs.

  9. A single herpesvirus protein can mediate vesicle formation in the nuclear envelope.

    PubMed

    Lorenz, Michael; Vollmer, Benjamin; Unsay, Joseph D; Klupp, Barbara G; García-Sáez, Ana J; Mettenleiter, Thomas C; Antonin, Wolfram

    2015-03-13

    Herpesviruses assemble capsids in the nucleus and egress by unconventional vesicle-mediated trafficking through the nuclear envelope. Capsids bud at the inner nuclear membrane into the nuclear envelope lumen. The resulting intralumenal vesicles fuse with the outer nuclear membrane, delivering the capsids to the cytoplasm. Two viral proteins are required for vesicle formation, the tail-anchored pUL34 and its soluble interactor, pUL31. Whether cellular proteins are involved is unclear. Using giant unilamellar vesicles, we show that pUL31 and pUL34 are sufficient for membrane budding and scission. pUL34 function can be bypassed by membrane tethering of pUL31, demonstrating that pUL34 is required for pUL31 membrane recruitment but not for membrane remodeling. pUL31 can inwardly deform membranes by oligomerizing on their inner surface to form buds that constrict to vesicles. Therefore, a single viral protein can mediate all events necessary for membrane budding and abscission. PMID:25605719

  10. Cell density-dependent nuclear/cytoplasmic localization of NORPEG (RAI14) protein

    SciTech Connect

    Kutty, R. Krishnan . E-mail: kuttyk@nei.nih.gov; Chen, Shanyi; Samuel, William; Vijayasarathy, Camasamudram; Duncan, Todd; Tsai, Jen-Yue; Fariss, Robert N.; Carper, Deborah; Jaworski, Cynthia; Wiggert, Barbara

    2006-07-14

    NORPEG (RAI14), a developmentally regulated gene induced by retinoic acid, encodes a 980 amino acid (aa) residue protein containing six ankyrin repeats and a long coiled-coil domain [Kutty et al., J. Biol. Chem. 276 (2001), pp. 2831-2840]. We have expressed aa residues 1-287 of NORPEG and used the recombinant protein to produce an anti-NORPEG polyclonal antibody. Confocal immunofluorescence analysis showed that the subcellular localization of NORPEG in retinal pigment epithelial (ARPE-19) cells varies with cell density, with predominantly nuclear localization in nonconfluent cells, but a cytoplasmic localization, reminiscent of cytoskeleton, in confluent cultures. Interestingly, an evolutionarily conserved putative monopartite nuclear localization signal (P{sup 27}KKRKAP{sup 276}) was identified by analyzing the sequences of NORPEG and its orthologs. GFP-NORPEG (2-287 aa), a fusion protein containing this signal, was indeed localized to nuclei when expressed in ARPE-19 or COS-7 cells. Deletion and mutation analysis indicated that the identified nuclear localization sequence is indispensable for nuclear targeting.

  11. Nuclear resonance vibrational spectroscopy (NRVS) of rubredoxin and MoFe protein crystals

    NASA Astrophysics Data System (ADS)

    Guo, Yisong; Brecht, Eric; Aznavour, Kristen; Nix, Jay C.; Xiao, Yuming; Wang, Hongxin; George, Simon J.; Bau, Robert; Keable, Stephen; Peters, John W.; Adams, Michael W. W.; , Francis E. Jenney, Jr.; Sturhahn, Wolfgang; Alp, Ercan E.; Zhao, Jiyong; Yoda, Yoshitaka; Cramer, Stephen P.

    2013-12-01

    We have applied 57Fe nuclear resonance vibrational spectroscopy (NRVS) for the first time to study the dynamics of Fe centers in Iron-sulfur protein crystals, including oxidized wild type rubredoxin crystals from Pyrococcus furiosus, and the MoFe protein of nitrogenase from Azotobacter vinelandii. Thanks to the NRVS selection rule, selectively probed vibrational modes have been observed in both oriented rubredoxin and MoFe protein crystals. The NRVS work was complemented by extended X-ray absorption fine structure spectroscopy (EXAFS) measurements on oxidized wild type rubredoxin crystals from Pyrococcus furiosus. The EXAFS spectra revealed the Fe-S bond length difference in oxidized Pf Rd protein, which is qualitatively consistent with the crystal structure.

  12. Identification of a functional nuclear export signal in the green fluorescent protein asFP499

    SciTech Connect

    Mustafa, Huseyin . E-mail: huseyinm@hotmail.com; Strasser, Bernd; Rauth, Sabine; Irving, Robert A.; Wark, Kim L.

    2006-04-21

    The green fluorescent protein (GFP) asFP499 from Anemonia sulcata is a distant homologue of the GFP from Aequorea victoria. We cloned the asFP499 gene into a mammalian expression vector and showed that this protein was expressed in the human lymphoblast cell line Ramos RA1 and in the embryonic kidney 293T cell line (HEK 293T). In HEK 293T cells, asFP499 was localized mainly in the cytoplasm, suggesting that the protein was excluded from the nucleus. We identified {sub 194}LRMEKLNI{sub 201} as a candidate nuclear export signal in asFP499 and mutated the isoleucine at position 201 to an alanine. Unlike the wildtype form, the mutant protein was distributed throughout the cytoplasm and nucleus. This is First report of a GFP that contains a functional NES.

  13. Nuclear resonance vibrational spectroscopy (NRVS) of rubredoxin and MoFe protein crystals

    PubMed Central

    Guo, Yisong; Brecht, Eric; Aznavour, Kristen; Nix, Jay C.; Xiao, Yuming; Wang, Hongxin; George, Simon J.; Bau, Robert; Keable, Stephen; Peters, John W.; Adams, Michael W.W.; Jenney, Francis; Sturhahn, Wolfgang; Alp, Ercan E.; Zhao, Jiyong; Yoda, Yoshitaka; Cramer, Stephen P.

    2014-01-01

    We have applied 57Fe nuclear resonance vibrational spectroscopy (NRVS) for the first time to study the dynamics of Fe centers in Fe-S protein crystals, including oxidized wild type rubredoxin crystals from Pyrococcus furiosus, and the MoFe protein of nitrogenase from Azotobacter vinelandii. Thanks to the NRVS selection rule, selectively probed vibrational modes have been observed in both oriented rubredoxin and MoFe protein crystals. The NRVS work was complemented by extended X-ray absorption fine structure spectroscopy (EXAFS) measurements on oxidized wild type rubredoxin crystals from Pyrococcus furiosus. The EXAFS spectra revealed the Fe-S bond length difference in oxidized Pf Rd protein, which is qualitatively consistent with the X-ray crystal structure. PMID:26052177

  14. SUMOylation regulates the nuclear mobility of CREB binding protein and its association with nuclear bodies in live cells

    SciTech Connect

    Ryan, Colm M.; Kindle, Karin B.; Collins, Hilary M.; Heery, David M.

    2010-01-01

    The lysine acetyltransferase CREB binding protein (CBP) is required for chromatin modification and transcription at many gene promoters. In fixed cells, a large proportion of CBP colocalises to PML or nuclear bodies. Using live cell imaging, we show here that YFP-tagged CBP expressed in HEK293 cells undergoes gradual accumulation in nuclear bodies, some of which are mobile and migrate towards the nuclear envelope. Deletion of a short lysine-rich domain that contains the major SUMO acceptor sites of CBP abrogated its ability to be SUMO modified, and prevented its association with endogenous SUMO-1/PML speckles in vivo. This SUMO-defective CBP showed enhanced ability to co-activate AML1-mediated transcription. Deletion mapping revealed that the SUMO-modified region was not sufficient for targeting CBP to PML bodies, as C-terminally truncated mutants containing this domain showed a strong reduction in accumulation at PML bodies. Fluorescence recovery after photo-bleaching (FRAP) experiments revealed that YFP-CBP{Delta}998-1087 had a retarded recovery time in the nucleus, as compared to YFP-CBP. These results indicate that SUMOylation regulates CBP function by influencing its shuttling between nuclear bodies and chromatin microenvironments.

  15. Nuclear importation of Mariner transposases among eukaryotes: motif requirements and homo-protein interactions.

    PubMed

    Demattei, Marie-Véronique; Hedhili, Sabah; Sinzelle, Ludivine; Bressac, Christophe; Casteret, Sophie; Moiré, Nathalie; Cambefort, Jeanne; Thomas, Xavier; Pollet, Nicolas; Gantet, Pascal; Bigot, Yves

    2011-01-01

    Mariner-like elements (MLEs) are widespread transposable elements in animal genomes. They have been divided into at least five sub-families with differing host ranges. We investigated whether the ability of transposases encoded by Mos1, Himar1 and Mcmar1 to be actively imported into nuclei varies between host belonging to different eukaryotic taxa. Our findings demonstrate that nuclear importation could restrict the host range of some MLEs in certain eukaryotic lineages, depending on their expression level. We then focused on the nuclear localization signal (NLS) in these proteins, and showed that the first 175 N-terminal residues in the three transposases were required for nuclear importation. We found that two components are involved in the nuclear importation of the Mos1 transposase: an SV40 NLS-like motif (position: aa 168 to 174), and a dimerization sub-domain located within the first 80 residues. Sequence analyses revealed that the dimerization moiety is conserved among MLE transposases, but the Himar1 and Mcmar1 transposases do not contain any conserved NLS motif. This suggests that other NLS-like motifs must intervene in these proteins. Finally, we showed that the over-expression of the Mos1 transposase prevents its nuclear importation in HeLa cells, due to the assembly of transposase aggregates in the cytoplasm. PMID:21876763

  16. An N-terminal nuclear localization sequence but not the calmodulin-binding domain mediates nuclear localization of nucleomorphin, a protein that regulates nuclear number in Dictyostelium

    SciTech Connect

    Myre, Michael A.; O'Day, Danton H. . E-mail: doday@utm.utoronto.ca

    2005-06-24

    Nucleomorphin is a novel nuclear calmodulin (CaM)-binding protein (CaMBP) containing an extensive DEED (glu/asp repeat) domain that regulates nuclear number. GFP-constructs of the 38 kDa NumA1 isoform localize as intranuclear patches adjacent to the inner nuclear membrane. The translocation of CaMBPs into nuclei has previously been shown by others to be mediated by both classic nuclear localization sequences (NLSs) and CaM-binding domains (CaMBDs). Here we show that NumA1 possesses a CaMBD ({sup 171}EDVSRFIKGKLLQKQQKIYKDLERF{sup 195}) containing both calcium-dependent-binding motifs and an IQ-like motif for calcium-independent binding. GFP-constructs containing only NumA1 residues 1-129, lacking the DEED and CaMBDs, still localized as patches at the internal periphery of nuclei thus ruling out a direct role for the CaMBD in nuclear import. These constructs contained the amino acid residues {sup 48}KKSYQDPEIIAHSRPRK{sup 64} that include both a putative bipartite and classical NLS. GFP-bipartite NLS constructs localized uniformly within nuclei but not as patches. As with previous work, removal of the DEED domain resulted in highly multinucleate cells. However as shown here, multinuclearity only occurred when the NLS was present allowing the protein to enter nuclei. Site-directed mutation analysis in which the NLS was changed to {sup 48}EF{sup 49} abolished the stability of the GFP fusion at the protein but not RNA level preventing subcellular analyses. Cells transfected with the {sup 48}EF{sup 49} construct exhibited slowed growth when compared to parental AX3 cells and other GFP-NumA1 deletion mutants. In addition to identifying an NLS that is sufficient for nuclear translocation of nucleomorphin and ruling out CaM-binding in this event, this work shows that the nuclear localization of NumA1 is crucial to its ability to regulate nuclear number in Dictyostelium.

  17. LaRbp38: A Leishmania amazonensis protein that binds nuclear and kinetoplast DNAs

    SciTech Connect

    Lira, C.B.B.; Siqueira Neto, J.L.; Giardini, M.A.; Winck, F.V.; Ramos, C.H.I.; Cano, M.I.N. . E-mail: micano@ibb.unesp.br

    2007-07-06

    Leishmania amazonensis causes a wide spectrum of leishmaniasis. There are no vaccines or adequate treatment for leishmaniasis, therefore there is considerable interest in the identification of new targets for anti-leishmania drugs. The central role of telomere-binding proteins in cell maintenance makes these proteins potential targets for new drugs. In this work, we used a combination of purification chromatographies to screen L. amazonensis proteins for molecules capable of binding double-stranded telomeric DNA. This approach resulted in the purification of a 38 kDa polypeptide that was identified by mass spectrometry as Rbp38, a trypanosomatid protein previously shown to stabilize mitochondrial RNA and to associate with nuclear and kinetoplast DNAs. Western blotting and supershift assays confirmed the identity of the protein as LaRbp38. Competition and chromatin immunoprecipitation assays confirmed that LaRbp38 interacted with kinetoplast and nuclear DNAs in vivo and suggested that LaRbp38 may have dual cellular localization and more than one function.

  18. A RanGTP-independent mechanism allows ribosomal protein nuclear import for ribosome assembly

    PubMed Central

    Schütz, Sabina; Fischer, Ute; Altvater, Martin; Nerurkar, Purnima; Peña, Cohue; Gerber, Michaela; Chang, Yiming; Caesar, Stefanie; Schubert, Olga T; Schlenstedt, Gabriel; Panse, Vikram G

    2014-01-01

    Within a single generation time a growing yeast cell imports ?14 million ribosomal proteins (r-proteins) into the nucleus for ribosome production. After import, it is unclear how these intrinsically unstable and aggregation-prone proteins are targeted to the ribosome assembly site in the nucleolus. Here, we report the discovery of a conserved nuclear carrier Tsr2 that coordinates transfer of the r-protein eS26 to the earliest assembling pre-ribosome, the 90S. In vitro studies revealed that Tsr2 efficiently dissociates importin:eS26 complexes via an atypical RanGTP-independent mechanism that terminates the import process. Subsequently, Tsr2 binds the released eS26, shields it from proteolysis, and ensures its safe delivery to the 90S pre-ribosome. We anticipate similar carriers—termed here escortins—to securely connect the nuclear import machinery with pathways that deposit r-proteins onto developing pre-ribosomal particles. DOI: http://dx.doi.org/10.7554/eLife.03473.001 PMID:25144938

  19. Acetylation-dependent nuclear arrangement and recruitment of BMI1 protein to UV-damaged chromatin.

    PubMed

    Sustá?ková, Gabriela; Kozubek, Stanislav; Stixová, Lenka; Legartová, So?a; Matula, Pavel; Orlova, Darya; Bártová, Eva

    2012-05-01

    Polycomb group (PcG) proteins, organized into Polycomb bodies, are important regulatory components of epigenetic processes involved in the heritable transcriptional repression of target genes. Here, we asked whether acetylation can influence the nuclear arrangement and function of the BMI1 protein, a core component of the Polycomb group complex, PRC1. We used time-lapse confocal microscopy, micro-irradiation by UV laser (355 nm) and GFP technology to study the dynamics and function of the BMI1 protein. We observed that BMI1 was recruited to UV-damaged chromatin simultaneously with decreased lysine acetylation, followed by the recruitment of heterochromatin protein HP1? to micro-irradiated regions. Pronounced recruitment of BMI1 was rapid, with half-time ? = 15 sec; thus, BMI1 is likely involved in the initiation step leading to the recognition of UV-damaged sites. Histone hyperacetylation, stimulated by HDAC inhibitor TSA, suppression of transcription by actinomycin D, and ATP-depletion prevented increased accumulation of BMI1 to ?H2AX-positive irradiated chromatin. Moreover, BMI1 had slight ability to recognize spontaneously occurring DNA breaks caused by other pathophysiological processes. Taken together, our data indicate that the dynamics of recognition of UV-damaged chromatin, and the nuclear arrangement of BMI1 protein can be influenced by acetylation and occur as an early event prior to the recruitment of HP? to UV-irradiated chromatin. PMID:21732356

  20. Nuclear localization of DMP1 proteins suggests a role in intracellular signaling

    SciTech Connect

    Siyam, Arwa; Department of Endodontology, Kornberg School of Dentistry, Temple University, 3223 North Broad Street, Philadelphia, PA 19140-5007 ; Wang, Suzhen; Qin, Chunlin; Mues, Gabriele; Stevens, Roy; D'Souza, Rena N.; Lu, Yongbo

    2012-08-03

    Highlights: Black-Right-Pointing-Pointer Nuclear localization of DMP1 in various cell lines. Black-Right-Pointing-Pointer Non-synchronized cells show either nuclear or cytoplasmic localization of DMP1. Black-Right-Pointing-Pointer Nuclear DMP1 is restricted to the nucleoplasm but absent in the nucleolus. -- Abstract: Dentin matrix protein 1 (DMP1) is highly expressed in odontoblasts and osteoblasts/osteocytes and plays an essential role in tooth and bone mineralization and phosphate homeostasis. It is debatable whether DMP1, in addition to its function in the extracellular matrix, can enter the nucleus and function as a transcription factor. To better understand its function, we examined the nuclear localization of endogenous and exogenous DMP1 in C3H10T1/2 mesenchymal cells, MC3T3-E1 preosteoblast cells and 17IIA11 odontoblast-like cells. RT-PCR analyses showed the expression of endogenous Dmp1 in all three cell lines, while Western-blot analysis detected a major DMP1 protein band corresponding to the 57 kDa C-terminal fragment generated by proteolytic processing of the secreted full-length DMP1. Immunofluorescent staining demonstrated that non-synchronized cells presented two subpopulations with either nuclear or cytoplasmic localization of endogenous DMP1. In addition, cells transfected with a construct expressing HA-tagged full-length DMP1 also showed either nuclear or cytoplasmic localization of the exogenous DMP1 when examined with an antibody against the HA tag. Furthermore, nuclear DMP1 was restricted to the nucleoplasm but was absent in the nucleolus. In conclusion, these findings suggest that, apart from its role as a constituent of dentin and bone matrix, DMP1 might play a regulatory role in the nucleus.

  1. Tristetraprolin Inhibits Poly(A)-Tail Synthesis in Nuclear mRNA that Contains AU-Rich Elements by Interacting with Poly(A)-Binding Protein Nuclear 1

    PubMed Central

    Chiang, Pei-Yu; Lin, Nien-Yi; Shen, Yu-Fang; Chang, Geen-Dong; Chang, Ching-Jin

    2012-01-01

    Background Tristetraprolin binds mRNA AU-rich elements and thereby facilitates the destabilization of mature mRNA in the cytosol. Methodology/Principal Findings To understand how tristetraprolin mechanistically functions, we biopanned with a phage-display library for proteins that interact with tristetraprolin and retrieved, among others, a fragment of poly(A)-binding protein nuclear 1, which assists in the 3'-polyadenylation of mRNA by binding to immature poly(A) tails and thereby increases the activity of poly(A) polymerase, which is directly responsible for polyadenylation. The tristetraprolin/poly(A)-binding protein nuclear 1 interaction was characterized using tristetraprolin and poly(A)-binding protein nuclear 1 deletion mutants in pull-down and co-immunoprecipitation assays. Tristetraprolin interacted with the carboxyl-terminal region of poly(A)-binding protein nuclear 1 via its tandem zinc finger domain and another region. Although tristetraprolin and poly(A)-binding protein nuclear 1 are located in both the cytoplasm and the nucleus, they interacted in vivo in only the nucleus. In vitro, tristetraprolin bound both poly(A)-binding protein nuclear 1 and poly(A) polymerase and thereby inhibited polyadenylation of AU-rich element–containing mRNAs encoding tumor necrosis factor ?, GM-CSF, and interleukin-10. A tandem zinc finger domain–deleted tristetraprolin mutant was a less effective inhibitor. Expression of a tristetraprolin mutant restricted to the nucleus resulted in downregulation of an AU-rich element–containing tumor necrosis factor ?/luciferase mRNA construct. Conclusion/Significance In addition to its known cytosolic mRNA–degrading function, tristetraprolin inhibits poly(A) tail synthesis by interacting with poly(A)-binding protein nuclear 1 in the nucleus to regulate expression of AU-rich element–containing mRNA. PMID:22844456

  2. Cysteine-Specific Labeling of Proteins with a Nitroxide Biradical for Dynamic Nuclear Polarization NMR.

    PubMed

    Voinov, Maxim A; Good, Daryl B; Ward, Meaghan E; Milikisiyants, Sergey; Marek, Antonin; Caporini, Marc A; Rosay, Melanie; Munro, Rachel A; Ljumovic, Milena; Brown, Leonid S; Ladizhansky, Vladimir; Smirnov, Alex I

    2015-08-13

    Dynamic nuclear polarization (DNP) enhances the signal in solid-state NMR of proteins by transferring polarization from electronic spins to the nuclear spins of interest. Typically, both the protein and an exogenous source of electronic spins, such as a biradical, are either codissolved or suspended and then frozen in a glycerol/water glassy matrix to achieve a homogeneous distribution. While the use of such a matrix protects the protein upon freezing, it also reduces the available sample volume (by ca. a factor of 4 in our experiments) and causes proportional NMR signal loss. Here we demonstrate an alternative approach that does not rely on dispersing the DNP agent in a glassy matrix. We synthesize a new biradical, ToSMTSL, which is based on the known DNP agent TOTAPOL, but also contains a thiol-specific methanethiosulfonate group to allow for incorporating this biradical into a protein in a site-directed manner. ToSMTSL was characterized by EPR and tested for DNP of a heptahelical transmembrane protein, Anabaena sensory rhodopsin (ASR), by covalent modification of solvent-exposed cysteine residues in two (15)N-labeled ASR mutants. DNP enhancements were measured at 400 MHz/263 GHz NMR/EPR frequencies for a series of samples prepared in deuterated and protonated buffers and with varied biradical/protein ratios. While the maximum DNP enhancement of 15 obtained in these samples is comparable to that observed for an ASR sample cosuspended with ~17 mM TOTAPOL in a glycerol-d8/D2O/H2O matrix, the achievable sensitivity would be 4-fold greater due to the gain in the filling factor. We anticipate that the DNP enhancements could be further improved by optimizing the biradical structure. The use of covalently attached biradicals would broaden the applicability of DNP NMR to structural studies of proteins. PMID:26230514

  3. Lectin-binding proteins in nuclear preparations from rat liver and malignant tumors.

    PubMed

    Burrus, G R; Schmidt, W N; Briggs, J A; Hnilica, L S; Briggs, R C

    1988-02-01

    Lectin binding [concanavalin A, biotinylated ricinus communis agglutinin, and biotinylated succinylated wheat germ agglutinin (B-SWGA)] was used to detect the glycosylated proteins associated with a residual protein fraction [insoluble in 4% sodium dodecyl sulfate and termed the nuclear residual fraction (NRF)] or with nuclear matrix preparations from normal rat liver, azo dye (3'-MeDAB)-induced rat hepatoma, and Walker 256 transplantable carcinosarcoma. One- and two-dimensional gel electrophoresis were used with lectins, polyclonal antisera, and monoclonal antibody binding to characterize some of the glycoconjugates. Two polypeptide bands with approximate molecular weights of 95,000 and 55,000, shown previously to be present only in the induced tumor cells and the Walker 256 tumor, were reactive with lectins. In addition, a Mr 62,000 protein reacted only with B-SWGA in the nuclear matrix fractions from normal rat liver and the induced hepatoma. A polypeptide band (approximate molecular weight, 213,000) in the Walker 256 NRF reacted with concanavalin A and biotinylated ricinus communis agglutinin. One polypeptide band (approximate molecular weight, 182,000) reacted with concanavalin A in all three tissues, with biotinylated ricinus communis agglutinin and B-SWGA in the Walker NRF, and with B-SWGA in the hepatoma NRF. Another polypeptide band (approximate molecular weight, 138,000), reactive with all three lectins, was present in all three tissues. Our findings are consistent with previous reports of lectin binding proteins in the eukaryotic cell nucleus and indicate that certain glycoproteins isolated in nuclear preparations are found specifically in 3'-MeDAB-induced hepatoma and Walker 256 transplantable carcinosarcoma. PMID:3335020

  4. Utilization of nuclear structural proteins for targeted therapy and detection of proliferative and differentiation disorders

    DOEpatents

    Lelievre, Sophie (Berkeley, CA); Bissell, Mina (Berkeley, CA)

    2001-01-01

    The localization of nuclear apparatus proteins (NUMA) is used to identify tumor cells and different stages in the tumor progression and differentiation processes. There is a characteristic organization of NuMA in tumor cells and in phenotypically normal cells. NuMA distribution patterns are significantly less diffuse in proliferating non-malignant cells compared to malignant cells. The technique encompasses cell immunostaining using a NuMA specific antibody, and microscopic analysis of NuMA distribution within each nucleus.

  5. Interaction of HTLV-1 Tax protein with calreticulin: implications for Tax nuclear export and secretion.

    PubMed

    Alefantis, Timothy; Flaig, Katherine E; Wigdahl, Brian; Jain, Pooja

    2007-05-01

    Human T cell leukemia virus type 1 (HTLV-1) is the etiologic agent of adult T cell leukemia (ATL) and HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). The HTLV-1 transcriptional transactivator protein Tax plays an integral role in virus replication and disease progression. Traditionally, Tax is described as a nuclear protein where it performs its primary role as a transcriptional transactivator. However, recent studies have clearly shown that Tax can also be localized to the cytoplasm where it has been shown to interact with a number of host transcription factors most notably NF-kappaB, constitutive expression of which is directly related to the T cell transforming properties of Tax in ATL patients. The presence of a functional nuclear export signal (NES) within Tax and the secretion of full-length Tax have also been demonstrated previously. Additionally, release of Tax from HTLV-1-infected cells and the presence of cell-free Tax was demonstrated in the CSF of HAM/TSP patients suggesting that the progression to HAM/TSP might be mediated by the ability of Tax to function as an extracellular cytokine. Therefore, in both ATL and HAM/TSP Tax nuclear export and nucleocytoplasmic shuttling may play a critical role, the mechanism of which remains unknown. In this study, we have demonstrated that the calcium binding protein calreticulin interacts with Tax by co-immunoprecipitation. This interaction was found to localize to a region at or near the nuclear membrane. In addition, differential expression of calreticulin was demonstrated in various cell types that correlated with their ability to retain cytoplasmic Tax, particularly in astrocytes. Finally, a comparison of a number of HTLV-1-infected T cell lines to non-infected T cells revealed higher expression of calreticulin in infected cells implicating a direct role for this protein in HTLV-1 infection. PMID:17395420

  6. Long isoform of ErbB3 binding protein, p48, mediates protein kinase B/Akt-dependent HDM2 stabilization and nuclear localization

    SciTech Connect

    Kim, Chung Kwon; Lee, Sang Bae; Nguyen, Truong L.X.; Lee, Kyung-Hoon; Center for Molecular Medicine, Samsung Biomedical Research Institute, Sungkyunkwan University School of Medicine, Suwon, 440-746 ; Um, Sung Hee; Center for Molecular Medicine, Samsung Biomedical Research Institute, Sungkyunkwan University School of Medicine, Suwon, 440-746 ; Kim, Jihoe; Ahn, Jee-Yin; Center for Molecular Medicine, Samsung Biomedical Research Institute, Sungkyunkwan University School of Medicine, Suwon, 440-746

    2012-01-15

    p48 is a long isoform of the ErbB3 binding protein that has oncogenic functions including promotion of carcinogenesis and induction of malignant transformation through negative regulation of tumor suppressor p53. Here, we show that high level of p48 protein expression leads to enhance HDM2 phosphorylation by Akt and inhibits the self-ubiquitination of HDM2 by up-regulation of Akt activity, thereby promoting its protein stability. Moreover, p48 expression leads to accumulated nuclear localization of HDM2, whereas p48 depletion disturbs its nuclear localization. Hence, higher expression of p48 in cancer cells reduces p53 levels through modulation of HDM2 nuclear localization and protein stability via regulation of its Akt-mediated phosphorylation.

  7. Mapping Functional Group Free Energy Patterns at Protein Occluded Sites: Nuclear Receptors and G-Protein Coupled Receptors

    PubMed Central

    2015-01-01

    Occluded ligand-binding pockets (LBP) such as those found in nuclear receptors (NR) and G-protein coupled receptors (GPCR) represent a significant opportunity and challenge for computer-aided drug design. To determine free energies maps of functional groups of these LBPs, a Grand-Canonical Monte Carlo/Molecular Dynamics (GCMC/MD) strategy is combined with the Site Identification by Ligand Competitive Saturation (SILCS) methodology. SILCS-GCMC/MD is shown to map functional group affinity patterns that recapitulate locations of functional groups across diverse classes of ligands in the LBPs of the androgen (AR) and peroxisome proliferator-activated-? (PPAR?) NRs and the metabotropic glutamate (mGluR) and ?2-adreneric (?2AR) GPCRs. Inclusion of protein flexibility identifies regions of the binding pockets not accessible in crystal conformations and allows for better quantitative estimates of relative ligand binding affinities in all the proteins tested. Differences in functional group requirements of the active and inactive states of the ?2AR LBP were used in virtual screening to identify high efficacy agonists targeting ?2AR in Airway Smooth Muscle (ASM) cells. Seven of the 15 selected ligands were found to effect ASM relaxation representing a 46% hit rate. Hence, the method will be of use for the rational design of ligands in the context of chemical biology and the development of therapeutic agents. PMID:25692383

  8. SINC, a type III secreted protein of Chlamydia psittaci, targets the inner nuclear membrane of infected cells and uninfected neighbors

    PubMed Central

    Mojica, Sergio A.; Hovis, Kelley M.; Frieman, Matthew B.; Tran, Bao; Hsia, Ru-ching; Ravel, Jacques; Jenkins-Houk, Clifton; Wilson, Katherine L.; Bavoil, Patrik M.

    2015-01-01

    SINC, a new type III secreted protein of the avian and human pathogen Chlamydia psittaci, uniquely targets the nuclear envelope of C. psittaci–infected cells and uninfected neighboring cells. Digitonin-permeabilization studies of SINC-GFP–transfected HeLa cells indicate that SINC targets the inner nuclear membrane. SINC localization at the nuclear envelope was blocked by importazole, confirming SINC import into the nucleus. Candidate partners were identified by proximity to biotin ligase-fused SINC in HEK293 cells and mass spectrometry (BioID). This strategy identified 22 candidates with high confidence, including the nucleoporin ELYS, lamin B1, and four proteins (emerin, MAN1, LAP1, and LBR) of the inner nuclear membrane, suggesting that SINC interacts with host proteins that control nuclear structure, signaling, chromatin organization, and gene silencing. GFP-SINC association with the native LEM-domain protein emerin, a conserved component of nuclear “lamina” structure, or with a complex containing emerin was confirmed by GFP pull down. Our findings identify SINC as a novel bacterial protein that targets the nuclear envelope with the capability of globally altering nuclear envelope functions in the infected host cell and neighboring uninfected cells. These properties may contribute to the aggressive virulence of C. psittaci. PMID:25788290

  9. BGLF4 Kinase Modulates the Structure and Transport Preference of the Nuclear Pore Complex To Facilitate Nuclear Import of Epstein-Barr Virus Lytic Proteins

    PubMed Central

    Chang, Chou-Wei; Lee, Chung-Pei; Su, Mei-Tzu; Tsai, Ching-Hwa

    2014-01-01

    ABSTRACT BGLF4 kinase, the only Ser/Thr protein kinase encoded by the Epstein-Barr virus (EBV) genome, phosphorylates multiple viral and cellular substrates to optimize the cellular environment for viral DNA replication and the nuclear egress of nucleocapsids. Previously, we found that nuclear targeting of BGLF4 is through direct interaction with the FG repeat-containing nucleoporins (FG-Nups) Nup62 and Nup153 independently of cytosolic transport factors. Here, we investigated the regulatory effects of BGLF4 on the structure and biological functions of the nuclear pore complex (NPC). In EBV-positive NA cells, the distribution of FG-Nups was modified during EBV reactivation. In transfected cells, BGLF4 changed the staining pattern of Nup62 and Nup153 in a kinase activity-dependent manner. Detection with anti-phospho-Ser/Thr-Pro MPM-2 antibody demonstrated that BGLF4 induced the phosphorylation of Nup62 and Nup153. The nuclear targeting of importin ? was attenuated in the presence of BGLF4, leading to inhibition of canonical nuclear localization signal (NLS)-mediated nuclear import. An in vitro nuclear import assay revealed that BGLF4 induced the nuclear import of larger molecules. Notably, we found that BGLF4 promoted the nuclear import of several non-NLS-containing EBV proteins, including the viral DNA-replicating enzymes BSLF1, BBLF2/3, and BBLF4 and the major capsid protein (VCA), in cotransfected cells. The data presented here suggest that BGLF4 interferes with the normal functions of Nup62 and Nup153 and preferentially helps the nuclear import of viral proteins for viral DNA replication and assembly. In addition, the nuclear import-promoting activity was found in cells expressing the BGLF4 homologs of another two gammaherpesviruses but not those from alpha- and betaherpesviruses. IMPORTANCE During lytic replication, many EBV genome-encoded proteins need to be transported into the nucleus, not only for viral DNA replication but also for the assembly of nucleocapsids. Because nuclear pore complexes are effective gateways that control nucleocytoplasmic traffic, most EBV proteins without canonical NLSs are retained in the cytoplasm until they form complexes with their NLS-containing partners for nuclear targeting. In this study, we found that EBV BGLF4 protein kinase interacts with the Nup62 and Nup153 and induces the redistribution of FG-Nups. BGLF4 modulates the function of the NPC to inhibit the nuclear import of host NLS-containing proteins. Simultaneously, the nuclear import of non-NLS-containing EBV lytic proteins was enhanced, possibly through phosphorylation of Nup62 and Nup153, nuclear pore dilation, or microtubule reorganization. Overall, our data suggest that BGLF4-induced modification of nuclear pore transport may block nuclear targeting of cellular proteins and increase the import of viral proteins to promote viral lytic replication. PMID:25410863

  10. Identification of a Karyopherin ?1/?2 Proline-Tyrosine Nuclear Localization Signal in Huntingtin Protein*

    PubMed Central

    Desmond, Carly R.; Atwal, Randy Singh; Xia, Jianrun; Truant, Ray

    2012-01-01

    Among the known pathways of protein nuclear import, the karyopherin ?2/transportin pathway is only the second to have a defined nuclear localization signal (NLS) consensus. Huntingtin, a 350-kDa protein, has defined roles in the nucleus, as well as a CRM1/exportin-dependent nuclear export signal; however, the NLS and exact pathway of import have remained elusive. Here, using a live cell assay and affinity chromatography, we show that huntingtin has a karyopherin ?2-dependent proline-tyrosine (PY)-NLS in the amino terminus of the protein. This NLS comprises three consensus components: a basic charged sequence, a downstream conserved arginine, and a PY sequence. Unlike the classic PY-NLS, which has an unstructured intervening sequence between the consensus components, we show that a ? sheet structured region separating the consensus elements is critical for huntingtin NLS function. The huntingtin PY-NLS is also capable of import through the importin/karyopherin ?1 pathway but was not functional in all cell types tested. We propose that this huntingtin PY-NLS may comprise a new class of multiple import factor-dependent NLSs with an internal structural component that may regulate NLS activity. PMID:23012356

  11. An RNA-protein complex links enhanced nuclear 3' processing with cytoplasmic mRNA stabilization.

    PubMed

    Ji, Xinjun; Kong, Jian; Liebhaber, Stephen A

    2011-07-01

    Post-transcriptional controls are critical to gene regulation. These controls are frequently based on sequence-specific binding of trans-acting proteins to cis-acting motifs on target RNAs. Prior studies have revealed that the KH-domain protein, ?CP, binds to a 3' UTR C-rich motif of h?-globin mRNA and contributes to its cytoplasmic stability. Here, we report that this 3' UTR ?CP complex regulates the production of mature ?-globin mRNA by enhancing 3' processing of the h?-globin transcript. We go on to demonstrate that this nuclear activity reflects enhancement of both the cleavage and the polyadenylation reactions and that ?CP interacts in vivo with core components of the 3' processing complex. Consistent with its nuclear processing activity, our studies reveal that ?CP assembles co-transcriptionally at the h?-globin chromatin locus and that this loading is selectively enriched at the 3' terminus of the gene. The demonstrated linkage of nuclear processing with cytoplasmic stabilization via a common RNA-protein complex establishes a basis for integration of sequential controls critical to robust and sustained expression of a target mRNA. PMID:21623344

  12. Nuclear Export of the Yeast Hexokinase 2 Protein Requires the Xpo1 (Crm1)-dependent Pathway*

    PubMed Central

    Peláez, Rafael; Herrero, Pilar; Moreno, Fernando

    2009-01-01

    Hexokinase 2 (Hxk2) from Saccharomyces cerevisiae was one of the first metabolic enzymes described as a multifunctional protein. Hxk2 has a double subcellular localization; it functions as a glycolytic enzyme in the cytoplasm and as a regulator of gene transcription of several Mig1-regulated genes in the nucleus. However, the mechanism by which Hxk2 enters and leaves the nucleus is still unknown. In low glucose conditions, Hxk2 is phosphorylated at serine 14, but how this phosphorylation may affect glucose signaling is also unknown at the moment. Here we report that the Hxk2 protein is an export substrate of the carrier protein Xpo1 (Crm1). We also show that the Hxk2 nuclear export and the binding of Hxk2 and Xpo1 involve two leucine-rich nuclear export signals (NES) located between leucine 23 and isoleucine 33 (NES1) and between leucine 310 and leucine 318 (NES2). We also show that the Hxk2 phosphorylation at serine 14 promotes Hxk2 export by facilitating the association of Hxk2 with Xpo1. Our study uncovers a new cargo for the Xpo1 yeast exportin and identifies Hxk2 phosphorylation at serine 14 as a regulatory mechanism that controls its nuclear exit in function of the glucose levels. PMID:19525230

  13. Chromatin decompaction by the nucleosomal binding protein HMGN5 impairs nuclear sturdiness

    NASA Astrophysics Data System (ADS)

    Furusawa, Takashi; Rochman, Mark; Taher, Leila; Dimitriadis, Emilios K.; Nagashima, Kunio; Anderson, Stasia; Bustin, Michael

    2015-01-01

    In most metazoan nuclei, heterochromatin is located at the nuclear periphery in contact with the nuclear lamina, which provides mechanical stability to the nucleus. We show that in cultured cells, chromatin decompaction by the nucleosome binding protein HMGN5 decreases the sturdiness, elasticity and rigidity of the nucleus. Mice overexpressing HMGN5, either globally or only in the heart, are normal at birth but develop hypertrophic heart with large cardiomyoctyes, deformed nuclei and disrupted lamina and die of cardiac malfunction. Chromatin decompaction is seen in cardiomyocytes of newborn mice but misshaped nuclei with disrupted lamina are seen only in adult cardiomyocytes, suggesting that loss of heterochromatin diminishes the ability of the nucleus to withstand the mechanical forces of the contracting heart. Thus, heterochromatin enhances the ability of the nuclear lamina to maintain the sturdiness and shape of the eukaryotic nucleus; a structural role for chromatin that is distinct from its genetic functions.

  14. Chromatin De-Compaction By The Nucleosomal Binding Protein HMGN5 Impairs Nuclear Sturdiness

    PubMed Central

    Furusawa, Takashi; Rochman, Mark; Taher, Leila; Dimitriadis, Emilios K.; Nagashima, Kunio; Anderson, Stasia; Bustin, Michael

    2014-01-01

    In most metazoan nuclei, heterochromatin is located at the nuclear periphery in contact with the nuclear lamina, which provides mechanical stability to the nucleus. We show that in cultured cells, chromatin de-compaction by the nucleosome binding protein HMGN5 decreases the sturdiness, elasticity, and rigidity of the nucleus. Mice overexpressing HMGN5, either globally or only in the heart, are normal at birth but develop hypertrophic heart with large cardiomyoctyes, deformed nuclei and disrupted lamina, and die of cardiac malfunction. Chromatin de-compaction is seen in cardiomyocytes of newborn mice but misshaped nuclei with disrupted lamina are seen only in adult cardiomyocytes, suggesting that loss of heterochromatin diminishes the ability of the nucleus to withstand the mechanical forces of the contracting heart. Thus, heterochromatin enhances the ability of the nuclear lamina to maintain the sturdiness and shape of the eukaryotic nucleus; a structural role for chromatin that is distinct from its genetic functions. PMID:25609380

  15. Retinoblastoma-binding Protein 4-regulated Classical Nuclear Transport Is Involved in Cellular Senescence.

    PubMed

    Tsujii, Akira; Miyamoto, Yoichi; Moriyama, Tetsuji; Tsuchiya, Yuko; Obuse, Chikashi; Mizuguchi, Kenji; Oka, Masahiro; Yoneda, Yoshihiro

    2015-12-01

    Nucleocytoplasmic trafficking is a fundamental cellular process in eukaryotic cells. Here, we demonstrated that retinoblastoma-binding protein 4 (RBBP4) functions as a novel regulatory factor to increase the efficiency of importin ?/?-mediated nuclear import. RBBP4 accelerates the release of importin ?1 from importin ? via competitive binding to the importin ?-binding domain of importin ? in the presence of RanGTP. Therefore, it facilitates importin ?/?-mediated nuclear import. We showed that the importin ?/? pathway is down-regulated in replicative senescent cells, concomitant with a decrease in RBBP4 level. Knockdown of RBBP4 caused both suppression of nuclear transport and induction of cellular senescence. This is the first report to identify a factor that competes with importin ?1 to bind to importin ?, and it demonstrates that the loss of this factor can trigger cellular senescence. PMID:26491019

  16. Targeting the Nuclear Export Protein XPO1/CRM1 Reverses Epithelial to Mesenchymal Transition.

    PubMed

    Azmi, Asfar S; Muqbil, Irfana; Wu, Jack; Aboukameel, Amro; Senapedis, William; Baloglu, Erkan; Bollig-Fischer, Aliccia; Dyson, Gregory; Kauffman, Michael; Landesman, Yosef; Shacham, Sharon; Philip, Philip A; Mohammad, Ramzi M

    2015-01-01

    Here we demonstrate for the first time that targeted inhibition of nuclear exporter protein exportin 1 (XPO1) also known as chromosome maintenance region 1 (CRM1) by Selective Inhibitor of Nuclear Export (SINE) compounds results in reversal of EMT in snail-transduced primary human mammary epithelial cells (HMECs). SINE compounds selinexor (KPT-330) and KPT-185, leptomycin B (LMB as +ve control) but not KPT-301 (-ve control) reverse EMT, suppress mesenchymal markers and consequently induce growth inhibition, apoptosis and prevent spheroid formation. SINE treatment resulted in nuclear retention of snail regulator FBXL5 that was concurrent with suppression of snail and down-regulation of mesenchymal markers. FBXL5 siRNA or transfection with cys528 mut-Xpo1 (lacking SINE binding site) markedly abrogated SINE activity highlighting an XPO1 and FBXL5 mediated mechanism of action. Silencing XPO1 or snail caused re-expression of FBXL5 as well as EMT reversal. Pathway analysis on SINE treated HMECs further verified the involvement of additional F-Box family proteins and confirmed the suppression of snail network. Oral administration of selinexor (15?mg/kg p.o. QoDx3/week for 3weeks) resulted in complete cures (no tumor rebound at 120 days) of HMLER-Snail xenografts. These findings raise the unique possibility of blocking EMT at the nuclear pore. PMID:26536918

  17. The plant TPX2 protein regulates prospindle assembly before nuclear envelope breakdown.

    PubMed

    Vos, Jan W; Pieuchot, Laurent; Evrard, Jean-Luc; Janski, Natacha; Bergdoll, Marc; de Ronde, Dryas; Perez, Laurent H; Sardon, Teresa; Vernos, Isabelle; Schmit, Anne-Catherine

    2008-10-01

    The Targeting Protein for Xklp2 (TPX2) is a central regulator of spindle assembly in vertebrate cells. The absence or excess of TPX2 inhibits spindle formation. We have defined a TPX2 signature motif that is present once in vertebrate sequences but twice in plants. Plant TPX2 is predominantly nuclear during interphase and is actively exported before nuclear envelope breakdown to initiate prospindle assembly. It localizes to the spindle microtubules but not to the interdigitating polar microtubules during anaphase or to the phragmoplast as it is rapidly degraded during telophase. We characterized the Arabidopsis thaliana TPX2-targeting domains and show that the protein is able to rescue microtubule assembly in TPX2-depleted Xenopus laevis egg extracts. Injection of antibodies to TPX2 into living plant cells inhibits the onset of mitosis. These results demonstrate that plant TPX2 already functions before nuclear envelope breakdown. Thus, plants have adapted nuclear-cytoplasmic shuttling of TPX2 to maintain proper spindle assembly without centrosomes. PMID:18941054

  18. Targeting the Nuclear Export Protein XPO1/CRM1 Reverses Epithelial to Mesenchymal Transition

    PubMed Central

    Azmi, Asfar S.; Muqbil, Irfana; Wu, Jack; Aboukameel, Amro; Senapedis, William; Baloglu, Erkan; Bollig-Fischer, Aliccia; Dyson, Gregory; Kauffman, Michael; Landesman, Yosef; Shacham, Sharon; Philip, Philip A.; Mohammad, Ramzi M.

    2015-01-01

    Here we demonstrate for the first time that targeted inhibition of nuclear exporter protein exportin 1 (XPO1) also known as chromosome maintenance region 1 (CRM1) by Selective Inhibitor of Nuclear Export (SINE) compounds results in reversal of EMT in snail-transduced primary human mammary epithelial cells (HMECs). SINE compounds selinexor (KPT-330) and KPT-185, leptomycin B (LMB as +ve control) but not KPT-301 (–ve control) reverse EMT, suppress mesenchymal markers and consequently induce growth inhibition, apoptosis and prevent spheroid formation. SINE treatment resulted in nuclear retention of snail regulator FBXL5 that was concurrent with suppression of snail and down-regulation of mesenchymal markers. FBXL5 siRNA or transfection with cys528 mut-Xpo1 (lacking SINE binding site) markedly abrogated SINE activity highlighting an XPO1 and FBXL5 mediated mechanism of action. Silencing XPO1 or snail caused re-expression of FBXL5 as well as EMT reversal. Pathway analysis on SINE treated HMECs further verified the involvement of additional F-Box family proteins and confirmed the suppression of snail network. Oral administration of selinexor (15?mg/kg p.o. QoDx3/week for 3weeks) resulted in complete cures (no tumor rebound at 120 days) of HMLER-Snail xenografts. These findings raise the unique possibility of blocking EMT at the nuclear pore. PMID:26536918

  19. The Plant TPX2 Protein Regulates Prospindle Assembly before Nuclear Envelope Breakdown[W

    PubMed Central

    Vos, Jan W.; Pieuchot, Laurent; Evrard, Jean-Luc; Janski, Natacha; Bergdoll, Marc; de Ronde, Dryas; Perez, Laurent H.; Sardon, Teresa; Vernos, Isabelle; Schmit, Anne-Catherine

    2008-01-01

    The Targeting Protein for Xklp2 (TPX2) is a central regulator of spindle assembly in vertebrate cells. The absence or excess of TPX2 inhibits spindle formation. We have defined a TPX2 signature motif that is present once in vertebrate sequences but twice in plants. Plant TPX2 is predominantly nuclear during interphase and is actively exported before nuclear envelope breakdown to initiate prospindle assembly. It localizes to the spindle microtubules but not to the interdigitating polar microtubules during anaphase or to the phragmoplast as it is rapidly degraded during telophase. We characterized the Arabidopsis thaliana TPX2-targeting domains and show that the protein is able to rescue microtubule assembly in TPX2-depleted Xenopus laevis egg extracts. Injection of antibodies to TPX2 into living plant cells inhibits the onset of mitosis. These results demonstrate that plant TPX2 already functions before nuclear envelope breakdown. Thus, plants have adapted nuclear–cytoplasmic shuttling of TPX2 to maintain proper spindle assembly without centrosomes. PMID:18941054

  20. Reverse Micelles As a Platform for Dynamic Nuclear Polarization in Solution NMR of Proteins

    PubMed Central

    2015-01-01

    Despite tremendous advances in recent years, solution NMR remains fundamentally restricted due to its inherent insensitivity. Dynamic nuclear polarization (DNP) potentially offers significant improvements in this respect. The basic DNP strategy is to irradiate the EPR transitions of a stable radical and transfer this nonequilibrium polarization to the hydrogen spins of water, which will in turn transfer polarization to the hydrogens of the macromolecule. Unfortunately, these EPR transitions lie in the microwave range of the electromagnetic spectrum where bulk water absorbs strongly, often resulting in catastrophic heating. Furthermore, the residence times of water on the surface of the protein in bulk solution are generally too short for efficient transfer of polarization. Here we take advantage of the properties of solutions of encapsulated proteins dissolved in low viscosity solvents to implement DNP in liquids. Such samples are largely transparent to the microwave frequencies required and thereby avoid significant heating. Nitroxide radicals are introduced into the reverse micelle system in three ways: attached to the protein, embedded in the reverse micelle shell, and free in the aqueous core. Significant enhancements of the water resonance ranging up to ??93 at 0.35 T were observed. We also find that the hydration properties of encapsulated proteins allow for efficient polarization transfer from water to the protein. These and other observations suggest that merging reverse micelle encapsulation technology with DNP offers a route to a significant increase in the sensitivity of solution NMR spectroscopy of proteins and other biomolecules. PMID:24456213

  1. Optimized expression, solubilization and purification of nuclear inclusion protein b of cardamom mosaic virus.

    PubMed

    Jebasingh, T; Jacob, T; Shah, M; Das, D; Krishnaswamy, S; Usha, R

    2008-04-01

    All RNA viruses encode an RNA-dependent RNA polymerase (RdRP) that is required for replication of the viral genome. Nuclear inclusion b (NIb) gene codes for the RdRp in Potyviridae viruses. In this study, expression, solubilization and purification of NIb protein of Cardamom mosaic virus (CdMV) is reported. The objective of the present study was to express and purify the NIb protein of CdMV on a large scale for structural characterization, as the structure of the RdRp from a plant virus is yet to be determined. However, the expression of NIb protein with hexa-histidine tag in Escherichia coli led to insoluble aggregates. Out of all the approaches [making truncated versions to reduce the size of protein; replacing an amino acid residue likely to be involved in hydrophobic intermolecular interactions with a hydrophilic one; expressing the protein along with chaperones; expression in Origami cells for proper disulphide bond formation, in E. coli as a fusion with maltose-binding protein (MBP) and in Nicotiana tabacum] to obtain the RdRp in a soluble form, only expression in E. coli as a fusion with MBP and its expression in N. tabacum were successful. The NIb expressed in plant or as a fusion with MBP in E. coli can be scaled up for further work. PMID:21086722

  2. Diffusion and retention are major determinants of protein targeting to the inner nuclear membrane.

    PubMed

    Ungricht, Rosemarie; Klann, Michael; Horvath, Peter; Kutay, Ulrike

    2015-06-01

    Newly synthesized membrane proteins are constantly sorted from the endoplasmic reticulum (ER) to various membranous compartments. How proteins specifically enrich at the inner nuclear membrane (INM) is not well understood. We have established a visual in vitro assay to measure kinetics and investigate requirements of protein targeting to the INM. Using human LBR, SUN2, and LAP2? as model substrates, we show that INM targeting is energy-dependent but distinct from import of soluble cargo. Accumulation of proteins at the INM relies on both a highly interconnected ER network, which is affected by energy depletion, and an efficient immobilization step at the INM. Nucleoporin depletions suggest that translocation through nuclear pore complexes (NPCs) is rate-limiting and restricted by the central NPC scaffold. Our experimental data combined with mathematical modeling support a diffusion-retention-based mechanism of INM targeting. We experimentally confirmed the sufficiency of diffusion and retention using an artificial reporter lacking natural sorting signals that recapitulates the energy dependence of the process in vivo. PMID:26056139

  3. Respiratory syncytial virus M2-1 protein induces the activation of nuclear factor kappa B

    SciTech Connect

    Reimers, Kerstin . E-mail: reimers.kerstin@mh-hannover.de; Buchholz, Katja; Werchau, Hermann

    2005-01-20

    Respiratory syncytial virus (RSV) induces the production of a number of cytokines and chemokines by activation of nuclear factor kappa B (NF-{kappa}B). The activation of NF-{kappa}B has been shown to depend on viral replication in the infected cells. In this study, we demonstrate that expression of RSV M2-1 protein, a transcriptional processivity and anti-termination factor, is sufficient to activate NF-{kappa}B in A549 cells. Electromobility shift assays show increased NF-{kappa}B complexes in the nuclei of M2-1-expressing cells. M2-1 protein is found in nuclei of M2-1-expressing cells and in RSV-infected cells. Co-immunoprecipitations of nuclear extracts of M2-1-expressing cells and of RSV-infected cells revealed an association of M2-1 with Rel A protein. Furthermore, the activation of NF-{kappa}B depends on the C-terminus of the RSV M2-1 protein, as shown by NF-{kappa}B-induced gene expression of a reporter gene construct.

  4. Protein Sub-Nuclear Localization Based on Effective Fusion Representations and Dimension Reduction Algorithm LDA

    PubMed Central

    Wang, Shunfang; Liu, Shuhui

    2015-01-01

    An effective representation of a protein sequence plays a crucial role in protein sub-nuclear localization. The existing representations, such as dipeptide composition (DipC), pseudo-amino acid composition (PseAAC) and position specific scoring matrix (PSSM), are insufficient to represent protein sequence due to their single perspectives. Thus, this paper proposes two fusion feature representations of DipPSSM and PseAAPSSM to integrate PSSM with DipC and PseAAC, respectively. When constructing each fusion representation, we introduce the balance factors to value the importance of its components. The optimal values of the balance factors are sought by genetic algorithm. Due to the high dimensionality of the proposed representations, linear discriminant analysis (LDA) is used to find its important low dimensional structure, which is essential for classification and location prediction. The numerical experiments on two public datasets with KNN classifier and cross-validation tests showed that in terms of the common indexes of sensitivity, specificity, accuracy and MCC, the proposed fusing representations outperform the traditional representations in protein sub-nuclear localization, and the representation treated by LDA outperforms the untreated one. PMID:26703574

  5. Diffusion and retention are major determinants of protein targeting to the inner nuclear membrane

    PubMed Central

    Ungricht, Rosemarie; Klann, Michael; Horvath, Peter

    2015-01-01

    Newly synthesized membrane proteins are constantly sorted from the endoplasmic reticulum (ER) to various membranous compartments. How proteins specifically enrich at the inner nuclear membrane (INM) is not well understood. We have established a visual in vitro assay to measure kinetics and investigate requirements of protein targeting to the INM. Using human LBR, SUN2, and LAP2? as model substrates, we show that INM targeting is energy-dependent but distinct from import of soluble cargo. Accumulation of proteins at the INM relies on both a highly interconnected ER network, which is affected by energy depletion, and an efficient immobilization step at the INM. Nucleoporin depletions suggest that translocation through nuclear pore complexes (NPCs) is rate-limiting and restricted by the central NPC scaffold. Our experimental data combined with mathematical modeling support a diffusion-retention–based mechanism of INM targeting. We experimentally confirmed the sufficiency of diffusion and retention using an artificial reporter lacking natural sorting signals that recapitulates the energy dependence of the process in vivo. PMID:26056139

  6. Protein Sub-Nuclear Localization Based on Effective Fusion Representations and Dimension Reduction Algorithm LDA.

    PubMed

    Wang, Shunfang; Liu, Shuhui

    2015-01-01

    An effective representation of a protein sequence plays a crucial role in protein sub-nuclear localization. The existing representations, such as dipeptide composition (DipC), pseudo-amino acid composition (PseAAC) and position specific scoring matrix (PSSM), are insufficient to represent protein sequence due to their single perspectives. Thus, this paper proposes two fusion feature representations of DipPSSM and PseAAPSSM to integrate PSSM with DipC and PseAAC, respectively. When constructing each fusion representation, we introduce the balance factors to value the importance of its components. The optimal values of the balance factors are sought by genetic algorithm. Due to the high dimensionality of the proposed representations, linear discriminant analysis (LDA) is used to find its important low dimensional structure, which is essential for classification and location prediction. The numerical experiments on two public datasets with KNN classifier and cross-validation tests showed that in terms of the common indexes of sensitivity, specificity, accuracy and MCC, the proposed fusing representations outperform the traditional representations in protein sub-nuclear localization, and the representation treated by LDA outperforms the untreated one. PMID:26703574

  7. Histone deacetylase 2 and N-Myc reduce p53 protein phosphorylation at serine 46 by repressing gene transcription of tumor protein 53-induced nuclear protein 1

    PubMed Central

    Shahbazi, Jeyran; Scarlett, Christopher J.; Norris, Murray D.; Liu, Bing; Haber, Michelle; Tee, Andrew E.; Carrier, Alice; Biankin, Andrew V.; London, Wendy B.; Marshall, Glenn M.; Lock, Richard B.; Liu, Tao

    2014-01-01

    Myc oncoproteins and histone deacetylases (HDACs) exert oncogenic effects by modulating gene transcription. Paradoxically, N-Myc induces p53 gene expression. Tumor protein 53-induced nuclear protein 1 (TP53INP1) phosphorylates p53 protein at serine 46, leading to enhanced p53 activity, transcriptional activation of p53 target genes and programmed cell death. Here we aimed to identify the mechanism through which N-Myc overexpressing p53 wild-type neuroblastoma cells acquired resistance to apoptosis. TP53INP1 was found to be one of the genes most significantly repressed by HDAC2 and N-Myc according to Affymetrix microarray gene expression datasets. HDAC2 and N-Myc reduced TP53INP1 gene expression by direct binding to the TP53INP1 gene promoter, leading to transcriptional repression of TP53INP1, p53 protein de-phosphorylation at serine 46, neuroblastoma cell proliferation and survival. Moreover, low levels of TP53INP1 expression in human neuroblastoma tissues correlated with high levels of N-Myc expression and poor patient outcome, and the BET bromodomain inhibitors JQ1 and I-BET151 reduced N-Myc expression and reactivated TP53INP1 expression in neuroblastoma cells. These findings identify TP53INP1 repression as an important co-factor for N-Myc oncogenesis, and provide further evidence for the potential application of BET bromodomain inhibitors in the therapy of N-Myc-induced neuroblastoma. PMID:24952595

  8. Identification of a nuclear localization sequence in the polyomavirus capsid protein VP2

    NASA Technical Reports Server (NTRS)

    Chang, D.; Haynes, J. I. 2nd; Brady, J. N.; Consigli, R. A.; Spooner, B. S. (Principal Investigator)

    1992-01-01

    A nuclear localization signal (NLS) has been identified in the C-terminal (Glu307-Glu-Asp-Gly-Pro-Gln-Lys-Lys-Lys-Arg-Arg-Leu318) amino acid sequence of the polyomavirus minor capsid protein VP2. The importance of this amino acid sequence for nuclear transport of newly synthesized VP2 was demonstrated by a genetic "subtractive" study using the constructs pSG5VP2 (expressing full-length VP2) and pSG5 delta 3VP2 (expressing truncated VP2, lacking amino acids Glu307-Leu318). These constructs were transfected into COS-7 cells, and the intracellular localization of the VP2 protein was determined by indirect immunofluorescence. These studies revealed that the full-length VP2 was localized in the nucleus, while the truncated VP2 protein was localized in the cytoplasm and not transported to the nucleus. A biochemical "additive" approach was also used to determine whether this sequence could target nonnuclear proteins to the nucleus. A synthetic peptide identical to VP2 amino acids Glu307-Leu318 was cross-linked to the nonnuclear proteins bovine serum albumin (BSA) or immunoglobulin G (IgG). The conjugates were then labeled with fluorescein isothiocyanate and microinjected into the cytoplasm of NIH 3T6 cells. Both conjugates localized in the nucleus of the microinjected cells, whereas unconjugated BSA and IgG remained in the cytoplasm. Taken together, these genetic subtractive and biochemical additive approaches have identified the C-terminal sequence of polyoma-virus VP2 (containing amino acids Glu307-Leu318) as the NLS of this protein.

  9. Identification of nuclear genes encoding chloroplast-localized proteins required for embryo development in Arabidopsis.

    PubMed

    Bryant, Nicole; Lloyd, Johnny; Sweeney, Colleen; Myouga, Fumiyoshi; Meinke, David

    2011-04-01

    We describe here the diversity of chloroplast proteins required for embryo development in Arabidopsis (Arabidopsis thaliana). Interfering with certain chloroplast functions has long been known to result in embryo lethality. What has not been reported before is a comprehensive screen for embryo-defective (emb) mutants altered in chloroplast proteins. From a collection of transposon and T-DNA insertion lines at the RIKEN chloroplast function database (http://rarge.psc.riken.jp/chloroplast/) that initially appeared to lack homozygotes and segregate for defective seeds, we identified 23 additional examples of EMB genes that likely encode chloroplast-localized proteins. Fourteen gene identities were confirmed with allelism tests involving duplicate mutant alleles. We then queried journal publications and the SeedGenes database (www.seedgenes.org) to establish a comprehensive dataset of 381 nuclear genes encoding chloroplast proteins of Arabidopsis associated with embryo-defective (119 genes), plant pigment (121 genes), gametophyte (three genes), and alternate (138 genes) phenotypes. Loci were ranked based on the level of certainty that the gene responsible for the phenotype had been identified and the protein product localized to chloroplasts. Embryo development is frequently arrested when amino acid, vitamin, or nucleotide biosynthesis is disrupted but proceeds when photosynthesis is compromised and when levels of chlorophyll, carotenoids, or terpenoids are reduced. Chloroplast translation is also required for embryo development, with genes encoding chloroplast ribosomal and pentatricopeptide repeat proteins well represented among EMB datasets. The chloroplast accD locus, which is necessary for fatty acid biosynthesis, is essential in Arabidopsis but not in Brassica napus or maize (Zea mays), where duplicated nuclear genes compensate for its absence or loss of function. PMID:21139083

  10. Mapping of nuclear import signal and importin {alpha}3 binding regions of 52K protein of bovine adenovirus-3

    SciTech Connect

    Paterson, Carolyn P.; Ayalew, Lisanework E.; Veterinary Microbiology, University of Saskatchewan, Saskatoon, SK S7N 5E3 S7N 5B4 Canada ; Tikoo, Suresh K.

    2012-10-10

    The L1 region of bovine adenovirus (BAdV)-3 encodes a non-structural protein designated 52K. Anti-52K serum detected a protein of 40 kDa, which localized to the nucleus but not to the nucleolus in BAdV-3-infected or transfected cells. Analysis of mutant 52K proteins suggested that three basic residues ({sup 105}RKR{sup 107}) of the identified domain (amino acids {sup 102}GMPRKRVLT{sup 110}) are essential for nuclear localization of 52K. The nuclear import of a GST-52K fusion protein utilizes the classical importin {alpha}/{beta}-dependent nuclear transport pathway. The 52K protein is preferentially bound to the cellular nuclear import receptor importin {alpha}3. Although deletion of amino acid 102-110 is sufficient to abrogate the nuclear localization of 52K, amino acid 90-133 are required for interaction with importin-{alpha}3 and localizing a cytoplasmic protein to the nucleus. These results suggest that 52K contains a bipartite NLS, which preferentially utilize an importin {alpha}3 nuclear import receptor-mediated pathway to transport 52K to the nucleus.

  11. Use of a protein-blotting procedure and a specific DNA probe to identify nuclear proteins that recognize the promoter region of the transferrin receptor gene.

    PubMed Central

    Miskimins, W K; Roberts, M P; McClelland, A; Ruddle, F H

    1985-01-01

    We describe a procedure for detecting high-affinity, sequence-specific DNA-binding proteins from crude nuclear extracts. The technique utilizes electrophoretic transfer of NaDodSO4/PAGE-fractionated proteins onto nitrocellulose filters. Incubation of the filters with a 5% (wt/vol) solution of nonfat dry milk effectively blocks nonspecific and low-affinity DNA-binding sites. Incubation of the blocked filters with radiolabeled DNA under optimal binding conditions and subsequent autoradiography reveals high-affinity DNA-protein interactions. We have used this procedure to identify proteins that bind specifically to the promoter region of the transferrin receptor gene. Images PMID:2995982

  12. Novel Nuclear Protein Complexes of Dystrophin 71 Isoforms in Rat Cultured Hippocampal GABAergic and Glutamatergic Neurons

    PubMed Central

    Alemán, Víctor; Osorio, Beatriz; Chávez-González, Oscar; Rendon, Alvaro; Martínez-Rojas, Dalila; Meraz-Ríos, Marco Antonio

    2015-01-01

    The precise functional role of the dystrophin 71 in neurons is still elusive. Previously, we reported that dystrophin 71d and dystrophin 71f are present in nuclei from cultured neurons. In the present work, we performed a detailed analysis of the intranuclear distribution of dystrophin 71 isoforms (Dp71d and Dp71f), during the temporal course of 7-day postnatal rats hippocampal neurons culture for 1h, 2, 4, 10, 15 and 21 days in vitro (DIV). By immunofluorescence assays, we detected the highest level of nuclear expression of both dystrophin Dp71 isoforms at 10 DIV, during the temporal course of primary culture. Dp71d and Dp71f were detected mainly in bipolar GABAergic (?60%) and multipolar Glutamatergic (?40%) neurons, respectively. We also characterized the existence of two nuclear dystrophin-associated protein complexes (DAPC): dystrophin 71d or dystrophin 71f bound to ?-dystroglycan, ?1-, ?-, ?2-dystrobrevins, ?-syntrophin, and syntrophin-associated protein nNOS (Dp71d-DAPC or Dp71f-DAPC, respectively), in the hippocampal neurons. Furthermore, both complexes were localized in interchromatin granule cluster structures (nuclear speckles) of neuronal nucleoskeleton preparations. The present study evinces that each Dp71’s complexes differ slightly in dystrobrevins composition. The results demonstrated that Dp71d-DAPC was mainly localized in bipolar GABAergic and Dp71f-DAPC in multipolar Glutamatergic hippocampal neurons. Taken together, our results show that dystrophin 71d, dystrophin 71f and DAP integrate protein complexes, and both complexes were associated to nuclear speckles structures. PMID:26378780

  13. Platelet-derived growth factor induces phosphorylation of a 64-kDa nuclear protein

    SciTech Connect

    Shawver, L.K.; Pierce, G.F.; Kawahara, R.S.; Deuel, T.F.

    1989-01-15

    The platelet-derived growth factor (PDGF) stimulated the phosphorylation of a nuclear protein of 64 kDa (pp64) in nuclei of nontransformed normal rat kidney (NRK) cells. Low levels of phosphorylation of pp64 were observed in nuclei of serum-starved NRK cells. Fetal calf serum (FCS), PDGF, and homodimeric v-sis and PDGF A-chain protein enhanced the incorporation of 32P into pp64 over 4-fold within 30 min and over 8-fold within 2 h of exposure of NRK cells to the growth factors. In contrast, constitutive phosphorylation of 32P-labeled pp64 in nuclei of NRK cells transformed by the simian sarcoma virus (SSV) was high and only minimally stimulated by PDGF and FCS. 32P-Labeled pp64 was isolated from nuclei of PDGF-stimulated nontransformed NRK cells; the 32P of pp64 was labile in 1 M KOH, and pp64 was not significantly recognized by anti-phosphotyrosine antisera, suggesting that the PDGF-induced phosphorylation of pp64 occurred on serine or on threonine residues. However, pp64 from SSV-transformed NRK cell nuclei was significantly stable to base hydrolysis and was immunoprecipitated with anti-phosphotyrosine antisera, suggesting that pp64 from SSV-transformed cell nuclei is phosphorylated also on tyrosine. FCS, PDGF, and PDGF A- and B-chain homodimers thus stimulate the rapid time-dependent phosphorylation of a 64-kDa nuclear protein shortly after stimulation of responsive cells. The growth factor-stimulated phosphorylation of pp64 and the constitutive high levels of pp64 phosphorylation in cells transformed by SSV suggest important roles for pp64 and perhaps regulated nuclear protein kinases and phosphatases in cell division and proliferation.

  14. Nuclear localization of Cdc25 is regulated by DNA damage and a 14-3-3 protein.

    PubMed

    Lopez-Girona, A; Furnari, B; Mondesert, O; Russell, P

    1999-01-14

    DNA damage activates a cell-cycle checkpoint that prevents mitosis while DNA repair is under way. The protein Chk1 enforces this checkpoint by phosphorylating the mitotic inducer Cdc25. Phosphorylation of Cdc25 by Chk1 creates a binding site in Cdc25 for 14-3-3 proteins, but it is not known how 14-3-3 proteins regulate Cdc25. Rad24 is a 14-3-3 protein that is important in the DNA-damage checkpoint in fission yeast. Here we show that Rad24 controls the intracellular distribution of Cdc25. Elimination of Rad24 causes nuclear accumulation of Cdc25. Activation of the DNA-damage checkpoint causes the net nuclear export of Cdc25 by a process that requires Chk1, Rad24 and nuclear-export machinery. Mutation of a putative nuclear-export signal in Rad24 impairs the nuclear exclusion of Rad24, the damage-induced nuclear export of Cdc25 and the damage checkpoint. Thus, Rad24 appears to function as an attachable nuclear-export signal that enhances the nuclear export of Cdc25 in response to DNA damage. PMID:9923681

  15. proper cell division ELYS is a dual nucleoporin/kinetochore protein required for nuclear pore assembly and

    E-print Network

    Forbes, Douglass

    cell extracts. Indeed, a large fraction of ELYS localizes to the nuclear pore complexes of HeLa cells depletion from HeLa cells leads to an increase in cytokinesis defects. Thus, we have identified an unexproper cell division ELYS is a dual nucleoporin/kinetochore protein required for nuclear pore

  16. EAST Organizes Drosophila Insulator Proteins in the Interchromosomal Nuclear Compartment and Modulates CP190 Binding to Chromatin

    PubMed Central

    Golovnin, Anton; Melnikova, Larisa; Shapovalov, Igor; Kostyuchenko, Margarita; Georgiev, Pavel

    2015-01-01

    Recent data suggest that insulators organize chromatin architecture in the nucleus. The best studied Drosophila insulator proteins, dCTCF (a homolog of the vertebrate insulator protein CTCF) and Su(Hw), are DNA-binding zinc finger proteins. Different isoforms of the BTB-containing protein Mod(mdg4) interact with Su(Hw) and dCTCF. The CP190 protein is a cofactor for the dCTCF and Su(Hw) insulators. CP190 is required for the functional activity of insulator proteins and is involved in the aggregation of the insulator proteins into specific structures named nuclear speckles. Here, we have shown that the nuclear distribution of CP190 is dependent on the level of EAST protein, an essential component of the interchromatin compartment. EAST interacts with CP190 and Mod(mdg4)-67.2 proteins in vitro and in vivo. Over-expression of EAST in S2 cells leads to an extrusion of the CP190 from the insulator bodies containing Su(Hw), Mod(mdg4)-67.2, and dCTCF. In consistent with the role of the insulator bodies in assembly of protein complexes, EAST over-expression led to a striking decrease of the CP190 binding with the dCTCF and Su(Hw) dependent insulators and promoters. These results suggest that EAST is involved in the regulation of CP190 nuclear localization. PMID:26489095

  17. DETECTION OF EXTRA-NUCLEAR HIGH MOBILITY GROUP BOX-1 PROTEIN IN A CANINE MODEL OF MYOCARDIAL INFARCTION

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The high mobility group box-1 protein (HMGB-1) is a well-characterized nuclear protein recently shown to be involved in endotoxin-induced inflammation and injury. Studies have linked HMGB-1 release to the production of pro-inflammatory cytokines; however, a role for HMGB-1 in other disorders involvi...

  18. The Novel Human Protein Arginine N-Methyltransferase PRMT6 Is a Nuclear Enzyme Displaying Unique Substrate Specificity*

    E-print Network

    Clarke, Steven

    The Novel Human Protein Arginine N-Methyltransferase PRMT6 Is a Nuclear Enzyme Displaying Unique arginine methylation is a prevalent posttrans- lational modification in eukaryotic cells that has been processes. In searching the human genome for protein arginine N-methyltransferase (PRMT) family members

  19. MicroRNA-205 promotes the tumorigenesis of nasopharyngeal carcinoma through targeting tumor protein p53-inducible nuclear protein 1

    PubMed Central

    NIE, GUOHUI; DUAN, HONGFANG; LI, XIAOQING; YU, ZHENDONG; LUO, LIANG; LU, RUIJING; JI, ZILIANG; ZHANG, WEI

    2015-01-01

    Nasopharyngeal carcinoma (NPC) is a common type of cancer in southern China, miRNAs have been shown to be involved in the tumorigenesis of multiple cancer types. The present study aimed to explore the potential role of miR-205 in NPC. Reverse transcription quantitative polymerase chain reaction was used to determine the expression levels of miR-205 in 20 fresh NPC specimens and 20 normal nasopharyngeal tissues. The function of miR-205 in the proliferation, migration, invasion and apoptosis of NPC-derived cells was detected by MTT assay, colony formation assay, wound healing assay, Transwell assay and flow cytometry. Furthermore, a target gene of miR-205 was identified using the luciferase reporter assay. The expression of miR-205 was increased in NPC tissues compared with that in normal tissues. Overexpression of miR-205 was found to promote the proliferation, migration and invasion of NPC-derived cells, while apoptosis was suppressed. Tumor protein p53-inducible nuclear protein 1 was identified as a target gene of miR-205. Overall, the present study demonstrated that miR-205 may function as an oncogene in NPC tumorigenesis. PMID:26252115

  20. Nuclear body formation and PML body remodeling by the human cytomegalovirus protein UL35

    SciTech Connect

    Salsman, Jayme; Wang Xueqi; Frappier, Lori

    2011-06-05

    The human cytomegalovirus (HCMV) UL35 gene encodes two proteins, UL35 and UL35a. Expression of UL35 in transfected cells results in the formation of UL35 nuclear bodies that associate with promyelocytic leukemia (PML) protein. PML forms the basis for PML nuclear bodies that are important for suppressing viral lytic gene expression. Given the important relationship between PML and viral infection, we have further investigated the association of UL35 with PML bodies. We demonstrate that UL35 bodies form independently of PML and subsequently recruit PML, Sp100 and Daxx. In contrast, UL35a did not form bodies; however, it could bind UL35 and inhibit the formation of UL35 bodies. The HCMV tegument protein pp71 promoted the formation of UL35 bodies and the cytoplasmic localization of UL35a. Similarly, UL35a shifted pp71 to the cytoplasm. These results indicate that the interplay between UL35, UL35a and pp71 affects their subcellular localization and likely their functions throughout infection.

  1. Concentration-dependent lamin assembly and its roles in the localization of other nuclear proteins

    PubMed Central

    Guo, Yuxuan; Kim, Youngjo; Shimi, Takeshi; Goldman, Robert D.; Zheng, Yixian

    2014-01-01

    The nuclear lamina (NL) consists of lamin polymers and proteins that bind to the polymers. Disruption of NL proteins such as lamin and emerin leads to developmental defects and human diseases. However, the expression of multiple lamins, including lamin-A/C, lamin-B1, and lamin-B2, in mammals has made it difficult to study the assembly and function of the NL. Consequently, it has been unclear whether different lamins depend on one another for proper NL assembly and which NL functions are shared by all lamins or are specific to one lamin. Using mouse cells deleted of all or different combinations of lamins, we demonstrate that the assembly of each lamin into the NL depends primarily on the lamin concentration present in the nucleus. When expressed at sufficiently high levels, each lamin alone can assemble into an evenly organized NL, which is in turn sufficient to ensure the even distribution of the nuclear pore complexes. By contrast, only lamin-A can ensure the localization of emerin within the NL. Thus, when investigating the role of the NL in development and disease, it is critical to determine the protein levels of relevant lamins and the intricate shared or specific lamin functions in the tissue of interest. PMID:24523288

  2. Autophagy-mediated degradation of nuclear envelope proteins during oncogene-induced senescence.

    PubMed

    Lenain, Christelle; Gusyatiner, Olga; Douma, Sirith; van den Broek, Bram; Peeper, Daniel S

    2015-11-01

    Cellular senescence is a largely irreversible form of cell cycle arrest triggered by various types of damage and stress, including oncogene expression (termed oncogene-induced senescence or OIS). We and others have previously demonstrated that OIS occurs in human benign lesions, acting as a potent tumor suppressor mechanism. Numerous phenotypic changes occur during OIS, both in the cytoplasm and in the nucleus. These include the activation of autophagy, a catabolic process operating in the cytoplasm and downregulation of lamin B1, a component of the nuclear lamina. However, it is unknown whether these changes relate to each other. We discovered that cells entering BRAF(V600E)- or H-RAS(G12V)-induced senescence downregulate not only lamin B1 but also lamin A, as well as several other nuclear envelope (NE) proteins, resulting in an altered NE morphology. Depletion of LMNB1 or LMNA/C was sufficient to recapitulate some OIS features, including cell cycle exit and downregulation of NE proteins. We further found that the global loss of NE proteins is a consequence of their degradation by the autophagy machinery, which occurs concomitantly with autophagy induction and increased lysosomal content and activity. Our study therefore reveals a previously unknown connection between autophagy and the disruption of NE integrity during OIS. PMID:26354777

  3. Adenovirus early region 4 34-kilodalton protein directs the nuclear localization of the early region 1B 55-kilodalton protein in primate cells.

    PubMed Central

    Goodrum, F D; Shenk, T; Ornelles, D A

    1996-01-01

    The localization of the adenovirus type 5 34-kDa E4 and 55-kDa E1B proteins was determined in the absence of other adenovirus proteins. When expressed by transfection in human, monkey, hamster, rat, and mouse cell lines, the E1B protein was predominantly cytoplasmic and typically was excluded from the nucleus. When expressed by transfection, the E4 protein accumulated in the nucleus. Strikingly, when coexpressed by transfection in human, monkey, or baby hamster kidney cells, the E1B protein colocalized in the nucleus with the E4 protein. A complex of the E4 and E1B proteins was identified by coimmunoprecipitation in transfected HeLa cells. By contrast to the interaction observed in primate and baby hamster kidney cells, the E4 protein failed to direct the E1B protein to the nucleus in rat and mouse cell lines as well as CHO and V79 hamster cell lines. This failure of the E4 protein to direct the nuclear localization of the E1B protein in REF-52 rat cells was overcome by fusion with HeLa cells. Within 4 h of heterokaryon formation and with protein synthesis inhibited, a portion of the E4 protein present in the REF-52 nuclei migrated to the HeLa nuclei. Simultaneously, the previously cytoplasmic E1B protein colocalized with the E4 protein in both human and rat cell nuclei. These results suggest that a primate cell-specific factor mediates the functional interaction of the E1B and E4 proteins of adenovirus. PMID:8709260

  4. The essential WD40 protein Cia1 is involved in a late step of cytosolic and nuclear iron-sulfur protein assembly.

    PubMed

    Balk, Janneke; Aguilar Netz, Daili J; Tepper, Katharina; Pierik, Antonio J; Lill, Roland

    2005-12-01

    The assembly of cytosolic and nuclear iron-sulfur (Fe/S) proteins in yeast is dependent on the iron-sulfur cluster assembly and export machineries in mitochondria and three recently identified extramitochondrial proteins, the P-loop NTPases Cfd1 and Nbp35 and the hydrogenase-like Nar1. However, the molecular mechanism of Fe/S protein assembly in the cytosol is far from being understood, and more components are anticipated to take part in this process. Here, we have identified and functionally characterized a novel WD40 repeat protein, designated Cia1, as an essential component required for Fe/S cluster assembly in vivo on cytosolic and nuclear, but not mitochondrial, Fe/S proteins. Surprisingly, Nbp35 and Nar1, themselves Fe/S proteins, could assemble their Fe/S clusters in the absence of Cia1, demonstrating that these components act before Cia1. Consequently, Cia1 is involved in a late step of Fe/S cluster incorporation into target proteins. Coimmunoprecipitation assays demonstrated a specific interaction between Cia1 and Nar1. In contrast to the mostly cytosolic Nar1, Cia1 is preferentially localized to the nucleus, suggesting an additional function of Cia1. Taken together, our results indicate that Cia1 is a new member of the cytosolic Fe/S protein assembly (CIA) machinery participating in a step after Nbp35 and Nar1. PMID:16314508

  5. Nuclear import strategies of high-risk HPV18 L2 minor capsid protein.

    PubMed

    Klucevsek, K; Daley, J; Darshan, M S; Bordeaux, J; Moroianu, J

    2006-08-15

    We have investigated the nuclear import strategies of high-risk HPV18 L2 minor capsid protein. HPV18 L2 interacts with Kap alpha2 adapter, and Kap beta2 and Kap beta3 nuclear import receptors. Moreover, binding of RanGTP to either Kap beta2 or Kap beta3 inhibits their interaction with L2, suggesting that these Kap beta/L2 complexes are import competent. Mapping studies show that HPV18 L2 contains two NLSs: in the N-terminus (nNLS) and in the C-terminus (cNLS), both of which can independently mediate nuclear import. Both nNLS and cNLS form a complex with Kap alpha2beta1 heterodimer and mediate nuclear import via a classical pathway. The nNLS is also essential for the interaction of HPV18 L2 with Kap beta2 and Kap beta3. Interestingly, both nNLS and cNLS interact with the viral DNA and this DNA binding occurs without nucleotide sequence specificity. Together, the data suggest that HPV18 L2 can interact via its NLSs with several Kaps and the viral DNA and may enter the nucleus via multiple import pathways mediated by Kap alpha2beta1 heterodimers, Kap beta2 and Kap beta3. PMID:16733063

  6. HIV-1 capsid undergoes coupled binding and isomerization by the nuclear pore protein NUP358

    PubMed Central

    2013-01-01

    Background Lentiviruses such as HIV-1 can be distinguished from other retroviruses by the cyclophilin A-binding loop in their capsid and their ability to infect non-dividing cells. Infection of non-dividing cells requires transport through the nuclear pore but how this is mediated is unknown. Results Here we present the crystal structure of the N-terminal capsid domain of HIV-1 in complex with the cyclophilin domain of nuclear pore protein NUP358. The structure reveals that HIV-1 is positioned to allow single-bond resonance stabilisation of exposed capsid residue P90. NMR exchange experiments demonstrate that NUP358 is an active isomerase, which efficiently catalyzes cis-trans isomerization of the HIV-1 capsid. In contrast, the distantly related feline lentivirus FIV can bind NUP358 but is neither isomerized by it nor requires it for infection. Conclusion Isomerization by NUP358 may be preserved by HIV-1 to target the nuclear pore and synchronize nuclear entry with capsid uncoating. PMID:23902822

  7. Nuclear import strategies of high-risk HPV18 L2 minor capsid protein

    SciTech Connect

    Klucevsek, K.; Daley, J.; Darshan, M.S.; Bordeaux, J.; Moroianu, J. . E-mail: moroianu@bc.edu

    2006-08-15

    We have investigated the nuclear import strategies of high-risk HPV18 L2 minor capsid protein. HPV18 L2 interacts with Kap {alpha}{sub 2} adapter, and Kap {beta}{sub 2} and Kap {beta}{sub 3} nuclear import receptors. Moreover, binding of RanGTP to either Kap {beta}{sub 2} or Kap {beta}{sub 3} inhibits their interaction with L2, suggesting that these Kap {beta}/L2 complexes are import competent. Mapping studies show that HPV18 L2 contains two NLSs: in the N-terminus (nNLS) and in the C-terminus (cNLS), both of which can independently mediate nuclear import. Both nNLS and cNLS form a complex with Kap {alpha}{sub 2}{beta}{sub 1} heterodimer and mediate nuclear import via a classical pathway. The nNLS is also essential for the interaction of HPV18 L2 with Kap {beta}{sub 2} and Kap {beta}{sub 3}. Interestingly, both nNLS and cNLS interact with the viral DNA and this DNA binding occurs without nucleotide sequence specificity. Together, the data suggest that HPV18 L2 can interact via its NLSs with several Kaps and the viral DNA and may enter the nucleus via multiple import pathways mediated by Kap {alpha}{sub 2}{beta}{sub 1} heterodimers, Kap {beta}{sub 2} and Kap {beta}{sub 3}.

  8. Information flow and protein dynamics: the interplay between nuclear magnetic resonance spectroscopy and molecular dynamics simulations.

    PubMed

    Pastor, Nina; Amero, Carlos

    2015-01-01

    Proteins participate in information pathways in cells, both as links in the chain of signals, and as the ultimate effectors. Upon ligand binding, proteins undergo conformation and motion changes, which can be sensed by the following link in the chain of information. Nuclear magnetic resonance (NMR) spectroscopy and molecular dynamics (MD) simulations represent powerful tools for examining the time-dependent function of biological molecules. The recent advances in NMR and the availability of faster computers have opened the door to more detailed analyses of structure, dynamics, and interactions. Here we briefly describe the recent applications that allow NMR spectroscopy and MD simulations to offer unique insight into the basic motions that underlie information transfer within and between cells. PMID:25999971

  9. Information flow and protein dynamics: the interplay between nuclear magnetic resonance spectroscopy and molecular dynamics simulations

    PubMed Central

    Pastor, Nina; Amero, Carlos

    2015-01-01

    Proteins participate in information pathways in cells, both as links in the chain of signals, and as the ultimate effectors. Upon ligand binding, proteins undergo conformation and motion changes, which can be sensed by the following link in the chain of information. Nuclear magnetic resonance (NMR) spectroscopy and molecular dynamics (MD) simulations represent powerful tools for examining the time-dependent function of biological molecules. The recent advances in NMR and the availability of faster computers have opened the door to more detailed analyses of structure, dynamics, and interactions. Here we briefly describe the recent applications that allow NMR spectroscopy and MD simulations to offer unique insight into the basic motions that underlie information transfer within and between cells. PMID:25999971

  10. Transcriptional activation of NAD{sup +}-dependent protein deacetylase SIRT1 by nuclear receptor TLX

    SciTech Connect

    Iwahara, Naotoshi; Hisahara, Shin; Hayashi, Takashi; Department of Neurology, Sapporo Medical University, Sapporo 060-8556 ; Horio, Yoshiyuki

    2009-09-04

    An orphan nuclear receptor TLX is a transcriptional repressor that promotes the proliferation and self-renewal of neural precursor cells (NPCs). SIRT1, an NAD{sup +}-dependent protein deacetylase, is highly expressed in the NPCs and participates in neurogenesis. Here, we found that TLX colocalized with SIRT1 and knockdown of TLX by small interfering RNAs decreased SIRT1 levels in NPCs. TLX increased the SIRT1 expression by binding to the newly identified TLX-activating element in the SIRT1 gene promoter in HEK293 cells. Thus, TLX is an inducer of SIRT1 and may contribute to neurogenesis both as a transactivator and as a repressor.

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

    SciTech Connect

    Nakagawa, Koji; Uehata, Yasuko; Natsuizaka, Mitsuteru; Kohara, Toshihisa; Darmanin, Stephanie; Asaka, Masahiro; Department of Cancer Preventive Medicine, Graduate School of Medicine, Hokkaido University, N15 W7, Kita-ku, Sapporo, Hokkaido 060-8638 ; Takeda, Hiroshi; Department of Gastroenterology and Hematology, Graduate School of Medicine, Hokkaido University, N15 W7, Kita-ku, Sapporo, Hokkaido 060-8638 ; Kobayashi, Masanobu; School of Nursing and Social Services, Health Sciences University of Hokkaido, Ishikari-Toubetsu, Hokkaido 061-0293

    2012-11-02

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

  12. Nuclear Pore Complex Protein Sequences Determine Overall Copolymer Brush Structure and Function?

    NASA Astrophysics Data System (ADS)

    Ando, David; Kim, Yongwoon; Zandi, Roya; Colvin, Michael; Rexach, Michael; Gopinathan, Ajay

    2015-03-01

    Disordered proteins are an interesting class of unfolded protein biopolymers which are functionally versatile. Their sequences are unconstrained by a sequence-structure relationship, and allow for a wide range of chemical and physical polymer properties. The Nuclear Pore Complex (NPC) contains over one hundred of such proteins (FG nups), which collectively function to regulate the exchange of all materials between the nucleus and cytoplasm. We perform coarse grained simulations of both individual FG nups and grafted rings of nups mimicking the in vivo geometry of the NPC, supplemented with polymer brush modeling. Our results indicate that different regions or ``blocks'' of an individual FG nup can have distinctly different forms of disorder, and that this property appears to be a conserved feature across eukarya. Furthermore, this block structure at the individual protein level is critical to the formation of a unique higher-order polymer brush architecture. Because the interactions between FG nups may be modulated by certain forms of transport factors, our results indicate that transitions between brush morphologies could play an important role in regulating transport across the NPC, suggesting novel forms of gated transport across membrane pores with wide biomimetic applicability.

  13. Smad nuclear interacting protein, SNIP1, mediates the ecdysteroid signal transduction in red crayfish Procambarus clarkii.

    PubMed

    Wang, Daojun; Peng, Tao; Yu, Yingying; Liu, Chao-Liang; Zhu, Bao-Jian

    2015-02-01

    A smad nuclear interacting protein 1 (SNIP1) homolog was identified in the crayfish Procambarus clarkii and this gene encoded a polypeptide of 288 amino acids. Phylogenic analysis showed that P. clarkii SNIP1 was highly homologous to that of invertebrates and shared a highly conserved FHA domain of SNIP proteins at the C-terminus. Quantitative real-time PCR (qRT-PCR) analysis showed that Pc-SNIP1 expression was higher in heart than that in other examined tissues. In addition, prokaryotic expression and purification of the recombinant SNIP1 protein were performed. SDS-PAGE and western blot analysis demonstrated that a 35 KDa recombinant protein was successfully expressed in Escherichia coli cells. The expression of Pc-SNIP1 was significantly up-regulated in hepatopancreas after 20-hydroxyecdysone induction. Knockdown of Pc-SNIP1 gene by small interfering RNA (siRNA) transfection had significant influence on the expression of some ecdysteroid-responsive genes in hepatopancreas, which were confirmed by qRT-PCR and western blot. All together, these results suggest that SNIP1 is involved in the physiological process regulated by ecdysteroid in P. clarkii. PMID:25678477

  14. Dystonin/Bpag1 is a necessary endoplasmic reticulum/nuclear envelope protein in sensory neurons

    SciTech Connect

    Young, Kevin G.; Kothary, Rashmi

    2008-09-10

    Dystonin/Bpag1 proteins are cytoskeletal linkers whose loss of function in mice results in a hereditary sensory neuropathy with a progressive loss of limb coordination starting in the second week of life. These mice, named dystonia musculorum (dt), succumb to the disease and die of unknown causes prior to sexual maturity. Previous evidence indicated that cytoskeletal defects in the axon are a primary cause of dt neurodegeneration. However, more recent data suggests that other factors may be equally important contributors to the disease process. In the present study, we demonstrate perikaryal defects in dorsal root ganglion (DRG) neurons at stages preceding the onset of loss of limb coordination in dt mice. Abnormalities include alterations in endoplasmic reticulum (ER) chaperone protein expression, indicative of an ER stress response. Dystonin in sensory neurons localized in association with the ER and nuclear envelope (NE). A fusion protein ofthe dystonin-a2 isoform, which harbors an N-terminal transmembrane domain, associated with and reorganized the ER in cell culture. This isoform also interacts with the NE protein nesprin-3{alpha}, but not nesprin-3{beta}. Defects in dt mice, as demonstrated here, may ultimately result in pathogenesis involving ER dysfunction and contribute significantly to the dt phenotype.

  15. Identification of a Novel Nuclear Localization Signal Sequence in Chlamydia trachomatis-Secreted Hypothetical Protein CT311

    PubMed Central

    Lei, Lei; Dong, Xiaohua; Li, Zhongyu; Zhong, Guangming

    2013-01-01

    We previously reported that Chlamydia trachomatis hypothetical protein CT311 was secreted out of chlamydial inclusion and into host cell cytosol. We now found that CT311 further entered host cell nucleus at the late stage of infection and continued to accumulate in the nucleus of C. trachomatis-infected cells. When CT311 was expressed via a transgene in mammalian cells, CT311 protein was exclusively detected in the nucleus, suggesting that CT311 by itself is sufficient for nuclear targeting. However, preexisting nuclear CT311 did not affect subsequent chlamydial infection. Using deletion constructs, we mapped a nuclear localization signal sequence of CT311 to residues 21 to 63 (21AVEGKPLSRAAQLRERRKDLHVSGKPSPRYALKKRALEAKKNK63). This sequence was sufficient for targeting a heterologous protein into mammalian cell nucleus and it contains two independent clusters of basic residues (34RERRK38 and 53KKRALEAKKNK63 respectively). Deletion or alanine substitution of the basic residues in either cluster led to loss of nuclear targeting activity, suggesting that both clusters are critical for the nuclear targeting function. These observations have demonstrated that the hypothetical protein CT311 possesses a novel nuclear localization signal sequence with dual modules of basic residues for targeting host cell nucleus during Chlamydia trachomatis infection. PMID:23717625

  16. Nuclear translocation of the cytoskeleton-associated protein, smALP, upon induction of skeletal muscle differentiation

    SciTech Connect

    Cambier, Linda; Pomies, Pascal

    2011-06-17

    Highlights: {yields} The cytoskeleton-associated protein, smALP, is expressed in differentiated skeletal muscle. {yields} smALP is translocated from the cytoplasm to the nucleus of C2C12 myoblasts upon induction of myogenesis. {yields} The differentiation-dependent nuclear translocation of smALP occurs in parallel with the nuclear accumulation of myogenin. {yields} The LIM domain of smALP is essential for the nuclear accumulation of the protein. {yields} smALP might act in the nucleus to control some critical aspect of the muscle differentiation process. -- Abstract: The skALP isoform has been shown to play a critical role in actin organization and anchorage within the Z-discs of skeletal muscles, but no data is available on the function of the smALP isoform in skeletal muscle cells. Here, we show that upon induction of differentiation a nuclear translocation of smALP from the cytoplasm to the nucleus of C2C12 myoblasts, concomitant to an up-regulation of the protein expression, occurs in parallel with the nuclear accumulation of myogenin. Moreover, we demonstrate that the LIM domain of smALP is essential for the nuclear translocation of the protein.

  17. KPNA2 is a nuclear export protein that contributes to aberrant localisation of key proteins and poor prognosis of breast cancer

    PubMed Central

    Alshareeda, A T; Negm, O H; Green, A R; Nolan, C C; Tighe, P; Albarakati, N; Sultana, R; Madhusudan, S; Ellis, I O; Rakha, E A

    2015-01-01

    Background: It is recognised that modulations of the nuclear import of macromolecules have a role in changing cellular phenotypes and carcinogenesis. We and others have noticed that aberrant subcellular localisation of DNA damage response (DDR) proteins in breast cancer (BC) is associated with loss-of-function phenotype. This study aims to investigate the biological and clinical significance of the nucleocytoplasmic transport protein karyopherin ?-2 (KPNA2), and its role in controlling DDR proteins subcellular localisation in BC. Methods: A large (n=1494) and well-characterised series of early-stage invasive BC with a long-term follow-up was assessed for KPNA2 protein by using immunohistochemistry. Results: KPNA2 expression was associated with the subcellular localisation of key DDR proteins that showed cytoplasmic expression including BRCA1, RAD51, SMC6L1, ?H2AX, BARD1, UBC9, PIAS1 and CHK1. High level of KPNA2 was associated not only with cytoplasmic localisation of these proteins but also with their low/negative nuclear expression. Positive KPNA2 expression was associated with negative oestrogen receptor and triple-negative phenotype. Survival analysis showed that KPNA2 was associated with poor outcome (P<0.0001), but this effect was not independent of other prognostic variables. Conclusions: This study provides further evidence for the complexity of DDR mechanism in BC, and that KNPA2 has a role in the aberrant subcellular localisation of DDR proteins with subsequent impaired function. PMID:25989275

  18. Conservation of inner nuclear membrane targeting sequences in mammalian Pom121 and yeast Heh2 membrane proteins

    PubMed Central

    Kralt, Annemarie; Jagalur, Noorjahan B.; van den Boom, Vincent; Lokareddy, Ravi K.; Steen, Anton; Cingolani, Gino; Fornerod, Maarten; Veenhoff, Liesbeth M.

    2015-01-01

    Endoplasmic reticulum–synthesized membrane proteins traffic through the nuclear pore complex (NPC) en route to the inner nuclear membrane (INM). Although many membrane proteins pass the NPC by simple diffusion, two yeast proteins, ScSrc1/ScHeh1 and ScHeh2, are actively imported. In these proteins, a nuclear localization signal (NLS) and an intrinsically disordered linker encode the sorting signal for recruiting the transport factors for FG-Nup and RanGTP-dependent transport through the NPC. Here we address whether a similar import mechanism applies in metazoans. We show that the (putative) NLSs of metazoan HsSun2, MmLem2, HsLBR, and HsLap2? are not sufficient to drive nuclear accumulation of a membrane protein in yeast, but the NLS from RnPom121 is. This NLS of Pom121 adapts a similar fold as the NLS of Heh2 when transport factor bound and rescues the subcellular localization and synthetic sickness of Heh2?NLS mutants. Consistent with the conservation of these NLSs, the NLS and linker of Heh2 support INM localization in HEK293T cells. The conserved features of the NLSs of ScHeh1, ScHeh2, and RnPom121 and the effective sorting of Heh2-derived reporters in human cells suggest that active import is conserved but confined to a small subset of INM proteins. PMID:26179916

  19. The SUN protein Mps3 controls Ndc1 distribution and function on the nuclear membrane

    PubMed Central

    Chen, Jingjing; Smoyer, Christine J.; Slaughter, Brian D.; Unruh, Jay R.

    2014-01-01

    In closed mitotic systems such as Saccharomyces cerevisiae, nuclear pore complexes (NPCs) and the spindle pole body (SPB) must assemble into an intact nuclear envelope (NE). Ndc1 is a highly conserved integral membrane protein involved in insertion of both complexes. In this study, we show that Ndc1 interacts with the SUN domain–containing protein Mps3 on the NE in live yeast cells using fluorescence cross-correlation spectroscopy. Genetic and molecular analysis of a series of new ndc1 alleles allowed us to understand the role of Ndc1–Mps3 binding at the NE. We show that the ndc1-L562S allele is unable to associate specifically with Mps3 and find that this mutant is lethal due to a defect in SPB duplication. Unlike other ndc1 alleles, the growth and Mps3 binding defect of ndc1-L562S is fully suppressed by deletion of POM152, which encodes a NPC component. Based on our data we propose that the Ndc1–Mps3 interaction is important for controlling the distribution of Ndc1 between the NPC and SPB. PMID:24515347

  20. Arabidopsis MDA1, a Nuclear-Encoded Protein, Functions in Chloroplast Development and Abiotic Stress Responses

    PubMed Central

    Robles, Pedro; Micol, José Luis; Quesada, Víctor

    2012-01-01

    Most chloroplast and mitochondrial proteins are encoded by nuclear genes, whose functions remain largely unknown because mutant alleles are lacking. A reverse genetics screen for mutations affecting the mitochondrial transcription termination factor (mTERF) family in Arabidopsis thaliana allowed us to identify 75 lines carrying T-DNA insertions. Two of them were homozygous for insertions in the At4g14605 gene, which we dubbed MDA1 (MTERF DEFECTIVE IN Arabidopsis1). The mda1 mutants exhibited altered chloroplast morphology and plant growth, and reduced pigmentation of cotyledons, leaves, stems and sepals. The mda1 mutations enhanced salt and osmotic stress tolerance and altered sugar responses during seedling establishment, possibly as a result of reduced ABA sensitivity. Loss of MDA1 function caused up-regulation of the RpoTp/SCA3 nuclear gene encoding a plastid RNA polymerase and modified the steady-state levels of chloroplast gene transcripts. Double mutant analyses indicated that MDA1 and the previously described mTERF genes SOLDAT10 and RUG2 act in different pathways. Our findings reveal a new role for mTERF proteins in the response to abiotic stress, probably through perturbed ABA retrograde signalling resulting from a disruption in chloroplast homeostasis. PMID:22905186

  1. Nuclear localization of the C2H2 zinc finger protein Msn2p is regulated by stress and protein kinase A activity.

    PubMed

    Görner, W; Durchschlag, E; Martinez-Pastor, M T; Estruch, F; Ammerer, G; Hamilton, B; Ruis, H; Schüller, C

    1998-02-15

    Msn2p and the partially redundant factor Msn4p are key regulators of stress-responsive gene expression in Saccharomyces cerevisiae. They are required for the transcription of a number of genes coding for proteins with stress-protective functions. Both Msn2p and Msn4p are Cys2His2 zinc finger proteins and bind to the stress response element (STRE). In vivo footprinting studies show that the occupation of STREs is enhanced in stressed cells and dependent on the presence of Msn2p and Msn4p. Both factors accumulate in the nucleus under stress conditions, such as heat shock, osmotic stress, carbon-source starvation, and in the presence of ethanol or sorbate. Stress-induced nuclear localization was found to be rapid, reversible, and independent of protein synthesis. Nuclear localization of Msn2p and Msn4p was shown to be correlated inversely to cAMP levels and protein kinase A (PKA) activity. A region with significant homologies shared between Msn2p and Msn4p is sufficient to confer stress-regulated localization to a SV40-NLS-GFP fusion protein. Serine to alanine or aspartate substitutions in a conserved PKA consensus site abolished cAMP-driven nuclear export and cytoplasmic localization in unstressed cells. We propose stress and cAMP-regulated intracellular localization of Msn2p to be a key step in STRE-dependent transcription and in the general stress response. PMID:9472026

  2. Nuclear diacylglycerol produced by phosphoinositide-specific phospholipase C is responsible for nuclear translocation of protein kinase C-alpha.

    PubMed

    Neri, L M; Borgatti, P; Capitani, S; Martelli, A M

    1998-11-01

    It is well established that an independent inositide cycle is present within the nucleus, where it is involved in the control of cell proliferation and differentiation. Previous results have shown that when Swiss 3T3 cells are treated with insulin-like growth factor-I (IGF-I) a rapid and sustained increase in mass of diacylglycerol (DAG) occurs within the nuclei, accompanied by a decrease in the levels of both phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate. However, it is unclear whether or not other lipids could contribute to this prolonged rise in DAG levels. We now report that the IGF-I-dependent increase in nuclear DAG production can be inhibited by the specific phosphatidylinositol phospholipase C inhibitor 1-O-octadeyl-2-O-methyl-sn-glycero-3-phosphocholine or by neomycin sulfate but not by the purported phosphatidylcholine-phospholipase C specific inhibitor D609 or by inhibitors of phospholipase D-mediated DAG generation. Treatment of cells with 1-O-octadeyl-2-O-methyl-sn-glycero-3-phosphocholine or neomycin sulfate inhibited translocation of protein kinase C-alpha to the nucleus. Moreover, exposure of cells to 1-O-octadeyl-2-O-methyl-sn-glycero-3-phosphocholine, but not to D609, dramatically reduced the number of cells entering S-phase upon stimulation with IGF-I. These results suggest that the only phospholipase responsible for generation of nuclear DAG after IGF-I stimulation of 3T3 cells is PI-PLC. When this activity is inhibited, neither DAG rise is seen nor PKC-alpha translocation to the nucleus occurs. Furthermore, this PI-PLC activity appears to be essential for the G0/G1 to S-phase transition. PMID:9792687

  3. Identification of a nuclear export signal sequence for bovine papillomavirus E1 protein

    SciTech Connect

    Rosas-Acosta, German; Wilson, Van G.

    2008-03-30

    Recent studies have demonstrated nuclear export by papillomavirus E1 proteins, but the requisite export sequence(s) for bovine papillomavirus (BPV) E1 were not defined. In this report we identify three functional nuclear export sequences (NES) present in BPV E1, with NES2 being the strongest in reporter assays. Nuclear localization of BPV1 E1 was modulated by over- or under-expression of CRM1, the major cellular exportin, and export was strongly reduced by the CRM1 inhibitor, Leptomycin B, indicating that E1 export occurs primarily through a CRM1-dependent process. Consistent with the in vivo functional results, E1 bound CRM1 in an in vitro pull-down assay. In addition, sumoylated E1 bound CRM1 more effectively than unmodified E1, suggesting that E1 export may be regulated by SUMO modification. Lastly, an E1 NES2 mutant accumulated in the nucleus to a greater extent than wild-type E1, yet was defective for viral origin replication in vivo. However, NES2 exhibited no intrinsic replication defect in an in vitro replication assay, implying that nucleocytoplasmic shuttling may be required to maintain E1 in a replication competent state.

  4. A Nuclear Factor of High Mobility Group Box Protein in Toxoplasma gondii

    PubMed Central

    Wang, Hui; Lei, Tao; Liu, Jing; Li, Muzi; Nan, Huizhu; Liu, Qun

    2014-01-01

    High mobility group box 1 (HMGB1) is a nuclear factor that usually binds DNA and modulates gene expression in multicellular organisms. Three HMGB1 orthologs were predicted in the genome of Toxoplasma gondii, an obligate intracellular protozoan pathogen, termed TgHMGB1a, b and c. Phylogenetic and bioinformatic analyses indicated that these proteins all contain a single HMG box and which shared in three genotypes. We cloned TgHMGB1a, a 33.9 kDa protein that can stimulates macrophages to release TNF-?, and, we demonstrated that the TgHMGB1a binds distorted DNA structures such as cruciform DNA in electrophoretic mobility shift assays (EMSA). Immunofluorescence assay indicated TgHMGB1a concentrated in the nucleus of intracellular tachyzoites but translocated into the cytoplasm while the parasites release to extracellular. There were no significant phenotypic changes when the TgHMGB1a B box was deleted, while transgenic parasites that overexpressed TgHMGB1a showed slower intracellular growth and caused delayed death in mouse, further quantitative RT-PCR analyses showed that the expression levels of many important genes, including virulence factors, increased when TgHMGB1a was overexpressed, but no significant changes were observed in TgHMGB1a B box-deficient parasites. Our findings demonstrated that TgHMGB1a is indeed a nuclear protein that maintains HMG box architectural functions and is a potential proinflammatory factor during the T.gondii infection. Further studies that clarify the functions of TgHMGB1s will increase our knowledge of transcriptional regulation and parasite virulence, and might provide new insight into host–parasite interactions for T. gondii infection. PMID:25369210

  5. Arrest of Nuclear Division in Plasmodium through Blockage of Erythrocyte Surface Exposed Ribosomal Protein P2

    PubMed Central

    Das, Sudipta; Basu, Himanish; Korde, Reshma; Tewari, Rita; Sharma, Shobhona

    2012-01-01

    Malaria parasites reside inside erythrocytes and the disease manifestations are linked to the growth inside infected erythrocytes (IE). The growth of the parasite is mostly confined to the trophozoite stage during which nuclear division occurs followed by the formation of cell bodies (schizogony). The mechanism and regulation of schizogony are poorly understood. Here we show a novel role for a Plasmodium falciparum 60S stalk ribosomal acidic protein P2 (PfP2) (PFC0400w), which gets exported to the IE surface for 6–8 hrs during early schizogony, starting around 26–28 hrs post-merozoite invasion. The surface exposure is demonstrated using multiple PfP2-specific monoclonal antibodies, and is confirmed through transfection using PfP2-GFP. The IE surface-exposed PfP2-protein occurs mainly as SDS-resistant P2-homo-tetramers. Treatment with anti-PfP2 monoclonals causes arrest of IEs at the first nuclear division. Upon removal of the antibodies, about 80–85% of synchronized parasites can be released even after 24 hrs of antibody treatment. It has been reported that a tubovesicular network (TVN) is set up in early trophozoites which is used for nutrient import. Anti-P2 monoclonal antibodies cause a complete fragmentation of TVN by 36 hrs, and impairs lipid import in IEs. These may be downstream causes for the cell-cycle arrest. Upon antibody removal, the TVN is reconstituted, and the cell division progresses. Each of the above properties is observed in the rodent malaria parasite species P. yoelii and P. berghei. The translocation of the P2 protein to the IE surface is therefore likely to be of fundamental importance in Plasmodium cell division. PMID:22912579

  6. A monoclonal antibody identifies a 215 000-dalton nuclear envelope protein restricted to certain cell types.

    PubMed

    Brown, G; Turner, B M; Morris, C J; Bahman, A M; Fisher, A G; Whitfield, W G; Davies, S; Barthakur, R; Johnson, G D

    1985-11-01

    The monoclonal antibody, AGF2.3, was isolated from mice immunised with the human promyeloid cell line HL60. By immunofluorescence and immunoelectron microscopy the antibody was shown to bind to the nuclear envelope in uninduced HL60 cells. Immunofluorescent staining was reduced to very low levels in HL60 cells induced to mature to monocytes or neutrophils by addition of 12-0-tetradecanoylphorbol-13-acetate or dimethyl sulfoxide respectively. Blood neutrophils did not express the antigen. Weak immunofluorescent staining of cell nuclei was observed in peripheral blood lymphocytes and in sections of normal human kidney, tonsil and skin epithelium. The AGF2.3 antigen was strongly expressed on the nuclei of 21/21 haemopoietic cell lines and 21/25 permanent non-haemopoietic cell lines representing various cell types. In contrast, the antigen was not expressed by any of six primary (untransformed) cell cultures. These included fibroblasts, endothelial cells and keratinocytes. The antigen was expressed in the Q10 SV-40 transformed cell line derived from a non-expressing primary fibroblast culture. AGF2.3 antibody precipitated a protein with an apparent subunit molecular weight of approximately 215 kDa from Triton X-100 extracts of HL60 and HeLa cells labelled with 35S-methionine. This protein was not detectable in extracts of primary skin fibroblasts prepared in parallel. We conclude that AGF2.3 antibody recognises a previously undescribed protein associated with the nuclear envelope which is expressed at high levels in most transformed cell lines but which is weakly expressed or absent in normal tissues and primary cell cultures. PMID:2417847

  7. Nuclear localization and regulation of erk- and rsk-encoded protein kinases.

    PubMed Central

    Chen, R H; Sarnecki, C; Blenis, J

    1992-01-01

    We demonstrate that members of the erk-encoded family of mitogen-activated protein (MAP) kinases (pp44/42mapk/erk) and members of the rsk-encoded protein kinases (RSKs or pp90rsk) are present in the cytoplasm and nucleus of HeLa cells. Addition of growth factors to serum-deprived cells results in increased tyrosine and threonine phosphorylation and in the activation of cytosolic and nuclear MAP kinases. Activated MAP kinases then phosphorylate (serine/threonine) and activate RSKs. Concurrently, a fraction of the activated MAP kinases and RSKs enter the nucleus. In addition, a distinct growth-regulated RSK-kinase activity (an enzyme[s] that phosphorylates recombinant RSK in vitro and that may be another member of the erk-encoded family of MAP kinases) was found associated with a postnuclear membrane fraction. Regulation of nuclear MAP kinase and RSK activities by growth factors and phorbol ester is coordinate with immediate-early gene expression. Indeed, in vitro, MAP kinase and/or RSK phosphorylates histone H3 and the recombinant c-Fos and c-Jun polypeptides, transcription factors phosphorylated in a variety of cells in response to growth stimuli. These in vitro studies raise the possibility that the MAP kinase/RSK signal transduction pathway represents a protein-Tyr/Ser/Thr phosphorylation cascade with the spatial distribution and temporal regulation that can account for the rapid transmission of growth-regulating information from the membrane, through the cytoplasm, and to the nucleus. Images PMID:1545823

  8. A Role for Timely Nuclear Translocation of Clock Repressor Proteins in Setting Circadian Clock Speed

    PubMed Central

    Lee, Euna

    2014-01-01

    By means of a circadian clock system, all the living organisms on earth including human beings can anticipate the environmental rhythmic changes such as light/dark and warm/cold periods in a daily as well as in a yearly manner. Anticipating such environmental changes provide organisms with survival benefits via manifesting behavior and physiology at an advantageous time of the day and year. Cell-autonomous circadian oscillators, governed by transcriptional feedback loop composed of positive and negative elements, are organized into a hierarchical system throughout the organisms and generate an oscillatory expression of a clock gene by itself as well as clock controlled genes (ccgs) with a 24 hr periodicity. In the feedback loop, hetero-dimeric transcription factor complex induces the expression of negative regulatory proteins, which in turn represses the activity of transcription factors to inhibit their own transcription. Thus, for robust oscillatory rhythms of the expression of clock genes as well as ccgs, the precise control of subcellular localization and/or timely translocation of core clock protein are crucial. Here, we discuss how sub-cellular localization and nuclear translocation are controlled in a time-specific manner focusing on the negative regulatory clock proteins. PMID:25258565

  9. Functions of the DExD/H-box proteins in nuclear pre-mRNA splicing.

    PubMed

    Chang, Tien-Hsien; Tung, Luh; Yeh, Fu-Lung; Chen, Jui-Hui; Chang, Shang-Lin

    2013-08-01

    In eukaryotes, many genes are transcribed as precursor messenger RNAs (pre-mRNAs) that contain exons and introns, the latter of which must be removed and exons ligated to form the mature mRNAs. This process is called pre-mRNA splicing, which occurs in the nucleus. Although the chemistry of pre-mRNA splicing is identical to that of the self-splicing Group II introns, hundreds of proteins and five small nuclear RNAs (snRNAs), U1, U2, U4, U5, and U6, are essential for executing pre-mRNA splicing. Spliceosome, arguably the most complex cellular machine made up of all those proteins and snRNAs, is responsible for carrying out pre-mRNA splicing. In contrast to the transcription and the translation machineries, spliceosome is formed anew onto each pre-mRNA and undergoes a series of highly coordinated reconfigurations to form the catalytic center. This amazing process is orchestrated by a number of DExD/H-proteins that are the focus of this article, which aims to review the field in general and to project the exciting challenges and opportunities ahead. This article is part of a Special Issue entitled: The Biology of RNA helicases - Modulation for life. PMID:23454554

  10. Towards a robust description of intrinsic protein disorder using nuclear magnetic resonance spectroscopy.

    PubMed

    Schneider, Robert; Huang, Jie-rong; Yao, Mingxi; Communie, Guillaume; Ozenne, Valéry; Mollica, Luca; Salmon, Loïc; Jensen, Malene Ringkjøbing; Blackledge, Martin

    2012-01-01

    In order to understand the conformational behaviour of Intrinsically Disordered Proteins (IDPs), it is essential to develop a molecular representation of the partially folded state. Due to the very large number of degrees of conformational freedom available to such a disordered system, this problem is highly underdetermined. Characterisation therefore requires extensive experimental data, and novel analytical tools are required to exploit the specific conformational sensitivity of different experimental parameters. In this review we concentrate on the use of nuclear magnetic resonance (NMR) spectroscopy for the study of conformational behaviour of IDPs at atomic resolution. Each experimental NMR parameter is sensitive to different aspects of the structural and dynamic behaviour of the disordered state and requires specific consideration of the relevant averaging properties of the physical interaction. In this review we present recent advances in the description of disordered proteins and the selection of representative ensembles on the basis of experimental data using statistical coil sampling from flexible-meccano and ensemble selection using ASTEROIDS. Using these tools we aim to develop a unified molecular representation of the disordered state, combining complementary data sets to extract a meaningful description of the conformational behaviour of the protein. PMID:21874206

  11. Cotyledon nuclear proteins bind to DNA fragments harboring regulatory elements of phytohemagglutinin genes.

    PubMed Central

    Riggs, C D; Voelker, T A; Chrispeels, M J

    1989-01-01

    The effects of deleting DNA sequences upstream from the phytohemagglutinin-L gene of Phaseolus vulgaris have been examined with respect to the level of gene product produced in the seeds of transgenic tobacco. Our studies indicate that several upstream regions quantitatively modulate expression. Between -1000 and -675, a negative regulatory element reduces expression approximately threefold relative to shorter deletion mutants that do not contain this region. Positive regulatory elements lie between -550 and -125 and, compared with constructs containing only 125 base pairs of upstream sequences (-125), the presence of these two regions can be correlated with a 25-fold and a 200-fold enhancement of phytohemagglutinin-L levels. These experiments were complemented by gel retardation assays, which demonstrated that two of the three regions bind cotyledon nuclear proteins from mid-mature seeds. One of the binding sites maps near a DNA sequence that is highly homologous to protein binding domains located upstream from the soybean seed lectin and Kunitz trypsin inhibitor genes. Competition experiments demonstrated that the upstream regions of a bean beta-phaseolin gene, the soybean seed lectin gene, and an oligonucleotide from the upstream region of the trypsin inhibitor gene can compete differentially for factor binding. We suggest that these legume genes may be regulated in part by evolutionarily conserved protein/DNA interactions. PMID:2535513

  12. Flowering and genome integrity control by a nuclear matrix protein in Arabidopsis

    PubMed Central

    Xu, Yifeng; Gan, Eng-Seng; He, Yuehui; Ito, Toshiro

    2013-01-01

    The matrix attachment regions (MARs) binding proteins could finely orchestrate temporal and spatial gene expression during development. In Arabidopsis, transposable elements (TEs) and TE-like repeat sequences are transcriptionally repressed or attenuated by the coordination of many key players including DNA methyltransferases, histone deacetylases, histone methyltransferases and the siRNA pathway, which help to protect genomic integrity and control multiple developmental processes such as flowering. We have recently reported that an AT-hook nuclear matrix binding protein, TRANSPOSABLE ELEMENT SILENCING VIA AT-HOOK (TEK), participates in a histone deacetylation (HDAC) complex to silence TEs and genes containing a TE-like sequence, including AtMu1, FWA and FLOWERING LOCUS C (FLC) in Ler background. We have shown that TEK knockdown causes increased histone acetylation, reduced H3K9me2 and moderate reduction of DNA methylation in the target loci, leading to the de-repression of FLC and FWA, as well as TE reactivation. Here we discuss the role of TEK as a putative MAR binding protein which functions in the maintenance of genome integrity and in flowering control by silencing TEs and repeat-containing genes. PMID:23836195

  13. Modulation of Epstein–Barr Virus Nuclear Antigen 2-dependent transcription by protein arginine methyltransferase 5

    SciTech Connect

    Liu, Cheng-Der; Cheng, Chi-Ping; Fang, Jia-Shih; Chen, Ling-Chih; Zhao, Bo; Kieff, Elliott; Peng, Chih-Wen

    2013-01-18

    Highlights: ? Catalytic active PRMT5 substantially binds to the EBNA2 RG domain. ? PRMT5 augments the EBNA2-dependent transcription. ? PRMT5 triggers the symmetric dimethylation of the EBNA2 RG domain. ? PRMT5 enhances the promoter occupancy of EBNA2 on its target promoters. -- Abstract: Epstein–Barr Virus Nuclear Antigen (EBNA) 2 features an Arginine–Glycine repeat (RG) domain at amino acid positions 335–360, which is a known target for protein arginine methyltransferaser 5 (PRMT5). In this study, we performed protein affinity pull-down assays to demonstrate that endogenous PRMT5 derived from lymphoblastoid cells specifically associated with the protein bait GST-E2 RG. Transfection of a plasmid expressing PRMT5 induced a 2.5- to 3-fold increase in EBNA2-dependent transcription of both the LMP1 promoter in AKATA cells, which contain the EBV genome endogenously, and a Cp-Luc reporter plasmid in BJAB cells, which are EBV negative. Furthermore, we showed that there was a 2-fold enrichment of EBNA2 occupancy in target promoters in the presence of exogenous PRMT5. Taken together, we show that PRMT5 triggers the symmetric dimethylation of EBNA2 RG domain to coordinate with EBNA2-mediated transcription. This modulation suggests that PRMT5 may play a role in latent EBV infection.

  14. Nuclear Factor I transcription factors regulate IGF binding protein 5 gene transcription in human osteoblasts

    PubMed Central

    Pérez-Casellas, Laura A.; Wang, Xiaoying; Howard, Kristy; Rehage, Mark W.; Strong, Donna D.; Linkhart, Thomas A.

    2009-01-01

    Insulin-like growth factor binding protein 5 (IGFBP5) is expressed in many cell types including osteoblasts and modulates IGF activities. IGFBP5 may affect osteoblasts and bone formation, in part by mechanisms independent of binding IGFs. The highly conserved IGFBP5 proximal promoter within 100 nucleotides of the start of transcription contains functional cis regulatory elements for C/EBP, Myb and AP-2. We report evidence for a functional Nuclear Factor I (NFI) cis element that mediates activation or repression of IGFBP5 transcription by the NFI gene family. All four NFI genes were expressed in human osteoblast cultures and osteosarcoma cell lines. Co-transfection with human IGFBP5 promoter luciferase reporter and murine Nfi expression vectors showed that Nfib was the most active in stimulating transcription. Nfix was less active and Nfia and Nfic were inhibitory. Knockdown of NFIB and NFIC expression using siRNA decreased and increased IGFBP5 expression, respectively. Analysis of IGFBP5 promoter deletion and mutation reporter constructs identified a functional NFI cis element. All four NFI proteins bound the NFI site in electrophoretic mobility shift experiments and NFI-B bound in Chromatin Immunoprecipitation assays. Results suggest that NFI proteins are important regulators of IGFBP5 expression in human osteoblasts and thus in modulating IGFBP5 functions in bone. PMID:18809517

  15. Identification of nuclear beta II protein kinase C as a mitotic lamin kinase.

    PubMed

    Goss, V L; Hocevar, B A; Thompson, L J; Stratton, C A; Burns, D J; Fields, A P

    1994-07-22

    Multisite phosphorylation of the nuclear lamins is thought to regulate the process of mitotic nuclear envelope breakdown in vivo. Here we investigate the involvement of two proposed human mitotic lamin kinases, beta II protein kinase C (PKC) and p34cdc2/cyclin B kinase, in human lamin B1 phosphorylation in vitro and in intact cells. We find that both kinases can phosphorylate purified soluble lamin B at similar rates. However, beta II PKC phosphorylates interphase nuclear envelope lamin B at more than 200 times the rate of human p34cdc2/cyclin B kinase. beta II PKC-mediated phosphorylation of lamin B is confined to two sites, Ser395 and Ser405, within the carboxyl-terminal domain, whereas human p34cdc2/cyclin B kinase phosphorylates a single site, Ser23, in the amino-terminal domain. A second potential p34cdc2/cyclin B kinase site within the carboxyl-terminal domain, Ser393, is not phosphorylated by human p34cdc2/cyclin B kinase. However, invertebrate p34cdc2/cyclin B kinase from sea star exhibits a different specificity, phosphorylating both amino- and carboxyl-terminal sites. Mitotic human lamin B from intact cells is phosphorylated predominantly in its carboxyl-terminal domain. Comparative tryptic phosphopeptide mapping demonstrates that the beta II PKC site, Ser405, is a prominent target of mitotic lamin B phosphorylation in vivo. beta II PKC translocates to the nucleus during the G2/M phase of cell cycle concomitant with phosphorylation of Ser405, indicating a physiologic role for nuclear beta II PKC activation in mitotic lamin B phosphorylation in vivo. The presence of phosphorylation sites within the carboxyl-terminal domain of mitotic lamin B which are not phosphorylated by either beta II PKC or p34cdc2/cyclin B kinase suggests the involvement of other lamin kinase(s) in G2/M phase lamin B phosphorylation. PMID:8034666

  16. The nuclear localization of low risk HPV11 E7 protein mediated by its zinc binding domain is independent of nuclear import receptors

    SciTech Connect

    Piccioli, Zachary; McKee, Courtney H.; Leszczynski, Anna; Onder, Zeynep; Hannah, Erin C.; Mamoor, Shahan; Crosby, Lauren; Moroianu, Junona

    2010-11-10

    We investigated the nuclear import of low risk HPV11 E7 protein using 1) transfection assays in HeLa cells with EGFP fusion plasmids containing 11E7 and its domains and 2) nuclear import assays in digitonin-permeabilized HeLa cells with GST fusion proteins containing 11E7 and its domains. The EGFP-11E7 and EGFP-11cE7{sub 39-98} localized mostly to the nucleus. The GST-11E7 and GST-11cE7{sub 39-98} were imported into the nuclei in the presence of either Ran-GDP or RanG19V-GTP mutant and in the absence of nuclear import receptors. This suggests that 11E7 enters the nucleus via a Ran-dependent pathway, independent of nuclear import receptors, mediated by a nuclear localization signal located in its C-terminal domain (cNLS). This cNLS contains the zinc binding domain consisting of two copies of Cys-X-X-Cys motif. Mutagenesis of Cys residues in these motifs changed the localization of the EGFP-11cE7/-11E7 mutants to cytoplasmic, suggesting that the zinc binding domain is essential for nuclear localization of 11E7.

  17. Nuclear localization of the NS3 protein of hepatitis C virus and factors affecting the localization.

    PubMed Central

    Muramatsu, S; Ishido, S; Fujita, T; Itoh, M; Hotta, H

    1997-01-01

    Subcellular localization of the NS2 and NS3 proteins of hepatitis C virus was analyzed. In stable Ltk transfectants inducibly expressing an NS2-NS3 polyprotein (amino acids [aa] 810 to 1463), processed full-size NS2 (aa 810 to 1026) was detected exclusively in a cytoplasmic membrane fraction. On the other hand, the other processed product, carboxy-truncated NS3 (NS3 deltaC1463; aa 1027 to 1463), was present in both cytoplasmic and nuclear fractions. To further analyze subcellular localization of NS3, NS3 deltaC1459 (aa 1027 to 1459), full-size NS3 (NS3F; aa 1027 to 1657), and both amino- and carboxy-truncated NS3 (NS3 deltaNdeltaC; aa 1201 to 1459) were expressed in HeLa cells by using a vaccinia virus-T7 hybrid expression system. NS3 deltaC1459 and NS3F accumulated in the nucleus as well as in the cytoplasm, exhibiting a dot-like staining pattern. On the other hand, NS3 deltaNdeltaC was localized predominantly in the cytoplasm, suggesting the presence of a nuclear localization signal(s) in the amino-terminal sequence of NS3. NS4A, a viral cofactor for the NS3 protease, inhibited nuclear transport of NS3 deltaC1459 and NS3F, with the latter inhibited to a lesser extent than was the former. Interestingly, wild-type p53 tumor suppressor augmented nuclear localization of NS3 deltaC1459 and NS3F, whereas mutant-type p53 inhibited nuclear localization and augmented cytoplasmic localization of NS3 deltaC1459. However, subcellular localization of NS3 deltaNdeltaC was not affected by either type of p53. Wild-type p53-mediated nuclear accumulation of NS3 deltaC1459 and NS3F was inhibited partially, but not completely, by coexpressed NS4A, with NS3F again affected less prominently than was NS3 deltaC1459. PMID:9188558

  18. The hepatitis B virus HBx protein is a dual specificity cytoplasmic activator of Ras and nuclear activator of transcription factors.

    PubMed Central

    Doria, M; Klein, N; Lucito, R; Schneider, R J

    1995-01-01

    The HBx protein of hepatitis B virus (HBV) is a transcriptional activator that is required for infection and may play an important role in HBV-associated hepatocarcinogenesis. Recently, we and others have shown that HBx stimulates the Ras-Raf-MAP kinase cascade, which leads to enhanced cell proliferation and the activation of transcription factors AP-1 and NF-kappa B. Other studies have shown that HBx can activate transcription by interacting directly with nuclear components of the transcription machinery. Therefore we examined the basis for the different reported activities of HBx. Here, we show that HBx is a complex protein, displaying independent activities in different intracellular locations. The intracellular distribution of HBx protein was first investigated using scanning confocal laser immunomicroscopy and by genetic studies. Our work has established that HBx expressed in cultured cells is found authentically in both the cytoplasm and the nucleus. HBx is not strongly associated with any intracellular structures, but some preferential accumulation was observed near the cell surface. Next, HBx variants were constructed containing a functional or mutant nuclear localization sequence. We show that when HBx is engineered to relocate exclusively to the nucleus, it no longer activates the Ras-Raf-MAP kinase cascade, nor does it activate transcription factors AP-1 and NF-kappa B. Surprisingly, nuclear HBx fully retains the ability to stimulate HBV enhancer I, which is activated independently of the Ras and protein kinase C pathways. Therefore HBx protein stimulates signal transduction pathways in the cytoplasm and transactivates transcription elements in the nucleus. Furthermore, SV40 T antigen is shown to induce the nuclear sequestration of HBx protein and to block its activation of NF-kappa B, demonstrating that HBx is regulated by proteins that alter its intracellular distribution. The conflicting functions of HBx protein in viral infection and possibly carcinoma may involve the regulation of its differential distribution in the cell. Images PMID:7588604

  19. Protein Kinase A Activation Enhances ?-Catenin Transcriptional Activity through Nuclear Localization to PML Bodies

    PubMed Central

    Zhang, Mei; Mahoney, Emilia; Zuo, Tao; Manchanda, Parmeet K.; Davuluri, Ramana V.; Kirschner, Lawrence S.

    2014-01-01

    The Protein Kinase A (PKA) and Wnt signaling cascades are fundamental pathways involved in cellular development and maintenance. In the osteoblast lineage, these pathways have been demonstrated functionally to be essential for the production of mineralized bone. Evidence for PKA-Wnt crosstalk has been reported both during tumorigenesis and during organogenesis, and the nature of the interaction is thought to rely on tissue and cell context. In this manuscript, we analyzed bone tumors arising from mice with activated PKA caused by mutation of the PKA regulatory subunit Prkar1a. In primary cells from these tumors, we observed relocalization of ?-catenin to intranuclear punctuate structures, which were identified as PML bodies. Cellular redistribution of ?-catenin could be recapitulated by pharmacologic activation of PKA. Using 3T3-E1 pre-osteoblasts as a model system, we found that PKA phosphorylation sites on ?-catenin were required for nuclear re-localization. Further, ?-catenin's transport to the nucleus was accompanied by an increase in canonical Wnt-dependent transcription, which also required the PKA sites. PKA-Wnt crosstalk in the cells was bi-directional, including enhanced interactions between ?-catenin and the cAMP-responsive element binding protein (CREB) and transcriptional crosstalk between the Wnt and PKA signaling pathways. Increases in canonical Wnt/?-catenin signaling were associated with a decrease in the activity of the non-canonical Wnt/Ror2 pathway, which has been shown to antagonize canonical Wnt signaling. Taken together, this study provides a new understanding of the complex regulation of the subcellular distribution of ?-catenin and its differential protein-protein interaction that can be modulated by PKA signaling. PMID:25299576

  20. C++ OPPS, a new software for the interpretation of protein dynamics from nuclear magnetic resonance measurements

    NASA Astrophysics Data System (ADS)

    Zerbetto, Mirco; Polimeno, Antonino; Meirovitch, Eva

    Nuclear magnetic resonance (NMR) is a powerful tool for elucidating protein dynamics because of the possibility to interpret nuclear spin relaxation properties in terms of microdynamic parameters. Magnetic relaxation times T1, T2, and NOE depend on dipolar and quadrupolar interactions, on chemical shift anisotropy and cross-correlation effects. Within the framework of given motional model, it is possible to express the NMR relaxation times as functions of spectral densities (Abragam, The Principles of Nuclear Magnetism; Oxford University Press: Clarendon, London, 1961), obtaining the connection between macroscopic observables and microscopic properties. In this context, recently Meirovitch et al. (Shapiro et al., Biochemistry 2002, 41, 6271, Meirovitch et al., J Phys Chem B 2006, 110, 20615, Meirovitch et al., J Phys Chem B 2007, 111, 12865) applied the dynamical model introduced by Polimeno and Freed (Polimeno and Freed, Adv Chem Phys 1993, 83, 89, Polimeno and Freed, J Phys Chem 1995, 99, 10995), known as the slowly relaxing local structure (SRLS) model, to the study of NMR data. The program C++OPPS (http://www.chimica.unipd.it/licc/), developed in our laboratory, implements the SRLS model in an user-friendly way with a graphical user interface (GUI), introduced to simplify the work to users who do not feel at ease with the complex mathematics of the model and the difficulties of command line based programs. The program is an evolution of the old FORTRAN 77 implementation COPPS (COupled Protein Probe Smoluchowski) and presents a number of new features: the presence of an easy to use GUI written in JAVA; high calculation performance thanks to features of C++ language, employment of BLAS (basic linear algebra subprograms) library (Blackford et al., Trans Math Soft 2002, 28, 135) in handling matrix-vector operations and parallelization of the code under the MPI (message passing interface) paradigm (Gropp et al., Parallel Comput 1996, 22, 789, Gropp and Lusk, User's Guide for mpich, a Portable Implementation of MPI Mathematics and Computer Science Division; Argonne National Laboratory, 1996); possibility to predict the diffusion tensor of the protein via a hydrodynamic approach (Barone et al., J Comp Chem, in press). A cluster version of C++OPPS was also developed, which can be easily accessed by users via the web.

  1. Nuclear (DNA, RNA, histone and non-histone protein) and nucleolar changes during growth and senescence of may apple leaves.

    PubMed

    Bhattacharya, P K; Pappelis, A J; Lee, S C; BeMiller, J N; Karagiannis, C S

    1996-12-20

    Quantitative interference microscopy was used to determine changes in nuclear and nucleolar indices (dry mass and cross-sectional area) in upper and lower epidermal cells and adjacent leaf-margin hair cells of the May apple (Podophyllum peltatum L.) leaves over a 42-day period (after leaves emerged above the ground litter). These indices decreased in a highly correlated manner. A ploidy variation may exist between epidermal cells and leaf-margin hair cells. Using the leaf-margin hair cells model, six nuclear macromolecule indices (total nucleic acid, DNA, RNA, total nuclear protein, histone and non-histone protein), nuclear volume, nucleolar volume and perinucleolar volume (measured using quantitative epifluorescence-phase contrast microscopy) all declined with age (42-day study) in a highly correlated manner. The degeneration of the nucleus and nucleolus in the three leaf locations studied followed the patterns observed for programmed cellular senescence and death (necrosis) in epidermal cells of onion leaf bases (stored tissue; leaf bases did not contain chlorophyll) and human epithelial cells (buccal; cervical). We conclude that the epidermal cells and leaf-margin hair cells from green leaves of the May Apple are ideal for the study of programmed cell senescence and death in plants, especially for the partitioning of this process into the study of: the point-of-no-return (solubilization of the karyoskeleton and loss of non-histone proteins and RNA associated with the karyoskeleton from the nucleus); nuclear pycnosis (loss of nuclear dry mass and volume and loss of nuclear internal support structure); chromatin condensation, margination along the inner nuclear envelope; and DNA-histone degeneration; degeneration of the nucleolus and loss of the perinucleolar zone of exclusion. The characterization of chlorenchyma cells during the 42-day period should now be undertaken (leaf senescence as indicated by the beginning of yellowing about 35 days after emergence) to determine whether these cells with functional chloroplasts undergo nuclear changes like those lacking functional chloroplasts. PMID:9080390

  2. Localization of the adenovirus early region 1B 55-kilodalton protein during lytic infection: association with nuclear viral inclusions requires the early region 4 34-kilodalton protein.

    PubMed Central

    Ornelles, D A; Shenk, T

    1991-01-01

    The distribution of the adenovirus early region 1B 55-kDa protein (E1B-55kDa) in lytically infected HeLa cells was determined. At the time of infection, when the E1B-55kDa protein facilitates the cytoplasmic accumulation of viral mRNA while simultaneously restricting the accumulation of most cellular mRNA, five distinct intracellular localizations of the protein were observed. Only one of these was disrupted when cells were infected with a mutant virus that fails to produce a second viral protein encoded by early region 4 (E4-34kDa). This protein normally forms a complex with the E1B-55kDa polypeptide, enabling it to influence RNA metabolism. This key localization of the E1B protein was within and about the periphery of nuclear viral inclusion bodies believed to be the site of viral DNA replication and transcription. In the absence of the E4-34kDa protein, the coincidence of E1B-55kDa-specific immunofluorescence and phase-dense viral inclusions was reduced compared with that in a wild-type infection. Similarly, by immunoelectron microscopy, the relative number of E1B-55kDa-specific immunogold particles associated with the clear fibrillar inclusion bodies was reduced. However, the E4-34kDa protein was not required for the close association of the early region 2A DNA binding protein with the viral inclusions. We propose that the viral 55-kDa-34-kDa protein complex interacts with a cellular factor required for cytoplasmic accumulation of mRNAs and directs it to the periphery of the transcriptionally active viral inclusion bodies. This model provides an explanation for the ability of these viral proteins to simultaneously enhance accumulation of viral mRNAs and inhibit accumulation of cellular mRNAs. Images PMID:1824641

  3. Nuclear matrix protein Matrin3 regulates alternative splicing and forms overlapping regulatory networks with PTB

    PubMed Central

    Coelho, Miguel B; Attig, Jan; Bellora, Nicolás; König, Julian; Hallegger, Martina; Kayikci, Melis; Eyras, Eduardo; Ule, Jernej; Smith, Christopher WJ

    2015-01-01

    Matrin3 is an RNA- and DNA-binding nuclear matrix protein found to be associated with neural and muscular degenerative diseases. A number of possible functions of Matrin3 have been suggested, but no widespread role in RNA metabolism has yet been clearly demonstrated. We identified Matrin3 by its interaction with the second RRM domain of the splicing regulator PTB. Using a combination of RNAi knockdown, transcriptome profiling and iCLIP, we find that Matrin3 is a regulator of hundreds of alternative splicing events, principally acting as a splicing repressor with only a small proportion of targeted events being co-regulated by PTB. In contrast to other splicing regulators, Matrin3 binds to an extended region within repressed exons and flanking introns with no sharply defined peaks. The identification of this clear molecular function of Matrin3 should help to clarify the molecular pathology of ALS and other diseases caused by mutations of Matrin3. PMID:25599992

  4. Nuclear basic protein changes during spermiogenesis in the longnose skate and the spiny dogfish.

    PubMed

    Bols, N C; Boliska, S A; Rainville, J B; Kasinsky, H E

    1980-06-01

    Nuclear shape and the organization of nuclei within the seminiferous follicles have been used to divide spermiogenesis in the longnose skate, Raja rhina, into eight stages and in the spiny dogfish, Squalus acanthias, into seven stages. Cytochemical techniques for basic proteins reveal that as spermatid nuclei begin to elongate they have the somatic histone complement, and as they complete elongation they contain a very arginine-rich, TCA-extractable complement, or the salmon sperm histones type. After the nuclei have developed their ultimate corkscrew shape the final transition takes place to a very arginine-rich, TCA-stable complement, or the mammalian sperm histone type. These nuclei were not rich in sulfhydryl groups, but they were extractable with sodium thioglycolate. In addition, a number of variables affecting TCA extraction and subsequent alkaline fast green staining are described. PMID:6161983

  5. The Inhibition of Heat Shock Protein 90 Facilitates the Degradation of Poly-Alanine Expanded Poly (A) Binding Protein Nuclear 1 via the Carboxyl Terminus of Heat Shock Protein 70-Interacting Protein

    PubMed Central

    Shi, Chao; Huang, Xuan; Zhang, Bin; Zhu, Dan; Luo, Huqiao; Lu, Quqin; Xiong, Wen-Cheng; Mei, Lin; Luo, Shiwen

    2015-01-01

    Background Since the identification of poly-alanine expanded poly(A) binding protein nuclear 1 (PABPN1) as the genetic cause of oculopharyngeal muscular dystrophy (OPMD), considerable progress has been made in our understanding of the pathogenesis of the disease. However, the molecular mechanisms that regulate the onset and progression of the disease remain unclear. Results In this study, we show that PABPN1 interacts with and is stabilized by heat shock protein 90 (HSP90). Treatment with the HSP90 inhibitor 17-AAG disrupted the interaction of mutant PABPN1 with HSP90 and reduced the formation of intranuclear inclusions (INIs). Furthermore, mutant PABPN1 was preferentially degraded in the presence of 17-AAG compared with wild-type PABPN1 in vitro and in vivo. The effect of 17-AAG was mediated through an increase in the interaction of PABPN1 with the carboxyl terminus of heat shock protein 70-interacting protein (CHIP). The overexpression of CHIP suppressed the aggregation of mutant PABPN1 in transfected cells. Conclusions Our results demonstrate that the HSP90 molecular chaperone system plays a crucial role in the selective elimination of abnormal PABPN1 proteins and also suggest a potential therapeutic application of the HSP90 inhibitor 17-AAG for the treatment of OPMD. PMID:26414348

  6. The GIP gamma-tubulin complex-associated proteins are involved in nuclear architecture in Arabidopsis thaliana

    PubMed Central

    Batzenschlager, Morgane; Masoud, Kinda; Janski, Natacha; Houlné, Guy; Herzog, Etienne; Evrard, Jean-Luc; Baumberger, Nicolas; Erhardt, Mathieu; Nominé, Yves; Kieffer, Bruno; Schmit, Anne-Catherine; Chabouté, Marie-Edith

    2013-01-01

    During interphase, the microtubular cytoskeleton of cycling plant cells is organized in both cortical and perinuclear arrays. Perinuclear microtubules (MTs) are nucleated from ?-Tubulin Complexes (?-TuCs) located at the surface of the nucleus. The molecular mechanisms of ?-TuC association to the nuclear envelope (NE) are currently unknown. The ?-TuC Protein 3 (GCP3)-Interacting Protein 1 (GIP1) is the smallest ?-TuC component identified so far. AtGIP1 and its homologous protein AtGIP2 participate in the localization of active ?-TuCs at interphasic and mitotic MT nucleation sites. Arabidopsis gip1gip2 mutants are impaired in establishing a fully functional mitotic spindle and exhibit severe developmental defects. In this study, gip1gip2 knock down mutants were further characterized at the cellular level. In addition to defects in both the localization of ?-TuC core proteins and MT fiber robustness, gip1gip2 mutants exhibited a severe alteration of the nuclear shape associated with an abnormal distribution of the nuclear pore complexes. Simultaneously, they showed a misorganization of the inner nuclear membrane protein AtSUN1. Furthermore, AtGIP1 was identified as an interacting partner of AtTSA1 which was detected, like the AtGIP proteins, at the NE. These results provide the first evidence for the involvement of a ?-TuC component in both nuclear shaping and NE organization. Functional hypotheses are discussed in order to propose a model for a GIP-dependent nucleo-cytoplasmic continuum. PMID:24348487

  7. SU-E-J-61: Electrodynamics and Nano-Scale Fluid Dynamics in Protein Localization of Nuclear Pore Complexes

    SciTech Connect

    Cunningham, J; Gatenby, R

    2014-06-01

    Purpose: To develop a simulation to catalyze a reevaluation of common assumptions about 3 dimensional diffusive processes and help cell biologists gain a more nuanced, intuitive understanding of the true physical hurdles of protein signaling cascades. Furthermore, to discuss the possibility of intracellular electrodynamics as a critical, unrecognized component of cellular biology and protein dynamics that is necessary for optimal information flow from the cell membrane to the nucleus. Methods: The Unity 3D gaming physics engine was used to build an accurate virtual scale model of the cytoplasm within a few hundred nanometers of the nuclear membrane. A cloud of simulated pERK proteins is controlled by the physics simulation, where diffusion is based on experimentally measured values and the electrodynamics are based on theoretical nano-fluid dynamics. The trajectories of pERK within the cytoplasm and through the 1250 nuclear pores on the nuclear surface is recorded and analyzed. Results: The simulation quickly demonstrates that pERKs moving solely by diffusion will rarely locate and come within capture distance of a nuclear pore. The addition of intracellular electrodynamics between charges on the nuclear pore complexes and on pERKs increases the number of successful translocations by allowing the electro-physical attractive effects to draw in pERKs from the cytoplasm. The effects of changes in intracellular shielding ion concentrations allowed for estimation of the “capture radius” under varying conditions. Conclusion: The simulation allows a shift in perspective that is paramount in attempting to communicate the scale and dynamics of intracellular protein cascade mechanics. This work has allowed researchers to more fully understand the parameters involved in intracellular electrodynamics, such as shielding anion concentration and protein charge. As these effects are still far below the spatial resolution of currently available measurement technology this simulation is invaluable to researchers to visualize and study current hypotheses concerning protein dynamics.

  8. Electron-nuclear interactions as probes of domain motion in proteins

    PubMed Central

    Shapira, Boaz; Prestegard, James H.

    2010-01-01

    Long range interactions between nuclear spins and paramagnetic ions can serve as a sensitive monitor of internal motion of various parts of proteins, including functional loops and separate domains. In the case of interdomain motion, the interactions between the ion and NMR-observable nuclei are modulated in direction and magnitude mainly by a combination of overall and interdomain motions. The effects on observable parameters such as paramagnetic relaxation enhancement (PRE) and pseudocontact shift (PCS) can, in principle, be used to characterize motion. These parameters are frequently used for the purpose of structural refinements. However, their use to probe actual domain motions is less common and is lacking a proper theoretical treatment from a motional perspective. In this work, a suitable spin Hamiltonian is incorporated in a two body diffusion model to produce the time correlation function for the nuclear spin–paramagnetic ion interactions. Simulated observables for nuclei in different positions with respect to the paramagnetic ion are produced. Based on these simulations, it demonstrated that both the PRE and the PCS can be very sensitive probes of domain motion. Results for different nuclei within the protein sense different aspects of the motions. Some are more sensitive to the amplitude of the internal motion, others are more sensitive to overall diffusion rates, allowing separation of these contributions. Experimentally, the interaction strength can also be tuned by substitution of different paramagnetic ions or by varying magnetic field strength (in the case of lanthanides) to allow the use of more detailed diffusion models without reducing the reliability of data fitting. PMID:20331317

  9. Electron-nuclear interactions as probes of domain motion in proteins

    NASA Astrophysics Data System (ADS)

    Shapira, Boaz; Prestegard, James H.

    2010-03-01

    Long range interactions between nuclear spins and paramagnetic ions can serve as a sensitive monitor of internal motion of various parts of proteins, including functional loops and separate domains. In the case of interdomain motion, the interactions between the ion and NMR-observable nuclei are modulated in direction and magnitude mainly by a combination of overall and interdomain motions. The effects on observable parameters such as paramagnetic relaxation enhancement (PRE) and pseudocontact shift (PCS) can, in principle, be used to characterize motion. These parameters are frequently used for the purpose of structural refinements. However, their use to probe actual domain motions is less common and is lacking a proper theoretical treatment from a motional perspective. In this work, a suitable spin Hamiltonian is incorporated in a two body diffusion model to produce the time correlation function for the nuclear spin-paramagnetic ion interactions. Simulated observables for nuclei in different positions with respect to the paramagnetic ion are produced. Based on these simulations, it demonstrated that both the PRE and the PCS can be very sensitive probes of domain motion. Results for different nuclei within the protein sense different aspects of the motions. Some are more sensitive to the amplitude of the internal motion, others are more sensitive to overall diffusion rates, allowing separation of these contributions. Experimentally, the interaction strength can also be tuned by substitution of different paramagnetic ions or by varying magnetic field strength (in the case of lanthanides) to allow the use of more detailed diffusion models without reducing the reliability of data fitting.

  10. Nuclear Inelastic Scattering and Mössbauer Spectroscopy as Local Probes for Ligand Binding Modes and Electronic Properties in Proteins: Vibrational Behavior of a Ferriheme Center Inside a ?-Barrel Protein

    PubMed Central

    Moeser, Beate; Janoschka, Adam; Wolny, Juliusz A.; Paulsen, Hauke; Filippov, Igor; Berry, Robert E.; Zhang, Hongjun; Chumakov, Alexander I.; Walker, F. Ann; Schünemann, Volker

    2012-01-01

    In this work we present a study of the influence of the protein matrix on its ability to tune the binding of small ligands such as NO, cyanide (CN-) and histamine to the ferric heme iron center in the NO-storage and -transport protein Nitrophorin 2 (NP2) from the salivary glands of the blood-sucking insect Rhodnius prolixus. Conventional Mössbauer spectroscopy shows a diamagnetic ground state of the NP2-NO complex and Type I and II electronic ground states of the NP2-CN- and NP2-histamine complex, respectively. The change in the vibrational signature of the protein upon ligand binding has been monitored by Nuclear Inelastic Scattering (NIS), also called Nuclear Resonant Vibrational Spectroscopy (NRVS). The NIS data thus obtained have also been calculated by quantum mechanical (QM) density functional theory (DFT) coupled with Molecular Mechanics (MM) methods. The calculations presented here show that the heme ruffling in NP2 is a consequence of the interaction with the protein matrix. Structure optimizations of the heme and its ligands with DFT retain the characteristic saddling and ruffling only if the protein matrix is taken into account. Furthermore, simulations of the NIS data by QM/MM calculations suggest that the pH dependence of the binding of NO, but not of CN– and histamine, might be a consequence of the protonation state of the heme carboxyls. PMID:22295945

  11. Identification of the methylation preference region in heterogeneous nuclear ribonucleoprotein K by protein arginine methyltransferase 1 and its implication in regulating nuclear/cytoplasmic distribution

    SciTech Connect

    Chang, Yuan-I; Hsu, Sheng-Chieh; Chau, Gar-Yang; Department of Surgery, School of Medicine, National Yang-Ming University, Taipei 112, Taiwan ; Huang, Chi-Ying F.; Institute of Clinical Medicine, National Yang-Ming University, Taipei 112, Taiwan ; Sung, Jung-Sung; Hua, Wei-Kai; Lin, Wey-Jinq; Department of Education and Research, Taipei City Hospital, Taipei 103, Taiwan

    2011-01-21

    Research highlights: {yields} Verifying by direct methylation assay the substrate sites of PRMT1 in the hnRNP K protein. {yields} Identifying the preferred PMRT1 methylation regions in hnRNP K by kinetic analysis. {yields} Linking methylation in regulating nuclear localization of hnRNP K. -- Abstract: Protein arginine methylation plays crucial roles in numerous cellular processes. Heterogeneous nuclear ribonucleoprotein K (hnRNP K) is a multi-functional protein participating in a variety of cellular functions including transcription and RNA processing. HnRNP K is methylated at multiple sites in the glycine- and arginine-rich (RGG) motif. Using various RGG domain deletion mutants of hnRNP K as substrates, here we show by direct methylation assay that protein arginine methyltransferase 1 (PRMT1) methylated preferentially in a.a. 280-307 of the RGG motif. Kinetic analysis revealed that deletion of a.a. 280-307, but not a.a. 308-327, significantly inhibited rate of methylation. Importantly, nuclear localization of hnRNP K was significantly impaired in mutant hnRNP K lacking the PRMT1 methylation region or upon pharmacological inhibition of methylation. Together our results identify preferred PRMT1 methylation sequences of hnRNP K by direct methylation assay and implicate a role of arginine methylation in regulating intracellular distribution of hnRNP K.

  12. A nuclear fraction of turnip crinkle virus capsid protein is important for elicitation of the host resistance response.

    PubMed

    Kang, Sung-Hwan; Qu, Feng; Morris, T Jack

    2015-12-01

    The N-terminal 25 amino acids (AAs) of turnip crinkle virus (TCV) capsid protein (CP) are recognized by the resistance protein HRT to trigger a hypersensitive response (HR) and systemic resistance to TCV infection. This same region of TCV CP also contains a motif that interacts with the transcription factor TIP, as well as a nuclear localization signal (NLS). However, it is not yet known whether nuclear localization of TCV CP is needed for the induction of HRT-mediated HR and resistance. Here we present new evidence suggesting a tight correlation between nuclear inclusions formed by CP and the manifestation of HR. We show that a fraction of TCV CP localized to cell nuclei to form discrete inclusion-like structures, and a mutated CP (R6A) known to abolish HR failed to form nuclear inclusions. Notably, TIP-CP interaction augments the inclusion-forming activity of CP by tethering inclusions to the nuclear membrane. This TIP-mediated augmentation is also critical for HR resistance, as another CP mutant (R8A) known to elicit a less restrictive HR, though still self-associated into nuclear inclusions, failed to direct inclusions to the nuclear membrane due to its inability to interact with TIP. Finally, exclusion of CP from cell nuclei abolished induction of HR. Together, these results uncovered a strong correlation between nuclear localization and nuclear inclusion formation by TCV CP and induction of HR, and suggest that CP nuclear inclusions could be the key trigger of the HRT-dependent, yet TIP-reinforced, resistance to TCV. PMID:26299399

  13. Functional Characterization of Nuclear Localization and Export Signals in Hepatitis C Virus Proteins and Their Role in the Membranous Web

    PubMed Central

    Levin, Aviad; Neufeldt, Christopher J.; Pang, Daniel; Wilson, Kristen; Loewen-Dobler, Darci; Joyce, Michael A.; Wozniak, Richard W.; Tyrrell, D. Lorne J

    2014-01-01

    The hepatitis C virus (HCV) is a positive strand RNA virus of the Flavivirus family that replicates in the cytoplasm of infected hepatocytes. Previously, several nuclear localization signals (NLS) and nuclear export signals (NES) have been identified in HCV proteins, however, there is little evidence that these proteins travel into the nucleus during infection. We have recently shown that nuclear pore complex (NPC) proteins (termed nucleoporins or Nups) are present in the membranous web and are required during HCV infection. In this study, we identify a total of 11 NLS and NES sequences in various HCV proteins. We show direct interactions between HCV proteins and importin ?5 (IPOA5/kap?1), importin ?3 (IPO5/kap ?3), and exportin 1 (XPO1/CRM1) both in-vitro and in cell culture. These interactions can be disrupted using peptides containing the specific NLS or NES sequences of HCV proteins. Moreover, using a synchronized infection system, we show that these peptides inhibit HCV infection during distinct phases of the HCV life cycle. The inhibitory effects of these peptides place them in two groups. The first group binds IPOA5 and inhibits infection during the replication stage of HCV life cycle. The second group binds IPO5 and is active during both early replication and early assembly. This work delineates the entire life cycle of HCV and the active involvement of NLS sequences during HCV replication and assembly. Given the abundance of NLS sequences within HCV proteins, our previous finding that Nups play a role in HCV infection, and the relocation of the NLS double-GFP reporter in HCV infected cells, this work supports our previous hypothesis that NPC-like structures and nuclear transport factors function in the membranous web to create an environment conducive to viral replication. PMID:25485706

  14. Rhinovirus 3C Protease Facilitates Specific Nucleoporin Cleavage and Mislocalisation of Nuclear Proteins in Infected Host Cells

    PubMed Central

    Walker, Erin J.; Younessi, Parisa; Fulcher, Alex J.; McCuaig, Robert; Thomas, Belinda J.; Bardin, Philip G.

    2013-01-01

    Human Rhinovirus (HRV) infection results in shut down of essential cellular processes, in part through disruption of nucleocytoplasmic transport by cleavage of the nucleoporin proteins (Nups) that make up the host cell nuclear pore. Although the HRV genome encodes two proteases (2A and 3C) able to cleave host proteins such as Nup62, little is known regarding the specific contribution of each. Here we use transfected as well as HRV-infected cells to establish for the first time that 3C protease is most likely the mediator of cleavage of Nup153 during HRV infection, while Nup62 and Nup98 are likely to be targets of HRV2A protease. HRV16 3C protease was also able to elicit changes in the appearance and distribution of the nuclear speckle protein SC35 in transfected cells, implicating it as a key mediator of the mislocalisation of SC35 in HRV16-infected cells. In addition, 3C protease activity led to the redistribution of the nucleolin protein out of the nucleolus, but did not affect nuclear localisation of hnRNP proteins, implying that complete disruption of nucleocytoplasmic transport leading to relocalisation of hnRNP proteins from the nucleus to the cytoplasm in HRV-infected cells almost certainly requires 2A in addition to 3C protease. Thus, a specific role for HRV 3C protease in cleavage and mislocalisation of host cell nuclear proteins, in concert with 2A, is implicated for the first time in HRV pathogenesis. PMID:23951130

  15. Targeted Mutation of Nuclear Bone Morphogenetic Protein 2 Impairs Secondary Immune Response in a Mouse Model

    PubMed Central

    Olsen, Daniel S.; Goar, Wesley A.; Nichols, Brandt A.; Bailey, K. Tyson; Christensen, S. Loyd; Merriam, Kayla R.; Reynolds, Paul R.; Wilson, Eric; Weber, K. Scott; Bridgewater, Laura C.

    2015-01-01

    We recently identified a nuclear variant of the BMP2 growth factor, called nBMP2. In an effort to understand the function of this variant protein, we generated a mouse line in which BMP2 is expressed and functions normally, but nBMP2 is excluded from the nucleus. This novel mutation allows the study of nBMP2 without compromising BMP2 function. To determine whether nBMP2 plays a role in immune function, we performed a series of experiments in which we compared mouse survival, organ weights, immune cells numbers, and bacterial load in wild type and nBmp2NLStm mice following primary and secondary challenges with Staphylococcus aureus. Following primary challenge with S. aureus, wild type and nBmp2NLStm mice showed no differences in survival or bacterial load and generated similar numbers and types of leukocytes, although mutant spleens were smaller than wild type. Secondary bacterial challenge with S. aureus, however, produced differences in survival, with increased mortality seen in nBmp2NLStm mice. This increased mortality corresponded to higher levels of bacteremia in nBmp2NLStm mice and to a reduced enlargement of mutant spleens in response to the secondary infection. Together, these results suggest that the recently described nuclear variant of BMP2 is necessary for efficient secondary immune responses. PMID:26491697

  16. Transmembrane protein Sun2 is involved in tethering mammalian meiotic telomeres to the nuclear envelope.

    PubMed

    Schmitt, Johannes; Benavente, Ricardo; Hodzic, Didier; Höög, Christer; Stewart, Colin L; Alsheimer, Manfred

    2007-05-01

    Dynamic repositioning of telomeres is a unique feature of meiotic prophase I that is highly conserved among eukaryotes. At least in fission yeast it was shown to be required for proper alignment and recombination of homologous chromosomes. On entry into meiosis telomeres attach to the nuclear envelope and transiently cluster at a limited area to form a chromosomal bouquet. Telomere clustering is thought to promote chromosome recognition and stable pairing of the homologs. However, the molecular basis of telomere attachment and movement is largely unknown. Here we report that mammalian SUN-domain protein Sun2 specifically localizes to the nuclear envelope attachment sites of meiotic telomeres. Sun2-telomere association is maintained throughout the dynamic movement of telomeres. This association does not require the assembly of chromosomal axial elements or the presence of A-type lamins. Detailed EM analysis revealed that Sun2 is part of a membrane-spanning fibrillar complex that interconnects attached telomeres with cytoplasmic structures. Together with recent findings in fission yeast, our study indicates that the molecular mechanisms required for tethering meiotic telomeres and their dynamic movements during bouquet formation are conserved among eukaryotes. PMID:17452644

  17. Evaluating nuclear protein-coding genes for phylogenetic utility in beetles.

    PubMed

    Wild, Alexander L; Maddison, David R

    2008-09-01

    Although nuclear protein-coding genes have proven broadly useful for phylogenetic inference, relatively few such genes are regularly employed in studies of Coleoptera, the most diverse insect order. We increase the number of loci available for beetle systematics by developing protocols for three genes previously unused in beetles (alpha-spectrin, RNA polymerase II and topoisomerase I) and by refining protocols for five genes already in use (arginine kinase, CAD, enolase, PEPCK and wingless). We evaluate the phylogenetic performance of each gene in a Bayesian framework against a presumably known test phylogeny. The test phylogeny covers 31 beetle specimens and two outgroup taxa of varying age, including three of the four extant beetle suborders and a denser sampling in Adephaga and in the carabid genus Bembidion. All eight genes perform well for Cenozoic divergences and accurately separate closely related species within Bembidion, but individual genes differ markedly in accuracy over the older Mesozoic and Permian divergences. The concatenated data reconstruct the test phylogeny with high support in both Bayesian and parsimony analyses, indicating that combining data from multiple nuclear loci will be a fruitful approach for assembling the beetle tree of life. PMID:18644735

  18. Nicotine Mediates Hypochlorous acid-Induced Nuclear Protein Damage in Mammalian Cells

    PubMed Central

    Salama, Samir A.; Arab, Hany H.; Omar, Hany A.; Maghrabi, Ibrahim A.; Snapka, Robert M.

    2014-01-01

    Activated neutrophils secrete hypochlorous acid (HOCl) into the extracellular space of inflamed tissues. Because of short diffusion distance in biological fluids, HOCl damaging effect is restricted to the extracellular compartment. The current study aimed at investigating the ability of nicotine, a component of tobacco and electronic cigarettes, to mediate HOCl-induced intracellular damage. We report, for the first time, that HOCl reacts with nicotine to produce nicotine chloramine (Nic-Cl). Nic-Cl caused dose-dependent damage to proliferating cell nuclear antigen (PCNA), a nuclear protein, in cultured mammalian lung and kidney cells. Vitamin C, Trolox, glutathione, and N-acetyl-L-cysteine inhibited the Nic-Cl-induced PCNA damage, implicating oxidation in PCNA damage. These findings point out the ability of nicotine to mediate HOCl-induced intracellular damage and suggest antioxidants as protective measures. The results also raise the possibility that Nic-Cl can be created in the inflamed tissues of tobacco and electronic cigarette smokers and may contribute to smoking-related diseases. PMID:24357417

  19. Nuclear localization of CPI-17, a protein phosphatase-1 inhibitor protein, affects histone H3 phosphorylation and corresponds to proliferation of cancer and smooth muscle cells

    SciTech Connect

    Eto, Masumi; Kirkbride, Jason A.; Chugh, Rishika; Karikari, Nana Kofi; Kim, Jee In; Cardiovascular Research Institute, Kyungpook National University School of Medicine, Daegu 700-422

    2013-04-26

    Highlights: •Non-canonical roles of the myosin phosphatase inhibitor (CPI-17) were studied. •CPI-17 is localized in the nucleus of hyperplastic cancer and smooth muscle cells. •CPI-17 Ser12 phosphorylation may regulate the nuclear import. •CPI-17 regulates histone H3 phosphorylation and cell proliferation. •The nuclear CPI-17-PP1 axis plays a proliferative role in cells. -- Abstract: CPI-17 (C-kinase-activated protein phosphatase-1 (PP1) inhibitor, 17 kDa) is a cytoplasmic protein predominantly expressed in mature smooth muscle (SM) that regulates the myosin-associated PP1 holoenzyme (MLCP). Here, we show CPI-17 expression in proliferating cells, such as pancreatic cancer and hyperplastic SM cells. Immunofluorescence showed that CPI-17 was concentrated in nuclei of human pancreatic cancer (Panc1) cells. Nuclear accumulation of CPI-17 was also detected in the proliferating vascular SM cell culture and cells at neointima of rat vascular injury model. The N-terminal 21-residue tail domain of CPI-17 was necessary for the nuclear localization. Phospho-mimetic Asp-substitution of CPI-17 at Ser12 attenuated the nuclear import. CPI-17 phosphorylated at Ser12 was not localized at nuclei, suggesting a suppressive role of Ser12 phosphorylation in the nuclear import. Activated CPI-17 bound to all three isoforms of PP1 catalytic subunit in Panc1 nuclear extracts. CPI-17 knockdown in Panc1 resulted in dephosphorylation of histone H3 at Thr3, Ser10 and Thr11, whereas it had no effects on the phosphorylation of myosin light chain and merlin, the known targets of MLCP. In parallel, CPI-17 knockdown suppressed Panc1 proliferation. We propose that CPI-17 accumulated in the nucleus through the N-terminal tail targets multiple PP1 signaling pathways regulating cell proliferation.

  20. Protein interactions at the higher plant nuclear envelope: evidence for a linker of nucleoskeleton and cytoskeleton complex

    PubMed Central

    Evans, David E.; Pawar, Vidya; Smith, Sarah J.; Graumann, Katja

    2014-01-01

    Following the description of SAD1/UNC84 (SUN) domain proteins in higher plants, evidence has rapidly increased that plants contain a functional linker of nucleoskeleton and cytoskeleton (LINC) complex bridging the nuclear envelope (NE). While the SUN domain proteins appear to be highly conserved across kingdoms, other elements of the complex are not and some key components and interactions remain to be identified. This mini review examines components of the LINC complex, including proteins of the SUN domain family and recently identified plant Klarsicht/Anc/Syne-1 homology (KASH) domain proteins. First of these to be described were WIPs (WPP domain interacting proteins), which act as protein anchors in the outer NE. The plant KASH homologs are C-terminally anchored membrane proteins with the extreme C-terminus located in the nuclear periplasm; AtWIPs contain a highly conserved X-VPT motif at the C-terminus in contrast to PPPX in opisthokonts. The role of the LINC complex in organisms with a cell wall, and description of further LINC complex components will be considered, together with other potential plant-specific functions. PMID:24847341

  1. Incorporation of the nuclear pore basket protein Nup153 into nuclear pore structures is dependent upon lamina assembly: evidence from cell-free extracts of Xenopus eggs

    PubMed Central

    Smythe, Carl; Jenkins, Hazel E.; Hutchison, Christopher J.

    2000-01-01

    In cell-free extracts of Xenopus eggs that support the assembly of replication-competent nuclei, we found that lamin B3 specifically associates with four polypeptides (termed SLAPs, soluble lamin associated proteins). Here, one SLAP is identified as the nuclear pore complex protein Nup153, one member of the F/GXFG motif-containing nucleoporins. In vitro translated Nup153 and lamin B3 co-immunoprecipitate, and lamin B3 interacts specifically with the C-terminal domain of Nup153. During nuclear envelope assembly, other F/GXFG-containing nucleoporins are incorporated into the nuclear envelope preceding lamina assembly. Incorporation of Nup153 occurs at the same time as lamina assembly. When lamina assembly is prevented using the dominant-negative mutant XlaminB?2+, Nup153 does not appear at the nuclear envelope, while other F/GXFG-containing nucleoporins and Nup93 are recruited normally. When the lamina of pre-assembled nuclei is disrupted using the same dominant-negative mutant, the distribution of other nucleoporins is unaffected. However, Nup153 recruitment at the nuclear envelope is lost. Our results indicate that both the recruitment and maintenance of Nup153 at the pore are dependent upon the integrity of the lamina. PMID:10921874

  2. Genome-wide promoter binding profiling of protein phosphatase-1 and its major nuclear targeting subunits.

    PubMed

    Verheyen, Toon; Görnemann, Janina; Verbinnen, Iris; Boens, Shannah; Beullens, Monique; Van Eynde, Aleyde; Bollen, Mathieu

    2015-07-13

    Protein phosphatase-1 (PP1) is a key regulator of transcription and is targeted to promoter regions via associated proteins. However, the chromatin binding sites of PP1 have never been studied in a systematic and genome-wide manner. Methylation-based DamID profiling in HeLa cells has enabled us to map hundreds of promoter binding sites of PP1 and three of its major nuclear interactors, i.e. RepoMan, NIPP1 and PNUTS. Our data reveal that the ?, ? and ? isoforms of PP1 largely bind to distinct subsets of promoters and can also be differentiated by their promoter binding pattern. PP1? emerged as the major promoter-associated isoform and shows an overlapping binding profile with PNUTS at dozens of active promoters. Surprisingly, most promoter binding sites of PP1 are not shared with RepoMan, NIPP1 or PNUTS, hinting at the existence of additional, largely unidentified chromatin-targeting subunits. We also found that PP1 is not required for the global chromatin targeting of RepoMan, NIPP1 and PNUTS, but alters the promoter binding specificity of NIPP1. Our data disclose an unexpected specificity and complexity in the promoter binding of PP1 isoforms and their chromatin-targeting subunits. PMID:25990731

  3. Genome-wide promoter binding profiling of protein phosphatase-1 and its major nuclear targeting subunits

    PubMed Central

    Verheyen, Toon; Görnemann, Janina; Verbinnen, Iris; Boens, Shannah; Beullens, Monique; Van Eynde, Aleyde; Bollen, Mathieu

    2015-01-01

    Protein phosphatase-1 (PP1) is a key regulator of transcription and is targeted to promoter regions via associated proteins. However, the chromatin binding sites of PP1 have never been studied in a systematic and genome-wide manner. Methylation-based DamID profiling in HeLa cells has enabled us to map hundreds of promoter binding sites of PP1 and three of its major nuclear interactors, i.e. RepoMan, NIPP1 and PNUTS. Our data reveal that the ?, ? and ? isoforms of PP1 largely bind to distinct subsets of promoters and can also be differentiated by their promoter binding pattern. PP1? emerged as the major promoter-associated isoform and shows an overlapping binding profile with PNUTS at dozens of active promoters. Surprisingly, most promoter binding sites of PP1 are not shared with RepoMan, NIPP1 or PNUTS, hinting at the existence of additional, largely unidentified chromatin-targeting subunits. We also found that PP1 is not required for the global chromatin targeting of RepoMan, NIPP1 and PNUTS, but alters the promoter binding specificity of NIPP1. Our data disclose an unexpected specificity and complexity in the promoter binding of PP1 isoforms and their chromatin-targeting subunits. PMID:25990731

  4. Fluctuation-based imaging of nuclear Rac1 activation by protein oligomerisation

    PubMed Central

    Hinde, Elizabeth; Yokomori, Kyoko; Gaus, Katharina; Hahn, Klaus M.; Gratton, Enrico

    2014-01-01

    Here we describe a fluctuation-based method to quantify how protein oligomerisation modulates signalling activity of a multifunctional protein. By recording fluorescence lifetime imaging microscopy (FLIM) data of a FRET biosensor in a format that enables concomitant phasor and cross Number and Brightness (cN&B) analysis, we measure the nuclear dynamics of a Rac1 FRET biosensor and assess how Rac1 homo-oligomers (N&B) regulate Rac1 activity (hetero-oligomerisation with the biosensor affinity reagent, PBD, by FLIM-FRET) or interaction with an unknown binding partner (cN&B). The high spatiotemporal resolution of this method allowed us to discover that upon DNA damage monomeric and active Rac1 in the nucleus is segregated from dimeric and inactive Rac1 in the cytoplasm. This reorganisation requires Rac1 GTPase activity and is associated with an importin-?2 redistribution. Only with this multiplexed approach can we assess the oligomeric state a molecular complex must form in order to regulate a complex signalling network. PMID:24573109

  5. Cell cycle-dependent SUMO-1 conjugation to nuclear mitotic apparatus protein (NuMA)

    SciTech Connect

    Seo, Jae Sung; Kim, Ha Na; Kim, Sun-Jick; Bang, Jiyoung; Kim, Eun-A; Sung, Ki Sa; Yoon, Hyun-Joo; Yoo, Hae Yong; Choi, Cheol Yong

    2014-01-03

    Highlights: •NuMA is modified by SUMO-1 in a cell cycle-dependent manner. •NuMA lysine 1766 is the primary target site for SUMOylation. •SUMOylation-deficient NuMA induces multiple spindle poles during mitosis. •SUMOylated NuMA induces microtubule bundling. -- Abstract: Covalent conjugation of proteins with small ubiquitin-like modifier 1 (SUMO-1) plays a critical role in a variety of cellular functions including cell cycle control, replication, and transcriptional regulation. Nuclear mitotic apparatus protein (NuMA) localizes to spindle poles during mitosis, and is an essential component in the formation and maintenance of mitotic spindle poles. Here we show that NuMA is a target for covalent conjugation to SUMO-1. We find that the lysine 1766 residue is the primary NuMA acceptor site for SUMO-1 conjugation. Interestingly, SUMO modification of endogenous NuMA occurs at the entry into mitosis and this modification is reversed after exiting from mitosis. Knockdown of Ubc9 or forced expression of SENP1 results in impairment of the localization of NuMA to mitotic spindle poles during mitosis. The SUMOylation-deficient NuMA mutant is defective in microtubule bundling, and multiple spindles are induced during mitosis. The mitosis-dependent dynamic SUMO-1 modification of NuMA might contribute to NuMA-mediated formation and maintenance of mitotic spindle poles during mitosis.

  6. Fluctuation-based imaging of nuclear Rac1 activation by protein oligomerisation

    NASA Astrophysics Data System (ADS)

    Hinde, Elizabeth; Yokomori, Kyoko; Gaus, Katharina; Hahn, Klaus M.; Gratton, Enrico

    2014-02-01

    Here we describe a fluctuation-based method to quantify how protein oligomerisation modulates signalling activity of a multifunctional protein. By recording fluorescence lifetime imaging microscopy (FLIM) data of a FRET biosensor in a format that enables concomitant phasor and cross Number and Brightness (cN&B) analysis, we measure the nuclear dynamics of a Rac1 FRET biosensor and assess how Rac1 homo-oligomers (N&B) regulate Rac1 activity (hetero-oligomerisation with the biosensor affinity reagent, PBD, by FLIM-FRET) or interaction with an unknown binding partner (cN&B). The high spatiotemporal resolution of this method allowed us to discover that upon DNA damage monomeric and active Rac1 in the nucleus is segregated from dimeric and inactive Rac1 in the cytoplasm. This reorganisation requires Rac1 GTPase activity and is associated with an importin-?2 redistribution. Only with this multiplexed approach can we assess the oligomeric state a molecular complex must form in order to regulate a complex signalling network.

  7. The HIV matrix protein p17 induces hepatic lipid accumulation via modulation of nuclear receptor transcriptoma

    PubMed Central

    Renga, Barbara; Francisci, Daniela; Carino, Adriana; Marchianò, Silvia; Cipriani, Sabrina; Chiara Monti, Maria; Del Sordo, Rachele; Schiaroli, Elisabetta; Distrutti, Eleonora; Baldelli, Franco; Fiorucci, Stefano

    2015-01-01

    Liver disease is the second most common cause of mortality in HIV-infected persons. Exactly how HIV infection per se affects liver disease progression is unknown. Here we have investigated mRNA expression of 49 nuclear hormone receptors (NRs) and 35 transcriptional coregulators in HepG2 cells upon stimulation with the HIV matrix protein p17. This viral protein regulated mRNA expression of some NRs among which LXR? and its transcriptional co-activator MED1 were highly induced at mRNA level. Dissection of p17 downstream intracellular pathway demonstrated that p17 mediated activation of Jak/STAT signaling is responsible for the promoter dependent activation of LXR. The treatment of both HepG2 as well as primary hepatocytes with HIV p17 results in the transcriptional activation of LXR target genes (SREBP1c and FAS) and lipid accumulation. These effects are lost in HepG2 cells pre-incubated with a serum from HIV positive person who underwent a vaccination with a p17 peptide as well as in HepG2 cells pre-incubated with the natural LXR antagonist gymnestrogenin. These results suggest that HIV p17 affects NRs and their related signal transduction thus contributing to the progression of liver disease in HIV infected patients. PMID:26469385

  8. Nuclear cap binding protein maps close to the xeroderma pigmentosum complementation group A (XPA) locus in human and mouse

    SciTech Connect

    Chadwick, B.P.; Obermayr, F.; Frischauf, A.M.

    1996-08-01

    This report describes the localization of the nuclear cap binding protein (NCBP) to human chromosome 9 and mouse chromosome 4 using somatic cell hybridization analysis. NCBP plays an important role in the splicing and transport of messenger-RNA. 11 refs., 1 fig.

  9. The nuclear basket proteins Mlp1p and Mlp2p are part of a dynamic interactome including Esc1p and the proteasome

    PubMed Central

    Niepel, Mario; Molloy, Kelly R.; Williams, Rosemary; Farr, Julia C.; Meinema, Anne C.; Vecchietti, Nicholas; Cristea, Ileana M.; Chait, Brian T.; Rout, Michael P.; Strambio-De-Castillia, Caterina

    2013-01-01

    The basket of the nuclear pore complex (NPC) is generally depicted as a discrete structure of eight protein filaments that protrude into the nucleoplasm and converge in a ring distal to the NPC. We show that the yeast proteins Mlp1p and Mlp2p are necessary components of the nuclear basket and that they also embed the NPC within a dynamic protein network, whose extended interactome includes the spindle organizer, silencing factors, the proteasome, and key components of messenger ribonucleoproteins (mRNPs). Ultrastructural observations indicate that the basket reduces chromatin crowding around the central transporter of the NPC and might function as a docking site for mRNP during nuclear export. In addition, we show that the Mlps contribute to NPC positioning, nuclear stability, and nuclear envelope morphology. Our results suggest that the Mlps are multifunctional proteins linking the nuclear transport channel to multiple macromolecular complexes involved in the regulation of gene expression and chromatin maintenance. PMID:24152732

  10. Constitutive Nuclear Expression of Dentin Matrix Protein 1 Fails to Rescue the Dmp1-null Phenotype*

    PubMed Central

    Lin, Shuxian; Zhang, Qi; Cao, Zhengguo; Lu, Yongbo; Zhang, Hua; Yan, Kevin; Liu, Ying; McKee, Marc D.; Qin, Chunlin; Chen, Zhi; Feng, Jian Q.

    2014-01-01

    Dentin matrix protein 1 (DMP1) plays multiple roles in bone, tooth, phosphate homeostasis, kidney, salivary gland, reproductive cycles, and the development of cancer. In vitro studies have indicated two different biological mechanisms: 1) as a matrix protein, DMP1 interacts with ?v?3 integrin and activates MAP kinase signaling; and 2) DMP1 serves as a transcription co-factor. In vivo studies have demonstrated its key role in osteocytes. This study attempted to determine whether DMP1 functions as a transcription co-factor and regulates osteoblast functions. For gene expression comparisons using adenovirus constructs, we targeted the expression of DMP1 either to the nucleus only by replacing the endogenous signal peptide with a nuclear localization signal (NLS) sequence (referred to as NLSDMP1) or to the extracellular matrix as the WT type (referred to as SPDMP1) in MC3T3 osteoblasts. High levels of DMP1 in either form greatly increased osteogenic gene expression in an identical manner. However, the targeted NLSDMP1 transgene driven by a 3.6-kb rat Col 1?1 promoter in the nucleus of osteoblasts and osteocytes failed to rescue the phenotyope of Dmp1-null mice, whereas the SPDMP1 transgene rescued the rickets defect. These studies support the notion that DMP1 functions as an extracellular matrix protein, rather than as a transcription co-factor in vivo. We also show that DMP1 continues its expression in osteoblasts during postnatal development and that the deletion of Dmp1 leads to an increase in osteoblast proliferation. However, poor mineralization in the metaphysis indicates a critical role for DMP1 in both osteoblasts and osteocytes. PMID:24917674

  11. Sde2: A novel nuclear protein essential for telomeric silencing and genomic stability in Schizosaccharomyces pombe

    SciTech Connect

    Sugioka-Sugiyama, Rie; Initiative for the Promotion of Young Scientists' Independent Research, University of Tsukuba, Tsukuba, Ibaraki 305-8577 ; Sugiyama, Tomoyasu; Initiative for the Promotion of Young Scientists' Independent Research, University of Tsukuba, Tsukuba, Ibaraki 305-8577; Precursory Research for Embryonic Science and Technology , Japan Science and Technology Agency , Kawaguchi, Saitama 332-0012

    2011-03-18

    Research highlights: {yields} Sde2 is essential for telomere silencing. {yields} Sde2 is involved in the maintenance of genomic stability. {yields} Sde2 promotes the recruitment of SHREC, a histone deacetylase complex, to telomeres. -- Abstract: Telomeres, specialized domains assembled at the ends of linear chromosomes, are essential for genomic stability in eukaryotes. The formation and maintenance of telomeres are governed by numerous factors such as telomeric repeats, telomere-binding proteins, heterochromatin proteins, and telomerase. Here, we report Sde2, a novel nuclear protein essential for telomeric silencing and genomic stability in the fission yeast Schizosaccharomyces pombe. A deficiency in sde2 results in the derepression of the ura4{sup +} gene inserted near telomeric repeats, and the noncoding transcripts from telomeric regions accumulate in sde2{Delta} cells. The loss of Sde2 function compromises transcriptional silencing at telomeres, and this silencing defect is accompanied by increased levels of acetylated histone H3K14 and RNA polymerase II occupancy at telomeres as well as reduced recruitment of the SNF2 ATPase/histone deacetylase-containing complex SHREC to telomeres. Deletion of sde2 also leads to a higher frequency of mitotic minichromosome loss, and sde2{Delta} cells often form asci that contain spores in abnormal numbers, shapes, or both. In addition, sde2{Delta} cells are highly sensitive to several stresses, including high/low temperatures, bleomycin, which induces DNA damage, and thiabendazole, a microtubule-destabilizing agent. Furthermore, Sde2 genetically interacts with the telomere regulators Taz1, Pof3, and Ccq1. These findings demonstrate that Sde2 cooperates with other telomere regulators to maintain functional telomeres, thereby preventing genomic instability.

  12. Identification of a nuclear export signal in the KSHV latent protein LANA2 mediating its export from the nucleus

    SciTech Connect

    Munoz-Fontela, C.; Collado, M.; Rodriguez, E.; Garcia, M.A.; Alvarez-Barrientos, A.; Arroyo, J.; Nombela, C.; Rivas, C. . E-mail: mdcrivas@farm.ucm.es

    2005-11-15

    LANA2 is a latent protein detected in Kaposi's sarcoma-associated herpesvirus (KSHV)-infected B cells that inhibits p53-dependent transcriptional transactivation and apoptosis and PKR-dependent apoptosis, suggesting an important role in the transforming activity of the virus. It has been reported that LANA2 localizes into the nucleus of both KSHV-infected B cells and transiently transfected HeLa cells. In this study, we show that LANA2 is a nucleocytoplasmic shuttling protein that requires a Rev-type nuclear export signal located in the C-terminus to direct the protein to the cytoplasm, through an association with the export receptor CRM1. In addition, a functional protein kinase B (PKB)/Akt phosphorylation motif partially overlapping with the nuclear export signal was identified. Nuclear exclusion of LANA2 was negatively regulated by the phosphorylation of threonine 564 by Akt. The ability of LANA2 to shuttle between nucleus and cytoplasm has implications for the function of this viral protein.

  13. Cloning of the cDNA for U1 small nuclear ribonucleoprotein particle 70K protein from Arabidopsis thaliana

    NASA Technical Reports Server (NTRS)

    Reddy, A. S.; Czernik, A. J.; An, G.; Poovaiah, B. W.

    1992-01-01

    We cloned and sequenced a plant cDNA that encodes U1 small nuclear ribonucleoprotein (snRNP) 70K protein. The plant U1 snRNP 70K protein cDNA is not full length and lacks the coding region for 68 amino acids in the amino-terminal region as compared to human U1 snRNP 70K protein. Comparison of the deduced amino acid sequence of the plant U1 snRNP 70K protein with the amino acid sequence of animal and yeast U1 snRNP 70K protein showed a high degree of homology. The plant U1 snRNP 70K protein is more closely related to the human counter part than to the yeast 70K protein. The carboxy-terminal half is less well conserved but, like the vertebrate 70K proteins, is rich in charged amino acids. Northern analysis with the RNA isolated from different parts of the plant indicates that the snRNP 70K gene is expressed in all of the parts tested. Southern blotting of genomic DNA using the cDNA indicates that the U1 snRNP 70K protein is coded by a single gene.

  14. Mutations within the nuclear localization signal of the porcine reproductive and respiratory syndrome virus nucleocapsid protein attenuate virus replication

    SciTech Connect

    Lee, Changhee; Hodgins, Douglas; Calvert, Jay G.; Welch, Siao-Kun W.; Jolie, Rika; Yoo, Dongwan . E-mail: dyoo@uoguelph.ca

    2006-03-01

    Porcine reproductive and respiratory syndrome virus (PRRSV) is an RNA virus replicating in the cytoplasm, but the nucleocapsid (N) protein is specifically localized to the nucleus and nucleolus in virus-infected cells. A 'pat7' motif of 41-PGKK(N/S)KK has previously been identified in the N protein as the functional nuclear localization signal (NLS); however, the biological consequences of N protein nuclear localization are unknown. In the present study, the role of N protein nuclear localization during infection was investigated in pigs using an NLS-null mutant virus. When two lysines at 43 and 44 at the NLS locus were substituted to glycines, the modified NLS with 41-PGGGNKK restricted the N protein to the cytoplasm. This NLS-null mutation was introduced into a full-length infectious cDNA clone of PRRSV. Upon transfection of cells, the NLS-null full-length clone induced cytopathic effects and produced infectious progeny. The NLS-null virus grew to a titer 100-fold lower than that of wild-type virus. To examine the response to NLS-null PRRSV in the natural host, three groups of pigs, consisting of seven animals per group, were intranasally inoculated with wild-type, placebo, or NLS-null virus, and the animals were maintained for 4 weeks. The NLS-null-infected pigs had a significantly shorter mean duration of viremia than wild-type-infected pigs but developed significantly higher titers of neutralizing antibodies. Mutations occurred at the NLS locus in one pig during viremia, and four types of mutations were identified: 41-PGRGNKK, 41-PGGRNKK, and 41-PGRRNKK, and 41-PGKKSKK. Both wild-type and NLS-null viruses persisted in the tonsils for at least 4 weeks, and the NLS-null virus persisting in the tonsils was found to be mutated to either 41-PGRGNKK or 41-PGGRNKK in all pigs. No other mutation was found in the N gene. All types of reversions which occurred during viremia and persistence were able to translocate the mutated N proteins to the nucleus, indicating a strong selection pressure for reversion at the NLS locus of the N protein in vivo. Reversions from NLS-null to functional NLS in the tonsils suggest a possible correlation of viral persistence with N protein nuclear localization. These results show that N protein nuclear localization is non-essential for PRRSV multiplication but may play an important role in viral attenuation and in pathogenesis in vivo.

  15. Characterization of a novel Dp71 dystrophin-associated protein complex (DAPC) present in the nucleus of HeLa cells: Members of the nuclear DAPC associate with the nuclear matrix

    SciTech Connect

    Fuentes-Mera, Lizeth; Rodriguez-Munoz, Rafael; Gonzalez-Ramirez, Ricardo; Garcia-Sierra, Francisco; Gonzalez, Everardo; Mornet, Dominique; Cisneros, Bulmaro . E-mail: bcisnero@cinvestav.mx

    2006-10-01

    Dystrophin is an essential component in the assembly and maintenance of the dystrophin-associated protein complex (DAPC), which includes members of the dystroglycan, syntrophin, sarcoglycan and dystrobrevin protein families. Distinctive complexes have been described in the cell membrane of different tissues and cultured cells. In this work, we report the identification and characterization of a novel DAPC present in the nuclei of HeLa cells, which contains dystrophin Dp71 as a key component. Using confocal microscopy and cell fractionation analyses, we found the presence of Dp71, {beta}-sarcoglycan, {beta}-dystroglycan, {alpha}- and {beta}-syntrophin, {alpha}1- and {beta}-dystrobrevin and nNOS in the nuclei of HeLa cells. Furthermore, we demonstrated by co-immunoprecipitation experiments that most of these proteins form a complex in the nuclear compartment. Next, we analyze the possible association of the nuclear DAPC with the nuclear matrix. We found the presence of Dp71, {beta}-dystroglycan, nNOS, {beta}-sarcoglycan, {alpha}/{beta} syntrophin, {alpha}1-dystrobrevin and {beta}-dystrobrevin in the nuclear matrix protein fractions and in situ nuclear matrix preparations from HeLa cells. Moreover, we found that Dp71, {beta}-dystroglycan and {beta}-dystrobrevin co-immunoprecipitated with the nuclear matrix proteins lamin B1 and actin. The association of members of the nuclear DAPC with the nuclear matrix indicates that they may work as scaffolding proteins involved in nuclear architecture.

  16. A model for the dynamic nuclear/nucleolar/cytoplasmic trafficking of the porcine reproductive and respiratory syndrome virus (PRRSV) nucleocapsid protein based on live cell imaging

    SciTech Connect

    You, Jae-Hwan; Howell, Gareth; Pattnaik, Asit K.; Osorio, Fernando A.; Hiscox, Julian A.

    2008-08-15

    Porcine reproductive and respiratory syndrome virus (PRRSV), an arterivirus, in common with many other positive strand RNA viruses, encodes a nucleocapsid (N) protein which can localise not only to the cytoplasm but also to the nucleolus in virus-infected cells and cells over-expressing N protein. The dynamic trafficking of positive strand RNA virus nucleocapsid proteins and PRRSV N protein in particular between the cytoplasm and nucleolus is unknown. In this study live imaging of permissive and non-permissive cell lines, in conjunction with photo-bleaching (FRAP and FLIP), was used to investigate the trafficking of fluorescent labeled (EGFP) PRRSV-N protein. The data indicated that EGFP-PRRSV-N protein was not permanently sequestered to the nucleolus and had equivalent mobility to cellular nucleolar proteins. Further the nuclear import of N protein appeared to occur faster than nuclear export, which may account for the observed relative distribution of N protein between the cytoplasm and the nucleolus.

  17. Nuclear localized protein-1 (Nulp1) increases cell death of human osteosarcoma cells and binds the X-linked inhibitor of apoptosis protein

    SciTech Connect

    Steen, Hakan; Lindholm, Dan

    2008-02-08

    Nuclear localized protein-1 (Nulp1) is a recently identified gene expressed in mouse and human tissues particularly during embryonic development. Nulp1 belongs to the family of basic helix-loop-helix (bHLH) proteins that are important in development. The precise function of Nulp1 in cells is however not known. We observed that overexpression of Nulp1 induces a large increase in cell death of human osteosarcoma Saos2 cells with DNA fragmentation. In mouse N2A neuroblastoma cells Nulp1 affected cell proliferation and sensitized cells towards death induced by staurosporine. Staining using a novel antibody localized Nulp1 mainly to the cell nucleus and to some extent to the cytoplasm. Nulp1 binds the X-linked inhibitor of apoptosis protein (XIAP) and this interaction was increased during cell death. These results indicate that Nulp1 plays a role in cell death control and may influence tumor growth.

  18. Levels of synthesis of primate-specific nuclear proteins differ between growth-arrested and proliferating cells

    SciTech Connect

    Celis, J.E.; Madsen, P.; Nielsen, S.; Ratz, G.P.; Lauridsen, J.B.; Celis, A.

    1987-02-01

    A monoclonal antibody that reacts specifically with the proliferation-sensitive nuclear proteins, isoelectric focusing (IEF) 8Z31 (molecular weight (MW), 76,000 charge variants, HeLa protein catalogue number) has been characterized. As determined by indirect immunofluorescence, the antibody stains the nucleolus and nucleoplasm of interphase-cultured cells of primate origin, but does not react with cells of other species. Proteins having similar MWs and isoelectric points as the human or monkey (primates) proteins were not observed in cultured cells of the following species: aves, bat, dog, dolphin, goat, hamster, mink, mouse, pisces, potoroo, rabbit and rat. Quantitative two-dimensional (2D) gel electrophoretic analysis of (/sup 35/S)methionine-labelled proteins synthesized by normal (quiescent, proliferating) and SV40-transformed human MRC-5 fibroblasts revealed significant differences in the levels of synthesis of both IEF 8Z30 and 8Z31. In quiescent cells the main labelled product corresponded to IEF 8Z31 (ratio IEF 8Z31/8Z30, 2.3), while in the transformed cells the major product was IEF 8Z30 (ratio, 0.62). Normal proliferating fibroblasts exhibited similar levels of both proteins (ratio, 1.21). Combined levels of synthesis of both proteins were 1.50 and 1.20 times as high in the transformed cells as in the quiescent and proliferating cells, respectively. Modulation of the levels of synthesis of these proteins may play a role in cell proliferation.

  19. Nuclear Magnetic Resonance Characterization of the Type III Secretion System Tip Chaperone Protein PcrG of Pseudomonas aeruginosa.

    PubMed

    Chaudhury, Sukanya; Nordhues, Bryce A; Kaur, Kawaljit; Zhang, Na; De Guzman, Roberto N

    2015-11-01

    Lung infection with Pseudomonas aeruginosa is the leading cause of death among cystic fibrosis patients. To initiate infection, P. aeruginosa assembles a protein nanomachine, the type III secretion system (T3SS), to inject bacterial proteins directly into target host cells. An important regulator of the P. aeruginosa T3SS is the chaperone protein PcrG, which forms a complex with the tip protein, PcrV. In addition to its role as a chaperone to the tip protein, PcrG also regulates protein secretion. PcrG homologues are also important in the T3SS of other pathogens such as Yersinia pestis, the causative agent of bubonic plague. The atomic structure of PcrG or any member of the family of tip protein chaperones is currently unknown. Here, we show by circular dichroism and nuclear magnetic resonance (NMR) spectroscopy that PcrG lacks a tertiary structure. However, it is not completely disordered but contains secondary structures dominated by two long ?-helices from residue 16 to 41 and from residue 55 to 76. The helices of PcrG are partially formed, have similar backbone dynamics, and are flexible. NMR titrations show that the entire length of PcrG residues from position 9 to 76 is involved in binding to PcrV. PcrG adds to the growing list of partially folded or unstructured proteins with important roles in type III secretion. PMID:26451841

  20. The L2 Minor Capsid Protein of Human Papillomavirus Type 16 Interacts with a Network of Nuclear Import Receptors

    PubMed Central

    Darshan, Medha S.; Lucchi, John; Harding, Emily; Moroianu, Junona

    2004-01-01

    The L2 minor capsid proteins enter the nucleus twice during viral infection: in the initial phase after virion disassembly and in the productive phase when, together with the L1 major capsid proteins, they assemble the replicated viral DNA into virions. In this study we investigated the interactions between the L2 protein of high-risk human papillomavirus type 16 (HPV16) and nuclear import receptors. We discovered that HPV16 L2 interacts directly with both Kap?2 and Kap?3. Moreover, binding of Ran-GTP to either Kap?2 or Kap?3 inhibits its interaction with L2, suggesting that the Kap?/L2 complex is import competent. In addition, we found that L2 forms a complex with the Kap?2?1 heterodimer via interaction with the Kap?2 adapter. In agreement with the binding data, nuclear import of L2 in digitonin-permeabilized cells could be mediated by either Kap?2?1 heterodimers, Kap?2, or Kap?3. Mapping studies revealed that HPV16 L2 contains two nuclear localization signals (NLSs), in the N terminus (nNLS) and C terminus (cNLS), that could mediate its nuclear import. Together the data suggest that HPV16 L2 interacts via its NLSs with a network of karyopherins and can enter the nucleus via several import pathways mediated by Kap?2?1 heterodimers, Kap?2, and Kap?3. PMID:15507604

  1. The l2 minor capsid protein of human papillomavirus type 16 interacts with a network of nuclear import receptors.

    PubMed

    Darshan, Medha S; Lucchi, John; Harding, Emily; Moroianu, Junona

    2004-11-01

    The L2 minor capsid proteins enter the nucleus twice during viral infection: in the initial phase after virion disassembly and in the productive phase when, together with the L1 major capsid proteins, they assemble the replicated viral DNA into virions. In this study we investigated the interactions between the L2 protein of high-risk human papillomavirus type 16 (HPV16) and nuclear import receptors. We discovered that HPV16 L2 interacts directly with both Kapbeta(2) and Kapbeta(3). Moreover, binding of Ran-GTP to either Kapbeta(2) or Kapbeta(3) inhibits its interaction with L2, suggesting that the Kapbeta/L2 complex is import competent. In addition, we found that L2 forms a complex with the Kapalpha(2)beta(1) heterodimer via interaction with the Kapalpha(2) adapter. In agreement with the binding data, nuclear import of L2 in digitonin-permeabilized cells could be mediated by either Kapalpha(2)beta(1) heterodimers, Kapbeta(2), or Kapbeta(3). Mapping studies revealed that HPV16 L2 contains two nuclear localization signals (NLSs), in the N terminus (nNLS) and C terminus (cNLS), that could mediate its nuclear import. Together the data suggest that HPV16 L2 interacts via its NLSs with a network of karyopherins and can enter the nucleus via several import pathways mediated by Kapalpha(2)beta(1) heterodimers, Kapbeta(2), and Kapbeta(3). PMID:15507604

  2. Protein arginine methyltransferase CARM1 attenuates the paraspeckle-mediated nuclear retention of mRNAs containing IRAlus.

    PubMed

    Hu, Shi-Bin; Xiang, Jian-Feng; Li, Xiang; Xu, Yefen; Xue, Wei; Huang, Min; Wong, Catharine C; Sagum, Cari A; Bedford, Mark T; Yang, Li; Cheng, Donghang; Chen, Ling-Ling

    2015-03-15

    In many cells, mRNAs containing inverted repeated Alu elements (IRAlus) in their 3' untranslated regions (UTRs) are inefficiently exported to the cytoplasm. Such nuclear retention correlates with paraspeckle-associated protein complexes containing p54(nrb). However, nuclear retention of mRNAs containing IRAlus is variable, and how regulation of retention and export is achieved is poorly understood. Here we show one mechanism of such regulation via the arginine methyltransferase CARM1 (coactivator-associated arginine methyltransferase 1). We demonstrate that disruption of CARM1 enhances the nuclear retention of mRNAs containing IRAlus. CARM1 regulates this nuclear retention pathway at two levels: CARM1 methylates the coiled-coil domain of p54(nrb), resulting in reduced binding of p54(nrb) to mRNAs containing IRAlus, and also acts as a transcription regulator to suppress NEAT1 transcription, leading to reduced paraspeckle formation. These actions of CARM1 work together synergistically to regulate the export of transcripts containing IRAlus from paraspeckles under certain cellular stresses, such as poly(I:C) treatment. This work demonstrates how a post-translational modification of an RNA-binding protein affects protein-RNA interaction and also uncovers a mechanism of transcriptional regulation of the long noncoding RNA NEAT1. PMID:25792598

  3. Interaction between the inner nuclear membrane lamin B receptor and the heterochromatic methyl binding protein, MeCP2

    SciTech Connect

    Guarda, Alessia; Bolognese, Fabrizio; Bonapace, Ian Marc; Badaracco, Gianfranco

    2009-07-01

    The nuclear membrane has an important role for the dynamic regulation of the genome, besides the well-established cytoskeletal function. The nuclear lamina is emerging as an important player in the organization of the position and functional state of interphase chromosomes. Epigenetic modifications such as DNA methylation and histone modifications are required for genome reprogramming during development, tissue-specific gene expression and global gene silencing. The Methyl-CpG binding protein MeCP2 binds methyl-CpG dinucleotides in the mammalian genome and functions as a transcriptional repressor in vivo by interacting with Sin3A, thereby recruiting histone deacetylases (HDAC). MeCP2 also mediates the formation of higher-order chromatin structures contributing to determine the architectural organization of the nucleus. In this paper, we show that MeCP2 interacts in vitro and in vivo with the inner nuclear membrane protein LBR and that the unstructured aminoacidic sequence linking the MBD and TRD domains of MeCP2 is responsible for this association. The formation of an LBR-MeCP2 protein complex might help providing a molecular explanation to the distribution of part of the heterochromatin at the nuclear periphery linked to inner membrane.

  4. Polyphyly of nuclear lamin genes indicates an early eukaryotic origin of the metazoan-type intermediate filament proteins

    PubMed Central

    Kollmar, Martin

    2015-01-01

    The nuclear lamina is a protein meshwork associated with the inner side of the nuclear envelope contributing structural, signalling and regulatory functions. Here, I report on the evolution of an important component of the lamina, the lamin intermediate filament proteins, across the eukaryotic tree of life. The lamins show a variety of protein domain and sequence motif architectures beyond the classical ?-helical rod, nuclear localisation signal, immunoglobulin domain and CaaX motif organisation, suggesting extension and adaptation of functions in many species. I identified lamin genes not only in metazoa and Amoebozoa as previously described, but also in other opisthokonts including Ichthyosporea and choanoflagellates, in oomycetes, a sub-family of Stramenopiles, and in Rhizaria, implying that they must have been present very early in eukaryotic evolution if not even the last common ancestor of all extant eukaryotes. These data considerably extend the current perception of lamin evolution and have important implications with regard to the evolution of the nuclear envelope. PMID:26024016

  5. Formation of nuclear bodies by the lncRNA Gomafu-associating proteins Celf3 and SF1

    PubMed Central

    Ishizuka, Akira; Hasegawa, Yuko; Ishida, Kentaro; Yanaka, Kaori; Nakagawa, Shinichi

    2014-01-01

    Gomafu/MIAT/Rncr2 is a long noncoding RNA that has been proposed to control retinal cell specification, stem cell differentiation and alternative splicing of schizophrenia-related genes. However, how Gomafu controls these biological processes at the molecular level has remained largely unknown. In this study, we identified the RNA-binding protein Celf3 as a novel Gomafu-associating protein. Knockdown of Celf3 led to the down-regulation of Gomafu, and cross-link RNA precipitation analysis confirmed specific binding between Celf3 and Gomafu. In the neuroblastoma cell line Neuro2A, Celf3 formed novel nuclear bodies (named CS bodies) that colocalized with SF1, another Gomafu-binding protein. Gomafu, however, was not enriched in the CS bodies; instead, it formed distinct nuclear bodies in separate regions in the nucleus. These observations suggest that Gomafu indirectly modulates the function of the splicing factors SF1 and Celf3 by sequestering these proteins into separate nuclear bodies. PMID:25145264

  6. Membrane proteins Bqt3 and -4 anchor telomeres to the nuclear envelope to ensure chromosomal bouquet formation

    PubMed Central

    Chikashige, Yuji; Yamane, Miho; Okamasa, Kasumi; Tsutsumi, Chihiro; Kojidani, Tomoko; Sato, Mamiko; Haraguchi, Tokuko

    2009-01-01

    In many organisms, telomeres cluster to form a bouquet arrangement of chromosomes during meiotic prophase. Previously, we reported that two meiotic proteins, Bqt1 and -2, are required for tethering telomeres to the spindle pole body (SPB) during meiotic prophase in fission yeast. This study has further identified two novel, ubiquitously expressed inner nuclear membrane (INM) proteins, Bqt3 and -4, which are required for bouquet formation. We found that in the absence of Bqt4, telomeres failed to associate with the nuclear membranes in vegetative cells and consequently failed to cluster to the SPB in meiotic prophase. In the absence of Bqt3, Bqt4 protein was degraded during meiosis, leading to a phenotype similar to that of the bqt4-null mutant. Collectively, these results show that Bqt4 anchors telomeres to the INM and that Bqt3 protects Bqt4 from protein degradation. Interestingly, the functional integrity of telomeres is maintained even when they are separated from the nuclear envelope in vegetative cells. PMID:19948484

  7. Molecular crowding affects diffusion and binding of nuclear proteins in heterochromatin and reveals the fractal organization of chromatin

    PubMed Central

    Bancaud, Aurélien; Huet, Sébastien; Daigle, Nathalie; Mozziconacci, Julien; Beaudouin, Joël; Ellenberg, Jan

    2009-01-01

    The nucleus of eukaryotes is organized into functional compartments, the two most prominent being heterochromatin and nucleoli. These structures are highly enriched in DNA, proteins or RNA, and thus thought to be crowded. In vitro, molecular crowding induces volume exclusion, hinders diffusion and enhances association, but whether these effects are relevant in vivo remains unclear. Here, we establish that volume exclusion and diffusive hindrance occur in dense nuclear compartments by probing the diffusive behaviour of inert fluorescent tracers in living cells. We also demonstrate that chromatin-interacting proteins remain transiently trapped in heterochromatin due to crowding induced enhanced affinity. The kinetic signatures of these crowding consequences allow us to derive a fractal model of chromatin organization, which explains why the dynamics of soluble nuclear proteins are affected independently of their size. This model further shows that the fractal architecture differs between heterochromatin and euchromatin, and predicts that chromatin proteins use different target-search strategies in the two compartments. We propose that fractal crowding is a fundamental principle of nuclear organization, particularly of heterochromatin maintenance. PMID:19927119

  8. The N-terminus of porcine circovirus type 2 replication protein is required for nuclear localization and ori binding activities

    SciTech Connect

    Lin, W.-L.; Chien, M.-S.; Du, Y.-W.; Wu, P.-C.; Huang Chienjin

    2009-02-20

    Porcine circovirus type 2 possesses a circular, single-stranded DNA genome that requires the replication protein (Rep) for virus replication. To characterize the DNA binding potential and the significant region that confers the nuclear localization of the Rep protein, the defined coding regions of rep gene were cloned and expressed. All of the recombinant proteins except for the N-terminal 110 residues deletion mutant could bind to the double-stranded minimal binding site of replication origin (ori). In addition, the N-terminal deletion mutant lacking 110 residues exhibited mainly cytoplasmic staining in the transfected cells in contrast to the others, which localized dominantly in the nucleus, suggesting that this N-terminal domain is essential for nuclear localization. Furthermore, a series of green fluorescence proteins (GFP) containing potential nuclear localization signal (NLS) sequences were tested for their cellular distribution. The ability of the utmost 20 residues of the N-terminal region to target the GFP to the nucleus confirmed its role as a functional NLS.

  9. Nuclear Factor 90, a cellular dsRNA binding protein inhibits the HIV Rev-export function

    PubMed Central

    Urcuqui-Inchima, Silvio; Castaño, Maria Eugenia; Hernandez-Verdun, Danièle; St-Laurent, Georges; Kumar, Ajit

    2006-01-01

    Background The HIV Rev protein is known to facilitate export of incompletely spliced and unspliced viral transcripts to the cytoplasm, a necessary step in virus life cycle. The Rev-mediated nucleo-cytoplasmic transport of nascent viral transcripts, dependents on interaction of Rev with the RRE RNA structural element present in the target RNAs. The C-terminal variant of dsRNA-binding nuclear protein 90 (NF90ctv) has been shown to markedly attenuate viral replication in stably transduced HIV-1 target cell line. Here we examined a mechanism of interference of viral life cycle involving Rev-NF90ctv interaction. Results Since Rev:RRE complex formations depend on protein:RNA and protein:protein interactions, we investigated whether the expression of NF90ctv might interfere with Rev-mediated export of RRE-containing transcripts. When HeLa cells expressed both NF90ctv and Rev protein, we observed that NF90ctv inhibited the Rev-mediated RNA transport. In particular, three regions of NF90ctv protein are involved in blocking Rev function. Moreover, interaction of NF90ctv with the RRE RNA resulted in the expression of a reporter protein coding sequences linked to the RRE structure. Moreover, Rev influenced the subcellular localization of NF90ctv, and this process is leptomycin B sensitive. Conclusion The dsRNA binding protein, NF90ctv competes with HIV Rev function at two levels, by competitive protein:protein interaction involving Rev binding to specific domains of NF90ctv, as well as by its binding to the RRE-RNA structure. Our results are consistent with a model of Rev-mediated HIV-1 RNA export that envisions Rev-multimerization, a process interrupted by NF90ctv. PMID:17125513

  10. Arsenic mediated disruption of promyelocytic leukemia protein nuclear bodies induces ganciclovir susceptibility in Epstein-Barr positive epithelial cells

    SciTech Connect

    Sides, Mark D.; Block, Gregory J.; Shan, Bin; Esteves, Kyle C.; Lin, Zhen; Flemington, Erik K.; Lasky, Joseph A.

    2011-06-20

    Promyelocytic leukemia protein nuclear bodies (PML NBs) have been implicated in host immune response to viral infection. PML NBs are targeted for degradation during reactivation of herpes viruses, suggesting that disruption of PML NB function supports this aspect of the viral life cycle. The Epstein-Barr virus (EBV) Latent Membrane Protein 1 (LMP1) has been shown to suppress EBV reactivation. Our finding that LMP1 induces PML NB immunofluorescence intensity led to the hypothesis that LMP1 may modulate PML NBs as a means of maintaining EBV latency. Increased PML protein and morphometric changes in PML NBs were observed in EBV infected alveolar epithelial cells and nasopharyngeal carcinoma cells. Treatment with low dose arsenic trioxide disrupted PML NBs, induced expression of EBV lytic proteins, and conferred ganciclovir susceptibility. This study introduces an effective modality to induce susceptibility to ganciclovir in epithelial cells with implications for the treatment of EBV associated pathologies.

  11. Actin-Related Protein Arp6 Influences H2A.Z-Dependent and -Independent Gene Expression and Links Ribosomal Protein Genes to Nuclear Pores

    PubMed Central

    Oma, Yukako; Kalck, Véronique; Akimura, Kazumi; Taddei, Angela; Iwahashi, Hitoshi; Kugou, Kazuto; Ohta, Kunihiro; Gasser, Susan M.; Harata, Masahiko

    2010-01-01

    Actin-related proteins are ubiquitous components of chromatin remodelers and are conserved from yeast to man. We have examined the role of the budding yeast actin-related protein Arp6 in gene expression, both as a component of the SWR1 complex (SWR-C) and in its absence. We mapped Arp6 binding sites along four yeast chromosomes using chromatin immunoprecipitation from wild-type and swr1 deleted (swr1?) cells. We find that a majority of Arp6 binding sites coincide with binding sites of Swr1, the catalytic subunit of SWR-C, and with the histone H2A variant Htz1 (H2A.Z) deposited by SWR-C. However, Arp6 binding detected at centromeres, the promoters of ribosomal protein (RP) genes, and some telomeres is independent of Swr1 and Htz1 deposition. Given that RP genes and telomeres both show association with the nuclear periphery, we monitored the ability of Arp6 to mediate the localization of chromatin to nuclear pores. Arp6 binding is sufficient to shift a randomly positioned locus to nuclear periphery, even in a swr1? strain. Arp6 is also necessary for the pore association of its targeted RP promoters possibly through cell cycle-dependent factors. Loss of Arp6, but not Htz1, leads to an up-regulation of these RP genes. In contrast, the pore-association of GAL1 correlates with Htz1 deposition, and loss of Arp6 reduces both GAL1 activation and peripheral localization. We conclude that Arp6 functions both together with the nucleosome remodeler Swr1 and also without it, to mediate Htz1-dependent and Htz1-independent binding of chromatin domains to nuclear pores. This association is shown to have modulating effects on gene expression. PMID:20419146

  12. Characterization of a nuclear localization signal in the C-terminus of the adeno-associated virus Rep68/78 proteins

    SciTech Connect

    Cassell, Geoffrey D.; Weitzman, Matthew D. . E-mail: weitzman@salk.edu

    2004-10-01

    Adeno-associated virus (AAV) replicates in the nucleus of infected cells, and therefore multiple nuclear import events are required for productive infection. We analyzed nuclear import of the viral Rep proteins and characterized a nuclear localization signal (NLS) in the C-terminus. We demonstrate that basic residues in this region constitute an NLS that is transferable and mediates interaction with the nuclear import receptor importin {alpha} in vitro. Mutant Rep proteins are predominantly cytoplasmic and are severely compromised for interactions with importin {alpha}, but retain their enzymatic functions in vitro. Interestingly, mutations of the NLS had significantly less effect on importin {alpha} interaction and replication in the context of Rep78 than when incorporated into the Rep68 protein. Together, our results demonstrate that a bipartite NLS exists in the shared part of Rep68 and Rep78, and suggest that an alternate entry mechanism may also contribute to nuclear localization of the Rep78 protein.

  13. Nuclear HMGA1 nonhistone chromatin proteins directly influence mitochondrial transcription, maintenance, and function

    SciTech Connect

    Dement, Gregory A.; Maloney, Scott C.; Reeves, Raymond . E-mail: reevesr@mail.wsu.edu

    2007-01-01

    We have previously demonstrated that HMGA1 proteins translocate from the nucleus to mitochondria and bind to mitochondrial DNA (mtDNA) at the D-loop control region [G.A. Dement, N.R. Treff, N.S. Magnuson, V. Franceschi, R. Reeves, Dynamic mitochondrial localization of nuclear transcription factor HMGA1, Exp. Cell Res. 307 (2005) 388-401.] [11]. To elucidate possible physiological roles for such binding, we employed methods to analyze mtDNA transcription, mitochondrial maintenance, and other organelle functions in transgenic human MCF-7 cells (HA7C) induced to over-express an HA-tagged HMGA1 protein and control (parental) MCF-7 cells. Quantitative real-time (RT) PCR analyses demonstrated that mtDNA levels were reduced approximately 2-fold in HMGA1 over-expressing HA7C cells and flow cytometric analyses further revealed that mitochondrial mass was significantly reduced in these cells. Cellular ATP levels were also reduced in HA7C cells and survival studies showed an increased sensitivity to killing by 2-deoxy-D-glucose, a glycolysis-specific inhibitor. Flow cytometric analyses revealed additional mitochondrial abnormalities in HA7C cells that are consistent with a cancerous phenotype: namely, increased reactive oxygen species (ROS) and increased mitochondrial membrane potential ({delta}{psi}{sub m}). Additional RT-PCR analyses demonstrated that gene transcripts from both the heavy (ND2, COXI, ATP6) and light (ND6) strands of mtDNA were up-regulated approximately 3-fold in HA7C cells. Together, these mitochondrial changes are consistent with many previous reports and reveal several possible mechanisms by which HMGA1 over-expression, a common feature of naturally occurring cancers, may affect tumor progression.

  14. Converging Nuclear Magnetic Shielding Calculations with Respect to Basis and System Size in Protein Systems

    PubMed Central

    Hartman, Joshua D.; Neubauer, Thomas J.; Caulkins, Bethany G.; Mueller, Leonard J.; Beran, Gregory J. O.

    2015-01-01

    Ab initio chemical shielding calculations greatly facilitate the interpretation of nuclear magnetic resonance (NMR) chemical shifts in biological systems, but the large sizes of these systems requires approximations in the chemical models used to represent them. Achieving good convergence in the predicted chemical shieldings is necessary before one can unravel how other complex structural and dynamical factors affect the NMR measurements. Here, we investigate how to balance trade-offs between using a better basis set or a larger cluster model for predicting the chemical shieldings of the substrates in two representative examples of protein-substrate systems involving different domains in tryptophan synthase: the N-(4?-trifluoromethoxybenzoyl)-2-aminoethyl phosphate (F9) ligand which binds in the ? active site, and the 2-aminophenol (2AP) quinonoid intermediate formed in the ? active site. We first demonstrate that a chemically intuitive three-layer, locally dense basis model that uses a large basis on the substrate, a medium triple-zeta basis to describe its hydrogen-bonding partners and/or surrounding van derWaals cavity, and a crude basis set for more distant atoms provides chemical shieldings in good agreement with much more expensive large basis calculations. Second, long-range quantum mechanical interactions are important, and one can accurately estimate them as a small-basis correction to larger-basis calculations on a smaller cluster. The combination of these approaches enables one to perform density functional theory NMR chemical shift calculations in protein systems that are well-converged with respect to both basis set and cluster size. PMID:25993979

  15. AMP-activated protein kinase (AMPK) activation inhibits nuclear translocation of Smad4 in mesangial cells and diabetic kidneys.

    PubMed

    Zhao, Jinghong; Miyamoto, Satoshi; You, Young-Hyun; Sharma, Kumar

    2015-05-15

    Diabetic nephropathy is characterized by diffuse mesangial matrix expansion and is largely dependent on the TGF-?/Smad signaling pathway. Smad4 is required for TGF-? signaling; however, its regulation has not been well characterized in diabetic kidney disease. Here, we report that high glucose is sufficient to stimulate nuclear translocation of Smad4 in mesangial cells and that stimulation of the major energy sensor AMP-activated protein kinase (AMPK) has a potent effect to block Smad4 nuclear translocation. Activation of AMPK by 5-aminoimidazole-4-carboxamide-1-?-d-ribofuranoside (AICAR) inhibited high glucose-induced and TGF-? stimulation of nuclear Smad4. To identify which of the catalytic ?-subunits may be involved, small interfering (si) RNA-based inhibition of AMPK ?1- or ?2-subunit was employed. Inhibition of either subunit reduced overall AMPK activity and contributed to Smad4 nuclear accumulation. In an animal model of early diabetic kidney disease, induction of diabetes was found to markedly stimulate Smad4 protein levels and enhance nuclear accumulation. AMPK activation with AICAR completely prevented the upregulation of Smad4 and reduced mesangial matrix accumulation. We conclude that stimulation of Smad4 in cell culture and in in vivo models of early diabetic kidney disease is dependent on AMPK. PMID:25428125

  16. Nuclear Relocalization of Polyadenylate Binding Protein during Rift Valley Fever Virus Infection Involves Expression of the NSs Gene

    PubMed Central

    Copeland, Anna Maria; Altamura, Louis A.; Van Deusen, Nicole M.

    2013-01-01

    Rift Valley fever virus (RVFV), an ambisense member of the family Bunyaviridae, genus Phlebovirus, is the causative agent of Rift Valley fever, an important zoonotic infection in Africa and the Middle East. Phlebovirus proteins are translated from virally transcribed mRNAs that, like host mRNA, are capped but, unlike host mRNAs, are not polyadenylated. Here, we investigated the role of PABP1 during RVFV infection of HeLa cells. Immunofluorescence studies of infected cells demonstrated a gross relocalization of PABP1 to the nucleus late in infection. Immunofluorescence microscopy studies of nuclear proteins revealed costaining between PABP1 and markers of nuclear speckles. PABP1 relocalization was sharply decreased in cells infected with a strain of RVFV lacking the gene encoding the RVFV nonstructural protein S (NSs). To determine whether PABP1 was required for RVFV infection, we measured the production of nucleocapsid protein (N) in cells transfected with small interfering RNAs (siRNAs) targeting PABP1. We found that the overall percentage of RVFV N-positive cells was not changed by siRNA treatment, indicating that PABP1 was not required for RVFV infection. However, when we analyzed populations of cells producing high versus low levels of PABP1, we found that the percentage of RVFV N-positive cells was decreased in cell populations producing physiologic levels of PABP1 and increased in cells with reduced levels of PABP1. Together, these results suggest that production of the NSs protein during RVFV infection leads to sequestration of PABP1 in the nuclear speckles, creating a state within the cell that favors viral protein production. PMID:23966414

  17. Lia1p, a novel protein required during nuclear differentiation for genome-wide DNA rearrangements in Tetrahymena thermophila.

    PubMed

    Rexer, Charles H; Chalker, Douglas L

    2007-08-01

    Extensive genome-wide rearrangements occur during somatic macronuclear development in Tetrahymena thermophila. These events are guided by RNA interference-directed chromatin modification including histone H3 lysine 9 methylation, which marks specific germ line-limited internal eliminated sequences (IESs) for excision. Several genes putatively involved in these developmental genome rearrangements were identified based on their proteins' localization to differentiating somatic nuclei, and here we demonstrate that one, LIA1, encodes a novel protein that is an essential component of the genome rearrangement machinery. A green fluorescent protein-Lia1 fusion protein exhibited dynamic nuclear localization during development that has striking similarity to that of the dual chromodomain-containing DNA rearrangement protein, Pdd1p. Coimmunoprecipitation experiments showed that Lia1p associates with Pdd1p and IES chromatin during macronuclear development. Cell lines in which we disrupted both the germ line and somatic copies of LIA1 (DeltaLIA1) grew normally but were unable to generate viable progeny, arresting late in development just prior to returning to vegetative growth. These mutant lines failed to properly form Pdd1p-containing nuclear structures and eliminate IESs despite showing normal levels of H3K9 methylation. These data indicate that Lia1p is required late in conjugation for the reorganization of the Tetrahymena genome. PMID:17586719

  18. Tip loci: six Chlamydomonas nuclear suppressors that permit the translocation of proteins with mutant thylakoid signal sequences.

    PubMed

    Bernd, K K; Kohorn, B D

    1998-07-01

    Mutations within the signal sequence of cytochrome f (cytf) in Chlamydomonas inhibit thylakoid membrane protein translocation and render cells nonphotosynthetic. Twenty-seven suppressors of the mutant signal sequences were selected for their ability to restore photoautotrophic growth and these describe six nuclear loci named tip1 through 6 for thylakoid insertion protein. The tip mutations restore the translocation of cytf and are not allele specific, as they suppress a number of different cytf signal sequence mutations. Tip5 and 2 may act early in cytf translocation, while Tip1, 3, 4, and 6 are engaged later. The tip mutations have no phenotype in the absence of a signal sequence mutation and there is genetic interaction between tip4, and tip5 suggesting an interaction of their encoded proteins. As there is overlap in the energetic, biochemical and genetic requirements for the translocation of nuclear and chloroplast-encoded thylakoid proteins, the tip mutations likely identify components of a general thylakoid protein translocation apparatus. PMID:9649521

  19. Nuclear transport of human immunodeficiency virus type 1, visna virus, and equine infectious anemia virus Rev proteins: identification of a family of transferable nuclear export signals.

    PubMed

    Meyer, B E; Meinkoth, J L; Malim, M H

    1996-04-01

    The human immunodeficiency virus type 1 Rev trans activator binds directly to unspliced viral mRNA in the nucleus and activates its transport to the cytoplasm. In additon to the sequences that confer RNA binding and nuclear localization, Rev has a carboxy-terminal region, the activation domain, whose integrity is essential for biological activity. Because it has been established that Rev constitutively exits and reenters the nucleus and that the activation domain is required for nuclear exit, it has been proposed that Rev's activation domain is a nuclear export signal (NES). Here, we used microinjection-based assays to demonstrate that the activation domain of human immunodeficiency virus type 1 Rev imparts rapid nuclear export after its transfer to heterologous substrates. NES- mediated export is specific, as it is sensitive both to inactivation by missense mutation and to selective inhibition by an excess of the wild-type, but not mutant, activation domain peptide. Examination of the Rev trans activators of two nonprimate lentiviruses, visna virus and equine infectious anemia virus, revealed that their activation domains are also potent NESs. Taken together, these data demonstrate that nuclear export can be determined by positively acting peptide motifs, namely, NESs, and suggest that Rev proteins activate viral RNA transport by providing export ribonucleoproteins with specific information that targets them to the cytoplasm. PMID:8642662

  20. A novel inhibitor of apoptosis protein (IAP)-interacting protein, Vestigial-like (Vgl)-4, counteracts apoptosis-inhibitory function of IAPs by nuclear sequestration

    SciTech Connect

    Jin, Hyung-Seung; Park, Hyung-Sun; Shin, Jun-Ha; Kim, Dong-Hwan; Translational Research Center for Protein Function Control, Yonsei University, Yonsei-ro 50, Seodaemoon-gu, Seoul 120-749 ; Jun, Sung-Hun; Lee, Chang-Jun; Lee, Tae H.

    2011-09-02

    Highlights: {yields} We identified a new IAP binding protein Vgl-4. {yields} Vgl-4 is expressed mainly in the nucleus and triggers a relocalization of IAPs from the cytoplasm to the nucleus. {yields} Vgl-4-mediated IAP nuclear localization was blocked by TRAF2 coexpression. {yields} Vgl-4 suppresses the ability of IAPs to prevent cell death, however TRAF2 can revere the effect of Vgl-4. {yields} Vgl-4 functions as an IAP regulator by binding to IAPs and altering their sub-cellular localization. -- Abstract: The inhibitors of apoptosis proteins (IAP), which include cIAP1, cIAP2 and XIAP, suppress apoptosis through the inhibition of caspases, and the activity of IAPs is regulated by a variety of IAP-binding proteins. Herein, we report the identification of a Vestigial-like 4 (Vgl-4), which functions as a transcription cofactor in cardiac myocytes, as a new IAP binding protein. Vgl-4 is expressed predominantly in the nucleus and its overexpression triggers a relocalization of IAPs from the cytoplasm to the nucleus. cIAP1/2-interacting protein TRAF2 (TNF receptor-associated factor 2) prevented the Vgl-4-driven nuclear localization of cIAP2. Accordingly, the forced relocation of IAPs to the nucleus by Vgl-4 significantly reduced their ability to prevent Bax- and TNF{alpha}-induced apoptosis, which can be recovered by co-expression with TRAF2. Our results suggest that Vgl-4 may play a role in the apoptotic pathways by regulating translocation of IAPs between different cell compartments.

  1. Repression of the Drosophila proliferating-cell nuclear antigen gene promoter by zerknuellt protein

    SciTech Connect

    Yamaguchi, Masamitsu; Hirose, Fumiko; Nishida, Yasuyoshi; Matsukage, Akio )

    1991-10-01

    A 631-bp fragment containing the 5{prime}-flanking region of the Drosophila melanogaster proliferating-cell nuclear antigen (PCNA) gene was placed upstream of the chloramphenicol acetyltransferase (CAT) gene of a CAT vector. A transient expression assay of CAT activity in Drosophila Kc cells transfected with this plasmid and a set of 5{prime}-deletion derivatives revealed that the promoter function resided within a 192-bp region. Cotransfection with a zerknuellt (zen)-expressing plasmid specifically repressed CAT expression. However, cotransfection with expression plasmids for a nonfunctional zen mutation, even skipped, or bicoid showed no significant effect on CAT expression. RNase protection analysis revealed that the repression by zen was at the transcription step. The target sequence of zen was mapped within the 34-bp region of the PCNA gene promoter, even though it lacked zen protein-binding sites. Transgenic flies carrying the PCNA gene regulatory region fused with lacZ were established. These results indicate that zen indirectly represses PCNA gene expression, probably by regulating the expression of some transcription factor(s) that binds to the PCNA gene promoter.

  2. Function of a TGF-beta inducible nuclear protein in the silk gland in Bombyx mori.

    PubMed

    Wang, J-L; Zhang, Y-P; Gu, Y-Y; Wang, J-X; Zhao, X-F

    2009-04-01

    A TGF-beta inducible nuclear protein 1 (BmTINP1) was cloned from silkworm, Bombyx mori. Polyclonal antibodies against BmTINP1 were produced and subsequently used in immunoblotting and immunohistochemistry analyses. The immunoblotting analyses demonstrated that BmTINP1 was specifically expressed in the anterior silk gland (ASG) and the middle silk gland (MSG) but not in the posterior silk gland (PSG). There were two bands that suggested the existence of an isoform of BmTINP1. The expression profiles of BmTINP1 in ASGs and MSGs were similar, and they manifested a high level of expression throughout the period during which silk gland grew exponentially. Immunohistochemistry results revealed that BmTINP1 was translocated from the nucleus into the cytoplasm when larvae developed from the 4th-HCS into the 5th instar. 20-hydroxyecdysone (20E) promotes the translocation, while the methoprene [a juvenile hormone (JH) analog] restrains the process. Our findings indicate that BmTINP1 is involved in silk produce along with the rapid growth of ASGs and MSGs during the last instar larvae, and the process could be regulated by hormones via control of BmTINP1 translocation from the nucleus to the cytoplasm. PMID:19220509

  3. The folate-coupled enzyme MTHFD2 is a nuclear protein and promotes cell proliferation

    PubMed Central

    Gustafsson Sheppard, Nina; Jarl, Lisa; Mahadessian, Diana; Strittmatter, Laura; Schmidt, Angelika; Madhusudan, Nikhil; Tegnér, Jesper; Lundberg, Emma K.; Asplund, Anna; Jain, Mohit; Nilsson, Roland

    2015-01-01

    Folate metabolism is central to cell proliferation and a target of commonly used cancer chemotherapeutics. In particular, the mitochondrial folate-coupled metabolism is thought to be important for proliferating cancer cells. The enzyme MTHFD2 in this pathway is highly expressed in human tumors and broadly required for survival of cancer cells. Although the enzymatic activity of the MTHFD2 protein is well understood, little is known about its larger role in cancer cell biology. We here report that MTHFD2 is co-expressed with two distinct gene sets, representing amino acid metabolism and cell proliferation, respectively. Consistent with a role for MTHFD2 in cell proliferation, MTHFD2 expression was repressed in cells rendered quiescent by deprivation of growth signals (serum) and rapidly re-induced by serum stimulation. Overexpression of MTHFD2 alone was sufficient to promote cell proliferation independent of its dehydrogenase activity, even during growth restriction. In addition to its known mitochondrial localization, we found MTHFD2 to have a nuclear localization and co-localize with DNA replication sites. These findings suggest a previously unknown role for MTHFD2 in cancer cell proliferation, adding to its known function in mitochondrial folate metabolism. PMID:26461067

  4. Nuclear protein I{kappa}B-{zeta} inhibits the activity of STAT3

    SciTech Connect

    Wu, Zhihao; Zhang, Xiaoai; Yang, Juntao; Wu, Guangzhou; Zhang, Ying; Yuan, Yanzhi; Jin, Chaozhi; Chang, Zhijie; Wang, Jian; Yang, Xiaoming; He, Fuchu; Institutes of Biomedical Sciences, Fudan University, Shanghai 200032

    2009-09-18

    STAT3 (Signal transducer and activator of transcription 3) is a key transcription factor of the JAK-STAT (Janus kinase/signal transducer and activator of transcription) pathway that regulates cell proliferation and apoptosis. Activation of STAT3 is under tight regulation, and yet the different signaling pathways and the mechanisms that regulate its activity remain to be elucidated. Using a yeast two-hybrid screening, we have identified a nuclear protein I{kappa}B-{zeta} that interacts in a novel way with STAT3. This physical interaction was further confirmed by co-immunoprecipitation assays. The interaction regions were mapped to the coiled-coil domain of STAT3 and the C-terminal of I{kappa}B-{zeta}. Overexpression of I{kappa}B-{zeta} inhibited the transcriptional activity of STAT3. It also suppressed cell growth and induced cell apoptosis in SRC-simulated cells, which is partially mediated by down-regulation of expression of a known STAT3 target gene, MCL1. Our results suggest that I{kappa}B-{zeta} is a negative regulator of STAT3, and demonstrate a novel mechanism in which a component of the NF-{kappa}B signaling pathway inhibits the activation of STAT3.

  5. Role for Tyrosine Phosphorylation of A-kinase Anchoring Protein 8 (AKAP8) in Its Dissociation from Chromatin and the Nuclear Matrix.

    PubMed

    Kubota, Sho; Morii, Mariko; Yuki, Ryuzaburo; Yamaguchi, Noritaka; Yamaguchi, Hiromi; Aoyama, Kazumasa; Kuga, Takahisa; Tomonaga, Takeshi; Yamaguchi, Naoto

    2015-04-24

    Protein-tyrosine phosphorylation regulates a wide variety of cellular processes at the plasma membrane. Recently, we showed that nuclear tyrosine kinases induce global nuclear structure changes, which we called chromatin structural changes. However, the mechanisms are not fully understood. In this study we identify protein kinase A anchoring protein 8 (AKAP8/AKAP95), which associates with chromatin and the nuclear matrix, as a nuclear tyrosine-phosphorylated protein. Tyrosine phosphorylation of AKAP8 is induced by several tyrosine kinases, such as Src, Fyn, and c-Abl but not Syk. Nucleus-targeted Lyn and c-Src strongly dissociate AKAP8 from chromatin and the nuclear matrix in a kinase activity-dependent manner. The levels of tyrosine phosphorylation of AKAP8 are decreased by substitution of multiple tyrosine residues on AKAP8 into phenylalanine. Importantly, the phenylalanine mutations of AKAP8 inhibit its dissociation from nuclear structures, suggesting that the association/dissociation of AKAP8 with/from nuclear structures is regulated by its tyrosine phosphorylation. Furthermore, the phenylalanine mutations of AKAP8 suppress the levels of nuclear tyrosine kinase-induced chromatin structural changes. In contrast, AKAP8 knockdown increases the levels of chromatin structural changes. Intriguingly, stimulation with hydrogen peroxide induces chromatin structural changes accompanied by the dissociation of AKAP8 from nuclear structures. These results suggest that AKAP8 is involved in the regulation of chromatin structural changes through nuclear tyrosine phosphorylation. PMID:25770215

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

    PubMed

    Rutz, Natalja; Heilbronn, Regine; Weger, Stefan

    2015-08-28

    Based on its specific interaction with cullin3 mediated by an N-terminal BTB/POZ homologous domain, KCTD5 has been proposed to function as substrate adapter for cullin3 based ubiquitin E3 ligases. In the present study we tried to validate this hypothesis through identification and characterization of additional KCTD5 interaction partners. For the replication protein MCM7, the zinc finger protein ZNF711 and FAM193B, a yet poorly characterized cytoplasmic protein, we could demonstrate specific interaction with KCTD5 both in yeast two-hybrid and co-precipitation studies in mammalian cells. Whereas trimeric complexes of cullin3 and KCTD5 with the respective KCTD5 binding partner were formed, KCTD5/cullin3 induced polyubiquitylation and/or proteasome-dependent degradation of these binding partners could not be demonstrated. On the contrary, KCTD5 or Cullin3 overexpression increased ZNF711 protein stability. PMID:26188516

  7. Regulation of Stress-Inducible Phosphoprotein 1 Nuclear Retention by Protein Inhibitor of Activated STAT PIAS1

    PubMed Central

    Soares, Iaci N.; Caetano, Fabiana A.; Pinder, Jordan; Rodrigues, Bruna Roz; Beraldo, Flavio H.; Ostapchenko, Valeriy G.; Durette, Chantal; Pereira, Grace Schenatto; Lopes, Marilene H.; Queiroz-Hazarbassanov, Nicolle; Cunha, Isabela W.; Sanematsu, Paulo I.; Suzuki, Sergio; Bleggi-Torres, Luiz F.; Schild-Poulter, Caroline; Thibault, Pierre; Dellaire, Graham; Martins, Vilma R.; Prado, Vania F.; Prado, Marco A. M.

    2013-01-01

    Stress-inducible phosphoprotein 1 (STI1), a cochaperone for Hsp90, has been shown to regulate multiple pathways in astrocytes, but its contributions to cellular stress responses are not fully understood. We show that in response to irradiation-mediated DNA damage stress STI1 accumulates in the nucleus of astrocytes. Also, STI1 haploinsufficiency decreases astrocyte survival after irradiation. Using yeast two-hybrid screenings we identified several nuclear proteins as STI1 interactors. Overexpression of one of these interactors, PIAS1, seems to be specifically involved in STI1 nuclear retention and in directing STI1 and Hsp90 to specific sub-nuclear regions. PIAS1 and STI1 co-immunoprecipitate and PIAS1 can function as an E3 SUMO ligase for STI. Using mass spectrometry we identified five SUMOylation sites in STI1. A STI1 mutant lacking these five sites is not SUMOylated, but still accumulates in the nucleus in response to increased expression of PIAS1, suggesting the possibility that a direct interaction with PIAS1 could be responsible for STI1 nuclear retention. To test this possibility, we mapped the interaction sites between PIAS1 and STI1 using yeast-two hybrid assays and surface plasmon resonance and found that a large domain in the N-terminal region of STI1 interacts with high affinity with amino acids 450–480 of PIAS1. Knockdown of PIAS1 in astrocytes impairs the accumulation of nuclear STI1 in response to irradiation. Moreover, a PIAS1 mutant lacking the STI1 binding site is unable to increase STI1 nuclear retention. Interestingly, in human glioblastoma multiforme PIAS1 expression is increased and we found a significant correlation between increased PIAS1 expression and STI1 nuclear localization. These experiments provide evidence that direct interaction between STI1 and PIAS1 is involved in the accumulation of nuclear STI1. This retention mechanism could facilitate nuclear chaperone activity. PMID:23938469

  8. Regulation of stress-inducible phosphoprotein 1 nuclear retention by protein inhibitor of activated STAT PIAS1.

    PubMed

    Soares, Iaci N; Caetano, Fabiana A; Pinder, Jordan; Rodrigues, Bruna Roz; Beraldo, Flavio H; Ostapchenko, Valeriy G; Durette, Chantal; Pereira, Grace Schenatto; Lopes, Marilene H; Queiroz-Hazarbassanov, Nicolle; Cunha, Isabela W; Sanematsu, Paulo I; Suzuki, Sergio; Bleggi-Torres, Luiz F; Schild-Poulter, Caroline; Thibault, Pierre; Dellaire, Graham; Martins, Vilma R; Prado, Vania F; Prado, Marco A M

    2013-11-01

    Stress-inducible phosphoprotein 1 (STI1), a cochaperone for Hsp90, has been shown to regulate multiple pathways in astrocytes, but its contributions to cellular stress responses are not fully understood. We show that in response to irradiation-mediated DNA damage stress STI1 accumulates in the nucleus of astrocytes. Also, STI1 haploinsufficiency decreases astrocyte survival after irradiation. Using yeast two-hybrid screenings we identified several nuclear proteins as STI1 interactors. Overexpression of one of these interactors, PIAS1, seems to be specifically involved in STI1 nuclear retention and in directing STI1 and Hsp90 to specific sub-nuclear regions. PIAS1 and STI1 co-immunoprecipitate and PIAS1 can function as an E3 SUMO ligase for STI. Using mass spectrometry we identified five SUMOylation sites in STI1. A STI1 mutant lacking these five sites is not SUMOylated, but still accumulates in the nucleus in response to increased expression of PIAS1, suggesting the possibility that a direct interaction with PIAS1 could be responsible for STI1 nuclear retention. To test this possibility, we mapped the interaction sites between PIAS1 and STI1 using yeast-two hybrid assays and surface plasmon resonance and found that a large domain in the N-terminal region of STI1 interacts with high affinity with amino acids 450-480 of PIAS1. Knockdown of PIAS1 in astrocytes impairs the accumulation of nuclear STI1 in response to irradiation. Moreover, a PIAS1 mutant lacking the STI1 binding site is unable to increase STI1 nuclear retention. Interestingly, in human glioblastoma multiforme PIAS1 expression is increased and we found a significant correlation between increased PIAS1 expression and STI1 nuclear localization. These experiments provide evidence that direct interaction between STI1 and PIAS1 is involved in the accumulation of nuclear STI1. This retention mechanism could facilitate nuclear chaperone activity. PMID:23938469

  9. Leishmania major telomerase TERT protein has a nuclear/mitochondrial eclipsed distribution that is affected by oxidative stress.

    PubMed

    Campelo, Riward; Díaz Lozano, Isabel; Figarella, Katherine; Osuna, Antonio; Ramírez, José Luis

    2015-01-01

    In its canonical role the reverse transcriptase telomerase recovers the telomeric repeats that are lost during DNA replication. Other locations and activities have been recently described for the telomerase protein subunit TERT in mammalian cells. In the present work, using biochemistry, molecular biology, and electron microscopy techniques, we found that in the human parasite Leishmania major, TERT (and telomerase activity) shared locations between the nuclear, mitochondrial, and cytoplasmic compartments. Also, some telomerase activity and TERT protein could be found in ? 100-nm nanovesicles. In the mitochondrial compartment, TERT appears to be mainly associated with the kinetoplast DNA. When Leishmania cells were exposed to H2O2, TERT changed its relative abundance and activity between the nuclear and mitochondrial compartments, with the majority of activity residing in the mitochondrion. Finally, overexpression of TERT in Leishmania transfected cells not only increased the parasitic cell growth rate but also increased their resistance to oxidative stress. PMID:25312950

  10. Leishmania major Telomerase TERT Protein Has a Nuclear/Mitochondrial Eclipsed Distribution That Is Affected by Oxidative Stress

    PubMed Central

    Campelo, Riward; Díaz Lozano, Isabel; Figarella, Katherine; Osuna, Antonio

    2014-01-01

    In its canonical role the reverse transcriptase telomerase recovers the telomeric repeats that are lost during DNA replication. Other locations and activities have been recently described for the telomerase protein subunit TERT in mammalian cells. In the present work, using biochemistry, molecular biology, and electron microscopy techniques, we found that in the human parasite Leishmania major, TERT (and telomerase activity) shared locations between the nuclear, mitochondrial, and cytoplasmic compartments. Also, some telomerase activity and TERT protein could be found in ?100-nm nanovesicles. In the mitochondrial compartment, TERT appears to be mainly associated with the kinetoplast DNA. When Leishmania cells were exposed to H2O2, TERT changed its relative abundance and activity between the nuclear and mitochondrial compartments, with the majority of activity residing in the mitochondrion. Finally, overexpression of TERT in Leishmania transfected cells not only increased the parasitic cell growth rate but also increased their resistance to oxidative stress. PMID:25312950

  11. Nucleocytoplasmic shuttling of the rabies virus P protein requires a nuclear localization signal and a CRM1-dependent nuclear export signal

    SciTech Connect

    Pasdeloup, David; Poisson, Nicolas; Raux, Helene; Gaudin, Yves; Ruigrok, Rob W.H. . E-mail: danielle.blondel@vms.cnrs-gif.fr

    2005-04-10

    Rabies virus P protein is a co-factor of the viral RNA polymerase. It has been shown previously that P mRNA directs the synthesis of four N-terminally truncated P products P2, P3, P4, and P5 due to translational initiation by a leaky scanning mechanism at internal Met codons. Whereas P and P2 are located in the cytoplasm, P3, P4, and P5 are found in the nucleus. Here, we have analyzed the molecular basis of the subcellular localization of these proteins. Using deletion mutants fused to GFP protein, we show the presence of a nuclear localization signal (NLS) in the C-terminal part of P (172-297). This domain contains a short lysine-rich stretch ({sup 211}KKYK{sup 214}) located in close proximity with arginine 260 as revealed by the crystal structure of P. We demonstrate the critical role of lysine 214 and arginine 260 in NLS activity. In the presence of Leptomycin B, P is retained in the nucleus indicating that it contains a CRM1-dependent nuclear export signal (NES). The subcellular distribution of P deletion mutants indicates that the domain responsible for export is the amino-terminal part of the protein. The use of fusion proteins that have amino terminal fragments of P fused to {beta}-galactosidase containing the NLS of SV40 T antigen allows us to identify a NES between residues 49 and 58. The localization of NLS and NES determines the cellular distribution of the P gene products.

  12. Depletion of the protein kinase VRK1 disrupts nuclear envelope morphology and leads to BAF retention on mitotic chromosomes.

    PubMed

    Molitor, Tyler P; Traktman, Paula

    2014-03-01

    Barrier to autointegration factor (BAF), which is encoded by the BANF1 gene, binds with high-affinity to double-stranded DNA and LEM domain-containing proteins at the nuclear periphery. A BANF1 mutation has recently been associated with a novel human progeria syndrome, and cells from these patients have aberrant nuclear envelopes. The interactions of BAF with its DNA- and protein-binding partners are known to be regulated by phosphorylation, and previously we validated BAF as a highly efficient substrate for the VRK1 protein kinase. Here we show that depletion of VRK1 in MCF10a and MDA-MB-231 cells results in aberrant nuclear architecture. The immobile fraction of green fluorescent protein (GFP)-BAF at the nuclear envelope (NE) is elevated, suggesting that prolonged interactions of BAF with its binding partners is likely responsible for the aberrant NE architecture. Because detachment of BAF from its binding partners is associated with NE disassembly, we performed live-imaging analysis of control and VRK1-depleted cells to visualize GFP-BAF dynamics during mitosis. In the absence of VRK1, BAF does not disperse but instead remains chromosome bound from the onset of mitosis. VRK1 depletion also increases the number of anaphase bridges and multipolar spindles. Thus phosphorylation of BAF by VRK1 is essential both for normal NE architecture and proper dynamics of BAF-chromosome interactions during mitosis. These results are consistent with previous studies of the VRK/BAF signaling axis in Caenorhabditis elegans and Drosophila melanogaster and validate VRK1 as a key regulator of NE architecture and mitotic chromosome dynamics in mammalian cells. PMID:24430874

  13. The human T-cell leukemia virus type 1 transactivator protein Tax colocalizes in unique nuclear structures with NF-kappaB proteins.

    PubMed Central

    Bex, F; McDowall, A; Burny, A; Gaynor, R

    1997-01-01

    The Tax protein of human T-cell leukemia virus type 1 (HTLV-1) is a potent activator of viral transcription. Tax also activates the expression of specific cellular genes involved in the control of T-lymphocyte growth via effects on cellular transcription factors, including members of the NF-kappaB/cRel family. Immunocytochemistry and electron microscopy were used to characterize the intracellular localization of Tax and identify cellular factors which are the potential targets for its transcriptional activity. These studies indicated that Tax localizes in discrete nuclear foci in T lymphocytes transformed by HTLV-1 and in cells transduced with Tax expression vectors. The Tax-containing foci are complex nuclear structures comprising a central core in which Tax colocalizes with splicing factor Sm. In addition to splicing factors Sm and SC-35, the Tax-containing nuclear structures also contain transcriptional components, including the largest subunit of RNA polymerase II and cyclin-dependent kinase CDK8. The inclusion of the two subunits of NF-kappaB, p50 and RelA, and the presence of the mRNA from a gene specifically activated by Tax through NF-kappaB binding sites suggest that these unique nuclear structures participate in Tax-mediated activation of gene expression via the NF-kappaB pathway. PMID:9094620

  14. Characterization of STIP, a multi-domain nuclear protein, highly conserved in metazoans, and essential for embryogenesis in Caenorhabditis elegans

    SciTech Connect

    Ji Qiongmei; Huang, C.-H. . E-mail: chuang@nybloodcenter.org; Peng Jianbin; Hashmi, Sarwar; Ye Tianzhang; Chen Ying

    2007-04-15

    We report here the identification and characterization of STIP, a multi-domain nuclear protein that contains a G-patch, a coiled-coil, and several short tryptophan-tryptophan repeats highly conserved in metazoan species. To analyze their functional role in vivo, we cloned nematode stip-1 genes and determined the spatiotemporal pattern of Caenorhabditis elegans STIP-1 protein. RNA analyses and Western blots revealed that stip-1 mRNA was produced via trans-splicing and translated as a 95-kDa protein. Using reporter constructs, we found STIP-1 to be expressed at all developmental stages and in many tissue/cell types including worm oocyte nuclei. We found that STIP-1 is targeted to the nucleus and forms large polymers with a rod-like shape when expressed in mammalian cells. Using deletion mutants, we mapped the regions of STIP-1 involved in nuclear import and polymer assembly. We further showed that knockdown of C. elegans stip-1 by RNA interference arrested development and resulted in morphologic abnormalities around the 16-cell stage followed by 100% lethality, suggesting its essential role in worm embryogenesis. Importantly, the embryonic lethal phenotype could be faithfully rescued with Drosophila and human genes via transgenic expression. Our data provide the first direct evidence that STIP have a conserved essential nuclear function across metazoans from worms to humans.

  15. Laminin isoforms: biological roles and effects on the intracellular distribution of nuclear proteins in intestinal epithelial cells

    SciTech Connect

    Turck, Natacha; Gross, Isabelle; Gendry, Patrick; Stutzmann, Jeanne; Freund, Jean-Noel; Kedinger, Michele; Simon-Assmann, Patricia; Launay, Jean-Francois . E-mail: Jean-Francois.Launay@inserm.u-strasbg.fr

    2005-02-15

    Laminins are structurally and functionally major components of the extracellular matrix. Four isoforms of laminins (laminin-1, -2, -5 and -10) are expressed in a specific pattern along the crypt-villus axis of the intestine. Previous works indicated that expression of these isoforms is developmentally regulated and that laminins could modulate the behaviour of intestinal cells, but the exact role of each isoform remained unclear. Here, we report the first systematic analysis of the cellular functions of the four isoforms using the human colon adenocarcinoma Caco2/TC7 cell line as a model. We compared the respective abilities of each isoform to modulate adhesion, proliferation and differentiation of intestinal epithelial cells. We found that the isoforms were functionally distinct, with laminin-10 being the most adhesive substratum, laminin-2, laminin-5 and laminin-10 enhancing cellular proliferation and at the opposite, laminin-1 stimulating intestinal cell differentiation. To begin to characterise the molecular events induced by the different isoforms, we examined by immunofluorescence the intracellular distribution of several nuclear proteins, recently highlighted by a nuclear proteomic approach. We observed clear nucleocytoplasmic redistribution of these proteins, which depended on the laminin isoform. These results provide evidence for a distinct functional role of laminins in intestinal cell functions characterised by specific localisation of nuclear proteins.

  16. Nuclear location of tumor suppressor protein maspin inhibits proliferation of breast cancer cells without affecting proliferation of normal epithelial cells

    PubMed Central

    2014-01-01

    Background Maspin, which is classified as a tumor suppressor protein, is downregulated in many types of cancer. Several studies have suggested potential anti-proliferative activity of maspin as well as sensitizing activity of maspin for therapeutic cytotoxic agents in breast cancer tissue culture and animal models. All of the experimental data gathered so far have been based on studies with maspin localized cytoplasmically, while maspin in breast cancer tumor cells may be located in the cytoplasm, nucleus or both. In this study, the effect of maspin cytoplasmic and nuclear location and expression level on breast cancer proliferation and patient survival was studied. Methods Tissue sections from 166 patients with invasive ductal breast cancer were stained by immunohistochemistry for maspin and Ki-67 protein. The localization and expression level of maspin were correlated with estimated patient overall survival and percent of Ki-67-positive cells. In further studies, we created constructs for transient transfection of maspin into breast cancer cells with targeted cytoplasmic and nuclear location. We analyzed the effect of maspin location in normal epithelial cell line MCF10A and three breast cancer cell lines - MCF-7, MDA-MB-231 and SKBR-3 - by immunofluorescence and proliferation assay. Results We observed a strong positive correlation between moderate and high nuclear maspin level and survival of patients. Moreover, a statistically significant negative relationship was observed between nuclear maspin and Ki-67 expression in patients with invasive ductal breast cancer. Spearman’s correlation analysis showed a negative correlation between level of maspin localized in nucleus and percentage of Ki-67 positive cells. No such differences were observed in cells with cytoplasmic maspin. We found a strong correlation between nuclear maspin and loss of Ki-67 protein in breast cancer cell lines, while there was no effect in normal epithelial cells from breast. The anti-proliferative effect of nuclear maspin on breast cancer cells was statistically significant in comparison to cytoplasmic maspin. Conclusions Our results suggest that nuclear maspin localization may be a prognostic factor in breast cancer and may have a strong therapeutic potential in gene therapy. Moreover, these data provide a new insight into the role of cytoplasmic and nuclear fractions of maspin in breast cancer. PMID:24581141

  17. Targeting of adenovirus E1A and E4-ORF3 proteins to nuclear matrix- associated PML bodies

    PubMed Central

    1995-01-01

    The PML protein was first identified as part of a fusion product with the retinoic acid receptor alpha (RAR alpha), resulting from the t(15;17) chromosomal translocation associated with acute promyelocytic leukemia (APL). It has been previously demonstrated that PML, which is tightly bound to the nuclear matrix, concentrates in discrete subnuclear compartments that are disorganized in APL cells due to the expression of the PML-RAR alpha hybrid. Here we report that adenovirus infection causes a drastic redistribution of PML from spherical nuclear bodies into fibrous structures. The product encoded by adenovirus E4- ORF3 is shown to be responsible for this reorganization and to colocalize with PML into these fibers. In addition, we demonstrate that E1A oncoproteins concentrate in the PML domains, both in infected and transiently transfected cells, and that this association requires the conserved amino acid motif (D)LXCXE, common to all viral oncoproteins that bind pRB or the related p107 and p130 proteins. The SV-40 large T antigen, another member of this oncoprotein family is also found in close association with the PML nuclear bodies. Taken together, the present data indicate that the subnuclear domains containing PML represent a preferential target for DNA tumor viruses, and therefore suggest a more general involvement of the PML nuclear bodies in oncogenic processes. PMID:7559785

  18. Nuclear Export of Smads by RanBP3L Regulates Bone Morphogenetic Protein Signaling and Mesenchymal Stem Cell Differentiation

    PubMed Central

    Chen, Fenfang; Lin, Xia; Xu, Pinglong; Zhang, Zhengmao; Chen, Yanzhen; Wang, Chao; Han, Jiahuai; Zhao, Bin; Xiao, Mu

    2015-01-01

    Bone morphogenetic proteins (BMPs) play vital roles in regulating stem cell maintenance and differentiation. BMPs can induce osteogenesis and inhibit myogenesis of mesenchymal stem cells. Canonical BMP signaling is stringently controlled through reversible phosphorylation and nucleocytoplasmic shuttling of Smad1, Smad5, and Smad8 (Smad1/5/8). However, how the nuclear export of Smad1/5/8 is regulated remains unclear. Here we report that the Ran-binding protein RanBP3L acts as a nuclear export factor for Smad1/5/8. RanBP3L directly recognizes dephosphorylated Smad1/5/8 and mediates their nuclear export in a Ran-dependent manner. Increased expression of RanBP3L blocks BMP-induced osteogenesis of mouse bone marrow-derived mesenchymal stem cells and promotes myogenic induction of C2C12 mouse myoblasts, whereas depletion of RanBP3L expression enhances BMP-dependent stem cell differentiation activity and transcriptional responses. In conclusion, our results demonstrate that RanBP3L, as a nuclear exporter for BMP-specific Smads, plays a critical role in terminating BMP signaling and regulating mesenchymal stem cell differentiation. PMID:25755279

  19. Nuclear Export of 60S Ribosomal Subunits Depends on Xpo1p and Requires a Nuclear Export Sequence-Containing Factor, Nmd3p, That Associates with the Large Subunit Protein Rpl10p

    PubMed Central

    Gadal, Olivier; Strauß, Daniela; Kessl, Jacques; Trumpower, Bernard; Tollervey, David; Hurt, Ed

    2001-01-01

    Nuclear export of ribosomes requires a subset of nucleoporins and the Ran system, but specific transport factors have not been identified. Using a large subunit reporter (Rpl25p-eGFP), we have isolated several temperature-sensitive ribosomal export (rix) mutants. One of these corresponds to the ribosomal protein Rpl10p, which interacts directly with Nmd3p, a conserved and essential protein associated with 60S subunits. We find that thermosensitive nmd3 mutants are impaired in large subunit export. Strikingly, Nmd3p shuttles between the nucleus and cytoplasm and is exported by the nuclear export receptor Xpo1p. Moreover, we show that export of 60S subunits is Xpo1p dependent. We conclude that nuclear export of 60S subunits requires the nuclear export sequence-containing nonribosomal protein Nmd3p, which directly binds to the large subunit protein Rpl10p. PMID:11313466

  20. Interaction of Heat Shock Protein Cpn10 with the Cyclin E/Cdk2 Substrate Nuclear Protein Ataxia-Telangiectasia (NPAT) Is Involved in Regulating Histone Transcription.

    PubMed

    Ling Zheng, Li; Wang, Fei Ya; Cong, Xiao Xia; Shen, Yue; Rao, Xi Sheng; Huang, Dao Sheng; Fan, Wei; Yi, Peng; Wang, Xin Bao; Zheng, Lei; Zhou, Yi Ting; Luo, Yan

    2015-12-01

    Precise modulation of histone gene transcription is critical for cell cycle progression. As a direct substrate of Cyclin E/CDK2, nuclear protein ataxia-telangiectasia (NPAT) is a crucial factor in regulating histone transcription and cell cycle progression. Here we identified that Cpn10/HSPE, a 10-kDa heat shock protein, is a novel interacting partner of NPAT. A pool of Cpn10 is colocalized with NPAT foci during G1 and S phases in nuclei. Gain- and loss-of-function experiments unraveled an essential role of Cpn10 in histone transcription. A conserved DLFD motif within Cpn10 was critical for targeting NPAT and modulating histone transcription. More importantly, knockdown of Cpn10 disrupted the focus formation of both NPAT and FADD-like interleukin-1?-converting enzyme-associated huge protein without affecting Coilin-positive Cajal bodies. Finally, Cpn10 is important for S phase progression and cell proliferation. Taken together, our finding revealed a novel role of Cpn10 in the spatial regulation of NPAT signaling and disclosed a previously unappreciated link between the heat shock protein and histone transcription regulation. PMID:26429916

  1. Purifying selection in mammalian mitochondrial protein-coding genes is highly effective and congruent with evolution of nuclear genes.

    PubMed

    Popadin, Konstantin Yu; Nikolaev, Sergey I; Junier, Thomas; Baranova, Maria; Antonarakis, Stylianos E

    2013-02-01

    The mammalian mitochondrial genomes differ from the nuclear genomes by maternal inheritance, absence of recombination, and higher mutation rate. All these differences decrease the effective population size of mitochondrial genome and make it more susceptible to accumulation of slightly deleterious mutations. It was hypothesized that mitochondrial genes, especially in species with low effective population size, irreversibly degrade leading to decrease of organismal fitness and even to extinction of species through the mutational meltdown. To interrogate this hypothesis, we compared the purifying selections acting on the representative set of mitochondrial (potentially degrading) and nuclear (potentially not degrading) protein-coding genes in species with different effective population size. For 21 mammalian species, we calculated the ratios of accumulation of slightly deleterious mutations approximated by Kn/Ks separately for mitochondrial and nuclear genomes. The 75% of variation in Kn/Ks is explained by two independent variables: type of a genome (mitochondrial or nuclear) and effective population size of species approximated by generation time. First, we observed that purifying selection is more effective in mitochondria than in the nucleus that implies strong evolutionary constraints of mitochondrial genome. Mitochondrial de novo nonsynonymous mutations have at least 5-fold more harmful effect when compared with nuclear. Second, Kn/Ks of mitochondrial and nuclear genomes is positively correlated with generation time of species, indicating relaxation of purifying selection with decrease of species-specific effective population size. Most importantly, the linear regression lines of mitochondrial and nuclear Kn/Ks's from generation times of species are parallel, indicating congruent relaxation of purifying selection in both genomes. Thus, our results reveal that the distribution of selection coefficients of de novo nonsynonymous mitochondrial mutations has a similar shape with the distribution of de novo nonsynonymous nuclear mutations, but its mean is five times smaller. The harmful effect of mitochondrial de novo nonsynonymous mutations triggers highly effective purifying selection, which maintains the fitness of the mammalian mitochondrial genome. PMID:22983951

  2. The human homologue of yeast ArgRIII protein is an inositol phosphate multikinase with predominantly nuclear localization.

    PubMed Central

    Nalaskowski, Marcus M; Deschermeier, Christina; Fanick, Werner; Mayr, Georg W

    2002-01-01

    The function of the transcription regulator ArgRIII in the expression of several genes involved in the metabolism of arginine in yeast has been well studied. It was previously reported that it is also an inositol phosphate multikinase and an important factor of the mRNA export pathway [reviewed by Shears (2000) Bioessays 22, 786-789]. In the present study we report the cloning of a full-length 1248-bp cDNA encoding a human inositol phosphate multikinase (IPMK). This protein has a calculated molecular mass of 47.219 kDa. Functionally important motifs [inositol phosphate-binding site, ATP-binding site, catalytically important SSLL (Ser-Ser-Leu-Leu) domain] are conserved between the human IPMK and yeast ArgRIII. Bacterially expressed protein demonstrated an inositol phosphate multikinase activity similar to that of yeast ArgRIII. Ins(1,4,5)P3 is phosphorylated at positions 3 and 6 up to Ins(1,3,4,5,6)P5. The human IPMK fused with a fluorescent protein tag is localized predominantly in the nucleus when transiently expressed in mammalian cells. A basic cluster in the protein's C-terminus is positively involved in nuclear targeting. These findings are consistent with the concept of a nuclear inositol phosphate signalling and phosphorylation pathway in mammalian cells. PMID:12027805

  3. A nuclear localization for Avr2 from Fusarium oxysporum is required to activate the tomato resistance protein I-2

    PubMed Central

    Ma, Lisong; Cornelissen, Ben J. C.; Takken, Frank L. W.

    2013-01-01

    Plant pathogens secrete effector proteins to promote host colonization. During infection of tomato xylem vessels, Fusarium oxysporum f. sp. lycopersici (Fol) secretes the Avr2 effector protein. Besides being a virulence factor, Avr2 is recognized intracellularly by the tomato I-2 resistance protein, resulting in the induction of host defenses. Here, we show that AVR2 is highly expressed in root- and xylem-colonizing hyphae three days post inoculation of roots. Co-expression of I-2 with AVR2 deletion constructs using agroinfiltration in Nicotiana benthamiana leaves revealed that, except for the N-terminal 17 amino acids, the entire AVR2 protein is required to trigger I-2-mediated cell death. The truncated Avr2 variants are still able to form homo-dimers, showing that the central region of Avr2 is required for dimerization. Simultaneous production of I-2 and Avr2 chimeras carrying various subcellular localization signals in N. benthamiana leaves revealed that a nuclear localization of Avr2 is required to trigger I-2-dependent cell death. Nuclear exclusion of Avr2 prevented its activation of I-2, suggesting that Avr2 is recognized by I-2 in the nucleus. PMID:23596453

  4. The involvement of nuclear factor-?appaB in the nuclear targeting and cyclin E1 upregulating activities of hepatoma upregulated protein.

    PubMed

    Chen, Jo-Mei Maureen; Chiu, Shao-Chih; Wei, Tong-You Wade; Lin, Shin-Yi; Chong, Cheong-Meng; Wu, Chi-Chen; Huang, Jiao-Ying; Yang, Shu-Ting; Ku, Chia-Feng; Hsia, Jiun-Yi; Yu, Chang-Tze Ricky

    2015-01-01

    Hepatoma upregulated protein (HURP) is originally isolated during the search for the genes associated with hepatoma. HURP is upregulated in many human cancers. Culture cells exhibit transformed and invasive phenotype when ectopic HURP is introduced, revealing HURP as an oncogene candidate. Our previous studies demonstrated that Aurora-A regulated the cell transforming activities of HURP by phosphorylating HURP at four serines. To unravel how the Aurora-A/HURP cascade contributes to cell transformation, we firstly noticed that HURP shuttled between cytoplasm and nucleus. The nuclear localization activity of HURP was promoted or abolished by overexpression or knockdown of Aurora-A. Similarly, the HURP phosphorylation mimicking mutant 4E had higher nuclear targeting activity than the phosphorylation deficient mutant 4A. The HURP 4E accelerated G1 progression and upregulated cyclin E1, and the cyclin E1 upregulating and cell transforming activities of HURP were diminished when the nuclear localization signal (NLS) was removed from HURP. Furthermore, HURP employed p38/nuclear factor-?B (NF-?B) cascade to stimulate cell growth. Interestingly, NF-?B trapped HURP in nucleus by interacting with HURP 4E. At last, the HURP/NF-?B complex activated the cyclin E1 promoter. Collectively, Aurora-A/HURP relays cell transforming signal to NF-?B, and the HURP/NF-?B complex is engaged in the regulation of cyclin E1 expression. PMID:25289861

  5. Essential Function for the Nuclear Protein Akirin2 in B Cell Activation and Humoral Immune Responses.

    PubMed

    Tartey, Sarang; Matsushita, Kazufumi; Imamura, Tomoko; Wakabayashi, Atsuko; Ori, Daisuke; Mino, Takashi; Takeuchi, Osamu

    2015-07-15

    Akirin2, an evolutionarily conserved nuclear protein, is an important factor regulating inflammatory gene transcription in mammalian innate immune cells by bridging the NF-?B and SWI/SNF complexes. Although Akirin is critical for Drosophila immune responses, which totally rely on innate immunity, the mammalian NF-?B system is critical not only for the innate but also for the acquired immune system. Therefore, we investigated the role of mouse Akirin2 in acquired immune cells by ablating Akirin2 function in B lymphocytes. B cell-specific Akirin2-deficient (Cd19(Cre/+)Akirin2(fl/fl)) mice showed profound decrease in the splenic follicular (FO) and peritoneal B-1, but not splenic marginal zone (MZ), B cell numbers. However, both Akirin2-deficient FO and MZ B cells showed severe proliferation defect and are prone to undergo apoptosis in response to TLR ligands, CD40, and BCR stimulation. Furthermore, B cell cycling was defective in the absence of Akirin2 owing to impaired expression of genes encoding cyclin D and c-Myc. Additionally, Brg1 recruitment to the Myc and Ccnd2 promoter was severely impaired in Akirin2-deficient B cells. Cd19(Cre/+)Akirin2(fl/fl) mice showed impaired in vivo immune responses to T-dependent and -independent Ags. Collectively, these results demonstrate that Akirin2 is critical for the mitogen-induced B cell cycle progression and humoral immune responses by controlling the SWI/SNF complex, further emphasizing the significant function of Akirin2 not only in the innate, but also in adaptive immune cells. PMID:26041538

  6. The quantitative assessment of the role played by basic amino acid clusters in the nuclear uptake of human ribosomal protein L7

    SciTech Connect

    Tai, Lin-Ru; Chou, Chang-Wei; Lee, I-Fang; Kirby, Ralph; Lin, Alan

    2013-02-15

    In this study, we used a multiple copy (EGFP){sub 3} reporter system to establish a numeric nuclear index system to assess the degree of nuclear import. The system was first validated by a FRAP assay, and then was applied to evaluate the essential and multifaceted nature of basic amino acid clusters during the nuclear import of ribosomal protein L7. The results indicate that the sequence context of the basic cluster determines the degree of nuclear import, and that the number of basic residues in the cluster is irrelevant; rather the position of the pertinent basic residues is crucial. Moreover, it also found that the type of carrier protein used by basic cluster has a great impact on the degree of nuclear import. In case of L7, importin ?2 or importin ?3 are preferentially used by clusters with a high import efficiency, notwithstanding that other importins are also used by clusters with a weaker level of nuclear import. Such a preferential usage of multiple basic clusters and importins to gain nuclear entry would seem to be a common practice among ribosomal proteins in order to ensure their full participation in high rate ribosome synthesis. - Highlights: ? We introduce a numeric index system that represents the degree of nuclear import. ? The rate of nuclear import is dictated by the sequence context of the basic cluster. ? Importin ?2 and ?3 were mainly responsible for the N4 mediated nuclear import.

  7. Nuclear Magnetic Resonance based Characterization of the Protein Binding Pocket using Hyperpolarized Ligand 

    E-print Network

    Min, Hlaing

    2014-08-04

    binding of the hyperpolarized ligand to the protein. Trypsin and benzamidine were chosen as models for the protein and the ligand because the binding of benzamidine to trypsin is well-known. Several enhanced NMR signals of trypsin appeared from...

  8. Proteins.

    ERIC Educational Resources Information Center

    Doolittle, Russell F.

    1985-01-01

    Examines proteins which give rise to structure and, by virtue of selective binding to other molecules, make genes. Binding sites, amino acids, protein evolution, and molecular paleontology are discussed. Work with encoding segments of deoxyribonucleic acid (exons) and noncoding stretches (introns) provides new information for hypotheses. (DH)

  9. Regulation of RNA processing and transport by a nuclear guanine nucleotide release protein and members of the Ras superfamily.

    PubMed Central

    Kadowaki, T; Goldfarb, D; Spitz, L M; Tartakoff, A M; Ohno, M

    1993-01-01

    The RCC1 gene of mammals encodes a guanine nucleotide release protein (GNRP). RCC1 and a homolog in Saccharomyces cerevisiae (MTR1/PRP20/SRM1) have previously been implicated in control of mRNA metabolism and export from the nucleus. We here demonstrate that a temperature-sensitive fission yeast mutant which has a mutation in a homologous gene, and two of three additional (mtr1/prp20/srm1) mutants accumulate nuclear poly(A)+ RNA at 37 degrees C. In S.cerevisiae, maturation of rRNA and tRNA is also inhibited at 37 degrees C. Nevertheless, studies with the corresponding BHK-21 cell mutant indicate that protein import into the nucleus continues. MTR1 homologs regulate RNA processing at a point which is distinct from their regulation of chromosome condensation since: (i) poly(A)+ RNA accumulation in the fission yeast mutant precedes chromosome condensation, and (ii) unlike chromosome condensation, accumulation of nuclear poly(A)+ RNA does not require p34cdc28 kinase activation or protein synthesis. Moreover, experiments involving inhibition of DNA synthesis indicate that the S.cerevisiae homolog does not govern cell cycle checkpoint control. Since RCC1p acts as GNRP for Ran, a small nuclear GTPase of the ras superfamily, we have identified two homologs of Ran in S.cerevisiae (CNR1 and CNR2). Only CNR1 is essential, but both code for proteins extremely similar to Ran and can suppress mtr1 mutations in allele-specific fashion. Thus, MTR1 and its homologs appear to act as GNRPs for a family of conserved GTPases in controlling RNA metabolism and transport. Their role in governing checkpoint control appears to be restricted to higher eukaryotes. Images PMID:7687541

  10. Overexpression of Nuclear Protein Kinase CK2 ? Catalytic Subunit (CK2?) as a Poor Prognosticator in Human Colorectal Cancer

    PubMed Central

    Hsu, Jung-Chin; Li, Chien-Feng; Fang, Chia-Lang; Lai, Hsi-Chin; Hseu, You-Cheng; Lin, Yi-Feng; Uen, Yih-Huei

    2011-01-01

    Background Colorectal cancer (CRC) is one of the most common malignancies but the current therapeutic approaches for advanced CRC are less efficient. Thus, novel therapeutic approaches are badly needed. The purpose of this study is to investigate the involvement of nuclear protein kinase CK2 ? subunit (CK2?) in tumor progression, and in the prognosis of human CRC. Methodology/Principal Findings Expression levels of nuclear CK2? were analyzed in 245 colorectal tissues from patients with CRC by immunohistochemistry, quantitative real-time PCR and Western blot. We correlated the expression levels with clinicopathologic parameters and prognosis in human CRC patients. Overexpression of nuclear CK2? was significantly correlated with depth of invasion, nodal status, American Joint Committee on Cancer (AJCC) staging, degree of differentiation, and perineural invasion. Patients with high expression levels of nuclear CK2? had a significantly poorer overall survival rate compared with patients with low expression levels of nuclear CK2?. In multi-variate Cox regression analysis, overexpression of nuclear CK2? was proven to be an independent prognostic marker for CRC. In addition, DLD-1 human colon cancer cells were employed as a cellular model to study the role of CK2? on cell growth, and the expression of CK2? in DLD-1 cells was inhibited by using siRNA technology. The data indicated that CK2?-specific siRNA treatment resulted in growth inhibition. Conclusions/Significance Taken together, overexpression of nuclear CK2? can be a useful marker for predicting the outcome of patients with CRC. PMID:21359197

  11. FoxO proteins' nuclear retention and BH3-only protein Bim induction evoke mitochondrial dysfunction-mediated apoptosis in berberine-treated HepG2 cells.

    PubMed

    Shukla, Shatrunajay; Rizvi, Fatima; Raisuddin, Sheikh; Kakkar, Poonam

    2014-11-01

    Mammalian forkhead-box family members belonging to the 'O' category (FoxO) manipulate a plethora of genes modulating a wide array of cellular functions including cell cycle regulation, apoptosis, DNA damage repair, and energy metabolism. FoxO overexpression and nuclear accumulation have been reported to show correlation with hindered tumor growth in vitro and size in vivo, while FoxO's downregulation via phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) pathway has been linked with tumor promotion. In this study, we have explored for the first time intervention of berberine, a plant-derived isoquinoline alkaloid, with FoxO family proteins in hepatoma cells. We observed that berberine significantly upregulated the mRNA expression of both FoxO1 and FoxO3a. Their phosphorylation-mediated cytoplasmic sequestration followed by degradation was prevented by berberine-induced downmodulation of the PI3K/Akt/mTOR pathway which promoted FoxO nuclear retention. PTEN, a tumor suppressor gene and negative regulator of the PI3K/Akt axis, was upregulated while phosphorylation of its Ser380 residue (possible mechanism of PTEN degradation) was significantly decreased in treated HepG2 cells. Exposure to berberine induced a significant increase in transcriptional activity of FoxO, as shown by GFP reporter assay. FoxO transcription factors effectively heightened BH3-only protein Bim expression, which in turn, being a direct activator of proapoptotic protein Bax, altered Bax/Bcl-2 ratio, culminating into mitochondrial dysfunction, caspases activation, and DNA fragmentation. The pivotal role of Bim in berberine-mediated cytotoxicity was further corroborated by knockdown experiments where Bim-silencing partially restored HepG2 cell viability during berberine exposure. In addition, a correlation between oxidative overload and FoxO's nuclear accumulation via JNK activation was evident as berberine treatment led to a pronounced increase in JNK phosphorylation together with enhanced ROS generation, lipid peroxidation, decreased activities of superoxide dismutase and catalase, and diminished glutathione levels. Thus, our findings suggest that the antiproliferative effect of berberine may in part be due to mitochondria-mediated apoptosis with Bim acting as a pivotal downstream factor of FoxO-induced transcriptional activation. PMID:25128467

  12. A recurrent polyalanine expansion in the transcription factor FOXL2 induces extensive nuclear and cytoplasmic protein aggregation

    PubMed Central

    Caburet, S; Demarez, A; Moumne, L; Fellous, M; De Baere, E; Veitia, R

    2004-01-01

    Blepharophimosis syndrome is an autosomal dominant disease characterised by eyelid malformations, associated or not with premature ovarian failure. It is caused by mutations in the FOXL2 gene, which encodes a forkhead transcription factor containing a polyalanine (polyAla) domain of 14 alanines. Expansions of the polyAla tract from 14 to 24 residues account for 30% of the reported mutations and lead mainly to isolated palpebral defects. We have transfected COS-7 cells with DNA constructs driving the expression of the wildtype and mutant FOXL2 proteins fused to the green fluorescent protein. The polyAla expansion was found to induce the formation of intranuclear aggregates and a mislocalisation of the protein due to extensive cytoplasmic aggregation. These findings were confirmed by immunofluorescence. Co-transfection experiments suggest that the wildtype and mutant proteins can co-aggregate. We propose that the mechanism for the molecular pathogenesis of the polyAla expansions of FOXL2 may be its mislocalisation concomitant with its inclusion into nuclear aggregates. This may diminish the pool of active protein. Potential effects of aggregation on cell viability are under study. PMID:15591279

  13. Mercuric chloride induces increases in both cytoplasmic and nuclear free calcium ions through a protein phosphorylation-linked mechanism.

    PubMed

    Parashar, A; Akhand, A A; Rawar, R; Furuno, T; Nakanishi, M; Kato, M; Suzuki, H; Nakashima, I

    1999-01-01

    The mechanism of the lymphocyte stimulatory action of sulfhydryl group-reactive mercuric ions was studied with respect to its potential ability to induce a protein tyrosine phosphorylation-linked signal for mobilization of free Ca2+ into cytoplasm and nucleus of the cell. Exposure of human leukamic T cell line (Jurkat) cells to high (1 mM) and low (0.01 mM) concentrations of HgCl2 induced tyrosine phosphorylation of multiple proteins in a concentration-dependent manner. Confocal microscopy directly visualized the time course localization of Ca2+ inside the cells after exposure to HgCl2. The onset and level of Ca2+ mobilization following HgCl2 exposure were in parallel to those of protein tyrosine phosphorylation. Interestingly, by either concentration of HgCl2, Ca2+ was mobilized in both cytoplasm and nucleus almost simultaneously, and the level of Ca2+ mobilization in the nucleus was more than that in the cytoplasm. All the HgCl2-mediated Ca2+ mobilization was prevented by addition of protein kinase inhibitor staurosporin prior to HgCl2. These results suggest that heavy metal stress triggers a protein tyrosine phosphorylation-linked signal that leads to a nuclear event-dominant Ca2+ mobilization. PMID:9890656

  14. Adenovirus Preterminal Protein Binds to the CAD Enzyme at Active Sites of Viral DNA Replication on the Nuclear Matrix

    PubMed Central

    Angeletti, Peter C.; Engler, Jeffrey A.

    1998-01-01

    Adenovirus (Ad) replicative complexes form at discrete sites on the nuclear matrix (NM) via an interaction mediated by the precursor of the terminal protein (pTP). The identities of cellular proteins involved in these complexes have remained obscure. We present evidence that pTP binds to a multifunctional pyrimidine biosynthesis enzyme found at replication domains on the NM. Far-Western blotting identified proteins of 150 and 240 kDa that had pTP binding activity. Amino acid sequencing of the 150-kDa band revealed sequence identity to carbamyl phosphate synthetase I (CPS I) and a high degree of homology to the related trifunctional enzyme known as CAD (for carbamyl phosphate synthetase, aspartate transcarbamylase, and dihydroorotase). Western blotting with an antibody directed against CAD detected a 240-kDa band that comigrated with that detected by pTP far-Western blotting. Binding experiments showed that a pTP-CAD complex was immunoprecipitable from cell extracts in which pTP was expressed by a vaccinia virus recombinant. Additionally, in vitro-translated epitope-tagged pTP and CAD were immunoprecipitable as a complex, indicating the occurrence of a protein-protein interaction. Confocal fluorescence microscopy of Ad-infected NM showed that pTP and CAD colocalized in nuclear foci. Both pTP and CAD were confirmed to colocalize with active sites of replication detected by bromodeoxyuridine incorporation. These data support the concept that the pTP-CAD interaction may allow anchorage of Ad replication complexes in the proximity of required cellular factors and may help to segregate replicated and unreplicated viral DNA. PMID:9525610

  15. Nuclear Degradation of Wilms Tumor 1-associating Protein and Survivin Splice Variant Switching Underlie IGF-1-mediated Survival*

    PubMed Central

    Small, Theodore W.; Pickering, J. Geoffrey

    2009-01-01

    WTAP (Wilms tumor 1-associating protein) is a recently identified nuclear protein that is essential for mouse embryo development. The Drosophila homolog of WTAP, Fl(2)d, regulates pre-mRNA splicing; however, the role of WTAP in mammalian cells is uncertain. To elucidate a context for WTAP action, we screened growth and survival factors for their effects on WTAP expression in vascular smooth muscle cells (SMCs), a cell type previously found to express WTAP dynamically. This revealed that insulin-like growth factor-1 (IGF-1) uniquely reduced WTAP abundance. This decline in WTAP proved to be necessary for IGF-1 to confer its antiapoptotic properties, which were blocked by transducing the WTAP gene into SMCs. WTAP down-regulation by IGF-1 was mediated by an IGF-1 receptor-phosphatidylinositol 3-kinase-Akt signaling axis that directed WTAP degradation via a nuclear 26 S proteasome. Moreover, by promoting the degradation of WTAP, IGF-1 shifted the pre-mRNA splicing program for the survival factor, survivin, to reduce expression of survivin-2B, which is proapoptotic, and increase expression of survivin, which is antiapoptotic. Knockdown of survivin-2B rescued the ability of IGF-1 to promote survival when WTAP was overexpressed. These data uncover a novel regulatory cascade for human SMC survival based on adjusting the nuclear abundance of WTAP to define the splice variant balance among survivin isoforms. PMID:19605357

  16. Electrostatic Energetics of Bacillus subtilis Ribonuclease P Protein Determined by Nuclear Magnetic Resonance-Based Histidine pKa Measurements.

    PubMed

    Mosley, Pamela L; Daniels, Kyle G; Oas, Terrence G

    2015-09-01

    The pKa values of ionizable groups in proteins report the free energy of site-specific proton binding and provide a direct means of studying pH-dependent stability. We measured histidine pKa values (H3, H22, and H105) in the unfolded (U), intermediate (I), and sulfate-bound folded (F) states of RNase P protein, using an efficient and accurate nuclear magnetic resonance-monitored titration approach that utilizes internal reference compounds and a parametric fitting method. The three histidines in the sulfate-bound folded protein have pKa values depressed by 0.21 ± 0.01, 0.49 ± 0.01, and 1.00 ± 0.01 units, respectively, relative to that of the model compound N-acetyl-l-histidine methylamide. In the unliganded and unfolded protein, the pKa values are depressed relative to that of the model compound by 0.73 ± 0.02, 0.45 ± 0.02, and 0.68 ± 0.02 units, respectively. Above pH 5.5, H22 displays a separate resonance, which we have assigned to I, whose apparent pKa value is depressed by 1.03 ± 0.25 units, which is ?0.5 units more than in either U or F. The depressed pKa values we observe are consistent with repulsive interactions between protonated histidine side chains and the net positive charge of the protein. However, the pKa differences between F and U are small for all three histidines, and they have little ionic strength dependence in F. Taken together, these observations suggest that unfavorable electrostatics alone do not account for the fact that RNase P protein is intrinsically unfolded in the absence of ligand. Multiple factors encoded in the P protein sequence account for its IUP property, which may play an important role in its function. PMID:26267651

  17. Electrostatic Energetics of Bacillus subtilis Ribonuclease P Protein Determined by Nuclear Magnetic Resonance-Based Histidine pKa Measurements

    PubMed Central

    Mosley, Pamela L.; Daniels, Kyle G.; Oas, Terrence G.

    2015-01-01

    The pKa values of ionizable groups in proteins report the free energy of site-specific proton binding and provide a direct means of studying pH-dependent stability. We measured histidine pKa values (H3, H22, and H105) in the unfolded (U), intermediate (I), and sulfate-bound folded (F) states of RNase P protein, using an efficient and accurate nuclear magnetic resonance-monitored titration approach that utilizes internal reference compounds and a parametric fitting method. The three histidines in the sulfate-bound folded protein have pKa values depressed by 0.21 ± 0.01, 0.49 ± 0.01, and 1.00 ± 0.01 units, respectively, relative to that of the model compound N-acetyl-L-histidine methylamide. In the unliganded and unfolded protein, the pKa values are depressed relative to that of the model compound by 0.73 ± 0.02, 0.45 ± 0.02, and 0.68 ± 0.02 units, respectively. Above pH 5.5, H22 displays a separate resonance, which we have assigned to I, whose apparent pKa value is depressed by 1.03 ± 0.25 units, which is ~0.5 units more than in either U or F. The depressed pKa values we observe are consistent with repulsive interactions between protonated histidine side chains and the net positive charge of the protein. However, the pKa differences between F and U are small for all three histidines, and they have little ionic strength dependence in F. Taken together, these observations suggest that unfavorable electrostatics alone do not account for the fact that RNase P protein is intrinsically unfolded in the absence of ligand. Multiple factors encoded in the P protein sequence account for its IUP property, which may play an important role in its function. PMID:26267651

  18. Protein

    MedlinePLUS

    ... Go lean with protein. • Choose lean meats and poultry. Lean beef cuts include round steaks (top loin, ... main dishes. • Use nuts to replace meat or poultry, not in addition to meat or poultry (i. ...

  19. Protein

    MedlinePLUS

    ... Fruits Fats and Cholesterol Types of Fat Cholesterol Dietary fat and disease Calcium and Milk Vitamins Healthy Drinks ... the high protein diet saw improvements in blood lipids and blood pressure. ( 11 ) A more recent study ...

  20. Interaction of nucleosome assembly proteins abolishes nuclear localization of DGK{zeta} by attenuating its association with importins

    SciTech Connect

    Okada, Masashi; Hozumi, Yasukazu; Ichimura, Tohru; Tanaka, Toshiaki; Hasegawa, Hiroshi; Yamamoto, Masakazu; Takahashi, Nobuya; Iseki, Ken; Yagisawa, Hitoshi; Shinkawa, Takashi; Isobe, Toshiaki; Goto, Kaoru

    2011-12-10

    Diacylglycerol kinase (DGK) is involved in the regulation of lipid-mediated signal transduction through the metabolism of a second messenger diacylglycerol. Of the DGK family, DGK{zeta}, which contains a nuclear localization signal, localizes mainly to the nucleus but translocates to the cytoplasm under pathological conditions. However, the detailed mechanism of translocation and its functional significance remain unclear. To elucidate these issues, we used a proteomic approach to search for protein targets that interact with DGK{zeta}. Results show that nucleosome assembly protein (NAP) 1-like 1 (NAP1L1) and NAP1-like 4 (NAP1L4) are identified as novel DGK{zeta} binding partners. NAP1Ls constitutively shuttle between the nucleus and the cytoplasm in transfected HEK293 cells. The molecular interaction of DGK{zeta} and NAP1Ls prohibits nuclear import of DGK{zeta} because binding of NAP1Ls to DGK{zeta} blocks import carrier proteins, Qip1 and NPI1, to interact with DGK{zeta}, leading to cytoplasmic tethering of DGK{zeta}. In addition, overexpression of NAP1Ls exerts a protective effect against doxorubicin-induced cytotoxicity. These findings suggest that NAP1Ls are involved in a novel molecular basis for the regulation of nucleocytoplasmic shuttling of DGK{zeta} and provide a clue to examine functional significance of its translocation under pathological conditions.

  1. The nuclear fraction of protein kinase CK2 binds to the upstream stimulatory factors (USFs) in the absence of DNA.

    PubMed

    Spohrer, Sarah; Dimova, Elitsa Y; Kietzmann, Thomas; Montenarh, Mathias; Götz, Claudia

    2016-02-01

    The functions of the upstream stimulatory factors USF1 and USF2 are, like those of other transcription factors, regulated by reversible phosphorylation. Besides many other kinases also protein kinase CK2 phosphorylates USF1 but not USF2. In a yeast-two-hybrid screen, however, the non-catalytic CK2? subunit of CK2 was identified as a binding partner of USF2. This surprising observation prompted us to investigate the CK2/USF interaction in more detail in the present study. By using immunofluorescence analyses as well as co-immunoprecipitations we found that USF1 and USF2 bound to CK2? and CK2? exclusively in the nucleus, though CK2? and to a minor amount CK2? were also present in the cytoplasm. Furthermore, we found that unlike other substrates the phosphorylation of USF1 required the presence of the regulatory CK2? subunit; the catalytic ?-subunit of CK2 alone was not able to phosphorylate USF1. Thus, the correct phosphorylation of USF1 is only guaranteed and strictly controlled in particular by nuclear CK2?. Although the data indicated that a nuclear subfraction of CK2 subunits associated with USF proteins, DNA pull down experiments revealed that the CK2 subunits did not co-localize with DNA bound USF proteins indicating that the USF/CK2 interaction has a pre- or post DNA binding function. PMID:26577526

  2. An Essential Nuclear Protein in Trypanosomes Is a Component of mRNA Transcription/Export Pathway

    PubMed Central

    Serpeloni, Mariana; Moraes, Carolina Borsoi; Muniz, João Renato Carvalho; Motta, Maria Cristina Machado; Ramos, Augusto Savio Peixoto; Kessler, Rafael Luis; Inoue, Alexandre Haruo; Duarte daRocha, Wanderson; Yamada-Ogatta, Sueli Fumie; Fragoso, Stenio Perdigão; Goldenberg, Samuel; Freitas-Junior, Lucio H.; Ávila, Andréa Rodrigues

    2011-01-01

    In eukaryotic cells, different RNA species are exported from the nucleus via specialized pathways. The mRNA export machinery is highly integrated with mRNA processing, and includes a different set of nuclear transport adaptors as well as other mRNA binding proteins, RNA helicases, and NPC-associated proteins. The protozoan parasite Trypanosoma cruzi is the causative agent of Chagas disease, a widespread and neglected human disease which is endemic to Latin America. Gene expression in Trypanosoma has unique characteristics, such as constitutive polycistronic transcription of protein-encoding genes and mRNA processing by trans-splicing. In general, post-transcriptional events are the major points for regulation of gene expression in these parasites. However, the export pathway of mRNA from the nucleus is poorly understood. The present study investigated the function of TcSub2, which is a highly conserved protein ortholog to Sub2/ UAP56, a component of the Transcription/Export (TREX) multiprotein complex connecting transcription with mRNA export in yeast/human. Similar to its orthologs, TcSub2 is a nuclear protein, localized in dispersed foci all over the nuclei —except the fibrillar center of nucleolus— and at the interface between dense and non-dense chromatin areas, proposing the association of TcSub2 with transcription/processing sites. These findings were analyzed further by BrUTP incorporation assays and confirmed that TcSub2 is physically associated with active RNA polymerase II (RNA pol II), but not RNA polymerase I (RNA pol I) or Spliced Leader (SL) transcription, demonstrating participation particularly in nuclear mRNA metabolism in T. cruzi. The double knockout of the TcSub2 gene is lethal in T. cruzi, suggesting it has an essential function. Alternatively, RNA interference assays were performed in Trypanosoma brucei. It allowed demonstrating that besides being an essential protein, its knockdown causes mRNA accumulation in the nucleus and decrease of translation levels, reinforcing that Trypanosoma-Sub2 (Tryp-Sub2) is a component of mRNA transcription/export pathway in trypanosomes. PMID:21687672

  3. Changes in nuclear and nucleolar protein content during the growth and differentiation of root parenchyma cells in plant species with different DNA-endoreplication dynamics.

    PubMed

    Marciniak, K; Bilecka, A

    1986-01-01

    Using cytophotometric procedures, we measured the nuclear and nucleolar protein content of successive zones of growth and differentiation in consecutive (1-7 mm) root segments obtained from eight species of the Angiospermae after staining the preparations with Feulgen-Naphthol Yellow S (F-NYS). In meristematic cells the nuclear and nucleolar protein content was found to double during the cell cycle. In species in which differentiation occurs at the same time as nuclear DNA endoreplication, i.e. Vicia faba subsp. minor, V. faba subsp. major, Pisum sativum, Hordeum vulgare and Amaryllis belladonna, the pool of nuclear proteins observed during the G2 phase of the cell cycle was seen in the differentiated zone in nuclei containing 8C DNA. Species in which differentiation is not accompanied by the process of nuclear DNA endoreplication, i.e. Levisticum officinale, Tulipa kaufmanniana and Haemanthus katharinae, exhibited the highest nuclear proteins content during the G2 phase of the cell cycle; comparably high values were not found in the differentiated zone. A decrease in nucleolar protein content was observed during the process of differentiation, this tendency being more evident in the studied species that do not exhibit endoreplication. PMID:3733472

  4. Nuclear Eg5 (kinesin spindle protein) expression predicts docetaxel response and prostate cancer aggressiveness

    PubMed Central

    Dezentjé, Vincent O.; Buijs, Jeroen T.; De Krijger, Ronald R.; Smit, Vincent T.H.B.M.; Van Weerden, Wytske M.; Gelderblom, Hans; van der Pluijm, Gabri

    2014-01-01

    Novel biomarkers predicting prostate cancer (PCa) aggressiveness and docetaxel therapy response of PCa patients are needed. In this study the correlation between nuclear Eg5-expression, PCa docetaxel response and PCa aggressiveness was assessed. Immunohistochemical staining for nuclear Eg5 was performed on 117 archival specimens from 110 PCa patients treated with docetaxel between 2004 and 2012. Samples were histologically categorized as positive/negative. Median follow-up time from diagnosis was 11.6 years. Nuclear Eg5-expression was significantly related to docetaxel response (p=0.036) in tissues acquired within three years before docetaxel initiation. Nuclear Eg5-expression was not related to Gleason-score (p=0.994). Survival of patients after docetaxel initiation did not differ based on nuclear Eg5-expression (p=0.540). Analyzing samples taken before hormonal therapy, overall survival and time to docetaxel use were significantly decreased in patients with nuclear Eg5-expressing tumors (p<0.01). Eg5-positive nuclei were found more frequently in T4-staged tumors (p=0.04), Gleason 8-10 tumors (p=0.08), and in metastasized tumors (p<0.01). Multivariate analyses indicated that nuclear Eg5-expression may be an independent parameter for tumor aggressiveness. Limitations of a retrospective analysis apply. In conclusion, nuclear Eg5-expression may be a predictive biomarker for docetaxel response in metastatic castrate-resistant PCa patients and a prognostic biomarker for hormone-naive PCa patients. Prospective validation studies are needed. PMID:25277178

  5. Nuclear Eg5 (kinesin spindle protein) expression predicts docetaxel response and prostate cancer aggressiveness.

    PubMed

    Wissing, Michel D; De Morrée, Ellen S; Dezentjé, Vincent O; Buijs, Jeroen T; De Krijger, Ronald R; Smit, Vincent T H B M; Van Weerden, Wytske M; Gelderblom, Hans; van der Pluijm, Gabri

    2014-09-15

    Novel biomarkers predicting prostate cancer (PCa) aggressiveness and docetaxel therapy response of PCa patients are needed. In this study the correlation between nuclear Eg5-expression, PCa docetaxel response and PCa aggressiveness was assessed. Immunohistochemical staining for nuclear Eg5 was performed on 117 archival specimens from 110 PCa patients treated with docetaxel between 2004 and 2012. Samples were histologically categorized as positive/negative. Median follow-up time from diagnosis was 11.6 years. Nuclear Eg5-expression was significantly related to docetaxel response (p=0.036) in tissues acquired within three years before docetaxel initiation. Nuclear Eg5-expression was not related to Gleason-score (p=0.994). Survival of patients after docetaxel initiation did not differ based on nuclear Eg5-expression (p=0.540). Analyzing samples taken before hormonal therapy, overall survival and time to docetaxel use were significantly decreased in patients with nuclear Eg5-expressing tumors (p<0.01). Eg5-positive nuclei were found more frequently in T4-staged tumors (p=0.04), Gleason 8-10 tumors (p=0.08), and in metastasized tumors (p<0.01). Multivariate analyses indicated that nuclear Eg5-expression may be an independent parameter for tumor aggressiveness. Limitations of a retrospective analysis apply. In conclusion, nuclear Eg5-expression may be a predictive biomarker for docetaxel response in metastatic castrate-resistant PCa patients and a prognostic biomarker for hormone-naive PCa patients. Prospective validation studies are needed. PMID:25277178

  6. Fibrillarin, a nucleolar protein, is required for normal nuclear morphology and cellular growth in HeLa cells

    SciTech Connect

    Amin, Mohammed Abdullahel; Matsunaga, Sachihiro; Ma, Nan; Takata, Hideaki; Yokoyama, Masami; Uchiyama, Susumu; Fukui, Kiichi . E-mail: kfukui@bio.eng.osaka-u.ac.jp

    2007-08-24

    Fibrillarin is a key small nucleolar protein in eukaryotes, which has an important role in pre-rRNA processing during ribosomal biogenesis. Though several functions of fibrillarin are known, its function during the cell cycle is still unknown. In this study, we confirmed the dynamic localization of fibrillarin during the cell cycle of HeLa cells and also performed functional studies by using a combination of immunofluorescence microscopy and RNAi technique. We observed that depletion of fibrillarin has almost no effect on the nucleolar structure. However, fibrillarin-depleted cells showed abnormal nuclear morphology. Moreover, fibrillarin depletion resulted in the reduction of the cellular growth and modest accumulation of cells with 4n DNA content. Our data suggest that fibrillarin would play a critical role in the maintenance of nuclear shape and cellular growth.

  7. Proton nuclear magnetic resonance assignments and secondary structure determination of the Co1E1 rop (rom) protein

    SciTech Connect

    Eberle, W. European Molecular Biology Lab., Heidelberg ); Klaus, W. ); Cesareni, G. ); Sander, C. ); Roesch, P. )

    1990-08-14

    The complete resonance assignment of the Co1E1 rop (rom) protein at pH 2.3 was obtained by two-dimensional (2D) proton nuclear magnetic resonance spectroscopy ({sup 1}H NMR) at 500 and 600 MHz using through-bond and through-space connectivities. Sequential assignments and elements of regular secondary structure were deduced by analysis of nuclear Overhauser enhancement spectroscopy (NOESY) experiments and {sup 3}J{sub HN{alpha}} coupling constants. One 7.2-kDa monomer of the homodimer consists of two antiparallel helices connected by a hairpin loop at residue 31. The C-terminal peptide consisting of amino acids 59-63 shows no stable conformation. The dimer forms a four-helix bundle with opposite polarization of neighboring elements in agreement with the x-ray structure.

  8. Differential level of DSB repair fidelity effected by nuclear protein extracts derived from radiosensitive and radioresistant human tumour cells.

    PubMed Central

    Britten, R. A.; Liu, D.; Kuny, S.; Allalunis-Turner, M. J.

    1997-01-01

    A cell-free plasmid reactivation assay was used to determine the fidelity of DNA double-strand break (DSB) repair in a panel of eight DSB repair-proficient human tumour cell lines. Nuclear protein extracts derived from radiosensitive tumour cells were less capable of correctly rejoining EcoRI-induced DSBs than were similar extracts from radioresistant tumour cells. Linear regression analysis suggests that there was a significant (r2 = 0.84, P = 0.001, d.f. = 6) correlation between the fidelity of DSB rejoining and the SF2 values of the cell lines studied. This cell-free assay is clearly sensitive to differences in the nuclear protein composition that reflect the clinically relevant radiosensitivity of these cell lines. The fact that our cell-free assay yielded similar results to previous studies that used intracellular plasmid reactivation assays suggests that those differences in DSB mis-rejoining frequencies in radiosensitive and radioresistant cell lines may be due to inherent differences in nuclear protein composition and are not directly attributable to differences in proliferation rates between cell lines. The underlying cause for this association between DSB mis-rejoining frequencies and radiosensitivity is presently unknown, however restriction endonuclease mapping and polymerase chain reaction (PCR) amplification analysis revealed that approximately 40% of the mis-rejoined DSBs arose as a result of the deletion of between 40 and 440 base pairs. These data raise the possibility that the radiosensitivity of DSB repair-proficient human tumour cell lines may be partly determined by the predisposition of these cell lines to activate non-conservative DSB rejoining pathways. PMID:9400940

  9. A nuclear localization of the infectious haematopoietic necrosis virus NV protein is necessary for optimal viral growth

    USGS Publications Warehouse

    Choi, M.K.; Moon, C.H.; Ko, M.S.; Lee, U.-H.; Cho, W.J.; Cha, S.J.; Do, J.W.; Heo, G.J.; Jeong, S.G.; Hahm, Y.S.; Harmache, A.; Bremont, M.; Kurath, G.; Park, J.-W.

    2011-01-01

    The nonvirion (NV) protein of infectious hematopoietic necrosis virus (IHNV) has been previously reported to be essential for efficient growth and pathogenicity of IHNV. However, little is known about the mechanism by which the NV supports the viral growth. In this study, cellular localization of NV and its role in IHNV growth in host cells was investigated. Through transient transfection in RTG-2 cells of NV fused to green fluorescent protein (GFP), a nuclear localization of NV was demonstrated. Deletion analyses showed that the 32EGDL35 residues were essential for nuclear localization of NV protein, and fusion of these 4 amino acids to GFP directed its transport to the nucleus. We generated a recombinant IHNV, rIHNV-NV-?EGDL in which the 32EGDL35 was deleted from the NV. rIHNVs with wild-type NV (rIHNV-NV) or with the NV gene replaced with GFP (rIHNV-?NV-GFP) were used as controls. RTG-2 cells infected with rIHNV-?NV-GFP and rIHNV-NV-?EGDL yielded 12- and 5-fold less infectious virion, respectively, than wild type rIHNV-infected cells at 48 h post-infection (p.i.). While treatment with poly I:C at 24 h p.i. did not inhibit replication of wild-type rIHNVs, replication rates of rIHNV-?NV-GFP and rIHNV-NV-?EGDL were inhibited by poly I:C. In addition, both rIHNV-?NV and rIHNV-NV-?EGDL induced higher levels of expressions of both IFN1 and Mx1 than wild-type rIHNV. These data suggest that the IHNV NV may support the growth of IHNV through inhibition of the INF system and the amino acid residues of 32EGDL35 responsible for nuclear localization are important for the inhibitory activity of NV.

  10. Nuclear Calcium/Calmodulin-dependent Protein Kinase II Signaling Enhances Cardiac Progenitor Cell Survival and Cardiac Lineage Commitment.

    PubMed

    Quijada, Pearl; Hariharan, Nirmala; Cubillo, Jonathan D; Bala, Kristin M; Emathinger, Jacqueline M; Wang, Bingyan J; Ormachea, Lucia; Bers, Donald M; Sussman, Mark A; Poizat, Coralie

    2015-10-16

    Ca(2+)/Calmodulin-dependent protein kinase II (CaMKII) signaling in the heart regulates cardiomyocyte contractility and growth in response to elevated intracellular Ca(2+). The ?B isoform of CaMKII is the predominant nuclear splice variant in the adult heart and regulates cardiomyocyte hypertrophic gene expression by signaling to the histone deacetylase HDAC4. However, the role of CaMKII? in cardiac progenitor cells (CPCs) has not been previously explored. During post-natal growth endogenous CPCs display primarily cytosolic CaMKII?, which localizes to the nuclear compartment of CPCs after myocardial infarction injury. CPCs undergoing early differentiation in vitro increase levels of CaMKII?B in the nuclear compartment where the kinase may contribute to the regulation of CPC commitment. CPCs modified with lentiviral-based constructs to overexpress CaMKII?B (CPCe?B) have reduced proliferative rate compared with CPCs expressing eGFP alone (CPCe). Additionally, stable expression of CaMKII?B promotes distinct morphological changes such as increased cell surface area and length of cells compared with CPCe. CPCe?B are resistant to oxidative stress induced by hydrogen peroxide (H2O2) relative to CPCe, whereas knockdown of CaMKII?B resulted in an up-regulation of cell death and cellular senescence markers compared with scrambled treated controls. Dexamethasone (Dex) treatment increased mRNA and protein expression of cardiomyogenic markers cardiac troponin T and ?-smooth muscle actin in CPCe?B compared with CPCe, suggesting increased differentiation. Therefore, CaMKII?B may serve as a novel modulatory protein to enhance CPC survival and commitment into the cardiac and smooth muscle lineages. PMID:26324717

  11. Transcriptome analysis reveals nuclear-encoded proteins for the maintenance of temporary plastids in the dinoflagellate Dinophysis acuminata

    PubMed Central

    2010-01-01

    Background Dinophysis is exceptional among dinoflagellates, possessing plastids derived from cryptophyte algae. Although Dinophysis can be maintained in pure culture for several months, the genus is mixotrophic and needs to feed either to acquire plastids (a process known as kleptoplastidy) or obtain growth factors necessary for plastid maintenance. Dinophysis does not feed directly on cryptophyte algae, but rather on a ciliate (Myrionecta rubra) that has consumed the cryptophytes and retained their plastids. Despite the apparent absence of cryptophyte nuclear genes required for plastid function, Dinophysis can retain cryptophyte plastids for months without feeding. Results To determine if this dinoflagellate has nuclear-encoded genes for plastid function, we sequenced cDNA from Dinophysis acuminata, its ciliate prey M. rubra, and the cryptophyte source of the plastid Geminigera cryophila. We identified five proteins complete with plastid-targeting peptides encoded in the nuclear genome of D. acuminata that function in photosystem stabilization and metabolite transport. Phylogenetic analyses show that the genes are derived from multiple algal sources indicating some were acquired through horizontal gene transfer. Conclusions These findings suggest that D. acuminata has some functional control of its plastid, and may be able to extend the useful life of the plastid by replacing damaged transporters and protecting components of the photosystem from stress. However, the dearth of plastid-related genes compared to other fully phototrophic algae suggests that D. acuminata does not have the nuclear repertoire necessary to maintain the plastid permanently. PMID:20537123

  12. Nucleoporin 62-Like Protein Activates Canonical Wnt Signaling through Facilitating the Nuclear Import of ?-Catenin in Zebrafish

    PubMed Central

    Yang, Xiaojie; Gu, Qilin; Lin, Li; Li, Shaoyang; Zhong, Shan

    2015-01-01

    Nucleoporin p62 (Nup62) localizes in the central channel of nuclear pore complexes (NPCs) and regulates nuclear pore permeability and nucleocytoplasmic transport. However, the developmental roles of Nup62 in vertebrates remain largely unclear. Zebrafish Nup62-like protein (Nup62l) is a homolog of mammalian Nup62. The nup62l gene is maternally expressed, but its transcripts are ubiquitously distributed during early embryogenesis and enriched in the head, pharynx, and intestine of developing embryos. Activation of the Wnt/?-catenin pathway positively modulates nup62l transcription, while Bmp signaling acts downstream of Wnt/?-catenin signaling to negatively regulate nup62l expression. Overexpression of nup62l dorsalized embryos and enhanced gastrula convergence and extension (CE) movements. In contrast, knockdown of Nup62l led to ventralized embryos, an impediment to CE movements, and defects in specification of midline organ progenitors. Mechanistically, Nup62l acts as an activator of Wnt/?-catenin signaling through interaction with and facilitation of nuclear import of ?-catenin-1/2 in zebrafish. Thus, Nup62l regulates dorsoventral patterning, gastrula CE movements, and proper specification of midline organ precursors through mediating the nuclear import of ?-catenins in zebrafish. PMID:25605329

  13. The Dedicated Chaperone Acl4 Escorts Ribosomal Protein Rpl4 to Its Nuclear Pre-60S Assembly Site.

    PubMed

    Pillet, Benjamin; García-Gómez, Juan J; Pausch, Patrick; Falquet, Laurent; Bange, Gert; de la Cruz, Jesús; Kressler, Dieter

    2015-10-01

    Ribosomes are the highly complex macromolecular assemblies dedicated to the synthesis of all cellular proteins from mRNA templates. The main principles underlying the making of ribosomes are conserved across eukaryotic organisms and this process has been studied in most detail in the yeast Saccharomyces cerevisiae. Yeast ribosomes are composed of four ribosomal RNAs (rRNAs) and 79 ribosomal proteins (r-proteins). Most r-proteins need to be transported from the cytoplasm to the nucleus where they get incorporated into the evolving pre-ribosomal particles. Due to the high abundance and difficult physicochemical properties of r-proteins, their correct folding and fail-safe targeting to the assembly site depends largely on general, as well as highly specialized, chaperone and transport systems. Many r-proteins contain universally conserved or eukaryote-specific internal loops and/or terminal extensions, which were shown to mediate their nuclear targeting and association with dedicated chaperones in a growing number of cases. The 60S r-protein Rpl4 is particularly interesting since it harbours a conserved long internal loop and a prominent C-terminal eukaryote-specific extension. Here we show that both the long internal loop and the C-terminal eukaryote-specific extension are strictly required for the functionality of Rpl4. While Rpl4 contains at least five distinct nuclear localization signals (NLS), the C-terminal part of the long internal loop associates with a specific binding partner, termed Acl4. Absence of Acl4 confers a severe slow-growth phenotype and a deficiency in the production of 60S subunits. Genetic and biochemical evidence indicates that Acl4 can be considered as a dedicated chaperone of Rpl4. Notably, Acl4 localizes to both the cytoplasm and nucleus and it has the capacity to capture nascent Rpl4 in a co-translational manner. Taken together, our findings indicate that the dedicated chaperone Acl4 accompanies Rpl4 from the cytoplasm to its pre-60S assembly site in the nucleus. PMID:26447800

  14. The Dedicated Chaperone Acl4 Escorts Ribosomal Protein Rpl4 to Its Nuclear Pre-60S Assembly Site

    PubMed Central

    Pillet, Benjamin; García-Gómez, Juan J.; Pausch, Patrick; Falquet, Laurent; Bange, Gert; de la Cruz, Jesús; Kressler, Dieter

    2015-01-01

    Ribosomes are the highly complex macromolecular assemblies dedicated to the synthesis of all cellular proteins from mRNA templates. The main principles underlying the making of ribosomes are conserved across eukaryotic organisms and this process has been studied in most detail in the yeast Saccharomyces cerevisiae. Yeast ribosomes are composed of four ribosomal RNAs (rRNAs) and 79 ribosomal proteins (r-proteins). Most r-proteins need to be transported from the cytoplasm to the nucleus where they get incorporated into the evolving pre-ribosomal particles. Due to the high abundance and difficult physicochemical properties of r-proteins, their correct folding and fail-safe targeting to the assembly site depends largely on general, as well as highly specialized, chaperone and transport systems. Many r-proteins contain universally conserved or eukaryote-specific internal loops and/or terminal extensions, which were shown to mediate their nuclear targeting and association with dedicated chaperones in a growing number of cases. The 60S r-protein Rpl4 is particularly interesting since it harbours a conserved long internal loop and a prominent C-terminal eukaryote-specific extension. Here we show that both the long internal loop and the C-terminal eukaryote-specific extension are strictly required for the functionality of Rpl4. While Rpl4 contains at least five distinct nuclear localization signals (NLS), the C-terminal part of the long internal loop associates with a specific binding partner, termed Acl4. Absence of Acl4 confers a severe slow-growth phenotype and a deficiency in the production of 60S subunits. Genetic and biochemical evidence indicates that Acl4 can be considered as a dedicated chaperone of Rpl4. Notably, Acl4 localizes to both the cytoplasm and nucleus and it has the capacity to capture nascent Rpl4 in a co-translational manner. Taken together, our findings indicate that the dedicated chaperone Acl4 accompanies Rpl4 from the cytoplasm to its pre-60S assembly site in the nucleus. PMID:26447800

  15. The Nuclear Pore Complex Function of Sec13 Protein Is Required for Cell Survival during Retinal Development*

    PubMed Central

    Niu, Xubo; Hong, Jian; Zheng, Xiaofeng; Melville, David B.; Knapik, Ela W.; Meng, Anming; Peng, Jinrong

    2014-01-01

    Sec13 is a dual function protein, being a core component of both the COPII coat, which mediates protein trafficking from the endoplasmic reticulum to the Golgi apparatus, and the nuclear pore complex (NPC), which facilitates nucleo-cytoplasmic traffic. Here, we present a genetic model to differentiate the roles of these two functions of Sec13 in vivo. We report that sec13sq198 mutant embryos develop small eyes that exhibit disrupted retinal lamination and that the mutant retina contains an excessive number of apoptotic cells. Surprisingly, we found that loss of COPII function by oligonucleotide-mediated gene knockdown of sec31a and sec31b or brefeldin A treatment did not disrupt retinal lamination, although it did result in digestive organ defects similar to those seen in sec13sq198, suggesting that the digestive organ defects observed in sec13sq198 are due to loss of COPII function, whereas the retinal lamination defects are due to loss of the NPC function. We showed that the retinal cells of sec13sq198 failed to form proper nuclear pores, leading to a nuclear accumulation of total mRNA and abnormal activation of the p53-dependent apoptosis pathway, causing the retinal defect in sec13sq198. Furthermore, we found that a mutant lacking Nup107, a key NPC-specific component, phenocopied the retinal lamination phenotype as observed in sec13sq198. Our results demonstrate a requirement for the nuclear pore function of Sec13 in development of the retina and provide the first genetic evidence to differentiate the contributions of the NPC and the COPII functions of Sec13 during organogenesis. PMID:24627485

  16. In Vitro Interaction of Nuclear Proteins with the Promoter of Soybean Heat Shock Gene Gmhsp17.5E1

    PubMed Central

    Czarnecka, Eva; Fox, Paul C.; Gurley, William B.

    1990-01-01

    Proteins present in crude nuclear extracts of soybean (Glycine max) plumules were shown to bind in vitro to the 5? flanking sequences of the soybean heat shock gene Gmhsp17.5E. The specificity of binding activity present in extracts from both control (28°C) and heat shocked (40°C) tissues was demonstrated by reciprocal competition experiments using gel mobility retardation assays. Footprinting experiments using DNase I with crude nuclear extracts indicated that a continuous stretch of 5? flanking sequences extending from ?40 to ?153 was protected from digestion in vitro. Nuclear proteins that were partially purified by heparin agarose chromatography were shown to bind specific TATA-proximal sequences containing the heat shock consensus elements (HSEs) (?73 to ?49; ?107 to ?84) and AT-rich motifs (?119 to ?153). Other binding sites within AT-rich sequences (?906 to ?888, ?868 to 863, ?859 to 853, and ?841 to ?830), distal HSE elements (?568 to ?532) and a TATA/dyad (?234 to ?207) were also identified by DNase I footprinting of TATA-distal probes. DNA binding activities specific for the HSE and AT-rich sequences were present in nuclear extracts from both control and heat shocked tissues. Both types of binding activity were increased after heat shock treatment; HSE binding increased from 1.8- to 2.7-fold, and binding to AT-rich sequences showed an increase from 1.3- to 1.7-fold. Images Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 PMID:16667874

  17. Determining the DUF55-domain structure of human thymocyte nuclear protein 1 from crystals partially twinned by tetartohedry

    SciTech Connect

    Yu, Feng; Song, Aixin; Xu, Chunyan; Sun, Lihua; Li, Jian; Tang, Lin; Yu, Minmin; Yeates, Todd O.; Hu, Hongyu; He, Jianhua

    2009-06-06

    Human thymocyte nuclear protein 1 (hTHYN1) contains a unique DUF55 domain of 167 residues (55-221), but its cellular function is unclear. Crystals of DUF55 belong to the trigonal space group P3{sub 1}, but twinning causes the data to approach an apparent 622 symmetry. Two datasets to 2.3 {angstrom} resolution were collected. Statistical analysis confirmed that both datasets were partially twinned by tetartohedry. Tetartohedral twin fractions were estimated. After the structure was determined, only one twofold axis of rotational pseudosymmetry was found in the crystal structure. Using the DALI program, a YTH domain, which is a potential RNA binding domain from human YTH domain-containing protein 2, was identified to have the most similar three-dimensional fold to DUF55. It is implied that DUF55 might be a potential RNA-related domain.

  18. Polymorphisms of the HNF1A Gene Encoding Hepatocyte Nuclear Factor-1? are Associated with C-Reactive Protein

    PubMed Central

    Reiner, Alexander P.; Barber, Mathew J.; Guan, Yongtao; Ridker, Paul M.; Lange, Leslie A.; Chasman, Daniel I.; Walston, Jeremy D.; Cooper, Gregory M.; Jenny, Nancy S.; Rieder, Mark J.; Durda, J. Peter; Smith, Joshua D.; Novembre, John; Tracy, Russell P.; Rotter, Jerome I.; Stephens, Matthew; Nickerson, Deborah A.; Krauss, Ronald M.

    2008-01-01

    Data from the Pharmacogenomics and Risk of Cardiovascular Disease (PARC) study and the Cardiovascular Health Study (CHS) provide independent and confirmatory evidence for association between common polymorphisms of the HNF1A gene encoding hepatocyte nuclear factor-1? and plasma C-reactive protein (CRP) concentration. Analyses with the use of imputation-based methods to combine genotype data from both studies and to test untyped SNPs from the HapMap database identified several SNPs within a 5 kb region of HNF1A intron 1 with the strongest evidence of association with CRP phenotype. PMID:18439552

  19. Expression of nuclear matrix proteins binding matrix attachment regions in prostate cancer. PARP-1: New player in tumor progression.

    PubMed

    Barboro, Paola; Ferrari, Nicoletta; Capaia, Matteo; Petretto, Andrea; Salvi, Sandra; Boccardo, Simona; Balbi, Cecilia

    2015-10-01

    Prostate cancer (PCa) displays infrequent point mutations, whereas genomic rearrangements are highly prevalent. In eukaryotes, the genome is compartmentalized into chromatin loop domains by the attachment to the nuclear matrix (NM), and it has been demonstrated that several recombination hot spots are situated at the base of loops. Here, we have characterized the binding between NM proteins and matrix attachment regions (MARs) in PCa. Nontumor and 44 PCa tissues were analyzed. More aggressive tumors were characterized by an increase in the complexity of the NM protein patterns that was synchronous with a decrease in the number of proteins binding the MAR sequences. PARP-1 was the protein that showed the most evident changes. The expression of the PARP-1 associated with NM increased and it was dependent on tumor aggressiveness. Immunohistochemical analysis showed that the protein was significantly overexpressed in tumor cells. To explore the role of PARP-1 in PCa progression, PCa cells were treated with the PARP inhibitor, ABT-888. In androgen-independent PC3 cells, PARP inhibition significantly decreased cell viability, migration, invasion, chromatin loop dimensions and histone acetylation. Collectively, our study provides evidence that MAR-binding proteins are involved in the development and progression of PCa. PARP could play a key role in the compartmentalization of chromatin and in the development of the more aggressive phenotype. Thus, PARP can no longer be viewed only as an enzyme involved in DNA repair, but that its role in chromatin modulation could provide the basis for a new therapeutic approach to the treatment of PCa. PMID:25808111

  20. Separation and identification of differentially expressed nuclear matrix proteins between human esophageal immortalized and carcinomatous cell lines

    PubMed Central

    Xiong, Xing-Dong; Li, En-Min; Xu, Li-Yan; Chen, Hai-Bin; Chen, Ling; Cai, Wei-Jia; Han, Ya-Li; Shen, Zhong-Ying; Zeng, Yi

    2003-01-01

    AIM: To separate and identify differentially expressed nuclear matrix proteins (NMPs) between the immortalized human esophageal epithelial cell line (SHEE) and the malignantly transformed esophageal carcinoma cell line (SHEEC), and to provide new ways for finding specific markers and the pathogenesis of esophageal carcinoma. METHODS: SHEE and SHEEC cell lines were used to extract NMPs. The quality of NMPs was monitored by Western blot analysis including DNA topoisomerase II?, proliferation cell nuclear antigen (PCNA) and histone. NMPs of SHEE and SHEEC were analyzed by two-dimensional electrophoresis (2-DE), silver staining and PDQuest6.2 image analysis software. Three spots in which the differentially expressed NMPs were more obvious, were selected and analyzed with matrix-assisted laser desorption/ionization time of flying mass spectrometry (MALDI-TOF-MS) and database search. RESULTS: Western blot analysis revealed that DNA topoisomerase II? and PCNA were detected, and the majority of histones were deleted in NMPs of SHEE and SHEEC. After 2-DE image analysis by PDQuest6.2 software, the 2-DE maps were detected with an average of 106 ± 7.1 spots in SHEE and 132 ± 5.0 spots in SHEEC. Most of them were matched one another (r = 0.72), only 16 protein spots were found differing in intensity. Three NMPs including cytoskeletal tropomyosin, FK506-binding protein 6, similar to retinoblastoma binding protein 8 were preliminarily identified by MALDI- TOF-MS. CONCLUSION: These differentially expressed NMPs may play an important role during malignant transformation from SHEE to SHEEC. Their separation and identification will contribute to searching for specific markers and probing into the pathogenesis of esophageal carcinoma. PMID:14562366

  1. Identification of two human nuclear proteins that recognise the cytosine-rich strand of human telomeres in vitro

    PubMed Central

    Lacroix, Laurent; Liénard, Hélène; Labourier, Emmanuel; Djavaheri-Mergny, Mojgan; Lacoste, Jérôme; Leffers, Henrik; Tazi, Jamal; Hélène, Claude; Mergny, Jean-Louis

    2000-01-01

    Most studies on the structure of DNA in telomeres have been dedicated to the double-stranded region or the guanosine-rich strand and consequently little is known about the factors that may bind to the telomere cytosine-rich (C-rich) strand. This led us to investigate whether proteins exist that can recognise C-rich sequences. We have isolated several nuclear factors from human cell extracts that specifically bind the C-rich strand of vertebrate telomeres [namely a d(CCCTAA)n repeat] with high affinity and bind double-stranded telomeric DNA with a 100× reduced affinity. A biochemical assay allowed us to characterise four proteins of apparent molecular weights 66–64, 45 and 35 kDa, respectively. To identify these polypeptides we screened a ?gt11-based cDNA expression library, obtained from human HeLa cells using a radiolabelled telomeric oligonucleotide as a probe. Two clones were purified and sequenced: the first corresponded to the hnRNP K protein and the second to the ASF/SF2 splicing factor. Confirmation of the screening results was obtained with recombinant proteins, both of which bind to the human telomeric C-rich strand in vitro. PMID:10710423

  2. Cmr1/WDR76 defines a nuclear genotoxic stress body linking genome integrity and protein quality control

    PubMed Central

    Gallina, Irene; Colding, Camilla; Henriksen, Peter; Beli, Petra; Nakamura, Kyosuke; Offman, Judith; Mathiasen, David P.; Silva, Sonia; Hoffmann, Eva; Groth, Anja; Choudhary, Chunaram; Lisby, Michael

    2015-01-01

    DNA replication stress is a source of genomic instability. Here we identify changed mutation rate 1 (Cmr1) as a factor involved in the response to DNA replication stress in Saccharomyces cerevisiae and show that Cmr1—together with Mrc1/Claspin, Pph3, the chaperonin containing TCP1 (CCT) and 25 other proteins—define a novel intranuclear quality control compartment (INQ) that sequesters misfolded, ubiquitylated and sumoylated proteins in response to genotoxic stress. The diversity of proteins that localize to INQ indicates that other biological processes such as cell cycle progression, chromatin and mitotic spindle organization may also be regulated through INQ. Similar to Cmr1, its human orthologue WDR76 responds to proteasome inhibition and DNA damage by relocalizing to nuclear foci and physically associating with CCT, suggesting an evolutionarily conserved biological function. We propose that Cmr1/WDR76 plays a role in the recovery from genotoxic stress through regulation of the turnover of sumoylated and phosphorylated proteins. PMID:25817432

  3. Immunolocalization in three dimensions: immunogold staining of cytoskeletal and nuclear matrix proteins in resinless electron microscopy sections.

    PubMed Central

    Nickerson, J A; Krockmalnic, G; He, D C; Penman, S

    1990-01-01

    We describe two methods for staining resinless thin sections with antibodies and gold-conjugated second antibodies. Immunolocalization of specific proteins is a powerful tool for cell structure studies but current techniques do not develop its full potential. Immunofluorescence provides only low-resolution localization, whereas conventional thin-section electron microscopy images and immunostains only the section surface. Resinless sections of extracted cell structures offer a simple and effective means of immuno-electron microscopy. Without embedding plastic or soluble proteins, the cell cytostructure produces high-contrast, three-dimensional images. Resinless sections of detergent-extracted cells are prepared by embedding in diethylene glycol distearate, sectioning, and removing diethylene glycol distearate before microscopy. In the first method of immunostaining, extracted cells were fixed and stained with antibodies before embedment, sectioning, removal of the embedding resin, and critical point drying. In the postembedment method, the sample was embedded and sectioned, the diethylene glycol distearate was removed, and the sample was rehydrated before antibody staining. With these techniques, specific proteins were localized with high resolution throughout the entire section. Stereoscopic micrographs of resinless sections revealed the precise localization of specific cytoskeleton and nuclear matrix proteins in three dimensions with unprecedented clarity. Images PMID:2315318

  4. Structures and Metal-Binding Properties of Helicobacter pylori Neutrophil-Activating Protein with a Di-Nuclear Ferroxidase Center

    PubMed Central

    Yokoyama, Hideshi; Fujii, Satoshi

    2014-01-01

    Helicobacter pylori causes severe diseases, such as chronic gastritis, peptic ulcers, and stomach cancers. H. pylori neutrophil-activating protein (HP-NAP) is an iron storage protein that forms a dodecameric shell, promotes the adhesion of neutrophils to endothelial cells, and induces the production of reactive oxygen radicals. HP-NAP belongs to the DNA-protecting proteins under starved conditions (Dps) family, which has significant structural similarities to the dodecameric ferritin family. The crystal structures of the apo form and metal-ion bound forms, such as iron, zinc, and cadmium, of HP-NAP have been determined. This review focused on the structures and metal-binding properties of HP-NAP. These metal ions bind at the di-nuclear ferroxidase center (FOC) by different coordinating patterns. In comparison with the apo structure, metal loading causes a series of conformational changes in conserved residues among HP-NAP and Dps proteins (Trp26, Asp52, and Glu56) at the FOC. HP-NAP forms a spherical dodecamer with 23 symmetry including two kinds of pores. Metal ions have been identified around one of the pores; therefore, the negatively-charged pore is suitable for the passage of metal ions. PMID:24971723

  5. A cell free protein fragment complementation assay for monitoring the core interaction of the human cytomegalovirus nuclear egress complex.

    PubMed

    Schnee, Margit; Wagner, Felicia M; Koszinowski, Ulrich H; Ruzsics, Zsolt

    2012-07-01

    Certain viral protein-protein interactions provide attractive targets for antiviral drug development. Recently, we described a ?-lactamase based protein fragment complementation assay (PCA) to study the core interaction of the nuclear egress complex (NEC) of different herpesviruses in cells. Now, to have a cell free assay for inhibitor screens, we expressed split ?-lactamase tagged interaction domains of the viral pUL50 and pUL53 proteins representing the NEC of human cytomegalovirus (HCMV) in bacteria. After validation and basic characterization of this NEC-PCA, we tested peptide inhibitors of the pUL50-pUL53 complex. We show that peptides resembling sequences of the first conserved region of pUL53 can inhibit the NEC-PCA. This, on one hand, indicated that the core interaction in the HCMV NEC is mediated by a linear motif. On the other hand it proved that this new pUL50-pUL53 interaction assay allows a simple cell free test for small molecular inhibitors. PMID:22580129

  6. Phosphorus nuclear magnetic resonance studies of lipid-protein interactions: Human erythrocyte glycophorin and phospholipids

    SciTech Connect

    Yeagle, P.L.; Kelsey, D. )

    1989-03-07

    Human erythrocyte glycophorin containing four molecules of phospholipid tightly bound to the protein was isolated from human red cell ghosts. This protein preparation was reconstituted into a digalactosyl diglyceride bilayer. The {sup 31}P NMR spectrum of this reconstituted membrane produced an axially symmetric powder pattern arising exclusively from the phospholipids bound to glycophorin. The width of the powder pattern, about 90 ppm, is about twice as broad as that normally exhibited by a phospholipid bilayer. The chemical shift tensor is perturbed relative to phospholipids in a bilayer. The spin-lattice relaxation rate of these protein-bound phospholipids is found to be nearly an order of magnitude faster than phospholipids in a bilayer. The results are consistent with phospholipids tightly bound to the membrane protein and undergoing rotational diffusion, perhaps as a complex of phospholipid and protein.

  7. The eukaryotic P loop NTPase Nbp35: an essential component of the cytosolic and nuclear iron-sulfur protein assembly machinery.

    PubMed

    Hausmann, Anja; Aguilar Netz, Daili J; Balk, Janneke; Pierik, Antonio J; Mühlenhoff, Ulrich; Lill, Roland

    2005-03-01

    Soluble P loop NTPases represent a large protein family and are involved in diverse cellular functions. Here, we functionally characterized the first member of the Mrp/Nbp35 subbranch of this family, the essential Nbp35p of Saccharomyces cerevisiae. The protein resides in the cytosol and nucleus and carries an Fe/S cluster at its N terminus. Assembly of the Fe/S cluster requires the mitochondrial Fe/S cluster (ISC)-assembly and -export machineries. Depletion of Nbp35p strongly impairs the activity of the cytosolic Fe/S protein, isopropylmalate isomerase (Leu1p), whereas mitochondrial Fe/S enzymes are unaffected. Moreover, defects in the de novo maturation of various cytosolic and nuclear Fe/S proteins were observed in the absence of Nbp35p, demonstrating the functional involvement of Nbp35p in the biogenesis of extramitochondrial Fe/S proteins. Furthermore, Nbp35p genetically interacts with the closely similar P loop NTPase, Cfd1p, and the hydrogenase-like Nar1p, both of which were recently shown to perform a crucial function in cytosolic and nuclear Fe/S protein biogenesis. Hence, our study suggests that eukaryotic Nbp35 NTPases function in Fe/S protein maturation. The findings provide strong evidence for the existence of a highly conserved and essential machinery dedicated to assembling cytosolic and nuclear Fe/S proteins. PMID:15728363

  8. Nuclear export of S6K1 II is regulated by protein kinase CK2 phosphorylation at Ser-17.

    PubMed

    Panasyuk, Ganna; Nemazanyy, Ivan; Zhyvoloup, Alexander; Bretner, Maria; Litchfield, David W; Filonenko, Valeriy; Gout, Ivan T

    2006-10-20

    Ribosomal S6 kinases (S6Ks) are principal players in the regulation of cell growth and energy metabolism. Signaling via phosphatidylinositol 3-kinase and mammalian target of rapamycin pathways mediates the activation of S6K in response to various mitogenic stimuli. The family of S6Ks consists of two forms, S6K1 and -2, that have cytoplasmic and nuclear splicing variants, S6K1 II and S6K1 I, respectively. Nuclear-cytoplasmic shuttling of both isoforms induced by mitogenic stimuli has been reported recently. Here we present the identification of protein kinase CK2 (CK2) as a novel binding and regulatory partner for S6K1 II. The interaction between S6K1 II and CK2beta regulatory subunit was initially identified in a yeast two-hybrid screen and further confirmed by co-immunoprecipitation of transiently expressed and endogenous proteins. The interaction between S6K1 II and CK2 was found to occur in serum-starved and serum-stimulated cells. In addition, we found that S6K1 II is a substrate for CK2. The localization of the CK2 phosphorylation site was narrowed down to Ser-17 in S6K1 II. Mutational analysis and the use of phosphospecific antibody indicate that Ser-17 is a major in vitro and in vivo phosphorylation site for CK2. Functional studies reveal that, in contrast to the wild type kinase, the phosphorylation-mimicking mutant of S6K1 II (S17E) retains its cytoplasmic localization in serum-stimulated cells. Treatment of cells with the nuclear export inhibitor leptomycin B revealed that the S17E mutant accumulates in the nucleus to the same extent as S6K1 II wild type. These results indicate that nuclear import of the S17E mutant is not affected, although the export is significantly enhanced. We also provide evidence that nuclear export of S6K1 is mediated by a CRM1-dependent mechanism. Taken together, this study establishes a functional link between S6K1 II and CK2 signaling, which involves the regulation of S6K1 II nuclear export by CK2-mediated phosphorylation of Ser-17. PMID:16895915

  9. Characterization of a synthetic peroxodiiron(III) protein model complex by nuclear resonance vibrational spectroscopy

    E-print Network

    Do, Loi Hung

    The vibrational spectrum of an ?[superscript 1],?[superscript 1]-1,2-peroxodiiron(III) complex was measured by nuclear resonance vibrational spectroscopy and fit using an empirical force field analysis. Isotopic 18O2 ...

  10. Dynamic nuclear polarization in biomolecular solid state NMR : methods and applications in peptides and membrane proteins

    E-print Network

    Bajaj, Vikram Singh

    2007-01-01

    Solid state NMR can probe structure and dynamics on length scales from the atomic to the supramolecular. However, low sensitivity limits its application in macromolecules. NMR sensitivity can be improved by dynamic nuclear ...

  11. The rice RING finger E3 ligase, OsHCI1, drives nuclear export of multiple substrate proteins and its heterogeneous overexpression enhances acquired thermotolerance

    PubMed Central

    Lim, Sung Don; Cho, Hyun Yong; Park, Yong Chan; Ham, Deok Jae; Lee, Ju Kyong; Jang, Cheol Seong

    2013-01-01

    Thermotolerance is very important for plant survival when plants are subjected to lethally high temperature. However, thus far little is known about the functions of RING E3 ligase in response to heat shock in plants. This study found that one rice gene encoding the RING finger protein was specifically induced by heat and cold stress treatments but not by salinity or dehydration and named it OsHCI1 (Oryza sativa heat and cold induced 1). Subcellular localization results showed that OsHCI1 was mainly associated with the Golgi apparatus and moved rapidly and extensively along the cytoskeleton. In contrast, OsHCI1 may have accumulated in the nucleus under high temperatures. OsHCI1 physically interacted with nuclear substrate proteins including a basic helix-loop-helix transcription factor. Transient co-overexpression of OsHCI1 and each of three nuclear proteins showed that their fluorescent signals moved into the cytoplasm as punctuate formations. Heterogeneous overexpression of OsHCI1 in Arabidopsis highly increased survival rate through acquired thermotolerance. It is proposed that OsHCI1 mediates nuclear–cytoplasmic trafficking of nuclear substrate proteins via monoubiquitination and drives an inactivation device for the nuclear proteins under heat shock. PMID:23698632

  12. Structural polymorphism within a regulatory element of the human KRAS promoter: formation of G4-DNA recognized by nuclear proteins

    PubMed Central

    Cogoi, Susanna; Paramasivam, Manikandan; Spolaore, Barbara; Xodo, Luigi E.

    2008-01-01

    The human KRAS proto-oncogene contains a critical nuclease hypersensitive element (NHE) upstream of the major transcription initiation site. In this article, we demonstrate by primer-extension experiments, PAGE, chemical footprinting, CD, UV and FRET experiments that the G-rich strand of NHE (32R) folds into intra-molecular G-quadruplex structures. Fluorescence data show that 32R in 100 mM KCl melts with a biphasic profile, showing the formation of two distinct G-quadruplexes with Tm of ?55°C (Q1) and ?72°C (Q2). DMS-footprinting and CD suggest that Q1 can be a parallel and Q2 a mixed parallel/antiparallel G-quadruplex. When dsNHE (32R hybridized to its complementary) is incubated with a nuclear extract from Panc-1 cells, three DNA–protein complexes are observed by EMSA. The complex of slower mobility is competed by quadruplex 32R, but not by mutant oligonucleotides, which cannot form a quadruplex structure. Using paramagnetic beads coupled with 32R, we pulled down from the Panc-1 extract proteins with affinity for quadruplex 32R. One of these is the heterogeneous nuclear ribonucleoprotein A1, which was previously reported to unfold quadruplex DNA. Our study suggests a role of quadruplex DNA in KRAS transcription and provides the basis for the rationale design of molecular strategies to inhibit the expression of KRAS. PMID:18490377

  13. Expression-level dependent perturbation of cell proteostasis and nuclear morphology by aggregation-prone polyglutamine proteins.

    PubMed

    Lu, Meng; Williamson, Neil; Boschetti, Chiara; Ellis, Tom; Yoshimi, Tatsuya; Tunnacliffe, Alan

    2015-09-01

    We describe a gene expression system for use in mammalian cells that yields reproducible, inducible gene expression that can be modulated within the physiological range. A synthetic promoter library was generated from which representatives were selected that gave weak, intermediate-strength or strong promoter activity. Each promoter resulted in a tight expression range when used to drive single-copy reporter genes integrated at the same genome location in stable cell lines, in contrast to the broad range of expression typical of transiently transfected cells. To test this new expression system in neurodegenerative disease models, we used each promoter type to generate cell lines carrying single-copy genes encoding polyglutamine-containing proteins. Expression over a period of up to three months resulted in a proportion of cells developing juxtanuclear aggresomes whose rate of formation, penetrance, and morphology were expression-level dependent. At the highest expression levels, fibrillar aggregates deposit close to the nuclear envelope, indicating that cell proteostasis is overwhelmed by misfolded protein species. We also observed expression-level dependent, abnormal nuclear morphology in cells containing aggresomes, with up to ?80% of cells affected. This system constitutes a valuable tool in gene regulation at different levels and allows the quantitative assessment of gene expression effects when developing disease models or investigating cell function through the introduction of gene constructs. PMID:25854808

  14. RNA-protein crosslinking identifies novel targets for the nuclear RNA surveillance machinery 

    E-print Network

    Wlotzka, Wiebke

    2011-06-27

    The RNA binding proteins Nrd1 and Nab3 function in transcription termination by RNA Pol II, acting via interactions with the CTD of the largest polymerase subunit, particularly on snRNA and snoRNA genes. They also ...

  15. Ligand binding proteins: roles in ligand transfer and activation of nuclear receptors 

    E-print Network

    Petrescu, Anca Daniela

    2004-09-30

    Cholesterol and fatty acyl-coenzymeA thioesters are signalling molecules with role in regulation of genes involved in lipid and glucose transport and metabolism. The studies described herein focused on three proteins that bind lipids and have...

  16. Differential roles of heat shock protein 70 in the in vitro nuclear import of glucocorticoid receptor and simian virus 40 large tumor antigen.

    PubMed Central

    Yang, J; DeFranco, D B

    1994-01-01

    Nuclear import of glucocorticoid receptors (GRs) was analyzed in vitro with digitonin-permeabilized cells (S. A. Adam, R. Sterne-Marr, and L. Gerace, J. Cell Biol. 111:807-816, 1990). Indirect immunofluorescence methods were used to monitor the transport of GRs from rat hepatoma and fibroblast cell cytosol into HeLa nuclei. In vitro nuclear import of GRs was shown to be hormone dependent and to require ATP and incubation at ambient temperatures (i.e., 30 degrees C). Hormone-dependent dissociation of GR-bound proteins, such as the 90-kDa heat shock protein, hsp90, is part of an activation process that is obligatory for the expression of the receptor's DNA-binding activity. Inhibition of in vitro GR activation by Na2MoO4 blocked hormone-dependent nuclear import, demonstrating that receptor activation is required for nuclear import. The addition to GR-containing cytosol of antiserum directed against the cytosolic 70-kDa heat shock protein, hsp70, while effective in blocking the nuclear import of simian virus 40 large tumor antigen (SV40 TAg), did not affect hormone-dependent nuclear import of endogenous, full-length GRs or an exogenously added truncated GR protein (i.e., XGR556) that lacks a hormone-binding domain but possesses a constitutively active nuclear localization signal sequence (NLS). Depletion of hsp70 from HeLa cell cytosol did not affect the nuclear import of exogenously added XGR556 but led to inhibition of SV40 TAg nuclear import. Thus, two closely related NLSs, one contained within GRs and the other contained within SV40 TAg, are distinguished by their differential requirements for hsp70 in vitro. Images PMID:8035791

  17. Characterization of the AT180 epitope of phosphorylated Tau protein by a combined nuclear magnetic resonance and fluorescence spectroscopy approach

    SciTech Connect

    Amniai, Laziza; Lippens, Guy; Landrieu, Isabelle

    2011-09-09

    Highlights: {yields} pThr231 of the Tau protein is necessary for the binding of the AT180 antibody. {yields} pSer235 of the Tau protein does not interfere with the AT180 recognition of pThr231. {yields} Epitope mapping is efficiently achieved by combining NMR and FRET spectroscopy. -- Abstract: We present here the characterization of the epitope recognized by the AT180 monoclonal antibody currently used to define an Alzheimer's disease (AD)-related pathological form of the phosphorylated Tau protein. Some ambiguity remains as to the exact phospho-residue(s) recognized by this monoclonal: pThr231 or both pThr231 and pSer235. To answer this question, we have used a combination of nuclear magnetic resonance (NMR) and fluorescence spectroscopy to characterize in a qualitative and quantitative manner the phospho-residue(s) essential for the epitope recognition. Data from the first step of NMR experiments are used to map the residues bound by the antibodies, which were found to be limited to a few residues. A fluorophore is then chemically attached to a cystein residue introduced close-by the mapped epitope, at arginine 221, by mutagenesis of the recombinant protein. The second step of Foerster resonance energy transfer (FRET) between the AT180 antibody tryptophanes and the phospho-Tau protein fluorophore allows to calculate a dissociation constant Kd of 30 nM. We show that the sole pThr231 is necessary for the AT180 recognition of phospho-Tau and that phosphorylation of Ser235 does not interfere with the binding.

  18. Nuclear factor of activated T cell (NFAT) transcription proteins regulate genes involved in adipocyte metabolism and lipolysis

    SciTech Connect

    Holowachuk, Eugene W. . E-mail: geneh@telenet.net

    2007-09-21

    NFAT involvement in adipocyte physiological processes was examined by treatment with CsA and/or GSK3{beta} inhibitors (Li{sup +} or TZDZ-8), which prevent or increase NFAT nuclear translocation, respectively. CsA treatment reduced basal and TNF{alpha}-induced rates of lipolysis by 50%. Adipocytes preincubated with Li{sup +} or TZDZ-8 prior to CsA and/or TNF{alpha}, exhibited enhanced basal rates of lipolysis and complete inhibition of CsA-mediated decreased rates of lipolysis. CsA treatment dramatically reduced the mRNA levels of adipocyte-specific genes (aP2, HSL, PPAR{gamma}, ACS and Adn), compared with control or TNF{alpha}-treatment, whereas Li{sup +} pretreatment blocked the inhibitory effects of CsA, and mRNA levels of aP2, HSL, PPAR{gamma}, and ACS were found at or above control levels. NFAT nuclear localization, assessed by EMSA, confirmed that CsA or Li{sup +} treatments inhibited or increased NFAT nuclear translocation, respectively. These results show that NFAT proteins in mature adipocytes participate in the transcriptional control of genes involved in adipocyte metabolism and lipolysis.

  19. Identification of a functional nuclear localization signal within the human USP22 protein

    SciTech Connect

    Xiong, Jianjun; Wang, Yaqin; Gong, Zhen; Liu, Jianyun; Li, Weidong

    2014-06-20

    Highlights: • USP22 was accumulated in nucleus. • We identified of a functional USP22 NLS. • The KRRK amino acid residues are indispensable in NLS. • The KRRK motif is conserved in USP22 homologues. - Abstract: Ubiquitin-specific processing enzyme 22 (USP22), a member of the deubiquitinase family, is over-expressed in most human cancers and has been implicated in tumorigenesis. Because it is an enzymatic subunit of the human SAGA transcriptional cofactor, USP22 deubiquitylates histone H2A and H2B in the nucleus, thus participating in gene regulation and cell-cycle progression. However, the mechanisms regulating its nuclear translocation have not yet been elucidated. It was here demonstrated that USP22 is imported into the nucleus through a mechanism mediated by nuclear localization signal (NLS). The bipartite NLS sequence KRELELLKHNPKRRKIT (aa152–168), was identified as the functional NLS for its nuclear localization. Furthermore, a short cluster of basic amino acid residues KRRK within this bipartite NLS plays the primary role in nuclear localization and is evolutionarily conserved in USP22 homologues. In the present study, a functional NLS and the minimal sequences required for the active targeting of USP22 to the nucleus were identified. These findings may provide a molecular basis for the mechanism underlying USP22 nuclear trafficking and function.

  20. Proteomic analysis of the nuclear matrix in the early stages of rat liver carcinogenesis: Identification of differentially expressed and MAR-binding proteins

    SciTech Connect

    Barboro, Paola; D'Arrigo, Cristina; Repaci, Erica; Bagnasco, Luca; Orecchia, Paola; Carnemolla, Barbara; Patrone, Eligio; Balbi, Cecilia

    2009-01-15

    Tumor progression is characterized by definite changes in the protein composition of the nuclear matrix (NM). The interactions of chromatin with the NM occur via specific DNA sequences called MARs (matrix attachment regions). In the present study, we applied a proteomic approach along with a Southwestern assay to detect both differentially expressed and MAR-binding NM proteins, in persistent hepatocyte nodules (PHN) in respect with normal hepatocytes (NH). In PHN, the NM undergoes changes both in morphology and in protein composition. We detected over 500 protein spots in each two dimensional map and 44 spots were identified. Twenty-three proteins were differentially expressed; among these, 15 spots were under-expressed and 8 spots were over-expressed in PHN compared to NH. These changes were synchronous with several modifications in both NM morphology and the ability of NM proteins to bind nuclear RNA and/or DNA containing MARs sequences. In PHN, we observed a general decrease in the expression of the basic proteins that bound nuclear RNA and the over-expression of two species of Mw 135 kDa and 81 kDa and pI 6.7-7.0 and 6.2-7.4, respectively, which exclusively bind to MARs. These results suggest that the deregulated expression of these species might be related to large-scale chromatin reorganization observed in the process of carcinogenesis by modulating the interaction between MARs and the scaffold structure.

  1. Nuclear Matrix protein SMAR1 represses HIV-1 LTR mediated transcription through chromatin remodeling

    SciTech Connect

    Sreenath, Kadreppa; Pavithra, Lakshminarasimhan; Singh, Sandeep; Sinha, Surajit; Dash, Prasanta K.; Siddappa, Nagadenahalli B.; Ranga, Udaykumar; Mitra, Debashis; Chattopadhyay, Samit

    2010-04-25

    Nuclear Matrix and MARs have been implicated in the transcriptional regulation of host as well as viral genes but their precise role in HIV-1 transcription remains unclear. Here, we show that > 98% of HIV sequences contain consensus MAR element in their promoter. We show that SMAR1 binds to the LTR MAR and reinforces transcriptional silencing by tethering the LTR MAR to nuclear matrix. SMAR1 associated HDAC1-mSin3 corepressor complex is dislodged from the LTR upon cellular activation by PMA/TNFalpha leading to an increase in the acetylation and a reduction in the trimethylation of histones, associated with the recruitment of RNA Polymerase II on the LTR. Overexpression of SMAR1 lead to reduction in LTR mediated transcription, both in a Tat dependent and independent manner, resulting in a decreased virion production. These results demonstrate the role of SMAR1 in regulating viral transcription by alternative compartmentalization of LTR between the nuclear matrix and chromatin.

  2. Nuclear and cytoplasmic p53 suppress cell invasion by inhibiting respiratory Complex-I activity via Bcl-2 family proteins

    PubMed Central

    Kim, Eun Mi; Park, Jong Kuk; Hwang, Sang-Gu; Kim, Wun-Jae; Liu, Zheng-Gang; Kang, Sang Won; Um, Hong-Duck

    2014-01-01

    Although the p53 tumor suppressor/transcription factor often accumulates in the cytoplasm of healthy cells, limited information is available on the cytoplasmic function of p53. Here, we show that cytoplasmic p53 suppresses cell invasion by reducing mitochondrial reactive oxygen species (ROS) levels. Analysis revealed that this function is mediated by Bcl-2 family proteins: Cytoplasmic p53 binds Bcl-w, liberating Bax, which then binds ND5, a subunit of respiratory complex-I, thereby suppressing complex-I activity and thus ROS production. The G13289A mutation of ND5, identified in cancer patients, prevents Bax/ND5 interactions and promotes ROS production and cell invasion. We also showed that Bcl-XL and Bak can substitute for Bcl-w and Bax, respectively, regulating complex-I activity and supporting the cytoplasmic function of p53; nuclear p53 also suppresses complex-I activity by inducing Bax expression. Studies in animal models support the notion that p53 and Bcl-2 family proteins exhibit these functions in vivo. This study demonstrates a link between p53 and Bcl-2 proteins as regulators of ROS production and cellular invasiveness, and reveals complex-I, especially ND5, as their functional target. PMID:25115399

  3. Pathway of B1-Alu expression in microinjected oocytes: Xenopus laevis proteins associated with nuclear precursor and processed cytoplasmic RNAs.

    PubMed Central

    Maraia, R; Zasloff, M; Plotz, P; Adeniyi-Jones, S

    1988-01-01

    We have previously characterized B1-Alu gene expression by microinjected Xenopus laevis oocytes. The transcription, endonucleolytic processing and its kinetics, nuclear transport kinetics, and subsequent cellular compartmentalization have been described previously (Adeniyi-Jones and Zasloff, Nature 317:81-84, 1985). Briefly, a B1-Alu gene is transcribed by RNA polymerase III to a 210-nucleotide (210nt) primary transcript which is processed to yield 135nt and 75nt RNAs. After processing, the 135nt RNA enters the cytoplasmic compartment, where it remains stable, while the 75nt RNA is degraded. In this report we characterize this pathway further and show that the RNAs involved are complexed with specific X. laevis proteins. The primary transcript was associated with an X. laevis protein of 63 kilodaltons (p63) as well as La, a protein known to be associated with RNA polymerase III transcripts. After processing, the cytoplasmic 135nt RNA remained associated only with the X. laevis p63 in the form of a small ribonucleoprotein. Human autoimmune antibodies were purified by affinity chromatography to X. laevis p63 and used to immunoprecipitate human ribonucleoprotein containing a 63-kilodalton polypeptide and small RNAs. These data suggest that Alu-analogous ribonucleoproteins and their metabolic pathways are conserved across species and provide insight as to their possible functions. Images PMID:2460743

  4. CLUH regulates mitochondrial biogenesis by binding mRNAs of nuclear-encoded mitochondrial proteins

    PubMed Central

    Gao, Jie; Schatton, Désirée; Martinelli, Paola; Hansen, Henriette; Pla-Martin, David; Barth, Esther; Becker, Christian; Altmueller, Janine; Frommolt, Peter; Sardiello, Marco

    2014-01-01

    Mitochondrial function requires coordination of two genomes for protein biogenesis, efficient quality control mechanisms, and appropriate distribution of the organelles within the cell. How these mechanisms are integrated is currently not understood. Loss of the Clu1/CluA homologue (CLUH) gene led to clustering of the mitochondrial network by an unknown mechanism. We find that CLUH is coregulated both with genes encoding mitochondrial proteins and with genes involved in ribosomal biogenesis and translation. Our functional analysis identifies CLUH as a cytosolic messenger ribonucleic acid (RNA; mRNA)–binding protein. RNA immunoprecipitation experiments followed by next-generation sequencing demonstrated that CLUH specifically binds a subset of mRNAs encoding mitochondrial proteins. CLUH depletion decreased the levels of proteins translated by target transcripts and caused mitochondrial clustering. A fraction of CLUH colocalizes with tyrosinated tubulin and can be detected close to mitochondria, suggesting a role in regulating transport or translation of target transcripts close to mitochondria. Our data unravel a novel mechanism linking mitochondrial biogenesis and distribution. PMID:25349259

  5. Expression dynamics and ultrastructural localization of epitope-tagged Abutilon mosaic virus nuclear shuttle and movement proteins in Nicotiana benthamiana cells

    SciTech Connect

    Kleinow, Tatjana; Tanwir, Fariha; Kocher, Cornelia; Krenz, Bjoern; Wege, Christina; Jeske, Holger

    2009-09-01

    The geminivirus Abutilon mosaic virus (AbMV) encodes two proteins which are essential for viral spread within plants. The nuclear shuttle protein (NSP) transfers viral DNA between the nucleus and cytoplasm, whereas the movement protein (MP) facilitates transport between cells through plasmodesmata and long-distance via phloem. An inducible overexpression system for epitope-tagged NSP and MP in plants yielded unprecedented amounts of both proteins. Western blots revealed extensive posttranslational modification and truncation for MP, but not for NSP. Ultrastructural examination of Nicotiana benthamiana tissues showed characteristic nucleopathic alterations, including fibrillar rings, when epitope-tagged NSP and MP were simultaneously expressed in leaves locally infected with an AbMV DNA A in which the coat protein gene was replaced by a green fluorescent protein encoding gene. Immunogold labelling localized NSP in the nucleoplasm and in the fibrillar rings. MP appeared at the cell periphery, probably the plasma membrane, and plasmodesmata.

  6. AKT3 controls mitochondrial biogenesis and autophagy via regulation of the major nuclear export protein CRM-1

    PubMed Central

    Corum, Daniel G.; Tsichlis, Philip N.; Muise-Helmericks, Robin C.

    2014-01-01

    Our previous work has shown that Akt3 is required for mitochondrial biogenesis in primary human endothelial cells (ECs) and in Akt3-null mice; Akt3 affects subcellular localization of peroxisome proliferator-activated receptor ? coactivator-1 (PGC-1?), the master regulator of mitochondrial biogenesis. The purpose of this study is to determine the mechanism by which Akt3 controls the subcellular distribution of PGC-1? and to explore the effect on mitochondrial biogenesis and turnover during angiogenesis. Here we use standard biochemical analyses and Akt3-knockdown strategies to show that Akt3 controls the stabilization of chromosome maintenance region-1 (CRM-1), the major nuclear export receptor. Site-directed mutagenesis and association analyses show that PGC-1? nuclear export is CRM-1 dependent. Akt3 knockdown and CRM-1 overexpression cause 3-fold reductions in PGC-1? target gene expression, compared to control levels. Akt3 inhibition causes autophagy, as measured by autophagosome formation, in a CRM-1-dependent, Akt1/mTOR-independent pathway. In vivo, Akt3-null and heterozygous mice show dose-dependent decreases in angiogenesis compared to wild-type littermates (?5- and 2.5-fold decreases, respectively), as assessed by Matrigel plug assays. This correlates with an ?1.5-fold decrease in mitochondrial Cox IV expression. Our studies suggest that Akt3 is a regulator of mitochondrial dynamics in the vasculature via regulation of CRM-1-dependent nuclear export.—Corum, D. G., Tsichlis, P. N., Muise-Helmericks, R. C. AKT3 controls mitochondrial biogenesis and autophagy via regulation of the major nuclear export protein CRM-1. PMID:24081905

  7. Identification of a novel nuclear localization signal and speckle-targeting sequence of tuftelin-interacting protein 11, a splicing factor involved in spliceosome disassembly

    SciTech Connect

    Tannukit, Sissada; Crabb, Tara L.; Hertel, Klemens J.; Wen, Xin; Jans, David A.; Paine, Michael L.

    2009-12-18

    Tuftelin-interacting protein 11 (TFIP11) is a protein component of the spliceosome complex that promotes the release of the lariat-intron during late-stage splicing through a direct recruitment and interaction with DHX15/PRP43. Expression of TFIP11 is essential for cell and organismal survival. TFIP11 contains a G-patch domain, a signature motif of RNA-processing proteins that is responsible for TFIP11-DHX15 interactions. No other functional domains within TFIP11 have been described. TFIP11 is localized to distinct speckled regions within the cell nucleus, although excluded from the nucleolus. In this study sequential C-terminal deletions and mutational analyses have identified two novel protein elements in mouse TFIP11. The first domain covers amino acids 701-706 (VKDKFN) and is an atypical nuclear localization signal (NLS). The second domain is contained within amino acids 711-735 and defines TFIP11's distinct speckled nuclear localization. The identification of a novel TFIP11 nuclear speckle-targeting sequence (TFIP11-STS) suggests that this domain directly interacts with additional spliceosomal components. These data help define the mechanism of nuclear/nuclear speckle localization of the splicing factor TFIP11, with implications for it's function.

  8. Identification of a novel nuclear localization signal and speckle-targeting sequence of tuftelin-interacting protein 11, a splicing factor involved in spliceosome disassembly

    PubMed Central

    Tannukit, Sissada; Crabb, Tara L.; Hertel, Klemens J.; Wen, Xin; Jans, David A.; Paine, Michael L.

    2009-01-01

    Tuftelin-interacting protein 11 (TFIP11) is a protein component of the spliceosome complex that promotes the release of the lariat-intron during late-stage splicing through a direct recruitment and interaction with DHX15/PRP43. Expression of TFIP11 is essential for cell and organismal survival. TFIP11 contains a G-patch domain, a signature motif of RNA-processing proteins that is responsible for TFIP11-DHX15 interactions. No other functional domains within TFIP11 have been described. TFIP11 is localized to distinct speckled regions within the cell nucleus, although excluded from the nucleolus. In this study sequential C-terminal deletions and mutational analyses have identified two novel protein elements in mouse TFIP11. The first domain covers amino acids 701–706 (VKDKFN) and is an atypical nuclear localization signal (NLS). The second domain is contained within amino acids 711–735 and defines TFIP11’s distinct speckled nuclear localization. The identification of a novel TFIP11 nuclear speckle targeting sequence (TFIP11-STS) suggests that this domain directly interacts with additional spliceosomal components. These data help define the mechanism of nuclear/nuclear speckle localization of the splicing factor TFIP11, with implications for it’s function. PMID:19857462

  9. Nuclear Resonance Vibrational Spectroscopy (NRVS) of Fe S model compounds, Fe S proteins, and nitrogenase

    NASA Astrophysics Data System (ADS)

    Cramer, Stephen P.; Xiao, Yuming; Wang, Hongxin; Guo, Yisong; Smith, Matt C.

    2006-06-01

    We have used nuclear resonance vibrational spectroscopy (NRVS) to examine the nature of the Fe S unit. Specifically, vibrational characteristics have been determined, and through incremental steps in model system complexity, applied to analysis of the enzyme nitrogenase. This stepwise strategy demonstrates NRVS as a viable bioinorganic tool, and will undoubtedly increase the application of synchrotron spectroscopy to biological problems.

  10. Herpes Simplex Virus ICP27 Protein Directly Interacts with the Nuclear Pore Complex through Nup62, Inhibiting Host Nucleocytoplasmic Transport Pathways*

    PubMed Central

    Malik, Poonam; Tabarraei, Alijan; Kehlenbach, Ralph H.; Korfali, Nadia; Iwasawa, Ryota; Graham, Sheila V.; Schirmer, Eric C.

    2012-01-01

    The herpes simplex virus ICP27 protein is important for the expression and nuclear export of viral mRNAs. Although several binding sites have been mapped along the ICP27 sequence for various RNA and protein partners, including the transport receptor TAP of the host cell nuclear transport machinery, several aspects of ICP27 trafficking through the nuclear pore complex remain unclear. We investigated if ICP27 could interact directly with the nuclear pore complex itself, finding that ICP27 directly binds the core nucleoporin Nup62. This is confirmed through co-immunoprecipitation and in vitro binding assays with purified components. Mapping with ICP27 deletion and point mutants further shows that the interaction requires sequences in both the N and C termini of ICP27. Expression of wild type ICP27 protein inhibited both classical, importin ?/?-dependent and transportin-dependent nuclear import. In contrast, an ICP27 point mutant that does not interact with Nup62 had no such inhibitory effect. We suggest that ICP27 association with Nup62 provides additional binding sites at the nuclear pore for ICP27 shuttling, thus supporting ICP27-mediated transport. We propose that ICP27 competes with some host cell transport receptors for binding, resulting in inhibition of those host transport pathways. PMID:22334672

  11. Characterization of nuclear proteins that bind to the regulatory TGATTGGC motif in the human immunodeficiency virus type 1 long terminal repeat.

    PubMed Central

    Schwartz, C; Canonne-Hergaux, F; Aunis, D; Schaeffer, E

    1997-01-01

    We have recently elucidated the nature and function of transcription factors present in Jurkat, glial and neuronal cells that interact with modulatory region B, the nuclear receptor responsive element, in the long terminal repeat of human immunodeficiency virus type 1 (HIV-1). Considering the key role that the combination of host cell proteins plays in HIV-1 gene transcription, it appears essential to characterize proteins interacting with the adjacent region A. In vitro experiments revealed that the 5'-TGATTGGC-3'motif of region A is the target for at least three distinct proteins, one belonging to the nuclear factor I family, while two others are related to the cAMP response element binding (CREB) protein family. One of these proteins, present in DNA-protein complex C2, is formed by distinct polypeptides of relative molecular mass 43 000 and 50 000. We have purified the 43 kDa protein, which is distinct from CREB-43, and have shown that renatured p43 is able to specifically interact with site A. Transient expression experiments with vectors containing wild-type or mutant motif A revealed that basal HIV-1 gene transcription in Jurkat cells is regulated by antagonistic effects of the site A binding proteins. PMID:9092627

  12. Identification and fine mapping of nuclear and nucleolar localization signals within the human ribosomal protein S17.

    PubMed

    Kenney, Scott P; Meng, Xiang-Jin

    2015-01-01

    Human ribosomal protein S17 (RPS17) is mutated in Diamond-Blackfan Anemia (DBA), a bone marrow disorder that fails to produce sufficient red blood cells leading to anemia. Recently, an RPS17 protein sequence was also found to be naturally inserted in the genome of hepatitis E virus (HEV) from patients chronically-infected by HEV. The role of RPS17 in HEV replication and pathogenesis remains unknown due to the lack of knowledge about how RPS17 functions at a molecular level. Understanding the biological function of RPS17 is critical for elucidating its role in virus infection and DBA disease processes. In this study we probed the subcellular distribution of normal and mutant RPS17 proteins in a human liver cell line (Huh7). RPS17 was primarily detected within the nucleus, and more specifically within the nucleoli. Using a transient expression system in which RPS17 or truncations were expressed as fusions with enhanced yellow fluorescent protein (eYFP), we were able to identify and map, for the first time, two separate nuclear localization signals (NLSs), one to the first 13 amino acids of the amino-terminus of RPS17 and the other within amino acids 30-60. Additionally, we mapped amino acid sequences required for nucleolar accumulation of RPS17 to amino acids 60-70. Amino acids 60-70 possess a di-RG motif that may be necessary for nucleolar retention of RPS17. The results from this study enhance our knowledge of RSP17 and will facilitate future mechanistic studies about the roles of RSP17 in hepatitis E and DBA disease processes. PMID:25853866

  13. Sorting of inner nuclear membrane-directed proteins at the endoplasmic reticulum membrane 

    E-print Network

    Saksena, Suraj

    2006-04-12

    protein ODV-E66 (E66) showed that E66 trafficking to the INM is mediated via an INM sorting signal (Sorting Motif or SM). In this study, using a site-specific crosslinking approach we demonstrate that following ER membrane integration, the SM is adjacent...

  14. Static and pulsed field gradient nuclear magnetic resonance studies of water diffusion in protein matrices

    NASA Astrophysics Data System (ADS)

    Rosenstihl, M.; Vogel, M.

    2011-10-01

    Static field gradient and pulsed field gradient NMR are used to study the temperature dependence of water diffusion in myoglobin and lysozyme matrices for low hydration levels of about 0.3 g/g. We show that in order to determine reliable self-diffusion coefficients D in a broad temperature range, it is very important to consider an exchange of magnetization between water and protein protons, often denoted as cross relaxation. Specifically, upon cooling, the observed stimulated-echo decays, which reflect water diffusion near ambient temperature, become more and more governed by cross relaxation. We demonstrate that comparison of experimental results for inhomogeneous and homogeneous magnetic fields enables successful separation of diffusion and relaxation contributions to the stimulated-echo decays. Making use of this possibility, we find that in the temperature range 230-300 K, the temperature-dependent diffusivities D exhibit a Vogel-Fulcher-Tammann behavior, where water diffusion in the studied protein matrices is substantially slower than in the bulk. By comparing present and previous data, we discuss relations between translational and rotational motions and between short-range and long-range water dynamics in protein matrices. In addition, we critically examine the significance of results from previous applications of NMR diffusometry to the temperature-dependent water diffusion in protein matrices.

  15. Protein structure determination in solution by two-dimensional and three-dimensional nuclear magnetic resonance spectroscopy.

    PubMed

    Gronenborn, A M; Clore, G M

    1990-01-01

    Over the last decade, nuclear magnetic resonance (NMR) spectroscopy has evolved into a powerful method for determining structures of biological macromolecules. This has opened a unique opportunity for obtaining high-resolution three-dimensional structures in solution, in contrast to the well-established methods of X-ray diffraction, which are applicable only to solids and in particular single crystals. This rapid development has been spurred by several key advances in the field, especially the introduction of two- and three-dimensional NMR experiments, high field spectrometers (500 and 600 MHz), and computational algorithms for converting NMR derived restraints into three-dimensional structures. This review outlines the methodology employed for solving protein structures in solution, describing the basic NMR experiments necessary as well as introducing the concepts upon which the computational algorithms are founded. A variety of examples is discussed, illustrating the present state of the art, and future possibilities are indicated. PMID:2301728

  16. Threonine 308 phosphorylated form of Akt translocates to the nucleus of PC12 cells under nerve growth factor stimulation and associates with the nuclear matrix protein nucleolin.

    PubMed

    Borgatti, Paola; Martelli, Alberto M; Tabellini, Giovanna; Bellacosa, Alfonso; Capitani, Silvano; Neri, Luca M

    2003-07-01

    We have examined the issue of whether or not in PC12 cells it may be observed a nerve growth factor (NGF) nuclear translocation of an active (phosphorylated) Akt. Western blot analysis with antibodies to either total or phosphorylated Akt showed a maximal nuclear translocation after 15 min of NGF stimulation. NGF increased rapidly and transiently the enzymatic activity of immunoprecipitable nuclear Akt and after 45 min the values returned to a level close to the basal one. Enzyme translocation was blocked by the selective phosphoinositide 3-kinase inhibitor, LY294002. Confocal microscopy of samples stained with antibody to Akt showed an evident increase in immunostaining intensity in the nuclear interior after NGF treatment. Treatment of cells with inhibitors of protein phosphatase PP2A, calyculin A, or okadaic acid, maintained the phosphorylation levels of nuclear Akt. Immunoprecipitation experiments revealed an association between Akt and PP2A that was maximal when nuclear Akt activity was decreased. Both total and active Akt associated with the nuclear matrix and, in particular, with the protein nucleolin, with which Akt co-immunoprecipitated. These findings strongly suggest that the intranuclear translocation of active Akt is an important step in the signaling pathways elicited by the neurotrophin NGF and that the intranuclear control of Akt is achieved through the action of PP2A. PMID:12767043

  17. AIRE-induced apoptosis is associated with nuclear translocation of stress sensor protein GAPDH

    SciTech Connect

    Liiv, Ingrid; Haljasorg, Uku; Kisand, Kai; Maslovskaja, Julia; Laan, Martti; Peterson, Paert

    2012-06-22

    Highlights: Black-Right-Pointing-Pointer AIRE induces apoptosis in epithelial cells. Black-Right-Pointing-Pointer CARD domain of AIRE is sufficient for apoptosis induction. Black-Right-Pointing-Pointer AIRE induced apoptosis involves GAPDH translocation to the nuclei. Black-Right-Pointing-Pointer Deprenyl inhibits AIRE induced apoptosis. -- Abstract: AIRE (Autoimmune Regulator) has a central role in the transcriptional regulation of self-antigens in medullary thymic epithelial cells, which is necessary for negative selection of autoreactive T cells. Recent data have shown that AIRE can also induce apoptosis, which may be linked to cross-presentation of these self-antigens. Here we studied AIRE-induced apoptosis using AIRE over-expression in a thymic epithelial cell line as well as doxycycline-inducible HEK293 cells. We show that the HSR/CARD domain in AIRE together with a nuclear localization signal is sufficient to induce apoptosis. In the nuclei of AIRE-positive cells, we also found an increased accumulation of a glycolytic enzyme, glyceraldehyde-3-phosphate (GAPDH) reflecting cellular stress and apoptosis. Additionally, AIRE-induced apoptosis was inhibited with an anti-apoptotic agent deprenyl that blocks GAPDH nitrosylation and nuclear translocation. We propose that the AIRE-induced apoptosis pathway is associated with GAPDH nuclear translocation and induction of NO-induced cellular stress in AIRE-expressing cells.

  18. The nuclear pore complex protein ALADIN is anchored via NDC1 but not via POM121 and GP210 in the nuclear envelope

    SciTech Connect

    Kind, Barbara; Koehler, Katrin; Lorenz, Mike; Huebner, Angela

    2009-12-11

    The nuclear pore complex (NPC) consists of {approx}30 different proteins and provides the only sites for macromolecular transport between cytoplasm and nucleus. ALADIN was discovered as a new member of the NPC. Mutations in ALADIN are known to cause triple A syndrome, a rare autosomal recessive disorder characterized by adrenal insufficiency, alacrima, and achalasia. The function and exact location of the nucleoporin ALADIN within the NPC multiprotein complex is still unclear. Using a siRNA-based approach we downregulated the three known membrane integrated nucleoporins NDC1, GP210, and POM121 in stably expressing GFP-ALADIN HeLa cells. We identified NDC1 but not GP210 and POM121 as the main anchor of ALADIN within the NPC. Solely the depletion of NDC1 caused mislocalization of ALADIN. Vice versa, the depletion of ALADIN led also to disappearance of NDC1 at the NPC. However, the downregulation of two further membrane-integral nucleoporins GP210 and POM121 had no effect on ALADIN localization. Furthermore, we could show a direct association of NDC1 and ALADIN in NPCs by fluorescence resonance energy transfer (FRET) measurements. Based on our findings we conclude that ALADIN is anchored in the nuclear envelope via NDC1 and that this interaction gets lost, if ALADIN is mutated. The loss of integration of ALADIN in the NPC is a main pathogenetic aspect for the development of the triple A syndrome and suggests that the interaction between ALADIN and NDC1 may be involved in the pathogenesis of the disease.

  19. Structure, localization and histone binding properties of nuclear-associated nucleosome assembly protein from Plasmodium falciparum

    PubMed Central

    2010-01-01

    Background Nucleosome assembly proteins (NAPs) are histone chaperones that are crucial for the shuttling and incorporation of histones into nucleosomes. NAPs participate in the assembly and disassembly of nucleosomes thus contributing to chromatin structure organization. The human malaria parasite Plasmodium falciparum contains two nucleosome assembly proteins termed PfNapL and PfNapS. Methods Three-dimensional crystal structure of PfNapS has been determined and analysed. Gene knockout and localization studies were also performed on PfNapS using transfection studies. Fluorescence spectroscopy was performed to identify histone-binding sites on PfNapS. Extensive sequence and structural comparisons were done with the crystal structures available for NAP/SET family of proteins. Results Crystal structure of PfNapS shares structural similarity with previous structures from NAP/SET family. Failed attempts to knock-out the gene for PfNapS from malaria parasite suggest essentiality in the parasite. GFP-fused PfNapS fusion protein targeting indicates cellular localization of PfNapS in the parasite nucleus. Fluorescence spectroscopy data suggest that PfNapS interacts with core histones (tetramer, octamer, H3, H4, H2A and H2B) at a different site from its interaction with linker histone H1. This analysis illustrates two regions on the PfNapS dimer as the possible sites for histone recognition. Conclusions This work presents a thorough analysis of the structural, functional and regulatory attributes of PfNapS from P. falciparum with respect to previously studied histone chaperones. PMID:20377878

  20. Measurement of Lens Protein Aggregation in Vivo Using Dynamic Light Scattering in a Guinea Pig/UVA Model for Nuclear Cataract

    PubMed Central

    Simpanya, M. Francis; Ansari, Rafat R.; Leverenz, Victor; Giblin, Frank J.

    2009-01-01

    The role of UVA radiation in the formation of human nuclear cataract is not well understood. We have previously shown that exposing guinea pigs for 5 months to a chronic low level of UVA light produces increased lens nuclear light scattering and elevated levels of protein disulfide. Here we have used the technique of dynamic light scattering (DLS) to investigate lens protein aggregation in vivo in the guinea pig/UVA model. DLS size distribution analysis conducted at the same location in the lens nucleus of control and UVA-irradiated animals showed a 28% reduction in intensity of small diameter proteins in experimental lenses compared with controls (P < 0.05). In addition, large diameter proteins in UVA-exposed lens nuclei increased five-fold in intensity compared to controls (P < 0.05). The UVA-induced increase in apparent size of lens nuclear small diameter proteins was three-fold (P < 0.01), and the size of large diameter aggregates was more than four-fold in experimental lenses compared with controls. The diameter of crystallin aggregates in the UVA-irradiated lens nucleus was estimated to be 350 nm, a size able to scatter light. No significant changes in protein size were detected in the anterior cortex of UVA-irradiated lenses. It is presumed that the presence of a UVA chromophore in the guinea pig lens (NADPH bound to zeta crystallin), as well as traces of oxygen, contributed to UVA-induced crystallin aggregation. The results indicate a potentially harmful role for UVA light in the lens nucleus. A similar process of UVA-irradiated protein aggregation may take place in the older human lens nucleus, accelerating the formation of human nuclear cataract. PMID:18627516

  1. The APC tumor suppressor binds to C-terminal binding protein to divert nuclear beta-catenin from TCF.

    PubMed

    Hamada, Fumihiko; Bienz, Mariann

    2004-11-01

    Adenomatous polyposis coli (APC) is an important tumor suppressor in the colon. APC antagonizes the transcriptional activity of the Wnt effector beta-catenin by promoting its nuclear export and its proteasomal destruction in the cytoplasm. Here, we show that a third function of APC in antagonizing beta-catenin involves C-terminal binding protein (CtBP). APC is associated with CtBP in vivo and binds to CtBP in vitro through its conserved 15 amino acid repeats. Failure of this association results in elevated levels of beta-catenin/TCF complexes and of TCF-mediated transcription. Notably, CtBP is neither associated with TCF in vivo nor does mutation of the CtBP binding motifs in TCF-4 alter its transcriptional activity. This questions the idea that CtBP is a direct corepressor of TCF. Our evidence indicates that APC is an adaptor between beta-catenin and CtBP and that CtBP lowers the availability of free nuclear beta-catenin for binding to TCF by sequestering APC/beta-catenin complexes. PMID:15525529

  2. Nuclear envelope precursor vesicle targeting to chromatin is stimulated by protein phosphatase 1 in Xenopus egg extracts

    SciTech Connect

    Ito, Hiromi; Koyama, Yuhei; Takano, Makoto; Ishii, Kohei; Maeno, Mitsugu; Furukawa, Kazuhiro; Horigome, Tsuneyoshi . E-mail: thori@chem.sc.niigata-u.ac.jp

    2007-05-15

    The mechanism underlying targeting of the nuclear membrane to chromatin at the end of mitosis was studied using an in vitro cell-free system comprising Xenopus egg membrane and cytosol fractions, and sperm chromatin. The mitotic phase membrane, which was separated from a mitotic phase extract of Xenopus eggs and could not bind to chromatin, became able to bind to chromatin on pretreatment with a synthetic phase cytosol fraction of Xenopus eggs. When the cytosol fraction was depleted of protein phosphatase 1 (PP1) with anti-Xenopus PP1{gamma}1 antibodies, this ability was lost. The addition of recombinant xPP1{gamma}1 to the PP1-depleted cytosol fraction restored the ability. These and other results suggested that dephosphorylation of mitotic phosphorylation sites on membranes by PP1 in the synthetic phase cytosol fraction promoted targeting of the membranes to chromatin. On the other hand, a fragment containing the chromatin-binding domain of lamin B receptor (LBR) but not emerin inhibited targeting of membrane vesicles. It was also shown that PP1 dephosphorylates a phosphate group(s) responsible for regulation of the binding of LBR to chromatin. A possible mechanism involving PP1 and LBR for the regulation of nuclear membrane targeting to chromatin was discussed.

  3. Human cyclin E, a nuclear protein essential for the G1-to-S phase transition.

    PubMed

    Ohtsubo, M; Theodoras, A M; Schumacher, J; Roberts, J M; Pagano, M

    1995-05-01

    Cyclin E was first identified by screening human cDNA libraries for genes that would complement G1 cyclin mutations in Saccharomyces cerevisiae and has subsequently been found to have specific biochemical and physiological properties that are consistent with it performing a G1 function in mammalian cells. Most significantly, the cyclin E-Cdk2 complex is maximally active at the G1/S transition, and overexpression of cyclin E decreases the time it takes the cell to complete G1 and enter S phase. We have now found that mammalian cells express two forms of cyclin E protein which differ from each other by the presence or absence of a 15-amino-acid amino-terminal domain. These proteins are encoded by alternatively spliced mRNAs and are localized to the nucleus during late G1 and early S phase. Fibroblasts engineered to constitutively overexpress either form of cyclin E showed elevated cyclin E-dependent kinase activity and a shortened G1 phase of the cell cycle. The overexpressed cyclin E protein was detected in the nucleus during all cell cycle phases, including G0. Although the cyclin E protein could be overexpressed in quiescent cells, the cyclin E-Cdk2 complex was inactive. It was not activated until 6 to 8 h after readdition of serum, 4 h earlier than the endogenous cyclin E-Cdk2. This premature activation of cyclin E-Cdk2 was consistent with the extent of G1 shortening caused by cyclin E overexpression. Microinjection of affinity-purified anti-cyclin E antibodies during G1 inhibited entry into S phase, whereas microinjection performed near the G1/S transition was ineffective. These results demonstrate that cyclin E is necessary for entry into S phase. Moreover, we found that cyclin E, in contrast to cyclin D1, was required for the G1/S transition even in cells lacking retinoblastoma protein function. Therefore, cyclins E and D1 control two different transitions within the human cell cycle. PMID:7739542

  4. Human cyclin E, a nuclear protein essential for the G1-to-S phase transition.

    PubMed Central

    Ohtsubo, M; Theodoras, A M; Schumacher, J; Roberts, J M; Pagano, M

    1995-01-01

    Cyclin E was first identified by screening human cDNA libraries for genes that would complement G1 cyclin mutations in Saccharomyces cerevisiae and has subsequently been found to have specific biochemical and physiological properties that are consistent with it performing a G1 function in mammalian cells. Most significantly, the cyclin E-Cdk2 complex is maximally active at the G1/S transition, and overexpression of cyclin E decreases the time it takes the cell to complete G1 and enter S phase. We have now found that mammalian cells express two forms of cyclin E protein which differ from each other by the presence or absence of a 15-amino-acid amino-terminal domain. These proteins are encoded by alternatively spliced mRNAs and are localized to the nucleus during late G1 and early S phase. Fibroblasts engineered to constitutively overexpress either form of cyclin E showed elevated cyclin E-dependent kinase activity and a shortened G1 phase of the cell cycle. The overexpressed cyclin E protein was detected in the nucleus during all cell cycle phases, including G0. Although the cyclin E protein could be overexpressed in quiescent cells, the cyclin E-Cdk2 complex was inactive. It was not activated until 6 to 8 h after readdition of serum, 4 h earlier than the endogenous cyclin E-Cdk2. This premature activation of cyclin E-Cdk2 was consistent with the extent of G1 shortening caused by cyclin E overexpression. Microinjection of affinity-purified anti-cyclin E antibodies during G1 inhibited entry into S phase, whereas microinjection performed near the G1/S transition was ineffective. These results demonstrate that cyclin E is necessary for entry into S phase. Moreover, we found that cyclin E, in contrast to cyclin D1, was required for the G1/S transition even in cells lacking retinoblastoma protein function. Therefore, cyclins E and D1 control two different transitions within the human cell cycle. PMID:7739542

  5. Production of rhesus monkey cloned embryos expressing monomeric red fluorescent protein by interspecies somatic cell nuclear transfer.

    PubMed

    Zhu, Hai-Ying; Kang, Jin-Dan; Li, Suo; Jin, Jun-Xue; Hong, Yu; Jin, Long; Guo, Qing; Gao, Qing-Shan; Yan, Chang-Guo; Yin, Xi-Jun

    2014-02-21

    Interspecies somatic cell nuclear transfer (iSCNT) is a promising method to clone endangered animals from which oocytes are difficult to obtain. Monomeric red fluorescent protein 1 (mRFP1) is an excellent selection marker for transgenically modified cloned embryos during somatic cell nuclear transfer (SCNT). In this study, mRFP-expressing rhesus monkey cells or porcine cells were transferred into enucleated porcine oocytes to generate iSCNT and SCNT embryos, respectively. The development of these embryos was studied in vitro. The percentage of embryos that underwent cleavage did not significantly differ between iSCNT and SCNT embryos (P>0.05; 71.53% vs. 80.30%). However, significantly fewer iSCNT embryos than SCNT embryos reached the blastocyst stage (2.04% vs. 10.19%, P<0.05). Valproic acid was used in an attempt to increase the percentage of iSCNT embryos that developed to the blastocyst stage. However, the percentages of embryos that underwent cleavage and reached the blastocyst stage were similar between untreated iSCNT embryos and iSCNT embryos treated with 2mM valproic acid for 24h (72.12% vs. 70.83% and 2.67% vs. 2.35%, respectively). These data suggest that porcine-rhesus monkey interspecies embryos can be generated that efficiently express mRFP1. However, a significantly lower proportion of iSCNT embryos than SCNT embryos reach the blastocyst stage. Valproic acid does not increase the percentage of porcine-rhesus monkey iSCNT embryos that reach the blastocyst stage. The mechanisms underling nuclear reprogramming and epigenetic modifications in iSCNT need to be investigated further. PMID:24491539

  6. Cloning of a protein arginine methyltransferase PRMT1 homologue from Schistosoma mansoni: Evidence for roles in nuclear receptor signaling and RNA metabolism

    SciTech Connect

    Mansure, Jose Joao; Furtado, Daniel Rodrigues; Bastos de Oliveira, Francisco Meirelles; Rumjanek, Franklin David; Franco, Gloria Regina; Fantappie, Marcelo Rosado . E-mail: fantappie@bioqmed.ufrj.br

    2005-10-07

    The most studied arginine methyltransferase is the type I enzyme, which catalyzes the transfer of an S-adenosyl-L-methionine to a broad spectrum of substrates, including histones, RNA-transporting proteins, and nuclear hormone receptor coactivators. We cloned a cDNA encoding a protein arginine methyltransferase in Schistosoma mansoni (SmPRMT1). SmPRMT1 is highly homologous to the vertebrate PRMT1 enzyme. In vitro methylation assays showed that SmPRMT1 recombinant protein was able to specifically methylate histone H4. Two schistosome proteins likely to be involved in RNA metabolism, SMYB1 and SmSmD3, that display a number of RGG motifs, were strongly methylated by SmPRMT1. In vitro GST pull-down assays showed that SMYB1 and SmSmD3 physically interacted with SmPRMT1. Additional GST pull-down assay suggested the occurrence of a ternary complex including SmPRMT1, SmRXR1 nuclear receptor, and the p160 (SRC-1) nuclear receptor coactivator. Together, these data suggest a mechanism by which SmPRMT1 plays a role in nuclear receptor-mediated chromatin remodeling and RNA transactions.

  7. The nuclear protein Sam68 is cleaved by the FMDV 3C protease redistributing Sam68 to the cytoplasm during FMDV infection of host cells

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Infection by a variety of viruses alters the nuclear-cytoplasmic trafficking of certain host cell proteins. In our continued search for interacting factors, we reported the re-localization of RNA helicase A (RHA) from the nucleus to the cytoplasm in cells infected with foot-and-mouth disease virus ...

  8. A ?XaV motif in the Rift Valley fever virus NSs protein is essential for degrading p62, forming nuclear filaments and virulence

    PubMed Central

    Cyr, Normand; de la Fuente, Cynthia; Lecoq, Lauriane; Guendel, Irene; Chabot, Philippe R.; Kehn-Hall, Kylene; Omichinski, James G.

    2015-01-01

    Rift Valley fever virus (RVFV) is a single-stranded RNA virus capable of inducing fatal hemorrhagic fever in humans. A key component of RVFV virulence is its ability to form nuclear filaments through interactions between the viral nonstructural protein NSs and the host general transcription factor TFIIH. Here, we identify an interaction between a ?XaV motif in NSs and the p62 subunit of TFIIH. This motif in NSs is similar to ?XaV motifs found in nucleotide excision repair (NER) factors and transcription factors known to interact with p62. Structural and biophysical studies demonstrate that NSs binds to p62 in a similar manner as these other factors. Functional studies in RVFV-infected cells show that the ?XaV motif is required for both nuclear filament formation and degradation of p62. Consistent with the fact that the RVFV can be distinguished from other Bunyaviridae-family viruses due to its ability to form nuclear filaments in infected cells, the motif is absent in the NSs proteins of other Bunyaviridae-family viruses. Taken together, our studies demonstrate that p62 binding to NSs through the ?XaV motif is essential for degrading p62, forming nuclear filaments and enhancing RVFV virulence. In addition, these results show how the RVFV incorporates a simple motif into the NSs protein that enables it to functionally mimic host cell proteins that bind the p62 subunit of TFIIH. PMID:25918396

  9. Interaction of transportin-SR2 with Ras-related nuclear protein (Ran) GTPase.

    PubMed

    Taltynov, Oliver; Demeulemeester, Jonas; Christ, Frauke; De Houwer, Stéphanie; Tsirkone, Vicky G; Gerard, Melanie; Weeks, Stephen D; Strelkov, Sergei V; Debyser, Zeger

    2013-08-30

    The human immunodeficiency virus type 1 (HIV-1) and other lentiviruses are capable of infecting non-dividing cells and, therefore, need to be imported into the nucleus before integration into the host cell chromatin. Transportin-SR2 (TRN-SR2, Transportin-3, TNPO3) is a cellular karyopherin implicated in nuclear import of HIV-1. A model in which TRN-SR2 imports the viral preintegration complex into the nucleus is supported by direct interaction between TRN-SR2 and HIV-1 integrase (IN). Residues in the C-terminal domain of HIV-1 IN that mediate binding to TRN-SR2 were recently delineated. As for most nuclear import cargoes, the driving force behind HIV-1 preintegration complex import is likely a gradient of the GDP- and GTP-bound forms of Ran, a small GTPase. In this study we offer biochemical and structural characterization of the interaction between TRN-SR2 and Ran. By size exclusion chromatography we demonstrate stable complex formation of TRN-SR2 and RanGTP in solution. Consistent with the behavior of normal nuclear import cargoes, HIV-1 IN is released from the complex with TRN-SR2 by RanGTP. Although in concentrated solutions TRN-SR2 by itself was predominantly present as a dimer, the TRN-SR2-RanGTP complex was significantly more compact. Further analysis supported a model wherein one monomer of TRN-SR2 is bound to one monomer of RanGTP. Finally, we present a homology model of the TRN-SR2-RanGTP complex that is in excellent agreement with the experimental small angle x-ray scattering data. PMID:23878195

  10. Oxidative Damage and Nuclear Factor Erythroid 2-Related Factor 2 Protein Expression in Normal Skin and Keloid Tissue

    PubMed Central

    Lee, Yoon Jin; Kwon, Sun Bum; Kim, Chul Han; Cho, Hyun Deuk; Nam, Hae Seon; Lee, Sang Han; Lee, Mi Woo; Nam, Doo Hyun; Choi, Chang Yong

    2015-01-01

    Background Reactive oxygen species (ROS) play an important role in the induction of apoptosis under pathological conditions. Recently, a significant increase in ROS production and disrupted apoptosis mechanisms in keloids have been reported. Nuclear factor erythroid 2-related factor 2 (Nrf2) represents one of the most important cellular defense mechanisms against oxidative stress and is implicated in the regulation of apoptosis. Recently, it has been reported that Nrf2 upregulates Bcl-2, an anti-apoptotic protein. Objective To compare Nrf2 protein expression in normal skin tissues to keloid tissues. Methods ROS generation in keloid tissues was evaluated with OxyBlot analysis. Western blotting and/or immunohistochemical staining approaches were used to study expression of Nrf2 or Bcl-2 in keloid and normal skin tissues. Cellular fractionation was performed to examine subcellular distribution of Nrf2. Transfection of fibroblasts with Nrf2-specific small interfering RNA (siRNA) was conducted to understand the relationship between Nrf2 expression and apoptosis induction. Results Protein oxidation, a marker of oxidative stress, is increased in keloid tissues. Western blot analysis clearly showed that Nrf2 and Bcl-2 are downregulated in keloid tissues. Immunohistochemical staining of Nrf2 confirmed the results of the western blot analysis. Transfection of fibroblasts with the Nrf2-specific siRNA results in increased apoptosis and decreased cell viability. Conclusion Collectively, our data indicate that Nrf2 expression is downregulated in keloid tissues, and that Nrf2 is involved in the development of apoptosis in Nrf2 siRNA-transfected fibroblasts. We propose that a defective antioxidant system and apoptotic dysregulation may participate in keloid pathogenesis. PMID:26512164

  11. Transcriptional Activity and Nuclear Localization of Cabut, the Drosophila Ortholog of Vertebrate TGF-?-Inducible Early-Response Gene (TIEG) Proteins

    PubMed Central

    Belacortu, Yaiza; Weiss, Ron; Kadener, Sebastian; Paricio, Nuria

    2012-01-01

    Background Cabut (Cbt) is a C2H2-class zinc finger transcription factor involved in embryonic dorsal closure, epithelial regeneration and other developmental processes in Drosophila melanogaster. Cbt orthologs have been identified in other Drosophila species and insects as well as in vertebrates. Indeed, Cbt is the Drosophila ortholog of the group of vertebrate proteins encoded by the TGF-ß-inducible early-response genes (TIEGs), which belong to Sp1-like/Krüppel-like family of transcription factors. Several functional domains involved in transcriptional control and subcellular localization have been identified in the vertebrate TIEGs. However, little is known of whether these domains and functions are also conserved in the Cbt protein. Methodology/Principal Findings To determine the transcriptional regulatory activity of the Drosophila Cbt protein, we performed Gal4-based luciferase assays in S2 cells and showed that Cbt is a transcriptional repressor and able to regulate its own expression. Truncated forms of Cbt were then generated to identify its functional domains. This analysis revealed a sequence similar to the mSin3A-interacting repressor domain found in vertebrate TIEGs, although located in a different part of the Cbt protein. Using ?-Galactosidase and eGFP fusion proteins, we also showed that Cbt contains the bipartite nuclear localization signal (NLS) previously identified in TIEG proteins, although it is non-functional in insect cells. Instead, a monopartite NLS, located at the amino terminus of the protein and conserved across insects, is functional in Drosophila S2 and Spodoptera exigua Sec301 cells. Last but not least, genetic interaction and immunohistochemical assays suggested that Cbt nuclear import is mediated by Importin-?2. Conclusions/Significance Our results constitute the first characterization of the molecular mechanisms of Cbt-mediated transcriptional control as well as of Cbt nuclear import, and demonstrate the existence of similarities and differences in both aspects of Cbt function between the insect and the vertebrate TIEG proteins. PMID:22359651

  12. Coordinate regulation/localization of the carbohydrate responsive binding protein (ChREBP) by two nuclear export signal sites: Discovery of a new leucine-rich nuclear export signal site

    SciTech Connect

    Fukasawa, Masashi; Ge, Qing; Wynn, R. Max; Ishii, Seiji; Uyeda, Kosaku; Dallas Veterans Affairs Medical Center, Dallas, TX 75216

    2010-01-08

    Carbohydrate response element binding protein (ChREBP) is responsible for conversion of dietary carbohydrate to storage fat in liver by coordinating expression of the enzymes that channel glycolytic pyruvate into lipogenesis. The activation of ChREBP in response to high glucose is nuclear localization and transcription, and the inactivation of ChREBP under low glucose involves export from the nucleus to the cytosol. Here we report a new nuclear export signal site ('NES1') of ChREBP. Together these signals provide ChREBP with two NES sequences, both the previously reported NES2 and now the new NES1 coordinate to interact together with CRM1 (exportin) for nuclear export of the carbohydrate response element binding protein.

  13. Candesartan ameliorates acute myocardial infarction in rats through inducible nitric oxide synthase, nuclear factor??B, monocyte chemoattractant protein?1, activator protein?1 and restoration of heat shock protein 72.

    PubMed

    Lin, Xuefeng; Wu, Min; Liu, Bo; Wang, Junkui; Guan, Gongchang; Ma, Aiqun; Zhang, Yong

    2015-12-01

    Candesartan, an angiotensin II type 1 receptor antagonist, has a variety of biological activities, including antioxidant, anti?inflammatory and anticancer activities, with specific pharmacological effects. The present study investigated the mechanisms and protective effect of candesartan on acute myocardial infarction in rats. Male Wistar rats (8?week?old) were induced as a model of acute myocardial infarction and treated with candesartan (0.25 mg/kg) for 2 weeks. The present study first measured the activities of casein kinase (CK), the MB isoenzyme of creatine kinase (CK?MB) and lactate dehydrogenase (LDH), the level of cardiac troponin T (cTnT) and infarct size. Subsequently, western blot analysis was performed to analyze the protein expression levels of inducible nitric oxide synthase (iNOS) and heat shock protein 72 (HSP72) in the rats. An enzyme linked immunosorbent assay was used to detect iNOS and nuclear factor??B (NF??B) activity. In addition, gene expression levels of monocyte chemotactic protein?1 (MCP?1) and activating protein?1 (AP?1) were determined using reverse transcription?quantitative polymerase chain reaction analysis. Finally, the activities of caspase?3 and caspase?9 were examined using colorimetric assay kits. In the serum of the rat model of acute myocardial infarction, candesartan significantly increased the activities of CK, CK?MB and LDH, and the level of cTnT. The infarction size was perfected by candesartan treatment. Candesartan significantly reduced the protein expression and activity of iNOS, the activity of NF??B p65, and the gene expression levels of MCP?1 and AP?1 in the rat model of acute myocardial infarction. Candesartan increased the protein expression of HSP?72 in the acute myocardial infarction rat model. However, candesartan did not effect the levels of caspase?3 or caspase?9 in the rat model of acute myocardial infarction. These results suggested that candesartan ameliorates acute myocardial infarction in rats through iNOS, NF??B, MCP?1 and AP?1, and the restoration of HSP72. PMID:26499133

  14. Significance of host cell kinases in herpes simplex virus type 1 egress and lamin-associated protein disassembly from the nuclear lamina

    PubMed Central

    Leach, Natalie R.; Roller, Richard J.

    2010-01-01

    The nuclear lamina is thought to be a steric barrier to the herpesvirus capsid. Disruption of the lamina accompanied by phosphorylation of lamina proteins is a conserved feature of herpesvirus infection. In HSV-1-infected cells, protein kinase C (PKC) alpha and delta isoforms are recruited to the nuclear membrane and PKC delta has been implicated in phosphorylation of emerin and lamin B. We tested two critical hypotheses about the mechanism and significance of lamina disruption. First, we show that chemical inhibition of all PKC isoforms reduced viral growth five-fold and inhibited capsid egress from the nucleus. However, specific inhibition of either conventional PKCs or PKC delta does not inhibit viral growth. Second, we show hyperphosphorylation of emerin by viral and cellular kinases is required for its disassociation from the lamina. These data support hypothesis that phosphorylation of lamina components mediates lamina disruption during HSV nuclear egress. PMID:20674954

  15. Protein nanocages for self-triggered nuclear delivery of DNA-targeted chemotherapeutics in Cancer Cells.

    PubMed

    Bellini, Michela; Mazzucchelli, Serena; Galbiati, Elisabetta; Sommaruga, Silvia; Fiandra, Luisa; Truffi, Marta; Rizzuto, Maria A; Colombo, Miriam; Tortora, Paolo; Corsi, Fabio; Prosperi, Davide

    2014-12-28

    A genetically engineered apoferritin variant consisting of 24 heavy-chain subunits (HFn) was produced to achieve a cumulative delivery of an antitumor drug, which exerts its cytotoxic action by targeting the DNA at the nucleus of human cancer cells with subcellular precision. The rationale of our approach is based on exploiting the natural arsenal of defense of cancer cells to stimulate them to recruit large amounts of HFn nanoparticles loaded with doxorubicin inside their nucleus in response to a DNA damage, which leads to a programmed cell death. After demonstrating the selectivity of HFn for representative cancer cells compared to healthy fibroblasts, doxorubicin-loaded HFn was used to treat the cancer cells. The results from confocal microscopy and DNA damage assays proved that loading of doxorubicin in HFn nanoparticles increased the nuclear delivery of the drug, thus enhancing doxorubicin efficacy. Doxorubicin-loaded HFn acts as a "Trojan Horse": HFn was internalized in cancer cells faster and more efficiently compared to free doxorubicin, then promptly translocated into the nucleus following the DNA damage caused by the partial release in the cytoplasm of encapsulated doxorubicin. This self-triggered translocation mechanism allowed the drug to be directly released in the nuclear compartment, where it exerted its toxic action. This approach was reliable and straightforward providing an antiproliferative effect with high reproducibility. The particular self-assembling nature of HFn nanocage makes it a versatile and tunable nanovector for a broad range of molecules suitable both for detection and treatment of cancer cells. PMID:25312541

  16. The Interaction of CRM1 and the Nuclear Pore Protein Tpr

    PubMed Central

    Moussavi-Baygi, Ruhollah; Mofrad, Mohammad R. K.

    2014-01-01

    While much has been devoted to the study of transport mechanisms through the nuclear pore complex (NPC), the specifics of interactions and binding between export transport receptors and the NPC periphery have remained elusive. Recent work has demonstrated a binding interaction between the exportin CRM1 and the unstructured carboxylic tail of Tpr, on the nuclear basket. Strong evidence suggests that this interaction is vital to the functions of CRM1. Using molecular dynamics simulations and a newly refined method for determining binding regions, we have identified nine candidate binding sites on CRM1 for C-Tpr. These include two adjacent to RanGTP – from which one is blocked in the absence of RanGTP – and three next to the binding region of the cargo Snurportin. We report two additional interaction sites between C-Tpr and Snurportin, suggesting a possible role for Tpr import into the nucleus. Using bioinformatics tools we have conducted conservation analysis and functional residue prediction investigations to identify which parts of the obtained binding sites are inherently more important and should be highlighted. Also, a novel measure based on the ratio of available solvent accessible surface (RASAS) is proposed for monitoring the ligand/receptor binding process. PMID:24722547

  17. Nuclear inheritance of erythromycin resistance in human cells: New class of mitochondrial protein synthesis mutants

    SciTech Connect

    Doersen, C.J.; Stanbridge, E.J.

    1982-06-01

    The characterization of two new erythromycin-resistant mutants of HeLa cells is described. The strains ERY2305 and ERY2309 both exhibited resistance to erythromycin in growth assays and cell-free mitochondrial protein synthesis assays. The erythromycin resistance phenotype could not be transferred by cybridization. The mutation appeared to be encoded in the nucleus and inherited as a recessive trait. These two mutants, therefore, represent a new class of erythromycin-resistant mutants in human cells that is distinct from the cytoplasmically inherited mutation in strain ERY2301 described previously.

  18. Angiogenin-induced protein kinase B/Akt activation is necessary for angiogenesis but is independent of nuclear translocation of angiogenin in HUVE cells

    SciTech Connect

    Kim, Hye-Mi; Kang, Dong-Ku; Kim, Hak Yong; Kang, Sang Sun; Chang, Soo-Ik . E-mail: sichang@cbnu.ac.kr

    2007-01-12

    Angiogenin, a potent angiogenic factor, binds to endothelial cells and is endocytosed and rapidly translocated to and concentrated in the nucleolus where it binds to DNA. In this study, we report that angiogenin induces transient phosphorylation of protein kinase B/Akt in cultured human umbilical vein endothelial (HUVE) cells. LY294002 inhibits the angiogenin-induced protein kinase B/Akt activation and also angiogenin-induced cell migration in vitro as well as angiogenesis in chick embryo chorioallantoic membrane in vivo without affecting nuclear translocation of angiogenin in HUVE cells. These results suggest that cross-talk between angiogenin and protein kinase B/Akt signaling pathways is essential for angiogenin-induced angiogenesis in vitro and in vivo, and that angiogenin-induced PKB/Akt activation is independent of nuclear translocation of angiogenin in HUVE cells.

  19. DAYSLEEPER: a nuclear and vesicular-localized protein that is expressed in proliferating tissues

    PubMed Central

    2013-01-01

    Background DAYSLEEPER is a domesticated transposase that is essential for development in Arabidopsis thaliana [Nature, 436:282–284, 2005]. It is derived from a hAT-superfamily transposon and contains many of the features found in the coding sequence of these elements [Nature, 436:282–284, 2005, Genetics, 158:949–957, 2001]. This work sheds light on the expression of this gene and localization of its product in protoplasts and in planta. Using deletion constructs, important domains in the protein were identified. Results DAYSLEEPER is predominantly expressed in meristems, developing flowers and siliques. The protein is mainly localized in the nucleus, but can also be seen in discrete foci in the cytoplasm. Using several vesicular markers, we found that these foci belong to vesicular structures of the trans-golgi network, multivesicular bodies (MVB’s) and late endosomes. The central region as well as both the N- and the C-terminus are essential to DAYSLEEPER function, since versions of DAYSLEEPER deleted for these regions are not able to complement the daysleeper phenotype. Like hAT-transposases, we show that DAYSLEEPER has a functionally conserved dimerization domain [J Biol Chem, 282:7563–7575, 2007]. Conclusions DAYSLEEPER has retained the global structure of hAT transposases and it seems that most of these conserved features are essential to DAYSLEEPER’s cellular function. Although structurally similar, DAYSLEEPER seems to have broadened its range of action beyond the nucleus in comparison to transposases. PMID:24330683

  20. Human genome-wide RNAi screen reveals a role for nuclear pore proteins in poxvirus morphogenesis

    PubMed Central

    Sivan, Gilad; Martin, Scott E.; Myers, Timothy G.; Buehler, Eugen; Szymczyk, Krysia H.; Ormanoglu, Pinar; Moss, Bernard

    2013-01-01

    Poxviruses are considered less dependent on host functions than other DNA viruses because of their cytoplasmic site of replication and large genomes, which encode enzymes for DNA and mRNA synthesis. Nevertheless, RNAi screens with two independent human genome-scale libraries have identified more than 500 candidate genes that significantly inhibited and a similar number that enhanced replication and spread of infectious vaccinia virus (VACV). Translational, ubiquitin-proteosome, and endoplasmic reticulum-to-Golgi transport functions, known to be important for VACV, were enriched in the siRNA-inhibiting group, and RNA polymerase II and associated functions were enriched in the siRNA-enhancing group. Additional findings, notably the inhibition of VACV spread by siRNAs to several nuclear pore genes, were unanticipated. Knockdown of nucleoporin 62 strongly inhibited viral morphogenesis, with only a modest effect on viral gene expression, recapitulating and providing insight into previous studies with enucleated cells. PMID:23401514

  1. Nuclear relocalisation of cytoplasmic poly(A)-binding proteins PABP1 and PABP4 in response to UV irradiation reveals mRNA-dependent export of metazoan PABPs

    PubMed Central

    Burgess, Hannah M.; Richardson, William A.; Anderson, Ross C.; Salaun, Christine; Graham, Sheila V.; Gray, Nicola K.

    2011-01-01

    Poly(A)-binding protein 1 (PABP1) has a fundamental role in the regulation of mRNA translation and stability, both of which are crucial for a wide variety of cellular processes. Although generally a diffuse cytoplasmic protein, it can be found in discrete foci such as stress and neuronal granules. Mammals encode several additional cytoplasmic PABPs that remain poorly characterised, and with the exception of PABP4, appear to be restricted in their expression to a small number of cell types. We have found that PABP4, similarly to PABP1, is a diffusely cytoplasmic protein that can be localised to stress granules. However, UV exposure unexpectedly relocalised both proteins to the nucleus. Nuclear relocalisation of PABPs was accompanied by a reduction in protein synthesis but was not linked to apoptosis. In examining the mechanism of PABP relocalisation, we found that it was related to a change in the distribution of poly(A) RNA within cells. Further investigation revealed that this change in RNA distribution was not affected by PABP knockdown but that perturbations that block mRNA export recapitulate PABP relocalisation. Our results support a model in which nuclear export of PABPs is dependent on ongoing mRNA export, and that a block in this process following UV exposure leads to accumulation of cytoplasmic PABPs in the nucleus. These data also provide mechanistic insight into reports that transcriptional inhibitors and expression of certain viral proteins cause relocation of PABP to the nucleus. PMID:21940797

  2. Proton nuclear magnetic resonance and fluorescence spectroscopic studies of segmental mobility in aequorin and a green fluorescent protein from Aequorea forskalea

    SciTech Connect

    Nageswara Rao, B.D.; Kemple, M.D.; Prendergast, F.G.

    1980-10-01

    Aequorin is a protein of low molecular weight (20,000) isolated from the jellyfish Aequorea forskalea which emits blue light upon the binding of Ca/sup 2 +/ ions. This bioluminescence requires neither exogenous oxygen nor any other cofactors. The light emission occurs from an excited state of a chromophore (an imidazolopyrazinone) which is tightly and noncovalently bound to the protein. Apparently the binding of Ca/sup 2 +/ by the protein induces changes in the protein conformation which allow oxygen, already bound or otherwise held by the protein, to react with and therein oxidize the chromophore. The resulting discharged protein remains intact, with the Ca/sup 2 +/ and the chromophore still bound, but is incapable of further luminescence. The fluorescence spectrum of this discharged protein and the bioluminescence spectrum of the original charged aequorin are identical. A green fluorescent protein (GFP) of approx. 30,000 mol wt isolated from the same organism, functions in vivo as an acceptor of energy from aequorin and subsequently emits green light. We are applying proton nuclear magnetic resonance (NMR) spectroscopy and fluorescence spectroscopy to examine structural details of, and fluctuations associated with the luminescent reaction of aequorin and the in vivo energy transfer from aequorin to the GFP.

  3. Isotope-detected 1H NMR studies of proteins: a general strategy for editing interproton nuclear Overhauser effects by heteronuclear decoupling, with application to phage lambda repressor.

    PubMed Central

    Weiss, M A; Redfield, A G; Griffey, R H

    1986-01-01

    A strategy for editing interproton nuclear Overhauser effects (NOEs) in proteins is proposed and illustrated. Selective incorporation of 13C- (or 15N)-labeled amino acids into a protein permits NOEs involving the labeled residues to be identified by heteronuclear difference decoupling. Such heteronuclear editing simplifies the NOE difference spectrum and avoids ambiguities due to spin diffusion. Isotope-detected 1H NMR thus opens to study proteins too large for conventional one- and two-dimensional NMR methods (20-75 kDa). We have applied this strategy to the N-terminal domain of phage lambda repressor, a protein of dimer molecular mass 23 kDa. A tertiary NOE from an internal aromatic ring (Phe-51) to a beta-13C-labeled alanine residue (Ala-62) is demonstrated. PMID:3006046

  4. Nuclear matrix-associated protein SMAR1 regulates alternative splicing via HDAC6-mediated deacetylation of Sam68.

    PubMed

    Nakka, Kiran Kumar; Chaudhary, Nidhi; Joshi, Shruti; Bhat, Jyotsna; Singh, Kulwant; Chatterjee, Subhrangsu; Malhotra, Renu; De, Abhijit; Santra, Manas Kumar; Dilworth, F Jeffrey; Chattopadhyay, Samit

    2015-06-30

    Pre-mRNA splicing is a complex regulatory nexus modulated by various trans-factors and their posttranslational modifications to create a dynamic transcriptome through alternative splicing. Signal-induced phosphorylation and dephosphorylation of trans-factors are known to regulate alternative splicing. However, the role of other posttranslational modifications, such as deacetylation/acetylation, methylation, and ubiquitination, that could modulate alternative splicing in either a signal-dependent or -independent manner remain enigmatic. Here, we demonstrate that Scaffold/matrix-associated region-binding protein 1 (SMAR1) negatively regulates alternative splicing through histone deacetylase 6 (HDAC6)-mediated deacetylation of RNA-binding protein Sam68 (Src-associated substrate during mitosis of 68 kDa). SMAR1 is enriched in nuclear splicing speckles and associates with the snRNAs that are involved in splice site recognition. ERK-MAPK pathway that regulates alternative splicing facilitates ERK-1/2-mediated phosphorylation of SMAR1 at threonines 345 and 360 and localizes SMAR1 to the cytoplasm, preventing its interaction with Sam68. We showed that endogenously, SMAR1 through HDAC6 maintains Sam68 in a deacetylated state. However, knockdown or ERK-mediated phosphorylation of SMAR1 releases the inhibitory SMAR1-HDAC6-Sam68 complex, facilitating Sam68 acetylation and alternative splicing. Furthermore, loss of heterozygosity at the Chr.16q24.3 locus in breast cancer cells, wherein the human homolog of SMAR1 (BANP) has been mapped, enhances Sam68 acetylation and CD44 variant exon inclusion. In addition, tail-vein injections in mice with human breast cancer MCF-7 cells depleted for SMAR1 showed increased CD44 variant exon inclusion and concomitant metastatic propensity, confirming the functional role of SMAR1 in regulation of alternative splicing. Thus, our results reveal the complex molecular mechanism underlying SMAR1-mediated signal-dependent and -independent regulation of alternative splicing via Sam68 deacetylation. PMID:26080397

  5. Nuclear matrix-associated protein SMAR1 regulates alternative splicing via HDAC6-mediated deacetylation of Sam68

    PubMed Central

    Nakka, Kiran Kumar; Chaudhary, Nidhi; Joshi, Shruti; Bhat, Jyotsna; Singh, Kulwant; Chatterjee, Subhrangsu; Malhotra, Renu; De, Abhijit; Santra, Manas Kumar; Dilworth, F. Jeffrey; Chattopadhyay, Samit

    2015-01-01

    Pre-mRNA splicing is a complex regulatory nexus modulated by various trans-factors and their posttranslational modifications to create a dynamic transcriptome through alternative splicing. Signal-induced phosphorylation and dephosphorylation of trans-factors are known to regulate alternative splicing. However, the role of other posttranslational modifications, such as deacetylation/acetylation, methylation, and ubiquitination, that could modulate alternative splicing in either a signal-dependent or -independent manner remain enigmatic. Here, we demonstrate that Scaffold/matrix-associated region-binding protein 1 (SMAR1) negatively regulates alternative splicing through histone deacetylase 6 (HDAC6)-mediated deacetylation of RNA-binding protein Sam68 (Src-associated substrate during mitosis of 68 kDa). SMAR1 is enriched in nuclear splicing speckles and associates with the snRNAs that are involved in splice site recognition. ERK–MAPK pathway that regulates alternative splicing facilitates ERK-1/2–mediated phosphorylation of SMAR1 at threonines 345 and 360 and localizes SMAR1 to the cytoplasm, preventing its interaction with Sam68. We showed that endogenously, SMAR1 through HDAC6 maintains Sam68 in a deacetylated state. However, knockdown or ERK-mediated phosphorylation of SMAR1 releases the inhibitory SMAR1–HDAC6–Sam68 complex, facilitating Sam68 acetylation and alternative splicing. Furthermore, loss of heterozygosity at the Chr.16q24.3 locus in breast cancer cells, wherein the human homolog of SMAR1 (BANP) has been mapped, enhances Sam68 acetylation and CD44 variant exon inclusion. In addition, tail-vein injections in mice with human breast cancer MCF-7 cells depleted for SMAR1 showed increased CD44 variant exon inclusion and concomitant metastatic propensity, confirming the functional role of SMAR1 in regulation of alternative splicing. Thus, our results reveal the complex molecular mechanism underlying SMAR1-mediated signal-dependent and -independent regulation of alternative splicing via Sam68 deacetylation. PMID:26080397

  6. Neutron scattering and nuclear magnetic resonance spectroscopy structural studies of protein-DNA complexes

    SciTech Connect

    Bradbury, E.M.; Catasti, P.; Chen, X.; Gupta, G.; Imai, B.; Moyzis, R.; Ratliff, R.; Velupillai, S.

    1996-03-01

    This is the final report of a one-year, Laboratory-Directed Research and Development (LDRD) project at the Los Alamos National Laboratory (LANL). The project sought to employ advanced biophysical measurements to study the structure of nucleosomes and the structure of origins of DNA replication. The fundamental repeating unit of human chromosomes is the nucleosome, which contains about 200 base pairs of DNA and 9 histone proteins. Genome replication is strictly associated with the reversible acetylations of histones that unfold chromatin to allow access of factors to origins of DNA replications. The authors have studied two major structural problems: (1) the effects of histone acetylation on nucleosome structure, and (2) the structure of DNA origins of replication. They have recently completed preliminary X-ray scattering experiments at Stanford on positioned nucleosomes with defined DNA sequence and length, histone composition and level of acetylation. These experiments have shown that lengths of the DNA and acetylations of the histone H4 result in nucleosome structural changes. To understand internucleosomal interactions and the roles of histone H1 the authors have made preliminary x-ray scatter studies on native dinucleosomes that have demonstrated the feasibility of these experiments. The DNA sequence of the yeast replication origin has been synthesized for structure determination by multi-dimensional NMR spectroscopy.

  7. Nuclear matrix binding protein SMAR1 regulates T-cell differentiation and allergic airway disease.

    PubMed

    Chemmannur, S V; Badhwar, A J; Mirlekar, B; Malonia, S K; Gupta, M; Wadhwa, N; Bopanna, R; Mabalirajan, U; Majumdar, S; Ghosh, B; Chattopadhyay, S

    2015-11-01

    Asthma is a complex airway allergic disease involving the interplay of various cell types, cytokines, and transcriptional factors. Though many factors contribute to disease etiology, the molecular control of disease phenotype and responsiveness is not well understood. Here we report an essential role of the matrix attachment region (MAR)-binding protein SMAR1 in regulating immune response during allergic airway disease. Conditional knockout of SMAR1 in T cells rendered the mice resistant to eosinophilic airway inflammation against ovalbumin (OVA) allergen with low immunoglobulin E (IgE) and interleukin-5 (IL-5) levels. Moreover, a lower IgE/IgG2a ratio and higher interferon-? (IFN-?) response suggested aberrant skewing of T-cell differentiation toward type 1 helper T cell (Th1) response. We show that SMAR1 functions as a negative regulator of Th1 and Th17 differentiation by interacting with two potential and similar MAR regions present on the promoters of T-bet and IL-17. Thus, we present SMAR1 as a regulator of T-cell differentiation that favors the establishment of Th2 cells by modulating Th1 and Th17 responses. PMID:25736456

  8. The extracellular release of Schistosoma mansoni HMGB1 nuclear protein is mediated by acetylation

    SciTech Connect

    Coutinho Carneiro, Vitor; Moraes Maciel, Renata de; Caetano de Abreu da Silva, Isabel; Furtado Madeira da Costa, Rodrigo; Neto Paiva, Claudia; Torres Bozza, Marcelo; Rosado Fantappie, Marcelo

    2009-12-25

    Schistosoma mansoni HMGB1 (SmHMGB1) was revealed to be a substrate for the parasite histone acetyltransferases SmGCN5 and SmCBP1. We found that full-length SmHMGB1, as well as its HMG-box B (but not HMG-box A) were acetylated in vitro by SmGCN5 and SmCBP1. However, SmCBP1 was able to acetylate both substrates more efficiently than SmGCN5. Interestingly, the removal of the C-terminal acidic tail of SmHMGB1 (SmHMGB1{Delta}C) resulted in increased acetylation of the protein. We showed by mammalian cell transfection assays that SmHMGB1 and SmHMGB1{Delta}C were transported from the nucleus to the cytoplasm after sodium butyrate (NaB) treatment. Importantly, after NaB treatment, SmHMGB1 was also present outside the cell. Together, our data suggest that acetylation of SmHMGB1 plays a role in cellular trafficking, culminating with its secretion to the extracellular milieu. The possible role of SmHMGB1 acetylation in the pathogenesis of schistosomiasis is discussed.

  9. Production of rhesus monkey cloned embryos expressing monomeric red fluorescent protein by interspecies somatic cell nuclear transfer

    SciTech Connect

    Zhu, Hai-Ying; Kang, Jin-Dan; Li, Suo; Jin, Jun-Xue; Hong, Yu; Jin, Long; Guo, Qing; Gao, Qing-Shan; Yan, Chang-Guo; Yin, Xi-Jun

    2014-02-21

    Highlights: • Rhesus monkey cells were electroporated with a plasmid containing mRFP1, and an mRFP1-expressing cell line was generated. • For the first time, mRFP1-expressing rhesus monkey cells were used as donor cells for iSCNT. • The effect of VPA on the development of embryos cloned using iSCNT was determined. - Abstract: Interspecies somatic cell nuclear transfer (iSCNT) is a promising method to clone endangered animals from which oocytes are difficult to obtain. Monomeric red fluorescent protein 1 (mRFP1) is an excellent selection marker for transgenically modified cloned embryos during somatic cell nuclear transfer (SCNT). In this study, mRFP-expressing rhesus monkey cells or porcine cells were transferred into enucleated porcine oocytes to generate iSCNT and SCNT embryos, respectively. The development of these embryos was studied in vitro. The percentage of embryos that underwent cleavage did not significantly differ between iSCNT and SCNT embryos (P > 0.05; 71.53% vs. 80.30%). However, significantly fewer iSCNT embryos than SCNT embryos reached the blastocyst stage (2.04% vs. 10.19%, P < 0.05). Valproic acid was used in an attempt to increase the percentage of iSCNT embryos that developed to the blastocyst stage. However, the percentages of embryos that underwent cleavage and reached the blastocyst stage were similar between untreated iSCNT embryos and iSCNT embryos treated with 2 mM valproic acid for 24 h (72.12% vs. 70.83% and 2.67% vs. 2.35%, respectively). These data suggest that porcine-rhesus monkey interspecies embryos can be generated that efficiently express mRFP1. However, a significantly lower proportion of iSCNT embryos than SCNT embryos reach the blastocyst stage. Valproic acid does not increase the percentage of porcine-rhesus monkey iSCNT embryos that reach the blastocyst stage. The mechanisms underling nuclear reprogramming and epigenetic modifications in iSCNT need to be investigated further.

  10. Proteomic analysis of the major cellular proteins of bovine trophectoderm cell lines derived from IVP, parthenogenetic, and nuclear transfer embryos

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Nuclear cloning of cattle is currently very inefficient in terms of the production and survival of nuclear cloned calves. Because of the great promise of using nuclear cloning technology in farm animals to genetically improve their production and quality traits, nuclear cloning in cattle, and other...

  11. Yeast Srp1, a nuclear protein related to Drosophila and mouse pendulin, is required for normal migration, division, and integrity of nuclei during mitosis.

    PubMed

    Küssel, P; Frasch, M

    1995-08-21

    This paper describes genes from yeast and mouse with significant sequence similarities to a Drosophila gene that encodes the blood cell tumor suppressor pendulin. The protein encoded by the yeast gene, Srp1p, and mouse pendulin share 42% and 51% amino acid identity with Drosophila pendulin, respectively. All three proteins consist of 10.5 degenerate tandem repeats of approximately 42 amino acids each. Similar repeats occur in a superfamily of proteins that includes the Drosophila Armadillo protein. All three proteins contain a consensus sequence for a bipartite nuclear localization signal (NLS) in the N-terminal domain, which is not part of the repeat structure. Confocal microscopic analysis of yeast cells stained with antibodies against Srp1p reveals that this protein is intranuclear throughout the cell cycle. Targeted gene disruption shows that SRP1 is an essential gene. Despite their sequence similarities, Drosophila and mouse pendulin are unable to rescue the lethality of an SRP1 disruption. We demonstrate that yeast cells depleted of Srp1p arrest in mitosis with a G2 content of DNA. Arrested cells display abnormal structures and orientations of the mitotic spindles, aberrant segregation of the chromatin and the nuclei, and threads of chromatin emanating from the bulk of nuclear DNA. This phenotype suggests that Srp1p is required for the normal function of microtubules and the spindle pole bodies, as well as for nuclear integrity. We suggest that Srp1p interacts with multiple components of the cell nucleus that are required for mitosis and discuss its functional similarities to, and differences from Drosophila pendulin. PMID:7565597

  12. A redundant nuclear protein binding site contributes to negative regulation of the mouse mammary tumor virus long terminal repeat.

    PubMed Central

    Bramblett, D; Hsu, C L; Lozano, M; Earnest, K; Fabritius, C; Dudley, J

    1995-01-01

    The tissue specificity of mouse mammary tumor virus (MMTV) expression is controlled by regulatory elements in the MMTV long terminal repeat (LTR). These regulatory elements include the hormone response element, located approximately between -200 and -75, as well as binding sites for NF-1, Oct-1 (OTF-1), and mammary gland enhancer factors. Naturally occurring MMTV deletion variants isolated from T-cell and kidney tumors, transgenic-mouse experiments with MMTV LTR deletions, and transient transfection assays with LTR constructs indicate that there are additional transcription regulatory elements, including a negative regulatory element (NRE), located upstream of the hormone response element. To further define this regulatory region, we have constructed a series of BAL 31 deletion mutants in the MMTV LTR for use in transient transfection assays. These assays indicated that deletion of two regions (referred to as promoter-distal and -proximal NREs) between -637 and -201 elevated basal MMTV promoter activity in the absence of glucocorticoids. The region between -637 and -264 was surveyed for the presence of nuclear protein binding sites by gel retardation assays. Only one type of protein complex (referred to as NRE-binding protein or NBP) bound exclusively to sites that mapped to the promoter-distal and -proximal NREs identified by BAL 31 mutations. The promoter-proximal binding site was mapped further by linker substitution mutations and transfection assays. Mutations that mapped to a region containing an inverted repeat beginning at -287 relative to the start of transcription elevated basal expression of a reporter gene driven by the MMTV LTR. A 59-bp DNA fragment from the distal NRE also bound the NBP complex. Gel retardation assays showed that mutations within both inverted repeats of the proximal NRE eliminated NBP binding and mutations within single repeats altered NBP binding. Intriguingly, the NBP complex was detected in extracts from T cells and lung cells but was absent from mammary gland cells. These results suggest that a factor contributing to high-level expression of MMTV in the mammary gland is the lack of negative regulation by NBP. PMID:7494299

  13. Nuclear-receptor–mediated regulation of drug– and bile-acid–transporter proteins in gut and liver

    PubMed Central

    Staudinger, Jeff L.; Woody, Sarah; Sun, Mengxi; Cui, Wenqi

    2015-01-01

    Adverse drug events (ADEs) are a common cause of patient morbidity and mortality and are classically thought to result, in part, from variation in expression and activity of hepatic enzymes of drug metabolism. It is now known that alterations in the expression of genes that encode drug- and bile-acid–transporter proteins in both the gut and liver play a previously unrecognized role in determining patient drug response and eventual clinical outcome. Four nuclear receptor (NR) superfamily members, including pregnane X receptor (PXR, NR1I2), constitutive androstane receptor (NR1I3), farnesoid X receptor (NR1H4), and vitamin D receptor (NR1I1), play pivotal roles in drug- and bile-acid– activated programs of gene expression to coordinately regulate drug- and bile-acid transport activity in the intestine and liver. This review focuses on the NR-mediated gene activation of drug and bile-acid transporters in these tissues as well as the possible underlying molecular mechanisms. PMID:23330541

  14. Nuclear Factor-?B, p38, and Stress-Activated Protein Kinase Mitogen-Activated Protein Kinase Signaling Pathways Regulate Proinflammatory Cytokines and Apoptosis in Human Placental Explants in Response to Oxidative Stress

    PubMed Central

    Cindrova-Davies, Tereza; Spasic-Boskovic, Olivera; Jauniaux, Eric; Charnock-Jones, D. Stephen; Burton, Graham J.

    2007-01-01

    Preeclampsia is a potentially fatal complication of human pregnancy characterized by hypertension, proteinuria, and edema. Placental oxidative stress is a key element in the pathogenesis of the syndrome and results in the release of a cocktail of factors, including proinflammatory cytokines and apoptotic debris, that in turn cause activation of the maternal endothelium. The intermediary molecular mechanisms underlying this release are unknown, but they represent a potential target for therapeutic interventions. We examined activation of signaling pathways during hypoxia-reoxygenation of villous explants in vitro. Hypoxia-reoxygenation activated the p38 and stress-activated protein kinase mitogen-activated protein kinase (MAPK) and the nuclear factor-?B pathways. Downstream consequences included increased tissue concentrations and secretion of tumor necrosis factor-? and interleukin-1?, increased expression of cyclooxygenase-2, and increased apoptosis. Administration of vitamins C and E to explants blocked activation of the p38 and stress-activated protein kinase MAPK and nuclear factor-?B pathways. Vitamin administration or p38 pathway inhibition also reduced cyclooxygenase-2 expression, tumor necrosis factor-? and interleukin-1? secretion, and the levels of apoptosis. We conclude that oxidative stress is a potent inducer of placental synthesis and release of proinflammatory factors. Most of these effects are mediated through the p38 MAPK and nuclear factor-?B pathways and can be effectively blocked by vitamins C and E in vitro. PMID:17456758

  15. An early function of the adenoviral E1B 55 kDa protein is required for the nuclear relocalization of the cellular p53 protein in adenovirus-infected normal human cells

    SciTech Connect

    Cardoso, F.M.; Kato, Sayuri E.M.; Huang Wenying; Flint, S. Jane; Gonzalez, Ramon A.

    2008-09-01

    It is well established that the human subgroup C adenovirus type 5 (Ad5) E1B 55 kDa protein can regulate the activity and concentration of the cellular tumor suppressor, p53. However, the contribution(s) of these functions of the E1B protein to viral reproduction remains unclear. To investigate this issue, we examined properties of p53 in normal human cells infected by E1B mutant viruses that display defective entry into the late phase or viral late mRNA export. The steady-state concentrations of p53 were significantly higher in cells infected by the E1B 55 kDa null mutant Hr6 or three mutants carrying small insertions in the E1B 55 kDa protein coding sequence than in Ad5-infected cells. Nevertheless, none of the mutants induced apoptosis in infected cells. Rather, the localization of p53 to E1B containing nuclear sites observed during infection by Ad5 was prevented by mutations that impair interaction of the E1B protein with p53 and/or with the E4 Orf6 protein. These results indicate that the E1B protein fulfills an early function that correlates efficient entry into the late phase with the localization of E1B and p53 in the nucleus of Ad5-infected normal human cells.

  16. G Run-mediated Recognition of Proteolipid Protein and DM20 5 Splice Sites by U1 Small Nuclear RNA Is Regulated by

    E-print Network

    Hertel, Klemens J.

    G Run-mediated Recognition of Proteolipid Protein and DM20 5 Splice Sites by U1 Small Nuclear RNARNP complexes (8). The recognition of the 5 splice site by the RNA moiety of the U1snRNP, through direct base pairing between the 5 end of the U1snRNA and the 5 splice site of the pre-RNA, commits the pre-RNA

  17. The use of additive and subtractive approaches to examine the nuclear localization sequence of the polyomavirus major capsid protein VP1

    NASA Technical Reports Server (NTRS)

    Chang, D.; Haynes, J. I. 2nd; Brady, J. N.; Consigli, R. A.; Spooner, B. S. (Principal Investigator)

    1992-01-01

    A nuclear localization signal (NLS) has been identified in the N-terminal (Ala1-Pro-Lys-Arg-Lys-Ser-Gly-Val-Ser-Lys-Cys11) amino acid sequence of the polyomavirus major capsid protein VP1. The importance of this amino acid sequence for nuclear transport of VP1 protein was demonstrated by a genetic "subtractive" study using the constructs pSG5VP1 (full-length VP1) and pSG5 delta 5'VP1 (truncated VP1, lacking amino acids Ala1-Cys11). These constructs were used to transfect COS-7 cells, and expression and intracellular localization of the VP1 protein was visualized by indirect immunofluorescence. These studies revealed that the full-length VP1 was expressed and localized in the nucleus, while the truncated VP1 protein was localized in the cytoplasm and not transported to the nucleus. These findings were substantiated by an "additive" approach using FITC-labeled conjugates of synthetic peptides homologous to the NLS of VP1 cross-linked to bovine serum albumin or immunoglobulin G. Both conjugates localized in the nucleus after microinjection into the cytoplasm of 3T6 cells. The importance of individual amino acids found in the basic sequence (Lys3-Arg-Lys5) of the NLS was also investigated. This was accomplished by synthesizing three additional peptides in which lysine-3 was substituted with threonine, arginine-4 was substituted with threonine, or lysine-5 was substituted with threonine. It was found that lysine-3 was crucial for nuclear transport, since substitution of this amino acid with threonine prevented nuclear localization of the microinjected, FITC-labeled conjugate.

  18. The Molecular Chaperone TRiC/CCT Binds to the Trp-Asp 40 (WD40) Repeat Protein WDR68 and Promotes Its Folding, Protein Kinase DYRK1A Binding, and Nuclear Accumulation*

    PubMed Central

    Miyata, Yoshihiko; Shibata, Takeshi; Aoshima, Masato; Tsubata, Takuichi; Nishida, Eisuke

    2014-01-01

    Trp-Asp (WD) repeat protein 68 (WDR68) is an evolutionarily conserved WD40 repeat protein that binds to several proteins, including dual specificity tyrosine phosphorylation-regulated protein kinase (DYRK1A), MAPK/ERK kinase kinase 1 (MEKK1), and Cullin4-damage-specific DNA-binding protein 1 (CUL4-DDB1). WDR68 affects multiple and diverse physiological functions, such as controlling anthocyanin synthesis in plants, tissue growth in insects, and craniofacial development in vertebrates. However, the biochemical basis and the regulatory mechanism of WDR68 activity remain largely unknown. To better understand the cellular function of WDR68, here we have isolated and identified cellular WDR68 binding partners using a phosphoproteomic approach. More than 200 cellular proteins with wide varieties of biochemical functions were identified as WDR68-binding protein candidates. Eight T-complex protein 1 (TCP1) subunits comprising the molecular chaperone TCP1 ring complex/chaperonin-containing TCP1 (TRiC/CCT) were identified as major WDR68-binding proteins, and phosphorylation sites in both WDR68 and TRiC/CCT were identified. Co-immunoprecipitation experiments confirmed the binding between TRiC/CCT and WDR68. Computer-aided structural analysis suggested that WDR68 forms a seven-bladed ?-propeller ring. Experiments with a series of deletion mutants in combination with the structural modeling showed that three of the seven ?-propeller blades of WDR68 are essential and sufficient for TRiC/CCT binding. Knockdown of cellular TRiC/CCT by siRNA caused an abnormal WDR68 structure and led to reduction of its DYRK1A-binding activity. Concomitantly, nuclear accumulation of WDR68 was suppressed by the knockdown of TRiC/CCT, and WDR68 formed cellular aggregates when overexpressed in the TRiC/CCT-deficient cells. Altogether, our results demonstrate that the molecular chaperone TRiC/CCT is essential for correct protein folding, DYRK1A binding, and nuclear accumulation of WDR68. PMID:25342745

  19. The Inner Nuclear Membrane Protein Src1 Is Required for Stable Post-Mitotic Progression into G1 in Aspergillus nidulans

    PubMed Central

    Osmani, Aysha H.; Osmani, Stephen A.

    2015-01-01

    How membranes and associated proteins of the nuclear envelope (NE) are assembled specifically and inclusively around segregated genomes during exit from mitosis is incompletely understood. Inner nuclear membrane (INM) proteins play key roles by providing links between DNA and the NE. In this study we have investigated the highly conserved INM protein Src1 in Aspergillus nidulans and have uncovered a novel cell cycle response during post mitotic formation of G1 nuclei. Live cell imaging indicates Src1 could have roles during mitotic exit as it preferentially locates to the NE abscission points during nucleokinesis and to the NE surrounding forming daughter G1 nuclei. Deletion analysis further supported this idea revealing that although Src1 is not required for interphase progression or mitosis it is required for stable post-mitotic G1 nuclear formation. This conclusion is based upon the observation that in the absence of Src1 newly formed G1 nuclei are structurally unstable and immediately undergo architectural modifications typical of mitosis. These changes include NPC modifications that stop nuclear transport as well as disassembly of nucleoli. More intriguingly, the newly generated G1 nuclei then cycle between mitotic- and interphase-like states. The findings indicate that defects in post-mitotic G1 nuclear formation caused by lack of Src1 promote repeated failed attempts to generate stable G1 nuclei. To explain this unexpected phenotype we suggest a type of regulation that promotes repetition of defective cell cycle transitions rather than preventing progression past the defective cell cycle transition. We suggest the term “reboot regulation” to define this mode of cell cycle regulation. The findings are discussed in relationship to recent studies showing the Cdk1 master oscillator can entrain subservient oscillators that when uncoupled cause cell cycle transitions to be repeated. PMID:26147902

  20. Hepatitis B Virus X Protein Stabilizes Cyclin D1 and Increases Cyclin D1 Nuclear Accumulation through ERK-Mediated Inactivation of GSK-3?.

    PubMed

    Chen, Xiangmei; Zhang, Ling; Zheng, Sujun; Zhang, Ting; Li, Meng; Zhang, Xiaolei; Zeng, Zhenzhen; McCrae, Malcolm A; Zhao, Jingmin; Zhuang, Hui; Lu, Fengmin

    2015-05-01

    The Hepatitis B virus X protein (HBx) contributes centrally to the pathogenesis of hepatocellular carcinoma (HCC). It has been suggested that the transcriptional activation of cyclin D1 by HBx is implicated in the development of HCC. However, numerous studies have shown that overexpression of cyclin D1 alone is not sufficient to drive oncogenic transformation. Herein, we investigated whether HBx can stabilize cyclin D1 and induce cyclin D1 protein nuclear accumulation, and thereby accelerate hepatocarcinogenesis. The effects of HBx on cyclin D1 stabilization were assessed in cell-based transfection, Western blot, immunoprecipitation, immunocytofluorescence staining, and flow-cytometric assays. The results demonstrated that ectopic expression of HBx in HCC cells could extend the half-life of cyclin D1 protein from 40-60 minutes to 80-110 minutes. HBx stabilized cyclin D1 primarily in the S phase of the cell cycle, in a manner dependent on the inactivation of GSK-3?, which was mediated by ERK activation. HBx also prompted the nuclear accumulation of cyclin D1, and cotransfection of the constitutively active mutant of GSK-3? along with HBx could reverse the nuclear accumulation and subsequent cell proliferation induced by HBx. Further, a positive correlation between HBx and nuclear cyclin D1 level was established in HCC specimens detected by an immunohistochemical assay. Taken together, our results indicated that HBx could stabilize and increase cyclin D1 nuclear accumulation through ERK-mediated inactivation of GSK-3?. This HBx-induced cyclin D1 upregulation might play an important role in HCC development and progression. PMID:25712050

  1. The Herpes Simplex Virus Protein pUL31 Escorts Nucleocapsids to Sites of Nuclear Egress, a Process Coordinated by Its N-Terminal Domain

    PubMed Central

    Nagel, Claus-Henning; Binz, Anne; Sodeik, Beate; Bauerfeind, Rudolf; Bailer, Susanne M.

    2015-01-01

    Progeny capsids of herpesviruses leave the nucleus by budding through the nuclear envelope. Two viral proteins, the membrane protein pUL34 and the nucleo-phosphoprotein pUL31 form the nuclear egress complex that is required for capsid egress out of the nucleus. All pUL31 orthologs are composed of a diverse N-terminal domain with 1 to 3 basic patches and a conserved C-terminal domain. To decipher the functions of the N-terminal domain, we have generated several Herpes simplex virus mutants and show here that the N-terminal domain of pUL31 is essential with basic patches being critical for viral propagation. pUL31 and pUL34 entered the nucleus independently of each other via separate routes and the N-terminal domain of pUL31 was required to prevent their premature interaction in the cytoplasm. Unexpectedly, a classical bipartite nuclear localization signal embedded in this domain was not required for nuclear import of pUL31. In the nucleus, pUL31 associated with the nuclear envelope and newly formed capsids. Viral mutants lacking the N-terminal domain or with its basic patches neutralized still associated with nucleocapsids but were unable to translocate them to the nuclear envelope. Replacing the authentic basic patches with a novel artificial one resulted in HSV1(17+)Lox-UL31-hbpmp1mp2, that was viable but delayed in nuclear egress and compromised in viral production. Thus, while the C-terminal domain of pUL31 is sufficient for the interaction with nucleocapsids, the N-terminal domain was essential for capsid translocation to sites of nuclear egress and a coordinated interaction with pUL34. Our data indicate an orchestrated sequence of events with pUL31 binding to nucleocapsids and escorting them to the inner nuclear envelope. We propose a common mechanism for herpesviral nuclear egress: pUL31 is required for intranuclear translocation of nucleocapsids and subsequent interaction with pUL34 thereby coupling capsid maturation with primary envelopment. PMID:26083367

  2. The Herpes Simplex Virus Protein pUL31 Escorts Nucleocapsids to Sites of Nuclear Egress, a Process Coordinated by Its N-Terminal Domain.

    PubMed

    Funk, Christina; Ott, Melanie; Raschbichler, Verena; Nagel, Claus-Henning; Binz, Anne; Sodeik, Beate; Bauerfeind, Rudolf; Bailer, Susanne M

    2015-06-01

    Progeny capsids of herpesviruses leave the nucleus by budding through the nuclear envelope. Two viral proteins, the membrane protein pUL34 and the nucleo-phosphoprotein pUL31 form the nuclear egress complex that is required for capsid egress out of the nucleus. All pUL31 orthologs are composed of a diverse N-terminal domain with 1 to 3 basic patches and a conserved C-terminal domain. To decipher the functions of the N-terminal domain, we have generated several Herpes simplex virus mutants and show here that the N-terminal domain of pUL31 is essential with basic patches being critical for viral propagation. pUL31 and pUL34 entered the nucleus independently of each other via separate routes and the N-terminal domain of pUL31 was required to prevent their premature interaction in the cytoplasm. Unexpectedly, a classical bipartite nuclear localization signal embedded in this domain was not required for nuclear import of pUL31. In the nucleus, pUL31 associated with the nuclear envelope and newly formed capsids. Viral mutants lacking the N-terminal domain or with its basic patches neutralized still associated with nucleocapsids but were unable to translocate them to the nuclear envelope. Replacing the authentic basic patches with a novel artificial one resulted in HSV1(17+)Lox-UL31-hbpmp1mp2, that was viable but delayed in nuclear egress and compromised in viral production. Thus, while the C-terminal domain of pUL31 is sufficient for the interaction with nucleocapsids, the N-terminal domain was essential for capsid translocation to sites of nuclear egress and a coordinated interaction with pUL34. Our data indicate an orchestrated sequence of events with pUL31 binding to nucleocapsids and escorting them to the inner nuclear envelope. We propose a common mechanism for herpesviral nuclear egress: pUL31 is required for intranuclear translocation of nucleocapsids and subsequent interaction with pUL34 thereby coupling capsid maturation with primary envelopment. PMID:26083367

  3. Cervix carcinoma is associated with an up-regulation and nuclear localization of the dual-specificity protein phosphatase VHR

    PubMed Central

    Henkens, Rachel; Delvenne, Philippe; Arafa, Mohammad; Moutschen, Michel; Zeddou, Mustapha; Tautz, Lutz; Boniver, Jacques; Mustelin, Tomas; Rahmouni, Souad

    2008-01-01

    Background The 21-kDa Vaccinia virus VH1-related (VHR) dual-specific protein phosphatase (encoded by the DUSP3 gene) plays a critical role in cell cycle progression and is itself regulated during the cell cycle. We have previously demonstrated using RNA interference that cells lacking VHR arrest in the G1 and G2 phases of the cell cycle and show signs of beginning of cell senescence. Methods In this report, we evaluated successfully the expression levels of VHR protein in 62 hysterectomy or conization specimens showing the various (pre) neoplastic cervical epithelial lesions and 35 additional cases of hysterectomy performed for non-cervical pathologies, from patients under 50 years of age. We used a tissue microarray and IHC technique to evaluate the expression of the VHR phosphatase. Immunofluorescence staining under confocal microscopy, Western blotting and RT-PCR methods were used to investigate the localization and expression levels of VHR. Results We report that VHR is upregulated in (pre) neoplastic lesions (squamous intraepithelial lesions; SILs) of the uterine cervix mainly in high grade SIL (H-SIL) compared to normal exocervix. In the invasive cancer, VHR is also highly expressed with nuclear localization in the majority of cells compared to normal tissue where VHR is always in the cytoplasm. We also report that this phosphatase is highly expressed in several cervix cancer cell lines such as HeLa, SiHa, CaSki, C33 and HT3 compared to primary keratinocytes. The immunofluorescence technique under confocal microscopy shows that VHR has a cytoplasmic localization in primary keratinocytes, while it localizes in both cytoplasm and nucleus of the cancer cell lines investigated. We report that the up-regulation of this phosphatase is mainly due to its post-translational stabilization in the cancer cell lines compared to primary keratinocytes rather than increases in the transcription of DUSP3 locus. Conclusion These results together suggest that VHR can be considered as a new marker for cancer progression in cervix carcinoma and potential new target for anticancer therapy. PMID:18505570

  4. A Fungal Sarcolemmal Membrane-Associated Protein (SLMAP) Homolog Plays a Fundamental Role in Development and Localizes to the Nuclear Envelope, Endoplasmic Reticulum, and Mitochondria

    PubMed Central

    Nordzieke, Steffen; Zobel, Thomas; Fränzel, Benjamin; Wolters, Dirk A.

    2014-01-01

    Sarcolemmal membrane-associated protein (SLMAP) is a tail-anchored protein involved in fundamental cellular processes, such as myoblast fusion, cell cycle progression, and chromosomal inheritance. Further, SLMAP misexpression is associated with endothelial dysfunctions in diabetes and cancer. SLMAP is part of the conserved striatin-interacting phosphatase and kinase (STRIPAK) complex required for specific signaling pathways in yeasts, filamentous fungi, insects, and mammals. In filamentous fungi, STRIPAK was initially discovered in Sordaria macrospora, a model system for fungal differentiation. Here, we functionally characterize the STRIPAK subunit PRO45, a homolog of human SLMAP. We show that PRO45 is required for sexual propagation and cell-to-cell fusion and that its forkhead-associated (FHA) domain is essential for these processes. Protein-protein interaction studies revealed that PRO45 binds to STRIPAK subunits PRO11 and SmMOB3, which are also required for sexual propagation. Superresolution structured-illumination microscopy (SIM) further established that PRO45 localizes to the nuclear envelope, endoplasmic reticulum, and mitochondria. SIM also showed that localization to the nuclear envelope requires STRIPAK subunits PRO11 and PRO22, whereas for mitochondria it does not. Taken together, our study provides important insights into fundamental roles of the fungal SLMAP homolog PRO45 and suggests STRIPAK-related and STRIPAK-unrelated functions. PMID:25527523

  5. Immune activation mediated by the late blight resistance protein R1 requires nuclear localization of R1 and the effector AVR1.

    PubMed

    Du, Yu; Berg, Jeroen; Govers, Francine; Bouwmeester, Klaas

    2015-08-01

    Resistance against oomycete pathogens is mainly governed by intracellular nucleotide-binding leucine-rich repeat (NLR) receptors that recognize matching avirulence (AVR) proteins from the pathogen, RXLR effectors that are delivered inside host cells. Detailed molecular understanding of how and where NLR proteins and RXLR effectors interact is essential to inform the deployment of durable resistance (R) genes. Fluorescent tags, nuclear localization signals (NLSs) and nuclear export signals (NESs) were exploited to determine the subcellular localization of the potato late blight protein R1 and the Phytophthora infestans RXLR effector AVR1, and to target these proteins to the nucleus or cytoplasm. Microscopic imaging revealed that both R1 and AVR1 occurred in the nucleus and cytoplasm, and were in close proximity. Transient expression of NLS- or NES-tagged R1 and AVR1 in Nicotiana benthamiana showed that activation of the R1-mediated hypersensitive response and resistance required localization of the R1/AVR1 pair in the nucleus. However, AVR1-mediated suppression of cell death in the absence of R1 was dependent on localization of AVR1 in the cytoplasm. Balanced nucleocytoplasmic partitioning of AVR1 seems to be a prerequisite. Our results show that R1-mediated immunity is activated inside the nucleus with AVR1 in close proximity and suggest that nucleocytoplasmic transport of R1 and AVR1 is tightly regulated. PMID:25760731

  6. Complete genome sequence and integrated protein localization and interaction map for alfalfa dwarf virus, which combines properties of both cytoplasmic and nuclear plant rhabdoviruses.

    PubMed

    Bejerman, Nicolás; Giolitti, Fabián; de Breuil, Soledad; Trucco, Verónica; Nome, Claudia; Lenardon, Sergio; Dietzgen, Ralf G

    2015-09-01

    We have determined the full-length 14,491-nucleotide genome sequence of a new plant rhabdovirus, alfalfa dwarf virus (ADV). Seven open reading frames (ORFs) were identified in the antigenomic orientation of the negative-sense, single-stranded viral RNA, in the order 3'-N-P-P3-M-G-P6-L-5'. The ORFs are separated by conserved intergenic regions and the genome coding region is flanked by complementary 3' leader and 5' trailer sequences. Phylogenetic analysis of the nucleoprotein amino acid sequence indicated that this alfalfa-infecting rhabdovirus is related to viruses in the genus Cytorhabdovirus. When transiently expressed as GFP fusions in Nicotiana benthamiana leaves, most ADV proteins accumulated in the cell periphery, but unexpectedly P protein was localized exclusively in the nucleus. ADV P protein was shown to have a homotypic, and heterotypic nuclear interactions with N, P3 and M proteins by bimolecular fluorescence complementation. ADV appears unique in that it combines properties of both cytoplasmic and nuclear plant rhabdoviruses. PMID:26004251

  7. Concentrations of copper-binding proteins in livers of bluegills from the cooling lake at the H. B. Robinson Nuclear Power Station

    SciTech Connect

    Harrison, F.L.; Lam, J.R.

    1982-05-01

    Bluegills collected from the cooling lake of the H.B. Robinson Nuclear Power Station near the effluent discharge, near the water intake to the cooling system, and from a control population in a local pond were examined for total copper in muscle and liver tissues and metalloproteins in different compartments of liver tissues. Much lower concentrations of copper were found in muscle than in liver tissue. Also, copper changes in the environment were reflected in liver but not in muscle tissue. Liver metalloproteins were separated into low molecular weight (LMW), intermediate molecular weight (IMW), and high molecular weight (HMW) protein fractions using high performance liquid chromatography. Large differences in kinds and quantities of metals associated with metalloproteins were found. Copper concentrations in the LMW proteins (metallothionein-like proteins) were highest in bluegills from the discharge site and lowest in those from the control pond. Evidence of overloading of the metallothionein-like protein detoxification system was found in bluegills at the discharge site. These data and that from related studies indicate that the labile copper released from the cooling system of the H.B. Robinson Nuclear Power Station may be implicated in the increased deformities and reduced reproductive capacity found in the bluegill population in the adjacent cooling lake.

  8. A novel ligand for an SH3 domain of the adaptor protein Nck bears an SH2 domain and nuclear signaling motifs.

    PubMed

    Matuoka, K; Miki, H; Takahashi, K; Takenawa, T

    1997-10-20

    Nck is a small protein composed of Src homology regions (SH) 2 and 3, paralleling the adaptors c-Crk and Grb2/Ash, but its function remains enigmatic. To clarify Nck signaling, a human brain cDNA library was searched for targets of the SH3 moiety of Nck. A novel molecule detected therefrom (referred to as Nck-, Ash- and phospholipase Cgamma-binding protein 4) contained proline-rich sequences and, through the function of one of them, interacted with the middle SH3 domain of Nck. A NAP4 fusion peptide exhibited an affinity for Nck, Ash and phospholipase Cgamma in whole cell lysates. NAP4 also had an SH2 domain, which could bind to activated EGF receptor. These intermolecular interactions imply the intricacy of Nck-mediated signaling around the receptor protein-tyrosine kinases. In addition, NAP4 bore a putative nuclear localization signal and a Q-run/P-run composite, both characteristic of nuclear proteins, and might therefore relate to the presence of Nck in the cellular nucleus. PMID:9344857

  9. Eukaryotic features of the Xanthomonas type III effector AvrBs3: protein domains involved in transcriptional activation and the interaction with nuclear import receptors from pepper.

    PubMed

    Szurek, B; Marois, E; Bonas, U; Van den Ackerveken, G

    2001-06-01

    The AvrBs3 protein of the phytopathogenic bacterium Xanthomonas campestris pv. vesicatoria is targeted to host-plant cells by the bacterial Hrp type III secretion system. In pepper plants containing the Bs3 resistance gene, AvrBs3 induces the hypersensitive response (HR). AvrBs3 recognition is thought to occur in the plant cell nucleus as HR induction is dependent on nuclear localization signals (NLSs) and an acidic transcription activation domain (AAD). In a search for AvrBs3-interacting pepper proteins using the yeast two-hybrid system, we have isolated eight different classes of cDNA inserts including two genes for importin alpha proteins. Importin alpha is part of the nuclear import machinery and interacts with AvrBs3 through an NLS in the carboxy-terminus of the protein, both in yeast and in vitro. The mechanism of AvrBs3 recognition was further studied by analysis of the C-terminal AAD. This putative transcription-activation domain was shown to be required for AvrBs3 HR-inducing activity, and could be functionally replaced with the VP16 AAD from the Herpes simplex virus. Our data support the model in which the AvrBs3 effector localizes to the nucleus, where the Bs3-mediated surveillance system of resistant plants detects AvrBs3 through its interference with host gene transcription. PMID:11439138

  10. Polypyrimidine tract-binding protein and heterogeneous nuclear ribonucleoprotein A1 bind to human T-cell leukemia virus type 2 RNA regulatory elements.

    PubMed Central

    Black, A C; Luo, J; Watanabe, C; Chun, S; Bakker, A; Fraser, J K; Morgan, J P; Rosenblatt, J D

    1995-01-01

    Efficient expression of human T-cell leukemia virus (HTLV) and human immunodeficiency virus structural proteins requires Rx and Rev proteins, respectively. Decreased expression of Gag and Env appears to be due, in part, to intragenic RNA sequences, termed cis-acting repressive sequences (CRS), and may be mediated by binding of specific cellular factors. We demonstrated previously that two cellular proteins, p60CRS and p40CRS, interact with HTLV type 2.5' long terminal repeat CRS RNA and that the interaction of both proteins with CRS RNA correlates with function (A. C. Black, C. T. Ruland, J. Luo, A. Bakker, J. K. Fraser, and J. D. Rosenblatt, Virology 200:29-41, 1994). By radioimmunoprecipitation of HeLa nuclear proteins UV cross-linked to CRS RNAs with murine monoclonal antibodies, we now show that p40CRS is heterogeneous nuclear ribonucleoprotein (hnRNP) A1 and p60CRS is polypyrimidine tract-binding protein or hnRNP I. These immunoprecipitation results were confirmed by an immunobinding assay with hnRNP I and hnRNP AI antibodies and by cross-competition electrophoretic mobility shift experiments. In addition, we mapped a putative hnRNP A1 binding site in U5 RNA and demonstrated that p40CRS (hnRNP A1) binding to that site correlates with CRS function. Since both hnRNP I and hnRNP A1 have been shown to influence splicing and potentially other steps in RNA processing, the binding of both hnRNP I and hnRNP A1 to HTLV RNA regulatory elements may alter retrovirus RNA processing and may be involved in regulation by Rex. PMID:7474099

  11. The superior colliculus of the camel: a neuronal-specific nuclear protein (NeuN) and neuropeptide study

    PubMed Central

    Mensah-Brown, E P K; Garey, L J

    2006-01-01

    In this study we examined the superior colliculus of the midbrain of the one-humped (dromedary) camel, Camelus dromedarius, using Nissl staining and anti-neuronal-specific nuclear protein (NeuN) immunohistochemistry for total neuronal population as well as for the enkephalins, somatostatin (SOM) and substance P (SP). It was found that, unlike in most mammals, the superior colliculus is much larger than the inferior colliculus. The superior colliculus is concerned with visual reflexes and the co-ordination of head, neck and eye movements, which are certainly of importance to this animal with large eyes, head and neck, and apparently good vision. The basic neuronal architecture and lamination of the superior colliculus are similar to that in other mammals. However, we describe for the first time an unusually large content of neurons in the superior colliculus with strong immunoreactivity for met-enkephalin, an endogenous opioid. We classified the majority of these neurons as small (perimeters of 40–50 µm), and localized diffusely throughout the superficial grey and stratum opticum. In addition, large pyramidal-like neurons with perimeters of 100 µm and above were present in the intermediate grey layer. Large unipolar cells were located immediately dorsal to the deep grey layer. By contrast, small neurons (perimeters of 40–50 µm) immunopositive to SOM and SP were located exclusively in the superficial grey layer. We propose that this system may be associated with a pain-inhibiting pathway that has been described from the periaqueductal grey matter, juxtaposing the deep layers of the superior colliculus, to the lower brainstem and spinal cord. Such pain inhibition could be important in relation to the camel's life in the harsh environment of its native deserts, often living in very high temperatures with no shade and a diet consisting largely of thorny branches. PMID:16441568

  12. The superior colliculus of the camel: a neuronal-specific nuclear protein (NeuN) and neuropeptide study.

    PubMed

    Mensah-Brown, E P K; Garey, L J

    2006-02-01

    In this study we examined the superior colliculus of the midbrain of the one-humped (dromedary) camel, Camelus dromedarius, using Nissl staining and anti-neuronal-specific nuclear protein (NeuN) immunohistochemistry for total neuronal population as well as for the enkephalins, somatostatin (SOM) and substance P (SP). It was found that, unlike in most mammals, the superior colliculus is much larger than the inferior colliculus. The superior colliculus is concerned with visual reflexes and the co-ordination of head, neck and eye movements, which are certainly of importance to this animal with large eyes, head and neck, and apparently good vision. The basic neuronal architecture and lamination of the superior colliculus are similar to that in other mammals. However, we describe for the first time an unusually large content of neurons in the superior colliculus with strong immunoreactivity for met-enkephalin, an endogenous opioid. We classified the majority of these neurons as small (perimeters of 40-50 microm), and localized diffusely throughout the superficial grey and stratum opticum. In addition, large pyramidal-like neurons with perimeters of 100 microm and above were present in the intermediate grey layer. Large unipolar cells were located immediately dorsal to the deep grey layer. By contrast, small neurons (perimeters of 40-50 microm) immunopositive to SOM and SP were located exclusively in the superficial grey layer. We propose that this system may be associated with a pain-inhibiting pathway that has been described from the periaqueductal grey matter, juxtaposing the deep layers of the superior colliculus, to the lower brainstem and spinal cord. Such pain inhibition could be important in relation to the camel's life in the harsh environment of its native deserts, often living in very high temperatures with no shade and a diet consisting largely of thorny branches. PMID:16441568

  13. The Nuclear Factor (Erythroid-derived 2)-like 2 and Proteasome Maturation Protein Axis Mediate Bortezomib Resistance in Multiple Myeloma.

    PubMed

    Li, Bingzong; Fu, Jinxiang; Chen, Ping; Ge, Xueping; Li, Yali; Kuiatse, Isere; Wang, Hua; Wang, Huihan; Zhang, Xingding; Orlowski, Robert Z

    2015-12-11

    Resistance to the proteasome inhibitor bortezomib is an emerging clinical problem whose mechanisms have not been fully elucidated. We considered the possibility that this could be associated with enhanced proteasome activity in part through the action of the proteasome maturation protein (POMP). Bortezomib-resistant myeloma models were used to examine the correlation between POMP expression and bortezomib sensitivity. POMP expression was then modulated using genetic and pharmacologic approaches to determine the effects on proteasome inhibitor sensitivity in cell lines and in vivo models. Resistant cell lines were found to overexpress POMP, and while its suppression in cell lines enhanced bortezomib sensitivity, POMP overexpression in drug-naive cells conferred resistance. Overexpression of POMP was associated with increased levels of nuclear factor (erythroid-derived 2)-like (NRF2), and NRF2 was found to bind to and activate the POMP promoter. Knockdown of NRF2 in bortezomib-resistant cells reduced POMP levels and proteasome activity, whereas its overexpression in drug-naive cells increased POMP and proteasome activity. The NRF2 inhibitor all-trans-retinoic acid reduced cellular NRF2 levels and increased the anti-proliferative and pro-apoptotic activities of bortezomib in resistant cells, while decreasing proteasome capacity. Finally, the combination of all-trans-retinoic acid with bortezomib showed enhanced activity against primary patient samples and in a murine model of bortezomib-resistant myeloma. Taken together, these studies validate a role for the NRF2/POMP axis in bortezomib resistance and identify NRF2 and POMP as potentially attractive targets for chemosensitization to this proteasome inhibitor. PMID:26483548

  14. Amyloid fibril structure of peptides and proteins by magic angle spinning NMR spectroscopy and dynamic nuclear polarization

    E-print Network

    Debelouchina, Galia Tzvetanova

    2011-01-01

    Amyloid fibrils are insoluble, non-crystalline protein filaments associated with a number of diseases such as Alzheimer's and Type Il diabetes. They can have a functional role in different organisms and many proteins and ...

  15. Structure and function of the Influenza membrane protein M2 by magic angle spinning NMR and dynamic nuclear polarization

    E-print Network

    Andreas, Loren B

    2014-01-01

    Determination of the 3D structure of membrane proteins is a frontier that is rapidly being explored due to the importance of membrane proteins in regulating cellular processes and because they are the target of many drugs. ...

  16. Yeast Integral Membrane Proteins Apq12, Brl1, and Brr6 Form a Complex Important for Regulation of Membrane Homeostasis and Nuclear Pore Complex Biogenesis

    PubMed Central

    Lone, Museer A.; Atkinson, Aaron E.; Hodge, Christine A.; Cottier, Stéphanie; Martínez-Montañés, Fernando; Maithel, Shelley; Mène-Saffrané, Laurent

    2015-01-01

    Proper functioning of intracellular membranes is critical for many cellular processes. A key feature of membranes is their ability to adapt to changes in environmental conditions by adjusting their composition so as to maintain constant biophysical properties, including fluidity and flexibility. Similar changes in the biophysical properties of membranes likely occur when intracellular processes, such as vesicle formation and fusion, require dramatic changes in membrane curvature. Similar modifications must also be made when nuclear pore complexes (NPCs) are constructed within the existing nuclear membrane, as occurs during interphase in all eukaryotes. Here we report on the role of the essential nuclear envelope/endoplasmic reticulum (NE/ER) protein Brl1 in regulating the membrane composition of the NE/ER. We show that Brl1 and two other proteins characterized previously—Brr6, which is closely related to Brl1, and Apq12—function together and are required for lipid homeostasis. All three transmembrane proteins are localized to the NE and can be coprecipitated. As has been shown for mutations affecting Brr6 and Apq12, mutations in Brl1 lead to defects in lipid metabolism, increased sensitivity to drugs that inhibit enzymes involved in lipid synthesis, and strong genetic interactions with mutations affecting lipid metabolism. Mutations affecting Brl1 or Brr6 or the absence of Apq12 leads to hyperfluid membranes, because mutant cells are hypersensitive to agents that increase membrane fluidity. We suggest that the defects in nuclear pore complex biogenesis and mRNA export seen in these mutants are consequences of defects in maintaining the biophysical properties of the NE. PMID:26432634

  17. Yeast Integral Membrane Proteins Apq12, Brl1, and Brr6 Form a Complex Important for Regulation of Membrane Homeostasis and Nuclear Pore Complex Biogenesis.

    PubMed

    Lone, Museer A; Atkinson, Aaron E; Hodge, Christine A; Cottier, Stéphanie; Martínez-Montañés, Fernando; Maithel, Shelley; Mène-Saffrané, Laurent; Cole, Charles N; Schneiter, Roger

    2015-12-01

    Proper functioning of intracellular membranes is critical for many cellular processes. A key feature of membranes is their ability to adapt to changes in environmental conditions by adjusting their composition so as to maintain constant biophysical properties, including fluidity and flexibility. Similar changes in the biophysical properties of membranes likely occur when intracellular processes, such as vesicle formation and fusion, require dramatic changes in membrane curvature. Similar modifications must also be made when nuclear pore complexes (NPCs) are constructed within the existing nuclear membrane, as occurs during interphase in all eukaryotes. Here we report on the role of the essential nuclear envelope/endoplasmic reticulum (NE/ER) protein Brl1 in regulating the membrane composition of the NE/ER. We show that Brl1 and two other proteins characterized previously-Brr6, which is closely related to Brl1, and Apq12-function together and are required for lipid homeostasis. All three transmembrane proteins are localized to the NE and can be coprecipitated. As has been shown for mutations affecting Brr6 and Apq12, mutations in Brl1 lead to defects in lipid metabolism, increased sensitivity to drugs that inhibit enzymes involved in lipid synthesis, and strong genetic interactions with mutations affecting lipid metabolism. Mutations affecting Brl1 or Brr6 or the absence of Apq12 leads to hyperfluid membranes, because mutant cells are hypersensitive to agents that increase membrane fluidity. We suggest that the defects in nuclear pore complex biogenesis and mRNA export seen in these mutants are consequences of defects in maintaining the biophysical properties of the NE. PMID:26432634

  18. Yeast Nab3 Protein Contains a Self-assembly Domain Found in Human Heterogeneous Nuclear Ribonucleoprotein-C (hnRNP-C) That Is Necessary for Transcription Termination*

    PubMed Central

    Loya, Travis J.; O'Rourke, Thomas W.; Reines, Daniel

    2013-01-01

    Nab3 is an RNA-binding protein whose function is important for terminating transcription by RNA polymerase II. It co-assembles with Nrd1, and the resulting heterodimer of these heterogeneous nuclear ribonucleoprotein-C (hnRNP)-like proteins interacts with the nascent transcript and RNA polymerase II. Previous genetic analysis showed that a short carboxyl-terminal region of Nab3 is functionally important for termination and is located far from the Nab3 RNA recognition domain in the primary sequence. The domain is structurally homologous to hnRNP-C from higher organisms. Here we provide biochemical evidence that this short region is sufficient to enable self-assembly of Nab3 into a tetrameric form in a manner similar to the cognate region of human hnRNP-C. Within this region, there is a stretch of low complexity protein sequence (16 glutamines) adjacent to a putative ?-helix that potentiates the ability of the conserved region to self-assemble. The glutamine stretch and the final 18 amino acids of Nab3 are both important for termination in living yeast cells. The findings herein describe an additional avenue by which these hnRNP-like proteins can polymerize on target transcripts. This process is independent of, but acts in concert with, the interactions of the proteins with RNA and RNA polymerase and extends the relationship of Nab3 as a functional orthologue of a higher eukaryotic hnRNP. PMID:23192344

  19. A nuclear-encoded chloroplast protein harboring a single CRM domain plays an important role in the Arabidopsis growth and stress response

    PubMed Central

    2014-01-01

    Background Although several chloroplast RNA splicing and ribosome maturation (CRM) domain-containing proteins have been characterized for intron splicing and rRNA processing during chloroplast gene expression, the functional role of a majority of CRM domain proteins in plant growth and development as well as chloroplast RNA metabolism remains largely unknown. Here, we characterized the developmental and stress response roles of a nuclear-encoded chloroplast protein harboring a single CRM domain (At4g39040), designated CFM4, in Arabidopsis thaliana. Results Analysis of CFM4-GFP fusion proteins revealed that CFM4 is localized to chloroplasts. The loss-of-function T-DNA insertion mutants for CFM4 (cfm4) displayed retarded growth and delayed senescence, suggesting that CFM4 plays a role in growth and development of plants under normal growth conditions. In addition, cfm4 mutants showed retarded seed germination and seedling growth under stress conditions. No alteration in the splicing patterns of intron-containing chloroplast genes was observed in the mutant plants, but the processing of 16S and 4.5S rRNAs was abnormal in the mutant plants. Importantly, CFM4 was determined to possess RNA chaperone activity. Conclusions These results suggest that the chloroplast-targeted CFM4, one of two Arabidopsis genes encoding a single CRM domain-containing protein, harbors RNA chaperone activity and plays a role in the Arabidopsis growth and stress response by affecting rRNA processing in chloroplasts. PMID:24739417

  20. Oestrogen facilitates the binding of ubiquitous and liver-enriched nuclear proteins to the apoVLDL II promoter in vivo.

    PubMed

    Wijnholds, J; Muller, E; Ab, G

    1991-01-11

    Using genomic and in vitro DNasel footprinting, we have analyzed protein-DNA interactions within the promoter region of the oestrogen-inducible gene encoding chicken apoVLDL II. The footprints coincide with previously detected guanosine-protein contacts in vivo. All footprints identified are present in the apoVLDL II-expressing liver exclusively and absent in hormone-naive liver, spleen and oviduct. They comprise recognition sites for the oestrogen receptor, the ubiquitous COUP-transcription factor, the liver-enriched C/EBP and/or DBP and the liver-specific LF-A1. In vitro, binding of protein to the oestrogen response element (ERE) is excluded by the prior binding of a protein, possibly C/EBP or DBP, to an adjacent element. The recognition sequence of the COUP-TF is also a target for LF-A1. The results suggests that oestrogen-dependent liver specific activation of the apoVLDL II promoter is established by the binding of the oestrogen receptor to EREs and multiple liver-enriched factors (C/EBP, DBP and LF-A1) to their nearby recognition sequences. Apparently, several DNA binding nuclear proteins cooperate to keep the promoter in a state that is accessible for the RNA polymerase complex. PMID:2011511

  1. Oestrogen facilitates the binding of ubiquitous and liver-enriched nuclear proteins to the apoVLDL II promoter in vivo.

    PubMed Central

    Wijnholds, J; Muller, E; Ab, G

    1991-01-01

    Using genomic and in vitro DNasel footprinting, we have analyzed protein-DNA interactions within the promoter region of the oestrogen-inducible gene encoding chicken apoVLDL II. The footprints coincide with previously detected guanosine-protein contacts in vivo. All footprints identified are present in the apoVLDL II-expressing liver exclusively and absent in hormone-naive liver, spleen and oviduct. They comprise recognition sites for the oestrogen receptor, the ubiquitous COUP-transcription factor, the liver-enriched C/EBP and/or DBP and the liver-specific LF-A1. In vitro, binding of protein to the oestrogen response element (ERE) is excluded by the prior binding of a protein, possibly C/EBP or DBP, to an adjacent element. The recognition sequence of the COUP-TF is also a target for LF-A1. The results suggests that oestrogen-dependent liver specific activation of the apoVLDL II promoter is established by the binding of the oestrogen receptor to EREs and multiple liver-enriched factors (C/EBP, DBP and LF-A1) to their nearby recognition sequences. Apparently, several DNA binding nuclear proteins cooperate to keep the promoter in a state that is accessible for the RNA polymerase complex. Images PMID:2011511

  2. The 21.5-kDa isoform of myelin basic protein has a non-traditional PY-nuclear-localization signal

    SciTech Connect

    Smith, Graham S.T.; Seymour, Lauren V.; Boggs, Joan M.; Harauz, George

    2012-06-15

    Highlights: Black-Right-Pointing-Pointer Full-length 21.5-kDa MBP isoform is translocated to the nucleus. Black-Right-Pointing-Pointer We hypothesized that the exon-II-encoded sequence contained the NLS. Black-Right-Pointing-Pointer We mutated this sequence in RFP-tagged constructs and transfected N19-cells. Black-Right-Pointing-Pointer Abolition of two key positively-charged residues resulted in loss of nuclear-trafficking. Black-Right-Pointing-Pointer The 21.5-kDa isoform of classic MBP contains a non-traditional PY-NLS. -- Abstract: The predominant 18.5-kDa classic myelin basic protein (MBP) is mainly responsible for compaction of the myelin sheath in the central nervous system, but is multifunctional, having numerous interactions with Ca{sup 2+}-calmodulin, actin, tubulin, and SH3-domains, and can tether these proteins to a lipid membrane in vitro. The full-length 21.5-kDa MBP isoform has an additional 26 residues encoded by exon-II of the classic gene, which causes it to be trafficked to the nucleus of oligodendrocytes (OLGs). We have performed site-directed mutagenesis of selected residues within this segment in red fluorescent protein (RFP)-tagged constructs, which were then transfected into the immortalized N19-OLG cell line to view protein localization using epifluorescence microscopy. We found that 21.5-kDa MBP contains two non-traditional PY-nuclear-localization signals, and that arginine and lysine residues within these motifs were involved in subcellular trafficking of this protein to the nucleus, where it may have functional roles during myelinogenesis.

  3. A Novel Gene SbSI-2 Encoding Nuclear Protein from a Halophyte Confers Abiotic Stress Tolerance in E. coli and Tobacco

    PubMed Central

    Yadav, Narendra Singh; Singh, Vijay Kumar; Singh, Dinkar; Jha, Bhavanath

    2014-01-01

    Salicornia brachiata is an extreme halophyte that grows luxuriantly in coastal marshes. Previously, we have reported isolation and characterization of ESTs from Salicornia with large number of novel/unknown salt-responsive gene sequences. In this study, we have selected a novel salt-inducible gene SbSI-2 (Salicornia brachiata salt-inducible-2) for functional characterization. Bioinformatics analysis revealed that SbSI-2 protein has predicted nuclear localization signals and a strong protein-protein interaction domain. Transient expression of the RFP:SbSI2 fusion protein confirmed that SbSI-2 is a nuclear-localized protein. Genomic organization study showed that SbSI-2 is intronless and has a single copy in Salicornia genome. Quantitative RT-PCR analysis revealed higher SbSI-2 expression under salt stress and desiccation conditions. The SbSI-2 gene was transformed in E. coli and tobacco for functional characterization. pET28a-SbSI-2 recombinant E. coli cells showed higher tolerance to desiccation and salinity compared to vector alone. Transgenic tobacco plants overexpressing SbSI-2 have improved salt- and osmotic tolerance, accompanied by better growth parameters, higher relative water content, elevated accumulation of compatible osmolytes, lower Na+ and ROS accumulation and lesser electrolyte leakage than the wild-type. Overexpression of the SbSI-2 also enhanced transcript levels of ROS-scavenging genes and some stress-related transcription factors under salt and osmotic stresses. Taken together, these results demonstrate that SbSI-2 might play an important positive modulation role in abiotic stress tolerance. This identifies SbSI-2 as a novel determinant of salt/osmotic tolerance and suggests that it could be a potential bioresource for engineering abiotic stress tolerance in crop plants. PMID:24999628

  4. Expression of EphA2 protein is positively associated with age, tumor size and Fuhrman nuclear grade in clear cell renal cell carcinomas

    PubMed Central

    Wang, Longxi