WorldWideScience

Sample records for box nuclear protein

  1. The VirD2 pilot protein of Agrobacterium-transferred DNA interacts with the TATA box-binding protein and a nuclear protein kinase in plants.

    Science.gov (United States)

    Bakó, László; Umeda, Masaaki; Tiburcio, Antonio F; Schell, Jeff; Koncz, Csaba

    2003-08-19

    The bacterial virulence protein VirD2 plays an important role in nuclear import and chromosomal integration of Agrobacterium-transferred DNA in fungal, plant, animal, and human cells. Here we show that in nuclei of alfalfa cells, VirD2 interacts with and is phosphorylated by CAK2Ms, a conserved plant ortholog of cyclin-dependent kinase-activating kinases. CAK2Ms binds to and phosphorylates the C-terminal regulatory domain of RNA polymerase II largest subunit, which can recruit the TATA box-binding protein. VirD2 is found in tight association with the TATA box-binding protein in vivo. These results indicate that recognition of VirD2 is mediated by widely conserved nuclear factors in eukaryotes.

  2. SCF Ubiquitin Ligase F-box Protein Fbx15 Controls Nuclear Co-repressor Localization, Stress Response and Virulence of the Human Pathogen Aspergillus fumigatus.

    Directory of Open Access Journals (Sweden)

    Bastian Jöhnk

    2016-09-01

    Full Text Available F-box proteins share the F-box domain to connect substrates of E3 SCF ubiquitin RING ligases through the adaptor Skp1/A to Cul1/A scaffolds. F-box protein Fbx15 is part of the general stress response of the human pathogenic mold Aspergillus fumigatus. Oxidative stress induces a transient peak of fbx15 expression, resulting in 3x elevated Fbx15 protein levels. During non-stress conditions Fbx15 is phosphorylated and F-box mediated interaction with SkpA preferentially happens in smaller subpopulations in the cytoplasm. The F-box of Fbx15 is required for an appropriate oxidative stress response, which results in rapid dephosphorylation of Fbx15 and a shift of the cellular interaction with SkpA to the nucleus. Fbx15 binds SsnF/Ssn6 as part of the RcoA/Tup1-SsnF/Ssn6 co-repressor and is required for its correct nuclear localization. Dephosphorylated Fbx15 prevents SsnF/Ssn6 nuclear localization and results in the derepression of gliotoxin gene expression. fbx15 deletion mutants are unable to infect immunocompromised mice in a model for invasive aspergillosis. Fbx15 has a novel dual molecular function by controlling transcriptional repression and being part of SCF E3 ubiquitin ligases, which is essential for stress response, gliotoxin production and virulence in the opportunistic human pathogen A. fumigatus.

  3. Chironex fleckeri (Box Jellyfish) Venom Proteins

    Science.gov (United States)

    Brinkman, Diane L.; Konstantakopoulos, Nicki; McInerney, Bernie V.; Mulvenna, Jason; Seymour, Jamie E.; Isbister, Geoffrey K.; Hodgson, Wayne C.

    2014-01-01

    The box jellyfish Chironex fleckeri produces extremely potent and rapid-acting venom that is harmful to humans and lethal to prey. Here, we describe the characterization of two C. fleckeri venom proteins, CfTX-A (∼40 kDa) and CfTX-B (∼42 kDa), which were isolated from C. fleckeri venom using size exclusion chromatography and cation exchange chromatography. Full-length cDNA sequences encoding CfTX-A and -B and a third putative toxin, CfTX-Bt, were subsequently retrieved from a C. fleckeri tentacle cDNA library. Bioinformatic analyses revealed that the new toxins belong to a small family of potent cnidarian pore-forming toxins that includes two other C. fleckeri toxins, CfTX-1 and CfTX-2. Phylogenetic inferences from amino acid sequences of the toxin family grouped CfTX-A, -B, and -Bt in a separate clade from CfTX-1 and -2, suggesting that the C. fleckeri toxins have diversified structurally and functionally during evolution. Comparative bioactivity assays revealed that CfTX-1/2 (25 μg kg−1) caused profound effects on the cardiovascular system of anesthetized rats, whereas CfTX-A/B elicited only minor effects at the same dose. Conversely, the hemolytic activity of CfTX-A/B (HU50 = 5 ng ml−1) was at least 30 times greater than that of CfTX-1/2. Structural homology between the cubozoan toxins and insecticidal three-domain Cry toxins (δ-endotoxins) suggests that the toxins have a similar pore-forming mechanism of action involving α-helices of the N-terminal domain, whereas structural diversification among toxin members may modulate target specificity. Expansion of the cnidarian toxin family therefore provides new insights into the evolutionary diversification of box jellyfish toxins from a structural and functional perspective. PMID:24403082

  4. A nuclear chocolate box: the periodic table of nuclear medicine.

    Science.gov (United States)

    Blower, Philip J

    2015-03-21

    Radioisotopes of elements from all parts of the periodic table find both clinical and research applications in radionuclide molecular imaging and therapy (nuclear medicine). This article provides an overview of these applications in relation to both the radiological properties of the radionuclides and the chemical properties of the elements, indicating past successes, current applications and future opportunities and challenges for inorganic chemistry.

  5. DEAD-box proteins as RNA helicases and chaperones

    Science.gov (United States)

    Jarmoskaite, Inga; Russell, Rick

    2010-01-01

    DEAD-box proteins are ubiquitous in RNA-mediated processes and function by coupling cycles of ATP binding and hydrolysis to changes in affinity for single-stranded RNA. Many DEAD-box proteins use this basic mechanism as the foundation for a version of RNA helicase activity, efficiently separating the strands of short RNA duplexes in a process that involves little or no translocation. This activity, coupled with mechanisms to direct different DEAD-box proteins to their physiological substrates, allows them to promote RNA folding steps and rearrangements and to accelerate remodeling of RNA-protein complexes. This review will describe the properties of DEAD-box proteins as RNA helicases and the current understanding of how the energy from ATPase activity is used to drive the separation of RNA duplex strands. It will then describe how the basic biochemical properties allow some DEAD-box proteins to function as chaperones by promoting RNA folding reactions, with a focus on the self-splicing group I and group II intron RNAs. PMID:21297876

  6. Cardiac nuclear high mobility group box 1 prevents the development of cardiac hypertrophy and heart failure.

    Science.gov (United States)

    Funayama, Akira; Shishido, Tetsuro; Netsu, Shunsuke; Narumi, Taro; Kadowaki, Shinpei; Takahashi, Hiroki; Miyamoto, Takuya; Watanabe, Tetsu; Woo, Chang-Hoon; Abe, Jun-ichi; Kuwahara, Koichiro; Nakao, Kazuwa; Takeishi, Yasuchika; Kubota, Isao

    2013-09-01

    High mobility group box 1 (HMGB1) is an abundant and ubiquitous nuclear DNA-binding protein that has multiple functions dependent on its cellular location. HMGB1 binds to DNA, facilitating numerous nuclear functions including maintenance of genome stability, transcription, and repair. However, little is known about the effects of nuclear HMGB1 on cardiac hypertrophy and heart failure. The aim of this study was to examine whether nuclear HMGB1 plays a role in the development of cardiac hypertrophy induced by pressure overload. Analysis of human biopsy samples by immunohistochemistry showed decreased nuclear HMGB1 expression in failing hearts compared with normal hearts. Nuclear HMGB1 decreased in response to both endothelin-1 (ET-1) and angiotensin II (Ang II) stimulation in neonatal rat cardiomyocytes, where nuclear HMGB1 was acetylated and translocated to the cytoplasm. Overexpression of nuclear HMGB1 attenuated ET-1 induced cardiomyocyte hypertrophy. Thoracic transverse aortic constriction (TAC) was performed in transgenic mice with cardiac-specific overexpression of HMGB1 (HMGB1-Tg) and wild-type (WT) mice. Cardiac hypertrophy after TAC was attenuated in HMGB1-Tg mice and the survival rate after TAC was higher in HMGB1-Tg mice than in WT mice. Induction of foetal cardiac genes was decreased in HMGB1-Tg mice compared with WT mice. Nuclear HMGB1 expression was preserved in HMGB1-Tg mice compared with WT mice and significantly attenuated DNA damage after TAC was attenuated in HMGB1-TG mice. These results suggest that the maintenance of stable nuclear HMGB1 levels prevents hypertrophy and heart failure by inhibiting DNA damage.

  7. Characterization of an AGAMOUS-like MADS Box Protein, a Probable Constituent of Flowering and Fruit Ripening Regulatory System in Banana

    Science.gov (United States)

    Roy Choudhury, Swarup; Roy, Sujit; Nag, Anish; Singh, Sanjay Kumar; Sengupta, Dibyendu N.

    2012-01-01

    The MADS-box family of genes has been shown to play a significant role in the development of reproductive organs, including dry and fleshy fruits. In this study, the molecular properties of an AGAMOUS like MADS box transcription factor in banana cultivar Giant governor (Musa sp, AAA group, subgroup Cavendish) has been elucidated. We have detected a CArG-box sequence binding AGAMOUS MADS-box protein in banana flower and fruit nuclear extracts in DNA-protein interaction assays. The protein fraction in the DNA-protein complex was analyzed by mass spectrometry and using this information we have obtained the full length cDNA of the corresponding protein. The deduced protein sequence showed ∼95% amino acid sequence homology with MA-MADS5, a MADS-box protein described previously from banana. We have characterized the domains of the identified AGAMOUS MADS-box protein involved in DNA binding and homodimer formation in vitro using full-length and truncated versions of affinity purified recombinant proteins. Furthermore, in order to gain insight about how DNA bending is achieved by this MADS-box factor, we performed circular permutation and phasing analysis using the wild type recombinant protein. The AGAMOUS MADS-box protein identified in this study has been found to predominantly accumulate in the climacteric fruit pulp and also in female flower ovary. In vivo and in vitro assays have revealed specific binding of the identified AGAMOUS MADS-box protein to CArG-box sequence in the promoters of major ripening genes in banana fruit. Overall, the expression patterns of this MADS-box protein in banana female flower ovary and during various phases of fruit ripening along with the interaction of the protein to the CArG-box sequence in the promoters of major ripening genes lead to interesting assumption about the possible involvement of this AGAMOUS MADS-box factor in banana fruit ripening and floral reproductive organ development. PMID:22984496

  8. Characterization of an AGAMOUS-like MADS box protein, a probable constituent of flowering and fruit ripening regulatory system in banana.

    Directory of Open Access Journals (Sweden)

    Swarup Roy Choudhury

    Full Text Available The MADS-box family of genes has been shown to play a significant role in the development of reproductive organs, including dry and fleshy fruits. In this study, the molecular properties of an AGAMOUS like MADS box transcription factor in banana cultivar Giant governor (Musa sp, AAA group, subgroup Cavendish has been elucidated. We have detected a CArG-box sequence binding AGAMOUS MADS-box protein in banana flower and fruit nuclear extracts in DNA-protein interaction assays. The protein fraction in the DNA-protein complex was analyzed by mass spectrometry and using this information we have obtained the full length cDNA of the corresponding protein. The deduced protein sequence showed ~95% amino acid sequence homology with MA-MADS5, a MADS-box protein described previously from banana. We have characterized the domains of the identified AGAMOUS MADS-box protein involved in DNA binding and homodimer formation in vitro using full-length and truncated versions of affinity purified recombinant proteins. Furthermore, in order to gain insight about how DNA bending is achieved by this MADS-box factor, we performed circular permutation and phasing analysis using the wild type recombinant protein. The AGAMOUS MADS-box protein identified in this study has been found to predominantly accumulate in the climacteric fruit pulp and also in female flower ovary. In vivo and in vitro assays have revealed specific binding of the identified AGAMOUS MADS-box protein to CArG-box sequence in the promoters of major ripening genes in banana fruit. Overall, the expression patterns of this MADS-box protein in banana female flower ovary and during various phases of fruit ripening along with the interaction of the protein to the CArG-box sequence in the promoters of major ripening genes lead to interesting assumption about the possible involvement of this AGAMOUS MADS-box factor in banana fruit ripening and floral reproductive organ development.

  9. Protein arginine methyltransferase 1 regulates herpes simplex virus replication through ICP27 RGG-box methylation

    Energy Technology Data Exchange (ETDEWEB)

    Yu, Jungeun; Shin, Bongjin; Park, Eui-Soon; Yang, Sujeong; Choi, Seunga [Department of Microbiology, Chungnam National University, 220 Gung-dong, Yuseong-gu, Daejon 305-764 (Korea, Republic of); BK21 Bio Brain Center, Chungnam National University, 220 Gung-dong, Yuseong-gu, Daejon 305-764 (Korea, Republic of); Kang, Misun [Department of Microbiology, Chungnam National University, 220 Gung-dong, Yuseong-gu, Daejon 305-764 (Korea, Republic of); Rho, Jaerang, E-mail: jrrho@cnu.ac.kr [Department of Microbiology, Chungnam National University, 220 Gung-dong, Yuseong-gu, Daejon 305-764 (Korea, Republic of); BK21 Bio Brain Center, Chungnam National University, 220 Gung-dong, Yuseong-gu, Daejon 305-764 (Korea, Republic of); GRAST, Chungnam National University, 220 Gung-dong, Yuseong-gu, Daejon 305-764 (Korea, Republic of)

    2010-01-01

    Protein arginine methylation is involved in viral infection and replication through the modulation of diverse cellular processes including RNA metabolism, cytokine signaling, and subcellular localization. It has been suggested previously that the protein arginine methylation of the RGG-box of ICP27 is required for herpes simplex virus type-1 (HSV-1) viral replication and gene expression in vivo. However, a cellular mediator for this process has not yet been identified. In our current study, we show that the protein arginine methyltransferase 1 (PRMT1) is a cellular mediator of the arginine methylation of ICP27 RGG-box. We generated arginine substitution mutants in this domain and examined which arginine residues are required for methylation by PRMT1. R138, R148 and R150 were found to be the major sites of this methylation but additional arginine residues serving as minor methylation sites are still required to sustain the fully methylated form of ICP27 RGG. We also demonstrate that the nuclear foci-like structure formation, SRPK interactions, and RNA-binding activity of ICP27 are modulated by the arginine methylation of the ICP27 RGG-box. Furthermore, HSV-1 replication is inhibited by hypomethylation of this domain resulting from the use of general PRMT inhibitors or arginine mutations. Our data thus suggest that the PRMT1 plays a key role as a cellular regulator of HSV-1 replication through ICP27 RGG-box methylation.

  10. Forkhead box protein A2 (FOXA2 protein stability and activity are regulated by sumoylation.

    Directory of Open Access Journals (Sweden)

    Narasimhaswamy S Belaguli

    Full Text Available The forkhead box protein A2 (FOXA2 is an important regulator of glucose and lipid metabolism and organismal energy balance. Little is known about how FOXA2 protein expression and activity are regulated by post-translational modifications. We have identified that FOXA2 is post-translationally modified by covalent attachment of a small ubiquitin related modifier-1 (SUMO-1 and mapped the sumoylation site to the amino acid lysine 6 (K6. Preventing sumoylation by mutating the SUMO acceptor K6 to arginine resulted in downregulation of FOXA2 protein but not RNA expression in INS-1E insulinoma cells. K6R mutation also downregulated FOXA2 protein levels in HepG2 hepatocellular carcinoma cells, HCT116 colon cancer cells and LNCaP and DU145 prostate cancer cells. Further, interfering with FOXA2 sumoylation through siRNA mediated knockdown of UBC9, an essential SUMO E2 conjugase, resulted in downregulation of FOXA2 protein levels. Stability of sumoylation deficient FOXA2K6R mutant protein was restored when SUMO-1 was fused in-frame. FOXA2 sumoylation and FOXA2 protein levels were increased by PIAS1 SUMO ligase but not a SUMO ligase activity deficient PIAS1 mutant. Although expressed at lower levels, sumoylation deficient FOXA2K6R mutant protein was detectable in the nucleus indicating that FOXA2 nuclear localization is independent of sumoylation. Sumoylation increased the transcriptional activity of FOXA2 on Pdx-1 area I enhancer. Together, our results show that sumoylation regulates FOXA2 protein expression and activity.

  11. Multiple modes of chromatin remodeling by Forkhead box proteins.

    Science.gov (United States)

    Lalmansingh, Avin S; Karmakar, Sudipan; Jin, Yetao; Nagaich, Akhilesh K

    2012-07-01

    Forkhead box (FOX) proteins represent a large family of transcriptional regulators unified by their DNA binding domain (DBD) known as a 'forkhead' or 'winged helix' domain. Over 40 FOX genes have been identified in the mammalian genome. FOX proteins share significant sequence similarities in the DBD which allow them to bind to a consensus DNA response element. However, their modes of action are quite diverse as they regulate gene expression by acting as pioneer factors, transcription factors, or both. This review focuses on the mechanisms of chromatin remodeling with an emphasis on three sub-classes-FOXA, FOXO, and FOXP members. FOXA proteins serve as pioneer factors to open up local chromatin structure and thereby increase accessibility of chromatin to factors regulating transcription. FOXP proteins, in contrast, function as classic transcription factors to recruit a variety of chromatin modifying enzymes to regulate gene expression. FOXO proteins represent a hybrid subclass having dual roles as pioneering factors and transcription factors. A subset of FOX proteins interacts with condensed mitotic chromatin and may function as 'bookmarking' agents to maintain transcriptional competence at specific genomic sites. The overall diversity in chromatin remodeling function by FOX proteins is related to unique structural motifs present within the DBD flanking regions that govern selective interactions with core histones and/or chromatin coregulatory proteins. This article is part of a Special Issue entitled: Chromatin in time and space. Published by Elsevier B.V.

  12. Phylogenetic Distribution and Evolutionary History of Bacterial DEAD-Box Proteins

    OpenAIRE

    López-Ramírez, Varinia; Alcaraz, Luis D; Moreno-Hagelsieb, Gabriel; Olmedo-Álvarez, Gabriela

    2011-01-01

    DEAD-box proteins are found in all domains of life and participate in almost all cellular processes that involve RNA. The presence of DEAD and Helicase_C conserved domains distinguish these proteins. DEAD-box proteins exhibit RNA-dependent ATPase activity in vitro, and several also show RNA helicase activity. In this study, we analyzed the distribution and architecture of DEAD-box proteins among bacterial genomes to gain insight into the evolutionary pathways that have shaped their history. W...

  13. Determining Nuclear Fingerprints: Glove Boxes, Radiation Protection, and the International Atomic Energy Agency.

    Science.gov (United States)

    Rentetzi, Maria

    2017-06-01

    In a nuclear laboratory, a glove box is a windowed, sealed container equipped with two flexible gloves that allow the user to manipulate nuclear materials from the outside in an ostensibly safe environment. As a routine laboratory device, it invites neglect from historians and storytellers of science. Yet, since especially the Gulf War, glove boxes have put the interdependence of science, diplomacy, and politics into clear relief. Standing at the intersection of history of science and international history, technological materials and devices such as the glove box can provide penetrating insight into the role of international diplomatic organizations to the global circulation and control of scientific knowledge. The focus here is on the International Atomic Energy Agency. Copyright © 2017 The Author. Published by Elsevier Ltd.. All rights reserved.

  14. Ovule-specific MADS box proteins have conserved protein-protein interactions in monocots and dicot plants

    NARCIS (Netherlands)

    Favaro, R.; Immink, R.G.H.; Ferioli, V.; Bernasconi, B.; Byzova, M.; Angenent, G.C.; Kater, M.; Colombo, L.

    2002-01-01

    OsMADS13 is a rice MADS-box gene that is specifically expressed in developing ovules. The amino acid sequence of OsMADS13 shows 74␜imilarity to those of FLORAL BINDING PROTEIN 7 (FBP7) and FBP11, the products of two MADS-box genes that are necessary and sufficient to determine ovule identity in

  15. Identification and characterization of a nuclear localization signal of TRIM28 that overlaps with the HP1 box

    Energy Technology Data Exchange (ETDEWEB)

    Moriyama, Tetsuji; Sangel, Percival [Laboratory of Nuclear Transport Dynamics, National Institutes of Biomedical Innovation, Health and Nutrition (NIBIOHN), Osaka 567-0085 (Japan); Yamaguchi, Hiroki [School of Medicine, Osaka University, Osaka 565-0871 (Japan); Obuse, Chikashi [Graduate School of Life Science, Hokkaido University, Sapporo 001-0021 (Japan); Miyamoto, Yoichi [Laboratory of Nuclear Transport Dynamics, National Institutes of Biomedical Innovation, Health and Nutrition (NIBIOHN), Osaka 567-0085 (Japan); Oka, Masahiro, E-mail: moka@nibiohn.go.jp [Laboratory of Nuclear Transport Dynamics, National Institutes of Biomedical Innovation, Health and Nutrition (NIBIOHN), Osaka 567-0085 (Japan); Laboratory of Biomedical Innovation, Graduate School of Pharmaceutical Sciences, Osaka University, Osaka 565-0871 (Japan); Yoneda, Yoshihiro, E-mail: y-yoneda@nibiohn.go.jp [National Institutes of Biomedical Innovation, Health and Nutrition (NIBIOHN), Osaka 567-0085 (Japan); Laboratory of Biomedical Innovation, Graduate School of Pharmaceutical Sciences, Osaka University, Osaka 565-0871 (Japan)

    2015-07-03

    Tripartite motif-containing 28 (TRIM28) is a transcription regulator, which forms a repressor complex containing heterochromatin protein 1 (HP1). Here, we report identification of a nuclear localization signal (NLS) within the 462-494 amino acid region of TRIM28 that overlaps with its HP1 binding site, HP1 box. GST-pulldown experiments revealed the interaction of the arginine-rich TRIM28 NLS with various importin α subtypes (α1, α2 and α4). In vitro transport assay demonstrated that nuclear localization of GFP-TRIM28 NLS is mediated by importin αs, in conjunction with importin β1 and Ran. Further, we demonstrated that HP1 and importin αs compete for binding to TRIM28. Together, our findings suggest that importin α has an essential role in the nuclear delivery and preferential HP1 interaction of TRIM28. - Highlights: • TRIM28 contains an NLS within the 462-494 amino acid region. • The nuclear import of TRIM28 is mediated by importin α/importin β1. • TRIM28 NLS overlaps with HP1 Box. • HP1 and importin α compete for binding to TRIM28.

  16. Crystal structure of the shrimp proliferating cell nuclear antigen: structural complementarity with WSSV DNA polymerase PIP-box.

    Science.gov (United States)

    Carrasco-Miranda, Jesus S; Lopez-Zavala, Alonso A; Arvizu-Flores, Aldo A; Garcia-Orozco, Karina D; Stojanoff, Vivian; Rudiño-Piñera, Enrique; Brieba, Luis G; Sotelo-Mundo, Rogerio R

    2014-01-01

    DNA replication requires processivity factors that allow replicative DNA polymerases to extend long stretches of DNA. Some DNA viruses encode their own replicative DNA polymerase, such as the white spot syndrome virus (WSSV) that infects decapod crustaceans but still require host replication accessory factors. We have determined by X-ray diffraction the three-dimensional structure of the Pacific white leg shrimp Litopenaeus vannamei Proliferating Cell Nuclear Antigen (LvPCNA). This protein is a member of the sliding clamp family of proteins, that binds DNA replication and DNA repair proteins through a motif called PIP-box (PCNA-Interacting Protein). The crystal structure of LvPCNA was refined to a resolution of 3 Å, and allowed us to determine the trimeric protein assembly and details of the interactions between PCNA and the DNA. To address the possible interaction between LvPCNA and the viral DNA polymerase, we docked a theoretical model of a PIP-box peptide from the WSSV DNA polymerase within LvPCNA crystal structure. The theoretical model depicts a feasible model of interaction between both proteins. The crystal structure of shrimp PCNA allows us to further understand the mechanisms of DNA replication processivity factors in non-model systems.

  17. Crystal structure of the shrimp proliferating cell nuclear antigen: structural complementarity with WSSV DNA polymerase PIP-box.

    Directory of Open Access Journals (Sweden)

    Jesus S Carrasco-Miranda

    Full Text Available DNA replication requires processivity factors that allow replicative DNA polymerases to extend long stretches of DNA. Some DNA viruses encode their own replicative DNA polymerase, such as the white spot syndrome virus (WSSV that infects decapod crustaceans but still require host replication accessory factors. We have determined by X-ray diffraction the three-dimensional structure of the Pacific white leg shrimp Litopenaeus vannamei Proliferating Cell Nuclear Antigen (LvPCNA. This protein is a member of the sliding clamp family of proteins, that binds DNA replication and DNA repair proteins through a motif called PIP-box (PCNA-Interacting Protein. The crystal structure of LvPCNA was refined to a resolution of 3 Å, and allowed us to determine the trimeric protein assembly and details of the interactions between PCNA and the DNA. To address the possible interaction between LvPCNA and the viral DNA polymerase, we docked a theoretical model of a PIP-box peptide from the WSSV DNA polymerase within LvPCNA crystal structure. The theoretical model depicts a feasible model of interaction between both proteins. The crystal structure of shrimp PCNA allows us to further understand the mechanisms of DNA replication processivity factors in non-model systems.

  18. RNA-binding specificity of Y-box protein 1.

    Science.gov (United States)

    Dong, Jinjiang; Akcakanat, Argun; Stivers, David N; Zhang, Jiexin; Kim, Doyil; Meric-Bernstam, Funda

    2009-01-01

    Y-box protein 1 (YB-1) is a multifunctional DNA/RNA-binding protein that regulates transcription and translation. The specificity of YB-1's RNA binding and its consequences are unknown. Because expression and subcellular localization of YB-1 have been reported to be important in breast cancer, we determined the specificity and functional impact of YB-1 mRNA-binding in MCF7 breast cancer cells. We used YB-1 antibodies to immunoprecipitate YB-1 and microarray profiling to compare YB-1-bound and total poly(A) RNA. We demonstrated that YB-1 mRNA-binding was preferential. Transcript sequences significantly associated with this binding had high GC content. Selected YB-1 mRNA-binding targets were confirmed by QRT-PCR. However, downregulation of YB-1 levels by siRNA did not affect their RNA or protein expression. Thus, YB-1 has RNA-binding specificity; however, YB-1 binding does not necessarily regulate the stability or translation of its mRNA targets. Further study is needed to determine the functional consequences of selective YB-1 mRNA binding.

  19. Regulating the ethylene response of a plant by modulation of F-box proteins

    Science.gov (United States)

    Guo, Hongwei [Beijing, CN; Ecker, Joseph R [Carlsbad, CA

    2014-01-07

    The relationship between F-box proteins and proteins invovled in the ethylene response in plants is described. In particular, F-box proteins may bind to proteins involved in the ethylene response and target them for degradation by the ubiquitin/proteasome pathway. The transcription factor EIN3 is a key transcription factor mediating ethylne-regulated gene expression and morphological responses. EIN3 is degraded through a ubiquitin/proteasome pathway mediated by F-box proteins EBF1 and EBF2. The link between F-box proteins and the ethylene response is a key step in modulating or regulating the response of a plant to ethylene. Described herein are transgenic plants having an altered sensitivity to ethylene, and methods for making transgenic plant haing an althered sensitivity to ethylene by modulating the level of activity of F-box proteins. Methods of altering the ethylene response in a plant by modulating the activity or expression of an F-box protein are described. Also described are methods of identifying compounds that modulate the ethylene response in plants by modulating the level of F-box protein expression or activity.

  20. Efficient ASK-assisted system for expression and purification of plant F-box proteins.

    Science.gov (United States)

    Li, Haiou; Yao, Ruifeng; Ma, Sui; Hu, Shuai; Li, Suhua; Wang, Yupei; Yan, Chun; Xie, Daoxin; Yan, Jianbin

    2017-09-05

    Ubiquitin-mediated protein degradation plays an essential role in plant growth and development as well as responses to environmental and endogenous signals. F-box protein is one of the key components of the SCF (SKP1-CUL1-F-box protein) E3 ubiquitin ligase complex, which recruit specific substrate proteins for subsequent ubiquitination and 26S proteasome-mediated degradation to regulate developmental processes and signaling networks. However, it is not easy to obtain purified F-box proteins with high activity due to their unstable protein structures. Here, we found that Arabidopsis SKP-like proteins (ASKs) can significantly improve soluble expression of F-box proteins and maintain their bioactivity. We established an efficient ASK-assisted method to express and purify plant F-box proteins. The method meets a broad range of criteria required for the biochemical analysis or protein crystallization of plant F-box proteins. © 2017 The Authors The Plant Journal © 2017 John Wiley & Sons Ltd.

  1. Contributions of high mobility group box protein in experimental and clinical acute lung injury.

    Science.gov (United States)

    Ueno, Hiroshi; Matsuda, Tomoyuki; Hashimoto, Satoru; Amaya, Fumimasa; Kitamura, Yoshihiro; Tanaka, Masaki; Kobayashi, Atsuko; Maruyama, Ikuro; Yamada, Shingo; Hasegawa, Naoki; Soejima, Junko; Koh, Hidefumi; Ishizaka, Akitoshi

    2004-12-15

    This study was performed to examine the putative role of high mobility group box (HMGB) protein in the pathogenesis of acute lung injury (ALI). Observations were made (1) in 21 patients who were septic with ALI and 15 patients with normal lung function and (2) in a mouse model 24 hours after intratracheal instillation of lipopolysaccharide (LPS). The concentrations of HMGB1 were increased in plasma and lung epithelial lining fluid of patients with ALI and mice instilled with LPS. LPS-induced ALI was mitigated by anti-HMGB1 antibody. Although this protein was not detected in the plasma of control humans or mice, the concentrations of HMGB1 in lung epithelial lining fluid or in bronchoalveolar lavage fluid were unexpectedly high. The nuclear expression of HMGB1 was apparent in epithelial cells surrounding terminal bronchioles in normal mice, whereas its nuclear and cytoplasmic expression was observed in alveolar macrophages in LPS-instilled mice. Lung instillation of HMGB2 did not cause as much inflammation as HMGB1. Extracellular HMGB1 may play a key role in the pathogenesis of clinical and experimental ALI. However, its expression in normal airways is noteworthy and suggests that it also plays a physiologic role in the lung.

  2. Nuclear Matrix Proteins in Human Colon Cancer

    Science.gov (United States)

    Keesee, Susan K.; Meneghini, Marc D.; Szaro, Robert P.; Wu, Ying-Jye

    1994-03-01

    The nuclear matrix is the nonchromatin scaffolding of the nucleus. This structure confers nuclear shape, organizes chromatin, and appears to contain important regulatory proteins. Tissue specific nuclear matrix proteins have been found in the rat, mouse, and human. In this study we compared high-resolution two-dimensional gel electropherograms of nuclear matrix protein patterns found in human colon tumors with those from normal colon epithelia. Tumors were obtained from 18 patients undergoing partial colectomy for adenocarcinoma of the colon and compared with tissue from 10 normal colons. We have identified at least six proteins which were present in 18 of 18 colon tumors and 0 of 10 normal tissues, as well as four proteins present in 0 of 18 tumors and in 10 of 10 normal tissues. These data, which corroborate similar findings of cancer-specific nuclear matrix proteins in prostate and breast, suggest that nuclear matrix proteins may serve as important markers for at least some types of cancer.

  3. Plant nuclear hormone receptors: a role for small molecules in protein-protein interactions.

    Science.gov (United States)

    Lumba, Shelley; Cutler, Sean; McCourt, Peter

    2010-01-01

    Plant hormones are a group of chemically diverse small molecules that direct processes ranging from growth and development to biotic and abiotic stress responses. Surprisingly, genome analyses suggest that classic animal nuclear hormone receptor homologs do not exist in plants. It now appears that plants have co-opted several protein families to perceive hormones within the nucleus. In one solution to the problem, the hormones auxin and jasmonate (JA) act as “molecular glue” that promotes protein-protein interactions between receptor F-boxes and downstream corepressor targets. In another solution, gibberellins (GAs) bind and elicit a conformational change in a novel soluble receptor family related to hormone-sensitive lipases. Abscisic acid (ABA), like GA, also acts through an allosteric mechanism involving a START-domain protein. The molecular identification of plant nuclear hormone receptors will allow comparisons with animal nuclear receptors and testing of fundamental questions about hormone function in plant development and evolution.

  4. DNA Radiation Environments Program Spring 1991 2-meter box experiments and analyses. [DEfense Nuclear Agency (DNA)

    Energy Technology Data Exchange (ETDEWEB)

    Santoro, R.T. (Oak Ridge National Lab., TN (United States)); Whitaker, S.Y. (Clark Atlanta Univ., GA (United States))

    1993-03-01

    This report summarizes the Spring 1991 2-m Box experiments that were performed at the Army Pulse Radiation Facility (APRF) at Aberdeen Proving Ground. These studies were sponsored by the Defense Nuclear Agency (DNA) under the Radiation Environments Program to obtain measured data for benchmarking the Adjoint Monte Carlo Code System, MASH, Version 1.0. The MASH code system was developed for the Department of Defense and NATO for calculating neutron and gamma-ray radiation fields and shielding protection factors for armored vehicles and military structures against nuclear weapon radiation. In the 2-m Box experiments, neutron and gamma-ray dose rates and reduction factors were measured in the free-field and as a function of position on an anthropomorphic phantom that was placed outside and inside a borated polyethylene lined steel-walled 2-m box. The data were acquired at a distance of 400-m from the APRF reactor. The purpose of these experiments was to measure the neutron and gamma-ray dose rates as a function of detector location on the phantom for cases when the phantom was in the free-field and inside of the box. Neutron measurements were made using a BD-100R bubble detector and gamma-ray measurements were made using thermoluminescent detectors (TLD). Calculated and measured data were compared in terms of the C/M ratio. The calculated and measured neutron and gamma-ray dose rates and reduction factors agreed on the average within the [plus minus]20% limits mandated by DNA and demonstrate the capability of the MASH code system in reproducing measured data in nominally shielded assemblies.

  5. High-mobility group box 1 protein and its role in severe acute pancreatitis

    Science.gov (United States)

    Shen, Xiao; Li, Wei-Qin

    2015-01-01

    The high mobility group box 1 (HMGB1), which belongs to the subfamily of HMG-1/-2, is a highly conserved single peptide chain consisting of 215 amino acid residues with a molecular weight of approximately 24894 Da. HMGB1 is a ubiquitous nuclear protein in mammals and plays a vital role in inflammatory diseases. Acute pancreatitis is one of the most common causes of acute abdominal pain with a poor prognosis. Acute pancreatitis is an acute inflammatory process of the pancreas (duration of less than six months), for which the severe form is called severe acute pancreatitis (SAP). More and more studies have shown that HMGB1 has a bidirectional effect in the pathogenesis of SAP. Extracellular HMGB1 can aggravate the pancreatic inflammatory process, whereas intracellular HMGB1 has a protective effect against pancreatitis. The mechanism of HMGB1 is multiple, mainly through the nuclear factor-κB pathway. Receptors for advanced glycation end-products and toll-like receptors (TLR), especially TLR-2 and TLR-4, are two major types of receptors mediating the inflammatory process triggered by HMGB1 and may be also the main mediators in the pathogenesis of SAP. HMGB1 inhibitors, such as ethyl pyruvate, pyrrolidine dithiocarbamate and Scolopendra subspinipes mutilans, can decrease the level of extracellular HMGB1 and are the promising targets in the treatment of SAP. PMID:25663762

  6. Thermodynamics of the fragile X mental retardation protein RGG box interactions with G quartet forming RNA.

    Science.gov (United States)

    Zanotti, Kimberly J; Lackey, Patrick E; Evans, Genevieve L; Mihailescu, Mihaela-Rita

    2006-07-11

    Fragile X syndrome, the most common form of inherited mental retardation, is the result of an unstable expansion of a CGG trinucleotide repeat in the 5' UTR of the fragile X mental retardation-1 (FMR1) gene. The abnormal hypermethylation of the expanded CGG repeats causes the transcriptional silencing of the FMR1 gene and, consequently, the loss of the fragile X mental retardation protein (FMRP). FMRP is an RNA binding protein that binds to G quartet forming RNA using its RGG box motif. In this study we have performed a thermodynamic analysis of the interactions between the FMRP RGG box domain and Sc1, an RNA molecule which had been previously shown to be bound with high affinity by both the full-length FMRP and by its RGG box domain. We have determined that the association between the FMRP RGG box and Sc1 RNA is dominated by hydrophobic and hydrogen bond interactions, with minor contributions from electrostatic interactions, and that the FMRP RGG box binding increases the stability of the G quartet RNA structure significantly. Interestingly, we found that the G quartet recognition is necessary but not sufficient for the FMRP RGG box binding to this RNA target, indicating that additional interactions of the peptide, possibly with the stem and/or stem-G quartet junction region, are required. Our results also indicate that the G quartet RNA recognition is not a general feature of the RGG box motif but rather carries some sequence, protein and/or RNA, specificity.

  7. Boxing and running lead to a rise in serum levels of S-100B protein.

    Science.gov (United States)

    Otto, M; Holthusen, S; Bahn, E; Söhnchen, N; Wiltfang, J; Geese, R; Fischer, A; Reimers, C D

    2000-11-01

    Permanent neurological dysfunction is the primary medical concern of boxing. Recently it was reported that patients presenting elevated levels of the glial protein S-100B in serum after minor head injuries are more prone to develop neuropsychological deficits than patients with lower levels of S-100B protein. We assessed this protein before and after amateur boxing competitions (n = 10) and sparring bouts (n = 15). In several control groups, we investigated S-100B levels of participants before and after a 25 km race (n = 11), jogging (10 km, n = 12), short-term running (n = 12), and heading footballs (n = 12). There was an increase in S-100B protein after boxing and the running disciplines but not after ergometer cycling or soft heading of footballs. The increase in S-100B protein concentrations due to competitive boxing and after the 25 km race was significantly higher than that after performing other disciplines (p < 0.001). There was no significant difference between the increases caused by sparring and the running disciplines (p = 0.21). The number and severity of the strikes to the head correlated significantly with the increase in the S-100B protein levels. Levels of S-100B protein known to be associated with neuropsychological deficits were not reached in our study. In professional boxing, much higher levels are to be expected and would be worthy of investigation.

  8. Baculovirus F-Box Protein LEF-7 Modifies the Host DNA Damage Response To Enhance Virus Multiplication

    Science.gov (United States)

    Mitchell, Jonathan K.; Byers, Nathaniel M.

    2013-01-01

    The DNA damage response (DDR) of a host organism represents an effective antiviral defense that is frequently manipulated and exploited by viruses to promote multiplication. We report here that the large DNA baculoviruses, which require host DDR activation for optimal replication, encode a conserved replication factor, LEF-7, that manipulates the DDR via a novel mechanism. LEF-7 suppresses DDR-induced accumulation of phosphorylated host histone variant H2AX (γ-H2AX), a critical regulator of the DDR. LEF-7 was necessary and sufficient to block γ-H2AX accumulation caused by baculovirus infection or DNA damage induced by means of pharmacological agents. Deletion of LEF-7 from the baculovirus genome allowed γ-H2AX accumulation during virus DNA synthesis and impaired both very late viral gene expression and production of infectious progeny. Thus, LEF-7 is essential for efficient baculovirus replication. We determined that LEF-7 is a nuclear F-box protein that interacts with host S-phase kinase-associated protein 1 (SKP1), suggesting that LEF-7 acts as a substrate recognition component of SKP1/Cullin/F-box (SCF) complexes for targeted protein polyubiquitination. Site-directed mutagenesis demonstrated that LEF-7's N-terminal F-box is necessary for γ-H2AX repression and Autographa californica multiple nucleopolyhedrovirus (AcMNPV) replication events. We concluded that LEF-7 expedites virus replication most likely by selective manipulation of one or more host factors regulating the DDR, including γ-H2AX. Thus, our findings indicate that baculoviruses utilize a unique strategy among viruses for hijacking the host DDR by using a newly recognized F-box protein. PMID:24027328

  9. Direct modulation of T-box riboswitch-controlled transcription by protein synthesis inhibitors.

    Science.gov (United States)

    Stamatopoulou, Vassiliki; Apostolidi, Maria; Li, Shuang; Lamprinou, Katerina; Papakyriakou, Athanasios; Zhang, Jinwei; Stathopoulos, Constantinos

    2017-09-29

    Recently, it was discovered that exposure to mainstream antibiotics activate numerous bacterial riboregulators that control antibiotic resistance genes including metabolite-binding riboswitches and other transcription attenuators. However, the effects of commonly used antibiotics, many of which exhibit RNA-binding properties, on the widespread T-box riboswitches, remain unknown. In Staphylococcus aureus, a species-specific glyS T-box controls the supply of glycine for both ribosomal translation and cell wall synthesis, making it a promising target for next-generation antimicrobials. Here, we report that specific protein synthesis inhibitors could either significantly increase T-box-mediated transcription antitermination, while other compounds could suppress it, both in vitro and in vivo. In-line probing of the full-length T-box combined with molecular modelling and docking analyses suggest that the antibiotics that promote transcription antitermination stabilize the T-box:tRNA complex through binding specific positions on stem I and the Staphylococcal-specific stem Sa. By contrast, the antibiotics that attenuate T-box transcription bind to other positions on stem I and do not interact with stem Sa. Taken together, our results reveal that the transcription of essential genes controlled by T-box riboswitches can be directly modulated by commonly used protein synthesis inhibitors. These findings accentuate the regulatory complexities of bacterial response to antimicrobials that involve multiple riboregulators. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  10. DEAD-box Helicases as Integrators of RNA, Nucleotide and Protein Binding

    Science.gov (United States)

    Putnam, Andrea A.

    2013-01-01

    DEAD-box helicases perform diverse cellular functions in virtually all steps of RNA metabolism from Bacteria to Humans. Although DEAD-box helicases share a highly conserved core domain, the enzymes catalyze a wide range of biochemical reactions. In addition to the well established RNA unwinding and corresponding ATPase activities, DEAD-box helicases promote duplex formation and displace proteins from RNA. They can also function as assembly platforms for larger ribonucleoprotein complexes, and as metabolite sensors. This review aims to provide a perspective on the diverse biochemical features of DEAD-box helicases and connections to structural information. We discuss these data in the context of a model that views the enzymes as integrators of RNA, nucleotide, and protein binding. PMID:23416748

  11. Paired box mutations in familial and sporadic aniridia predicts truncated aniridia proteins

    Energy Technology Data Exchange (ETDEWEB)

    Martha, A.; Saunders, G.F. (Univ. of Texas M.D. Anderson Cancer Center, Houston, TX (United States)); Mintz-Hittner, H. (Baylor College of Medicine, Houston, TX (United States)); Lyons, L.A. (Univ. of Pittsburgh, PA (United States))

    1994-05-01

    Aniridia, an autosomal dominant ocular disorder characterized by iris hypoplasia, results from mutations in the PAX6 gene, which encodes paired box and homeobox motifs. In this report the authors describe five new mutations in the paired box region of the human PAX6 gene that are associated with aniridia. The paired box mutations detected were in both familial (three) and sporadic (two cases) cases. All five mutations predict truncated PAX6 proteins. This study indicates that early premature translational termination mutations in the PAX6 gene result in haploinsufficiency and generate the aniridia phenotype. 32 refs., 6 figs., 2 tabs.

  12. Nuclear matrix proteins and hereditary diseases.

    Science.gov (United States)

    Sjakste, N; Sjakste, T

    2005-03-01

    The review summarizes literature data on alterations of structure or expression of different nuclear matrix proteins in hereditary syndromes. From the point of view of involvement of nuclear matrix proteins in etiology and pathogenesis of the disease hereditary pathologies can be classified in pathologies with pathogenesis associated with defects of nuclear matrix proteins and pathologies associated to changes of the nuclear matrix protein spectrum. The first group includes laminopathies, hereditary diseases with abnormal nuclear-matrix associated proteins and triplet extension diseases associated with accumulation of abnormal proteins in the nuclear matrix. Laminopathies are hereditary diseases coupled to structural defects of the nuclear lamina. These diseases include Emery-Dreifuss muscular dystrophy, limb girdle muscular dystrophy, dilated cardiomyopathy (DCM) with conduction system disease, familial partial lipodystrophy (FPLD), autosomal recessive axonal neuropathy (Charcot-Marie-Tooth disorder type 2, CMT2), mandibuloacral dysplasia (MAD), Hutchison Gilford Progeria syndrome (HGS), Greenberg Skeletal Dysplasia, and Pelger-Huet anomaly (PHA). Most of them are due to mutations in the lamin A/C gene, one - to mutations in emerin gene, some are associated with mutations in Lamin B receptor gene. In Werner's, Bloom's, Cockayne's syndromes, Fanconi anemia, multiple carboxylase deficiency mutations in nuclear matrix protein or enzyme gene lead to deficient DNA repair, abnormal regulation of cell growth and differentiation or other specific metabolic functions. Proteins with a long polyglutamic tract synthesized in the cells of patients with dentato-rubral and pallido-luysian atrophy, myotonic dystrophy and Huntington disease interfere with transcription on the nuclear matrix. Down's syndrome is a representative of the group of diseases with altered nuclear matrix protein spectrum.

  13. A Novel Function of F-Box Protein FBXO17 in Negative Regulation of Type I IFN Signaling by Recruiting PP2A for IFN Regulatory Factor 3 Deactivation.

    Science.gov (United States)

    Peng, Di; Wang, Zining; Huang, Anfei; Zhao, Yong; Qin, F Xiao-Feng

    2017-01-15

    The F-box proteins were originally identified as the key component of SKP1-Cullin1-F-box E3 ligase complexes that control the stability of their specific downstream substrates essential for cell growth and survival. However, the involvement of these proteins in type I IFN (IFN-I) signaling during innate immunity has not been investigated. In this study we report that the F-box protein FBXO17 negatively regulates IFN-I signaling triggered by double-strand DNA, RNA, or viral infection. We found that FBXO17 specifically interacts with IFN regulatory factor 3 (IRF3) and decreases its dimerization and nuclear translocation. The decrease of IRF3 dimerization and nuclear translocation is due to the recruitment of protein phosphatase 2 (PP2A) mediated by FBXO17, resulting in IRF3 dephosphorylation. Interestingly, PP2A recruitment does not require the F-box domain but instead the F-box associated region of the protein; thus, the recruitment is independent of the canonical function of the SKP1-Cullin1-F-box family of E3 ligase. Together, our studies identify a previously unreported role of FBXO17 in regulating IFN-I signaling and further demonstrate a novel mechanism for IRF3 deactivation by F-box protein-mediated recruitment of PP2A. Copyright © 2017 by The American Association of Immunologists, Inc.

  14. RNA Remodeling Activity of DEAD Box Proteins Tuned by Protein Concentration, RNA Length, and ATP.

    Science.gov (United States)

    Kim, Younghoon; Myong, Sua

    2016-09-01

    DEAD box RNA helicases play central roles in RNP biogenesis. We reported earlier that LAF-1, a DEAD box RNA helicase in C. elegans, dynamically interacts with RNA and that the interaction likely contributes to the fluidity of RNP droplets. Here we investigate the molecular basis of the interaction of RNA with LAF-1 and its human homolog, DDX3X. We show that both LAF-1 and DDX3X, at low concentrations, are monomers that induce tight compaction of single-stranded RNA. At high concentrations, the proteins are multimeric and dynamically interact with RNA in an RNA length-dependent manner. The dynamic LAF-1-RNA interaction stimulates RNA annealing activity. ATP adversely affects the RNA remodeling ability of LAF-1 by suppressing the affinity, dynamics, and annealing activity of LAF-1, suggesting that ATP may promote disassembly of the RNP complex. Based on our results, we postulate a plausible molecular mechanism underlying the dynamic equilibrium of the LAF-1 RNP complex. Copyright © 2016 Elsevier Inc. All rights reserved.

  15. Molecular Cloning of a cDNA Encoding for Taenia solium TATA-Box Binding Protein 1 (TsTBP1 and Study of Its Interactions with the TATA-Box of Actin 5 and Typical 2-Cys Peroxiredoxin Genes.

    Directory of Open Access Journals (Sweden)

    Oscar Rodríguez-Lima

    Full Text Available TATA-box binding protein (TBP is an essential regulatory transcription factor for the TATA-box and TATA-box-less gene promoters. We report the cloning and characterization of a full-length cDNA that encodes a Taenia solium TATA-box binding protein 1 (TsTBP1. Deduced amino acid composition from its nucleotide sequence revealed that encodes a protein of 238 residues with a predicted molecular weight of 26.7 kDa, and a theoretical pI of 10.6. The NH2-terminal domain shows no conservation when compared with to pig and human TBP1s. However, it shows high conservation in size and amino acid identity with taeniids TBP1s. In contrast, the TsTBP1 COOH-terminal domain is highly conserved among organisms, and contains the amino acids involved in interactions with the TATA-box, as well as with TFIIA and TFIIB. In silico TsTBP1 modeling reveals that the COOH-terminal domain forms the classical saddle structure of the TBP family, with one α-helix at the end, not present in pig and human. Native TsTBP1 was detected in T. solium cysticerci´s nuclear extract by western blot using rabbit antibodies generated against two synthetic peptides located in the NH2 and COOH-terminal domains of TsTBP1. These antibodies, through immunofluorescence technique, identified the TBP1 in the nucleus of cells that form the bladder wall of cysticerci of Taenia crassiceps, an organism close related to T. solium. Electrophoretic mobility shift assays using nuclear extracts from T. solium cysticerci and antibodies against the NH2-terminal domain of TsTBP1 showed the interaction of native TsTBP1 with the TATA-box present in T. solium actin 5 (pAT5 and 2-Cys peroxiredoxin (Ts2-CysPrx gene promoters; in contrast, when antibodies against the anti-COOH-terminal domain of TsTBP1 were used, they inhibited the binding of TsTBP1 to the TATA-box of the pAT5 promoter gene.

  16. Effects of Quercetin Supplementation on Lipid and Protein Metabolism after Classic Boxing Training

    Science.gov (United States)

    Demirci, Nevzat

    2017-01-01

    The metabolic fitness (MF) is a component of athletes' physical conditioning. This study aims to investigate the effects of quercetin supplementation on Turkish Junior athletes' lipid and protein metabolism relating to MF after one month classic boxing training. Totally 20 voluntary junior male athletes were separated into two equal groups as the…

  17. Compound Heterozygosity for Y Box Proteins Causes Sterility Due to Loss of Translational Repression.

    Directory of Open Access Journals (Sweden)

    Elizabeth Snyder

    2015-12-01

    Full Text Available The Y-box proteins YBX2 and YBX3 bind RNA and DNA and are required for metazoan development and fertility. However, possible functional redundancy between YBX2 and YBX3 has prevented elucidation of their molecular function as RNA masking proteins and identification of their target RNAs. To investigate possible functional redundancy between YBX2 and YBX3, we attempted to construct Ybx2-/-;Ybx3-/- double mutants using a previously reported Ybx2-/- model and a newly generated global Ybx3-/- model. Loss of YBX3 resulted in reduced male fertility and defects in spermatid differentiation. However, homozygous double mutants could not be generated as haploinsufficiency of both Ybx2 and Ybx3 caused sterility characterized by extensive defects in spermatid differentiation. RNA sequence analysis of mRNP and polysome occupancy in single and compound Ybx2/3 heterozygotes revealed loss of translational repression almost exclusively in the compound Ybx2/3 heterozygotes. RNAseq analysis also demonstrated that Y-box protein dose-dependent loss of translational regulation was inversely correlated with the presence of a Y box recognition target sequence, suggesting that Y box proteins bind RNA hierarchically to modulate translation in a range of targets.

  18. F-BOX proteins in cancer cachexia and muscle wasting: Emerging regulators and therapeutic opportunities.

    Science.gov (United States)

    Sukari, Ammar; Muqbil, Irfana; Mohammad, Ramzi M; Philip, Philip A; Azmi, Asfar S

    2016-02-01

    Cancer cachexia is a debilitating metabolic syndrome accounting for fatigue, an impairment of normal activities, loss of muscle mass associated with body weight loss eventually leading to death in majority of patients with advanced disease. Cachexia patients undergoing skeletal muscle atrophy show consistent activation of the SCF ubiquitin ligase (F-BOX) family member Atrogin-1 (also known as MAFBx/FBXO32) alongside the activation of the muscle ring finger protein1 (MuRF1). Other lesser known F-BOX family members are also emerging as key players supporting muscle wasting pathways. Recent work highlights a spectrum of different cancer signaling mechanisms impacting F-BOX family members that feed forward muscle atrophy related genes during cachexia. These novel players provide unique opportunities to block cachexia induced skeletal muscle atrophy by therapeutically targeting the SCF protein ligases. Conversely, strategies that induce the production of proteins may be helpful to counter the effects of these F-BOX proteins. Through this review, we bring forward some novel targets that promote atrogin-1 signaling in cachexia and muscle wasting and highlight newer therapeutic opportunities that can help in the better management of patients with this devastating and fatal disorder. Copyright © 2016 Elsevier Ltd. All rights reserved.

  19. Compound Heterozygosity for Y Box Proteins Causes Sterility Due to Loss of Translational Repression.

    Science.gov (United States)

    Snyder, Elizabeth; Soundararajan, Ramani; Sharma, Manju; Dearth, Andrea; Smith, Benjamin; Braun, Robert E

    2015-12-01

    The Y-box proteins YBX2 and YBX3 bind RNA and DNA and are required for metazoan development and fertility. However, possible functional redundancy between YBX2 and YBX3 has prevented elucidation of their molecular function as RNA masking proteins and identification of their target RNAs. To investigate possible functional redundancy between YBX2 and YBX3, we attempted to construct Ybx2-/-;Ybx3-/- double mutants using a previously reported Ybx2-/- model and a newly generated global Ybx3-/- model. Loss of YBX3 resulted in reduced male fertility and defects in spermatid differentiation. However, homozygous double mutants could not be generated as haploinsufficiency of both Ybx2 and Ybx3 caused sterility characterized by extensive defects in spermatid differentiation. RNA sequence analysis of mRNP and polysome occupancy in single and compound Ybx2/3 heterozygotes revealed loss of translational repression almost exclusively in the compound Ybx2/3 heterozygotes. RNAseq analysis also demonstrated that Y-box protein dose-dependent loss of translational regulation was inversely correlated with the presence of a Y box recognition target sequence, suggesting that Y box proteins bind RNA hierarchically to modulate translation in a range of targets.

  20. The Nuclear Protein Database (NPD): sub-nuclear localisation and functional annotation of the nuclear proteome

    Science.gov (United States)

    Dellaire, G.; Farrall, R.; Bickmore, W.A.

    2003-01-01

    The Nuclear Protein Database (NPD) is a curated database that contains information on more than 1300 vertebrate proteins that are thought, or are known, to localise to the cell nucleus. Each entry is annotated with information on predicted protein size and isoelectric point, as well as any repeats, motifs or domains within the protein sequence. In addition, information on the sub-nuclear localisation of each protein is provided and the biological and molecular functions are described using Gene Ontology (GO) terms. The database is searchable by keyword, protein name, sub-nuclear compartment and protein domain/motif. Links to other databases are provided (e.g. Entrez, SWISS-PROT, OMIM, PubMed, PubMed Central). Thus, NPD provides a gateway through which the nuclear proteome may be explored. The database can be accessed at http://npd.hgu.mrc.ac.uk and is updated monthly. PMID:12520015

  1. The Nuclear Protein Database (NPD): sub-nuclear localisation and functional annotation of the nuclear proteome.

    Science.gov (United States)

    Dellaire, G; Farrall, R; Bickmore, W A

    2003-01-01

    The Nuclear Protein Database (NPD) is a curated database that contains information on more than 1300 vertebrate proteins that are thought, or are known, to localise to the cell nucleus. Each entry is annotated with information on predicted protein size and isoelectric point, as well as any repeats, motifs or domains within the protein sequence. In addition, information on the sub-nuclear localisation of each protein is provided and the biological and molecular functions are described using Gene Ontology (GO) terms. The database is searchable by keyword, protein name, sub-nuclear compartment and protein domain/motif. Links to other databases are provided (e.g. Entrez, SWISS-PROT, OMIM, PubMed, PubMed Central). Thus, NPD provides a gateway through which the nuclear proteome may be explored. The database can be accessed at http://npd.hgu.mrc.ac.uk and is updated monthly.

  2. F-box protein specificity for g1 cyclins is dictated by subcellular localization.

    Directory of Open Access Journals (Sweden)

    Benjamin D Landry

    Full Text Available Levels of G1 cyclins fluctuate in response to environmental cues and couple mitotic signaling to cell cycle entry. The G1 cyclin Cln3 is a key regulator of cell size and cell cycle entry in budding yeast. Cln3 degradation is essential for proper cell cycle control; however, the mechanisms that control Cln3 degradation are largely unknown. Here we show that two SCF ubiquitin ligases, SCF(Cdc4 and SCF(Grr1, redundantly target Cln3 for degradation. While the F-box proteins (FBPs Cdc4 and Grr1 were previously thought to target non-overlapping sets of substrates, we find that Cdc4 and Grr1 each bind to all 3 G1 cyclins in cell extracts, yet only Cln3 is redundantly targeted in vivo, due in part to its nuclear localization. The related cyclin Cln2 is cytoplasmic and exclusively targeted by Grr1. However, Cdc4 can interact with Cdk-phosphorylated Cln2 and target it for degradation when cytoplasmic Cdc4 localization is forced in vivo. These findings suggest that Cdc4 and Grr1 may share additional redundant targets and, consistent with this possibility, grr1Δ cdc4-1 cells demonstrate a CLN3-independent synergistic growth defect. Our findings demonstrate that structurally distinct FBPs are capable of interacting with some of the same substrates; however, in vivo specificity is achieved in part by subcellular localization. Additionally, the FBPs Cdc4 and Grr1 are partially redundant for proliferation and viability, likely sharing additional redundant substrates whose degradation is important for cell cycle progression.

  3. Identification, cloning and sequencing of two major venom proteins from the box jellyfish, Chironex fleckeri.

    Science.gov (United States)

    Brinkman, Diane; Burnell, James

    2007-11-01

    Two of the most abundant proteins found in the nematocysts of the box jellyfish Chironex fleckeri have been identified as C. fleckeri toxin-1 (CfTX-1) and toxin-2 (CfTX-2). The molecular masses of CfTX-1 and CfTX-2, as determined by SDS-PAGE, are approximately 43 and 45 kDa, respectively, and both proteins are strongly antigenic to commercially available box jellyfish antivenom and rabbit polyclonal antibodies raised against C. fleckeri nematocyst extracts. The amino acid sequences of mature CfTX-1 and CfTX-2 (436 and 445 residues, respectively) share significant homology with three known proteins: CqTX-A from Chiropsalmus quadrigatus, CrTXs from Carybdea rastoni and CaTX-A from Carybdea alata, all of which are lethal, haemolytic box jellyfish toxins. Multiple sequence alignment of the five jellyfish proteins has identified several short, but highly conserved regions of amino acids that coincide with a predicted transmembrane spanning region, referred to as TSR1, which may be involved in a pore-forming mechanism of action. Furthermore, remote protein homology predictions for CfTX-2 and CaTX-A suggest weak structural similarities to pore-forming insecticidal delta-endotoxins Cry1Aa, Cry3Bb and Cry3A.

  4. Overexpression of Forkhead Box M1 transcription factor and nuclear factor-κB in laryngeal squamous cell carcinoma: a potential indicator for poor prognosis.

    Science.gov (United States)

    Jiang, Li-Zhu; Wang, Peng; Deng, Bi; Huang, Chuang; Tang, Wei-Xue; Lu, Hong-Yi; Chen, Hong-Yan

    2011-08-01

    The Forkhead Box M1 transcription factor and nuclear factor-κB have been shown to play important roles in the development and progression of human cancers. However, the functional significance of Forkhead Box M1 transcription factor in laryngeal squamous cell carcinoma and the correlation between Forkhead Box M1 transcription factor and nuclear factor-κB remain unclear. In the current study, we have shown that Forkhead Box M1 transcription factor and nuclear factor-κB were significantly overexpressed in laryngeal squamous cell carcinoma tissues and precancerous lesions, compared with adjacent normal tissues (both P Box M1 transcription factor was significantly associated with histologic differentiation (rs = 0.321, P = .002), T stage (rs = 0.276, P = .009), lymph node metastasis (rs = 0.266, P = .012), and clinical stage (rs = 0.272, P = .010); overexpression of nuclear factor-κB was significantly associated with T stage (rs = 0.404, P Box M1 transcription factor and nuclear factor-κB were associated with worse overall survival (P = .041 and P Cox regression analysis showed that T stage, lymph node metastasis, and nuclear factor-κB were independent prognostic factors for laryngeal squamous cell carcinoma (P = .038, P = .014, and P = .005, respectively). Furthermore, a significant correlation was observed between Forkhead Box M1 transcription factor and nuclear factor-κB (rs = 0.683, P Box M1 transcription factor and nuclear factor-κB and the possible interaction between them may play important roles in the development and progression of laryngeal squamous cell carcinoma, and Forkhead Box M1 transcription factor and nuclear factor-κB may serve as useful prognostic markers for laryngeal squamous cell carcinoma. Crown Copyright © 2011. Published by Elsevier Inc. All rights reserved.

  5. Biochemical function of typical and variant Arabidopsis thaliana U-box E3 ubiquitin-protein ligases

    DEFF Research Database (Denmark)

    Wiborg, Jakob; O'Shea, Charlotte; Skriver, Karen

    2008-01-01

    respectively, but no productive interaction was observed with the UBC15 E2 tested. The activity of AtPUB54 [Arabidopsis thaliana (thale cress) plant U-box 54 protein] was dependent on Trp(266) in the E2-binding cleft, and the E2 selectivity was changed by substitution of this position. The function...... of the distant U-box protein, AtPUB49, representing a large family of eukaryotic proteins containing a U-box linked to a cyclophilin-like peptidyl-prolyl cis-trans isomerase domain, was characterized biochemically. AtPUB49 functioned both as a prolyl isomerase and a chaperone by catalysing cis.......g. co-existence or interactions with additional domains. The biochemical functions of AtPUB49 suggest that the protein can be involved in folding or degradation of protein substrates. Similar functions can also be retained within a protein complex with separate chaperone and U-box proteins....

  6. Nuclear factor ETF specifically stimulates transcription from promoters without a TATA box.

    Science.gov (United States)

    Kageyama, R; Merlino, G T; Pastan, I

    1989-09-15

    Transcription factor ETF stimulates the expression of the epidermal growth factor receptor (EGFR) gene which does not have a TATA box in the promoter region. Here, we show that ETF recognizes various GC-rich sequences including stretches of deoxycytidine or deoxyguanosine residues and GC boxes with similar affinities. ETF also binds to TATA boxes but with a lower affinity. ETF stimulated in vitro transcription from several promoters without TATA boxes but had little or no effect on TATA box-containing promoters even though they had strong ETF-binding sites. These inactive ETF-binding sites became functional when placed upstream of the EGFR promoter whose own ETF-binding sites were removed. Furthermore, when a TATA box was introduced into the EGFR promoter, the responsiveness to ETF was abolished. These results indicate that ETF is a specific transcription factor for promoters which do not contain TATA elements.

  7. Nuclear roles for cilia-associated proteins.

    Science.gov (United States)

    McClure-Begley, Tristan D; Klymkowsky, Michael W

    2017-01-01

    Cilia appear to be derived, evolutionarily, from structures present in the ancestral (pre-ciliary) eukaryote, such as microtubule-based vesicle trafficking and chromosome segregation systems. Experimental observations suggest that the ciliary gate, the molecular complex that mediates the selective molecular movement between cytoplasmic and ciliary compartments, shares features with nuclear pores. Our hypothesis is that this shared transport machinery is at least partially responsible for the observation that a number of ciliary and ciliogenesis-associated proteins are found within nuclei where they play roles in the regulation of gene expression, DNA repair, and nuclear import and export. Recognizing the potential for such nuclear roles is critical when considering the phenotypic effects that arise from the mutational modification of ciliary proteins.

  8. Structural Analysis of the G-Box Domain of the Microcephaly Protein CPAP Suggests a Role in Centriole Architecture

    Science.gov (United States)

    Hatzopoulos, Georgios N.; Erat, Michèle C.; Cutts, Erin; Rogala, Kacper B.; Slater, Leanne M.; Stansfeld, Philip J.; Vakonakis, Ioannis

    2013-01-01

    Summary Centrioles are evolutionarily conserved eukaryotic organelles composed of a protein scaffold surrounded by sets of microtubules organized with a 9-fold radial symmetry. CPAP, a centriolar protein essential for microtubule recruitment, features a C-terminal domain of unknown structure, the G-box. A missense mutation in the G-box reduces affinity for the centriolar shuttling protein STIL and causes primary microcephaly. Here, we characterize the molecular architecture of CPAP and determine the G-box structure alone and in complex with a STIL fragment. The G-box comprises a single elongated β sheet capable of forming supramolecular assemblies. Structural and biophysical studies highlight the conserved nature of the CPAP-STIL complex. We propose that CPAP acts as a horizontal “strut” that joins the centriolar scaffold with microtubules, whereas G-box domains form perpendicular connections. PMID:24076405

  9. Monte Carlo simulation of a clearance box monitor used for nuclear power plant decommissioning.

    Science.gov (United States)

    Bochud, François O; Laedermann, Jean-Pascal; Bailat, Claude J; Schuler, Christoph

    2009-05-01

    When decommissioning a nuclear facility it is important to be able to estimate activity levels of potentially radioactive samples and compare with clearance values defined by regulatory authorities. This paper presents a method of calibrating a clearance box monitor based on practical experimental measurements and Monte Carlo simulations. Adjusting the simulation for experimental data obtained using a simple point source permits the computation of absolute calibration factors for more complex geometries with an accuracy of a bit more than 20%. The uncertainty of the calibration factor can be improved to about 10% when the simulation is used relatively, in direct comparison with a measurement performed in the same geometry but with another nuclide. The simulation can also be used to validate the experimental calibration procedure when the sample is supposed to be homogeneous but the calibration factor is derived from a plate phantom. For more realistic geometries, like a small gravel dumpster, Monte Carlo simulation shows that the calibration factor obtained with a larger homogeneous phantom is correct within about 20%, if sample density is taken as the influencing parameter. Finally, simulation can be used to estimate the effect of a contamination hotspot. The research supporting this paper shows that activity could be largely underestimated in the event of a centrally-located hotspot and overestimated for a peripherally-located hotspot if the sample is assumed to be homogeneously contaminated. This demonstrates the usefulness of being able to complement experimental methods with Monte Carlo simulations in order to estimate calibration factors that cannot be directly measured because of a lack of available material or specific geometries.

  10. An Arabidopsis F-box protein regulates tapetum degeneration and pollen maturation during anther development.

    Science.gov (United States)

    Kim, Ok-Kyoung; Jung, Jae-Hoon; Park, Chung-Mo

    2010-07-01

    The Arabidopsis anther has a bilateral symmetry with four lobes, each consisting of four distinct layers of somatic cells from the outer to inner side: epidermis, endothecium, middle layer and tapetum. The tapetum is a layer of cells comprising the inner surface of the pollen wall. It plays an important role in anther development by providing enzymes, materials and nutrients required for pollen maturation. Genes and molecular mechanisms underlying tapetum formation and pollen wall biosynthesis have been studied in Arabidopsis. However, tapetum degeneration and anther dehiscence have not been well characterized at the molecular level. Here, we report that an Arabidopsis gene, designated reduced male fertility (RMF), regulates degeneration of tapetum and middle layer during anther development. The Arabidopsis dominant mutant rmf-1D overexpressing the RMF gene exhibited pleiotropic phenotypes, including dwarfed growth with small, dark-green leaves and low male fertility. Tapetum development and subsequent degeneration were impaired in the mutant. Accordingly, pollen maturation was disturbed, reducing the male fertility. In contrast, tapetum degeneration was somewhat accelerated in the RMF RNAi plants. The RMF gene was expressed predominantly in the anther, particularly in the pollen grains. Notably, the RMF protein contains an F-box motif and is localized to the nucleus. It physically interacts with the Arabidopsis-Skp1-like1 protein via the F-box motif. These observations indicate that the RMF gene encodes an F-box protein functioning in tapetum degeneration during anther development.

  11. Roles of F-box proteins in human digestive system tumors (Review).

    Science.gov (United States)

    Gong, Jian; Lv, Liang; Huo, Jirong

    2014-12-01

    F-box proteins (FBPs), the substrate-recognition subunit of E3 ubiquitin (Ub) ligase, are the important components of Ub proteasome system (UPS). FBPs are involved in multiple cellular processes through ubiquitylation and subsequent degradation of their target proteins. Many studies have described the roles of FBPs in human cancers. Digestive system tumors account for a large proportion of all the tumors, and their mortality is very high. This review summarizes for the first time the roles of FBPs in digestive system tumorige-nesis and tumor progression, aiming at finding new routes for the rational design of targeted anticancer therapies in digestive system tumors.

  12. Partial purification of cytolytic venom proteins from the box jellyfish, Chironex fleckeri.

    Science.gov (United States)

    Brinkman, Diane; Burnell, James

    2008-04-01

    Venom proteins from the nematocysts of Chironex fleckeri were fractionated by size-exclusion and cation-exchange chromatography. Using sheep erythrocyte haemolysis as an indicator of cytolytic activity, two major cytolysins, with native molecular masses of approximately 370 and 145kDa, and one minor cytolysin ( approximately 70kDa) were isolated. SDS-PAGE and western blot protein profiles revealed that the 370kDa haemolysin is composed of CfTX-1 and CfTX-2 subunits ( approximately 43 and 45kDa, respectively); the most abundant proteins found in C. fleckeri nematocyst extracts. The 145kDa haemolysin predominately contains two other major proteins ( approximately 39 and 41kDa), which are not antigenic towards commercially available box jellyfish antivenom or rabbit polyclonal antibodies raised against whole C. fleckeri nematocyst extracts or CfTX-1 and -2. The kinetics of CfTX-1 and -2 haemolytic activities are temperature dependent and characterised by a pre-lytic lag phase ( approximately 6-7min) prior to initiation of haemolysis. Significant amino acid sequence homology between the CfTX proteins and other box jellyfish toxins suggest that CfTX-1 and -2 may also be lethal and dermonecrotic. Therefore, further in vivo and in vitro studies are required to investigate the potential roles of CfTX-1 and -2 in the lethal effects of C. fleckeri venom.

  13. Related F-box proteins control cell death in Caenorhabditis elegans and human lymphoma

    Science.gov (United States)

    Chiorazzi, Michael; Rui, Lixin; Yang, Yandan; Ceribelli, Michele; Tishbi, Nima; Maurer, Carine W.; Ranuncolo, Stella M.; Zhao, Hong; Xu, Weihong; Chan, Wing-Chung C.; Jaffe, Elaine S.; Gascoyne, Randy D.; Campo, Elias; Rosenwald, Andreas; Ott, German; Delabie, Jan; Rimsza, Lisa M.; Shaham, Shai; Staudt, Louis M.

    2013-01-01

    Cell death is a common metazoan cell fate, and its inactivation is central to human malignancy. In Caenorhabditis elegans, apoptotic cell death occurs via the activation of the caspase CED-3 following binding of the EGL-1/BH3-only protein to the antiapoptotic CED-9/BCL2 protein. Here we report a major alternative mechanism for caspase activation in vivo involving the F-box protein DRE-1. DRE-1 functions in parallel to EGL-1, requires CED-9 for activity, and binds to CED-9, suggesting that DRE-1 promotes apoptosis by inactivating CED-9. FBXO10, a human protein related to DRE-1, binds BCL2 and promotes its degradation, thereby initiating cell death. Moreover, some human diffuse large B-cell lymphomas have inactivating mutations in FBXO10 or express FBXO10 at low levels. Our results suggest that DRE-1/FBXO10 is a conserved regulator of apoptosis. PMID:23431138

  14. Identification and expression analysis of the SQUAMOSA promoter-binding protein (SBP)-box gene family in Prunus mume.

    Science.gov (United States)

    Xu, Zongda; Sun, Lidan; Zhou, Yuzhen; Yang, Weiru; Cheng, Tangren; Wang, Jia; Zhang, Qixiang

    2015-10-01

    SQUAMOSA promoter-binding protein (SBP)-box family genes encode plant-specific transcription factors that play crucial roles in plant development, especially flower and fruit development. However, little information on this gene family is available for Prunus mume, an ornamental and fruit tree widely cultivated in East Asia. To explore the evolution of SBP-box genes in Prunus and explore their functions in flower and fruit development, we performed a genome-wide analysis of the SBP-box gene family in P. mume. Fifteen SBP-box genes were identified, and 11 of them contained an miR156 target site. Phylogenetic and comprehensive bioinformatics analyses revealed that different groups of SBP-box genes have undergone different evolutionary processes and varied in their length, structure, and motif composition. Purifying selection has been the main selective constraint on both paralogous and orthologous SBP-box genes. In addition, the sequences of orthologous SBP-box genes did not diverge widely after the split of P. mume and Prunus persica. Expression analysis of P. mume SBP-box genes revealed their diverse spatiotemporal expression patterns. Three duplicated SBP-box genes may have undergone subfunctionalization in Prunus. Most of the SBP-box genes showed high transcript levels in flower buds and young fruit. The four miR156-nontargeted genes were upregulated during fruit ripening. Together, these results provide information about the evolution of SBP-box genes in Prunus. The expression analysis lays the foundation for further research on the functions of SBP-box genes in P. mume and other Prunus species, especially during flower and fruit development.

  15. How the ankyrin and SOCS box protein, ASB9, binds to creatine kinase.

    Science.gov (United States)

    Balasubramaniam, Deepa; Schiffer, Jamie; Parnell, Jonathan; Mir, Stephan P; Amaro, Rommie E; Komives, Elizabeth A

    2015-03-03

    The ankyrin repeat and SOCS box (ASB) family is composed of 18 proteins and belongs to the suppressor of cytokine signaling (SOCS) box protein superfamily. The ASB proteins function as the substrate-recognition subunits of ECS-type (ElonginBC-Cullin-SOCS-box) Cullin RING E3 ubiquitin ligase (CRL) complexes that specifically transfer ubiquitin to cellular proteins targeting them for degradation by the proteasome. ASB9 binds to creatine kinase (CK) and targets it for degradation; however, the way in which ASB9 interacts with CK is not yet known. We present a complete characterization of the binding of ASB9 to CK. One ASB9 molecule binds to a dimer of CK. The binding affinity of ASB9(1-252) was extremely tight, and no dissociation could be observed. Deletion of the 34 N-terminal amino acids forming ASB9(35-252) resulted in weakening of the binding, so that a binding affinity of 2.6 nM could be measured. Amide hydrogen-deuterium exchange (HDXMS) experiments showed that both ASB9(1-252) and ASB9(35-252) protected the same region of CK, residues 182-203, which forms one side of the active site. The HDXMS experiments indicated that the N-terminal disordered region and first ankyrin repeat of ASB9 are protected from exchange in the complex. Molecular docking yielded a structural model consistent with all of the data that suggested the N-terminal residues of ASB9(1-252) may lie in one CK active site. This model was corroborated by enzymatic activity assays and mutational analysis.

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

    Science.gov (United States)

    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

  17. Synchronization of cytoplasmic and transferred mitochondrial ribosomal protein gene expression in land plants is linked to Telo-box motif enrichment

    Directory of Open Access Journals (Sweden)

    Zhu Xin-Guang

    2011-06-01

    Full Text Available Abstract Background Chloroplasts and mitochondria evolved from the endosymbionts of once free-living eubacteria, and they transferred most of their genes to the host nuclear genome during evolution. The mechanisms used by plants to coordinate the expression of such transferred genes, as well as other genes in the host nuclear genome, are still poorly understood. Results In this paper, we use nuclear-encoded chloroplast (cpRPGs, as well as mitochondrial (mtRPGs and cytoplasmic (euRPGs ribosomal protein genes to study the coordination of gene expression between organelles and the host. Results show that the mtRPGs, but not the cpRPGs, exhibit strongly synchronized expression with euRPGs in all investigated land plants and that this phenomenon is linked to the presence of a telo-box DNA motif in the promoter regions of mtRPGs and euRPGs. This motif is also enriched in the promoter regions of genes involved in DNA replication. Sequence analysis further indicates that mtRPGs, in contrast to cpRPGs, acquired telo-box from the host nuclear genome. Conclusions Based on our results, we propose a model of plant nuclear genome evolution where coordination of activities in mitochondria and chloroplast and other cellular functions, including cell cycle, might have served as a strong selection pressure for the differential acquisition of telo-box between mtRPGs and cpRPGs. This research also highlights the significance of physiological needs in shaping transcriptional regulatory evolution.

  18. Forkhead box O1 and muscle RING finger 1 protein expression in atrophic and hypertrophic denervated mouse skeletal muscle

    Science.gov (United States)

    2014-01-01

    Background Forkhead box O (FoxO) transcription factors and E3 ubiquitin ligases such as Muscle RING finger 1 (MuRF1) are believed to participate in the regulation of skeletal muscle mass. The function of FoxO transcription factors is regulated by post-translational modifications such as phosphorylation and acetylation. In the present study FoxO1 protein expression, phosphorylation and acetylation as well as MuRF1 protein expression, were examined in atrophic and hypertrophic denervated skeletal muscle. Methods Protein expression, phosphorylation and acetylation were studied semi-quantitatively using Western blots. Muscles studied were 6-days denervated mouse hind-limb muscles (anterior tibial as well as pooled gastrocnemius and soleus muscles, all atrophic), 6-days denervated mouse hemidiaphragm muscles (hypertrophic) and innervated control muscles. Total muscle homogenates were used as well as separated nuclear and cytosolic fractions of innervated and 6-days denervated anterior tibial and hemidiaphragm muscles. Results Expression of FoxO1 and MuRF1 proteins increased 0.3-3.7-fold in all 6-days denervated muscles studied, atrophic as well as hypertrophic. Phosphorylation of FoxO1 at S256 increased about 0.8-1-fold after denervation in pooled gastrocnemius and soleus muscles and in hemidiaphragm but not in unfractionated anterior tibial muscle. A small (0.2-fold) but statistically significant increase in FoxO1 phosphorylation was, however, observed in cytosolic fractions of denervated anterior tibial muscle. A statistically significant increase in FoxO1 acetylation (0.8-fold) was observed only in denervated anterior tibial muscle. Increases in total FoxO1 and in phosphorylated FoxO1 were only seen in cytosolic fractions of denervated atrophic anterior tibial muscle whereas in denervated hypertrophic hemidiaphragm both total FoxO1 and phosphorylated FoxO1 increased in cytosolic as well as in nuclear fractions. MuRF1 protein expression increased in cytosolic as well

  19. Nopaline-type Ti plasmid of Agrobacterium encodes a VirF-like functional F-box protein.

    Science.gov (United States)

    Lacroix, Benoît; Citovsky, Vitaly

    2015-11-20

    During Agrobacterium-mediated genetic transformation of plants, several bacterial virulence (Vir) proteins are translocated into the host cell to facilitate infection. One of the most important of such translocated factors is VirF, an F-box protein produced by octopine strains of Agrobacterium, which presumably facilitates proteasomal uncoating of the invading T-DNA from its associated proteins. The presence of VirF also is thought to be involved in differences in host specificity between octopine and nopaline strains of Agrobacterium, with the current dogma being that no functional VirF is encoded by nopaline strains. Here, we show that a protein with homology to octopine VirF is encoded by the Ti plasmid of the nopaline C58 strain of Agrobacterium. This protein, C58VirF, possesses the hallmarks of functional F-box proteins: it contains an active F-box domain and specifically interacts, via its F-box domain, with SKP1-like (ASK) protein components of the plant ubiquitin/proteasome system. Thus, our data suggest that nopaline strains of Agrobacterium have evolved to encode a functional F-box protein VirF.

  20. Regulatory T Cell and Forkhead Box Protein 3 as Modulators of Immune Homeostasis

    Directory of Open Access Journals (Sweden)

    Leonn Mendes Soares Pereira

    2017-05-01

    Full Text Available The transcription factor forkhead box protein 3 (FOXP3 is an essential molecular marker of regulatory T cell (Treg development in different microenvironments. Tregs are cells specialized in the suppression of inadequate immune responses and the maintenance of homeostatic tolerance. Studies have addressed and elucidated the role played by FOXP3 and Treg in countless autoimmune and infectious diseases as well as in more specific cases, such as cancer. Within this context, the present article reviews aspects of the immunoregulatory profile of FOXP3 and Treg in the management of immune homeostasis, including issues relating to pathology as well as immune tolerance.

  1. Regulatory T Cell and Forkhead Box Protein 3 as Modulators of Immune Homeostasis.

    Science.gov (United States)

    Pereira, Leonn Mendes Soares; Gomes, Samara Tatielle Monteiro; Ishak, Ricardo; Vallinoto, Antonio Carlos Rosário

    2017-01-01

    The transcription factor forkhead box protein 3 (FOXP3) is an essential molecular marker of regulatory T cell (Treg) development in different microenvironments. Tregs are cells specialized in the suppression of inadequate immune responses and the maintenance of homeostatic tolerance. Studies have addressed and elucidated the role played by FOXP3 and Treg in countless autoimmune and infectious diseases as well as in more specific cases, such as cancer. Within this context, the present article reviews aspects of the immunoregulatory profile of FOXP3 and Treg in the management of immune homeostasis, including issues relating to pathology as well as immune tolerance.

  2. Essential Role of X-Box Binding Protein-1 during Endoplasmic Reticulum Stress in Podocytes.

    Science.gov (United States)

    Hassan, Hossam; Tian, Xuefei; Inoue, Kazunori; Chai, Nathan; Liu, Chang; Soda, Keita; Moeckel, Gilbert; Tufro, Alda; Lee, Ann-Hwee; Somlo, Stefan; Fedeles, Sorin; Ishibe, Shuta

    2016-04-01

    Podocytes are terminally differentiated epithelial cells that reside along the glomerular filtration barrier. Evidence suggests that after podocyte injury, endoplasmic reticulum stress response is activated, but the molecular mechanisms involved are incompletely defined. In a mouse model, we confirmed that podocyte injury induces endoplasmic reticulum stress response and upregulated unfolded protein response pathways, which have been shown to mitigate damage by preventing the accumulation of misfolded proteins in the endoplasmic reticulum. Furthermore, simultaneous podocyte-specific genetic inactivation of X-box binding protein-1 (Xbp1), a transcription factor activated during endoplasmic reticulum stress and critically involved in the untranslated protein response, and Sec63, a heat shock protein-40 chaperone required for protein folding in the endoplasmic reticulum, resulted in progressive albuminuria, foot process effacement, and histology consistent with ESRD. Finally, loss of both Sec63 and Xbp1 induced apoptosis in podocytes, which associated with activation of the JNK pathway. Collectively, our results indicate that an intact Xbp1 pathway operating to mitigate stress in the endoplasmic reticulum is essential for the maintenance of a normal glomerular filtration barrier. Copyright © 2016 by the American Society of Nephrology.

  3. Alterations in the expression of DEAD-box and other RNA binding proteins during HIV-1 replication

    Directory of Open Access Journals (Sweden)

    Zeichner Steven L

    2004-12-01

    Full Text Available Abstract Recent results showed that certain DEAD box protein RNA helicases, DDX3 and DDX1, play an important role in the HIV infection cycle by facilitating the export of long, singly spliced or unspliced HIV RNAs from the nucleus via the CRM1-Rev pathway. Close examination of an extensive microarray expression profiling dataset obtained from cells latently infected with HIV induced to undergo lytic viral replication indicated that additional DEAD box proteins, beyond DDX3 and DDX1, exhibit differential expression during lytic HIV replication, and in latently infected cells prior to induction into active replication. This finding provides additional evidence that the involvement of DEAD box proteins and other RNA-binding proteins may play roles in active HIV replication and in the control of viral latency. Agents targeting these functions may offer new approaches to antiretroviral therapy and the therapeutic manipulation of HIV latency.

  4. Guideline for making application dossiers for novel proteins; Novel food dossiers: from black box to tool box

    NARCIS (Netherlands)

    Wagenberg, van C.P.A.; Janssens, B.; Kalk, C.; Sluis, van der A.; Noordam, M.Y.; Spiegel, van der M.

    2014-01-01

    This document describes when a new protein intended to market for human consumption is considered to be a novel food protein (novel protein) and when it is not, when an application dossier for authorisation must be made and when, in general, an application dossier for notification might be

  5. TBP-like protein (TLP) interferes with Taspase1-mediated processing of TFIIA and represses TATA box gene expression.

    Science.gov (United States)

    Suzuki, Hidefumi; Isogai, Momoko; Maeda, Ryo; Ura, Kiyoe; Tamura, Taka-Aki

    2015-07-27

    TBP-TFIIA interaction is involved in the potentiation of TATA box-driven promoters. TFIIA activates transcription through stabilization of TATA box-bound TBP. The precursor of TFIIA is subjected to Taspase1-directed processing to generate α and β subunits. Although this processing has been assumed to be required for the promoter activation function of TFIIA, little is known about how the processing is regulated. In this study, we found that TBP-like protein (TLP), which has the highest affinity to TFIIA among known proteins, affects Taspase1-driven processing of TFIIA. TLP interfered with TFIIA processing in vivo and in vitro, and direct binding of TLP to TFIIA was essential for inhibition of the processing. We also showed that TATA box promoters are specifically potentiated by processed TFIIA. Processed TFIIA, but not unprocessed TFIIA, associated with the TATA box. In a TLP-knocked-down condition, not only the amounts of TATA box-bound TFIIA but also those of chromatin-bound TBP were significantly increased, resulting in the stimulation of TATA box-mediated gene expression. Consequently, we suggest that TLP works as a negative regulator of the TFIIA processing and represses TFIIA-governed and TATA-dependent gene expression through preventing TFIIA maturation. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

  6. Anabolic Properties of High Mobility Group Box Protein-1 in Human Periodontal Ligament Cells In Vitro

    Directory of Open Access Journals (Sweden)

    Michael Wolf

    2014-01-01

    Full Text Available High mobility group box protein-1 (HMGB1 is mainly recognized as a chemoattractant for macrophages in the initial phase of host response to pathogenic stimuli. However, recent findings provide evidence for anabolic properties in terms of enhanced proliferation, migration, and support of wound healing capacity of mesenchymal cells suggesting a dual role of the cytokine in the regulation of immune response and subsequent regenerative processes. Here, we examined potential anabolic effects of HMGB1 on human periodontal ligament (PDL cells in the regulation of periodontal remodelling, for example, during orthodontic tooth movement. Preconfluent human PDL cells (hPDL were exposed to HMGB1 protein and the influence on proliferation, migration, osteogenic differentiation, and biomineralization was determined by MTS assay, real time PCR, immunofluorescence cytochemistry, ELISA, and von Kossa staining. HMGB1 protein increased hPDL cell proliferation, migration, osteoblastic marker gene expression, and protein production as well as mineralized nodule formation significantly. The present findings support the dual character of HMGB1 with anabolic therapeutic potential that might support the reestablishment of the structural and functional integrity of the periodontium following periodontal trauma such as orthodontic tooth movement.

  7. Cutting edge: extracellular high mobility group box-1 protein is a proangiogenic cytokine.

    Science.gov (United States)

    Mitola, Stefania; Belleri, Mirella; Urbinati, Chiara; Coltrini, Daniela; Sparatore, Bianca; Pedrazzi, Marco; Melloni, Edon; Presta, Marco

    2006-01-01

    The chromosomal high mobility group box-1 (HMGB1) protein acts as a proinflammatory cytokine when released in the extracellular environment by necrotic and inflammatory cells. In the present study, we show that HMGB1 exerts proangiogenic effects by inducing MAPK ERK1/2 activation, cell proliferation, and chemotaxis in endothelial cells of different origin. Accordingly, HMGB1 stimulates membrane ruffling and repair of a mechanically wounded endothelial cell monolayer and causes endothelial cell sprouting in a three-dimensional fibrin gel. In keeping with its in vitro properties, HMGB1 stimulates neovascularization when applied in vivo on the top of the chicken embryo chorioallantoic membrane whose blood vessels express the HMGB1 receptor for advanced glycation end products (RAGE). Accordingly, RAGE blockade by neutralizing Abs inhibits HMGB1-induced neovascularization in vivo and endothelial cell proliferation and membrane ruffling in vitro. Taken together, the data identify HMGB1/RAGE interaction as a potent proangiogenic stimulus.

  8. Regulation of Autophagy-Related Protein and Cell Differentiation by High Mobility Group Box 1 Protein in Adipocytes

    Directory of Open Access Journals (Sweden)

    Huanhuan Feng

    2016-01-01

    Full Text Available High mobility group box 1 protein (HMGB1 is a molecule related to the development of inflammation. Autophagy is vital to maintain cellular homeostasis and protect against inflammation of adipocyte injury. Our recent work focused on the relationship of HMGB1 and autophagy in 3T3-L1 cells. In vivo experimental results showed that, compared with the normal-diet group, the high-fat diet mice displayed an increase in adipocyte size in the epididymal adipose tissues. The expression levels of HMGB1 and LC3II also increased in epididymal adipose tissues in high-fat diet group compared to the normal-diet mice. The in vitro results indicated that HMGB1 protein treatment increased LC3II formation in 3T3-L1 preadipocytes in contrast to that in the control group. Furthermore, LC3II formation was inhibited through HMGB1 knockdown by siRNA. Treatment with the HMGB1 protein enhanced LC3II expression after 2 and 4 days but decreased the expression after 8 and 10 days among various differentiation stages of adipocytes. By contrast, FABP4 expression decreased on the fourth day and increased on the eighth day. Hence, the HMGB1 protein modulated autophagy-related proteins and lipid-metabolism-related genes in adipocytes and could be a new target for treatment of obesity and related metabolic diseases.

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

    Energy Technology Data Exchange (ETDEWEB)

    Busch, Albert; Kiel, Tilman; Heupel, Wolfgang-M. [University of Wuerzburg, Institute of Anatomy and Cell Biology, Koellikerstrasse 6, 97070 Wuerzburg (Germany); Wehnert, Manfred [Institute of Human Genetics, University of Greifswald, Greifswald (Germany); Huebner, Stefan, E-mail: stefan.huebner@mail.uni-wuerzburg.de [University of Wuerzburg, Institute of Anatomy and Cell Biology, Koellikerstrasse 6, 97070 Wuerzburg (Germany)

    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.

  10. The Y-Box Binding Protein 1 Suppresses Alzheimer's Disease Progression in Two Animal Models.

    Directory of Open Access Journals (Sweden)

    N V Bobkova

    Full Text Available The Y-box binding protein 1 (YB-1 is a member of the family of DNA- and RNA binding proteins. It is involved in a wide variety of DNA/RNA-dependent events including cell proliferation and differentiation, stress response, and malignant cell transformation. Previously, YB-1 was detected in neurons of the neocortex and hippocampus, but its precise role in the brain remains undefined. Here we show that subchronic intranasal injections of recombinant YB-1, as well as its fragment YB-11-219, suppress impairment of spatial memory in olfactory bulbectomized (OBX mice with Alzheimer's type degeneration and improve learning in transgenic 5XFAD mice used as a model of cerebral amyloidosis. YB-1-treated OBX and 5XFAD mice showed a decreased level of brain β-amyloid. In OBX animals, an improved morphological state of neurons was revealed in the neocortex and hippocampus; in 5XFAD mice, a delay in amyloid plaque progression was observed. Intranasally administered YB-1 penetrated into the brain and could enter neurons. In vitro co-incubation of YB-1 with monomeric β-amyloid (1-42 inhibited formation of β-amyloid fibrils, as confirmed by electron microscopy. This suggests that YB-1 interaction with β-amyloid prevents formation of filaments that are responsible for neurotoxicity and neuronal death. Our data are the first evidence for a potential therapeutic benefit of YB-1 for treatment of Alzheimer's disease.

  11. ADP-ribosylation of nuclear proteins in mouse testis

    OpenAIRE

    Faraone Mennella, Maria R.; Quesada, Piera; Farina, Benedetta; Leone, Enzo; Jones, Roy

    1982-01-01

    The nuclear acceptor proteins for poly(ADP-ribose) were investigated in mouse liver and testis. In liver, histones are ribosylated preferentially, whereas in testis the major acceptors are non-histone proteins. An analysis of the purified testicular acceptor proteins suggests that they are high- and low-mobility-group-like proteins.

  12. F-box protein FBXL2 targets cyclin D2 for ubiquitination and degradation to inhibit leukemic cell proliferation

    Science.gov (United States)

    Chen, Bill B.; Glasser, Jennifer R.; Coon, Tiffany A.; Zou, Chunbin; Miller, Hannah L.; Fenton, Moon; McDyer, John F.; Boyiadzis, Michael

    2012-01-01

    Hematologic maligancies exhibit a growth advantage by up-regulation of components within the molecular apparatus involved in cell-cycle progression. The SCF (Skip-Cullin1-F-box protein) E3 ligase family provides homeostatic feedback control of cell division by mediating ubiquitination and degradation of cell-cycle proteins. By screening several previously undescribed E3 ligase components, we describe the behavior of a relatively new SCF subunit, termed FBXL2, that ubiquitinates and destabilizes cyclin D2 protein leading to G0 phase arrest and apoptosis in leukemic and B-lymphoblastoid cell lines. FBXL2 expression was strongly suppressed, and yet cyclin D2 protein levels were robustly expressed in acute myelogenous leukemia (AML) and acute lymphoblastic leukemia (ALL) patient samples. Depletion of endogenous FBXL2 stabilized cyclin D2 levels, whereas ectopically expressed FBXL2 decreased cyclin D2 lifespan. FBXL2 did not bind a phosphodegron within its substrate, which is typical of other F-box proteins, but uniquely targeted a calmodulin-binding signature within cyclin D2 to facilitate its polyubiquitination. Calmodulin competes with the F-box protein for access to this motif where it bound and protected cyclin D2 from FBXL2. Calmodulin reversed FBXL2-induced G0 phase arrest and attenuated FBXL2-induced apoptosis of lymphoblastoid cells. These results suggest an antiproliferative effect of SCFFBXL2 in lymphoproliferative malignancies. PMID:22323446

  13. Scolopendra subspinipes mutilans protected the cerulein-induced acute pancreatitis by inhibiting high-mobility group box protein-1.

    Science.gov (United States)

    Jo, Il-Joo; Bae, Gi-Sang; Park, Kyoung-Chel; Choi, Sun Bok; Jung, Won-Seok; Jung, Su-Young; Cho, Jung-Hee; Choi, Mee-Ok; Song, Ho-Joon; Park, Sung-Joo

    2013-03-14

    To evaluate the inhibitory effects of Scolopendra subspinipes mutilans (SSM) on cerulein-induced acute pancreatitis (AP) in a mouse model. SSM water extract (0.1, 0.5, or 1 g/kg) was administrated intraperitoneally 1 h prior to the first injection of cerulein. Once AP developed, the stable cholecystokinin analogue, cerulein was injected hourly, over a 6 h period. Blood samples were taken 6 h later to determine serum amylase, lipase, and cytokine levels. The pancreas and lungs were rapidly removed for morphological examination, myeloperoxidase assay, and real-time reverse transcription polymerase chain reaction. To specify the role of SSM in pancreatitis, the pancreatic acinar cells were isolated using collagenase method. Then the cells were pre-treated with SSM, then stimulated with cerulein. The cell viability, cytokine productions and high-mobility group box protein-1 (HMGB-1) were measured. Furthermore, the regulating mechanisms of SSM action were evaluated. The administration of SSM significantly attenuated the severity of pancreatitis and pancreatitis associated lung injury, as was shown by the reduction in pancreatic edema, neutrophil infiltration, vacuolization and necrosis. SSM treatment also reduced pancreatic weight/body weight ratio, serum amylase, lipase and cytokine levels, and mRNA expression of multiple inflammatory mediators such as tumor necrosis factor-α and interleukin-1β. In addition, treatment with SSM inhibited HMGB-1 expression in the pancreas during AP. In accordance with in vivo data, SSM inhibited the cerulein-induced acinar cell death, cytokine, and HMGB-1 release. SSM also inhibited the activation of c-Jun NH2-terminal kinase, p38 and nuclear factor (NF)-κB. These results suggest that SSM plays a protective role during the development of AP and pancreatitis associated lung injury via deactivating c-Jun NH2-terminal kinase, p38 and NF-κB.

  14. Increased serum levels of high mobility group box 1 protein in patients with autistic disorder.

    Science.gov (United States)

    Emanuele, Enzo; Boso, Marianna; Brondino, Natascia; Pietra, Stefania; Barale, Francesco; Ucelli di Nemi, Stefania; Politi, Pierluigi

    2010-05-30

    High mobility group box 1 (HMGB1) is a highly conserved, ubiquitous protein that functions as an activator for inducing the immune response and can be released from neurons after glutamate excitotoxicity. The objective of the present study was to measure serum levels of HMGB1 in patients with autistic disorder and to study their relationship with clinical characteristics. We enrolled 22 adult patients with autistic disorder (mean age: 28.1+/-7.7 years) and 28 age- and gender-matched healthy controls (mean age: 28.7+/-8.1 years). Serum levels of HMGB1 were measured by enzyme-linked immunosorbent assay (ELISA). Compared with healthy subjects, serum levels of HMGB1 were significantly higher in patients with autistic disorder (10.8+/-2.6 ng/mL versus 5.6+/-2.5 ng/mL, respectively, Pautistic disorder. Increased HMGB1 may be a biological correlate of the impaired reciprocal social interactions in this neurodevelopmental disorder. Copyright 2010 Elsevier Inc. All rights reserved.

  15. Nuclear localization signal regulates porcine circovirus type 2 capsid protein nuclear export through phosphorylation.

    Science.gov (United States)

    Hou, Qiang; Hou, Shaohua; Chen, Qing; Jia, Hong; Xin, Ting; Jiang, Yitong; Guo, Xiaoyu; Zhu, Hongfei

    2018-02-15

    The open reading frame 2 (ORF2) of Porcine circovirus type 2 (PCV2) encodes the major Capsid (Cap) protein, which self-assembles into virus-like particle (VLP) of similar morphology to the PCV2 virion and accumulates in the nucleus through the N-terminal arginine-rich nuclear localization signal (NLS). In this study, PCV2 Cap protein and its derivates were expressed via the baculovirus expression system, and the cellular localization of the recombinant proteins were investigated using anti-Cap mAb by imaging flow cytometry. Analysis of subcellular localization of Cap protein and its variants demonstrated that NLS mediated Cap protein nuclear export as well as nuclear import, and a phosphorylation site (S17) was identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS) in the NLS domain to regulate Cap protein nuclear export. Phosphorylation of NLS regulating the PCV2 Cap protein nuclear export was also demonstrated in PK15 cells by fluorescence microscopy. Moreover, the influence of Rep and Rep' protein on Cap protein subcellular localization was investigated in PK15 cells. Phosphorylation of NLS regulating Cap protein nuclear export provides more detailed knowledge of the PCV2 viral life cycle. Copyright © 2018 Elsevier B.V. All rights reserved.

  16. FOXO3a Expression Regulated by ERK Signaling is Inversely Correlated With Y-Box Binding Protein-1 Expression in Prostate Cancer.

    Science.gov (United States)

    Imada, Kenjiro; Shiota, Masaki; Kuroiwa, Kentaro; Sugimoto, Masaaki; Abe, Tatsuro; Kohashi, Kenichi; Yokomizo, Akira; Eto, Masatoshi; Naito, Seiji; Oda, Yoshinao

    2017-02-01

    FOXO3a is a member of the forkhead O transcription factors. FOXO3a induces the factors that contribute to cell cycle arrest and is considered a tumor suppressor in several malignant tumors. Y-box binding protein-1 (YB-1) is a multifunctional protein whose high expression is correlated with poor prognoses in various malignant tumors. In the current study, we investigated the relationship between FOXO3a and YB-1 to validate their functional roles in prostate cancer. Western blotting and cytotoxicity assays were conducted in prostate cancer cells, LNCaP, and 22Rv1 cells. We also evaluated the protein expressions of FOXO3a and YB-1 in human prostate cancer tissues, using radical prostatectomy specimens. Then, we investigated the correlations between protein expressions and clinicopathologic parameters. We found that both FOXO3a and YB-1 proteins were phosphorylated by ERK signaling, resulting in FOXO3a inactivation and YB-1 activation in LNCaP and 22Rv1 cells. Inversely, inhibition of MEK or treatment with metformin activated FOXO3a through inactivation of ERK signaling and suppressed the viability of LNCaP and 22Rv1 cells in a dose-dependent manner. In immunohistochemical analysis, FOXO3a nuclear expression was inversely correlated with YB-1 nuclear expression (P < 0.0001). Furthermore, high FOXO3a nuclear expression was inversely correlated with a higher Gleason grade (P < 0.0001) and higher preoperative PSA (P = 0.0437). These results showed that in prostate cancer, FOXO3a, and YB-1 play inverse reciprocal roles as a tumor-suppressor gene and oncogene, respectively, through their master regulator ERK. Prostate 77:145-153, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  17. Autoantibodies to box A of high mobility group box 1 in systemic lupus erythematosus.

    Science.gov (United States)

    Schaper, F; de Leeuw, K; Horst, G; Maas, F; Bootsma, H; Heeringa, P; Limburg, P C; Westra, J

    2017-06-01

    Autoantibodies to nuclear structures are a hallmark of systemic lupus erythematosus (SLE), including autoantibodies to nuclear protein high mobility group box 1 (HMGB1). HMGB1 consists of three separate domains: box A, box B and an acidic tail. Recombinant box A acts as a competitive antagonist for HMGB1 and might be an interesting treatment option in SLE. However, antibodies to box A might interfere. Therefore, levels of anti-box A were examined in SLE patients in association with disease activity and clinical parameters. Serum anti-box A was measured in 86 SLE patients and 44 age- and sex-matched healthy controls (HC). Serum samples of 28 patients with primary Sjögren's syndrome and 32 patients with rheumatoid arthritis were included as disease controls. Anti-HMGB1 and anti-box B levels were also measured by enzyme-linked immunosorbent assay during quiescent disease [SLE Disease Activity Index (SLEDAI) ≤ 4, n = 47] and active disease (SLEDAI ≥ 5, n = 39). Anti-box A levels in active SLE patients were higher compared to quiescent patients, and were increased significantly compared to HC and disease controls. Anti-box A levels correlated positively with SLEDAI and anti-dsDNA levels and negatively with complement C3 levels. Increased levels of anti-box A antibodies were present in the majority of patients with nephritic (73%) and non-nephritic exacerbations (71%). Antibodies to the box A domain of HMGB1 might be an interesting new biomarker, as these had a high specificity for SLE and were associated with disease activity. Longitudinal studies should be performed to evaluate whether these antibodies perform better in predicting an exacerbation, especially non-nephritic exacerbations. © 2017 British Society for Immunology.

  18. An anther development F-box (ADF) protein regulated by tapetum degeneration retardation (TDR) controls rice anther development.

    Science.gov (United States)

    Li, Li; Li, Yixing; Song, Shufeng; Deng, Huafeng; Li, Na; Fu, Xiqin; Chen, Guanghui; Yuan, Longping

    2015-01-01

    In this study, we reported that a F-box protein, OsADF, as one of the direct targets of TDR , plays a critical role in rice tapetum cell development and pollen formation. The tapetum, the innermost sporophytic tissue of anther, plays an important supportive role in male reproduction in flowering plants. After meiosis, tapetal cells undergo programmed cell death (PCD) and provide nutrients for pollen development. Previously we showed that tapetum degeneration retardation (TDR), a basic helix-loop-helix transcription factor, can trigger tapetal PCD and control pollen wall development during anther development. However, the comprehensive regulatory network of TDR remains to be investigated. In this study, we cloned and characterized a panicle-specific expression F-box protein, anther development F-box (OsADF). By qRT-PCR and RNA in situ hybridization, we further confirmed that OsADF expressed specially in tapetal cells from stage 9 to stage 12 during anther development. In consistent with this specific expression pattern, the RNAi transgenic lines of OsADF exhibited abnormal tapetal degeneration and aborted microspores development, which eventually grew pollens with reduced fertility. Furthermore, we demonstrated that the TDR, a key regulator in controlling rice anther development, could regulate directly the expression of OsADF by binding to E-box motifs of its promoter. Therefore, this work highlighted the possible regulatory role of TDR, which regulates tapetal cell development and pollen formation via triggering the possible ADF-mediated proteolysis pathway.

  19. Prediction of nuclear proteins using SVM and HMM models

    Directory of Open Access Journals (Sweden)

    Raghava Gajendra PS

    2009-01-01

    Full Text Available Abstract Background The nucleus, a highly organized organelle, plays important role in cellular homeostasis. The nuclear proteins are crucial for chromosomal maintenance/segregation, gene expression, RNA processing/export, and many other processes. Several methods have been developed for predicting the nuclear proteins in the past. The aim of the present study is to develop a new method for predicting nuclear proteins with higher accuracy. Results All modules were trained and tested on a non-redundant dataset and evaluated using five-fold cross-validation technique. Firstly, Support Vector Machines (SVM based modules have been developed using amino acid and dipeptide compositions and achieved a Mathews correlation coefficient (MCC of 0.59 and 0.61 respectively. Secondly, we have developed SVM modules using split amino acid compositions (SAAC and achieved the maximum MCC of 0.66. Thirdly, a hidden Markov model (HMM based module/profile was developed for searching exclusively nuclear and non-nuclear domains in a protein. Finally, a hybrid module was developed by combining SVM module and HMM profile and achieved a MCC of 0.87 with an accuracy of 94.61%. This method performs better than the existing methods when evaluated on blind/independent datasets. Our method estimated 31.51%, 21.89%, 26.31%, 25.72% and 24.95% of the proteins as nuclear proteins in Saccharomyces cerevisiae, Caenorhabditis elegans, Drosophila melanogaster, mouse and human proteomes respectively. Based on the above modules, we have developed a web server NpPred for predicting nuclear proteins http://www.imtech.res.in/raghava/nppred/. Conclusion This study describes a highly accurate method for predicting nuclear proteins. SVM module has been developed for the first time using SAAC for predicting nuclear proteins, where amino acid composition of N-terminus and the remaining protein were computed separately. In addition, our study is a first documentation where exclusively nuclear

  20. Determination of Structures of Proteins in Solution using Nuclear ...

    Indian Academy of Sciences (India)

    Home; Journals; Resonance – Journal of Science Education; Volume 8; Issue 8. Determination of Structures of Proteins in Solution using Nuclear Magnetic Resonance. Siddhartha P Sarma. Research News Volume 8 Issue 8 August 2003 pp 86-99 ...

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

    Science.gov (United States)

    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

    2015-11-05

    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.

  2. TATA box-binding protein (TBP)-related factor 2 (TRF2), a third member of the TBP family

    OpenAIRE

    Rabenstein, Mark D.; Zhou, Sharleen; Lis, John T.; Tjian, Robert

    1999-01-01

    The TATA box-binding protein (TBP) is an essential component of the RNA polymerase II transcription apparatus in eukaryotic cells. Until recently, it was thought that the general transcriptional machinery was largely invariant and relied on a single TBP, whereas a large and diverse collection of activators and repressors were primarily responsible for imparting specificity to transcription initiation. However, it now appears that the “basal” transcriptional machinery also contributes to speci...

  3. Successful immunotherapy of autoimmune cholangitis by adoptive transfer of forkhead box protein 3+ regulatory T cells

    Science.gov (United States)

    Tanaka, H; Zhang, W; Yang, G-X; Ando, Y; Tomiyama, T; Tsuneyama, K; Leung, P; Coppel, R L; Ansari, A A; Lian, Z X; Ridgway, W M; Joh, T; Gershwin, M E

    2014-01-01

    Treatment of primary biliary cirrhosis (PBC) has lagged behind that of other autoimmune diseases. In this study we have addressed the potential utility of immunotherapy using regulatory T cells (Treg) to treat murine autoimmune cholangitis. In particular, we have taken advantage of our ability to produce portal inflammation and bile duct cell loss by transfer of CD8+ T cells from the dominant negative form of transforming growth factor beta receptor type II (dnTGF-βRII) mice to recombination-activating gene (Rag)1–/– recipients. We then used this robust established adoptive transfer system and co-transferred CD8+ T cells from dnTGF-βRII mice with either C57BL/6 or dnTGF-βRII forkhead box protein 3 (FoxP3+) T cells. Recipient mice were monitored for histology, including portal inflammation and intralobular biliary cell damage, and also included a study of the phenotypical changes in recipient lymphoid populations and local and systemic cytokine production. Importantly, we report herein that adoptive transfer of Treg from C57BL/6 but not dnTGF-βRII mice significantly reduced the pathology of autoimmune cholangitis, including decreased portal inflammation and bile duct damage as well as down-regulation of the secondary inflammatory response. Further, to define the mechanism of action that explains the differential ability of C57BL/6 Treg versus dnTGF-βRII Treg on the ability to down-regulate autoimmune cholangitis, we noted significant differential expression of glycoprotein A repetitions predominant (GARP), CD73, CD101 and CD103 and a functionally significant increase in interleukin (IL)-10 in Treg from C57BL/6 compared to dnTGF-βRII mice. Our data reflect the therapeutic potential of wild-type CD4+ FoxP3+ Treg in reducing the excessive T cell responses of autoimmune cholangitis, which has significance for the potential immunotherapy of PBC. PMID:25041369

  4. Successful immunotherapy of autoimmune cholangitis by adoptive transfer of forkhead box protein 3(+) regulatory T cells.

    Science.gov (United States)

    Tanaka, H; Zhang, W; Yang, G-X; Ando, Y; Tomiyama, T; Tsuneyama, K; Leung, P; Coppel, R L; Ansari, A A; Lian, Z X; Ridgway, W M; Joh, T; Gershwin, M E

    2014-11-01

    Treatment of primary biliary cirrhosis (PBC) has lagged behind that of other autoimmune diseases. In this study we have addressed the potential utility of immunotherapy using regulatory T cells (Treg ) to treat murine autoimmune cholangitis. In particular, we have taken advantage of our ability to produce portal inflammation and bile duct cell loss by transfer of CD8(+) T cells from the dominant negative form of transforming growth factor beta receptor type II (dnTGF-βRII) mice to recombination-activating gene (Rag)1(-/-) recipients. We then used this robust established adoptive transfer system and co-transferred CD8(+) T cells from dnTGF-βRII mice with either C57BL/6 or dnTGF-βRII forkhead box protein 3 (FoxP3(+) ) T cells. Recipient mice were monitored for histology, including portal inflammation and intralobular biliary cell damage, and also included a study of the phenotypical changes in recipient lymphoid populations and local and systemic cytokine production. Importantly, we report herein that adoptive transfer of Treg from C57BL/6 but not dnTGF-βRII mice significantly reduced the pathology of autoimmune cholangitis, including decreased portal inflammation and bile duct damage as well as down-regulation of the secondary inflammatory response. Further, to define the mechanism of action that explains the differential ability of C57BL/6 Treg versus dnTGF-βRII Treg on the ability to down-regulate autoimmune cholangitis, we noted significant differential expression of glycoprotein A repetitions predominant (GARP), CD73, CD101 and CD103 and a functionally significant increase in interleukin (IL)-10 in Treg from C57BL/6 compared to dnTGF-βRII mice. Our data reflect the therapeutic potential of wild-type CD4(+) FoxP3(+) Treg in reducing the excessive T cell responses of autoimmune cholangitis, which has significance for the potential immunotherapy of PBC. © 2014 British Society for Immunology.

  5. Recent advances in understanding plant nuclear envelope proteins involved in nuclear morphology.

    Science.gov (United States)

    Tamura, Kentaro; Goto, Chieko; Hara-Nishimura, Ikuko

    2015-03-01

    The nuclear envelope (NE) is a fundamental structure of the nucleus and plays an important role in nuclear morphology through the strict regulation of NE protein function. Beyond its physical barrier function between nucleoplasm and cytoplasm, recent studies of the plant NE have provided novel insights into basic aspects of nuclear morphology as well as cellular organization. In this review, we focus on plant NE proteins that have emerged from recent studies in nuclear morphology, and we discuss their physiological functions in cellular activities. A better understanding of the NE protein functions should provide key insights into the physiological significance of proper nuclear structure in plants. © The Author 2015. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.

  6. Function of nuclear membrane proteins in shaping the nuclear envelope integrity during closed mitosis.

    Science.gov (United States)

    Yang, Hui-Ju; Iwamoto, Masaaki; Hiraoka, Yasushi; Haraguchi, Tokuko

    2017-06-01

    The nuclear envelope (NE) not only protects the genome from being directly accessed by detrimental agents but also regulates genome organization. Breaches in NE integrity threaten genome stability and impede cellular function. Nonetheless, the NE constantly remodels, and NE integrity is endangered in dividing or differentiating cells. Specifically, in unicellular eukaryotes undergoing closed mitosis, the NE expands instead of breaking down during chromosome segregation. The newly assembling nuclear pore complexes (NPCs) penetrate the existing NE in interphase. A peculiar example of NE remodelling during nuclear differentiation in Tetrahymena involves formation of the redundant NE and clustered NPCs. Even under these conditions, the NE remains intact. Many recent studies on unicellular organisms have revealed that nuclear membrane proteins, such as LEM-domain proteins, play a role in maintaining NE integrity. This review summarizes and discusses how nuclear membrane proteins participate in NE integrity. © The Authors 2017. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.

  7. Induction of Chromosome Instability by Activation of Yes-Associated Protein and Forkhead Box M1 in Liver Cancer.

    Science.gov (United States)

    Weiler, Sofia M E; Pinna, Federico; Wolf, Thomas; Lutz, Teresa; Geldiyev, Aman; Sticht, Carsten; Knaub, Maria; Thomann, Stefan; Bissinger, Michaela; Wan, Shan; Rössler, Stephanie; Becker, Diana; Gretz, Norbert; Lang, Hauke; Bergmann, Frank; Ustiyan, Vladimir; Kalin, Tatiana V; Singer, Stephan; Lee, Ju-Seog; Marquardt, Jens U; Schirmacher, Peter; Kalinichenko, Vladimir V; Breuhahn, Kai

    2017-06-01

    Many different types of cancer cells have chromosome instability. The hippo pathway leads to phosphorylation of the transcriptional activator yes-associated protein 1 (YAP1, YAP), which regulates proliferation and has been associated with the development of liver cancer. We investigated the effects of hippo signaling via YAP on chromosome stability and hepatocarcinogenesis in humans and mice. We analyzed transcriptome data from 242 patients with hepatocellular carcinoma (HCC) to search for gene signatures associated with chromosomal instability (CIN); we investigated associations with overall survival time and cancer recurrence using Kaplan-Meier curves. We analyzed changes in expression of these signature genes, at mRNA and protein levels, after small interfering RNA-mediated silencing of YAP in Sk-Hep1, SNU182, HepG2, or pancreatic cancer cells, as well as incubation with thiostrepton (an inhibitor of forkhead box M1 [FOXM1]) or verteporfin (inhibitor of the interaction between YAP and TEA domain transcription factor 4 [TEAD4]). We performed co-immunoprecipitation and chromatin immunoprecipitation experiments. We collected liver tissues from mice that express a constitutively active form of YAP (YAP(S127A)) and analyzed gene expression signatures and histomorphologic parameters associated with chromosomal instability. Mice were given injections of thiostrepton and livers were collected and analyzed by immunoblotting, immunohistochemistry, histology, and real-time polymerase chain reaction. We performed immunohistochemical analyses on tissue microarrays of 105 HCCs and 7 nontumor liver tissues. Gene expression patterns associated with chromosome instability, called CIN25 and CIN70, were detected in HCCs from patients with shorter survival time or early cancer recurrence. TEAD4 and YAP were required for CIN25 and CIN70 signature expression via induction and binding of FOXM1. Disrupting the interaction between YAP and TEAD4 with verteporfin, or inhibiting FOXM1

  8. A photo-responsive F-box protein FOF2 regulates floral initiation by promoting FLC expression in Arabidopsis.

    Science.gov (United States)

    He, Reqing; Li, Xinmei; Zhong, Ming; Yan, Jindong; Ji, Ronghuan; Li, Xu; Wang, Qin; Wu, Dan; Sun, Mengsi; Tang, Dongying; Lin, Jianzhong; Li, Hongyu; Liu, Bin; Liu, Hongtao; Liu, Xuanming; Zhao, Xiaoying; Lin, Chentao

    2017-09-01

    Floral initiation is regulated by various genetic pathways in response to light, temperature, hormones and developmental status; however, the molecular mechanisms underlying the interactions between different genetic pathways are not fully understood. Here, we show that the photoresponsive gene FOF2 (F-box of flowering 2) negatively regulates flowering. FOF2 encodes a putative F-box protein that interacts specifically with ASK14, and its overexpression results in later flowering under both long-day and short-day photoperiods. Conversely, transgenic plants expressing the F-box domain deletion mutant of FOF2 (FOF2ΔF), or double loss of function mutant of FOF2 and FOL1 (FOF2-LIKE 1) present early flowering phenotypes. The late flowering phenotype of the FOF2 overexpression lines is suppressed by the flc-3 loss-of-function mutation. Furthermore, FOF2 mRNA expression is regulated by autonomous pathway gene FCA, and the repressive effect of FOF2 in flowering can be overcome by vernalization. Interestingly, FOF2 expression is regulated by light. The protein level of FOF2 accumulates in response to light, whereas it is degraded under dark conditions via the 26S proteasome pathway. Our findings suggest a possible mechanistic link between light conditions and the autonomous floral promotion pathway in Arabidopsis. © 2017 The Authors The Plant Journal © 2017 John Wiley & Sons Ltd.

  9. Protein expression profiling of nuclear membrane protein reveals potential biomarker of human hepatocellular carcinoma

    OpenAIRE

    Khan, Rizma; Zahid, Saadia; Wan, Yu-Jui; Forster, Jameson; Karim, A-Bashar; Nawabi, Atta M; Azhar, Abid; Rahman, M; Ahmed, Nikhat

    2013-01-01

    Abstract Background Complex molecular events lead to development and progression of liver cirrhosis to HCC. Differentially expressed nuclear membrane associated proteins are responsible for the functional and structural alteration during the progression from cirrhosis to carcinoma. Although alterations/ post translational modifications in protein expression have been extensively quantified, complementary analysis of nuclear membrane proteome changes h...

  10. Control of nuclear organization by F-actin binding proteins.

    Science.gov (United States)

    Pfisterer, Karin; Jayo, Asier; Parsons, Maddy

    2017-03-04

    The regulation of nuclear shape and deformability is a key factor in controlling diverse events from embryonic development to cancer cell metastasis, but the mechanisms governing this process are still unclear. Our recent study demonstrated an unexpected role for the F-actin bundling protein fascin in controlling nuclear plasticity through a direct interaction with Nesprin-2. Nesprin-2 is a component of the LINC complex that is known to couple the F-actin cytoskeleton to the nuclear envelope. We demonstrated that fascin, which is predominantly associated with peripheral F-actin rich filopodia, binds directly to Nesprin-2 at the nuclear envelope in a range of cell types. Depleting fascin or specifically blocking the fascin-Nesprin-2 complex leads to defects in nuclear polarization, movement and cell invasion. These studies reveal a novel role for an F-actin bundling protein in control of nuclear plasticity and underline the importance of defining nuclear-associated roles for F-actin binding proteins in future.

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

    Energy Technology Data Exchange (ETDEWEB)

    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.

  12. Protein interactions of MADS box transcription factors involved in flowering in Lolium perenne

    NARCIS (Netherlands)

    Ciannamea, S.; Kaufmann, K.; Frau, M.; Nougalli Tonaco, I.A.; Petersen, K.; Nielsen, K.K.; Angenent, G.C.; Immink, G.H.

    2006-01-01

    Regulation of flowering time is best understood in the dicot model species Arabidopsis thaliana. Molecular analyses revealed that genes belonging to the MADS box transcription factor family play pivotal regulatory roles in both the vernalization- and photoperiod-regulated flowering pathways. Here

  13. Nuclear proteins hijacked by mammalian cytoplasmic plus strand RNA viruses

    Energy Technology Data Exchange (ETDEWEB)

    Lloyd, Richard E., E-mail: rlloyd@bcm.edu

    2015-05-15

    Plus strand RNA viruses that replicate in the cytoplasm face challenges in supporting the numerous biosynthetic functions required for replication and propagation. Most of these viruses are genetically simple and rely heavily on co-opting cellular proteins, particularly cellular RNA-binding proteins, into new roles for support of virus infection at the level of virus-specific translation, and building RNA replication complexes. In the course of infectious cycles many nuclear-cytoplasmic shuttling proteins of mostly nuclear distribution are detained in the cytoplasm by viruses and re-purposed for their own gain. Many mammalian viruses hijack a common group of the same factors. This review summarizes recent gains in our knowledge of how cytoplasmic RNA viruses use these co-opted host nuclear factors in new functional roles supporting virus translation and virus RNA replication and common themes employed between different virus groups. - Highlights: • Nuclear shuttling host proteins are commonly hijacked by RNA viruses to support replication. • A limited group of ubiquitous RNA binding proteins are commonly hijacked by a broad range of viruses. • Key virus proteins alter roles of RNA binding proteins in different stages of virus replication.

  14. A comparison of high-mobility group-box 1 protein, lipopolysaccharide-binding protein and procalcitonin in severe community-acquired infections and bacteraemia: a prospective study

    DEFF Research Database (Denmark)

    Gaïni, Shahin; Koldkjaer, Ole G; Møller, Holger J

    2008-01-01

    INTRODUCTION: High-mobility group box-1 protein (HMGB1) has been known as a chromosomal protein for many years. HMGB1 has recently been shown to be a proinflammatory cytokine with a role in the immunopathogenesis of sepsis. Lipopolysaccharide-binding protein (LBP) has a central role in the innate...... cell count and neutrophils) were measured with commercially available laboratory techniques. RESULTS: A total of 185 adult patients were included in the study; 154 patients fulfilled our definition of infection. Levels of HMGB1, LBP and PCT were higher in infected patients compared with a healthy...

  15. Overexpression of a stress-responsive U-box protein gene VaPUB affects the accumulation of resistance related proteins in Vitis vinifera 'Thompson Seedless'.

    Science.gov (United States)

    Jiao, Li; Zhang, Yali; Lu, Jiang

    2017-03-01

    Many U-box proteins have been identified and characterized as important factors against environmental stresses such as chilling, heat, salinity and pathogen attack in plant. Our previous research reported the cloning of a novel U-box protein gene VaPUB from Vitis amurensis 'Zuoshanyi' grape and suggested a function of it in related to cold stress in the model plant Arabidopsis system. In this study, the role of VaPUB in response to biotic and abiotic stress was further analyzed in the homologous grapevine system by studying the transcript regulation and the protein accumulation in VaPUB transgenic vines. The expression analysis assay shown that VaPUB was significantly up-regulated 6 h after cold treatment and as early as 2 h post inoculation with Plasmopara viticola, a pathogen causing downy mildew disease in grapevine. Over-expressing VaPUB in V. Vinifera 'Thompson Seedless' affected the microstructure of leaves. The proteome assay shown that the accumulation of pathogenesis-related protein PR10 and many proteins involved in carbon and energy metabolism, oxidation reaction and protein metabolism were significantly altered in transgenic vines. In comparison with wild type plants, the expression level of PR10 family genes was significantly decreased in VaPUB transgenic vines under P. viticola treatment or cold stress. Results from this study showed that the U-box protein gene PUB quickly responded to both biotic stress and abiotic stress and significantly influenced the accumulation of resistance related proteins in grapevine. Copyright © 2016. Published by Elsevier Masson SAS.

  16. Paraformaldehyde Fixation May Lead to Misinterpretation of the Subcellular Localization of Plant High Mobility Group Box Proteins.

    Science.gov (United States)

    Li, Man-Wah; Zhou, Liang; Lam, Hon-Ming

    2015-01-01

    Arabidopsis High Mobility Group Box (HMBG) proteins were previously found associated with the interphase chromatin but not the metaphase chromosome. However, these studies are usually based on immunolocalization analysis involving paraformaldehyde fixation. Paraformaldehyde fixation has been widely adapted to preserved cell morphology before immunofluorescence staining. On one hand, the processed cells are no longer living. On the other hand, the processing may lead to misinterpretation of localization. HMGBs from Arabidopsis were fused with enhanced green fluorescence protein (EGFP) and transformed into tobacco BY-2 cells. Basically, the localization of these HMGB proteins detected with EGFP fluorescence in interphase agreed with previous publications. Upon 4% paraformaldehyde fixation, AtHMGB1 was found associated with interphase but not the metaphase chromosomes as previously reported. However, when EGFP fluorescence signal was directly observed under confocal microscope without fixation, association of AtHMGB1 with metaphase chromosomes can be detected. Paraformaldehyde fixation led to dissociation of EGFP tagged AtHMBG1 protein from metaphase chromosomes. This kind of pre-processing of live specimen may lead to dissociation of protein-protein or protein-nucleic acid interaction. Therefore, using of EGFP fusion proteins in live specimen is a better way to determine the correct localization and interaction of proteins.

  17. JFK, a Kelch domain-containing F-box protein, links the SCF complex to p53 regulation.

    Science.gov (United States)

    Sun, Luyang; Shi, Lei; Li, Wenqian; Yu, Wenhua; Liang, Jing; Zhang, Hua; Yang, Xiaohan; Wang, Yan; Li, Ruifang; Yao, Xingrong; Yi, Xia; Shang, Yongfeng

    2009-06-23

    The p53 tumor suppressor plays a central role in integrating cellular responses to various stresses. Tight regulation of p53 is thus essential for the maintenance of genome integrity and normal cell proliferation. Currently, several ubiquitin ligases, including the single-subunit RING-finger types--MDM2, Pirh2, and COP1--and the HECT-domain type--ARF-BP1--have been reported to target p53 for degradation. Here, we report the identification of a human Kelch domain-containing F-box protein, JFK. We showed that JFK promotes ubiquitination and degradation of p53. But unlike MDM2, Pirh2, COP1, and ARF-BP1, all of which possess an intrinsic ubiquitin ligase activity, JFK destabilizes p53 through the assembly of a Skp1-Cul1-F-box complex. Significantly, JFK inhibits p53-dependent transcription, and depletion of JFK stabilizes p53, promotes cell apoptosis, arrests cells in the G(1) phase, and sensitizes cells to ionizing radiation-induced cell death. These data indicate that JFK is a critical negative regulator of p53 and represents a pathway for the maintenance of p53 levels in unstressed cells. Our experiments link the Skp1-Cul1-F-box system to p53 regulation.

  18. Nuclear spectrin-like proteins are structural actin-binding proteins in plants.

    Science.gov (United States)

    Pérez-Munive, Clara; Moreno Díaz de la Espina, Susana

    2011-03-01

    Although actin is a relevant component of the plant nucleus, only three nuclear ABPs (actin-binding proteins) have been identified in plants to date: cofilin, profilin and nuclear myosin I. Although plants lack orthologues of the main structural nuclear ABPs in animals, such as lamins, lamin-associated proteins and nesprins, their genome does contain sequences with spectrin repeats and N-terminal calponin homology domains for actin binding that might be distant relatives of spectrin. We investigated here whether spectrin-like proteins could act as structural nuclear ABPs in plants. We have investigated the presence of spectrins in Allium cepa meristematic nuclei by Western blotting, confocal and electron microscopy, using antibodies against α- and β-spectrin chains that cross-react in plant nuclei. Their role as nuclear ABPs was analysed by co-immunoprecipitation and IF (immunofluorescence) co-localization and their association with the nuclear matrix was investigated by sequential extraction of nuclei with non-ionic detergent, and in low- and high-salt buffers after nuclease digestion. Our results demonstrate the existence of several spectrin-like proteins in the nucleus of onion cells that have different intranuclear distributions in asynchronous meristematic populations and associate with the nuclear matrix. These nuclear proteins co-immunoprecipitate and co-localize with actin. These results reveal that the plant nucleus contains spectrin-like proteins that are structural nuclear components and function as ABPs. Their intranuclear distribution suggests that plant nuclear spectrin-like proteins could be involved in multiple nuclear functions.

  19. An Arabidopsis F-box protein acts as a transcriptional co-factor to regulate floral development.

    Science.gov (United States)

    Chae, Eunyoung; Tan, Queenie K-G; Hill, Theresa A; Irish, Vivian F

    2008-04-01

    Plants flower in response to both environmental and endogenous signals. The Arabidopsis LEAFY (LFY) transcription factor is crucial in integrating these signals, and acts in part by activating the expression of multiple floral homeotic genes. LFY-dependent activation of the homeotic APETALA3 (AP3) gene requires the activity of UNUSUAL FLORAL ORGANS (UFO), an F-box component of an SCF ubiquitin ligase, yet how this regulation is effected has remained unclear. Here, we show that UFO physically interacts with LFY both in vitro and in vivo, and this interaction is necessary to recruit UFO to the AP3 promoter. Furthermore, a transcriptional repressor domain fused to UFO reduces endogenous LFY activity in plants, supporting the idea that UFO acts as part of a transcriptional complex at the AP3 promoter. Moreover, chemical or genetic disruption of proteasome activity compromises LFY-dependent AP3 activation, indicating that protein degradation is required to promote LFY activity. These results define an unexpected role for an F-box protein in functioning as a DNA-associated transcriptional co-factor in regulating floral homeotic gene expression. These results suggest a novel mechanism for promoting flower development via protein degradation and concomitant activation of the LFY transcription factor. This mechanism may be widely conserved, as homologs of UFO and LFY have been identified in a wide array of plant species.

  20. The C. elegans F-box proteins LIN-23 and SEL-10 antagonize centrosome duplication by regulating ZYG-1 levels.

    Science.gov (United States)

    Peel, Nina; Dougherty, Michael; Goeres, Jacqueline; Liu, Yan; O'Connell, Kevin F

    2012-08-01

    The correct segregation of DNA during cell division requires formation of a bipolar spindle, organized at each pole by a centrosome. The regulation of centrosome duplication such that each mitotic cell has exactly two centrosomes is therefore of central importance to cell division. Deregulation of centrosome duplication causes the appearance of supernumerary centrosomes, which are a hallmark of many cancer cells and can contribute to tumorigenesis. Overexpression of the kinase Plk4, which is required for centrosome duplication, causes the formation of extra centrosomes, and aberrant Plk4 expression levels are associated with cancer. Data from Drosophila and human cells show that Plk4 levels are regulated by the SCF ubiquitin ligase and proteasomal degradation. Recognition of Plk4 by the SCF complex is mediated by the F-box protein Slimb/βTrCP. We show that levels of the C. elegans Plk4 homolog ZYG-1 are elevated by impairing proteasome or SCF function, indicating that ZYG-1 is regulated by a conserved mechanism. In C. elegans, similar to Drosophila and humans, we find that the Slimb/βTrCP homolog LIN-23 regulates ZYG-1 levels. In addition, we show that a second F-box protein, SEL-10, also contributes to ZYG-1 regulation. Co-depletion of LIN-23 and SEL-10 suggests these proteins function cooperatively. Because SEL-10 is the homolog of human FBW7, which is frequently mutated in cancer, our findings have implications for understanding tumorigenesis.

  1. JFK, a Kelch domain-containing F-box protein, links the SCF complex to p53 regulation

    OpenAIRE

    Sun, Luyang; Shi, Lei; Li, Wenqian; Yu, Wenhua; LIANG, Jing; Zhang, Hua; Yang, Xiaohan; Wang, Yan; Li, Ruifang; Yao, Xingrong; Yi, Xia; Shang, Yongfeng

    2009-01-01

    The p53 tumor suppressor plays a central role in integrating cellular responses to various stresses. Tight regulation of p53 is thus essential for the maintenance of genome integrity and normal cell proliferation. Currently, several ubiquitin ligases, including the single-subunit RING-finger types—MDM2, Pirh2, and COP1—and the HECT-domain type—ARF-BP1—have been reported to target p53 for degradation. Here, we report the identification of a human Kelch domain-containing F-box protein, JFK. We ...

  2. A Role for the F-Box Protein HAWAIIAN SKIRT in Plant microRNA Function.

    Science.gov (United States)

    Lang, Patricia L M; Christie, Michael D; Dogan, Ezgi S; Schwab, Rebecca; Hagmann, Jörg; van de Weyer, Anna-Lena; Scacchi, Emanuele; Weigel, Detlef

    2018-01-01

    As regulators of gene expression in multicellular organisms, microRNAs (miRNAs) are crucial for growth and development. Although a plethora of factors involved in their biogenesis and action in Arabidopsis (Arabidopsis thaliana) has been described, these processes and their fine-tuning are not fully understood. Here, we used plants expressing an artificial miRNA target mimic (MIM) to screen for negative regulators of miR156. We identified a new mutant allele of the F-box gene HAWAIIAN SKIRT (HWS; At3G61590), hws-5, as a suppressor of the MIM156-induced developmental and molecular phenotypes. In hws plants, levels of some endogenous miRNAs are increased and their mRNA targets decreased. Plants constitutively expressing full-length HWS-but not a truncated version lacking the F-box domain-display morphological and molecular phenotypes resembling those of mutants defective in miRNA biogenesis and activity. In combination with such mutants, hws loses its delayed floral organ abscission ("skirt") phenotype, suggesting epistasis. Also, the hws transcriptome profile partially resembles those of well-known miRNA mutants hyl1-2, se-3, and ago1-27, pointing to a role in a common pathway. We thus propose HWS as a novel, F-box dependent factor involved in miRNA function. © 2018 American Society of Plant Biologists. All Rights Reserved.

  3. Forkhead box protein O3 transcription factor negatively regulates autophagy in human cancer cells by inhibiting forkhead box protein O1 expression and cytosolic accumulation.

    Directory of Open Access Journals (Sweden)

    Wan Long Zhu

    Full Text Available FoxO proteins are important regulators in cellular metabolism and are recognized to be nodes in multiple signaling pathways, most notably those involving PI3K/AKT and mTOR. FoxO proteins primarily function as transcription factors, but recent study suggests that cytosolic FoxO1 participates in the regulation of autophagy. In the current study, we find that cytosolic FoxO1 indeed stimulates cellular autophagy in multiple cancer cell lines, and that it regulates not only basal autophagy but also that induced by rapamycin and that in response to nutrient deprivation. These findings illustrate the importance of FoxO1 in cell metabolism regulation independent of its transcription factor function. In contrast to FoxO1, we find the closely related FoxO3a is a negative regulator of autophagy in multiple cancer cell lines, a previously unrecognized function for this protein, different from its function in benign fibroblast and muscle cells. The induction of autophagy by the knockdown of FoxO3a was found not to be mediated through the suppression of mTORC1 signaling; rather, the regulatory role of FoxO3a on autophagy was determined to be through its ability to transcriptionally suppress FoxO1. This complicated interplay of FoxO1 and FoxO3a suggests a complex checks- and balances-relationship between FoxO3a and FoxO1 in regulating autophagy and cell metabolism.

  4. Identification of novel putative-binding proteins for cellular prion protein and a specific interaction with the STIP1 homology and U-Box-containing protein 1.

    Science.gov (United States)

    Gimenez, Ana Paula Lappas; Richter, Larissa Morato Luciani; Atherino, Mariana Campos; Beirão, Breno Castello Branco; Fávaro, Celso; Costa, Michele Dietrich Moura; Zanata, Silvio Marques; Malnic, Bettina; Mercadante, Adriana Frohlich

    2015-01-01

    Prion diseases involve the conversion of the endogenous cellular prion protein, PrP(C), into a misfolded infectious isoform, PrP(Sc). Several functions have been attributed to PrP(C), and its role has also been investigated in the olfactory system. PrP(C) is expressed in both the olfactory bulb (OB) and olfactory epithelium (OE) and the nasal cavity is an important route of transmission of diseases caused by prions. Moreover, Prnp(-/-) mice showed impaired behavior in olfactory tests. Given the high PrP(C) expression in OE and its putative role in olfaction, we screened a mouse OE cDNA library to identify novel PrP(C)-binding partners. Ten different putative PrP(C) ligands were identified, which were involved in functions such as cellular proliferation and apoptosis, cytoskeleton and vesicle transport, ubiquitination of proteins, stress response, and other physiological processes. In vitro binding assays confirmed the interaction of PrP(C) with STIP1 homology and U-Box containing protein 1 (Stub1) and are reported here for the first time. Stub1 is a co-chaperone with ubiquitin E3-ligase activity, which is associated with neurodegenerative diseases characterized by protein misfolding and aggregation. Physiological and pathological implications of PrP(C)-Stub1 interaction are under investigation. The PrP(C)-binding proteins identified here are not exclusive to the OE, suggesting that these interactions may occur in other tissues and play general biological roles. These data corroborate the proposal that PrP(C) is part of a multiprotein complex that modulates several cellular functions and provide a platform for further studies on the physiological and pathological roles of prion protein.

  5. Theory of box-model hyperfine couplings and transport signatures of long-range nuclear-spin coherence in a quantum-dot spin valve

    Science.gov (United States)

    Chesi, Stefano; Coish, W. A.

    2015-06-01

    We have theoretically analyzed coherent nuclear-spin dynamics induced by electron transport through a quantum-dot spin valve. The hyperfine interaction between electron and nuclear spins in a quantum dot allows for the transfer of angular momentum from spin-polarized electrons injected from ferromagnetic or half-metal leads to the nuclear spin system under a finite voltage bias. Accounting for a local nuclear-spin dephasing process prevents the system from becoming stuck in collective dark states, allowing a large nuclear polarization to be built up in the long-time limit. After reaching a steady state, reversing the voltage bias induces a transient current response as the nuclear polarization is reversed. Long-range nuclear-spin coherence leads to a strong enhancement of spin-flip transition rates (by an amount proportional to the number of nuclear spins) and is revealed by an intense current burst, analogous to superradiant light emission. The crossover to a regime with incoherent spin flips occurs on a relatively long-time scale, on the order of the single-nuclear-spin dephasing time, which can be much longer than the time scale for the superradiant current burst. This conclusion is confirmed through a general master equation. For the two limiting regimes (coherent/incoherent spin flips), the general master equation recovers our simpler treatment based on rate equations, but is also applicable at intermediate dephasing. Throughout this work, we assume uniform hyperfine couplings, which yield the strongest coherent enhancement. We propose realistic strategies, based on isotopic modulation and wave-function engineering in core-shell nanowires, to realize this analytically solvable "box-model" of hyperfine couplings.

  6. Iron-regulatory proteins DmdR1 and DmdR2 of Streptomyces coelicolor form two different DNA-protein complexes with iron boxes.

    OpenAIRE

    Flores, Francisco J; Martín, Juan F

    2004-01-01

    In high G+C Gram-positive bacteria, the control of expression of genes involved in iron metabolism is exerted by a DmdR [divalent (bivalent) metal-dependent regulatory protein] in the presence of Fe2+ or other bivalent ions. The dmdR1 and dmdR2 genes of Streptomyces coelicolor were overexpressed in Escherichia coli and the DmdR1 and DmdR2 proteins were purified to homogeneity. Electrophoretic mobility-shift assays showed that both DmdR1 and DmdR2 bind to the 19-nt tox and desA iron boxes form...

  7. Adducin family proteins possess different nuclear export potentials.

    Science.gov (United States)

    Liu, Chia-Mei; Hsu, Wen-Hsin; Lin, Wan-Yi; Chen, Hong-Chen

    2017-05-10

    The adducin (ADD) family proteins, namely ADD1, ADD2, and ADD3, are actin-binding proteins that play important roles in the stabilization of membrane cytoskeleton and cell-cell junctions. All the ADD proteins contain a highly conserved bipartite nuclear localization signal (NLS) at the carboxyl termini, but only ADD1 can localize to the nucleus. The reason for this discrepancy is not clear. To avoid the potential effect of cell-cell junctions on the distribution of ADD proteins, HA epitope-tagged ADD proteins and mutants were transiently expressed in NIH3T3 fibroblasts and their distribution in the cytoplasm and nucleus was examined by immunofluorescence staining. Several nuclear proteins were identified to interact with ADD1 by mass spectrometry, which were further verified by co-immunoprecipitation. In this study, we found that ADD1 was detectable both in the cytoplasm and nucleus, whereas ADD2 and ADD3 were detected only in the cytoplasm. However, ADD2 and ADD3 were partially (~40%) sequestered in the nucleus by leptomycin B, a CRM1/exportin1 inhibitor. Upon the removal of leptomycin B, ADD2 and ADD3 re-distributed to the cytoplasm. These results indicate that ADD2 and ADD3 possess functional NLS and are quickly transported to the cytoplasm upon entering the nucleus. Indeed, we found that ADD2 and ADD3 possess much higher potential to counteract the activity of the NLS derived from Simian virus 40 large T-antigen than ADD1. All the ADD proteins appear to contain multiple nuclear export signals mainly in their head and neck domains. However, except for the leucine-rich motif ( 377 FEALMRMLDWLGYRT 391 ) in the neck domain of ADD1, no other classic nuclear export signal was identified in the ADD proteins. In addition, the nuclear retention of ADD1 facilitates its interaction with RNA polymerase II and zinc-finger protein 331. Our results suggest that ADD2 and ADD3 possess functional NLS and shuttle between the cytoplasm and nucleus. The discrepancy in the

  8. Overlapping CRE and E-box promoter elements can independently regulate COX-2 gene transcription in macrophages.

    Science.gov (United States)

    Mestre, J R; Rivadeneira, D E; Mackrell, P J; Duff, M; Stapleton, P P; Mack-Strong, V; Maddali, S; Smyth, G P; Tanabe, T; Daly, J M

    2001-05-11

    Macrophage cyclooxygenase-2 (COX-2) transcription is mediated through the collaboration of different promoter elements. Here, the role of an overlapping cyclic AMP responsive element (CRE)/E-box was investigated. Nuclear proteins bound both the CRE and E-box, which synergized with other promoter elements to induce COX-2 transcription. Endotoxin induced binding of nuclear proteins to the CRE and E-box and each element independently induced higher COX-2 transcription levels than the overlapping CRE/E-box. Transcription factors associated with the CRE binding complex included c-Jun and CRE binding protein and with the E-box binding complex USF-1; their overexpression significantly induced COX-2 transcription. Therefore, both CRE and E-box promoter elements regulate COX-2 transcription in macrophages.

  9. In vitro nuclear interactome of the HIV-1 Tat protein.

    LENUS (Irish Health Repository)

    Gautier, Virginie W

    2009-01-01

    BACKGROUND: One facet of the complexity underlying the biology of HIV-1 resides not only in its limited number of viral proteins, but in the extensive repertoire of cellular proteins they interact with and their higher-order assembly. HIV-1 encodes the regulatory protein Tat (86-101aa), which is essential for HIV-1 replication and primarily orchestrates HIV-1 provirus transcriptional regulation. Previous studies have demonstrated that Tat function is highly dependent on specific interactions with a range of cellular proteins. However they can only partially account for the intricate molecular mechanisms underlying the dynamics of proviral gene expression. To obtain a comprehensive nuclear interaction map of Tat in T-cells, we have designed a proteomic strategy based on affinity chromatography coupled with mass spectrometry. RESULTS: Our approach resulted in the identification of a total of 183 candidates as Tat nuclear partners, 90% of which have not been previously characterised. Subsequently we applied in silico analysis, to validate and characterise our dataset which revealed that the Tat nuclear interactome exhibits unique signature(s). First, motif composition analysis highlighted that our dataset is enriched for domains mediating protein, RNA and DNA interactions, and helicase and ATPase activities. Secondly, functional classification and network reconstruction clearly depicted Tat as a polyvalent protein adaptor and positioned Tat at the nexus of a densely interconnected interaction network involved in a range of biological processes which included gene expression regulation, RNA biogenesis, chromatin structure, chromosome organisation, DNA replication and nuclear architecture. CONCLUSION: We have completed the in vitro Tat nuclear interactome and have highlighted its modular network properties and particularly those involved in the coordination of gene expression by Tat. Ultimately, the highly specialised set of molecular interactions identified will

  10. Yeast DEAD Box Protein Mss116p Is a Transcription Elongation Factor That Modulates the Activity of Mitochondrial RNA Polymerase

    Science.gov (United States)

    Wojtas, Ireneusz D.; Tessitore, Kassandra; Henderson, Simmone; McAllister, William T.

    2014-01-01

    DEAD box proteins have been widely implicated in regulation of gene expression. Here, we show that the yeast Saccharomyces cerevisiae DEAD box protein Mss116p, previously known as a mitochondrial splicing factor, also acts as a transcription factor that modulates the activity of the single-subunit mitochondrial RNA polymerase encoded by RPO41. Binding of Mss116p stabilizes paused mitochondrial RNA polymerase elongation complexes in vitro and favors the posttranslocated state of the enzyme, resulting in a lower concentration of nucleotide substrate required to escape the pause; this mechanism of action is similar to that of elongation factors that enhance the processivity of multisubunit RNA polymerases. In a yeast strain in which the RNA splicing-related functions of Mss116p are dispensable, overexpression of RPO41 or MSS116 increases cell survival from colonies that were exposed to low temperature, suggesting a role for Mss116p in enhancing the efficiency of mitochondrial transcription under stress conditions. PMID:24732805

  11. Stepwise bending of DNA by a single TATA box binding protein

    DEFF Research Database (Denmark)

    Tolic-Nørrelykke, Simon F; Rasmussen, Mette B; Pavone, Francesco S

    2006-01-01

    cerevisiae TBPs interacting with single DNA molecules containing a TATA-box. Using video microscopy, we observed the Brownian motion of the beads tethered by short surface-bound DNA. When TBP binds to and bends the DNA, the conformation of the DNA changes and the amplitude of Brownian motion of the tehtered......-defined free and bound classes of Brownian motion, we observed another two classes of motion. These extra classes were identified with intermediate states of a three-step, linear binding pathway. Biological implications of the intermediate states are discussed....

  12. Orientia tsutsugamushi Strain Ikeda Ankyrin Repeat-Containing Proteins Recruit SCF1 Ubiquitin Ligase Machinery via Poxvirus-Like F-Box Motifs

    Science.gov (United States)

    Beyer, Andrea R.; VieBrock, Lauren; Rodino, Kyle G.; Miller, Daniel P.; Tegels, Brittney K.; Marconi, Richard T.

    2015-01-01

    ABSTRACT A rising theme among intracellular microbes is the delivery of ankyrin repeat-containing effectors (Anks) that interact with target proteins to co-opt host cell functions. Orientia tsutsugamushi, an obligate intracellular bacterium and the etiologic agent of scrub typhus, encodes one of the largest Ank repertoires of any sequenced microorganism. They have been previously identified as type 1 secretion system substrates. Here, in silico and manual sequence analyses revealed that a large proportion of O. tsutsugamushi strain Ikeda Anks bear a eukaryotic/poxvirus-like F-box motif, which is known to recruit host cell SCF1 ubiquitin ligase machinery. We assessed the Anks for the ability to serve as F-box proteins. Coimmunoprecipitation assays demonstrated that F-box-containing Anks interact with overexpressed and/or endogenous SCF1 components. When coexpressed with FLAG-Ank4_01 or FLAG-Ank9, a glutathione S-transferase (GST)-tagged version of the SCF1 component SKP1 localized to subcellular sites of FLAG-Ank accumulation. The abilities of recombinant Anks to interact and colocalize with SKP1 were F-box dependent. GST-SKP1 precipitated O. tsutsugamushi-derived Ank9 from infected host cells, verifying both that the pathogen expresses Ank9 during infection and the protein's capability to bind SKP1. Aligning O. tsutsugamushi, poxviral, and eukaryotic F-box sequences delineated three F-box residues that are highly conserved and likely to be functionally important. Substitution of these residues ablated the ability of GFP-Ank9 to interact with GST-SKP1. These results demonstrate that O. tsutsugamushi strain Ikeda Anks can co-opt host cell polyubiquitination machinery, provide the first evidence that an O. tsutsugamushi Ank does so during infection, and advance overall understanding of microbial F-box proteins. IMPORTANCE Ankyrin repeat-containing proteins (Anks) are important virulence factors of intracellular bacteria that mediate protein-protein interactions with

  13. Adducin family proteins possess different nuclear export potentials

    OpenAIRE

    Liu, Chia-Mei; Hsu, Wen-Hsin; Lin, Wan-Yi; Chen, Hong-Chen

    2017-01-01

    Background The adducin (ADD) family proteins, namely ADD1, ADD2, and ADD3, are actin-binding proteins that play important roles in the stabilization of membrane cytoskeleton and cell-cell junctions. All the ADD proteins contain a highly conserved bipartite nuclear localization signal (NLS) at the carboxyl termini, but only ADD1 can localize to the nucleus. The reason for this discrepancy is not clear. Methods To avoid the potential effect of cell-cell junctions on the distribution of ADD prot...

  14. Identification and characterization of proteins involved in nuclear organization using Drosophila GFP protein trap lines.

    Directory of Open Access Journals (Sweden)

    Margaret Rohrbaugh

    Full Text Available Strains from a collection of Drosophila GFP protein trap lines express GFP in the normal tissues where the endogenous protein is present. This collection can be used to screen for proteins distributed in the nucleus in a non-uniform pattern.We analyzed four lines that show peripheral or punctate nuclear staining. One of these lines affects an uncharacterized gene named CG11138. The CG11138 protein shows a punctate distribution in the nuclear periphery similar to that of Drosophila insulator proteins but does not co-localize with known insulators. Interestingly, mutations in Lamin proteins result in alterations in CG11138 localization, suggesting that this protein may be a novel component of the nuclear lamina. A second line affects the Decondensation factor 31 (Df31 gene, which encodes a protein with a unique nuclear distribution that appears to segment the nucleus into four different compartments. The X-chromosome of males is confined to one of these compartments. We also find that Drosophila Nucleoplasmin (dNlp is present in regions of active transcription. Heat shock leads to loss of dNlp from previously transcribed regions of polytene chromosome without redistribution to the heat shock genes. Analysis of Stonewall (Stwl, a protein previously found to be necessary for the maintenance of germline stem cells, shows that Stwl is present in a punctate pattern in the nucleus that partially overlaps with that of known insulator proteins. Finally we show that Stwl, dNlp, and Df31 form part of a highly interactive network. The properties of other components of this network may help understand the role of these proteins in nuclear biology.These results establish screening of GFP protein trap alleles as a strategy to identify factors with novel cellular functions. Information gained from the analysis of CG11138 Stwl, dNlp, and Df31 sets the stage for future studies of these proteins.

  15. Regulation of Nuclear Localization of Signaling Proteins by Cytokinin

    Energy Technology Data Exchange (ETDEWEB)

    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.

  16. A novel protein toxin from the deadly box jellyfish (Sea Wasp, Habu-kurage) Chiropsalmus quadrigatus.

    Science.gov (United States)

    Nagai, Hiroshi; Takuwa-Kuroda, Kyoko; Nakao, Masahiro; Oshiro, Naomasa; Iwanaga, Setsuko; Nakajima, Terumi

    2002-01-01

    The deadly box jellyfish (Sea Wasp, Habu-kurage in Japanese) Chiropsalmus quadrigatus Haeckel (Cubozoa) is distributed widely in the tropical Pacific region. In Japan, three fatal cases due to stings from this species have been reported officially. We successfully isolated C. quadrigatus toxin-A (CqTX-A, 44 kDa), a major proteinaceous toxin, for the first time, from the nematocysts of C. quadrigatus. CqTX-A showed lethal toxicity to crayfish when administered via intraperitoneal injection (LD50 = 80 microg/kg) and hemolytic activity toward 0.8% sheep red blood cells (ED50 = 160 ng/ml). Furthermore, we sequenced the cDNA encoding CqTX-A. The deduced amino acid sequence of CqTX-A (462 amino acids) showed 25.2% and 21.6% sequence similarity to Carybdea rastoni toxins (CrTXs) and Carybdea alata toxin-A (CrTX-A), respectively, which are Cubozoan jellyfish toxins.

  17. Effect of Y-box binding protein 1 overexpression on the prognosis of patients with intrahepatic cholangiocarcinoma undergoing postoperative adjuvant chemotherapy

    Directory of Open Access Journals (Sweden)

    LIU Heng

    2017-02-01

    Full Text Available ObjectiveTo investigate the association between Y-box binding protein 1 (YB-1 overexpression and the prognosis of patients with intrahepatic cholangiocarcinoma (ICC undergoing postoperative adjuvant chemotherapy. MethodsThe paraffin-embedded specimens were collected from 58 patients with ICC who underwent surgical treatment and postoperative adjuvant chemotherapy in The First Affiliated Hospital of Anhui Medical University from January 2010 to January 2015. Immunohistochemistry was used to measure the expression of YB-1 in ICC tissue; after ICC cells were transfected with YB-1 plasmid, the thiazolyl blue method was used to observe the change in gemcitabine sensitivity, and qPCR was used to observe the changes in the expression of multidrug resistance genes. The independent samples t-test was used for comparison of continuous data between two groups and a one-way analysis of variance was used for comparison of continuous data between multiple groups; the chi-square test was used for comparison of categorical data between groups. The Kaplan-Meier method was used to calculate survival rates and the log-rank test was used for survival difference analysis. ResultsAmong the 58 patients, 44 (75.9% had high expression of YB-1 in the cytoplasm of ICC cells (cytoplasm YB-1 positive group and 14 (24.1% had no expression of YB-1 in the cytoplasm of ICC cells (cytoplasm YB-1 negative group. Of all patients in the cytoplasm YB-1 positive group, 18 (40.9% also had positive nuclear expression of YB-1 (nuclear YB-1 positive group; the other 40 patients had no nuclear expression of YB-1 (nuclear YB-1 negative group. The nuclear YB-1 negative group had a significantly longer survival time than the nuclear YB-1 positive group (63 months vs 28 months, χ2=17.99, P<0.05. In the control plasmid group, the half-maximal inhibitory concentration (IC50 of gemcitabine in HCCC-9810 cells was 0.054 μmol, and in the pSG5-YB-1 plasma transfection group, IC50 increased to 0

  18. Spectrophotometric assessment of nuclear proteins: a preliminary study

    Directory of Open Access Journals (Sweden)

    A Ubiali

    2009-06-01

    Full Text Available Qualitative evaluation of protein content in formalin fixed, paraffin-embedded tissues is usually performed by means of cytofluorimetric analysis. On the other hand, several studies underline the opportunity to measure the concentration of nuclear proteins, which is often accomplished by using complex techniques and instrumentation. In the present work, we suggest a new application for the spectrophotometric evaluation of protein content on extracted and isolated nuclei, based on EDTA treatment of specimens and chemical extraction of proteins, followed by direct spectrophotometric measurement at UV wavelengths. We also demonstrate how this parameter correlates with other diagnostic factors, such as the proliferation index (MIB-1 and the DNA content (ploidy of cells. This method is simple and effective, yet less expensive than other protein quantitation protocols.

  19. Karyopherin binding interactions and nuclear import mechanism of nuclear pore complex protein Tpr

    Directory of Open Access Journals (Sweden)

    Frosst Phyllis D

    2009-10-01

    Full Text Available Abstract Background Tpr is a large protein with an extended coiled-coil domain that is localized within the nuclear basket of the nuclear pore complex. Previous studies 1 involving antibody microinjection into mammalian cells suggested a role for Tpr in nuclear export of proteins via the CRM1 export receptor. In addition, Tpr was found to co-immunoprecipitate with importins α and β from Xenopus laevis egg extracts 2, although the function of this is unresolved. Yeast Mlp1p and Mlp2p, which are homologous to vertebrate Tpr, have been implicated in mRNA surveillance to retain unspliced mRNAs in the nucleus34. To augment an understanding of the role of Tpr in nucleocytoplasmic trafficking, we explored the interactions of recombinant Tpr with the karyopherins CRM1, importin β and importin α by solid phase binding assays. We also investigated the conditions required for nuclear import of Tpr using an in vitro assay. Results We found that Tpr binds strongly and specifically to importin α, importin β, and a CRM1 containing trimeric export complex, and that the binding sites for importins α and β are distinct. We also determined that the nuclear import of Tpr is dependent on cytosolic factors and energy and is efficiently mediated by the importin α/β import pathway. Conclusion Based on the binding and nuclear import assays, we propose that Tpr is imported into the nucleus by the importin α/β heterodimer. In addition, we suggest that Tpr can serve as a nucleoporin binding site for importin β during import of importin β cargo complexes and/or importin β recycling. Our finding that Tpr bound preferentially to CRM1 in an export complex strengthens the notion that Tpr is involved in protein export.

  20. Virtual box

    DEFF Research Database (Denmark)

    Stougaard, Malthe Kirkhoff

    2007-01-01

    . This paper reports on the design, implementation and initial evaluation of Virtual Box. Virtual Box attempts to create a physical and engaging context in order to support reciprocal interactions with expressive content. An implemented version of Virtual Box is evaluated in a location-aware environment...

  1. Cyclin-like F-box protein plays a role in growth and development of the three model species Medicago truncatula, Lotus japonicus, and Arabidopsis thaliana

    Directory of Open Access Journals (Sweden)

    Boycheva I

    2015-08-01

    Full Text Available Irina Boycheva,1 Valya Vassileva,2 Miglena Revalska,1 Grigor Zehirov,2 Anelia Iantcheva1 1Department of Functional Genetics Legumes, 2AgroBioInstitute, Department of Plant Stress Molecular Biology, Institute of Plant Physiology and Genetics, Sofia, Bulgaria Abstract: In eukaryotes, F-box proteins are one of the main components of the SCF complex that belongs to the family of ubiquitin E3 ligases, which catalyze protein ubiquitination and maintain the balance between protein synthesis and degradation. In the present study, we clarified the role and function of the gene encoding cyclin-like F-box protein from Medicago truncatula using transgenic plants of the model species M. truncatula, Lotus japonicas, and Arabidopsis thaliana generated by Agrobacterium-mediated transformation. Morphological and transcriptional analyses combined with flow cytometry and histochemistry demonstrated the participation of this protein in many aspects of plant growth and development, including processes of indirect somatic embryogenesis and symbiotic nodulation. The cyclin-like F-box gene showed expression in all plant organs and tissues comprised of actively dividing cells. The observed variations in root and hypocotyl growth, leaf and silique development, ploidy levels, and leaf parameters in the obtained transgenic lines demonstrated the effects of this gene on organ development. Furthermore, knockdown of cyclin-like F-box led to accumulation of higher levels of the G2/M transition-specific gene cyclin B1:1 (CYCB1:1, suggesting its possible role in cell cycle control. Together, the collected data suggest a similar role of the cyclin-like F-box protein in the three model species, providing evidence for the functional conservation of the studied gene. Keywords: cyclin-like F-box, model legumes, Arabidopsis thaliana, plant growth, plant development, cell cycle

  2. Forkhead Box Protein 1 (FoxO1) Inhibits Accelerated β Cell Aging in Pancreas-specific SMAD7 Mutant Mice.

    Science.gov (United States)

    Xiao, Xiangwei; Chen, Congde; Guo, Ping; Zhang, Ting; Fischbach, Shane; Fusco, Joseph; Shiota, Chiyo; Prasadan, Krishna; Dong, Henry; Gittes, George K

    2017-02-24

    The mechanisms underlying the effects of exocrine dysfunction on the development of diabetes remain largely unknown. Here we show that pancreatic depletion of SMAD7 resulted in age-dependent increases in β cell dysfunction with accelerated glucose intolerance, followed by overt diabetes. The accelerated β cell dysfunction and loss of proliferation capacity, two features of β cell aging, appeared to be non-cell-autonomous, secondary to the adjacent exocrine failure as a "bystander effect." Increased Forkhead box protein 1 (FoxO1) acetylation and nuclear retention was followed by progressive FoxO1 loss in β cells that marked the onset of diabetes. Moreover, forced FoxO1 expression in β cells prevented β cell dysfunction and loss in this model. Thus, we present a model of accelerated β cell aging that may be useful for studying the mechanisms underlying β cell failure in diabetes. Moreover, we provide evidence highlighting a critical role of FoxO1 in maintaining β cell identity in the context of SMAD7 failure. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  3. Forkhead Box Protein 1 (FoxO1) Inhibits Accelerated β Cell Aging in Pancreas-specific SMAD7 Mutant Mice*

    Science.gov (United States)

    Xiao, Xiangwei; Chen, Congde; Guo, Ping; Zhang, Ting; Fischbach, Shane; Fusco, Joseph; Shiota, Chiyo; Prasadan, Krishna; Dong, Henry; Gittes, George K.

    2017-01-01

    The mechanisms underlying the effects of exocrine dysfunction on the development of diabetes remain largely unknown. Here we show that pancreatic depletion of SMAD7 resulted in age-dependent increases in β cell dysfunction with accelerated glucose intolerance, followed by overt diabetes. The accelerated β cell dysfunction and loss of proliferation capacity, two features of β cell aging, appeared to be non-cell-autonomous, secondary to the adjacent exocrine failure as a “bystander effect.” Increased Forkhead box protein 1 (FoxO1) acetylation and nuclear retention was followed by progressive FoxO1 loss in β cells that marked the onset of diabetes. Moreover, forced FoxO1 expression in β cells prevented β cell dysfunction and loss in this model. Thus, we present a model of accelerated β cell aging that may be useful for studying the mechanisms underlying β cell failure in diabetes. Moreover, we provide evidence highlighting a critical role of FoxO1 in maintaining β cell identity in the context of SMAD7 failure. PMID:28057752

  4. Poxvirus K7 protein adopts a Bcl-2 fold: biochemical mapping of its interactions with human DEAD box RNA helicase DDX3.

    Science.gov (United States)

    Kalverda, Arnout P; Thompson, Gary S; Vogel, Andre; Schröder, Martina; Bowie, Andrew G; Khan, Amir R; Homans, Steve W

    2009-01-23

    Poxviruses have evolved numerous strategies to evade host innate immunity. Vaccinia virus K7 is a 149-residue protein with previously unknown structure that is highly conserved in the orthopoxvirus family. K7 bears sequence and functional similarities to A52, which interacts with interleukin receptor-associated kinase 2 and tumor necrosis factor receptor-associated factor 6 to suppress nuclear factor kappaB activation and to stimulate the secretion of the anti-inflammatory cytokine interleukin-10. In contrast to A52, K7 forms a complex with DEAD box RNA helicase DDX3, thereby suppressing DDX3-mediated ifnb promoter induction. We determined the NMR solution structure of K7 to provide insight into the structural basis for poxvirus antagonism of innate immune signaling. The structure reveals an alpha-helical fold belonging to the Bcl-2 family despite an unrelated primary sequence. NMR chemical-shift mapping studies have localized the binding surface for DDX3 on a negatively charged face of K7. Furthermore, thermodynamic studies have mapped the K7-binding region to a 30-residue N-terminal fragment of DDX3, ahead of the core RNA helicase domains.

  5. Interactions of the G quartet forming semaphorin 3F RNA with the RGG box domain of the fragile X protein family.

    Science.gov (United States)

    Menon, Lakshmi; Mihailescu, Mihaela-Rita

    2007-01-01

    Fragile X syndrome, the most common cause of inherited mental retardation, is caused by the transcriptional silencing of the fmr1 gene due to an unstable expansion of a CGG trinucleotide repeat and its subsequent hypermethylation in its 5' UTR. This gene encodes for the fragile X mental retardation protein (FMRP), an RNA-binding protein that has been shown to use its RGG box domain to bind to G quartet-forming RNA. In this study, we performed a detailed analysis of the interactions between the FMRP RGG box domain and one of its proposed RNA targets, human semaphorin 3F (S3F) RNA by using biophysical methods such as fluorescence, UV and circular dichroism spectroscopy. We show that this RNA forms a G quartet-containing structure, which is recognized with high affinity and specificity by the FMRP RGG box. In addition, we analyzed the interactions of human S3F RNA with the RGG box and RG cluster of the two FMRP autosomal paralogs, the FXR1P and FXR2P. We found that this RNA is bound with high affinity and specificity only by the FXR1P RGG box, but not by the FXR2P RG cluster. Both FMRP and FXR1P RGG box are able to unwind the G quartet structure of S3F RNA, however, the peptide concentrations required in this process are very different: a ratio of 1:6 RNA:FMRP RGG box versus 1:2 RNA:FXR1P RGG box.

  6. Atrogin-1, A Muscle-Specific F-Box Protein Highly Expressed during Muscle Atrophy

    National Research Council Canada - National Science Library

    Marcelo D. Gomes; Stewart H. Lecker; R. Thomas Jagoe; Ami Navon; Alfred L. Goldberg

    2001-01-01

    Muscle wasting is a debilitating consequence of fasting, inactivity, cancer, and other systemic diseases that results primarily from accelerated protein degradation by the ubiquitin-proteasome pathway...

  7. Evaluation of cyclooxygenase protein expression in traumatized versus normal tissues from eastern box turtles (Terrapene carolina carolina).

    Science.gov (United States)

    Royal, Lillian W; Lascelles, B Duncan X; Lewbart, Gregory A; Correa, Maria T; Jones, Samuel L

    2012-06-01

    This pilot study was designed to determine whether cyclooxygenase (COX)-1, COX-2, or both are expressed in normal turtle tissues and whether level of expression changes when tissue becomes inflamed. Five eastern box turtles, Terrapene carolina carolina, that either died or were euthanatized due to disease or injuries were used for this work. Tissues were obtained from the five turtles. Western blot analysis was used to evaluate tissues for COX-1 and COX-2 proteins. Densiometric analysis was used to compare Western blot bands within each turtle. COX-1 and COX-2 were found in the liver, kidney, grossly normal muscle, and grossly traumatized (inflamed) muscle of all study turtles. In all cases, COX-1 and COX-2 proteins were increased in traumatized muscle over grossly normal nontraumatized muscle. The highest levels of COX-1 and COX-2 proteins were found in kidney and liver. There was no statistical difference between the amount of COX-1 protein in liver and kidney, but traumatized muscle compared with grossly normal muscle had significantly greater COX-1 but not COX 2 protein concentrations. There was no statistical difference between the amount of COX-2 protein in liver and kidney. Traumatized muscle expressed nonstatistically significant greater amounts of COX-2 compared with grossly normal muscle. COX-1 and COX-2 proteins are expressed in turtle tissues, and both isoforms are upregulated during inflammation of muscle tissue. Traditional nonsteroidal anti-inflammatory drugs (NSAIDs) that block both COX isoforms might be more efficacious than COX-2-selective drugs. This work suggests that NSAIDs should be evaluated for potential liver and kidney toxicity in turtles.

  8. RNA polymerase II components and Rrn7 form a preinitiation complex on the HomolD box to promote ribosomal protein gene expression in Schizosaccharomyces pombe.

    Science.gov (United States)

    Montes, Matías; Moreira-Ramos, Sandra; Rojas, Diego A; Urbina, Fabiola; Käufer, Norbert F; Maldonado, Edio

    2017-02-01

    In Schizosaccharomyces pombe, ribosomal protein gene (RPG) promoters contain a TATA box analog, the HomolD box, which is bound by the Rrn7 protein. Despite the importance of ribosome biogenesis for cell survival, the mechanisms underlying RPG transcription remain unknown. In this study, we found that components of the RNA polymerase II (RNAPII) system, consisting of the initiation or general transcription factors (GTFs) TFIIA, IIB, IIE, TATA-binding protein (TBP) and the RNAPII holoenzyme, interacted directly with Rrn7 in vitro, and were able to form a preinitiation complex (PIC) on the HomolD box. PIC complex formation follows an ordered pathway on these promoters. The GTFs and RNAPII can also be cross-linked to HomolD-containing promoters in vivo. In an in vitro reconstituted transcription system, RNAPII components and Rrn7 were necessary for HomolD-directed transcription. The Mediator complex was required for basal transcription from those promoters in whole cell extract (WCE). The Med17 subunit of Mediator also can be cross-linked to the promoter region of HomolD-containing promoters in vivo, suggesting the presence of the Mediator complex on HomolD box-containing promoters. Together, these data show that components of the RNAPII machinery and Rrn7 participate in the PIC assembly on the HomolD box, thereby directing RPG transcription. © 2017 Federation of European Biochemical Societies.

  9. Prm3p is a pheromone-induced peripheral nuclear envelope protein required for yeast nuclear fusion.

    Science.gov (United States)

    Shen, Shu; Tobery, Cynthia E; Rose, Mark D

    2009-05-01

    Nuclear membrane fusion is the last step in the mating pathway of the yeast Saccharomyces cerevisiae. We adapted a bioinformatics approach to identify putative pheromone-induced membrane proteins potentially required for nuclear membrane fusion. One protein, Prm3p, was found to be required for nuclear membrane fusion; disruption of PRM3 caused a strong bilateral defect, in which nuclear congression was completed but fusion did not occur. Prm3p was localized to the nuclear envelope in pheromone-responding cells, with significant colocalization with the spindle pole body in zygotes. A previous report, using a truncated protein, claimed that Prm3p is localized to the inner nuclear envelope. Based on biochemistry, immunoelectron microscopy and live cell microscopy, we find that functional Prm3p is a peripheral membrane protein exposed on the cytoplasmic face of the outer nuclear envelope. In support of this, mutations in a putative nuclear localization sequence had no effect on full-length protein function or localization. In contrast, point mutations and deletions in the highly conserved hydrophobic carboxy-terminal domain disrupted both protein function and localization. Genetic analysis, colocalization, and biochemical experiments indicate that Prm3p interacts directly with Kar5p, suggesting that nuclear membrane fusion is mediated by a protein complex.

  10. The Arabidopsis thaliana F-box protein FBL17 is essential for progression through the second mitosis during pollen development.

    Directory of Open Access Journals (Sweden)

    Andi Gusti

    Full Text Available In fungi and metazoans, the SCF-type Ubiquitin protein ligases (E3s play a critical role in cell cycle regulation by degrading negative regulators, such as cell cycle-dependent kinase inhibitors (CKIs at the G1-to-S-phase checkpoint. Here we report that FBL17, an Arabidopsis thaliana F-box protein, is involved in cell cycle regulation during male gametogenesis. FBL17 expression is strongly enhanced in plants co-expressing E2Fa and DPa, transcription factors that promote S-phase entry. FBL17 loss-of-function mutants fail to undergo pollen mitosis II, which generates the two sperm cells in mature A. thaliana pollen. Nonetheless, the single sperm cell-like cell in fbl17 mutants is functional but will exclusively fertilize the egg cell of the female gametophyte, giving rise to an embryo that will later abort, most likely due to the lack of functional endosperm. Seed abortion can, however, be overcome by mutations in FIE, a component of the Polycomb group complex, overall resembling loss-of-function mutations in the A. thaliana cyclin-dependent kinase CDKA;1. Finally we identified ASK11, as an SKP1-like partner protein of FBL17 and discuss a possible mechanism how SCF(FBL17 may regulate cell division during male gametogenesis.

  11. Isolation and characterization of a novel protein toxin from the Hawaiian box jellyfish (sea wasp) Carybdea alata.

    Science.gov (United States)

    Nagai, H; Takuwa, K; Nakao, M; Sakamoto, B; Crow, G L; Nakajima, T

    2000-08-28

    The box jellyfish (sea wasp) Carybdea alata Reynaud, 1830 (Cubozoa) is distributed widely in the tropics. The sting of C. alata causes severe pain and cutaneous inflammation in humans. We successfully isolated C. alata toxin-A (CaTX-A, 43 kDa) and -B (CaTX-B, 45 kDa) for the first time from the tentacle of C. alata collected at a site along the Hawaiian shore. The experimental results showed that CaTX-A, but not CaTX-B, is present in the nematocyst, the organ responsible for stinging. Both CaTX-A and -B showed potent hemolytic activity, with CaTX-A being lethally toxic to crayfish when administered via intraperitoneal injection. Furthermore, we sequenced the cDNA encoding CaTX-A. The deduced amino acid sequence of CaTX-A (463 amino acids) showed 43.7% homology to Carybdea rastoni toxins (CrTXs) but not with any other known proteins. Therefore, these jellyfish toxins potentially represent a novel class of bioactive proteins. Secondary structure analysis of CaTX-A and CrTXs suggested the presence of amphiphilic alpha-helices, which are also seen in several known hemolytic or cytolytic protein toxins, including peptide toxins. Copyright 2000 Academic Press.

  12. Model of the Ankyrin and SOCS Box Protein, ASB9, E3 Ligase Reveals a Mechanism for Dynamic Ubiquitin Transfer.

    Science.gov (United States)

    Schiffer, Jamie M; Malmstrom, Robert D; Parnell, Jonathan; Ramirez-Sarmiento, Cesar; Reyes, Javiera; Amaro, Rommie E; Komives, Elizabeth A

    2016-08-02

    Cullin-RING E3 ligases (CRLs) are elongated and bowed protein complexes that transfer ubiquitin over 60 Å to proteins targeted for proteasome degradation. One such CRL contains the ankyrin repeat and SOCS box protein 9 (ASB9), which binds to and partially inhibits creatine kinase (CK). While current models for the ASB9-CK complex contain some known interface residues, the overall structure and precise interface of the ASB9-CK complex remains unknown. Through an integrative modeling approach, we report a third-generation model that reveals precisely the interface interactions and also fits the shape of the ASB9-CK complex as determined by small-angle X-ray scattering. We constructed an atomic model for the entire CK-targeting CRL to uncover dominant modes of motion that could permit ubiquitin transfer. Remarkably, only the correctly docked CK-containing E3 ligase and not incorrectly docked structures permitted close approach of ubiquitin to the CK substrate. Copyright © 2016 Elsevier Ltd. All rights reserved.

  13. The D3 F-box protein is a key component in host strigolactone responses essential for arbuscular mycorrhizal symbiosis.

    Science.gov (United States)

    Yoshida, Satoko; Kameoka, Hiromu; Tempo, Misaki; Akiyama, Kohki; Umehara, Mikihisa; Yamaguchi, Shinjiro; Hayashi, Hideo; Kyozuka, Junko; Shirasu, Ken

    2012-12-01

    Arbuscular mycorrhiza (AM) represents an ancient endosymbiosis between plant roots and Glomeromycota fungi. Strigolactones (SLs), plant-derived terpenoid lactones, activate hyphal branching of AM fungi before physical contact. Lack of SL biosynthesis results in lower colonization of AM fungi. The F-box protein, DWARF3 (D3), and the hydrolase family protein DWARF14 (D14) are crucial for SL responses in rice. Here we conducted AM fungal colonization assays with the SL-insensitive d3 and d14 mutants. The d3 mutant exhibited strong defects in AM fungal colonization, whereas the d14 mutant showed higher AM fungal colonization. As D14 has a homologous protein, D14-LIKE, we generated D14-LIKE knockdown lines by RNA interference in the wildtype and d14 background. D14 and D14-LIKE double knockdown lines exhibited similar colonization rates as those of the d14-1 mutant. D3 is crucial for establishing AM symbiosis in rice, whereas D14 and D14-LIKE are not. Our results suggest distinct roles for these SL-related components in AM symbiosis. © 2012 The Authors. New Phytologist © 2012 New Phytologist Trust.

  14. 70-kDa Heat Shock Cognate Protein hsc70 Mediates Calmodulin-dependent Nuclear Import of the Sex-determining Factor SRY*

    Science.gov (United States)

    Kaur, Gurpreet; Lieu, Kim G.; Jans, David A.

    2013-01-01

    We recently showed that the developmentally important family of SOX (SRY (sex determining region on the Y chromosome)-related high mobility group (HMG) box) proteins require the calcium-binding protein calmodulin (CaM) for optimal nuclear accumulation, with clinical mutations in SRY that specifically impair nuclear accumulation via this pathway resulting in XY sex reversal. However, the mechanism by which CaM facilitates nuclear accumulation is unknown. Here, we show, for the first time, that the 70-kDa heat shock cognate protein hsc70 plays a key role in CaM-dependent nuclear import of SRY. Using a reconstituted nuclear import assay, we show that antibodies to hsc70 significantly reduce nuclear accumulation of wild type SRY and mutant derivatives thereof that retain CaM-dependent nuclear import, with an increased rate of nuclear accumulation upon addition of both CaM and hsc70, in contrast to an SRY mutant derivative with impaired CaM binding. siRNA knockdown of hsc70 in intact cells showed similar results, indicating clear dependence upon hsc70 for CaM-dependent nuclear import. Analysis using the technique of fluorescence recovery after photobleaching indicated that hsc70 is required for the maximal rate of SRY nuclear import in living cells but has no impact upon SRY nuclear retention/nuclear dynamics. Finally, we demonstrate direct binding of hsc70 to the SRY·CaM complex, with immunoprecipitation experiments from cell extracts showing association of hsc70 with wild type SRY, but not with a mutant derivative with impaired CaM binding, dependent on Ca2+. Our novel findings strongly implicate hsc70 in CaM-dependent nuclear import of SRY. PMID:23235156

  15. Distinctive Properties of the Nuclear Localization Signals of Inner Nuclear Membrane Proteins Heh1 and Heh2

    NARCIS (Netherlands)

    Lokareddy, Ravi K.; Hapsari, Rizqiya A.; van Rheenen, Mathilde; Pumroy, Ruth A.; Bhardwaj, Anshul; Steen, Anton; Veenhoff, Liesbeth M.; Cingolani, Gino

    2015-01-01

    Targeting of ER-synthesized membrane proteins to the inner nuclear membrane (INM) has long been explained by the diffusion-retention model. However, several INM proteins contain non-classical nuclear localization signal (NLS) sequences, which, in a few instances, have been shown to promote importin

  16. Proximity Utilizing Biotinylation of Nuclear Proteins in vivo

    Directory of Open Access Journals (Sweden)

    Arman Kulyyassov

    2015-06-01

    Full Text Available Introduction. The human genome consists of roughly 30,000 genes coding for over 500,000 different proteins, of which more than 10,000 proteins can be produced by the cell at any given time (the cellular “proteome”. It has been estimated that over 80% of proteins do not operate alone, but in complexes. These protein-protein interactions (PPI are regulated by several mechanisms. For example, post-translational modifications (methylation, acetylation, phosphorylation, or ubiquitination or metal-binding can lead to conformational changes that alter the affinity and kinetic parameters of the interaction. Many PPIs are part of larger cellular networks of interactions or interactomes. Indeed, these interactions are at the core of the entire interactomics system of any living cell, and so, aberrant PPIs are the basis of multiple diseases, such as neurodegenerative diseases and cancer. The objective of this study was to develop a method of monitoring protein-protein interactions and proximity dependence in vivo.Methods. The biotin ligase BirA was fused to the protein of interest, and the Biotin Acceptor Peptide (BAP was fused to an interacting partner to make the detection of its biotinylation possible by western blot or mass spectrometry.Results. Using several experimental systems (BirA.A + BAP.B, we showed that the biotinylation is interaction/proximity dependent. Here, A and B are the next nuclear proteins used in the experiments – 3 paralogues of heterochromatin protein HP1a (CBX5, HP1b (CBX1, HP1g (CBX3, wild type and transcription mutant factor Kap1, translesion DNA polymerase PolH and E3, ubiquitin ligase RAD18, Proliferative Cell Nuclear Antigen (PCNA, ubiquitin Ub, SUMO-2/3, different types and isoforms of histones H2A, H2Az, H3.1, H3.3, CenpA, H2A.BBD, and macroH2A. The variant of this approach is termed PUB-NChIP (Proximity Utilizing Biotinylation with Native Chromatin Immuno-precipitation and is designed to purify and study the protein

  17. Functional characterization of the ER stress induced X-box-binding protein-1 (Xbp-1 in the porcine system

    Directory of Open Access Journals (Sweden)

    Jin Dong-Il

    2011-05-01

    Full Text Available Abstract Background The unfolded protein response (UPR is an evolutionary conserved adaptive reaction for increasing cell survival under endoplasmic reticulum (ER stress conditions. X-box-binding protein-1 (Xbp1 is a key transcription factor of UPR that activates genes involved in protein folding, secretion, and degradation to restore ER function. The UPR induced by ER stress was extensively studied in diseases linked to protein misfolding and aggregations. However, in the porcine system, genes in the UPR pathway were not investigated. In this study, we isolated and characterized the porcine Xbp1 (pXbp1 gene in ER stress using porcine embryonic fibroblast (PEF cells and porcine organs. ER stress was induced by the treatment of tunicamycin and cell viability was investigated by the MTT assay. For cloning and analyzing the expression pattern of pXbp1, RT-PCR analysis and Western blot were used. Knock-down of pXbp1 was performed by the siRNA-mediated gene silencing. Results We found that the pXbp1 mRNA was the subject of the IRE1α-mediated unconventional splicing by ER stress. Knock-down of pXbp1 enhanced ER stress-mediated cell death in PEF cells. In adult organs, pXbp1 mRNA and protein were expressed and the spliced forms were detected. Conclusions It was first found that the UPR mechanisms and the function of pXbp1 in the porcine system. These results indicate that pXbp1 plays an important role during the ER stress response like other animal systems and open a new opportunity for examining the UPR pathway in the porcine model system.

  18. The Agrobacterium tumefaciens virulence protein VirE3 is a transcriptional activator of the F-box gene VBF.

    Science.gov (United States)

    Niu, Xiaolei; Zhou, Meiliang; Henkel, Christiaan V; van Heusden, G Paul H; Hooykaas, Paul J J

    2015-12-01

    During Agrobacterium tumefaciens-mediated transformation of plant cells a part of the tumour-inducing plasmid, T-DNA, is integrated into the host genome. In addition, a number of virulence proteins are translocated into the host cell. The virulence protein VirE3 binds to the Arabidopsis thaliana pBrp protein, a plant-specific general transcription factor of the TFIIB family. To study a possible role for VirE3 in transcriptional regulation, we stably expressed virE3 in A. thaliana under control of a tamoxifen-inducible promoter. By RNA sequencing we showed that upon expression of virE3 the RNA levels of 607 genes were increased more than three-fold and those of 132 genes decreased more than three-fold. One of the strongly activated genes was that encoding VBF (At1G56250), an F-box protein that may affect the levels of the VirE2 and VIP1 proteins. Using Arabidopsis cell suspension protoplasts we showed that VirE3 stimulates the VBF promoter, especially when co-expressed with pBrp. Although pBrp is localized at the external surface of plastids, co-expression of VirE3 and pBrp in Arabidopsis cell suspension protoplasts resulted in the accumulation of pBrp in the nucleus. Our results suggest that VirE3 affects the transcriptional machinery of the host cell to favour the transformation process. © 2015 The Authors The Plant Journal © 2015 John Wiley & Sons Ltd.

  19. Dysregulation of In Vitro Decidualization of Human Endometrial Stromal Cells by Insulin via Transcriptional Inhibition of Forkhead Box Protein O1.

    Directory of Open Access Journals (Sweden)

    Dorina Ujvari

    Full Text Available Insulin resistance and compensatory hyperinsulinemia are characteristic features of obesity and polycystic ovary syndrome, and both are associated with reduced fertility and implantation. There is little knowledge about the effect of insulin on the decidualization process and previous findings are contradictory. We investigated the effect of insulin on the regulation of forkhead box protein O1 (FOXO1, one of the most important transcription factors during decidualization. Endometrial stromal cells were isolated from six healthy, regularly menstruating women and decidualized in vitro. Gene expression levels of six putative FOXO1 target genes (including insulin-like growth factor binding protein-1 (IGFBP1 and prolactin (PRL were measured with Real-Time PCR following FOXO1 inhibition or insulin treatment. PI3K inhibition was used to identify the possible mechanism behind regulation. Subcellular localization of FOXO1 was analyzed with immunofluorescence. All the genes (IGFBP1, CTGF, INSR, DCN, LEFTY2, except prolactin, were evaluated as FOXO1 target genes in decidualizing stromal cells. Insulin caused a significant dose-dependent inhibition of the verified FOXO1 target genes. It was also demonstrated that insulin regulated FOXO1 target genes by transcriptional inactivation and nuclear export of FOXO1 via PI3K pathway. However, insulin did not inhibit the morphological transformation of endometrial stromal cells via transcriptional inactivation of FOXO1. This study provides new insights on the action of insulin on the endometrium via regulation of FOXO1. It is suggested that hyperinsulinemia results in dysregulation of a high number of FOXO1 controlled genes that may contribute to endometrial dysfunction and reproductive failure. Our findings may illuminate possible reasons for unexplained infertility.

  20. Chironex fleckeri (box jellyfish) venom proteins: expansion of a cnidarian toxin family that elicits variable cytolytic and cardiovascular effects.

    Science.gov (United States)

    Brinkman, Diane L; Konstantakopoulos, Nicki; McInerney, Bernie V; Mulvenna, Jason; Seymour, Jamie E; Isbister, Geoffrey K; Hodgson, Wayne C

    2014-02-21

    The box jellyfish Chironex fleckeri produces extremely potent and rapid-acting venom that is harmful to humans and lethal to prey. Here, we describe the characterization of two C. fleckeri venom proteins, CfTX-A (∼40 kDa) and CfTX-B (∼42 kDa), which were isolated from C. fleckeri venom using size exclusion chromatography and cation exchange chromatography. Full-length cDNA sequences encoding CfTX-A and -B and a third putative toxin, CfTX-Bt, were subsequently retrieved from a C. fleckeri tentacle cDNA library. Bioinformatic analyses revealed that the new toxins belong to a small family of potent cnidarian pore-forming toxins that includes two other C. fleckeri toxins, CfTX-1 and CfTX-2. Phylogenetic inferences from amino acid sequences of the toxin family grouped CfTX-A, -B, and -Bt in a separate clade from CfTX-1 and -2, suggesting that the C. fleckeri toxins have diversified structurally and functionally during evolution. Comparative bioactivity assays revealed that CfTX-1/2 (25 μg kg(-1)) caused profound effects on the cardiovascular system of anesthetized rats, whereas CfTX-A/B elicited only minor effects at the same dose. Conversely, the hemolytic activity of CfTX-A/B (HU50 = 5 ng ml(-1)) was at least 30 times greater than that of CfTX-1/2. Structural homology between the cubozoan toxins and insecticidal three-domain Cry toxins (δ-endotoxins) suggests that the toxins have a similar pore-forming mechanism of action involving α-helices of the N-terminal domain, whereas structural diversification among toxin members may modulate target specificity. Expansion of the cnidarian toxin family therefore provides new insights into the evolutionary diversification of box jellyfish toxins from a structural and functional perspective.

  1. Distinct roles for key karyogamy proteins during yeast nuclear fusion.

    Science.gov (United States)

    Melloy, Patricia; Shen, Shu; White, Erin; Rose, Mark D

    2009-09-01

    During yeast mating, cell fusion is followed by the congression and fusion of the two nuclei. Proteins required for nuclear fusion are found at the surface (Prm3p) and within the lumen (Kar2p, Kar5p, and Kar8p) of the nuclear envelope (NE). Electron tomography (ET) of zygotes revealed that mutations in these proteins block nuclear fusion with different morphologies, suggesting that they act in different steps of fusion. Specifically, prm3 zygotes were blocked before formation of membrane bridges, whereas kar2, kar5, and kar8 zygotes frequently contained them. Membrane bridges were significantly larger and occurred more frequently in kar2 and kar8, than in kar5 mutant zygotes. The kinetics of NE fusion in prm3, kar5, and kar8 mutants, measured by live-cell fluorescence microscopy, were well correlated with the size and frequency of bridges observed by ET. However the kar2 mutant was defective for transfer of NE lumenal GFP, but not diffusion within the lumen, suggesting that transfer was blocked at the NE fusion junction. These observations suggest that Prm3p acts before initiation of outer NE fusion, Kar5p may help dilation of the initial fusion pore, and Kar2p and Kar8p act after outer NE fusion, during inner NE fusion.

  2. Protein Interaction Screening for the Ankyrin Repeats and Suppressor of Cytokine Signaling (SOCS) Box (ASB) Family Identify Asb11 as a Novel Endoplasmic Reticulum Resident Ubiquitin Ligase

    DEFF Research Database (Denmark)

    Andresen, Christina Aaen; Smedegaard, Stine; Sylvestersen, Kathrine Beck

    2014-01-01

    The Ankyrin and SOCS (Suppressor of Cytokine Signaling) box (ASB) family of proteins function as the substrate recognition subunit in a subset of Elongin-Cullin-SOCS (ECS) E3 ubiquitin ligases. Despite counting with 18 members in humans, the identity of the physiological targets of the Asb protei...

  3. The Y-Box Binding Protein 1 Suppresses Alzheimer’s Disease Progression in Two Animal Models

    Science.gov (United States)

    Bobkova, N. V.; Lyabin, D. N.; Medvinskaya, N. I.; Samokhin, A. N.; Nekrasov, P. V.; Nesterova, I. V.; Aleksandrova, I. Y.; Tatarnikova, O. G.; Bobylev, A. G.; Vikhlyantsev, I. M.; Kukharsky, M. S.; Ustyugov, A. A.; Polyakov, D. N.; Eliseeva, I. A.; Kretov, D. A.; Guryanov, S. G.; Ovchinnikov, L. P.

    2015-01-01

    The Y-box binding protein 1 (YB-1) is a member of the family of DNA- and RNA binding proteins. It is involved in a wide variety of DNA/RNA-dependent events including cell proliferation and differentiation, stress response, and malignant cell transformation. Previously, YB-1 was detected in neurons of the neocortex and hippocampus, but its precise role in the brain remains undefined. Here we show that subchronic intranasal injections of recombinant YB-1, as well as its fragment YB-11−219, suppress impairment of spatial memory in olfactory bulbectomized (OBX) mice with Alzheimer’s type degeneration and improve learning in transgenic 5XFAD mice used as a model of cerebral amyloidosis. YB-1-treated OBX and 5XFAD mice showed a decreased level of brain β-amyloid. In OBX animals, an improved morphological state of neurons was revealed in the neocortex and hippocampus; in 5XFAD mice, a delay in amyloid plaque progression was observed. Intranasally administered YB-1 penetrated into the brain and could enter neurons. In vitro co-incubation of YB-1 with monomeric β-amyloid (1–42) inhibited formation of β-amyloid fibrils, as confirmed by electron microscopy. This suggests that YB-1 interaction with β-amyloid prevents formation of filaments that are responsible for neurotoxicity and neuronal death. Our data are the first evidence for a potential therapeutic benefit of YB-1 for treatment of Alzheimer’s disease. PMID:26394155

  4. Cell-penetrable mouse forkhead box protein 3 alleviates experimental arthritis in mice by up-regulating regulatory T cells.

    Science.gov (United States)

    Liu, Xia; Ji, Baoju; Sun, Mengyi; Wu, Weijiang; Huang, Lili; Sun, Aihua; Zong, Yangyong; Xia, Sheng; Shi, Liyun; Qian, Hui; Xu, Wenrong; Shao, Qixiang

    2015-07-01

    Regulatory T cells (T(regs)) have potential applications in clinical disease therapy, such as autoimmune diseases and transplant rejection. However, their numbers are limited. Forkhead box protein 3 (FoxP3) is a key transcription factor that controls T(reg) development and function. Here, we generated a cell-permeable fusion protein, protein transduction domain (PTD)-conjugated mouse FoxP3 protein (PTD-mFoxP3), and evaluated whether PTD-mFoxp3 can alleviate rheumatoid arthritis (RA) in the collagen-induced arthritis (CIA) mouse model. As expected, PTD-mFoxP3 was transduced into cells effectively, and inhibited T cell activation and attenuated the cell proliferation. It decreased interleukin (IL) 2 and interferon (IFN)-γ expression, and increased IL-10 expression in activated CD4(+)CD25(-) T cells. PTD-mFoxP3-transduced CD4(+)CD25(-) T cells attenuated proliferation of activated CD4(+)CD25(-) T cells. In addition, PTD-mFoxP3 blocked the Th17 differentiation programme in vitro and down-regulated IL-17 production from T cells by modulating induction and levels of retinoid-related orphan receptor gamma t (RORγt). Intra-articular delivery of PTD-mFoxP3 delayed disease incidence remarkably and alleviated autoimmune symptoms of CIA mice. Moreover, protective effects of PTD-mFoxP3 were associated with regulating the balance of T helper type 17 (Th17) and T(regs). These results suggest that PTD-mFoxP3 may be a candidate for RA therapy. © 2015 British Society for Immunology.

  5. Evaluation of the Schistosoma mansoni Y-box-binding protein (SMYB1 potential as a vaccine candidate against schistosomiasis

    Directory of Open Access Journals (Sweden)

    Silvia Regina Costa Dias

    2014-06-01

    Full Text Available Schistosomiasis is a neglected tropical disease, and after malaria, is the second most important tropical disease in public health. A vaccine that reduces parasitemia is desirable to achieve mass treatment with a low cost. Although potential antigens have been identified and tested in clinical trials, no effective vaccine against schistosomiasis is available. Y-box-binding proteins (YBPs regulate gene expression and participate in a variety of cellular processes, including transcriptional and translational regulation, DNA repair, cellular proliferation, drug resistance and stress responses. The Schistosoma mansoni ortholog of the human YB-1, SMYB1, is expressed in all stages of the parasite life cycle. Although SMYB1 binds to DNA or RNA oligonucleotides, immunohistochemistry assays demonstrated that it is primarily localized in the cytoplasm of parasite cells. In addition, SMYB1 interacts with a protein involved in mRNA processing, suggesting that SMYB1 functions in the turnover, transport and/or stabilization of RNA molecules during post-transcriptional gene regulation. Here we report the potential of SMYB1 as a vaccine candidate. We demonstrate that recombinant SMYB1 stimulates the production of high levels of specific IgG1 antibodies in a mouse model. The observed levels of specific IgG1 and IgG2a antibodies indicate an actual protection against cercariae challenge. Animals immunized with rSMYB1 exhibited a 26% reduction in adult worm burden and a 28% reduction in eggs retained in the liver. Although proteins from the worm tegument are considered optimal targets for vaccine development, this study demonstrates that unexposed cytoplasmic proteins can reduce the load of intestinal worms and the number of eggs retained in the liver.

  6. Evaluation of the Schistosoma mansoni Y-box-binding protein (SMYB1) potential as a vaccine candidate against schistosomiasis.

    Science.gov (United States)

    Dias, Sílvia R C; Boroni, Mariana; Rocha, Elizângela A; Dias, Thomaz L; de Laet Souza, Daniela; Oliveira, Fabrício M S; Bitar, Mainá; Macedo, Andrea M; Machado, Carlos R; Caliari, Marcelo V; Franco, Glória R

    2014-01-01

    Schistosomiasis is a neglected tropical disease, and after malaria, is the second most important tropical disease in public health. A vaccine that reduces parasitemia is desirable to achieve mass treatment with a low cost. Although potential antigens have been identified and tested in clinical trials, no effective vaccine against schistosomiasis is available. Y-box-binding proteins (YBPs) regulate gene expression and participate in a variety of cellular processes, including transcriptional and translational regulation, DNA repair, cellular proliferation, drug resistance, and stress responses. The Schistosoma mansoni ortholog of the human YB-1, SMYB1, is expressed in all stages of the parasite life cycle. Although SMYB1 binds to DNA or RNA oligonucleotides, immunohistochemistry assays demonstrated that it is primarily localized in the cytoplasm of parasite cells. In addition, SMYB1 interacts with a protein involved in mRNA processing, suggesting that SMYB1 functions in the turnover, transport, and/or stabilization of RNA molecules during post-transcriptional gene regulation. Here we report the potential of SMYB1 as a vaccine candidate. We demonstrate that recombinant SMYB1 stimulates the production of high levels of specific IgG1 antibodies in a mouse model. The observed levels of specific IgG1 and IgG2a antibodies indicate an actual protection against cercariae challenge. Animals immunized with rSMYB1 exhibited a 26% reduction in adult worm burden and a 28% reduction in eggs retained in the liver. Although proteins from the worm tegument are considered optimal targets for vaccine development, this study demonstrates that unexposed cytoplasmic proteins can reduce the load of intestinal worms and the number of eggs retained in the liver.

  7. Bento Boxes

    Science.gov (United States)

    Hasio, Cindy

    2010-01-01

    Bento boxes are common objects in Japanese culture, designed to hold enough lunch for one person. They have individual compartments and sometimes multiple tiers for rice, vegetables, and other side dishes. They are made of materials ranging from wood, cloth, aluminum, or plastic. In general, the greater the number of foods, the better the box is…

  8. Analysis of CFB, a cytokinin-responsive gene of Arabidopsis thaliana encoding a novel F-box protein regulating sterol biosynthesis.

    Science.gov (United States)

    Brenner, Wolfram G; Leuendorf, Jan Erik; Cortleven, Anne; Martin, Laetitia B B; Schaller, Hubert; Schmülling, Thomas

    2017-05-17

    Protein degradation by the ubiquitin-26S proteasome pathway is important for the regulation of cellular processes, but the function of most F-box proteins relevant to substrate recognition is unknown. We describe the analysis of the gene Cytokinin-induced F-box encoding (CFB, AT3G44326), identified in a meta-analysis of cytokinin-related transcriptome studies as one of the most robust cytokinin response genes. F-box domain-dependent interaction with the E3 ubiquitin ligase complex component ASK1 classifies CFB as a functional F-box protein. Apart from F-box and transmembrane domains, CFB contains no known functional domains. CFB is expressed in all plant tissues, predominantly in root tissue. A ProCFB:GFP-GUS fusion gene showed strongest expression in the lateral root cap and during lateral root formation. CFB-GFP fusion proteins were mainly localized in the nucleus and the cytosol but also at the plasma membrane. cfb mutants had no discernible phenotype, but CFB overexpressing plants showed several defects, such as a white upper inflorescence stem, similar to the hypomorphic cycloartenol synthase mutant cas1-1. Both CFB overexpressing plants and cas1-1 mutants accumulated the CAS1 substrate 2,3-oxidosqualene in the white stem tissue, the latter even more after cytokinin treatment, indicating impairment of CAS1 function. This suggests that CFB may link cytokinin and the sterol biosynthesis pathway. © The Author 2017. Published by Oxford University Press on behalf of the Society for Experimental Biology.

  9. Nuclear localization of bradykinin B(2) receptors reflects binding to the nuclear envelope protein lamin C.

    Science.gov (United States)

    Takano, Masaoki; Kanoh, Akira; Amako, Katsumi; Otani, Mieko; Sano, Keiji; Kanazawa-Hamada, Michiko; Matsuyama, Shogo

    2014-01-15

    The mechanism of action of bradykinin (BK), a pro-inflammatory mediator, is thought to be mediated by specific cell surface membrane bradykinin B2 receptors. Some evidence suggests that there are both intracellular and nuclear bradykinin B2 receptors. This study identified proteins that interact with the C-terminus of the bradykinin B2 receptor (in particular, the nuclear membrane protein lamin C), using the yeast two-hybrid system. The motif of the C-terminal domain (CT) mutant 303-320 in bradykinin B2 receptor was identified as a lamin C protein binding motif. Immunohistochemistry revealed colocalization of FLAG- bradykinin B2 receptor with HA-lamin C in the nucleus of HEK 293T cells. In situ proximity ligation assay (PLA) showed that FLAG-bradykinin B2 receptor formed heterodimers with HA-lamin C in the nucleus. In addition, live cell fluorescence imaging showed that bradykinin B2 receptor-EGFP was located in the nucleus and co-localized with HcRed-lamin C. Interestingly, neither BK addition nor bradykinin B2 receptor CT mutation reduced the binding to lamin C or changed the distribution of bradykinin B2 receptor. Taken together, these findings demonstrate that bradykinin B2 receptor-lamin C heterodimers form in the nucleus independent of BK stimulation and CT mutation. We propose that heterodimerization of bradykinin B2 receptor with lamin C is essential to nuclear localization of bradykinin B2 receptor and plays an important role in cell signaling and function. © 2013 Elsevier B.V. All rights reserved.

  10. Sry HMG box protein 9-positive (Sox9+) epithelial cell adhesion molecule-negative (EpCAM-) biphenotypic cells derived from hepatocytes are involved in mouse liver regeneration.

    Science.gov (United States)

    Tanimizu, Naoki; Nishikawa, Yuji; Ichinohe, Norihisa; Akiyama, Haruhiko; Mitaka, Toshihiro

    2014-03-14

    It has been shown that mature hepatocytes compensate tissue damages not only by proliferation and/or hypertrophy but also by conversion into cholangiocyte-like cells. We found that Sry HMG box protein 9-positive (Sox9(+)) epithelial cell adhesion molecule-negative (EpCAM(-)) hepatocyte nuclear factor 4α-positive (HNF4α(+)) biphenotypic cells showing hepatocytic morphology appeared near EpCAM(+) ductular structures in the livers of mice fed 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC)-containing diet. When Mx1-Cre:ROSA mice, which were injected with poly(I:C) to label mature hepatocytes, were fed with the DDC diet, we found LacZ(+)Sox9(+) cells near ductular structures. Although Sox9(+)EpCAM(-) cells adjacent to expanding ducts likely further converted into ductular cells, the incidence was rare. To know the cellular characteristics of Sox9(+)EpCAM(-) cells, we isolated them as GFP(+)EpCAM(-) cells from DDC-injured livers of Sox9-EGFP mice. Sox9(+)EpCAM(-) cells proliferated and could differentiate to functional hepatocytes in vitro. In addition, Sox9(+)EpCAM(-) cells formed cysts with a small central lumen in collagen gels containing Matrigel® without expressing EpCAM. These results suggest that Sox9(+)EpCAM(-) cells maintaining biphenotypic status can establish cholangiocyte-type polarity. Interestingly, we found that some of the Sox9(+) cells surrounded luminal spaces in DDC-injured liver while they expressed HNF4α. Taken together, we consider that in addition to converting to cholangiocyte-like cells, Sox9(+)EpCAM(-) cells provide luminal space near expanded ductular structures to prevent deterioration of the injuries and potentially supply new hepatocytes to repair damaged tissues.

  11. Human Cytomegalovirus Nuclear Capsids Associate with the Core Nuclear Egress Complex and the Viral Protein Kinase pUL97.

    Science.gov (United States)

    Milbradt, Jens; Sonntag, Eric; Wagner, Sabrina; Strojan, Hanife; Wangen, Christina; Lenac Rovis, Tihana; Lisnic, Berislav; Jonjic, Stipan; Sticht, Heinrich; Britt, William J; Schlötzer-Schrehardt, Ursula; Marschall, Manfred

    2018-01-13

    The nuclear phase of herpesvirus replication is regulated through the formation of regulatory multi-component protein complexes. Viral genomic replication is followed by nuclear capsid assembly, DNA encapsidation and nuclear egress. The latter has been studied intensely pointing to the formation of a viral core nuclear egress complex (NEC) that recruits a multimeric assembly of viral and cellular factors for the reorganization of the nuclear envelope. To date, the mechanism of the association of human cytomegalovirus (HCMV) capsids with the NEC, which in turn initiates the specific steps of nuclear capsid budding, remains undefined. Here, we provide electron microscopy-based data demonstrating the association of both nuclear capsids and NEC proteins at nuclear lamina budding sites. Specifically, immunogold labelling of the core NEC constituent pUL53 and NEC-associated viral kinase pUL97 suggested an intranuclear NEC-capsid interaction. Staining patterns with phospho-specific lamin A/C antibodies are compatible with earlier postulates of targeted capsid egress at lamina-depleted areas. Important data were provided by co-immunoprecipitation and in vitro kinase analyses using lysates from HCMV-infected cells, nuclear fractions, or infectious virions. Data strongly suggest that nuclear capsids interact with pUL53 and pUL97. Combined, the findings support a refined concept of HCMV nuclear trafficking and NEC-capsid interaction.

  12. Nuclear protein in testis carcinoma of the mediastinum: a case report.

    Science.gov (United States)

    Boleto, Gonçalo; Perotin, Jeanne-Marie; Launois, Claire; Uro-Coste, Emmanuelle; Birembaut, Philippe; Dury, Sandra; Vallerand, Hervé; Lebargy, François; Deslée, Gaëtan; Vella-Boucaud, Juliette

    2017-06-09

    Nuclear protein in testis carcinoma is a rare and very aggressive undifferentiated cancer which characteristically arises in the midline of the head, neck, and mediastinum. We describe the case of a 46-year-old white woman admitted for superior vena cava syndrome revealing a mediastinal tumor. Pathological examination of specimens obtained by mediastinoscopy revealed an undifferentiated tumor with solid growth and positive immunoreactivity for p40 and negative immunoreactivity for cytokeratin markers. Immunohistochemical staining was positive for nuclear protein in testis, allowing the diagnosis of nuclear protein in testis midline carcinoma of the mediastinum. We present a rare case of mediastinal nuclear protein in testis carcinoma with diagnosis based on nuclear protein in testis protein positivity and atypical immunohistochemical features including p40 positivity and anti-cytokeratin negativity. Physicians must remain aware of the possibility of nuclear protein in testis carcinoma especially in young patients with thoracic symptoms and suspicion of neoplasm.

  13. The F-box-containing protein UFO and AGAMOUS participate in antagonistic pathways governing early petal development in Arabidopsis.

    Science.gov (United States)

    Durfee, Tim; Roe, Judith L; Sessions, R Allen; Inouye, Carla; Serikawa, Kyle; Feldmann, Kenneth A; Weigel, Detlef; Zambryski, Patricia C

    2003-07-08

    The UNUSUAL FLORAL ORGANS (UFO) gene is required for multiple processes in the developing Arabidopsis flower, including the proper patterning and identity of both petals and stamens. The gene encodes an F-box-containing protein, UFO, which interacts physically and genetically with the Skp1 homolog, ASK1. In this report, we describe four ufo alleles characterized by the absence of petals, which uncover another role for UFO in promoting second whorl development. This UFO-dependent pathway is required regardless of the second whorl organ to be formed, arguing that it affects a basic process acting in parallel with those establishing organ identity. However, the pathway is dispensable in the absence of AGAMOUS (AG), a known inhibitor of petal development. In situ hybridization results argue that AG is not transcribed in the petal region, suggesting that it acts non-cell-autonomously to inhibit second whorl development in ufo mutants. These results are combined into a genetic model explaining early second whorl initiation/proliferation, in which UFO functions to inhibit an AG-dependent activity.

  14. Correlation between Serum Levels of High Mobility Group Box-1 Protein and Pancreatitis: A Meta-Analysis

    Directory of Open Access Journals (Sweden)

    Yan Lin

    2015-01-01

    Full Text Available Background. Aberrant expression of high mobility group box-1 protein (HMGB1 contributes to the progression of various inflammatory diseases. This meta-analysis focused on the clinical significance of serum HMGB1 levels in pancreatitis patients, with the goal of building a novel diagnostic score model. Method. We conducted a meta-analysis by searching in the PubMed, Embase, Web of Science, Cochrane Library, CISCOM, CINAHL, Google Scholar, China BioMedicine (CBM, and China National Knowledge Infrastructure (CNKI databases without any language restrictions. Studies were pooled and standard mean difference (SMD and its corresponding 95% confidence intervals (95% CIs were calculated. Version 12.0 STATA software was used for statistical analysis. Results. We performed a final analysis of 841 subjects from 12 clinical case-control studies. The meta-analysis results showed a positive association between serum HMGB1 levels and the progression of pancreatitis. In the subgroup analysis by country, high serum level of HMGB1 may be related to pancreatitis progression in China, Korea, Hungary, and Japan populations (all P<0.05. Conclusion. The present meta-analysis indicated that serum HMGB1 level was statistically elevated in patients with pancreatitis, and thus serum levels of HMGB1 could be determined to be a useful biomarker for pancreatitis patients.

  15. Cellular localization of Y-box binding protein 1 in brain tissue of rats, macaques, and humans

    Directory of Open Access Journals (Sweden)

    Horn Anja

    2009-03-01

    Full Text Available Abstract Background The Y-box binding protein 1 (YB-1 is considered to be one of the key regulators of transcription and translation. However, so far only limited knowledge exists regarding its cellular distribution in the adult brain. Results Analysis of YB-1 immunolabelling as well as double-labelling with the neuronal marker NeuN in rat brain tissue revealed a predominant neuronal expression in the dentate gyrus, the cornu ammonis pyramidal cell layer, layer III of the piriform cortex as well as throughout all layers of the parahippocampal cortex. In the hilus of the hippocampus single neurons expressed YB-1. The neuronal expression pattern was comparable in the hippocampus and parahippocampal cortex of adult macaques and humans. Double-labelling of YB-1 with the endothelial cell marker Glut-1, the multidrug transporter P-glycoprotein, and the astrocytic marker GFAP did not indicate a co-localization. Following status epilepticus in rats, no induction of YB-1 occurred in brain capillary endothelial cells and neurons. Conclusion In conclusion, our study demonstrates that YB-1 is predominantly expressed in neurons in the adult brain of rats, macaques and humans. Lack of a co-localization with Glut-1 and P-glycoprotein argues against a direct role of YB-1 in the regulation of blood-brain barrier P-glycoprotein.

  16. T-bet regulates differentiation of forkhead box protein 3+ regulatory T cells in programmed cell death-1-deficient mice

    Science.gov (United States)

    Tahara, M; Kondo, Y; Yokosawa, M; Tsuboi, H; Takahashi, S; Shibayama, S; Matsumoto, I; Sumida, T

    2015-01-01

    Programmed cell death-1 (PD-1) plays an important role in peripheral T cell tolerance, but whether or not it affects the differentiation of helper T cell subsets remains elusive. Here we describe the importance of PD-1 in the control of T helper type 1 (Th1) cell activation and development of forkhead box protein 3 (FoxP3+) regulatory T cells (Tregs). PD-1-deficient T cell-specific T-bet transgenic (P/T) mice showed growth retardation, and the majority died within 10 weeks. P/T mice showed T-bet over-expression, increased interferon (IFN)-γ production by CD4+ T cells and significantly low FoxP3+ Treg cell percentage. P/T mice developed systemic inflammation, which was probably induced by augmented Th1 response and low FoxP3+ Treg count. The study identified a unique, previously undescribed role for PD-1 in Th1 and Treg differentiation, with potential implication in the development of Th1 cell-targeted therapy. PMID:25219397

  17. Circulating pathogen-associated molecular pattern - binding proteins and High Mobility Group Box protein 1 in nascent metabolic syndrome: implications for cellular Toll-like receptor activity.

    Science.gov (United States)

    Jialal, I; Rajamani, U; Adams-Huet, B; Kaur, H

    2014-09-01

    The Metabolic Syndrome, (MetS) a global epidemic, is a state of low grade chronic inflammation and confers an increased risk for diabetes and CVD. We have previously reported increased activity of the pathogen recognition receptors, Toll-like receptors (TLRs), TLR2 and TLR4 in MetS. We hypothesized that increased TLR activity in MetS is due in part to increased levels of circulating PAMP-binding proteins, soluble CD14 (sCD14), lipopolysaccharide binding protein (LBP) and the damage associated molecular pattern (DAMP), High Mobility Group Box protein 1 (HMGB-1). We measured sCD14, LBP and HMGB-1 in fasting plasma from nascent MetS (n = 37) and healthy control subjects (n = 32) by ELISA. We also investigated the effects of sCD14 and LBP on TLR4 activity in human aortic endothelial cells (HAECs). Following adjustment for body mass index and waist circumference, sCD14, LBP and HMGB-1 levels remained significantly increased in MetS. Also their levels increased with increasing numbers of MetS risk factors. Only sCD14 correlated significantly with monocyte TLR4 protein and activity. None of these soluble biomarkers correlated with TLR2 protein. Both sCD14 and HMGB-1 correlated significantly with HOMA-IR. In LPS primed HAECs, sCD14 compared to LBP, resulted in a greater increase in both TLR4 abundance and inflammatory biomediators (NF-κB, IL-1β, IL-8 and TNF-α). Thus, we make the novel observation that sCD14 reflects increased monocyte TLR4 protein and activity in nascent MetS and by contributing to increased cellular inflammation could explain, in part, the increased risk for diabetes and CVD. Published by Elsevier Ireland Ltd.

  18. Identification of a bipartite nuclear localization signal in the silkworm Masc protein.

    Science.gov (United States)

    Sugano, Yudai; Kokusho, Ryuhei; Ueda, Masamichi; Fujimoto, Masaru; Tsutsumi, Nobuhiro; Shimada, Toru; Kiuchi, Takashi; Katsuma, Susumu

    2016-07-01

    The silkworm Masculinizer (Masc) gene encodes a CCCH-tandem zinc finger protein that controls both masculinization and dosage compensation. Masc protein is a nuclear protein, but the mechanism underlying the transport of this protein into the nucleus has not yet been elucidated. Here, we identified a functional bipartite nuclear localization signal (NLS) located between residues 274 and 290 of the Masc protein. Sequence comparison revealed that this bipartite NLS is evolutionarily conserved in Masc proteins from other lepidopteran insects. Furthermore, we showed that the degree of nuclear localization is not associated with the masculinizing activity of the Masc protein. © 2016 Federation of European Biochemical Societies.

  19. Mechanism of nuclear calcium signaling by inositol 1,4,5-trisphosphate produced in the nucleus, nuclear located protein kinase C and cyclic AMP-dependent protein kinase.

    Science.gov (United States)

    Klein, Christian; Malviya, Anant N

    2008-01-01

    Nuclear phospholipase C-gamma 1 can be phosphorylated by nuclear membrane located epidermal growth factor receptor sequel to epidermal growth factor-mediated signaling to the nucleus. The function of mouse liver phospholipase C-gamma 1 is attributed to a 120 kDa protein fragment which has been found to be a proteolytic product of the 150 kDa native nuclear enzyme. The tyrosine-phosphorylated 120 kDa protein band interacts with activated EGFR, binds phosphatidyl-3-OH kinase enhancer, and activates nuclear phosphatidylinositol-3-OH-kinase, and is capable of generating diacylglycerol in response to the epidermal growth factor signal to the nucleus in vivo. Thus a mechanism for nuclear production of inositol-1,4,5-trisphophate is unraveled. Nuclear generated inositol-1,4,5-trisphophate interacts with the inner membrane located inositol-1,4,5-trisphophate receptor and sequesters calcium into the nucleoplasm. Nuclear inositol-1,4,5-trisphophate receptor is phosphorylated by native nuclear protein kinase C which enhances the receptor-ligand interaction. Nuclear calcium-ATPase and inositol-1,3,4,5-tetrakisphophate receptor are located on the outer nuclear membrane, thus facilitating calcium transport into the nuclear envelope lumen either by ATP or inositol-1,3,4,5-tetrakisphophate depending upon the external free calcium concentrations. Nuclear calcium ATPase is phosphorylated by cyclic AMP-dependent protein kinase with enhanced calcium pumping activity. A holistic picture emerges here where tyrosine phosphorylation compliments serine phosphorylation of key moieties regulating nuclear calcium signaling. Evidence are forwarded in favor of proteolysis having a profound implications in nuclear calcium homeostasis in particular and signal transduction in general.

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

    Energy Technology Data Exchange (ETDEWEB)

    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.

  1. Dendritic cell nuclear protein-1, a novel depression-related protein, upregulates corticotropin-releasing hormone expression

    NARCIS (Netherlands)

    Zhou, Tian; Wang, Shanshan; Ren, Haigang; Qi, Xin-Rui; Luchetti, Sabina; Kamphuis, Willem; Zhou, Jiang-Ning; Wang, Guanghui; Swaab, Dick F.

    2010-01-01

    The recently discovered dendritic cell nuclear protein-1 is the product of a novel candidate gene for major depression. The A allele encodes full-length dendritic cell nuclear protein-1, while the T allele encodes a premature termination of translation at codon number 117 on chromosome 5. In the

  2. Serum high mobility group box 1 protein levels are not associated with either histological severity or treatment response in children and adults with nonalcoholic fatty liver disease

    OpenAIRE

    Yates, Katherine P.; Ross Deppe; Megan Comerford; Howard Masuoka; Cummings, Oscar W.; James Tonascia; Naga Chalasani; Raj Vuppalanchi

    2017-01-01

    Aim Serum high mobility group box 1 protein (HMGB1) is a proinflammatory molecule that could potentially serve as a biomarker for non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH) due to its correlation with degree of liver fibrosis. The aim of the current study was to examine the cross-sectional and longitudinal relationships between serum HMGB1 levels and liver histology in adults and children with NAFLD participating in two large randomized controlled trial...

  3. Porphyromonas gingivalis attenuates the insulin-induced phosphorylation and translocation of forkhead box protein O1 in human hepatocytes.

    Science.gov (United States)

    Takamura, Haruna; Yoshida, Kaya; Okamura, Hirohiko; Fujiwara, Natsumi; Ozaki, Kazumi

    2016-09-01

    Porphyromonas gingivalis (P. gingivalis) is a pathogen involved in periodontal disease. Recently, periodontal disease has been demonstrated to increase the risk of developing diabetes mellitus, although the molecular mechanism is not fully understood. Forkhead box protein O1 (FoxO1) is a transcriptional factor that regulates gluconeogenesis in the liver. Gluconeogenesis is a key process in the induction of diabetes mellitus; however, little is known regarding the relationship between periodontal disease and gluconeogenesis. In this study, to investigate whether periodontal disease influences hepatic gluconeogenesis, we examined the effects of P. gingivalis on the phosphorylation and translocation of FoxO1 in insulin-induced human hepatocytes. The human hepatocyte HepG2 was treated with insulin and Akt and FoxO1 phosphorylation was detected by western blot analysis. The localization of phosphorylated FoxO1 was detected by immunocytochemistry and western blot analysis. HepG2 cells were treated with SNAP26b-tagged P. gingivalis (SNAP-P.g.) before insulin stimulation, and then the changes in Akt and FoxO1 were determined by western blot analysis and immunocytochemistry. Insulin (100nM) induced FoxO1 phosphorylation 60min after treatment in HepG2 cells. Phosphorylated FoxO1 translocated to the cytoplasm. SNAP-P.g. internalized into HepG2 cells and decreased Akt and FoxO1 phosphorylation induced by insulin. The effect of insulin on FoxO1 translocation was also attenuated by SNAP-P.g. Our study shows that P. gingivalis decreases the phosphorylation and translocation of FoxO induced by insulin in HepG2 cells. Our results suggest that periodontal disease may increase hepatic gluconeogenesis by reducing the effects of insulin on FoxO1. Copyright © 2016 Elsevier Ltd. All rights reserved.

  4. Structure of the SPRY domain of the human RNA helicase DDX1, a putative interaction platform within a DEAD-box protein

    Energy Technology Data Exchange (ETDEWEB)

    Kellner, Julian N.; Meinhart, Anton, E-mail: anton.meinhart@mpimf-heidelberg.mpg.de [Max Planck Institute for Medical Research, Jahnstrasse 29, 69120 Heidelberg (Germany)

    2015-08-25

    The structure of the SPRY domain of the human RNA helicase DDX1 was determined at 2.0 Å resolution. The SPRY domain provides a putative protein–protein interaction platform within DDX1 that differs from other SPRY domains in its structure and conserved regions. The human RNA helicase DDX1 in the DEAD-box family plays an important role in RNA processing and has been associated with HIV-1 replication and tumour progression. Whereas previously described DEAD-box proteins have a structurally conserved core, DDX1 shows a unique structural feature: a large SPRY-domain insertion in its RecA-like consensus fold. SPRY domains are known to function as protein–protein interaction platforms. Here, the crystal structure of the SPRY domain of human DDX1 (hDSPRY) is reported at 2.0 Å resolution. The structure reveals two layers of concave, antiparallel β-sheets that stack onto each other and a third β-sheet beneath the β-sandwich. A comparison with SPRY-domain structures from other eukaryotic proteins showed that the general β-sandwich fold is conserved; however, differences were detected in the loop regions, which were identified in other SPRY domains to be essential for interaction with cognate partners. In contrast, in hDSPRY these loop regions are not strictly conserved across species. Interestingly, though, a conserved patch of positive surface charge is found that may replace the connecting loops as a protein–protein interaction surface. The data presented here comprise the first structural information on DDX1 and provide insights into the unique domain architecture of this DEAD-box protein. By providing the structure of a putative interaction domain of DDX1, this work will serve as a basis for further studies of the interaction network within the hetero-oligomeric complexes of DDX1 and of its recruitment to the HIV-1 Rev protein as a viral replication factor.

  5. The human mucin MUC4 is transcriptionally regulated by caudal-related homeobox, hepatocyte nuclear factors, forkhead box A, and GATA endodermal transcription factors in epithelial cancer cells.

    Science.gov (United States)

    Jonckheere, Nicolas; Vincent, Audrey; Perrais, Michaël; Ducourouble, Marie-Paule; Male, Anita Korteland-van; Aubert, Jean-Pierre; Pigny, Pascal; Carraway, Kermit L; Freund, Jean-Noël; Renes, Ingrid B; Van Seuningen, Isabelle

    2007-08-03

    The human gene MUC4 encodes a large transmembrane mucin that is developmentally regulated and expressed along the undifferentiated pseudostratified epithelium, as early as 6.5 weeks during fetal development. Immunohistochemical analysis of Muc4 expression in developing mouse lung and gastrointestinal tract showed a different spatio-temporal pattern of expression before and after cytodifferentiation. The molecular mechanisms governing MUC4 expression during development are, however, unknown. Hepatocyte nuclear factors (HNF), forkhead box A (FOXA), GATA, and caudal-related homeobox transcription factors (TFs) are known to control cell differentiation of gut endoderm derived-tissues during embryonic development. They also control the expression of cell- and tissue-specific genes and may thus control MUC4 expression. To test this hypothesis, we studied and deciphered the molecular mechanisms responsible for MUC4 transcriptional regulation by these TFs. Experiments using small interfering RNA, cell co-transfection, and site-directed mutagenesis indicated that MUC4 is regulated at the transcriptional level by CDX-1 and -2, HNF-1 alpha and -1 beta, FOXA1/A2, HNF-4 alpha and -4 gamma, and GATA-4, -5, and -6 factors in a cell-specific manner. Binding of TFs was assessed by chromatin immunoprecipitation, and gel-shift assays. Altogether, these results demonstrate that MUC4 is a target gene of endodermal TFs and thus point out an important role for these TFs in regulating MUC4 expression during epithelial differentiation during development, cancer, and repair.

  6. The transport of integral membrane proteins across the nuclear pore complex

    NARCIS (Netherlands)

    Meinema, Anne C.; Poolman, Bert; Veenhoff, Liesbeth M.

    2012-01-01

    The nuclear envelope protects and organizes the genome. The nuclear pore complexes embedded in the nuclear envelope allow selective transport of macromolecules between the cytosol and nucleoplasm, and as such help to control the flow of information from DNA to RNA to proteins. A growing list of

  7. Novel nuclear matrix protein HET binds to and influences activity of the HSP27 promoter in human breast cancer cells.

    Science.gov (United States)

    Oesterreich, S; Lee, A V; Sullivan, T M; Samuel, S K; Davie, J R; Fuqua, S A

    1997-11-01

    Since the small heat shock protein hsp27 enhances both growth and drug resistance in breast cancer cells, and is a bad prognostic factor in certain subsets of breast cancer patients, we have characterized the transcriptional regulation of hsp27, with the long-term goal of targeting its expression clinically. The majority of the promoter activity resides in the most proximal 200 bp. This region contains an imperfect estrogen response element (ERE) that is separated by a 13-bp spacer that contains a TATA box. Gel-shift analysis revealed the binding of a protein (termed HET for Hsp27-ERE-TATA-binding protein) to this region that was neither the estrogen receptor nor TATA-binding protein. We cloned a complete cDNA (2.9 kb) for HET from an MCF-7 cDNA library. To confirm the identity of the HET clone, we expressed a partial HET clone as a glutathione S-transferase fusion protein, and showed binding to the hsp27 promoter fragment in gel-retardation assays. The HET clone is almost identical to a recently published scaffold attachment factor (SAF-B) cloned from a HeLa cell cDNA library. Scaffold attachment factors are a subset of nuclear matrix proteins (NMP) that interact with matrix attachment regions. Analyzing how HET could act as a regulator of hsp27 transcription and as a SAF/NMP, we studied its subnuclear localization and its effect on hsp27 transcription in human breast cancer cells. We were able to show that HET is localized in the nuclear matrix in various breast cancer cell lines. Furthermore, in transient transfection assays using hsp27 promoter-luciferase reporter constructs, HET overexpression resulted in a dose-dependent decrease of hsp27 promoter activity in several cell lines.

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

    Science.gov (United States)

    Zhou, Xiao; Meier, Iris

    2014-08-12

    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.

  9. Fate of the inner nuclear membrane protein lamin B receptor and nuclear lamins in herpes simplex virus type 1 infection.

    Science.gov (United States)

    Scott, E S; O'Hare, P

    2001-09-01

    During herpesvirus egress, capsids bud through the inner nuclear membrane. Underlying this membrane is the nuclear lamina, a meshwork of intermediate filaments with which it is tightly associated. Details of alterations to the lamina and the inner nuclear membrane during infection and the mechanisms involved in capsid transport across these structures remain unclear. Here we describe the fate of key protein components of the nuclear envelope and lamina during herpes simplex virus type 1 (HSV-1) infection. We followed the distribution of the inner nuclear membrane protein lamin B receptor (LBR) and lamins A and B(2) tagged with green fluorescent protein (GFP) in live infected cells. Together with additional results from indirect immunofluorescence, our studies reveal major morphologic distortion of nuclear-rim LBR and lamins A/C, B(1), and B(2). By 8 h p.i., we also observed a significant redistribution of LBR-GFP to the endoplasmic reticulum, where it colocalized with a subpopulation of cytoplasmic glycoprotein B by immunofluorescence. In addition, analysis by fluorescence recovery after photobleaching reveals that LBR-GFP exhibited increased diffusional mobility within the nuclear membrane of infected cells. This is consistent with the disruption of interactions between LBR and the underlying lamina. In addition to studying stably expressed GFP-lamins by fluorescence microscopy, we studied endogenous A- and B-type lamins in infected cells by Western blotting. Both approaches reveal a loss of lamins associated with virus infection. These data indicate major disruption of the nuclear envelope and lamina of HSV-1-infected cells and are consistent with a virus-induced dismantling of the nuclear lamina, possibly in order to gain access to the inner nuclear membrane.

  10. Melanoma antigen gene protein-A11 (MAGE-11) F-box links the androgen receptor NH2-terminal transactivation domain to p160 coactivators.

    Science.gov (United States)

    Askew, Emily B; Bai, Suxia; Hnat, Andrew T; Minges, John T; Wilson, Elizabeth M

    2009-12-11

    Androgen-dependent transcriptional activity by the androgen receptor (AR) and its coregulators is required for male reproductive development and function. In humans and other primates, melanoma antigen gene protein-A11 (MAGE-11) is an AR selective coregulator that increases AR transcriptional activity. Here we show that the interaction between AR and MAGE-11 is mediated by AR NH(2)-terminal FXXLF motif binding to a highly conserved MAGE-11 F-box in the MAGE homology domain, and is modulated by serum stimulation of mitogen-activated protein kinase phosphorylation of MAGE-11 Ser-174. The MAGE-11-dependent increase in AR transcriptional activity is mediated by a direct interaction between MAGE-11 and transcriptional intermediary factor 2 (TIF2) through the NH(2)-terminal region of TIF2, and by a MAGE-11 FXXIF motif interaction with an F-box-like region in activation domain 1 of TIF2. The results suggest that MAGE-11 functions as a bridging factor to recruit AR coactivators through a novel FXX(L/I)F motif-F-box interaction paradigm.

  11. Melanoma Antigen Gene Protein-A11 (MAGE-11) F-box Links the Androgen Receptor NH2-terminal Transactivation Domain to p160 Coactivators*

    Science.gov (United States)

    Askew, Emily B.; Bai, Suxia; Hnat, Andrew T.; Minges, John T.; Wilson, Elizabeth M.

    2009-01-01

    Androgen-dependent transcriptional activity by the androgen receptor (AR) and its coregulators is required for male reproductive development and function. In humans and other primates, melanoma antigen gene protein-A11 (MAGE-11) is an AR selective coregulator that increases AR transcriptional activity. Here we show that the interaction between AR and MAGE-11 is mediated by AR NH2-terminal FXXLF motif binding to a highly conserved MAGE-11 F-box in the MAGE homology domain, and is modulated by serum stimulation of mitogen-activated protein kinase phosphorylation of MAGE-11 Ser-174. The MAGE-11-dependent increase in AR transcriptional activity is mediated by a direct interaction between MAGE-11 and transcriptional intermediary factor 2 (TIF2) through the NH2-terminal region of TIF2, and by a MAGE-11 FXXIF motif interaction with an F-box-like region in activation domain 1 of TIF2. The results suggest that MAGE-11 functions as a bridging factor to recruit AR coactivators through a novel FXX(L/I)F motif-F-box interaction paradigm. PMID:19828458

  12. Quantitative Analysis of Membrane Protein Transport Across the Nuclear Pore Complex

    NARCIS (Netherlands)

    Meinema, Anne C.; Poolman, Bert; Veenhoff, Liesbeth M.

    Nuclear transport of the Saccharomyces cerevisiae membrane proteins Src1/Heh1 and Heh2 across the NPC is facilitated by a long intrinsically disordered linker between the nuclear localization signal (NLS) and the transmembrane domain. The import of reporter proteins derived from Heh2 is dependent on

  13. Early localization of NPA58, a rat nuclear pore-associated protein, to ...

    Indian Academy of Sciences (India)

    The targeting of NPA58 to the reforming nuclear envelope in early telophase coincides with the recruitment of a well-characterized class of nuclear pore proteins recognized by the antibody mAb 414, and occurs prior to the incorporation of lamin B1 into the envelope. Significant protein import activity is detectable only after ...

  14. Banana Ovate family protein MaOFP1 and MADS-box protein MuMADS1 antagonistically regulated banana fruit ripening.

    Directory of Open Access Journals (Sweden)

    Juhua Liu

    Full Text Available The ovate family protein named MaOFP1 was identified in banana (Musa acuminata L.AAA fruit by a yeast two-hybrid (Y2H method using the banana MADS-box gene MuMADS1 as bait and a 2 day postharvest (DPH banana fruit cDNA library as prey. The interaction between MuMADS1 and MaOFP1 was further confirmed by Y2H and Bimolecular Fluorescence Complementation (BiFC methods, which showed that the MuMADS1 K domain interacted with MaOFP1. Real-time quantitative PCR evaluation of MuMADS1 and MaOFP1 expression patterns in banana showed that they are highly expressed in 0 DPH fruit, but present in low levels in the stem, which suggests that simultaneous but different expression patterns exist for both MuMADS1 and MaOFP1 in different tissues and developing fruits. Meanwhile, MuMADS1 and MaOFP1 expression was highly stimulated and greatly suppressed, respectively, by exogenous ethylene. In contrast, MaOFP1 expression was highly stimulated while MuMADS1 was greatly suppressed by the ethylene competitor 1-methylcyclopropene (1-MCP. These results indicate that MuMADS1 and MaOFP1 are antagonistically regulated by ethylene and might play important roles in postharvest banana fruit ripening.

  15. Characterization of a nuclear pore protein sheds light on the roles and composition of the Toxoplasma gondii nuclear pore complex.

    Science.gov (United States)

    Courjol, Flavie; Mouveaux, Thomas; Lesage, Kevin; Saliou, Jean-Michel; Werkmeister, Elisabeth; Bonabaud, Maurine; Rohmer, Marine; Slomianny, Christian; Lafont, Franck; Gissot, Mathieu

    2017-06-01

    The nuclear pore is a key structure in eukaryotes regulating nuclear-cytoplasmic transport as well as a wide range of cellular processes. Here, we report the characterization of the first Toxoplasma gondii nuclear pore protein, named TgNup302, which appears to be the orthologue of the mammalian Nup98-96 protein. We produced a conditional knock-down mutant that expresses TgNup302 under the control of an inducible tetracycline-regulated promoter. Under ATc treatment, a substantial decrease of TgNup302 protein in inducible knock-down (iKD) parasites was observed, causing a delay in parasite proliferation. Moreover, the nuclear protein TgENO2 was trapped in the cytoplasm of ATc-treated mutants, suggesting that TgNup302 is involved in nuclear transport. Fluorescence in situ hybridization revealed that TgNup302 is essential for 18S RNA export from the nucleus to the cytoplasm, while global mRNA export remains unchanged. Using an affinity tag purification combined with mass spectrometry, we identified additional components of the nuclear pore complex, including proteins potentially interacting with chromatin. Furthermore, reverse immunoprecipitation confirmed their interaction with TgNup302, and structured illuminated microscopy confirmed the NPC localization of some of the TgNup302-interacting proteins. Intriguingly, facilitates chromatin transcription complex (FACT) components were identified, suggesting the existence of an NPC-chromatin interaction in T. gondii. Identification of TgNup302-interacting proteins also provides the first glimpse at the NPC structure in Apicomplexa, suggesting a structural conservation of the NPC components between distant eukaryotes.

  16. The nuclear import of ribosomal proteins is regulated by mTOR

    Science.gov (United States)

    Kazyken, Dubek; Kaz, Yelimbek; Kiyan, Vladimir; Zhylkibayev, Assylbek A.; Chen, Chien-Hung; Agarwal, Nitin K.; Sarbassov, Dos D.

    2014-01-01

    Mechanistic target of rapamycin (mTOR) is a central component of the essential signaling pathway that regulates cell growth and proliferation by controlling anabolic processes in cells. mTOR exists in two distinct mTOR complexes known as mTORC1 and mTORC2 that reside mostly in cytoplasm. In our study, the biochemical characterization of mTOR led to discovery of its novel localization on nuclear envelope where it associates with a critical regulator of nuclear import Ran Binding Protein 2 (RanBP2). We show that association of mTOR with RanBP2 is dependent on the mTOR kinase activity that regulates the nuclear import of ribosomal proteins. The mTOR kinase inhibitors within thirty minutes caused a substantial decrease of ribosomal proteins in the nuclear but not cytoplasmic fraction. Detection of a nuclear accumulation of the GFP-tagged ribosomal protein rpL7a also indicated its dependence on the mTOR kinase activity. The nuclear abundance of ribosomal proteins was not affected by inhibition of mTOR Complex 1 (mTORC1) by rapamycin or deficiency of mTORC2, suggesting a distinctive role of the nuclear envelope mTOR complex in the nuclear import. Thus, we identified that mTOR in association with RanBP2 mediates the active nuclear import of ribosomal proteins. PMID:25294810

  17. Effect of the linkers between the zinc fingers in zinc finger protein 809 on gene silencing and nuclear localization

    Energy Technology Data Exchange (ETDEWEB)

    Ichida, Yu, E-mail: ichida-y@ncchd.go.jp; Utsunomiya, Yuko; Onodera, Masafumi

    2016-03-18

    Zinc finger protein 809 (ZFP809) belongs to the Kruppel-associated box-containing zinc finger protein (KRAB-ZFP) family and functions in repressing the expression of Moloney murine leukemia virus (MoMLV). ZFP809 binds to the primer-binding site (PBS)located downstream of the MoMLV-long terminal repeat (LTR) and induces epigenetic modifications at integration sites, such as repressive histone modifications and de novo DNA methylation. KRAB-ZFPs contain consensus TGEKP linkers between C2H2 zinc fingers. The phosphorylation of threonine residues within linkers leads to the inactivation of zinc finger binding to target sequences. ZFP809 also contains consensus linkers between zinc fingers. However, the function of ZFP809 linkers remains unknown. In the present study, we constructed ZFP809 proteins containing mutated linkers and examined their ability to silence transgene expression driven by MLV, binding ability to MLV PBS, and cellular localization. The results of the present study revealed that the linkers affected the ability of ZFP809 to silence transgene expression. Furthermore, this effect could be partly attributed to changes in the localization of ZFP809 proteins containing mutated linkers. Further characterization of ZFP809 linkers is required for understanding the functions and features of KRAB-ZFP-containing linkers. - Highlights: • ZFP809 has three consensus linkers between the zinc fingers. • Linkers are required for ZFP809 to silence transgene expression driven by MLV-LTR. • Linkers affect the precise nuclear localization of ZFP809.

  18. Epstein-Barr virus nuclear antigen 1 (EBNA1) protein induction of epithelial-mesenchymal transition in nasopharyngeal carcinoma cells.

    Science.gov (United States)

    Wang, Lu; Tian, Wen-Dong; Xu, Xia; Nie, Biao; Lu, Juan; Liu, Xiong; Zhang, Bao; Dong, Qi; Sunwoo, John B; Li, Gang; Li, Xiang-Ping

    2014-02-01

    The Epstein-Barr virus (EBV)-encoded EB nuclear antigen 1 (EBNA1) protein is required for maintenance and transmission of the viral episome in EBV-infected cells. The objective of this study was to investigate the role of EBNA1 protein in nasopharyngeal carcinoma (NPC). Tissue samples from 48 patients with NPC and 12 patients with chronic nasopharyngitis were subjected to immunohistochemical analysis of EBNA1 expression. EBNA1 combinational DNA was used to overexpress EBNA1 protein in NPC cell lines to assess tumor cell epithelial-mesenchymal transition (EMT), colony formation, migration and invasion, and gene expression. EBNA1 protein was highly expressed in NPC tissue specimens, and its expression was associated with NPC lymph node metastasis. EBNA1 expression affected NPC cell morphology and the expression of EMT markers in vitro. Furthermore, overexpression of EBNA1 inhibited the expression of microRNA 200a (miR-200a) and miR-200b and, in turn, up-regulated expression of their target genes, zinc finger E-box binding homeobox 1 ( ZEB1) and ZEB2, which are well known mediators of EMT. In addition, EBNA1-regulated miR-200a and miR-200b expression was mediated by transforming growth factor-β1. The current findings provided novel insight into the vital role of EBNA1 in manipulating a molecular switch of EMT in EBV-positive NPC cells. © 2013 American Cancer Society.

  19. The inner nuclear membrane protein lamin B receptor forms distinct microdomains and links epigenetically marked chromatin to the nuclear envelope.

    Science.gov (United States)

    Makatsori, Dimitra; Kourmouli, Niki; Polioudaki, Hara; Shultz, Leonard D; McLean, Kelvin; Theodoropoulos, Panayiotis A; Singh, Prim B; Georgatos, Spyros D

    2004-06-11

    Using heterochromatin-enriched fractions, we have detected specific binding of mononucleosomes to the N-terminal domain of the inner nuclear membrane protein lamin B receptor. Mass spectrometric analysis reveals that LBR-associated particles contain complex patterns of methylated/acetylated histones and are devoid of "euchromatic" epigenetic marks. LBR binds heterochromatin as a higher oligomer and forms distinct nuclear envelope microdomains in vivo. The organization of these membrane assemblies is affected significantly in heterozygous ic (ichthyosis) mutants, resulting in a variety of structural abnormalities and nuclear defects.

  20. RNAa Induced by TATA Box-Targeting MicroRNAs.

    Science.gov (United States)

    Zhang, Yijun; Zhang, Hui

    2017-01-01

    Recent studies reveal that some nuclear microRNAs (miRNA) and synthesized siRNAs target gene promoters to activate gene transcription (RNAa). Interestingly, our group identified a novel HIV-1-encoded miRNA, miR-H3, which targets specifically the core promoter TATA box of HIV-1 and activates viral gene expression. Depletion of miR-H3 significantly impaired the replication of HIV-1. miR-H3 mimics could activate viruses from CD4(+) T cells isolated from patients receiving suppressive highly active antiretroviral therapy, which is very intriguing for reducing HIV-1 latent reservoir. Further study revealed that many cellular miRNAs also function like miR-H3. For instance, let-7i targets the TATA box of the interleukin-2 (IL-2) promoter and upregulates IL-2 expression in T-lymphocytes. In RNAa induced by TATA box-targeting miRNAs, Argonaute (AGO) proteins are needed, but there is no evidence for the involvement of promoter-associated transcripts or epigenetic modifications. We propose that the binding of small RNA-AGO complex to TATA box could facilitate the assembly of RNA Polymerase II transcription preinitiation complex. In addition, synthesized small RNAs targeting TATA box can also efficiently activate transcription of interested genes, such as insulin, IL-2, and c-Myc. The discovery of RNAa induced by TATA box-targeting miRNA provides an easy-to-use tool for activating gene expression.

  1. Understanding renal nuclear protein accumulation: an in vitro approach to explain an in vivo phenomenon.

    Science.gov (United States)

    Luks, Lisanne; Maier, Marcia Y; Sacchi, Silvia; Pollegioni, Loredano; Dietrich, Daniel R

    2017-11-01

    Proper subcellular trafficking is essential to prevent protein mislocalization and aggregation. Transport of the peroxisomal enzyme D-amino acid oxidase (DAAO) appears dysregulated by specific pharmaceuticals, e.g., the anti-overactive bladder drug propiverine or a norepinephrine/serotonin reuptake inhibitor (NSRI), resulting in massive cytosolic and nuclear accumulations in rat kidney. To assess the underlying molecular mechanism of the latter, we aimed to characterize the nature of peroxisomal and cyto-nuclear shuttling of human and rat DAAO overexpressed in three cell lines using confocal microscopy. Indeed, interference with peroxisomal transport via deletion of the PTS1 signal or PEX5 knockdown resulted in induced nuclear DAAO localization. Having demonstrated the absence of active nuclear import and employing variably sized mCherry- and/or EYFP-fusion proteins of DAAO and catalase, we showed that peroxisomal proteins ≤134 kDa can passively diffuse into mammalian cell nuclei-thereby contradicting the often-cited 40 kDa diffusion limit. Moreover, their inherent nuclear presence and nuclear accumulation subsequent to proteasome inhibition or abrogated peroxisomal transport suggests that nuclear localization is a characteristic in the lifecycle of peroxisomal proteins. Based on this molecular trafficking analysis, we suggest that pharmaceuticals like propiverine or an NSRI may interfere with peroxisomal protein targeting and import, consequently resulting in massive nuclear protein accumulation in vivo.

  2. Exploring the evolution of the proteins of the plant nuclear envelope.

    Science.gov (United States)

    Poulet, Axel; Probst, Aline V; Graumann, Katja; Tatout, Christophe; Evans, David

    2017-01-02

    In this study, we explore the plasticity during evolution of proteins of the higher plant nuclear envelope (NE) from the most ancestral plant species to advanced angiosperms. The higher plant NE contains a functional Linker of Nucleoskeleton and Cytoskeleton (LINC) complex based on conserved Sad1-Unc84 (SUN) domain proteins and plant specific Klarsicht/Anc1/Syne homology (KASH) domain proteins. Recent evidence suggests the presence of a plant lamina underneath the inner membrane and various coiled-coil proteins have been hypothesized to be associated with it including Crowded Nuclei (CRWN; also termed LINC and NMCP), Nuclear Envelope Associated Protein (NEAP) protein families as well as the CRWN binding protein KAKU4. SUN domain proteins appear throughout with a key role for mid-SUN proteins suggested. Evolution of KASH domain proteins has resulted in increasing complexity, with some appearing in all species considered, while other KASH proteins are progressively gained during evolution. Failure to identify CRWN homologs in unicellular organisms included in the study and their presence in plants leads us to speculate that convergent evolution may have occurred in the formation of the lamina with each kingdom having new proteins such as the Lamin B receptor (LBR) and Lamin-Emerin-Man1 (LEM) domain proteins (animals) or NEAPs and KAKU4 (plants). Our data support a model in which increasing complexity at the nuclear envelope occurred through the plant lineage and suggest a key role for mid-SUN proteins as an early and essential component of the nuclear envelope.

  3. UNcleProt (Universal Nuclear Protein database of barley): The first nuclear protein database that distinguishes proteins from different phases of the cell cycle

    Czech Academy of Sciences Publication Activity Database

    Blavet, Nicolas; Uřinovská, J.; Jeřábková, Hana; Chamrád, I.; Vrána, Jan; Lenobel, R.; Beinhauer, D.; Šebela, M.; Doležel, Jaroslav; Petrovská, Beáta

    2017-01-01

    Roč. 8, č. 1 (2017), s. 70-80 ISSN 1949-1034 R&D Projects: GA ČR(CZ) GA14-28443S; GA MŠk(CZ) LO1204 Institutional support: RVO:61389030 Keywords : cicer-arietinum l. * rice oryza-sativa * chromatin-associated proteins * proteomic analysis * mitotic chromosomes * dehydration * localization * chickpea * network * phosphoproteome * barley * cell cycle * database * flow-cytometry * localization * mass spectrometry * nuclear proteome * nucleus Subject RIV: CE - Biochemistry Impact factor: 2.387, year: 2016

  4. Firing the sting: chemically induced discharge of cnidae reveals novel proteins and peptides from box jellyfish (Chironex fleckeri) venom.

    Science.gov (United States)

    Jouiaei, Mahdokht; Casewell, Nicholas R; Yanagihara, Angel A; Nouwens, Amanda; Cribb, Bronwen W; Whitehead, Darryl; Jackson, Timothy N W; Ali, Syed A; Wagstaff, Simon C; Koludarov, Ivan; Alewood, Paul; Hansen, Jay; Fry, Bryan G

    2015-03-18

    Cnidarian venom research has lagged behind other toxinological fields due to technical difficulties in recovery of the complex venom from the microscopic nematocysts. Here we report a newly developed rapid, repeatable and cost effective technique of venom preparation, using ethanol to induce nematocyst discharge and to recover venom contents in one step. Our model species was the Australian box jellyfish (Chironex fleckeri), which has a notable impact on public health. By utilizing scanning electron microscopy and light microscopy, we examined nematocyst external morphology before and after ethanol treatment and verified nematocyst discharge. Further, to investigate nematocyst content or "venom" recovery, we utilized both top-down and bottom-up transcriptomics-proteomics approaches and compared the proteome profile of this new ethanol recovery based method to a previously reported high activity and recovery protocol, based upon density purified intact cnidae and pressure induced disruption. In addition to recovering previously characterized box jellyfish toxins, including CfTX-A/B and CfTX-1, we recovered putative metalloproteases and novel expression of a small serine protease inhibitor. This study not only reveals a much more complex toxin profile of Australian box jellyfish venom but also suggests that ethanol extraction method could augment future cnidarian venom proteomics research efforts.

  5. Firing the Sting: Chemically Induced Discharge of Cnidae Reveals Novel Proteins and Peptides from Box Jellyfish (Chironex fleckeri Venom

    Directory of Open Access Journals (Sweden)

    Mahdokht Jouiaei

    2015-03-01

    Full Text Available Cnidarian venom research has lagged behind other toxinological fields due to technical difficulties in recovery of the complex venom from the microscopic nematocysts. Here we report a newly developed rapid, repeatable and cost effective technique of venom preparation, using ethanol to induce nematocyst discharge and to recover venom contents in one step. Our model species was the Australian box jellyfish (Chironex fleckeri, which has a notable impact on public health. By utilizing scanning electron microscopy and light microscopy, we examined nematocyst external morphology before and after ethanol treatment and verified nematocyst discharge. Further, to investigate nematocyst content or “venom” recovery, we utilized both top-down and bottom-up transcriptomics–proteomics approaches and compared the proteome profile of this new ethanol recovery based method to a previously reported high activity and recovery protocol, based upon density purified intact cnidae and pressure induced disruption. In addition to recovering previously characterized box jellyfish toxins, including CfTX-A/B and CfTX-1, we recovered putative metalloproteases and novel expression of a small serine protease inhibitor. This study not only reveals a much more complex toxin profile of Australian box jellyfish venom but also suggests that ethanol extraction method could augment future cnidarian venom proteomics research efforts.

  6. A Role for the F-Box Protein HAWAIIAN SKIRT in Plant microRNA Function1[OPEN

    Science.gov (United States)

    Christie, Michael D.; Dogan, Ezgi S.; Schwab, Rebecca; Hagmann, Jörg; van de Weyer, Anna-Lena; Scacchi, Emanuele

    2018-01-01

    As regulators of gene expression in multicellular organisms, microRNAs (miRNAs) are crucial for growth and development. Although a plethora of factors involved in their biogenesis and action in Arabidopsis (Arabidopsis thaliana) has been described, these processes and their fine-tuning are not fully understood. Here, we used plants expressing an artificial miRNA target mimic (MIM) to screen for negative regulators of miR156. We identified a new mutant allele of the F-box gene HAWAIIAN SKIRT (HWS; At3G61590), hws-5, as a suppressor of the MIM156-induced developmental and molecular phenotypes. In hws plants, levels of some endogenous miRNAs are increased and their mRNA targets decreased. Plants constitutively expressing full-length HWS—but not a truncated version lacking the F-box domain—display morphological and molecular phenotypes resembling those of mutants defective in miRNA biogenesis and activity. In combination with such mutants, hws loses its delayed floral organ abscission (“skirt”) phenotype, suggesting epistasis. Also, the hws transcriptome profile partially resembles those of well-known miRNA mutants hyl1-2, se-3, and ago1-27, pointing to a role in a common pathway. We thus propose HWS as a novel, F-box dependent factor involved in miRNA function. PMID:29114080

  7. The nuclear IκB family of proteins controls gene regulation and immune homeostasis.

    Science.gov (United States)

    MaruYama, Takashi

    2015-10-01

    The inhibitory IκB family of proteins is subdivided into two groups based on protein localization in the cytoplasm or in the nucleus. These proteins interact with NF-κB, a major transcription factor regulating the expression of many inflammatory cytokines, by modulating its transcriptional activity. However, nuclear IκB family proteins not only interact with NF-κB to change its transcriptional activity, but they also bind to chromatin and control gene expression. This review provides an overview of nuclear IκB family proteins and their role in immune homeostasis. Copyright © 2015 Elsevier B.V. All rights reserved.

  8. Structure and expression of MdFBCP1, encoding an F-box-containing protein 1, during Fuji apple (Malus domestica Borkh.) fruit ripening.

    Science.gov (United States)

    Han, Sang Eun; Seo, Young Sam; Heo, Seong; Kim, Daeil; Sung, Soon-Kee; Kim, Woo Taek

    2008-08-01

    From database comparisons of 1,117 expressed sequence tags (ESTs) generated from ripened Fuji apple fruits, we identified ten ubiquitin (Ub)-related genes. RNA gel-blot analysis suggests that these Ub-related genes are induced by at least four distinct signaling pathways in fruits. In this study, we analyzed structure and expression of MdFBCP1, encoding an F-box-containing protein 1, in Fuji apples. MdFBCP1 transcript was predominantly expressed in the fully ripened climacteric fruits, in which serge of ethylene production occurred. The MdFBCP1 gene was also activated effectively in response to exogenous ethylene treatment, with the induction pattern being comparable to those of ACC oxidase and beta-cyanoalanine synthase. Thus, it seems likely that the expression of MdFBCP1 is closely associated with a climacteric ethylene production and ACC oxidase activity and, hence, MdFBCP1 may play a role in the ripening process of Fuji apple fruits. Yeast two hybrid and in vitro pull-down assays revealed that MdFBCP1 physically interacted with MdSkp1 and N-terminal F-box motif was essential for this interaction. These results suggest that MdFBCP1 indeed functions as an F-box-containing protein and participates in the formation of SCF complex, which acts as E3 Ub ligase. Genomic Southern blot analysis showed that MdFBCP1 exhibited different pattern of restriction enzyme digestion in three cultivars (Tsugaru, Golden Delicious and Fuji) that produce different amount of ethylene, suggesting that the MdFBCP1 gene is organized in a cultivar specific manner. Collectively, our data suggest that Ub degradation pathway may play an important role in the ripening of Fuji apple fruits.

  9. Analysis of nuclear export using photoactivatable GFP fusion proteins and interspecies heterokaryons.

    Science.gov (United States)

    Nakrieko, Kerry-Ann; Ivanova, Iordanka A; Dagnino, Lina

    2010-01-01

    In this chapter, we review protocols for the analysis of nucleocytoplasmic shuttling of transcription factors and nuclear proteins, using two different approaches. The first involves the use of photoactivatable forms of the protein of interest by fusion to photoactivatable green fluorescent protein to follow its movement out of the nucleus by live-cell confocal microscopy. This methodology allows for the kinetic characterization of protein movements as well as measurement of steady-state levels. In a second procedure to assess the ability of a nuclear protein to move into and out of the nucleus, we describe the use of interspecies heterokaryon assays, which provide a measurement of steady-state distribution. These technologies are directly applicable to the analysis of nucleocytoplasmic movements not only of transcription factors, but also other nuclear proteins.

  10. An L1 box binding protein, GbML1, interacts with GbMYB25 to control cotton fibre development.

    Science.gov (United States)

    Zhang, Fei; Zuo, Kaijing; Zhang, Jieqiong; Liu, Xiang; Zhang, Lida; Sun, Xiaofen; Tang, Kexuan

    2010-08-01

    Transcription factors play key roles in plant development through their interaction with cis-elements and/or other transcription factors. A HD-Zip IV family transcription factor, Gossypium barbadense Meristem Layer 1 (GbML1) has been identified and characterized here. GbML1 specifically bound to the L1 box and the promoters of GbML1 and GbRDL1. GbML1 physically interacted with a key regulator of cotton fibre development, GbMYB25. Truncated and point mutation assays indicated the START-SAD domain was required for the binding to the C terminal domain (CTD) of GbMYB25. GbML1 overexpression in Arabidopsis increased the number of trichomes on stems and leaves and increased the accumulation of anthocyanin in leaves. Taken together, the L1 box binding protein, GbML1 was identified as the first partner for GbMYB25 and the role of START domain was discovered to be a protein binding domain in plants. Our findings will help the improvement of cotton fibre production and the understanding of the key role of HD-Zip family and MYB family in plants.

  11. ANP32B is a nuclear target of henipavirus M proteins.

    Directory of Open Access Journals (Sweden)

    Anja Bauer

    Full Text Available Membrane envelopment and budding of negative strand RNA viruses (NSVs is mainly driven by viral matrix proteins (M. In addition, several M proteins are also known to be involved in host cell manipulation. Knowledge about the cellular targets and detailed molecular mechanisms, however, is poor for many M proteins. For instance, Nipah Virus (NiV M protein trafficking through the nucleus is essential for virus release, but nuclear targets of NiV M remain unknown. To identify cellular interactors of henipavirus M proteins, tagged Hendra Virus (HeV M proteins were expressed and M-containing protein complexes were isolated and analysed. Presence of acidic leucine-rich nuclear phosphoprotein 32 family member B (ANP32B in the complex suggested that this protein represents a direct or indirect interactor of the viral matrix protein. Over-expression of ANP32B led to specific nuclear accumulation of HeV M, providing a functional link between ANP32B and M protein. ANP32B-dependent nuclear accumulation was observed after plasmid-driven expression of HeV and NiV matrix proteins and also in NiV infected cells. The latter indicated that an interaction of henipavirus M protein with ANP32B also occurs in the context of virus replication. From these data we conclude that ANP32B is a nuclear target of henipavirus M that may contribute to virus replication. Potential effects of ANP32B on HeV nuclear shuttling and host cell manipulation by HeV M affecting ANP32B functions in host cell survival and gene expression regulation are discussed.

  12. Type B lamins remain associated with the integral nuclear envelope protein p58 during mitosis: implications for nuclear reassembly.

    Science.gov (United States)

    Meier, J; Georgatos, S D

    1994-04-15

    p58 (also referred to as the lamin B receptor) is an integral membrane protein of the nuclear envelope known to form a multimeric complex with the lamins and other nuclear proteins during interphase. To examine the fate of this complex during mitosis, we have investigated the partitioning and the molecular interactions of p58 in dividing chicken hepatoma (DU249) cells. Using confocal microscopy and double immunolabelling, we show here that lamins B1 and B2 co-localize with p58 during all phases of mitosis and co-assemble around reforming nuclei. A close juxtaposition of p58/lamin B-containing vesicles and chromosomes is already detectable in metaphase; however, p58 and lamin reassembly proceeds slowly and is completed in late telophase--G1. Flotation of mitotic membranes in sucrose density gradients and analysis of mitotic vesicles by immunoelectron microscopy confirms that p58 and most of the type B lamins reside in the same compartment. Co-immunoprecipitation of both proteins by affinity-purified anti-p58 antibodies shows that they are physically associated in the context of a mitotic p58 'sub-complex'. This sub-assembly does not include the type A lamins which are fully solubilized during mitosis. Our data provide direct, in vivo and in vitro evidence that the majority of type B lamins remain connected to nuclear membrane 'receptors' during mitosis. The implications of these findings in nuclear envelope reassembly are discussed below.

  13. Evidence for ubiquitin-regulated nuclear and subnuclear trafficking among Paramyxovirinae matrix proteins.

    Directory of Open Access Journals (Sweden)

    Mickey Pentecost

    2015-03-01

    Full Text Available 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

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

    Directory of Open Access Journals (Sweden)

    Renouard Sullivan

    2012-01-01

    Full Text Available Abstract 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 μg 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.

  15. Efficient and dynamic nuclear localization of green fluorescent protein via RNA binding

    Energy Technology Data Exchange (ETDEWEB)

    Kitamura, Akira; Nakayama, Yusaku; Kinjo, Masataka, E-mail: kinjo@sci.hokudai.ac.jp

    2015-07-31

    Classical nuclear localization signal (NLS) sequences have been used for artificial localization of green fluorescent protein (GFP) in the nucleus as a positioning marker or for measurement of the nuclear-cytoplasmic shuttling rate in living cells. However, the detailed mechanism of nuclear retention of GFP-NLS remains unclear. Here, we show that a candidate mechanism for the strong nuclear retention of GFP-NLS is via the RNA-binding ability of the NLS sequence. GFP tagged with a classical NLS derived from Simian virus 40 (GFP-NLS{sup SV40}) localized not only in the nucleoplasm, but also to the nucleolus, the nuclear subdomain in which ribosome biogenesis takes place. GFP-NLS{sup SV40} in the nucleolus was mobile, and intriguingly, the diffusion coefficient, which indicates the speed of diffusing molecules, was 1.5-fold slower than in the nucleoplasm. Fluorescence correlation spectroscopy (FCS) analysis showed that GFP-NLS{sup SV40} formed oligomers via RNA binding, the estimated molecular weight of which was larger than the limit for passive nuclear export into the cytoplasm. These findings suggest that the nuclear localization of GFP-NLS{sup SV40} likely results from oligomerization mediated via RNA binding. The analytical technique used here can be applied for elucidating the details of other nuclear localization mechanisms, including those of several types of nuclear proteins. In addition, GFP-NLS{sup SV40} can be used as an excellent marker for studying both the nucleoplasm and nucleolus in living cells. - Highlights: • Nuclear localization signal-tagged GFP (GFP-NLS) showed clear nuclear localization. • The GFP-NLS dynamically localized not only in the nucleoplasm, but also to the nucleolus. • The nuclear localization of GFP-NLS results from transient oligomerization mediated via RNA binding. • Our NLS-tagging procedure is ideal for use in artificial sequestration of proteins in the nucleus.

  16. Cell differentiation by interaction of two HMG-box proteins: Mat1-Mc activates M cell-specific genes in S.pombe by recruiting the ubiquitous transcription factor Ste11 to weak binding sites

    DEFF Research Database (Denmark)

    Kjaerulff, S; Dooijes, D; Clevers, H

    1997-01-01

    The Schizosaccharomyces pombe mfm1 gene is expressed in an M cell-specific fashion. This regulation requires two HMG-box proteins: the ubiquitous Ste11 transcription factor and the M cell-controlling protein Mat1-Mc. Here we report that the mfm1 promoter contains a single, weak Stell-binding site...

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

    NARCIS (Netherlands)

    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

  18. High mobility group box-1 protein in patients with suspected community-acquired infections and sepsis: a prospective study

    DEFF Research Database (Denmark)

    Gaïni, Shahin; Pedersen, Svend Stenvang; Koldkjaer, Ole Graesbøll

    2008-01-01

    -infected patients and all infected patients, the area under the curve for HMGB1 was 0.59 (P white blood cell count, neutrophils, C-reactive protein, interleukin-6, procalcitonin, and lipopolysaccharide-binding protein (P

  19. The N-terminal domain of y-box binding protein-1 induces cell cycle arrest in g2/m phase by binding to cyclin d1.

    Science.gov (United States)

    Khandelwal, Payal; Padala, Mythili K; Cox, John; Guntaka, Ramareddy V

    2009-01-01

    Y-box binding protein YB-1 is a multifunctional protein involved in cell proliferation, regulation of transcription and translation. Our previous study indicated that disruption of one allele of Chk-YB-1b gene in DT-40 cells resulted in major defects in the cell cycle. The abnormalities seen in heterozygous mutants could be attributed to a dominant negative effect exerted by the disrupted YB-1 allele product. To test this hypothesis the N-terminal sequence of the YB-1 was fused with the third helix of antennapedia and the green fluorescent protein. These purified fusion proteins were introduced into rat hepatoma cells and their effect on cell proliferation was studied. Results indicate that the N-terminal 77 amino acid domain of the YB-1 protein induced the cells to arrest in G2/M phase of the cell cycle and undergo apoptosis. Additional deletion analysis indicated that as few as 26 amino acids of the N-terminus of YB-1 can cause these phenotypic changes. We further demonstrated that this N-terminal 77 amino acid domain of YB-1 sequesters cyclin D1 in the cytoplasm of cells at G2/M phase of cell cycle. We conclude that the N-terminal domain of YB-1 plays a major role in cell cycle progression through G2/M phase of cell cycle.

  20. The N-Terminal Domain of Y-Box Binding Protein-1 Induces Cell Cycle Arrest in G2/M Phase by Binding to Cyclin D1

    Directory of Open Access Journals (Sweden)

    Payal Khandelwal

    2009-01-01

    Full Text Available Y-box binding protein YB-1 is a multifunctional protein involved in cell proliferation, regulation of transcription and translation. Our previous study indicated that disruption of one allele of Chk-YB-1b gene in DT-40 cells resulted in major defects in the cell cycle. The abnormalities seen in heterozygous mutants could be attributed to a dominant negative effect exerted by the disrupted YB-1 allele product. To test this hypothesis the N-terminal sequence of the YB-1 was fused with the third helix of antennapedia and the green fluorescent protein. These purified fusion proteins were introduced into rat hepatoma cells and their effect on cell proliferation was studied. Results indicate that the N-terminal 77 amino acid domain of the YB-1 protein induced the cells to arrest in G2/M phase of the cell cycle and undergo apoptosis. Additional deletion analysis indicated that as few as 26 amino acids of the N-terminus of YB-1 can cause these phenotypic changes. We further demonstrated that this N-terminal 77 amino acid domain of YB-1 sequesters cyclin D1 in the cytoplasm of cells at G2/M phase of cell cycle. We conclude that the N-terminal domain of YB-1 plays a major role in cell cycle progression through G2/M phase of cell cycle.

  1. The F-box Protein KIB1 Mediates Brassinosteroid-Induced Inactivation and Degradation of GSK3-like Kinases in Arabidopsis.

    Science.gov (United States)

    Zhu, Jia-Ying; Li, Yuyao; Cao, Dong-Mei; Yang, Hongjuan; Oh, Eunkyoo; Bi, Yang; Zhu, Shengwei; Wang, Zhi-Yong

    2017-06-01

    The glycogen synthase kinase-3 (GSK3) family kinases are central cellular regulators highly conserved in all eukaryotes. In Arabidopsis, the GSK3-like kinase BIN2 phosphorylates a range of proteins to control broad developmental processes, and BIN2 is degraded through unknown mechanism upon receptor kinase-mediated brassinosteroid (BR) signaling. Here we identify KIB1 as an F-box E3 ubiquitin ligase that promotes the degradation of BIN2 while blocking its substrate access. Loss-of-function mutations of KIB1 and its homologs abolished BR-induced BIN2 degradation and caused severe BR-insensitive phenotypes. KIB1 directly interacted with BIN2 in a BR-dependent manner and promoted BIN2 ubiquitination in vitro. Expression of an F-box-truncated KIB1 caused BIN2 accumulation but dephosphorylation of its substrate BZR1 and activation of BR responses because KIB1 blocked BIN2 binding to BZR1. Our study demonstrates that KIB1 plays an essential role in BR signaling by inhibiting BIN2 through dual mechanisms of blocking substrate access and promoting degradation. Copyright © 2017 Elsevier Inc. All rights reserved.

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

    Science.gov (United States)

    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.

  3. An electrophoretic investigation of mammalian spermatid-specific nuclear proteins.

    Science.gov (United States)

    Lanneau, M; Loir, M

    1982-05-01

    Using standardized methods for protein extraction and analysis, the testes of rams, bulls, goats, boars, stallions, rats, cats, hedgehogs, European mink and ferrets were examined for basic spermatid nucleoproteins by electrophoresis. The results suggest that differences exist in the total number of these proteins as well as in the number and amount of the cross-linked cystein-containing proteins. These differences appear to be more family-specific than species-specific.

  4. Identification of a functional, CRM-1-dependent nuclear export signal in hepatitis C virus core protein.

    Directory of Open Access Journals (Sweden)

    Andrea Cerutti

    Full Text Available Hepatitis C virus (HCV infection is a major cause of chronic liver disease worldwide. HCV core protein is involved in nucleocapsid formation, but it also interacts with multiple cytoplasmic and nuclear molecules and plays a crucial role in the development of liver disease and hepatocarcinogenesis. The core protein is found mostly in the cytoplasm during HCV infection, but also in the nucleus in patients with hepatocarcinoma and in core-transgenic mice. HCV core contains nuclear localization signals (NLS, but no nuclear export signal (NES has yet been identified.We show here that the aa(109-133 region directs the translocation of core from the nucleus to the cytoplasm by the CRM-1-mediated nuclear export pathway. Mutagenesis of the three hydrophobic residues (L119, I123 and L126 in the identified NES or in the sequence encoding the mature core aa(1-173 significantly enhanced the nuclear localisation of the corresponding proteins in transfected Huh7 cells. Both the NES and the adjacent hydrophobic sequence in domain II of core were required to maintain the core protein or its fragments in the cytoplasmic compartment. Electron microscopy studies of the JFH1 replication model demonstrated that core was translocated into the nucleus a few minutes after the virus entered the cell. The blockade of nucleocytoplasmic export by leptomycin B treatment early in infection led to the detection of core protein in the nucleus by confocal microscopy and coincided with a decrease in virus replication.Our data suggest that the functional NLS and NES direct HCV core protein shuttling between the cytoplasmic and nuclear compartments, with at least some core protein transported to the nucleus. These new properties of HCV core may be essential for virus multiplication and interaction with nuclear molecules, influence cell signaling and the pathogenesis of HCV infection.

  5. Identification of a functional, CRM-1-dependent nuclear export signal in hepatitis C virus core protein.

    Science.gov (United States)

    Cerutti, Andrea; Maillard, Patrick; Minisini, Rosalba; Vidalain, Pierre-Olivier; Roohvand, Farzin; Pecheur, Eve-Isabelle; Pirisi, Mario; Budkowska, Agata

    2011-01-01

    Hepatitis C virus (HCV) infection is a major cause of chronic liver disease worldwide. HCV core protein is involved in nucleocapsid formation, but it also interacts with multiple cytoplasmic and nuclear molecules and plays a crucial role in the development of liver disease and hepatocarcinogenesis. The core protein is found mostly in the cytoplasm during HCV infection, but also in the nucleus in patients with hepatocarcinoma and in core-transgenic mice. HCV core contains nuclear localization signals (NLS), but no nuclear export signal (NES) has yet been identified.We show here that the aa(109-133) region directs the translocation of core from the nucleus to the cytoplasm by the CRM-1-mediated nuclear export pathway. Mutagenesis of the three hydrophobic residues (L119, I123 and L126) in the identified NES or in the sequence encoding the mature core aa(1-173) significantly enhanced the nuclear localisation of the corresponding proteins in transfected Huh7 cells. Both the NES and the adjacent hydrophobic sequence in domain II of core were required to maintain the core protein or its fragments in the cytoplasmic compartment. Electron microscopy studies of the JFH1 replication model demonstrated that core was translocated into the nucleus a few minutes after the virus entered the cell. The blockade of nucleocytoplasmic export by leptomycin B treatment early in infection led to the detection of core protein in the nucleus by confocal microscopy and coincided with a decrease in virus replication.Our data suggest that the functional NLS and NES direct HCV core protein shuttling between the cytoplasmic and nuclear compartments, with at least some core protein transported to the nucleus. These new properties of HCV core may be essential for virus multiplication and interaction with nuclear molecules, influence cell signaling and the pathogenesis of HCV infection.

  6. Nuclear transport factor directs localization of protein synthesis during mitosis

    NARCIS (Netherlands)

    Bogaart, Geert van den; Meinema, Anne C.; Krasnikov, Viktor; Veenhoff, Liesbeth M.; Poolman, Bert

    Export of messenger RNA from the transcription site in the nucleus and mRNA targeting to the translation site in the cytoplasm are key regulatory processes in protein synthesis. In yeast, the mRNA-binding proteins Nab2p and Nab4p/Hrp1p accompany transcripts to their translation site, where the

  7. Differential Utilization of TATA Box-binding Protein (TBP) and TBP-related Factor 1 (TRF1) at Different Classes of RNA Polymerase III Promoters*

    Science.gov (United States)

    Verma, Neha; Hung, Ko-Hsuan; Kang, Jin Joo; Barakat, Nermeen H.; Stumph, William E.

    2013-01-01

    In the fruit fly Drosophila melanogaster, RNA polymerase III transcription was found to be dependent not upon the canonical TATA box-binding protein (TBP) but instead upon the TBP-related factor 1 (TRF1) (Takada, S., Lis, J. T., Zhou, S., and Tjian, R. (2000) Cell 101, 459–469). Here we confirm that transcription of fly tRNA genes requires TRF1. However, we unexpectedly find that U6 snRNA gene promoters are occupied primarily by TBP in cells and that knockdown of TBP, but not TRF1, inhibits U6 transcription in cells. Moreover, U6 transcription in vitro effectively utilizes TBP, whereas TBP cannot substitute for TRF1 to promote tRNA transcription in vitro. Thus, in fruit flies, different classes of RNA polymerase III promoters differentially utilize TBP and TRF1 for the initiation of transcription. PMID:23955442

  8. Differential utilization of TATA box-binding protein (TBP) and TBP-related factor 1 (TRF1) at different classes of RNA polymerase III promoters.

    Science.gov (United States)

    Verma, Neha; Hung, Ko-Hsuan; Kang, Jin Joo; Barakat, Nermeen H; Stumph, William E

    2013-09-20

    In the fruit fly Drosophila melanogaster, RNA polymerase III transcription was found to be dependent not upon the canonical TATA box-binding protein (TBP) but instead upon the TBP-related factor 1 (TRF1) (Takada, S., Lis, J. T., Zhou, S., and Tjian, R. (2000) Cell 101, 459-469). Here we confirm that transcription of fly tRNA genes requires TRF1. However, we unexpectedly find that U6 snRNA gene promoters are occupied primarily by TBP in cells and that knockdown of TBP, but not TRF1, inhibits U6 transcription in cells. Moreover, U6 transcription in vitro effectively utilizes TBP, whereas TBP cannot substitute for TRF1 to promote tRNA transcription in vitro. Thus, in fruit flies, different classes of RNA polymerase III promoters differentially utilize TBP and TRF1 for the initiation of transcription.

  9. Nuclear and mitochondrial RNA editing systems have opposite effects on protein diversity.

    Science.gov (United States)

    Sloan, Daniel B

    2017-08-01

    RNA editing can yield protein products that differ from those directly encoded by genomic DNA. This process is pervasive in the mitochondria of many eukaryotes, where it predominantly results in the restoration of ancestral protein sequences. Nuclear mRNAs in metazoans also undergo editing (adenosine-to-inosine or 'A-to-I' substitutions), and most of these edits appear to be nonadaptive 'misfirings' of adenosine deaminases. However, recent analysis of cephalopod transcriptomes found that many editing sites are shared by anciently divergent lineages within this group, suggesting they play some adaptive role. Recent discoveries have also revealed that some fungi have an independently evolved A-to-I editing mechanism, resulting in extensive recoding of their nuclear mRNAs. Here, phylogenetic comparisons were used to determine whether RNA editing generally restores ancestral protein sequences or creates derived variants. Unlike in mitochondrial systems, RNA editing in metazoan and fungal nuclear transcripts overwhelmingly leads to novel sequences not found in inferred ancestral proteins. Even for the subset of RNA editing sites shared by deeply divergent cephalopod lineages, the primary effect of nuclear editing is an increase-not a decrease-in protein divergence. These findings suggest fundamental differences in the forces responsible for the evolution of RNA editing in nuclear versus mitochondrial systems. © 2017 The Author(s).

  10. Single domain antibodies for the knockdown of cytosolic and nuclear proteins.

    Science.gov (United States)

    Böldicke, Thomas

    2017-05-01

    Single domain antibodies (sdAbs) from camels or sharks comprise only the variable heavy chain domain. Human sdAbs comprise the variable domain of the heavy chain (VH) or light chain (VL) and can be selected from human antibodies. SdAbs are stable, nonaggregating molecules in vitro and in vivo compared to complete antibodies and scFv fragments. They are excellent novel inhibitors of cytosolic/nuclear proteins because they are correctly folded inside the cytosol in contrast to scFv fragments. SdAbs are unique because of their excellent specificity and possibility to target posttranslational modifications such as phosphorylation sites, conformers or interaction regions of proteins that cannot be targeted with genetic knockout techniques and are impossible to knockdown with RNAi. The number of inhibiting cytosolic/nuclear sdAbs is increasing and usage of synthetic single pot single domain antibody libraries will boost the generation of these fascinating molecules without the need of immunization. The most frequently selected antigenic epitopes belong to viral and oncogenic proteins, followed by toxins, proteins of the nervous system as well as plant- and drosophila proteins. It is now possible to select functional sdAbs against virtually every cytosolic/nuclear protein and desired epitope. The development of new endosomal escape protein domains and cell-penetrating peptides for efficient transfection broaden the application of inhibiting sdAbs. Last but not least, the generation of relatively new cell-specific nanoparticles such as polymersomes and polyplexes carrying cytosolic/nuclear sdAb-DNA or -protein will pave the way to apply cytosolic/nuclear sdAbs for inhibition of viral infection and cancer in the clinic. © 2017 The Protein Society.

  11. The Caenorhabditis elegans F-box protein SEL-10 promotes female development and may target FEM-1 and FEM-3 for degradation by the proteasome.

    Science.gov (United States)

    Jäger, Sibylle; Schwartz, Hillel T; Horvitz, H Robert; Conradt, Barbara

    2004-08-24

    The Caenorhabditis elegans F-box protein SEL-10 and its human homolog have been proposed to regulate LIN-12 Notch signaling by targeting for ubiquitin-mediated proteasomal degradation LIN-12 Notch proteins and SEL-12 PS1 presenilins, the latter of which have been implicated in Alzheimer's disease. We found that sel-10 is the same gene as egl-41, which previously had been defined by gain-of-function mutations that semidominantly cause masculinization of the hermaphrodite soma. Our results demonstrate that mutations causing loss-of-function of sel-10 also have masculinizing activity, indicating that sel-10 functions to promote female development. Genetically, sel-10 acts upstream of the genes fem-1, fem-2, and fem-3 and downstream of her-1 and probably tra-2. When expressed in mammalian cells, SEL-10 protein coimmunoprecipitates with FEM-1, FEM-2, and FEM-3, which are required for masculinization, and FEM-1 and FEM-3 are targeted by SEL-10 for proteasomal degradation. We propose that SEL-10-mediated proteolysis of FEM-1 and FEM-3 is required for normal hermaphrodite development.

  12. The Caenorhabditis elegans F-box protein SEL-10 promotes female development and may target FEM-1 and FEM-3 for degradation by the proteasome

    Science.gov (United States)

    Jäger, Sibylle; Schwartz, Hillel T.; Horvitz, H. Robert; Conradt, Barbara

    2004-01-01

    The Caenorhabditis elegans F-box protein SEL-10 and its human homolog have been proposed to regulate LIN-12 Notch signaling by targeting for ubiquitin-mediated proteasomal degradation LIN-12 Notch proteins and SEL-12 PS1 presenilins, the latter of which have been implicated in Alzheimer's disease. We found that sel-10 is the same gene as egl-41, which previously had been defined by gain-of-function mutations that semidominantly cause masculinization of the hermaphrodite soma. Our results demonstrate that mutations causing loss-of-function of sel-10 also have masculinizing activity, indicating that sel-10 functions to promote female development. Genetically, sel-10 acts upstream of the genes fem-1, fem-2, and fem-3 and downstream of her-1 and probably tra-2. When expressed in mammalian cells, SEL-10 protein coimmunoprecipitates with FEM-1, FEM-2, and FEM-3, which are required for masculinization, and FEM-1 and FEM-3 are targeted by SEL-10 for proteasomal degradation. We propose that SEL-10-mediated proteolysis of FEM-1 and FEM-3 is required for normal hermaphrodite development. PMID:15306688

  13. Serum levels of high mobility group box 1 protein and its association with quality of life and psychological and functional status in patients with fibromyalgia.

    Science.gov (United States)

    Oktayoglu, Pelin; Tahtasiz, Mehmet; Bozkurt, Mehtap; Em, Serda; Ucar, Demet; Yazmalar, Levent; Mete, Nuriye; Nas, Kemal; Gezer, Orhan

    2013-08-01

    High mobility group box 1 protein (HMGB1) is a proinflammatory cytokine. Previous studies have suggested that HMGB1 can play an important role in the pathogenesis of many rheumatic diseases. The purpose of this study was to investigate the serum levels of HMGB1 in patients with fibromyalgia (FM) and its association with quality of life and psychological and functional status in these patients. Twenty-nine patients who met the 1990 American College of Rheumatology (ACR) criteria for the classification of FM and 29 healthy controls (HC) were included in the present study. Serum samples were collected from both the patients and the HC, and HMGB1 levels were measured by enzyme-linked immunosorbent assay (ELISA). The Fibromyalgia Impact Questionnaire (FIQ) was used to assess the disease severity and functional status in patients with FM. Furthermore, the Nottingham Health Profile was used to assess quality of life in all subjects, as well as the Hospital Anxiety and Depression Scale (HADS) to assess depression and anxiety. The serum levels of HMGB1 protein were positively correlated with the FIQ scores in patients with FM (P = 0.002). Mean serum levels of HMGB1 were higher in patients with FM than in HC but this difference was not statistically significant. HMGB1 protein might be a good laboratory-sourced candidate for the assessment of functional status and disease severity in patients with FM. © 2013 Asia Pacific League of Associations for Rheumatology and Wiley Publishing Asia Pty Ltd.

  14. Prediction of an Epitope-based Computational Vaccine Strategy for Gaining Concurrent Immunization Against the Venom Proteins of Australian Box Jellyfish.

    Science.gov (United States)

    Alam, Md Jibran; Ashraf, Kutub Uddin Muhammad

    2013-09-01

    Australian Box Jellyfish (C. fleckeri) has the most rapid acting venom known to in the arena of toxicological research and is capable enough of killing a person in less than 5 minutes inflicting painful, debilitating and potentially life-threatening stings in humans. It has been understood that C. fleckeri venom proteins CfTX-1, 2 and HSP70-1 contain cardiotoxic, neurotoxic and highly dermatonecrotic components that can cause itchy bumpy rash and cardiac arrest. As there is no effective drug available, novel approaches regarding epitope prediction for vaccine development were performed in this study. Peptide fragments as nonamers of these antigenic venom proteins were analyzed by using computational tools that would elicit humoral and cell mediated immunity, were focused for attempting vaccine design. By ranking the peptides according to their proteasomal cleavage sites, TAP scores and IC50<250 nM, the predictions were scrutinized. Furthermore, the epitope sequences were examined by in silico docking simulation with different specific HLA receptors. Interestingly, to our knowledge, this is the maiden hypothetical immunization that predicts the promiscuous epitopes with potential contributions to the tailored design of improved safe and effective vaccines against antigenic venom proteins of C. fleckeri which would be effective especially for the Australian population. Although the computational approaches executed here are based on concrete confidence which demands more validation and in vivo experiments to validate such in silico approach.

  15. Genome-wide survey of B-box proteins in potato (Solanum tuberosum-Identification, characterization and expression patterns during diurnal cycle, etiolation and de-etiolation.

    Directory of Open Access Journals (Sweden)

    Urszula Talar

    Full Text Available Plant B-box domain proteins (BBX mediate many light-influenced developmental processes including seedling photomorphogenesis, seed germination, shade avoidance and photoperiodic regulation of flowering. Despite the wide range of potential functions, the current knowledge regarding BBX proteins in major crop plants is scarce. In this study, we identify and characterize the StBBX gene family in potato, which is composed of 30 members, with regard to structural properties and expression profiles under diurnal cycle, etiolation and de-etiolations. Based on domain organization and phylogenetic relationships, StBBX genes have been classified into five groups. Using real-time quantitative PCR, we found that expression of most of them oscillates following a 24-h rhythm; however, large differences in expression profiles were observed between the genes regarding amplitude and position of the maximal and minimal expression levels in the day/night cycle. On the basis of the time-of-day/time-of-night, we distinguished three expression groups specifically expressed during the light and two during the dark phase. In addition, we showed that the expression of several StBBX genes is under the control of the circadian clock and that some others are specifically associated with the etiolation and de-etiolation conditions. Thus, we concluded that StBBX proteins are likely key players involved in the complex diurnal and circadian networks regulating plant development as a function of light conditions and day duration.

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

    Energy Technology Data Exchange (ETDEWEB)

    Ryan, Colm M.; Kindle, Karin B.; Collins, Hilary M. [Gene Regulation Group, Centre for Biomolecular Sciences, School of Pharmacy, University of Nottingham, University Park, Nottingham NG7 2RD (United Kingdom); Heery, David M., E-mail: david.heery@nottingham.ac.uk [Gene Regulation Group, Centre for Biomolecular Sciences, School of Pharmacy, University of Nottingham, University Park, Nottingham NG7 2RD (United Kingdom)

    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.

  17. Functional conservation and divergence of four ginger AP1/AGL9 MADS-box genes revealed by analysis of their expression and protein-protein interaction, and ectopic expression of AhFUL gene in Arabidopsis.

    Directory of Open Access Journals (Sweden)

    Xiumei Li

    Full Text Available Alpinia genus are known generally as ginger-lilies for showy flowers in the ginger family, Zingiberaceae, and their floral morphology diverges from typical monocotyledon flowers. However, little is known about the functions of ginger MADS-box genes in floral identity. In this study, four AP1/AGL9 MADS-box genes were cloned from Alpinia hainanensis, and protein-protein interactions (PPIs and roles of the four genes in floral homeotic conversion and in floral evolution are surveyed for the first time. AhFUL is clustered to the AP1 lineage, AhSEP4 and AhSEP3b to the SEP lineage, and AhAGL6-like to the AGL6 lineage. The four genes showed conserved and divergent expression patterns, and their encoded proteins were localized in the nucleus. Seven combinations of PPI (AhFUL-AhSEP4, AhFUL-AhAGL6-like, AhFUL-AhSEP3b, AhSEP4-AhAGL6-like, AhSEP4-AhSEP3b, AhAGL6-like-AhSEP3b, and AhSEP3b-AhSEP3b were detected, and the PPI patterns in the AP1/AGL9 lineage revealed that five of the 10 possible combinations are conserved and three are variable, while conclusions cannot yet be made regarding the other two. Ectopic expression of AhFUL in Arabidopsis thaliana led to early flowering and floral organ homeotic conversion to sepal-like or leaf-like. Therefore, we conclude that the four A. hainanensis AP1/AGL9 genes show functional conservation and divergence in the floral identity from other MADS-box genes.

  18. The role of nuclear protein high mobility group box-1 (HMGB1) in the pathogenesis of systemic lupus erythematosus (SLE)

    NARCIS (Netherlands)

    Abdulahad Al-Qas Alias, Deena Abib

    2013-01-01

    Systemische Lupus Erythematosus (SLE) is een auto-immuunziekte met ontstekingen in allerlei organen, waaronder de nieren. UMCG-promovendus Deena Abdulahad toonde aan dat het eiwit HMGB1 verhoogd aanwezig is in het bloed bij ziekteactiviteit in de nieren. Het aantonen van HMGB1 in de urine kan helpen

  19. The overexpression of nuclear envelope protein Lap2β induces endoplasmic reticulum reorganisation via membrane stacking

    Directory of Open Access Journals (Sweden)

    Ekaterina G. Volkova

    2012-06-01

    Some nuclear envelope proteins are localised to both the nuclear envelope and the endoplasmic reticulum; therefore, it seems plausible that even small amounts of these proteins can influence the organisation of the endoplasmic reticulum. A simple method to study the possible effects of nuclear envelope proteins on endoplasmic reticulum organisation is to analyze nuclear envelope protein overexpression. Here, we demonstrate that Lap2β overexpression can induce the formation of cytoplasmic vesicular structures derived from endoplasmic reticulum membranes. Correlative light and electron microscopy demonstrated that these vesicular structures were composed of a series of closely apposed membranes that were frequently arranged in a circular fashion. Although stacked endoplasmic reticulum cisternae were highly ordered, Lap2β could readily diffuse into and out of these structures into the surrounding reticulum. It appears that low-affinity interactions between cytoplasmic domains of Lap2β can reorganise reticular endoplasmic reticulum into stacked cisternae. Although the effect of one protein may be insignificant at low concentrations, the cumulative effect of many non-specialised proteins may be significant.

  20. Co-expression and co-purification of archaeal and eukaryal box C/D RNPs.

    Directory of Open Access Journals (Sweden)

    Yu Peng

    Full Text Available Box C/D ribonucleoprotein particles (RNPs are 2'-O-methylation enzymes required for maturation of ribosomal and small nuclear RNA. Previous biochemical and structural studies of the box C/D RNPs were limited by the unavailability of purified intact RNPs. We developed a bacterial co-expression strategy based on the combined use of a multi-gene expression system and a tRNA-scaffold construct that allowed the expression and purification of homogeneous archaeal and human box C/D RNPs. While the co-expressed and co-purified archaeal box C/D RNP was found to be fully active in a 2'-O-methylation assay, the intact human U14 box C/D RNP showed no detectable catalytic activity, consistent with the earlier findings that assembly of eukaryotic box C/D RNPs is nonspontaneous and requires additional protein factors. Our systems provide a means for further biochemical and structural characterization of box C/D RNPs and their assembly factors.

  1. Multidimensional profiling of cell surface proteins and nuclear markers

    Energy Technology Data Exchange (ETDEWEB)

    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.

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

    Energy Technology Data Exchange (ETDEWEB)

    Xiang, Qi-Wang; Zou, Jin-Feng; Wang, Xin [Department of Preventive Veterinary Medicine, College of Veterinary Medicine, Shandong Agricultural University, Shandong, Taian 271018 (China); Shandong Provincial Key Laboratory of Animal Biotechnology and Disease Control and Prevention, Shandong, Taian 271018 (China); Sun, Ya-Ni [College of Veterinary Medicine, Northwest A and F University, Shanxi, Yangling 712100 (China); Gao, Ji-Ming; Xie, Zhi-Jing [Department of Preventive Veterinary Medicine, College of Veterinary Medicine, Shandong Agricultural University, Shandong, Taian 271018 (China); Shandong Provincial Key Laboratory of Animal Biotechnology and Disease Control and Prevention, Shandong, Taian 271018 (China); Wang, Yu [Department of Basic Medical Sciences, Taishan Medical College, Shandong, Taian 271000 (China); Zhu, Yan-Li [Department of Preventive Veterinary Medicine, College of Veterinary Medicine, Shandong Agricultural University, Shandong, Taian 271018 (China); Shandong Provincial Key Laboratory of Animal Biotechnology and Disease Control and Prevention, Shandong, Taian 271018 (China); Jiang, Shi-Jin, E-mail: sjjiang@sdau.edu.cn [Department of Preventive Veterinary Medicine, College of Veterinary Medicine, Shandong Agricultural University, Shandong, Taian 271018 (China); Shandong Provincial Key Laboratory of Animal Biotechnology and Disease Control and Prevention, Shandong, Taian 271018 (China)

    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. Acetylation dynamics of human nuclear proteins during the ionizing radiation-induced DNA damage response

    DEFF Research Database (Denmark)

    Bennetzen, Martin V; Larsen, Dorthe Helena; Dinant, Christoffel

    2013-01-01

    -dependent posttranslational modifications (PTMs). To complement our previous analysis of IR-induced temporal dynamics of nuclear phosphoproteome, we now identify a range of human nuclear proteins that are dynamically regulated by acetylation, and predominantly deacetylation, during IR-induced DDR by using mass spectrometry...... to genotoxic insults. Overall, these results present a resource of temporal profiles of a spectrum of protein acetylation sites during DDR and provide further insights into the highly dynamic nature of regulatory PTMs that help orchestrate the maintenance of genome integrity.......Genotoxic insults, such as ionizing radiation (IR), cause DNA damage that evokes a multifaceted cellular DNA damage response (DDR). DNA damage signaling events that control protein activity, subcellular localization, DNA binding, protein-protein interactions, etc. rely heavily on time...

  4. Laminopathy-inducing lamin A mutants can induce redistribution of lamin binding proteins into nuclear aggregates.

    Science.gov (United States)

    Hübner, S; Eam, J E; Hübner, A; Jans, D A

    2006-01-15

    Lamins, members of the family of intermediate filaments, form a supportive nucleoskeletal structure underlying the nuclear envelope and can also form intranuclear structures. Mutations within the A-type lamin gene cause a variety of degenerative diseases which are collectively referred to as laminopathies. At the molecular level, laminopathies have been shown to be linked to a discontinuous localization pattern of A-type lamins, with some laminopathies containing nuclear lamin A aggregates. Since nuclear aggregate formation could lead to the mislocalization of proteins interacting with A-type lamins, we set out to examine the effects of FLAG-lamin A N195K and R386K protein aggregate formation on the subnuclear distribution of the retinoblastoma protein (pRb) and the sterol responsive element binding protein 1a (SREBP1a) after coexpression as GFP-fusion proteins in HeLa cells. We observed strong recruitment of both proteins into nuclear aggregates. Nuclear aggregate recruitment of the NPC component nucleoporin NUP153 was also observed and found to be dependent on the N-terminus. That these effects were specific was implied by the fact that a number of other coexpressed karyophilic GFP-fusion proteins, such as the nucleoporin NUP98 and kanadaptin, did not coaggregate with FLAG-lamin A N195K or R386K. Immunofluorescence analysis further indicated that the precursor form of lamin A, pre-lamin A, could be found in intranuclear aggregates. Our results imply that redistribution into lamin A-/pre-lamin A-containing aggregates of proteins such as pRb and SREBP1a could represent a key aspect underlying the molecular pathogenesis of certain laminopathies.

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

    Directory of Open Access Journals (Sweden)

    Enrico Giampieri

    Full Text Available 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.

  6. Pandora's Box

    DEFF Research Database (Denmark)

    Andalib, Sasan; Divani, Afshin A.; Michel, Tanja M.

    2017-01-01

    Ischaemic heart disease and stroke are vascular events with serious health consequences worldwide. Recent genetic and epigenetic techniques have revealed many genetic determinants of these vascular events and simplified the approaches to research focused on ischaemic heart disease and stroke....... The pathogenetic mechanisms of ischaemic heart disease and stroke are complex, with mitochondrial involvement (partially or entirely) recently gaining substantial support. Not only can mitochondrial reactive oxygen species give rise to ischaemic heart disease and stroke by production of oxidised low......-density lipoprotein and induction of apoptosis, but the impact on pericytes contributes directly to the pathogenesis. Over the past two decades, publications implicate the causative role of nuclear genes in the development of ischaemic heart disease and stroke, in contrast to the potential role of mitochondrial DNA...

  7. Identification of a classical bipartite nuclear localization signal in the Drosophila TEA/ATTS protein scalloped.

    Directory of Open Access Journals (Sweden)

    Adam C Magico

    Full Text Available Drosophila melanogaster wing development has been shown to rely on the activity of a complex of two proteins, Scalloped (Sd and Vestigial (Vg. Within this complex, Sd is known to provide DNA binding though its TEA/ATTS domain, while Vg modulates this binding and provides transcriptional activation through N- and C-terminal activation domains. There is also evidence that Sd is required for the nuclear translocation of Vg. Indeed, a candidate sequence which shows consensus to the bipartite family of nuclear localization signals (NLSs has been identified within Sd previously, though it is not known if it is functional, or if additional unpredicted signals that mediate nuclear transport exist within the protein. By expressing various enhanced green fluorescent protein (eGFP tagged constructs within Drosophila S2 cells, we demonstrate that this NLS is indeed functional and necessary for the proper nuclear localization of Sd. Additionally, the region containing the NLS is critical for the wildtype function of ectopically expressed Sd, in the context of wing development. Using site-directed mutagenesis, we have identified a group of five amino acids within this NLS which is critical for its function, as well as another group of two which is of lesser importance. Together with data that suggests that this sequence mediates interactions with Importin-α3, we conclude that the identified NLS is likely a classical bipartite signal. Further dissection of Sd has also revealed that a large portion of the C-terminal domain of the protein is required its proper nuclear localization. Finally, a Leptomycin B (LB sensitive signal which appears to facilitate nuclear export is identified, raising the possibility that Sd also contains a nuclear export signal (NES.

  8. Identification of a classical bipartite nuclear localization signal in the Drosophila TEA/ATTS protein scalloped.

    Science.gov (United States)

    Magico, Adam C; Bell, John B

    2011-01-01

    Drosophila melanogaster wing development has been shown to rely on the activity of a complex of two proteins, Scalloped (Sd) and Vestigial (Vg). Within this complex, Sd is known to provide DNA binding though its TEA/ATTS domain, while Vg modulates this binding and provides transcriptional activation through N- and C-terminal activation domains. There is also evidence that Sd is required for the nuclear translocation of Vg. Indeed, a candidate sequence which shows consensus to the bipartite family of nuclear localization signals (NLSs) has been identified within Sd previously, though it is not known if it is functional, or if additional unpredicted signals that mediate nuclear transport exist within the protein. By expressing various enhanced green fluorescent protein (eGFP) tagged constructs within Drosophila S2 cells, we demonstrate that this NLS is indeed functional and necessary for the proper nuclear localization of Sd. Additionally, the region containing the NLS is critical for the wildtype function of ectopically expressed Sd, in the context of wing development. Using site-directed mutagenesis, we have identified a group of five amino acids within this NLS which is critical for its function, as well as another group of two which is of lesser importance. Together with data that suggests that this sequence mediates interactions with Importin-α3, we conclude that the identified NLS is likely a classical bipartite signal. Further dissection of Sd has also revealed that a large portion of the C-terminal domain of the protein is required its proper nuclear localization. Finally, a Leptomycin B (LB) sensitive signal which appears to facilitate nuclear export is identified, raising the possibility that Sd also contains a nuclear export signal (NES).

  9. Nuclear localization and secretion competence are conserved among henipavirus matrix proteins.

    Science.gov (United States)

    McLinton, Elisabeth C; Wagstaff, Kylie M; Lee, Alexander; Moseley, Gregory W; Marsh, Glenn A; Wang, Lin-Fa; Jans, David A; Lieu, Kim G; Netter, Hans J

    2017-04-01

    Viruses of the genus Henipavirus of the family Paramyxoviridae are zoonotic pathogens, which have emerged in Southeast Asia, Australia and Africa. Nipah virus (NiV) and Hendra virus are highly virulent pathogens transmitted from bats to animals and humans, while the henipavirus Cedar virus seems to be non-pathogenic in infection studies. The full replication cycle of the Paramyxoviridae occurs in the host cell's cytoplasm, where viral assembly is orchestrated by the matrix (M) protein. Unexpectedly, the NiV-M protein traffics through the nucleus as an essential step to engage the plasma membrane in preparation for viral budding/release. Comparative studies were performed to assess whether M protein nuclear localization is a common feature of the henipaviruses, including the recently sequenced (although not yet isolated) Ghanaian bat henipavirus (Kumasi virus, GH-M74a virus) and Mojiang virus. Live-cell confocal microscopy revealed that nuclear translocation of GFP-fused M protein is conserved between henipaviruses in both human- and bat-derived cell lines. However, the efficiency of M protein nuclear localization and virus-like particle budding competency varied. Additionally, Cedar virus-, Kumasi virus- and Mojiang virus-M proteins were mutated in a bipartite nuclear localization signal, indicating that a key lysine residue is essential for nuclear import, export and induction of budding events, as previously reported for NiV-M. The results of this study suggest that the M proteins of henipaviruses may utilize a similar nucleocytoplasmic trafficking pathway as an essential step during viral replication in both humans and bats.

  10. High-mobility group box-1 protein, lipopolysaccharide-binding protein, interleukin-6 and C-reactive protein in children with community acquired infections and bacteraemia: a prospective study

    Directory of Open Access Journals (Sweden)

    Kalnins Imants

    2010-02-01

    Full Text Available Abstract Introduction Even though sepsis is one of the common causes of children morbidity and mortality, specific inflammatory markers for identifying sepsis are less studied in children. The main aim of this study was to compare the levels of high-mobility group box-1 protein (HMGB1, Lipopolysaccharide-binding protein (LBP, Interleukin-6 (IL-6 and C-reactive protein (CRP between infected children without systemic inflammatory response syndrome (SIRS and children with severe and less severe sepsis. The second aim was to examine HMGB1, LBP, IL6 and CRP as markers for of bacteraemia. Methods Totally, 140 children with suspected or proven infections admitted to the Children's Clinical University Hospital of Latvia during 2008 and 2009 were included. Clinical and demographical information as well as infection focus were assessed in all patients. HMGB1, LBP, IL-6 and CRP blood samples were determined. Children with suspected or diagnosed infections were categorized into three groups of severity of infection: (i infected without SIRS (n = 36, (ii sepsis (n = 91 and, (iii severe sepsis (n = 13. They were furthermore classified according bacteraemia into (i bacteremia (n = 30 and (ii no bacteraemia (n = 74. Results There was no statistically significant difference in HMGB1 levels between children with different levels of sepsis or with and without bacteraemia. The levels of LBP, IL-6 and CRP were statistically significantly higher among patients with sepsis compared to those infected but without SIRS (p p Conclusion Elevated levels of LBP, IL-6 and CRP were associated with a more severe level of infection in children. Whereas LBP, IL-6 and CRP seem to be good markers to detect patients with bacteraemia, HMGB1 seem to be of minor importance. LBP, IL-6 and CRP levels may serve as good biomarkers for identifying children with severe sepsis and bacteraemia and, thus, may be routinely used in clinical practice.

  11. Effect of nitric oxide on gene transcription - S-Nitrosylation of nuclear proteins

    Directory of Open Access Journals (Sweden)

    Alexander eMengel

    2013-08-01

    Full Text Available Nitric oxide plays an important role in many different physiological processes in plants. It mainly acts by post-translationally modifying proteins. Modification of cysteine residues termed as S-nitrosylation is believed to be the most important mechanism for transduction of bioactivity of NO. The first proteins found to be nitrosylated were mainly of cytoplasmic origin or isolated from mitochondria and peroxisomes. Interestingly, it was shown that redox-sensitive transcription factors are also nitrosylated and that NO influences the redox-dependent nuclear transport of some proteins. This implies that NO plays a role in regulating transcription and/or general nuclear metabolism which is a fascinating new aspect of nitric oxide signaling in plants. In this review we will discuss the impact of S-nitrosylation on nuclear plant proteins with a focus on transcriptional regulation, describe the function of this modification and draw also comparisons to the animal system in which S-nitrosylation of nuclear proteins is a well characterized concept.

  12. Nuclear protein synthesis in animal and vegetal hemispheres of Xenopus oocytes

    Energy Technology Data Exchange (ETDEWEB)

    Feldherr, C.M.; Hodges, P. (Univ. of Florida, Gainesville (USA)); Paine, P.L. (Michigan Cancer Foundation, Detroit (USA))

    1988-12-01

    Experiments were conducted to determine if nuclear proteins are preferentially synthesized in the vicinity of the nucleus, a factor which could facilitate nucleocytoplasmic exchange. Using Xenopus oocytes, animal and vegetal hemispheres were separated by bisecting the cells in paraffin oil. It was initially established that protein synthesis is not affected by the bisecting procedure. To determine if nuclear protein synthesis is restricted to the animal hemisphere (which contains the nucleus), vegetal halves and enucleated animal halves were injected with ({sup 3}H)leucine and incubated in oil for 90 min. The labeled cell halves were then fused with unlabeled, nucleated animal hemispheres that had been previously injected with puromycin in amounts sufficient to prevent further protein synthesis. Thus, labeled polypeptides which subsequently entered the nuclei were synthesized before fusion. Three hours after fusion, the nuclei were isolated, run on two-dimensional gels, and fluorographed. Approximately 200 labeled nuclear polypeptides were compared, and only 2 were synthesized in significantly different amounts in the animal and vegetal hemispheres. The results indicate that nuclear protein synthesis is not restricted to the cytoplasm adjacent to the nucleus.

  13. Efficient and dynamic nuclear localization of green fluorescent protein via RNA binding.

    Science.gov (United States)

    Kitamura, Akira; Nakayama, Yusaku; Kinjo, Masataka

    2015-07-31

    Classical nuclear localization signal (NLS) sequences have been used for artificial localization of green fluorescent protein (GFP) in the nucleus as a positioning marker or for measurement of the nuclear-cytoplasmic shuttling rate in living cells. However, the detailed mechanism of nuclear retention of GFP-NLS remains unclear. Here, we show that a candidate mechanism for the strong nuclear retention of GFP-NLS is via the RNA-binding ability of the NLS sequence. GFP tagged with a classical NLS derived from Simian virus 40 (GFP-NLS(SV40)) localized not only in the nucleoplasm, but also to the nucleolus, the nuclear subdomain in which ribosome biogenesis takes place. GFP-NLS(SV40) in the nucleolus was mobile, and intriguingly, the diffusion coefficient, which indicates the speed of diffusing molecules, was 1.5-fold slower than in the nucleoplasm. Fluorescence correlation spectroscopy (FCS) analysis showed that GFP-NLS(SV40) formed oligomers via RNA binding, the estimated molecular weight of which was larger than the limit for passive nuclear export into the cytoplasm. These findings suggest that the nuclear localization of GFP-NLS(SV40) likely results from oligomerization mediated via RNA binding. The analytical technique used here can be applied for elucidating the details of other nuclear localization mechanisms, including those of several types of nuclear proteins. In addition, GFP-NLS(SV40) can be used as an excellent marker for studying both the nucleoplasm and nucleolus in living cells. Copyright © 2015 Elsevier Inc. All rights reserved.

  14. Increased plasma levels of the high mobility group box 1 protein (HMGB1) are associated with a higher score of gastrointestinal dysfunction in individuals with autism.

    Science.gov (United States)

    Babinská, K; Bucová, M; Ďurmanová, V; Lakatošová, S; Jánošíková, D; Bakoš, J; Hlavatá, A; Ostatníková, D

    2014-01-01

    Autism is a disorder of neural development characterized by impairments in communication, social interaction, restricted interests and repetitive behavior. The etiology of autism is poorly understood, the evidence indicates that inflammation may play a key role. In autism a high prevalence of gastrointestinal disturbances is reported, that are linked to a low-grade chronic inflammation of the intestinal mucosa. High mobility group box 1 protein (HMGB1) is an intranuclear protein that can be passively released from necrotic cells or actively secreted under inflammatory conditions as alarmin or late proinflammatory cytokine. The objective of this study was to measure plasma levels of HMGB1 in individuals with autism and to analyze their association with gastrointestinal symptoms. The study involved 31 subjects with low-functioning autistic disorder aged 2-22 years and 16 healthy controls. Plasma HMGB1 levels were significantly higher in individuals with autism than in controls (13.8+/-11.7 ng/ml vs. 7.90+/-4.0 ng/ml, pautism and its possible association with GI symptoms.

  15. Formation of C-terminally truncated version of the Taz1 protein employs cleavage-box structure in mRNA

    Energy Technology Data Exchange (ETDEWEB)

    Gunisova, Stanislava; Bartosova, Zdenka [Department of Genetics, Comenius University, Faculty of Natural Sciences, 842 15 Bratislava (Slovakia); Kramara, Juraj; Nosek, Jozef [Department of Biochemistry, Comenius University, Faculty of Natural Sciences, 842 15 Bratislava (Slovakia); Tomaska, Lubomir, E-mail: tomaska@fns.uniba.sk [Department of Genetics, Comenius University, Faculty of Natural Sciences, 842 15 Bratislava (Slovakia)

    2010-02-12

    When expressed in various hosts the taz1{sup +} gene encoding the fission yeast telomere-binding protein produces two forms of polypeptides: full-length (Taz1p) and truncated (Taz1p{Delta}C) version lacking almost entire Myb-domain. Whereas Taz1p binds telomeric DNA in vitro, Taz1p{Delta}C forms long filaments unable of DNA binding. The formation of Taz1p{Delta}C is a result of neither site-specific proteolysis, nor premature termination of transcription. In silico analysis of the taz1{sup +} RNA transcript revealed a stem-loop structure at the site of cleavage (cleavage box; CB). In order to explore whether it possesses inherent destabilizing effects, we cloned CB sequence into the open reading frame (ORF) of glutathione-S-transferase (GST) and observed that when expressed in Escherichia coli the engineered gene produced two forms of the reporter protein. The formation of the truncated version of GST was abolished, when CB was replaced with recoded sequence containing synonymous codons thus indicating that the truncation is based on structural properties of taz1{sup +} mRNA.

  16. Identification of Y-box binding protein 1 as a core regulator of MEK/ERK pathway-dependent gene signatures in colorectal cancer cells.

    Directory of Open Access Journals (Sweden)

    Karsten Jürchott

    2010-12-01

    Full Text Available Transcriptional signatures are an indispensible source of correlative information on disease-related molecular alterations on a genome-wide level. Numerous candidate genes involved in disease and in factors of predictive, as well as of prognostic, value have been deduced from such molecular portraits, e.g. in cancer. However, mechanistic insights into the regulatory principles governing global transcriptional changes are lagging behind extensive compilations of deregulated genes. To identify regulators of transcriptome alterations, we used an integrated approach combining transcriptional profiling of colorectal cancer cell lines treated with inhibitors targeting the receptor tyrosine kinase (RTK/RAS/mitogen-activated protein kinase pathway, computational prediction of regulatory elements in promoters of co-regulated genes, chromatin-based and functional cellular assays. We identified commonly co-regulated, proliferation-associated target genes that respond to the MAPK pathway. We recognized E2F and NFY transcription factor binding sites as prevalent motifs in those pathway-responsive genes and confirmed the predicted regulatory role of Y-box binding protein 1 (YBX1 by reporter gene, gel shift, and chromatin immunoprecipitation assays. We also validated the MAPK-dependent gene signature in colorectal cancers and provided evidence for the association of YBX1 with poor prognosis in colorectal cancer patients. This suggests that MEK/ERK-dependent, YBX1-regulated target genes are involved in executing malignant properties.

  17. Alternative splicing determines the interaction of SMRT isoforms with nuclear receptor-DNA complexes.

    Science.gov (United States)

    Faist, Flavie; Short, Stephen; Kneale, G Geoff; Sharpe, Colin R

    2009-06-01

    Signalling by small molecules, such as retinoic acid, is mediated by heterodimers comprising a class II nuclear receptor and an RXR (retinoid X receptor) subunit. The receptors bind to DNA response elements and act as ligand-dependent transcription factors, but, in the absence of signal, the receptors bind the co-repressors SMRT [silencing mediator for RAR (retinoic acid receptor) and TR (thyroid hormone receptor)] and NCoR (nuclear receptor co-repressor) and repress gene expression. Alternative splicing of the SMRT transcript in mammals generates six isoforms containing 1, 2 or 3 CoRNR (co-repressor for nuclear receptor) box motifs which are responsible for the interactions with nuclear receptors. We show that human cell lines express all six SMRT isoforms and then determine the binding affinity of mouse SMRT isoforms for RAR/RXR and three additional class II nuclear receptor-DNA complexes. This approach demonstrates the importance of the full complement of CoRNR boxes within each SMRT protein, rather than the identity of individual CoRNR boxes, in directing the interaction of SMRT with nuclear receptors. Each class of SMRT isoform displays a distinct feature, as the 1-box isoform discriminates between DNA response elements, the 2-box isoforms promote high-affinity binding to TR complexes and the 3-box isoforms show differential binding to nuclear receptors. Consequently, the differential deployment of SMRT isoforms observed in vivo could significantly expand the regulatory capacity of nuclear receptor signalling.

  18. Box model of radionuclide dispersion and radiation risk estimation for population in case of radioactivity release from nuclear submarine {number_sign}601 dumped in the Kara Sea

    Energy Technology Data Exchange (ETDEWEB)

    Yefimov, E.I.; Pankratov, D.V.; Ignatiev, S.V. [Inst. of Physics and Power Engineering, Obninsk (Russian Federation)

    1997-12-31

    When ships with nuclear reactors or nuclear materials aboard suffer shipwreck or in the case of burial or dumping of radioactive wastes, atmospheric fallout, etc., radionuclides may be released and spread in the sea, contaminating the sea water and the sea bottom. When a nuclear submarine (NS) is dumped this spread of activity may occur due to gradual core destruction by corrosion over many years. The objective of this paper is to develop a mathematical model of radionuclide dispersion and to assess the population dose and radiation risk for radionuclide release from the NS No. 601, with Pb-Bi coolant that was dumped in the Kara Sea.

  19. Ubc9 mediates nuclear localization and growth suppression of BRCA1 and BRCA1a proteins.

    Science.gov (United States)

    Qin, Yunlong; Xu, Jingyao; Aysola, Kartik; Begum, Nurjahan; Reddy, Vaishali; Chai, Yuli; Grizzle, William E; Partridge, Edward E; Reddy, E Shyam P; Rao, Veena N

    2011-12-01

    BRCA1 gene mutations are responsible for hereditary breast and ovarian cancers. In sporadic breast tumors, BRCA1 dysfunction or aberrant subcellular localization is thought to be common. BRCA1 is a nuclear-cytoplasm shuttling protein and the reason for cytoplasmic localization of BRCA1 in young breast cancer patients is not yet known. We have previously reported BRCA1 proteins unlike K109R and cancer-predisposing mutant C61G to bind Ubc9 and modulate ER-α turnover. In the present study, we have examined the consequences of altered Ubc9 binding and knockdown on the subcellular localization and growth inhibitory function of BRCA1 proteins. Our results using live imaging of YFP, GFP, RFP-tagged BRCA1, BRCA1a and BRCA1b proteins show enhanced cytoplasmic localization of K109 R and C61G mutant BRCA1 proteins in normal and cancer cells. Furthermore, down-regulation of Ubc9 in MCF-7 cells using Ubc9 siRNA resulted in enhanced cytoplasmic localization of BRCA1 protein and exclusive cytoplasmic retention of BRCA1a and BRCA1b proteins. These mutant BRCA1 proteins were transforming and impaired in their capacity to inhibit growth of MCF-7 and CAL51 breast cancer cells. Interestingly, cytoplasmic BRCA1a mutants showed more clonogenicity in soft agar and higher levels of expression of Ubc9 than parental MCF7 cells. This is the first report demonstrating the physiological link between cytoplasmic mislocalization of mutant BRCA1 proteins, loss of ER-α repression, loss of ubiquitin ligase activity and loss of growth suppression of BRCA1 proteins. Thus, binding of BRCA1 proteins to nuclear chaperone Ubc9 provides a novel mechanism for nuclear import and control of tumor growth. Copyright © 2011 Wiley-Liss, Inc.

  20. Quality control of inner nuclear membrane proteins by the Asi complex.

    Science.gov (United States)

    Foresti, Ombretta; Rodriguez-Vaello, Victoria; Funaya, Charlotta; Carvalho, Pedro

    2014-11-07

    Misfolded proteins in the endoplasmic reticulum (ER) are eliminated by a quality control system called ER-associated protein degradation (ERAD). However, it is unknown how misfolded proteins in the inner nuclear membrane (INM), a specialized ER subdomain, are degraded. We used a quantitative proteomics approach to reveal an ERAD branch required for INM protein quality control in yeast. This branch involved the integral membrane proteins Asi1, Asi2, and Asi3, which assembled into an Asi complex. Besides INM misfolded proteins, the Asi complex promoted the degradation of functional regulators of sterol biosynthesis. Asi-mediated ERAD was required for ER homeostasis, which suggests that spatial segregation of protein quality control systems contributes to ER function. Copyright © 2014, American Association for the Advancement of Science.

  1. Identification of nuclear protein targets for six leukemogenic tyrosine kinases governed by post-translational regulation.

    Directory of Open Access Journals (Sweden)

    Andrew Pierce

    Full Text Available Mutated tyrosine kinases are associated with a number of different haematological malignancies including myeloproliferative disorders, lymphoma and acute myeloid leukaemia. The potential commonalities in the action of six of these leukemogenic proteins on nuclear proteins were investigated using systematic proteomic analysis. The effects on over 3600 nuclear proteins and 1500 phosphopeptide sites were relatively quantified in seven isogenic cell lines. The effects of the kinases were diverse although some commonalities were found. Comparison of the nuclear proteomic data with transcriptome data and cytoplasmic proteomic data indicated that the major changes are due to post-translational mechanisms rather than changes in mRNA or protein distribution. Analysis of the promoter regions of genes whose protein levels changed in response to the kinases showed the most common binding site found was that for NFκB whilst other sites such as those for the glucocorticoid receptor were also found. Glucocorticoid receptor levels and phosphorylation were decreased by all 6 PTKs. Whilst Glucocorticoid receptor action can potentiate NFκB action those proteins where genes have NFκB binding sites were in often regulated post-translationally. However all 6 PTKs showed evidence of NFkB pathway modulation via activation via altered IkB and NFKB levels. Validation of a common change was also undertaken with PMS2, a DNA mismatch repair protein. PMS2 nuclear levels were decreased in response to the expression of all 6 kinases, with no concomitant change in mRNA level or cytosolic protein level. Response to thioguanine, that requires the mismatch repair pathway, was modulated by all 6 oncogenic kinases. In summary common targets for 6 oncogenic PTKs have been found that are regulated by post-translational mechanisms. They represent potential new avenues for therapies but also demonstrate the post-translational regulation is a key target of leukaemogenic kinases.

  2. Solid state nuclear magnetic resonance studies of prion peptides and proteins

    Energy Technology Data Exchange (ETDEWEB)

    Heller, Jonathan [Univ. of California, Berkeley, CA (United States)

    1997-08-01

    High-resolution structural studies using x-ray diffraction and solution nuclear magnetic resonance (NMR) are not feasible for proteins of low volubility and high tendency to aggregate. Solid state NMR (SSNMR) is in principle capable of providing structural information in such systems, however to do this efficiently and accurately, further SSNMR tools must be developed This dissertation describes the development of three new methods and their application to a biological system of interest, the priori protein (PrP).

  3. A protein antigenically related to nuclear lamin B mediates the association of intermediate filaments with desmosomes

    OpenAIRE

    1990-01-01

    Desmosomes are specialized domains of epithelial cell plasma membranes engaged in the anchoring of intermediate filaments (IF). So far, the desmosomal component(s) responsible for this binding has not been unambiguously identified. In the present work, we have examined bovine muzzle epidermis desmosomes for the presence of protein(s) structurally and functionally related to lamin B, the major receptor for IF in the nuclear envelope (Georgatos, S. D., and G. Blobel. 1987. J. Cell Biol. 105:105...

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

    Science.gov (United States)

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

    2014-06-09

    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. © 2014 Zhou et al.

  5. The Oncogenic Fusion Proteins SET-Nup214 and Sequestosome-1 (SQSTM1)-Nup214 Form Dynamic Nuclear Bodies and Differentially Affect Nuclear Protein and Poly(A)+ RNA Export.

    Science.gov (United States)

    Port, Sarah A; Mendes, Adélia; Valkova, Christina; Spillner, Christiane; Fahrenkrog, Birthe; Kaether, Christoph; Kehlenbach, Ralph H

    2016-10-28

    Genetic rearrangements are a hallmark of several forms of leukemia and can lead to oncogenic fusion proteins. One example of an affected chromosomal region is the gene coding for Nup214, a nucleoporin that localizes to the cytoplasmic side of the nuclear pore complex (NPC). We investigated two such fusion proteins, SET-Nup214 and SQSTM1 (sequestosome)-Nup214, both containing C-terminal portions of Nup214. SET-Nup214 nuclear bodies containing the nuclear export receptor CRM1 were observed in the leukemia cell lines LOUCY and MEGAL. Overexpression of SET-Nup214 in HeLa cells leads to the formation of similar nuclear bodies that recruit CRM1, export cargo proteins, and certain nucleoporins and concomitantly affect nuclear protein and poly(A) + RNA export. SQSTM1-Nup214, although mostly cytoplasmic, also forms nuclear bodies and inhibits nuclear protein but not poly(A) + RNA export. The interaction of the fusion proteins with CRM1 is RanGTP-dependent, as shown in co-immunoprecipitation experiments and binding assays. Further analysis revealed that the Nup214 parts mediate the inhibition of nuclear export, whereas the SET or SQSTM1 part determines the localization of the fusion protein and therefore the extent of the effect. SET-Nup214 nuclear bodies are highly mobile structures, which are in equilibrium with the nucleoplasm in interphase and disassemble during mitosis or upon treatment of cells with the CRM1-inhibitor leptomycin B. Strikingly, we found that nucleoporins can be released from nuclear bodies and reintegrated into existing NPC. Our results point to nuclear bodies as a means of preventing the formation of potentially insoluble and harmful protein aggregates that also may serve as storage compartments for nuclear transport factors. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  6. An enzyme-linked immunosorbent assay for autoantibodies against the nuclear protein Scl-70

    DEFF Research Database (Denmark)

    Geisler, C; Høier-Madsen, M

    1985-01-01

    This paper describes the development of an enzyme-linked immunosorbent assay (ELISA) for the detection and quantitation of autoantibodies against the nuclear protein Scl-70. The isolation of Scl-70 from rat livers and the conditions for the ELISA are described. Compared with the already established...... double diffusion in gel for detection of anti-Scl-70 antibodies this ELISA has advantages....

  7. Nuclear localization of human DNA mismatch repair protein exonuclease 1 (hEXO1)

    DEFF Research Database (Denmark)

    Knudsen, Nina Østergaard; Nielsen, Finn Cilius; Vinther, Lena

    2007-01-01

    Human exonuclease 1 (hEXO1) is implicated in DNA mismatch repair (MMR) and mutations in hEXO1 may be associated with hereditary nonpolyposis colorectal cancer (HNPCC). Since the subcellular localization of MMR proteins is essential for proper MMR function, we characterized possible nuclear locali...

  8. Forkhead box protein P1 as a downstream target of transforming growth factor-β induces collagen synthesis and correlates with a more stable plaque phenotype.

    Science.gov (United States)

    Bot, Pieter T; Grundmann, Sebastian; Goumans, Marie-José; de Kleijn, Dominique; Moll, Frans; de Boer, Onno; van der Wal, Allard C; van Soest, Alex; de Vries, Jean-Paul; van Royen, Niels; Piek, Jan J; Pasterkamp, Gerard; Hoefer, Imo E

    2011-09-01

    Atherosclerosis is an inflammatory disease, modulated by plaque stabilizing and de-stabilizing cell populations such as infiltrating monocytes and vascular smooth muscle cells (vSMCs). Transcription factors regulating proliferation and differentiation of atherosclerosis relevant cell types are of interest in this context. The forkhead box transcription factor FoxP1 modulates monocyte differentiation. We studied FoxP1 expression in atherosclerotic tissue, correlated FoxP1 expression with plaque characteristics and identified associations between FoxP1 and plaque proteins. 116 Atherosclerotic plaques from carotid endarterectomy samples were histologically classified (fibrous, fibroatheromatous, atheromatous) and subjected to semi-quantitative protein analysis. Macrophage, SMC content and intraplaque thrombus amount were determined histologically. FoxP1 expression was investigated by western blotting and immunohistochemistry. In addition FoxP1 was overexpressed in vitro to identify causal relations between FoxP1 and plaque proteins. FoxP1 expression was observed in SMCs, macrophages, endothelial cells and T-cells within the plaque. High SMC and collagen content correlated with increased FoxP1 isoform (72 kD and 95 kD) levels. 72 kD FoxP1 expression was lower in plaques containing intraplaque thrombus. FoxP1 correlated with active intraplaque TGFβ signaling. In vitro stimulation of SMCs with TGFβ resulted in increased FoxP1 levels. 72 kD FoxP1 correlated with expression of pro-fibrotic EGR-1 and increased Col1A1 expression. FoxP1 is expressed by different cell types in atherosclerotic lesions and associated with more stable plaque characteristics and intraplaque TGFβ signaling. FoxP1 expression in vitro is induced by TGFβ, resulting in increased collagen and EGR-1 expression, providing a mechanism for the observed association with a more stable plaque phenotype. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

  9. Decreased cytoplasmic X-box binding protein-1 expression is associated with poor prognosis and overall survival in patients with oral squamous cell carcinoma.

    Science.gov (United States)

    Hsu, Hui-Ting; Hsing, Ming-Tai; Yeh, Chung-Min; Chen, Chih-Jung; Yang, Jia-Sin; Yeh, Kun-Tu

    2018-01-02

    Squamous cell carcinoma is the most common cancer of the oral cavity. In spite of advancements in surgical, chemoradiological and targeted therapies, these therapeutic strategies still have had little impact on survival rates. X-box binding protein-1 (XBP-1) is a potent transcription factor that is involved in the unfolded protein response (UPR) pathway, which itself is activated in response to endoplasmic reticulum stress as a method to restore cellular homeostasis. The role XBP-1 plays in oral squamous cell carcinoma (OSCC) has yet to be determined. In this study, we used molecular and immunohistochemical analyses to investigate the role of XBP-1 protein playing in the OSCC carcinogenesis. We used immunohistochemical analyses to investigate XBP-1 expression in 255 OSCC tissue specimens, as well as migration and invasion assays with XBP-1 siRNA transfection of oral cancer cell lines to confirm its role in OSCC. The XBP-1 immunostaining was dichotomized as low-level expression and high-level expression. We found that low-level cytoplasmic XBP-1expression was significantly correlated with larger tumor size (p=0.047), more advanced clinical stage (pCox regression analysis revealed that cytoplasmic XBP-1 expression was a prognostic factor for overall survival of patients with OSCC. We also found that inhibition of XBP-1 promoted OSCC cell migration and invasion. Our results suggest that XBP-1 expression may play an essential role in the pathogenesis of OSCC and that targeting XBP-1 may be a sound therapeutic strategy. Copyright © 2018 Elsevier B.V. All rights reserved.

  10. The inner nuclear membrane protein Src1 associates with subtelomeric genes and alters their regulated gene expression

    OpenAIRE

    Grund, S.E.; Fischer, Tomás; Cabal, G.G.; Antúnez Temporal, Oreto; Pérez Ortín, José Enrique; Hurt, E.

    2008-01-01

    Inner nuclear membrane proteins containing a LEM (LAP2, emerin, and MAN1) domain participate in different processes, including chromatin organization, gene expression, and nuclear envelope biogenesis. In this study, we identify a robust genetic interaction between transcription export (TREX) factors and yeast Src1, an integral inner nuclear membrane protein that is homologous to vertebrate LEM2. DNA macroarray analysis revealed that the expression of the phosphate-regulated genes PHO11 ,PHO12...

  11. Evolutionary gradient of predicted nuclear localization signals (NLS)-bearing proteins in genomes of family Planctomycetaceae.

    Science.gov (United States)

    Guo, Min; Yang, Ruifu; Huang, Chen; Liao, Qiwen; Fan, Guangyi; Sun, Chenghang; Lee, Simon Ming-Yuen

    2017-04-04

    The nuclear envelope is considered a key classification marker that distinguishes prokaryotes from eukaryotes. However, this marker does not apply to the family Planctomycetaceae, which has intracellular spaces divided by lipidic intracytoplasmic membranes (ICMs). Nuclear localization signal (NLS), a short stretch of amino acid sequence, destines to transport proteins from cytoplasm into nucleus, and is also associated with the development of nuclear envelope. We attempted to investigate the NLS motifs in Planctomycetaceae genomes to demonstrate the potential molecular transition in the development of intracellular membrane system. In this study, we identified NLS-like motifs that have the same amino acid compositions as experimentally identified NLSs in genomes of 11 representative species of family Planctomycetaceae. A total of 15 NLS types and 170 NLS-bearing proteins were detected in the 11 strains. To determine the molecular transformation, we compared NLS-bearing protein abundances in the 11 representative Planctomycetaceae genomes with them in genomes of 16 taxonomically varied microorganisms: nine bacteria, two archaea and five fungi. In the 27 strains, 29 NLS types and 1101 NLS-bearing proteins were identified, principal component analysis showed a significant transitional gradient from bacteria to Planctomycetaceae to fungi on their NLS-bearing protein abundance profiles. Then, we clustered the 993 non-redundant NLS-bearing proteins into 181 families and annotated their involved metabolic pathways. Afterwards, we aligned the ten types of NLS motifs from the 13 families containing NLS-bearing proteins among bacteria, Planctomycetaceae or fungi, considering their diversity, length and origin. A transition towards increased complexity from non-planctomycete bacteria to Planctomycetaceae to archaea and fungi was detected based on the complexity of the 10 types of NLS-like motifs in the 13 NLS-bearing proteins families. The results of this study reveal that

  12. A carboxyl leucine-rich region of parathyroid hormone-related protein is critical for nuclear export.

    Science.gov (United States)

    Pache, Jared C; Burton, Douglas W; Deftos, Leonard J; Hastings, Randolph H

    2006-02-01

    PTHrP is an oncofetal protein with distinct proliferative and antiapoptotic roles that are affected by nucleocytoplasmic shuttling. The protein's nuclear export is sensitive to leptomycin B, consistent with a chromosome region maintenance protein 1-dependent pathway. We determined that the 109-139 region of PTHrP was involved in its nuclear export by demonstrating that a C-terminal truncation mutant, residues 1-108, exports at a reduced rate, compared with the wild-type 139 amino acid isoform. We searched for potential nuclear export sequences within the 109-139 region, which is leucine rich. Comparisons with established nuclear export sequences identified a putative consensus signal at residues 126-136. Deletion of this region resulted in nuclear export characteristics that closely matched those of the C-terminal truncation mutant. Confocal microscopic analyses of transfected 293, COS-1, and HeLa cells showed that steady-state nuclear levels of the truncated and deletion mutants were significantly greater than levels of wild-type PTHrP and were unaffected by leptomycin B, unlike the wild-type protein. In addition, both mutants demonstrated greatly reduced nuclear export with assays using nuclear preparations and intact cells. Based on these results, we conclude that the 126-136 amino acid sequence closely approximates the structure of a chromosome region maintenance protein 1-dependent leucine-rich nuclear export signal and is critical for nuclear export of PTHrP.

  13. Efficient nuclear export of p65-IkappaBalpha complexes requires 14-3-3 proteins.

    Science.gov (United States)

    Aguilera, Cristina; Fernández-Majada, Vanessa; Inglés-Esteve, Julia; Rodilla, Verónica; Bigas, Anna; Espinosa, Lluís

    2006-09-01

    IkappaB are responsible for maintaining p65 in the cytoplasm under non-stimulating conditions and promoting the active export of p65 from the nucleus following NFkappaB activation to terminate the signal. We now show that 14-3-3 proteins regulate the NFkappaB signaling pathway by physically interacting with p65 and IkappaBalpha proteins. We identify two functional 14-3-3 binding domains in the p65 protein involving residues 38-44 and 278-283, and map the interaction region of IkappaBalpha in residues 60-65. Mutation of these 14-3-3 binding domains in p65 or IkappaBalpha results in a predominantly nuclear distribution of both proteins. TNFalpha treatment promotes recruitment of 14-3-3 and IkappaBalpha to NFkappaB-dependent promoters and enhances the binding of 14-3-3 to p65. Disrupting 14-3-3 activity by transfection with a dominant-negative 14-3-3 leads to the accumulation of nuclear p65-IkappaBalpha complexes and the constitutive association of p65 with the chromatin. In this situation, NFkappaB-dependent genes become unresponsive to TNFalpha stimulation. Together our results indicate that 14-3-3 proteins facilitate the nuclear export of IkappaBalpha-p65 complexes and are required for the appropriate regulation of NFkappaB signaling.

  14. Down-regulation of Forkhead box protein A1 (FOXA1) leads to cancer stem cell-like properties in tamoxifen-resistant breast cancer cells through induction of interleukin-6.

    Science.gov (United States)

    Yamaguchi, Noritaka; Nakayama, Yuji; Yamaguchi, Naoto

    2017-05-19

    The selective estrogen receptor (ER) modulator tamoxifen inhibits ER signaling in breast cancer cells, and it is used for the treatment of ER-positive breast cancer. However, this type of cancer often acquires resistance to tamoxifen, and a better understanding of the molecular mechanisms underlying tamoxifen resistance is required. In this study, we established tamoxifen-resistant (TAM-R) breast cancer cells by long-term tamoxifen treatment of ER-positive breast cancer MCF7 cells. In TAM-R cells, expression of not only ERα, a major form of ER in breast cancer, but also its transcriptional partner forkhead box protein A1 (FOXA1) was found to be reduced. In contrast, activation of the transcription factor nuclear factor-κB (NF-κB) and expression of its target IL6 were increased in these cells. Stable expression of FOXA1, but not ERα, reduced the expression of IL6 in the FOXA1- and ERα-negative breast cancer MDA-MB-231 cells and TAM-R cells, without affecting the activation of the NF-κB signaling pathways. Conversely, FOXA1 knockdown induced IL6 expression in MCF7 cells. Chromatin immunoprecipitation assays revealed that FOXA1 bound to the promoter region of IL6 and repressed recruitment of the NF-κB complex to this region. TAM-R cells were found to have high mammosphere-forming activity, characteristics of cancer stem cells, and this activity was suppressed by NF-κB and IL6 signaling inhibitors. Taken together, these results suggest that FOXA1 suppresses expression of IL6 through inhibition of NF-κB recruitment to the IL6 promoter in an ERα-independent manner and that reduction in FOXA1 expression induces IL6 expression and contributes to cancer stem cell-like properties in TAM-R cells. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

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

    Energy Technology Data Exchange (ETDEWEB)

    Ostlund, Cecilia [Department of Medicine, College of Physicians and Surgeons, Columbia University, New York, NY 10032 (United States); Department of Pathology and Cell Biology, College of Physicians and Surgeons, Columbia University, New York, NY 10032 (United States); Guan, Tinglu [Department of Cell Biology, Scripps Research Institute, La Jolla, CA 92037 (United States); Figlewicz, Denise A. [Department of Neurology, University of Michigan, Ann Arbor, MI 48109 (United States); Hays, Arthur P. [Department of Pathology and Cell Biology, College of Physicians and Surgeons, Columbia University, New York, NY 10032 (United States); Worman, Howard J. [Department of Medicine, College of Physicians and Surgeons, Columbia University, New York, NY 10032 (United States); Department of Pathology and Cell Biology, College of Physicians and Surgeons, Columbia University, New York, NY 10032 (United States); Gerace, Larry [Department of Cell Biology, Scripps Research Institute, La Jolla, CA 92037 (United States); Schirmer, Eric C., E-mail: e.schirmer@ed.ac.uk [Department of Cell Biology, Scripps Research Institute, La Jolla, CA 92037 (United States); Wellcome Trust Centre for Cell Biology, University of Edinburgh, Edinburgh EH9 3JR (United Kingdom)

    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.

  16. Acetylation of the SUN protein Mps3 by Eco1 regulates its function in nuclear organization

    Science.gov (United States)

    Ghosh, Suman; Gardner, Jennifer M.; Smoyer, Christine J.; Friederichs, Jennifer M.; Unruh, Jay R.; Slaughter, Brian D.; Alexander, Richard; Chisholm, Robert D.; Lee, Kenneth K.; Workman, Jerry L.; Jaspersen, Sue L.

    2012-01-01

    The Saccharomyces cerevisiae SUN-domain protein Mps3 is required for duplication of the yeast centrosome-equivalent organelle, the spindle pole body (SPB), and it is involved in multiple aspects of nuclear organization, including telomere tethering and gene silencing at the nuclear membrane, establishment of sister chromatid cohesion, and repair of certain types of persistent DNA double-stranded breaks. How these diverse SUN protein functions are regulated is unknown. Here we show that the Mps3 N-terminus is a substrate for the acetyltransferase Eco1/Ctf7 in vitro and in vivo and map the sites of acetylation to three lysine residues adjacent to the Mps3 transmembrane domain. Mutation of these residues shows that acetylation is not essential for growth, SPB duplication, or distribution in the nuclear membrane. However, analysis of nonacetylatable mps3 mutants shows that this modification is required for accurate sister chromatid cohesion and for chromosome recruitment to the nuclear membrane. Acetylation of Mps3 by Eco1 is one of the few regulatory mechanisms known to control nuclear organization. PMID:22593213

  17. Effects of spectator ligands on the specific recognition of intrastrand platinum-DNA cross-links by high mobility group box and TATA-binding proteins.

    Science.gov (United States)

    Wei, M; Cohen, S M; Silverman, A P; Lippard, S J

    2001-10-19

    The results presented describe the effects of various spectator ligands, attached to a platinum 1,2-intrastand d(GpG) cross-link in duplex DNA, on the binding of high mobility group box (HMGB) domains and the TATA-binding protein (TBP). In addition to cisplatin-modified DNA, 15-base pair DNA probes modified by [Pt(1R,2R-diaminocyclohexane)](2+), cis-[Pt(NH(3))(cyclohexylamine)](2+), [Pt(ethylenediamine)](2+), cis-[Pt(NH(3))(cyclobutylamine)](2+), and cis-[Pt(NH(3))(2-picoline)](2+) were examined. Electrophoretic mobility shift assays show that both the A and B domains of HMGB1 as well as TBP discriminate between different platinum-DNA adducts. HMGB1 domain A is the most sensitive to the nature of the spectator ligands on platinum. The effect of the spectator ligands on protein binding also depends highly on the base pairs flanking the platinated d(GpG) site. Double-stranded oligonucleotides containing the AG*G*C sequence, where the asterisks denote the sites of platination, with different spectator ligands are only moderately discriminated by the HMGB proteins and TBP, but the recognition of dsTG*G*A is highly dependent on the ligands. The effects of HMGB1 overexpression in a BG-1 ovarian cancer cell line, induced by steroid hormones, on the sensitivity of cells treated with [Pt(1R,2R-diaminocyclohexane)Cl(2)] and cis-[Pt(NH(3))(cyclohexylamine)Cl(2)] were also examined. The results suggest that HMGB1 protein levels influence the cellular processing of cis-[Pt(NH(3))- (cyclohexylamine)](2+), but not [Pt((1R,2R)-diaminocyclohexane)](2+), DNA lesions. This result is consistent with the observed binding of HMGB1a to platinum-modified dsTG*G*A probes but not with the binding affinity of HMGB1a and HMGB1 to platinum-damaged dsAG*G*C oligonucleotides. These experiments reinforce the importance of sequence context in platinum-DNA lesion recognition by cellular proteins.

  18. TIF1alpha: a possible link between KRAB zinc finger proteins and nuclear receptors

    DEFF Research Database (Denmark)

    Le Douarin, B; You, J; Nielsen, Anders Lade

    1998-01-01

    family of proteins which contains an N-terminal RBCC (RING finger-B boxes-coiled coil) motif and a C-terminal bromodomain preceded by a PHD finger. In addition to these conserved domains present in a number of transcriptional regulatory proteins, TIF1alpha was found to contain several protein......1alpha and MOD1, which both are heterochromatinic proteins. Finally, TIF1alpha also has a binding site for KRAB silencing domains of C2H2 zinc finger proteins. TIF1beta, another member of the TIF1 gene family, has some interacting partners in common with TIF1alpha. TIF1beta can interact with HP1......alpha, MOD1 and KRAB domains, but apparently not with NRs. Both TIF1alpha and TIF1beta repress transcription when fused to a DNA binding domain in transiently transfected mammalian cells. A model discussing the potential function(s) of TIF1s in the control of transcription at the level of the chromatin...

  19. Calcium/Calmodulin-Dependent Protein Kinase is Involved in the Release of High Mobility Group Box 1 Via the Interferon-β Signaling Pathway

    Science.gov (United States)

    Ma, Lijuan; Kim, Seon-Ju

    2012-01-01

    Previously, we have reported that high mobility group box 1 (HMGB1), a proinflammatory mediator in sepsis, is released via the IFN-β-mediated JAK/STAT pathway. However, detailed mechanisms are still unclear. In this study, we dissected upstream signaling pathways of HMGB1 release using various molecular biology methods. Here, we found that calcium/calmodulin-dependent protein kinase (CaM kinase, CaMK) is involved in HMGB1 release by regulating IFN-β production. CaMK inhibitor, STO609, treatment inhibits LPS-induced IFN-β production, which is correlated with the phosphorylation of interferon regulatory factor 3 (IRF3). Additionally, we show that CaMK-I plays a major role in IFN-β production although other CaMK members also seem to contribute to this event. Furthermore, the CaMK inhibitor treatment reduced IFN-β production in a murine endotoxemia. Our results suggest CaMKs contribute to HMGB1 release by enhancing IFN-β production in sepsis. PMID:23091438

  20. Y-box Protein-1 Regulates the Expression of Collagen I in Hepatic Progenitor Cells via PDGFR-β/ERK/p90RSK Signalling

    Directory of Open Access Journals (Sweden)

    Fei Li

    2017-01-01

    Full Text Available Y-box protein-1 (YB-1 is a highly conserved transcription factor that is involved in multiple biological processes via transcriptional regulation of several genes, including p53, cyclin D1, and EGFR. YB-1 has been reported to be overexpressed in injured livers. This study aims to explore the functions of YB-1 in hepatic progenitor cells (HPCs. Herein, chromatin immunoprecipitation sequencing (ChIP-sequencing and RNA-sequencing assays identified that YB-1 participated in the biological adhesion process and ECM-receptor interactions in HPCs. Further study demonstrated that YB-1 modulated the expression of extracellular matrix components in HPCs. ChIP-sequencing assays established that PDGFR-β was a target gene of YB-1, and luciferase reporter assays confirmed that YB-1 negatively regulated PDGFR-β promoter activity in HPCs. In addition, PDGFR-β can regulate the expression of collagen I through ERK/p90RSK signalling, and disruption of the signalling pathway with a PDGFR-β inhibitor or ERK1/2 inhibitor abolished the regulatory effect of PDGFR-β on collagen I expression in HPCs. Conclusively, YB-1 can modulate the expression of collagen I in HPCs via direct binding to the PDGFR-β promoter, negatively regulating its expression. In addition, the ERK/p90RSK axis serves as the downstream signalling pathway of PDGFR-β.

  1. Brief Report: Endothelial-Specific X-Box Binding Protein 1 Deficiency Limits Tumor Necrosis Factor-Induced Leukocyte Recruitment and Vasculitis.

    Science.gov (United States)

    Ziogas, Athanasios; Muders, Michael H; Economopoulou, Matina; Sprott, David; Grossklaus, Sylvia; Siegert, Gabriele; Baretton, Gustavo B; Mitroulis, Ioannis; Chavakis, Triantafyllos

    2015-12-01

    Endothelial cell activation by tumor necrosis factor (TNF) and associated leukocyte infiltration are hallmarks of vasculitis. The aim of this study was to investigate the potential role of the cellular stress-associated endothelial X-box binding protein 1 (XBP-1) transcription factor in TNF-induced endothelial cell inflammation and vasculitis. Mice with an endothelial cell-specific XBP-1 deficiency were used in a modified local Shwartzman reaction (LSR) model of TNF-induced small vessel vasculitis. To address the contribution of XBP-1 to the TNF-mediated inflammatory response in endothelial cells, we examined the activation of XBP-1 expression by TNF as well as the effect of XBP-1 knockdown in endothelial cells on TNF-induced signaling, proinflammatory gene expression, and leukocyte-endothelial cell adhesion. The active spliced form of XBP-1 in endothelial cells was triggered by TNF. In addition, endothelial XBP-1 contributed to the sustained TNF-triggered NF-κB-dependent transcriptional activation of proinflammatory molecules, which was associated with leukocyte-endothelial cell adhesion. In the LSR model, endothelial cell-specific XBP-1-deficient mice displayed significantly less vascular damage, accompanied by reduced perivascular neutrophil infiltration, as compared with wild-type mice. Endothelial XBP-1 is activated by TNF and regulates leukocyte-endothelial cell adhesion in vitro as well as neutrophil infiltration and vascular damage in murine vasculitis. © 2015, American College of Rheumatology.

  2. COX-2 correlates with F-box protein, Skp2 expression and prognosis in human gastric carcinoma.

    Science.gov (United States)

    Honjo, Soichiro; Kase, Satoru; Osaki, Mitsuhiko; Ardyanto, Tonang Dwi; Kaibara, Nobuaki; Ito, Hisao

    2005-02-01

    The expression of cyclooxygenase (COX)-2 is induced by growth factors, tumor promoters and cytokines, and is correlated with carcinogenesis, tumor progression and inhibition of apoptosis. To clarify the pathological significance of COX-2, we examined the effect of a selective COX-2 inhibitor, NS398, on two human gastric carcinoma cell lines, MKN-45 and KATO-III, and the expression of Skp2, P27/Kip1 and COX-2 protein in human gastric carcinomas. NS398 inhibited cell growth in a time- and dose-dependent manner and exerted cell cycle arrest in the G0/G1 phase without induction of apoptosis in MKN-45, but had no effect in KATO-III. In MKN-45, NS398 induced up-regulation of P27/Kip1 and down-regulation of COX-2, cyclin D1 and Skp2. Immunohistochemistry using 63 surgically resected gastric carcinomas disclosed that COX-2 expression was correlated with Skp2 expression and that P27/Kip1 expression was inversely correlated with COX-2 and Skp2 expression. High levels of COX-2 or Skp2 were significantly correlated with poor survival (P=0.02 and P=0.004). Our results suggested that: a) NS398 induced inhibition of cell proliferation through cell cycle arrest and suppressed the expression of Skp2 in COX-2-expressing gastric carcinoma cells, and b) COX-2 contributes to the expression of Skp2 and poor survival in human gastric carcinomas.

  3. The clinicopathological significance of forkhead box P1 and forkhead box O3a in pancreatic ductal adenocarcinomas.

    Science.gov (United States)

    Luo, Xin; Yang, Zhulin; Liu, Xiao; Liu, Ziru; Miao, Xiongying; Li, Daiqiang; Zou, Qiong; Yuan, Yuan

    2017-05-01

    Pancreatic ductal adenocarcinoma is a highly malignant tumor with poor prognosis, and the biomarkers for the early diagnosis, targeting therapy, and prognosis are still not clinically available. This study investigated the expression of forkhead box P1 and forkhead box O3a proteins in human pancreatic ductal adenocarcinoma tumor tissues and pancreatic tissues with and without benign lesions using immunohistochemical staining. Results showed that the positive rates of forkhead box P1 and forkhead box O3a protein expression were significantly lower in pancreatic ductal adenocarcinoma tumors compared to peritumoral tissues, benign pancreatic tissues, and normal pancreatic tissues (p box P1 and forkhead box O3a protein expression exhibited dysplasia or intraepithelial neoplasia. The positive rates of forkhead box P1 and forkhead box O3a expression were significantly lower in cases with tumor mass >5 cm, lymph node metastasis, invasion to surrounding tissues and organs, and tumor-node-metastasis III + IV stage disease compared to cases with tumor mass ⩽5 cm (p box P1 and forkhead box O3a expression survived significantly shorter than patients with positive forkhead box P1 and forkhead box O3a expression (p = 0.000). Cox multivariate analysis revealed that negative forkhead box P1 and forkhead box O3a expression was an independent poor prognosis factor in pancreatic ductal adenocarcinoma patients. The area under the curve of a receiver operating characteristic curve was 0.642 for forkhead box P1 (95% confidence interval: 0.553-0.730) and 0.655 for forkhead box O3a (95% confidence interval: 0.6568-0.742). Loss of forkhead box P1 and forkhead box O3a protein expression is associated with carcinogenesis, progression, and poor prognosis in patients with pancreatic ductal adenocarcinomas.

  4. Comparative Analysis of the 15.5kD Box C/D snoRNP Core Protein in the Primitive Eukaryote Giardia lamblia Reveals Unique Structural and Functional Features

    Energy Technology Data Exchange (ETDEWEB)

    Biswas, Shyamasri; Buhrman, Greg; Gagnon, Keith; Mattos, Carla; Brown, II, Bernard A.; Maxwell, E. Stuart (NCSU); (UTSMC)

    2012-07-11

    Box C/D ribonucleoproteins (RNP) guide the 2'-O-methylation of targeted nucleotides in archaeal and eukaryotic rRNAs. The archaeal L7Ae and eukaryotic 15.5kD box C/D RNP core protein homologues initiate RNP assembly by recognizing kink-turn (K-turn) motifs. The crystal structure of the 15.5kD core protein from the primitive eukaryote Giardia lamblia is described here to a resolution of 1.8 {angstrom}. The Giardia 15.5kD protein exhibits the typical {alpha}-{beta}-{alpha} sandwich fold exhibited by both archaeal L7Ae and eukaryotic 15.5kD proteins. Characteristic of eukaryotic homologues, the Giardia 15.5kD protein binds the K-turn motif but not the variant K-loop motif. The highly conserved residues of loop 9, critical for RNA binding, also exhibit conformations similar to those of the human 15.5kD protein when bound to the K-turn motif. However, comparative sequence analysis indicated a distinct evolutionary position between Archaea and Eukarya. Indeed, assessment of the Giardia 15.5kD protein in denaturing experiments demonstrated an intermediate stability in protein structure when compared with that of the eukaryotic mouse 15.5kD and archaeal Methanocaldococcus jannaschii L7Ae proteins. Most notable was the ability of the Giardia 15.5kD protein to assemble in vitro a catalytically active chimeric box C/D RNP utilizing the archaeal M. jannaschii Nop56/58 and fibrillarin core proteins. In contrast, a catalytically competent chimeric RNP could not be assembled using the mouse 15.5kD protein. Collectively, these analyses suggest that the G. lamblia 15.5kD protein occupies a unique position in the evolution of this box C/D RNP core protein retaining structural and functional features characteristic of both archaeal L7Ae and higher eukaryotic 15.5kD homologues.

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

    Energy Technology Data Exchange (ETDEWEB)

    Siyam, Arwa [Department of Biomedical Sciences, Baylor College of Dentistry, Texas A and M Health Science Center, 3302 Gaston Ave., Dallas, TX 75246-2013 (United States); Department of Endodontology, Kornberg School of Dentistry, Temple University, 3223 North Broad Street, Philadelphia, PA 19140-5007 (United States); Wang, Suzhen; Qin, Chunlin; Mues, Gabriele [Department of Biomedical Sciences, Baylor College of Dentistry, Texas A and M Health Science Center, 3302 Gaston Ave., Dallas, TX 75246-2013 (United States); Stevens, Roy [Department of Endodontology, Kornberg School of Dentistry, Temple University, 3223 North Broad Street, Philadelphia, PA 19140-5007 (United States); D' Souza, Rena N. [Department of Biomedical Sciences, Baylor College of Dentistry, Texas A and M Health Science Center, 3302 Gaston Ave., Dallas, TX 75246-2013 (United States); Lu, Yongbo, E-mail: ylu@bcd.tamhsc.edu [Department of Biomedical Sciences, Baylor College of Dentistry, Texas A and M Health Science Center, 3302 Gaston Ave., Dallas, TX 75246-2013 (United States)

    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.

  6. Expression of Leukemia-Associated Nup98 Fusion Proteins Generates an Aberrant Nuclear Envelope Phenotype.

    Science.gov (United States)

    Fahrenkrog, Birthe; Martinelli, Valérie; Nilles, Nadine; Fruhmann, Gernot; Chatel, Guillaume; Juge, Sabine; Sauder, Ursula; Di Giacomo, Danika; Mecucci, Cristina; Schwaller, Jürg

    2016-01-01

    Chromosomal translocations involving the nucleoporin NUP98 have been described in several hematopoietic malignancies, in particular acute myeloid leukemia (AML). In the resulting chimeric proteins, Nup98's N-terminal region is fused to the C-terminal region of about 30 different partners, including homeodomain (HD) transcription factors. While transcriptional targets of distinct Nup98 chimeras related to immortalization are relatively well described, little is known about other potential cellular effects of these fusion proteins. By comparing the sub-nuclear localization of a large number of Nup98 fusions with HD and non-HD partners throughout the cell cycle we found that while all Nup98 chimeras were nuclear during interphase, only Nup98-HD fusion proteins exhibited a characteristic speckled appearance. During mitosis, only Nup98-HD fusions were concentrated on chromosomes. Despite the difference in localization, all tested Nup98 chimera provoked morphological alterations in the nuclear envelope (NE), in particular affecting the nuclear lamina and the lamina-associated polypeptide 2α (LAP2α). Importantly, such aberrations were not only observed in transiently transfected HeLa cells but also in mouse bone marrow cells immortalized by Nup98 fusions and in cells derived from leukemia patients harboring Nup98 fusions. Our findings unravel Nup98 fusion-associated NE alterations that may contribute to leukemogenesis.

  7. SMRT has tissue-specific isoform profiles that include a form containing one CoRNR box.

    Science.gov (United States)

    Short, Stephen; Malartre, Marianne; Sharpe, Colin

    2005-09-02

    SMRT acts as a corepressor for a range of transcription factors. The amino-terminal part of the protein includes domains that mainly mediate transcriptional repression whilst the carboxy-terminal part includes domains that interact with nuclear receptors using up to three motifs called CoRNR boxes. The region of the SMRT primary transcript encoding the interaction domains is subject to alternative splicing that varies the inclusion of the third CoRNR box. The profile in mice includes an abundant, novel SMRT isoform that possesses just one CoRNR box. Mouse tissues therefore express SMRT isoforms containing one, two or three CoRNR boxes. In frogs, the SMRT isoform profile is tissue-specific. The mouse also shows distinct profiles generated by differential expression levels of the SMRT transcript isoforms. The formation of multiple SMRT isoforms and their tissue-specific regulation indicates a mechanism, whereby cells can define the repertoire of transcription factors regulated by SMRT.

  8. Molecular decoy to the Y-box binding protein-1 suppresses the growth of breast and prostate cancer cells whilst sparing normal cell viability.

    Directory of Open Access Journals (Sweden)

    Jennifer H Law

    2010-09-01

    Full Text Available The Y-box binding protein-1 (YB-1 is an oncogenic transcription/translation factor that is activated by phosphorylation at S102 whereby it induces the expression of growth promoting genes such as EGFR and HER-2. We recently illustrated by an in vitro kinase assay that a novel peptide to YB-1 was highly phosphorylated by the serine/threonine p90 S6 kinases RSK-1 and RSK-2, and to a lesser degree PKCα and AKT. Herein, we sought to develop this decoy cell permeable peptide (CPP as a cancer therapeutic. This 9-mer was designed as an interference peptide that would prevent endogenous YB-1(S102 phosphorylation based on molecular docking. In cancer cells, the CPP blocked P-YB-1(S102 and down-regulated both HER-2 and EGFR transcript level and protein expression. Further, the CPP prevented YB-1 from binding to the EGFR promoter in a gel shift assay. Notably, the growth of breast (SUM149, MDA-MB-453, AU565 and prostate (PC3, LNCap cancer cells was inhibited by ∼90% with the CPP. Further, treatment with this peptide enhanced sensitivity and overcame resistance to trastuzumab in cells expressing amplified HER-2. By contrast, the CPP had no inhibitory effect on the growth of normal immortalized breast epithelial (184htert cells, primary breast epithelial cells, nor did it inhibit differentiation of hematopoietic progenitors. These data collectively suggest that the CPP is a novel approach to suppressing the growth of cancer cells while sparing normal cells and thereby establishes a proof-of-concept that blocking YB-1 activation is a new course of cancer therapeutics.

  9. The extracellular release of Schistosoma mansoni HMGB1 nuclear protein is mediated by acetylation

    Energy Technology Data Exchange (ETDEWEB)

    Coutinho Carneiro, Vitor; Moraes Maciel, Renata de; Caetano de Abreu da Silva, Isabel; Furtado Madeira da Costa, Rodrigo [Instituto de Bioquimica Medica, Programa de Biotecnologia e Biologia Molecular, Universidade Federal do Rio de Janeiro, CCS, Ilha do Fundao, Rio de Janeiro 21941-590 (Brazil); Neto Paiva, Claudia; Torres Bozza, Marcelo [Departamento de Imunologia, Instituto de Microbiologia Professor Paulo de Goes, Universidade Federal do Rio de Janeiro, CCS, Ilha do Fundao, Rio de Janeiro 21941-590 (Brazil); Rosado Fantappie, Marcelo, E-mail: fantappie@bioqmed.ufrj.br [Instituto de Bioquimica Medica, Programa de Biotecnologia e Biologia Molecular, Universidade Federal do Rio de Janeiro, CCS, Ilha do Fundao, Rio de Janeiro 21941-590 (Brazil)

    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.

  10. Vaccinia virus G8R protein: a structural ortholog of proliferating cell nuclear antigen (PCNA.

    Directory of Open Access Journals (Sweden)

    Melissa Da Silva

    Full Text Available 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.

  11. Nuclear entry of hepatitis B virus capsids involves disintegration to protein dimers followed by nuclear reassociation to capsids.

    Directory of Open Access Journals (Sweden)

    Birgit Rabe

    2009-08-01

    Full Text Available Assembly and disassembly of viral capsids are essential steps in the viral life cycle. Studies on their kinetics are mostly performed in vitro, allowing application of biochemical, biophysical and visualizing techniques. In vivo kinetics are poorly understood and the transferability of the in vitro models to the cellular environment remains speculative. We analyzed capsid disassembly of the hepatitis B virus in digitonin-permeabilized cells which support nuclear capsid entry and subsequent genome release. Using gradient centrifugation, size exclusion chromatography and immune fluorescence microscopy of digitonin-permeabilized cells, we showed that capsids open and close reversibly. In the absence of RNA, capsid re-assembly slows down; the capsids remain disintegrated and enter the nucleus as protein dimers or irregular polymers. Upon the presence of cellular RNA, capsids re-assemble in the nucleus. We conclude that reversible genome release from hepatitis B virus capsids is a unique strategy different from that of other viruses, which employs irreversible capsid destruction for genome release. The results allowed us to propose a model of HBV genome release in which the unique environment of the nuclear pore favors HBV capsid disassembly reaction, while both cytoplasm and nucleus favor capsid assembly.

  12. Drosophila proteins involved in metabolism of uracil-DNA possess different types of nuclear localization signals.

    Science.gov (United States)

    Merényi, Gábor; Kónya, Emese; Vértessy, Beáta G

    2010-05-01

    Adequate transport of large proteins that function in the nucleus is indispensable for cognate molecular events within this organelle. Selective protein import into the nucleus requires nuclear localization signals (NLS) that are recognized by importin receptors in the cytoplasm. Here we investigated the sequence requirements for nuclear targeting of Drosophila proteins involved in the metabolism of uracil-substituted DNA: the recently identified uracil-DNA degrading factor, dUTPase, and the two uracil-DNA glycosylases present in Drosophila. For the uracil-DNA degrading factor, NLS prediction identified two putative NLS sequences [PEKRKQE(320-326) and PKRKKKR(347-353)]. Truncation and site-directed mutagenesis using YFP reporter constructs showed that only one of these basic stretches is critically required for efficient nuclear localization in insect cells. This segment corresponds to the well-known prototypic NLS of SV40 T-antigen. An almost identical NLS segment is also present in the Drosophila thymine-DNA glycosylase, but no NLS elements were predicted in the single-strand-specific monofunctional uracil-DNA glycosylase homolog protein. This latter protein has a molecular mass of 31 kDa, which may allow NLS-independent transport. For Drosophila dUTPase, two isoforms with distinct features regarding molecular mass and subcellular distribution were recently described. In this study, we characterized the basic PAAKKMKID(10-18) segment of dUTPase, which has been predicted to be a putative NLS by in silico analysis. Deletion studies, using YFP reporter constructs expressed in insect cells, revealed the importance of the PAA(10-12) tripeptide and the ID(17-18) dipeptide, as well as the role of the PAAK(10-13) segment in nuclear localization of dUTPase. We constructed a structural model that shows the molecular basis of such recognition in three dimensions.

  13. The DEAD-box Protein Rok1 Orchestrates 40S and 60S Ribosome Assembly by Promoting the Release of Rrp5 from Pre-40S Ribosomes to Allow for 60S Maturation.

    Science.gov (United States)

    Khoshnevis, Sohail; Askenasy, Isabel; Johnson, Matthew C; Dattolo, Maria D; Young-Erdos, Crystal L; Stroupe, M Elizabeth; Karbstein, Katrin

    2016-06-01

    DEAD-box proteins are ubiquitous regulators of RNA biology. While commonly dubbed "helicases," their activities also include duplex annealing, adenosine triphosphate (ATP)-dependent RNA binding, and RNA-protein complex remodeling. Rok1, an essential DEAD-box protein, and its cofactor Rrp5 are required for ribosome assembly. Here, we use in vivo and in vitro biochemical analyses to demonstrate that ATP-bound Rok1, but not adenosine diphosphate (ADP)-bound Rok1, stabilizes Rrp5 binding to 40S ribosomes. Interconversion between these two forms by ATP hydrolysis is required for release of Rrp5 from pre-40S ribosomes in vivo, thereby allowing Rrp5 to carry out its role in 60S subunit assembly. Furthermore, our data also strongly suggest that the previously described accumulation of snR30 upon Rok1 inactivation arises because Rrp5 release is blocked and implicate a previously undescribed interaction between Rrp5 and the DEAD-box protein Has1 in mediating snR30 accumulation when Rrp5 release from pre-40S subunits is blocked.

  14. O2 sensing associated glycosylation exposes the F-box combining site of the Dictyostelium Skp1 subunit in E3 ubiquitin ligases.

    Science.gov (United States)

    Sheikh, M Osman; Thieker, David; Chalmers, Gordon; Schafer, Christopher M; Ishihara, Mayumi; Azadi, Parastoo; Woods, Robert J; Glushka, John N; Bendiak, Brad; Prestegard, James H; West, Christopher M

    2017-09-19

    Skp1 is a conserved protein linking cullin-1 to F-box proteins in SCF (Skp1-Cullin1-F-box) E3 ubiquitin ligases, which modify protein substrates with polyubiquitin chains that typically target them for 26S proteasome-mediated degradation. In Dictyostelium (a social amoeba), Toxoplasma gondii (the agent for human toxoplasmosis), and other protists, Skp1 is regulated by a unique pentasaccharide attached to hydroxylated Pro-143 within its C-terminal F-box binding domain. Prolyl hydroxylation of Skp1 contributes to O2-dependent Dictyostelium development, but full glycosylation at that position is required for optimal O2 sensing. Previous studies have shown that the glycan promotes organization of the F-box binding region in Skp1, and aids in Skp1's association with F-box proteins. Here, nuclear magnetic resonance and mass spectrometry approaches were used to determine the glycan structure, and then a combination of NMR and molecular dynamics simulations were employed to characterize the impact of the glycan on the conformation and motions of the intrinsically flexible F-box binding domain of Skp1. MD trajectories of glycosylated Skp1 whose calculated monosaccharide relaxation kinetics and rotational correlation times agreed with the NMR data indicated that the glycan interacts with the loop connecting two alpha-helices of the F-box combining site. In these trajectories, the helices separated from one another to create a more accessible and dynamic F-box interface. These results offer an unprecedented view of how a glycan modification influences a disordered region of a full-length protein. The increased sampling of an open Skp1 conformation can explain how glycosylation enhances interactions with F-box proteins in cells. Copyright © 2017, The American Society for Biochemistry and Molecular Biology.

  15. C-reactive protein reacts with the U1 small nuclear ribonucleoprotein

    Energy Technology Data Exchange (ETDEWEB)

    Du Clos, T.W. (VA Medical Center, Albuquerque, NM (USA))

    1989-10-15

    C-reactive protein (CRP) was found to produce a small, discrete, speckled fluorescence pattern in the nucleus of HEp-2 cells. Double staining with anti-RNP serum and CRP produced very similar staining patterns. By counterimmunoelectrophoresis CRP was bound to extractable nuclear antigens found in rabbit thymus extract. The reactive components of the extract were only partially sensitive to treatment with RNase. CRP immunoprecipitated the U1 RNA species from ({sup 32}P)labeled HeLa cells and the protein bands of the Sm/RNP complex from ({sup 35}S)-methionine-labeled HeLa cells. By blotting, CRP bound to several discrete bands in a calcium-dependent, PC-inhibitable manner. Two of the bands comigrated with the 70K protein band associated with the U1 snRNP, and its major breakdown product. Binding to these bands was inhibited by both EDTA and PC indicating that CRP binds these proteins through the PC-binding site. Binding to the 70K protein of the U1 snRNP was confirmed by reactivity with the recombinant 70K protein in a dot blot. These findings indicate the CRP binds to the U1-RNP snRNP particle. Considering the ability of CRP to inhibit antibody responses to its ligands and its ability to activate C and promote phagocytosis it is suggested that CRP may play a role in the regulation of autoantibody responses to nuclear Ag.

  16. Enhancement of protein transduction-mediated nuclear delivery by interaction with dynein/microtubules.

    Science.gov (United States)

    Moseley, Gregory W; Leyton, Denisse L; Glover, Dominic J; Filmer, Richard P; Jans, David A

    2010-02-01

    Nucleocytoplasmic trafficking is a major consideration for the design of vehicles for the delivery of drug/DNA cargo to cell nuclei for cancer and gene therapies. Recent data indicate that efficient nuclear import can involve the microtubule (MT)/dynein network, such that nuclear delivery of exogenous cargo could be enhanced by attachment to peptide modules mediating association with dynein components, but this has not been investigated. Here, we report that the nuclear delivery of an exogenous cargo that enters the cell by protein transduction can be enhanced by attachment to a dynein-association sequence, with dependence on the MT network. This indicates that dynein/MT-association modules may provide useful modules for DNA/drug delivery approaches. Copyright 2009 Elsevier B.V. All rights reserved.

  17. Inhibition of CRM1-mediated nuclear export of transcription factors by leukemogenic NUP98 fusion proteins.

    Science.gov (United States)

    Takeda, Akiko; Sarma, Nayan J; Abdul-Nabi, Anmaar M; Yaseen, Nabeel R

    2010-05-21

    NUP98 is a nucleoporin that plays complex roles in the nucleocytoplasmic trafficking of macromolecules. Rearrangements of the NUP98 gene in human leukemia result in the expression of numerous fusion oncoproteins whose effect on nucleocytoplasmic trafficking is poorly understood. The present study was undertaken to determine the effects of leukemogenic NUP98 fusion proteins on CRM1-mediated nuclear export. NUP98-HOXA9, a prototypic NUP98 fusion, inhibited the nuclear export of two known CRM1 substrates: mutated cytoplasmic nucleophosmin and HIV-1 Rev. In vitro binding assays revealed that NUP98-HOXA9 binds CRM1 through the FG repeat motif in a Ran-GTP-dependent manner similar to but stronger than the interaction between CRM1 and its export substrates. Two NUP98 fusions, NUP98-HOXA9 and NUP98-DDX10, whose fusion partners are structurally and functionally unrelated, interacted with endogenous CRM1 in myeloid cells as shown by co-immunoprecipitation. These leukemogenic NUP98 fusion proteins interacted with CRM1, Ran, and the nucleoporin NUP214 in a manner fundamentally different from that of wild-type NUP98. NUP98-HOXA9 and NUP98-DDX10 formed characteristic aggregates within the nuclei of a myeloid cell line and primary human CD34+ cells and caused aberrant localization of CRM1 to these aggregates. These NUP98 fusions caused nuclear accumulation of two transcription factors, NFAT and NFkappaB, that are regulated by CRM1-mediated export. The nuclear entrapment of NFAT and NFkappaB correlated with enhanced transcription from promoters responsive to these transcription factors. Taken together, the results suggest a new mechanism by which NUP98 fusions dysregulate transcription and cause leukemia, namely, inhibition of CRM1-mediated nuclear export with aberrant nuclear retention of transcriptional regulators.

  18. Immunocytochemical staining of endogenous nuclear proteins with the HIS-1 anti-poly-histidine monoclonal antibody: a potential source of error in His-tagged protein detection.

    Science.gov (United States)

    Chilumuri, Amrutha; Markiv, Anatoliy; Milton, Nathaniel G N

    2014-07-01

    Histidine-tagged proteins are widely used in biochemical studies and frequently detected with antibodies specific for the histidine tag. Immunocytochemistry is widely used in studies with overexpressed proteins to determine cellular localization and in the case of histidine-tagged proteins can be carried out with anti-polyhistidine antibodies. Recent studies have suggested that polyhistidine sequences are present within a small number of human proteins and may direct expression to the nucleus and nuclear speckles compartments of the cell. In this study immunocytochemical staining of human SH-SY5Y neuroblastoma cell lines with the HIS-1 anti-polyhistidine monoclonal antibody were determined. Results showed that the HIS-1 anti-polyhistidine monoclonal antibody stained endogenous nuclear proteins in SH-SY5Y cells. The stained proteins were contained within the nuclear membrane, but were not directly linked to DNA. In a histidine-tagged catalase overexpressing cell line the HIS-1 anti-polyhistidine monoclonal antibody showed nuclear staining, whilst staining with the CAT-505 anti-catalase monoclonal antibody showed primarily cytoplasmic staining. These results suggest that anti-polyhistidine antibody staining shows significant cross-reactivity with endogenous nuclear proteins in SH-SY5Y neuroblastoma cells and may not be suitable for localization studies of histidine-tagged proteins. Immunocytochemical studies with anti-polyhistidine antibodies and localization of histidine-tagged proteins must be confirmed with protein specific antibodies or other methodology. Copyright © 2014 Elsevier GmbH. All rights reserved.

  19. Nuclear Protein Sam68 Interacts with the Enterovirus 71 Internal Ribosome Entry Site and Positively Regulates Viral Protein Translation.

    Science.gov (United States)

    Zhang, Hua; Song, Lei; Cong, Haolong; Tien, Po

    2015-10-01

    Enterovirus 71 (EV71) recruits various cellular factors to assist in the replication and translation of its genome. Identification of the host factors involved in the EV71 life cycle not only will enable a better understanding of the infection mechanism but also has the potential to be of use in the development of antiviral therapeutics. In this study, we demonstrated that the cellular factor 68-kDa Src-associated protein in mitosis (Sam68) acts as an internal ribosome entry site (IRES) trans-acting factor (ITAF) that binds specifically to the EV71 5' untranslated region (5'UTR). Interaction sites in both the viral IRES (stem-loops IV and V) and the heterogeneous nuclear ribonucleoprotein K homology (KH) domain of Sam68 protein were further mapped using an electrophoretic mobility shift assay (EMSA) and biotin RNA pulldown assay. More importantly, dual-luciferase (firefly) reporter analysis suggested that overexpression of Sam68 positively regulated IRES-dependent translation of virus proteins. In contrast, both IRES activity and viral protein translation significantly decreased in Sam68 knockdown cells compared with the negative-control cells treated with short hairpin RNA (shRNA). However, downregulation of Sam68 did not have a significant inhibitory effect on the accumulation of the EV71 genome. Moreover, Sam68 was redistributed from the nucleus to the cytoplasm and interacts with cellular factors, such as poly(rC)-binding protein 2 (PCBP2) and poly(A)-binding protein (PABP), during EV71 infection. The cytoplasmic relocalization of Sam68 in EV71-infected cells may be involved in the enhancement of EV71 IRES-mediated translation. Since Sam68 is known to be a RNA-binding protein, these results provide direct evidence that Sam68 is a novel ITAF that interacts with EV71 IRES and positively regulates viral protein translation. The nuclear protein Sam68 is found as an additional new host factor that interacts with the EV71 IRES during infection and could potentially

  20. RNA helicase DEAD box protein 5 regulates Polycomb repressive complex 2/Hox transcript antisense intergenic RNA function in hepatitis B virus infection and hepatocarcinogenesis.

    Science.gov (United States)

    Zhang, Hao; Xing, Zheng; Mani, Saravana Kumar Kailasam; Bancel, Brigitte; Durantel, David; Zoulim, Fabien; Tran, Elizabeth J; Merle, Philippe; Andrisani, Ourania

    2016-10-01

    Chronic hepatitis B virus (HBV) infection is a major factor in hepatocellular carcinoma (HCC) pathogenesis by a mechanism not yet understood. Elucidating mechanisms of HBV-mediated hepatocarcinogenesis is needed to gain insights into classification and treatment of HCC. In HBV replicating cells, including virus-associated HCCs, suppressor of zeste 12 homolog (SUZ12), a core subunit of Polycomb repressive complex2 (PRC2), undergoes proteasomal degradation. This process requires the long noncoding RNA, Hox transcript antisense intergenic RNA (HOTAIR). Intriguingly, HOTAIR interacts with PRC2 and also binds RNA-binding E3 ligases, serving as a ubiquitination scaffold. Herein, we identified the RNA helicase, DEAD box protein 5 (DDX5), as a regulator of SUZ12 stability and PRC2-mediated gene repression, acting by regulating RNA-protein complexes formed with HOTAIR. Specifically, knockdown of DDX5 and/or HOTAIR enabled reexpression of PRC2-repressed genes epithelial cell adhesion molecule (EpCAM) and pluripotency genes. Also, knockdown of DDX5 enhanced transcription from the HBV minichromosome. The helicase activity of DDX5 stabilized SUZ12- and PRC2-mediated gene silencing, by displacing the RNA-binding E3 ligase, Mex-3 RNA-binding family member B (Mex3b), from HOTAIR. Conversely, ectopic expression of Mex3b ubiquitinated SUZ12, displaced DDX5 from HOTAIR, and induced SUZ12 down-regulation. In G2 phase of cells expressing the HBV X protein (HBx), SUZ12 preferentially associated with Mex3b, but not DDX5, resulting in de-repression of PRC2 targets, including EpCAM and pluripotency genes. Significantly, liver tumors from HBx/c-myc bitransgenic mice and chronically HBV-infected patients exhibited a strong negative correlation between DDX5 messenger RNA levels, pluripotency gene expression, and liver tumor differentiation. Notably, chronically infected HBV patients with HCC expressing reduced DDX5 exhibited poor prognosis after tumor resection, identifying DDX5 as an

  1. Mechanism for G2 phase-specific nuclear export of the kinetochore protein CENP-F.

    Science.gov (United States)

    Loftus, Kyle M; Cui, Heying; Coutavas, Elias; King, David S; Ceravolo, Amanda; Pereiras, Dylan; Solmaz, Sozanne R

    2017-08-03

    Centromere protein F (CENP-F) is a component of the kinetochore and a regulator of cell cycle progression. CENP-F recruits the dynein transport machinery and orchestrates several cell cycle-specific transport events, including transport of the nucleus, mitochondria and chromosomes. A key regulatory step for several of these functions is likely the G2 phase-specific export of CENP-F from the nucleus to the cytosol, where the cytoplasmic dynein transport machinery resides; however, the molecular mechanism of this process is elusive. Here, we have identified 3 phosphorylation sites within the bipartite classical nuclear localization signal (cNLS) of CENP-F. These sites are specific for cyclin-dependent kinase 1 (Cdk1), which is active in G2 phase. Phosphomimetic mutations of these residues strongly diminish the interaction of the CENP-F cNLS with its nuclear transport receptor karyopherin α. These mutations also diminish nuclear localization of the CENP-F cNLS in cells. Notably, the cNLS is phosphorylated in the -1 position, which is important to orient the adjacent major motif for binding into its pocket on karyopherin α. We propose that localization of CENP-F is regulated by a cNLS, and a nuclear export pathway, resulting in nuclear localization during most of interphase. In G2 phase, the cNLS is weakened by phosphorylation through Cdk1, likely resulting in nuclear export of CENP-F via the still active nuclear export pathway. Once CENP-F resides in the cytosol, it can engage in pathways that are important for cell cycle progression, kinetochore assembly and the faithful segregation of chromosomes into daughter cells.

  2. Nuclear protein in testis carcinoma of the mediastinum: a case report

    OpenAIRE

    Boleto, Gonçalo; Perotin, Jeanne-Marie; Launois, Claire; Uro-Coste, Emmanuelle; Birembaut, Philippe,; Dury, Sandra; Vallerand, Hervé; Lebargy, François; Deslée, Gaëtan; Vella-Boucaud, Juliette

    2016-01-01

    International audience; AbstractBackgroundNuclear protein in testis carcinoma is a rare and very aggressive undifferentiated cancer which characteristically arises in the midline of the head, neck, and mediastinum.Case presentationWe describe the case of a 46-year-old white woman admitted for superior vena cava syndrome revealing a mediastinal tumor. Pathological examination of specimens obtained by mediastinoscopy revealed an undifferentiated tumor with solid growth and positive immunoreacti...

  3. Crystal structure of the human eIF4AIII–CWC22 complex shows how a DEAD-box protein is inhibited by a MIF4G domain

    Science.gov (United States)

    Buchwald, Gretel; Schüssler, Steffen; Basquin, Claire; Le Hir, Hervé; Conti, Elena

    2013-01-01

    DEAD-box proteins are involved in all aspects of RNA processing. They bind RNA in an ATP-dependent manner and couple ATP hydrolysis to structural and compositional rearrangements of ribonucleoprotein particles. Conformational control is a major point of regulation for DEAD-box proteins to act on appropriate substrates and in a timely manner in vivo. Binding partners containing a middle domain of translation initiation factor 4G (MIF4G) are emerging as important regulators. Well-known examples are eIF4G and Gle1, which bind and activate the DEAD-box proteins eIF4A and Dbp5. Here, we report the mechanism of an inhibiting MIF4G domain. We determined the 2.0-Å resolution structure of the complex of human eIF4AIII and the MIF4G domain of the splicing factor Complexed With Cef1 (CWC22), an essential prerequisite for exon junction complex assembly by the splicing machinery. The CWC22 MIF4G domain binds both RecA domains of eIF4AIII. The mode of RecA2 recognition is similar to that observed in the activating complexes, yet is specific for eIF4AIII. The way the CWC22 MIF4G domain latches on the eIF4AIII RecA1 domain is markedly different from activating complexes. In the CWC22–eIF4AIII complex, the RNA-binding and ATP-binding motifs of the two RecA domains do not face each other, as would be required in the active state, but are in diametrically opposite positions. The binding mode of CWC22 to eIF4AIII reveals a facet of how MIF4G domains use their versatile structural frameworks to activate or inhibit DEAD-box proteins. PMID:24218557

  4. Crystal structure of the human eIF4AIII-CWC22 complex shows how a DEAD-box protein is inhibited by a MIF4G domain.

    Science.gov (United States)

    Buchwald, Gretel; Schüssler, Steffen; Basquin, Claire; Le Hir, Hervé; Conti, Elena

    2013-11-26

    DEAD-box proteins are involved in all aspects of RNA processing. They bind RNA in an ATP-dependent manner and couple ATP hydrolysis to structural and compositional rearrangements of ribonucleoprotein particles. Conformational control is a major point of regulation for DEAD-box proteins to act on appropriate substrates and in a timely manner in vivo. Binding partners containing a middle domain of translation initiation factor 4G (MIF4G) are emerging as important regulators. Well-known examples are eIF4G and Gle1, which bind and activate the DEAD-box proteins eIF4A and Dbp5. Here, we report the mechanism of an inhibiting MIF4G domain. We determined the 2.0-Å resolution structure of the complex of human eIF4AIII and the MIF4G domain of the splicing factor Complexed With Cef1 (CWC22), an essential prerequisite for exon junction complex assembly by the splicing machinery. The CWC22 MIF4G domain binds both RecA domains of eIF4AIII. The mode of RecA2 recognition is similar to that observed in the activating complexes, yet is specific for eIF4AIII. The way the CWC22 MIF4G domain latches on the eIF4AIII RecA1 domain is markedly different from activating complexes. In the CWC22-eIF4AIII complex, the RNA-binding and ATP-binding motifs of the two RecA domains do not face each other, as would be required in the active state, but are in diametrically opposite positions. The binding mode of CWC22 to eIF4AIII reveals a facet of how MIF4G domains use their versatile structural frameworks to activate or inhibit DEAD-box proteins.

  5. Promyelocytic leukemia-nuclear body proteins: herpesvirus enemies, accomplices, or both?

    Science.gov (United States)

    Saffert, Ryan T; Kalejta, Robert F

    2008-05-01

    The promyelocytic leukemia (PML) protein gathers other cellular proteins, such as Daxx and Sp100, to form subnuclear structures termed PML-nuclear bodies (PML-NBs) or ND10 domains. Many infecting viral genomes localize to PML-NBs, leading to speculation that these structures may represent the most efficient subnuclear location for viral replication. Conversely, many viral proteins modify or disrupt PML-NBs, suggesting that viral replication may be more efficient in the absence of these structures. Thus, a debate remains as to whether PML-NBs inhibit or enhance viral replication. Here we review and discuss recent data indicating that for herpesviruses, PML-NB proteins inhibit viral replication in cell types where productive, lytic replication occurs, while at the same time may enhance the establishment of lifelong latent infections in other cell types.

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

    Energy Technology Data Exchange (ETDEWEB)

    Guo, Yisong [University of California, Department of Applied Science (United States); Brecht, Eric [Montana State University, Department of Chemistry and Biochemistry (United States); Aznavour, Kristen [University of Southern California, Department of Chemistry (United States); Nix, Jay C. [Lawrence Berkeley National Laboratory, Physical Biosciences Division (United States); Xiao, Yuming; Wang, Hongxin [University of California, Department of Applied Science (United States); George, Simon J. [Lawrence Berkeley National Laboratory, Physical Biosciences Division (United States); Bau, Robert [University of Southern California, Department of Chemistry (United States); Keable, Stephen; Peters, John W. [Montana State University, Department of Chemistry and Biochemistry (United States); Adams, Michael W. W. [University of Georgia, Department of Biochemistry and Molecular Biology (United States); Jenney, Francis E. Jr. [Georgia Campus, Philadelphia College of Osteopathic Medicine (United States); Sturhahn, Wolfgang; Alp, Ercan E.; Zhao, Jiyong [Argonne National Laboratory, Advanced Photon Source (United States); Yoda, Yoshitaka [JASRI (Japan); Cramer, Stephen P., E-mail: spcramer@lbl.gov [University of California, Department of Applied Science (United States)

    2013-12-15

    We have applied {sup 57}Fe 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.

  7. Temporal association of protamine 1 with the inner nuclear membrane protein lamin B receptor during spermiogenesis.

    Science.gov (United States)

    Mylonis, Ilias; Drosou, Victoria; Brancorsini, Stefano; Nikolakaki, Eleni; Sassone-Corsi, Paolo; Giannakouros, Thomas

    2004-03-19

    During mammalian spermiogenesis, histones are replaced by transition proteins, which are in turn replaced by protamines P1 and P2. P1 protamine contains a short arginine/serine-rich (RS) domain that is highly phosphorylated before being deposited into sperm chromatin and almost completely dephosphorylated during sperm maturation. We now demonstrate that, in elongating spermatids, this phosphorylation is required for the temporal association of P1 protamine with lamin B receptor (LBR), an inner nuclear membrane protein that also possesses a stretch of RS dipeptides at its nucleoplasmic NH(2)-terminal domain. Previous studies have shown that the cellular protein p32 also binds tightly to the unmodified RS domain of LBR. Extending those findings, we now present evidence that p32 prevents phosphorylation of LBR and furthermore that dissociation of this protein precedes P1 protamine association. Our data suggest that docking of protamine 1 to the nuclear envelope is an important intermediate step in spermiogenesis and reveal a novel role for SR protein kinases and p32.

  8. Serum high mobility group box 1 protein levels are not associated with either histological severity or treatment response in children and adults with nonalcoholic fatty liver disease.

    Directory of Open Access Journals (Sweden)

    Katherine P Yates

    Full Text Available Serum high mobility group box 1 protein (HMGB1 is a proinflammatory molecule that could potentially serve as a biomarker for non-alcoholic fatty liver disease (NAFLD and non-alcoholic steatohepatitis (NASH due to its correlation with degree of liver fibrosis. The aim of the current study was to examine the cross-sectional and longitudinal relationships between serum HMGB1 levels and liver histology in adults and children with NAFLD participating in two large randomized controlled trials.Serum HMGB1 levels were measured at various time points in adults and children with NAFLD, who participated in PIVENS and TONIC clinical trials respectively. PIVENS trial compared vitamin E or pioglitazone to placebo in adults whereas TONIC trial compared vitamin E or metformin to placebo in children. Participants had liver biopsies at baseline and the end of treatment (96 weeks, and liver histology was reviewed by a central committee of study pathologists.In the cross-sectional analyses (n = 205 for PIVENS and 109 for TONIC, there was no significant relationship between serum HMGB1 levels and histological features such as steatosis, ballooning, inflammation, fibrosis, or presence of steatohepatitis in either adults or children. Serum HMGB1 levels did not change significantly during treatment either with placebo, vitamin E therapy (P = 0.81 or pioglitazone (P = 0.09 in the PIVENS trial. Similarly, serum HMGB1 levels did not change significantly during treatment either with placebo, metformin (P = 0.15 or vitamin E (P = 0.23 in the TONIC trial. In the longitudinal analyses (n = 105 for PIVENS and 109 for TONIC, changes in serum HMGB1 levels did not correlate with histologic improvement or resolution of NASH in either adults or children. There was no relationship between serum HMGB1 and ALT levels in either adults or children with NAFLD.Serum HMGB1 levels were not associated with histological severity or treatment response in either children or adults with NAFLD.

  9. High-Mobility Group Box-1 Protein Serum Levels Do Not Reflect Monocytic Function in Patients with Sepsis-Induced Immunosuppression

    Directory of Open Access Journals (Sweden)

    Nadine Unterwalder

    2010-01-01

    Full Text Available Background. High-mobility group box-1 (HMGB-1 protein is released during “late sepsis” by activated monocytes. We investigated whether systemic HMGB-1 levels are associated with indices of monocytic activation/function in patients with sepsis-induced immunosuppression. Methodology. 36 patients (31 male, 64±14 years with severe sepsis/septic shock and monocytic deactivation (reduced mHLA-DR expression and TNF-α release were assessed in a subanalysis of a placebo-controlled immunostimulatory trial using GM-CSF. HMGB-1 levels were assessed over a 9-day treatment interval. Data were compared to standardized biomarkers of monocytic immunity (mHLA-DR expression, TNF-α release. Principle findings. HMGB-1 levels were enhanced in sepsis but did not differ between treatment and placebo groups at baseline (14.6 ± 13.5 versus 12.5 ± 11.5 ng/ml, P=.62. When compared to controls, HMGB-1 level increased transiently in treated patients at day 5 (27.8±21.7 versus 11.0±14.9, P=.01. Between group differences were not noted at any other point of assessment. HMGB-1 levels were not associated with markers of monocytic function or clinical disease severity. Conclusions. GM-CSF treatment for sepsis-induced immunosuppression induces a moderate but only transient increase in systemic HMGB-1 levels. HMGB-1 levels should not be used for monitoring of monocytic function in immunostimulatory trials as they do not adequately portray contemporary changes in monocytic immunity.

  10. An episomally replicating vector binds to the nuclear matrix protein SAF-A in vivo

    Science.gov (United States)

    Jenke, Bok Hee C.; Fetzer, Christian P.; Stehle, Isa M.; Jönsson, Franziska; Fackelmayer, Frank O.; Conradt, Harald; Bode, Jürgen; Lipps, Hans J.

    2002-01-01

    pEPI-1, a vector in which a chromosomal scaffold/matrix-attached region (S/MAR) is linked to the simian virus 40 origin of replication, is propagated episomally in CHO cells in the absence of the virally encoded large T-antigen and is stably maintained in the absence of selection pressure. It has been suggested that mitotic stability is provided by a specific interaction of this vector with components of the nuclear matrix. We studied the interactions of pEPI-1 by crosslinking with cis-diamminedichloroplatinum II, after which it is found to copurify with the nuclear matrix. In a south-western analysis, the vector shows exclusive binding to hnRNP-U/SAF-A, a multifunctional scaffold/matrix specific factor. Immunoprecipitation of the crosslinked DNA–protein complex demonstrates that pEPI-1 is bound to this protein in vivo. These data provide the first experimental evidence for the binding of an artificial episome to a nuclear matrix protein in vivo and the basis for understanding the mitotic stability of this novel vector class. PMID:11897664

  11. A set of enhanced green fluorescent protein concatemers for quantitative determination of nuclear localization signal strength.

    Science.gov (United States)

    Böhm, Jennifer; Thavaraja, Ramya; Giehler, Susanne; Nalaskowski, Marcus M

    2017-09-15

    Regulated transport of proteins between nucleus and cytoplasm is an important process in the eukaryotic cell. In most cases, active nucleo-cytoplasmic protein transport is mediated by nuclear localization signal (NLS) and/or nuclear export signal (NES) motifs. In this study, we developed a set of vectors expressing enhanced GFP (EGFP) concatemers ranging from 2 to 12 subunits (2xEGFP to 12xEGFP) for analysis of NLS strength. As shown by in gel GFP fluorescence analysis and αGFP Western blotting, EGFP concatemers are expressed as fluorescent full-length proteins in eukaryotic cells. As expected, nuclear localization of concatemeric EGFPs decreases with increasing molecular weight. By oligonucleotide ligation this set of EGFP concatemers can be easily fused to NLS motifs. After determination of intracellular localization of EGFP concatemers alone and fused to different NLS motifs we calculated the size of a hypothetic EGFP concatemer showing a defined distribution of EGFP fluorescence between nucleus and cytoplasm (n/c ratio = 2). Clear differences of the size of the hypothetic EGFP concatemer depending on the fused NLS motif were observed. Therefore, we propose to use the size of this hypothetic concatemer as quantitative indicator for comparing strength of different NLS motifs. Copyright © 2017 Elsevier Inc. All rights reserved.

  12. Nuclear envelope breakdown induced by herpes simplex virus type 1 involves the activity of viral fusion proteins

    Energy Technology Data Exchange (ETDEWEB)

    Maric, Martina; Haugo, Alison C. [Department of Microbiology, University of Iowa, Iowa City, IA 52242 (United States); Dauer, William [Department of Neurology, University of Michigan, Ann Arbor, MI 48109 (United States); Johnson, David [Department of Microbiology and Immunology, Oregon Health Sciences University, Portland, OR 97201 (United States); Roller, Richard J., E-mail: richard-roller@uiowa.edu [Department of Microbiology, University of Iowa, Iowa City, IA 52242 (United States)

    2014-07-15

    Herpesvirus infection reorganizes components of the nuclear lamina usually without loss of integrity of the nuclear membranes. We report that wild-type HSV infection can cause dissolution of the nuclear envelope in transformed mouse embryonic fibroblasts that do not express torsinA. Nuclear envelope breakdown is accompanied by an eight-fold inhibition of virus replication. Breakdown of the membrane is much more limited during infection with viruses that lack the gB and gH genes, suggesting that breakdown involves factors that promote fusion at the nuclear membrane. Nuclear envelope breakdown is also inhibited during infection with virus that does not express UL34, but is enhanced when the US3 gene is deleted, suggesting that envelope breakdown may be enhanced by nuclear lamina disruption. Nuclear envelope breakdown cannot compensate for deletion of the UL34 gene suggesting that mixing of nuclear and cytoplasmic contents is insufficient to bypass loss of the normal nuclear egress pathway. - Highlights: • We show that wild-type HSV can induce breakdown of the nuclear envelope in a specific cell system. • The viral fusion proteins gB and gH are required for induction of nuclear envelope breakdown. • Nuclear envelope breakdown cannot compensate for deletion of the HSV UL34 gene.

  13. Degradation of HaloTag-fused nuclear proteins using bestatin-HaloTag ligand hybrid molecules.

    Science.gov (United States)

    Tomoshige, Shusuke; Naito, Mikihiko; Hashimoto, Yuichi; Ishikawa, Minoru

    2015-10-14

    We have developed a protein knockdown technology using hybrid small molecules designed as conjugates of a ligand for the target protein and a ligand for ubiquitin ligase cellular inhibitor of apoptosis protein 1 (cIAP1). However, this technology has several limitations. Here, we report the development of a novel protein knockdown system to address these limitations. In this system, target proteins are fused with HaloTag to provide a common binding site for a degradation inducer. We designed and synthesized small molecules consisting of alkyl chloride as the HaloTag-binding degradation inducer, which binds to HaloTag, linked to BE04 (2), which binds to cIAP1. Using this system, we successfully knocked down HaloTag-fused cAMP responsive element binding protein 1 (HaloTag-CREB1) and HaloTag-fused c-jun (HaloTag-c-jun), which are ligand-unknown nuclear proteins, in living cells. HaloTag-binding degradation inducers can be synthesized easily, and are expected to be useful as biological tools for pan-degradation of HaloTag-fused proteins.

  14. The Etl-1 gene encodes a nuclear protein differentially expressed during early mouse development.

    Science.gov (United States)

    Schoor, M; Schuster-Gossler, K; Gossler, A

    1993-07-01

    Recently, we isolated a novel mouse gene, Etl-1 (Enhancer-trap-locus-1), whose deduced amino acid sequence shows in its C-terminal portion striking homology to the brahma protein (BRM), a transcriptional regulator of homeotic genes in Drosophila, and to SNF2/SWI2, a transcriptional regulator of various genes in Saccharomyces cerevisiae. Here we report the generation of antibodies against the Etl-1 gene product (ETL-1) and describe the subcellular localization as well as the expression and distribution of the ETL-1 protein during mouse pre- and early post-implantation development. ETL-1 is a nuclear protein and is expressed in a biphasic manner during early embryogenesis. Moderate levels of ETL-1 were detected in unfertilized and fertilized eggs but in the latter the protein was not concentrated in the pronuclei and seemed evenly distributed throughout the cytoplasm. In two-cell embryos nuclear ETL-1 protein accumulated transiently and levels decreased during subsequent cleavage development. After the morula stage, ETL-1 levels increased again; in blastocysts high levels of ETL-1 were present in inner cell mass cells whereas trophectoderm cells contained little or no ETL-1. During subsequent development essentially all cell types except parietal endoderm and trophoblast cells contained high levels of ETL-1. Our results imply that nuclear ETL-1 is dispensable for the progression to the two cell stage, and suggest that during cleavage ETL-1 might be needed at the onset of embryonic transcription. In blastocysts ETL-1 function might be specifically required in cells of the inner cell mass and later in most cells of the embryo proper and extraembryonic ectoderm lineage.

  15. Cytoplasmic Targeting of Mutant Poly(A)‐Binding Protein Nuclear 1 Suppresses Protein Aggregation and Toxicity in Oculopharyngeal Muscular Dystrophy

    National Research Council Canada - National Science Library

    Abu‐Baker, Aida; Laganiere, Simon; Fan, Xueping; Laganiere, Janet; Brais, Bernard; Rouleau, Guy A

    2005-01-01

    ...–17 residues, located at the N‐terminus of the poly(A)‐binding protein nuclear 1 (PABPN1). A distinct pathological hallmark of OPMD is the presence of filamentous intranuclear aggregates in patients' skeletal muscle cells...

  16. Aberrant distributions of nuclear pore complex proteins in ALS mice and ALS patients.

    Science.gov (United States)

    Shang, Jingwei; Yamashita, Toru; Nakano, Yumiko; Morihara, Ryuta; Li, Xianghong; Feng, Tian; Liu, Xia; Huang, Yong; Fukui, Yusuke; Hishikawa, Nozomi; Ohta, Yasuyuki; Abe, Koji

    2017-05-14

    Nuclear pore complexes (NPCs) play important roles in traffic of molecules between the nucleus and cytoplasm, aberrant distributions of components of NPCs were demonstrated in C9orf72 amyotrophic lateral sclerosis (C9-ALS) patients, but it is elusive whether such abnormities are also the case with other cause of ALS disease. In the present study, we investigated the spatiotemporal distributions of RanGAP1 and 4 representative nucleoporins (GP210, NUP205, NUP107 and NUP50) of NPCs in human Cu/Zn superoxide dismutase-1 mutation transgenic (SOD1-Tg) mice and sporadic ALS patients. Compared with wild type (WT), these proteins displayed age-dependent and progressive nuclear precipitations, and cytoplasmic aberrant expressions in motor neurons of lumbar cord in SOD1-Tg mice from 10 to 18weeks (W). Double immunofluorescent analysis showed abnormal nuclear retention and apparent co-localizations of RanGAPl with NUP205 and NUP205 with NUPl07, meanwhile, GP210 with NUP205 mainly co-localized in the nuclear envelope (NE) of motor neurons. Furthermore, RanGAP1, GP210 and NUP50 showed similarly abnormal nuclear precipitations and cytoplasmic upregulations in SOD1-Tg mice and ALS patients, moreover, aberrant co-localizations of RanGAP1 with TDP-43 and NUP205 with TDP-43 were also observed in motor neurons. The present study indicated that the mislocalization of these proteins of NPCs may underlie the pathogenesis of ALS both in SOD1-Tg mice and human sporadic ALS patients, and these dysfunctions may be a fundamental pathway for ALS that is not specific only in C9-ALS but also in SOD1-ALS, which may be amenable to pharmacotherapeutic intervention. Copyright © 2017 IBRO. Published by Elsevier Ltd. All rights reserved.

  17. Successful Recovery of Nuclear Protein-Coding Genes from Small Insects in Museums Using Illumina Sequencing

    Science.gov (United States)

    Dasenko, Mark A.

    2015-01-01

    In this paper we explore high-throughput Illumina sequencing of nuclear protein-coding, ribosomal, and mitochondrial genes in small, dried insects stored in natural history collections. We sequenced one tenebrionid beetle and 12 carabid beetles ranging in size from 3.7 to 9.7 mm in length that have been stored in various museums for 4 to 84 years. Although we chose a number of old, small specimens for which we expected low sequence recovery, we successfully recovered at least some low-copy nuclear protein-coding genes from all specimens. For example, in one 56-year-old beetle, 4.4 mm in length, our de novo assembly recovered about 63% of approximately 41,900 nucleotides in a target suite of 67 nuclear protein-coding gene fragments, and 70% using a reference-based assembly. Even in the least successfully sequenced carabid specimen, reference-based assembly yielded fragments that were at least 50% of the target length for 34 of 67 nuclear protein-coding gene fragments. Exploration of alternative references for reference-based assembly revealed few signs of bias created by the reference. For all specimens we recovered almost complete copies of ribosomal and mitochondrial genes. We verified the general accuracy of the sequences through comparisons with sequences obtained from PCR and Sanger sequencing, including of conspecific, fresh specimens, and through phylogenetic analysis that tested the placement of sequences in predicted regions. A few possible inaccuracies in the sequences were detected, but these rarely affected the phylogenetic placement of the samples. Although our sample sizes are low, an exploratory regression study suggests that the dominant factor in predicting success at recovering nuclear protein-coding genes is a high number of Illumina reads, with success at PCR of COI and killing by immersion in ethanol being secondary factors; in analyses of only high-read samples, the primary significant explanatory variable was body length, with small beetles

  18. Successful Recovery of Nuclear Protein-Coding Genes from Small Insects in Museums Using Illumina Sequencing.

    Directory of Open Access Journals (Sweden)

    Kojun Kanda

    Full Text Available In this paper we explore high-throughput Illumina sequencing of nuclear protein-coding, ribosomal, and mitochondrial genes in small, dried insects stored in natural history collections. We sequenced one tenebrionid beetle and 12 carabid beetles ranging in size from 3.7 to 9.7 mm in length that have been stored in various museums for 4 to 84 years. Although we chose a number of old, small specimens for which we expected low sequence recovery, we successfully recovered at least some low-copy nuclear protein-coding genes from all specimens. For example, in one 56-year-old beetle, 4.4 mm in length, our de novo assembly recovered about 63% of approximately 41,900 nucleotides in a target suite of 67 nuclear protein-coding gene fragments, and 70% using a reference-based assembly. Even in the least successfully sequenced carabid specimen, reference-based assembly yielded fragments that were at least 50% of the target length for 34 of 67 nuclear protein-coding gene fragments. Exploration of alternative references for reference-based assembly revealed few signs of bias created by the reference. For all specimens we recovered almost complete copies of ribosomal and mitochondrial genes. We verified the general accuracy of the sequences through comparisons with sequences obtained from PCR and Sanger sequencing, including of conspecific, fresh specimens, and through phylogenetic analysis that tested the placement of sequences in predicted regions. A few possible inaccuracies in the sequences were detected, but these rarely affected the phylogenetic placement of the samples. Although our sample sizes are low, an exploratory regression study suggests that the dominant factor in predicting success at recovering nuclear protein-coding genes is a high number of Illumina reads, with success at PCR of COI and killing by immersion in ethanol being secondary factors; in analyses of only high-read samples, the primary significant explanatory variable was body length

  19. Delivering Single-Walled Carbon Nanotubes to the Nucleus Using Engineered Nuclear Protein Domains.

    Science.gov (United States)

    Boyer, Patrick D; Ganesh, Sairaam; Qin, Zhao; Holt, Brian D; Buehler, Markus J; Islam, Mohammad F; Dahl, Kris Noel

    2016-02-10

    Single-walled carbon nanotubes (SWCNTs) have great potential for cell-based therapies due to their unique intrinsic optical and physical characteristics. Consequently, broad classes of dispersants have been identified that individually suspend SWCNTs in water and cell media in addition to reducing nanotube toxicity to cells. Unambiguous control and verification of the localization and distribution of SWCNTs within cells, particularly to the nucleus, is needed to advance subcellular technologies utilizing nanotubes. Here we report delivery of SWCNTs to the nucleus by noncovalently attaching the tail domain of the nuclear protein lamin B1 (LB1), which we engineer from the full-length LMNB1 cDNA. More than half of this low molecular weight globular protein is intrinsically disordered but has an immunoglobulin-fold composed of a central hydrophobic core, which is highly suitable for associating with SWCNTs, stably suspending SWCNTs in water and cell media. In addition, LB1 has an exposed nuclear localization sequence to promote active nuclear import of SWCNTs. These SWCNTs-LB1 dispersions in water and cell media display near-infrared (NIR) absorption spectra with sharp van Hove peaks and an NIR fluorescence spectra, suggesting that LB1 individually disperses nanotubes. The dispersing capability of SWCNTs by LB1 is similar to that by albumin proteins. The SWCNTs-LB1 dispersions with concentrations ≥150 μg/mL (≥30 μg/mL) in water (cell media) remain stable for ≥75 days (≥3 days) at 4 °C (37 °C). Further, molecular dynamics modeling of association of LB1 with SWCNTs reveal that the exposure of the nuclear localization sequence is independent of LB1 binding conformation. Measurements from confocal Raman spectroscopy and microscopy, NIR fluorescence imaging of SWCNTs, and fluorescence lifetime imaging microscopy show that millions of these SWCNTs-LB1 complexes enter HeLa cells, localize to the nucleus of cells, and interact with DNA. We postulate that the

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

    Directory of Open Access Journals (Sweden)

    Rafael Rodríguez-Muñoz

    Full Text Available 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 β-dystroglycan, α1-, β-, α2-dystrobrevins, α-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.

  1. Role of the nuclear migration protein Lis1 in cell morphogenesis in Ustilago maydis

    Science.gov (United States)

    Valinluck, Michael; Ahlgren, Sara; Sawada, Mizuho; Locken, Kristopher; Banuett, Flora

    2010-01-01

    Ustilago maydis is a basidiomycete fungus that exhibits a yeast-like and a filamentous form. Growth of the fungus in the host leads to additional morphological transitions. The different morphologies are characterized by distinct nuclear movements. Dynein and α-tubulin are required for nuclear movements and for cell morphogenesis of the yeast-like form. Lis1 is a microtubule plus-end tracking protein (+TIPs) conserved in eukaryotes and required for nuclear migration and spindle positioning. Defects in nuclear migration result in altered cell fate and aberrant development in metazoans, slow growth in fungi and disease in humans (e.g. lissencephaly). Here we investigate the role of the human LIS1 homolog in U. maydis and demonstrate that it is essential for cell viability, not previously seen in other fungi. With a conditional null mutation we show that lis1 is necessary for nuclear migration in the yeast-like cell and during the dimorphic transition. Studies of asynchronous exponentially growing cells and time-lapse microscopy uncovered novel functions of lis1: It is necessary for cell morphogenesis, positioning of the septum and cell wall integrity. lis1-depleted cells exhibit altered axes of growth and loss of cell polarity leading to grossly aberrant cells with clusters of nuclei and morphologically altered buds devoid of nuclei. Altered septum positioning and cell wall deposition contribute to the aberrant morphology. lis1-depleted cells lyse, indicative of altered cell wall properties or composition. We also demonstrate, with indirect immunofluorescence to visualize tubulin, that lis1 is necessary for the normal organization of the microtubule cytoskeleton: lis1-depleted cells contain more and longer microtubules that can form coils perpendicular to the long axis of the cell. We propose that lis1 controls microtubule dynamics and thus the regulated delivery of vesicles to growth sites and other cell domains that govern nuclear movements. PMID:20524583

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

    Directory of Open Access Journals (Sweden)

    Shunfang Wang

    2015-12-01

    Full Text Available 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.

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

    Science.gov (United States)

    Wang, Shunfang; Liu, Shuhui

    2015-12-19

    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.

  4. Characterization of a family of novel cysteine- serine-rich nuclear proteins (CSRNP.

    Directory of Open Access Journals (Sweden)

    Sébastien Gingras

    2007-08-01

    Full Text Available Gene array analysis has been widely used to identify genes induced during T cell activation. Our studies identified an immediate early gene that is strongly induced in response to IL-2 in mouse T cells which we named cysteine- serine-rich nuclear protein-1 (CSRNP-1. The human ortholog was previously identified as an AXIN1 induced gene (AXUD1. The protein does not contain sequence defined domains or motifs annotated in public databases, however the gene is a member of a family of three mammalian genes that share conserved regions, including cysteine- and serine-rich regions and a basic domain, they encode nuclear proteins, possess transcriptional activation domain and bind the sequence AGAGTG. Consequently we propose the nomenclature of CSRNP-1, -2 and -3 for the family. To elucidate the physiological functions of CSRNP-1, -2 and -3, we generated mice deficient for each of these genes by homologous recombination in embryonic stem cells. Although the CSRNP proteins have the hallmark of transcription factors and CSRNP-1 expression is highly induced by IL-2, deletion of the individual genes had no obvious consequences on normal mouse development, hematopoiesis or T cell functions. However, combined deficiencies cause partial neonatal lethality suggesting that the genes have redundant functions.

  5. Nuclear Envelope Protein SUN2 Promotes Cyclophilin-A-Dependent Steps of HIV Replication

    Science.gov (United States)

    Lahaye, Xavier; Satoh, Takeshi; Gentili, Matteo; Cerboni, Silvia; Silvin, Aymeric; Conrad, Cécile; Ahmed-Belkacem, Abdelhakim; Rodriguez, Elisa C.; Guichou, Jean-François; Bosquet, Nathalie; Piel, Matthieu; Le Grand, Roger; King, Megan C.; Pawlotsky, Jean-Michel; Manel, Nicolas

    2016-01-01

    Summary During the early phase of replication, HIV reverse transcribes its RNA and crosses the nuclear envelope while escaping host antiviral defenses. The host factor Cyclophilin A (CypA) is essential for these steps and binds the HIV capsid; however, the mechanism underlying this effect remains elusive. Here, we identify related capsid mutants in HIV-1, HIV-2, and SIVmac that are restricted by CypA. This antiviral restriction of mutated viruses is conserved across species and prevents nuclear import of the viral cDNA. Importantly, the inner nuclear envelope protein SUN2 is required for the antiviral activity of CypA. We show that wild-type HIV exploits SUN2 in primary CD4+ T cells as an essential host factor that is required for the positive effects of CypA on reverse transcription and infection. Altogether, these results establish essential CypA-dependent functions of SUN2 in HIV infection at the nuclear envelope. PMID:27149839

  6. Nuclear Envelope Protein SUN2 Promotes Cyclophilin-A-Dependent Steps of HIV Replication

    Directory of Open Access Journals (Sweden)

    Xavier Lahaye

    2016-04-01

    Full Text Available During the early phase of replication, HIV reverse transcribes its RNA and crosses the nuclear envelope while escaping host antiviral defenses. The host factor Cyclophilin A (CypA is essential for these steps and binds the HIV capsid; however, the mechanism underlying this effect remains elusive. Here, we identify related capsid mutants in HIV-1, HIV-2, and SIVmac that are restricted by CypA. This antiviral restriction of mutated viruses is conserved across species and prevents nuclear import of the viral cDNA. Importantly, the inner nuclear envelope protein SUN2 is required for the antiviral activity of CypA. We show that wild-type HIV exploits SUN2 in primary CD4+ T cells as an essential host factor that is required for the positive effects of CypA on reverse transcription and infection. Altogether, these results establish essential CypA-dependent functions of SUN2 in HIV infection at the nuclear envelope.

  7. Protein kinases responsible for the phosphorylation of the nuclear egress core complex of human cytomegalovirus.

    Science.gov (United States)

    Sonntag, Eric; Milbradt, Jens; Svrlanska, Adriana; Strojan, Hanife; Häge, Sigrun; Kraut, Alexandra; Hesse, Anne-Marie; Amin, Bushra; Sonnewald, Uwe; Couté, Yohann; Marschall, Manfred

    2017-10-01

    Nuclear egress of herpesvirus capsids is mediated by a multi-component nuclear egress complex (NEC) assembled by a heterodimer of two essential viral core egress proteins. In the case of human cytomegalovirus (HCMV), this core NEC is defined by the interaction between the membrane-anchored pUL50 and its nuclear cofactor, pUL53. NEC protein phosphorylation is considered to be an important regulatory step, so this study focused on the respective role of viral and cellular protein kinases. Multiply phosphorylated pUL50 varieties were detected by Western blot and Phos-tag analyses as resulting from both viral and cellular kinase activities. In vitro kinase analyses demonstrated that pUL50 is a substrate of both PKCα and CDK1, while pUL53 can also be moderately phosphorylated by CDK1. The use of kinase inhibitors further illustrated the importance of distinct kinases for core NEC phosphorylation. Importantly, mass spectrometry-based proteomic analyses identified five major and nine minor sites of pUL50 phosphorylation. The functional relevance of core NEC phosphorylation was confirmed by various experimental settings, including kinase knock-down/knock-out and confocal imaging, in which it was found that (i) HCMV core NEC proteins are not phosphorylated solely by viral pUL97, but also by cellular kinases; (ii) both PKC and CDK1 phosphorylation are detectable for pUL50; (iii) no impact of PKC phosphorylation on NEC functionality has been identified so far; (iv) nonetheless, CDK1-specific phosphorylation appears to be required for functional core NEC interaction. In summary, our findings provide the first evidence that the HCMV core NEC is phosphorylated by cellular kinases, and that the complex pattern of NEC phosphorylation has functional relevance.

  8. Shaping 3-D boxes

    DEFF Research Database (Denmark)

    Stenholt, Rasmus; Madsen, Claus B.

    2011-01-01

    Enabling users to shape 3-D boxes in immersive virtual environments is a non-trivial problem. In this paper, a new family of techniques for creating rectangular boxes of arbitrary position, orientation, and size is presented and evaluated. These new techniques are based solely on position data......F) docking experiment against an existing technique, which requires the user to perform the rotation and scaling of the box explicitly. The precision of the users' box construction is evaluated by a novel error metric measuring the difference between two boxes. The results of the experiment strongly indicate...... that for precision docking of 9 DoF boxes, some of the proposed techniques are significantly better than ones with explicit rotation and scaling. Another interesting result is that the number of DoF simultaneously controlled by the user significantly influences the precision of the docking....

  9. Gammaherpesviral Tegument Proteins, PML-Nuclear Bodies and the Ubiquitin-Proteasome System

    Directory of Open Access Journals (Sweden)

    Florian Full

    2017-10-01

    Full Text Available Gammaherpesviruses like Epstein-Barr virus (EBV and Kaposi’s sarcoma-associated herpesvirus (KSHV subvert the ubiquitin proteasome system for their own benefit in order to facilitate viral gene expression and replication. In particular, viral tegument proteins that share sequence homology to the formylglycineamide ribonucleotide amidotransferase (FGARAT, or PFAS, an enzyme in the cellular purine biosynthesis, are important for disrupting the intrinsic antiviral response associated with Promyelocytic Leukemia (PML protein-associated nuclear bodies (PML-NBs by proteasome-dependent and independent mechanisms. In addition, all herpesviruses encode for a potent ubiquitin protease that can efficiently remove ubiquitin chains from proteins and thereby interfere with several different cellular pathways. In this review, we discuss mechanisms and functional consequences of virus-induced ubiquitination and deubiquitination for early events in gammaherpesviral infection.

  10. FE65 regulates and interacts with the Bloom syndrome protein in dynamic nuclear spheres - potential relevance to Alzheimer's disease.

    Science.gov (United States)

    Schrötter, Andreas; Mastalski, Thomas; Nensa, Fabian M; Neumann, Martin; Loosse, Christina; Pfeiffer, Kathy; Magraoui, Fouzi El; Platta, Harald W; Erdmann, Ralf; Theiss, Carsten; Uszkoreit, Julian; Eisenacher, Martin; Meyer, Helmut E; Marcus, Katrin; Müller, Thorsten

    2013-06-01

    The intracellular domain of the amyloid precursor protein (AICD) is generated following cleavage of the precursor by the γ-secretase complex and is involved in membrane to nucleus signaling, for which the binding of AICD to the adapter protein FE65 is essential. Here we show that FE65 knockdown causes a downregulation of the protein Bloom syndrome protein (BLM) and the minichromosome maintenance (MCM) protein family and that elevated nuclear levels of FE65 result in stabilization of the BLM protein in nuclear mobile spheres. These spheres are able to grow and fuse, and potentially correspond to the nuclear domain 10. BLM plays a role in DNA replication and repair mechanisms and FE65 was also shown to play a role in DNA damage response in the cell. A set of proliferation assays in our work revealed that FE65 knockdown in HEK293T cells reduced cell replication. On the basis of these results, we hypothesize that nuclear FE65 levels (nuclear FE65/BLM containing spheres) may regulate cell cycle re-entry in neurons as a result of increased interaction of FE65 with BLM and/or an increase in MCM protein levels. Thus, FE65 interactions with BLM and MCM proteins may contribute to the neuronal cell cycle re-entry observed in brains affected by Alzheimer's disease.

  11. Complex chromatin condensation patterns and nuclear protein transitions during spermiogenesis: examples from mollusks.

    Science.gov (United States)

    Chiva, M; Saperas, N; Ribes, E

    2011-12-01

    In this paper we review and analyze the chromatin condensation pattern during spermiogenesis in several species of mollusks. Previously, we had described the nuclear protein transitions during spermiogenesis in these species. The results of our study show two types of condensation pattern: simple patterns and complex patterns, with the following general characteristics: (a) When histones (always present in the early spermatid nucleus) are directly replaced by SNBP (sperm nuclear basic proteins) of the protamine type, the spermiogenic chromatin condensation pattern is simple. However, if the replacement is not direct but through intermediate proteins, the condensation pattern is complex. (b) The intermediate proteins found in mollusks are precursor molecules that are processed during spermiogenesis to the final protamine molecules. Some of these final protamines represent proteins with the highest basic amino acid content known to date, which results in the establishment of a very strong electrostatic interaction with DNA. (c) In some instances, the presence of complex patterns of chromatin condensation clearly correlates with the acquisition of specialized forms of the mature sperm nuclei. In contrast, simple condensation patterns always lead to rounded, oval or slightly cylindrical nuclei. (d) All known cases of complex spermiogenic chromatin condensation patterns are restricted to species with specialized sperm cells (introsperm). At the time of writing, we do not know of any report on complex condensation pattern in species with external fertilization and, therefore, with sperm cells of the primitive type (ect-aquasperm). (e) Some of the mollusk an spermiogenic chromatin condensation patterns of the complex type are very similar (almost identical) to those present in other groups of animals. Interestingly, the intermediate proteins involved in these cases can be very different.In this study, we discuss the biological significance of all these features and

  12. Identification of nuclear and cytoplasmic mRNA targets for the shuttling protein SF2/ASF.

    Science.gov (United States)

    Sanford, Jeremy R; Coutinho, Pedro; Hackett, Jamie A; Wang, Xin; Ranahan, William; Caceres, Javier F

    2008-10-08

    The serine and arginine-rich protein family (SR proteins) are highly conserved regulators of pre-mRNA splicing. SF2/ASF, a prototype member of the SR protein family, is a multifunctional RNA binding protein with roles in pre-mRNA splicing, mRNA export and mRNA translation. These observations suggest the intriguing hypothesis that SF2/ASF may couple splicing and translation of specific mRNA targets in vivo. Unfortunately the paucity of endogenous mRNA targets for SF2/ASF has hindered testing of this hypothesis. Here, we identify endogenous mRNAs directly cross-linked to SF2/ASF in different sub-cellular compartments. Cross-Linking Immunoprecipitation (CLIP) captures the in situ specificity of protein-RNA interaction and allows for the simultaneous identification of endogenous RNA targets as well as the locations of binding sites within the RNA transcript. Using the CLIP method we identified 326 binding sites for SF2/ASF in RNA transcripts from 180 protein coding genes. A purine-rich consensus motif was identified in binding sites located within exon sequences but not introns. Furthermore, 72 binding sites were occupied by SF2/ASF in different sub-cellular fractions suggesting that these binding sites may influence the splicing or translational control of endogenous mRNA targets. We demonstrate that ectopic expression of SF2/ASF regulates the splicing and polysome association of transcripts derived from the SFRS1, PABC1, NETO2 and ENSA genes. Taken together the data presented here indicate that SF2/ASF has the capacity to co-regulate the nuclear and cytoplasmic processing of specific mRNAs and provide further evidence that the nuclear history of an mRNA may influence its cytoplasmic fate.

  13. Crystallization and preliminary X-ray analysis of the splice variant of human ankyrin repeat and suppressor of cytokine signaling box protein 9 (hASB9-2).

    Science.gov (United States)

    Fei, Xiangwei; Zhang, Yong; Gu, Xing; Qiu, Rui; Mao, Yumin; Ji, Chaoneng

    2008-01-01

    Human ankyrin repeat and suppressor of cytokine signaling box protein 9 (hASB9), a subunit of an Elongin C-cullin-SOCS box (ECS) E3 ubiquitin ligase complex, is believed to be involved in specific substrate-recognition for ubiquitination and degradation. In fact, this specific substrate-recognition is determined by the ankyrin repeats of hASB9 protein. Here, we have cloned and overexpressed the hASB9-2, the splice variant of hASB9 with only one ankyrin repeat domain, as a 6His-tagged recombinant protein in Escherichia coli. The purified hASB9-2 protein was crystallized by the hanging-drop vapor-diffusion technique and diffracted to 2.2A resolution. The data showed that the cubic hASB9-2 crystal belongs to space group P4(3)32 with unit-cell parameters (a=b=c=129.25A, alpha=beta=gamma=90 degrees ). An asymmetric unit in the crystal was assumed to contain one protein molecule giving the Matthews Coefficient factor of 2.81 and the solvent content of 56.3%.

  14. Differentiation inducing factor-1 (DIF-1) induces gene and protein expression of the Dictyostelium nuclear calmodulin-binding protein nucleomorphin.

    Science.gov (United States)

    O'Day, Danton H; Poloz, Yekaterina; Myre, Michael A

    2009-02-01

    The nucleomorphin gene numA1 from Dictyostelium codes for a multi-domain, calmodulin binding protein that regulates nuclear number. To gain insight into the regulation of numA, we assessed the effects of the stalk cell differentiation inducing factor-1 (DIF-1), an extracellular signalling molecule, on the expression of numA1 RNA and protein. For comparison, the extracellular signalling molecules cAMP (mediates chemotaxis, prestalk and prespore differentiation) and ammonia (NH(3)/NH(4)(+); antagonizes DIF) were also studied. Starvation, which is a signal for multicellular development, results in a greater than 80% decrease in numA1 mRNA expression within 4 h. Treatment with ammonium chloride led to a greater than 90% inhibition of numA1 RNA expression within 2 h. In contrast, the addition of DIF-1 completely blocked the decrease in numA1 gene expression caused by starvation. Treatment of vegetative cells with cAMP led to decreases in numA1 RNA expression that were equivalent to those seen with starvation. Western blotting after various morphogen treatments showed that the maintenance of vegetative levels of numA1 RNA by DIF-1 in starved cells was reflected in significantly increased numA1 protein levels. Treatment with cAMP and/or ammonia led to decreased protein expression and each of these morphogens suppressed the stimulatory effects of DIF-1. Protein expression levels of CBP4a, a calcium-dependent binding partner of numA1, were regulated in the same manner as numA1 suggesting this potential co-regulation may be related to their functional relationship. NumA1 is the first calmodulin binding protein shown to be regulated by developmental morphogens in Dictyostelium being upregulated by DIF-1 and down-regulated by cAMP and ammonia.

  15. Virus-Induced Chaperone-Enriched (VICE domains function as nuclear protein quality control centers during HSV-1 infection.

    Directory of Open Access Journals (Sweden)

    Christine M Livingston

    2009-10-01

    Full Text Available Virus-Induced Chaperone-Enriched (VICE domains form adjacent to nuclear viral replication compartments (RC during the early stages of HSV-1 infection. Between 2 and 3 hours post infection at a MOI of 10, host protein quality control machinery such as molecular chaperones (e.g. Hsc70, the 20S proteasome and ubiquitin are reorganized from a diffuse nuclear distribution pattern to sequestration in VICE domains. The observation that VICE domains contain putative misfolded proteins suggests that they may be similar to nuclear inclusion bodies that form under conditions in which the protein quality control machinery is overwhelmed by the presence of misfolded proteins. The detection of Hsc70 in VICE domains, but not in nuclear inclusion bodies, indicates that Hsc70 is specifically reorganized by HSV-1 infection. We hypothesize that HSV-1 infection induces the formation of nuclear protein quality control centers to remodel or degrade aberrant nuclear proteins that would otherwise interfere with productive infection. Detection of proteolytic activity in VICE domains suggests that substrates may be degraded by the 20S proteasome in VICE domains. FRAP analysis reveals that GFP-Hsc70 is dynamically associated with VICE domains, suggesting a role for Hsc70 in scanning the infected nucleus for misfolded proteins. During 42 degrees C heat shock, Hsc70 is redistributed from VICE domains into RC perhaps to remodel viral replication and regulatory proteins that have become insoluble in these compartments. The experiments presented in this paper suggest that VICE domains are nuclear protein quality control centers that are modified by HSV-1 to promote productive infection.

  16. HIV-1 uncoating: connection to nuclear entry and regulation by host proteins

    Energy Technology Data Exchange (ETDEWEB)

    Ambrose, Zandrea, E-mail: zaa4@pitt.edu [Division of Infectious Diseases, Department of Medicine, University of Pittsburgh, School of Medicine, Pittsburgh, PA 15261 (United States); Aiken, Christopher [Department of Pathology, Microbiology and Immunology, Vanderbilt University, School of Medicine, Nashville, TN 37232 (United States)

    2014-04-15

    The RNA genome of human immunodeficiency virus type 1 (HIV-1) is enclosed by a capsid shell that dissociates within the cell in a multistep process known as uncoating, which influences completion of reverse transcription of the viral genome. Double-stranded viral DNA is imported into the nucleus for integration into the host genome, a hallmark of retroviral infection. Reverse transcription, nuclear entry, and integration are coordinated by a capsid uncoating process that is regulated by cellular proteins. Although uncoating is not well understood, recent studies have revealed insights into the process, particularly with respect to nuclear import pathways and protection of the viral genome from DNA sensors. Understanding uncoating will be valuable toward developing novel antiretroviral therapies for HIV-infected individuals.

  17. Control of Paternally Expressed Imprinted UPWARD CURLY LEAF1, a Gene Encoding an F-Box Protein That Regulates CURLY LEAF Polycomb Protein, in the Arabidopsis Endosperm.

    Directory of Open Access Journals (Sweden)

    Cheol Woong Jeong

    Full Text Available Genomic imprinting, an epigenetic process in mammals and flowering plants, refers to the differential expression of alleles of the same genes in a parent-of-origin-specific manner. In Arabidopsis, imprinting occurs primarily in the endosperm, which nourishes the developing embryo. Recent high-throughput sequencing analyses revealed that more than 200 loci are imprinted in Arabidopsis; however, only a few of these imprinted genes and their imprinting mechanisms have been examined in detail. Whereas most imprinted loci characterized to date are maternally expressed imprinted genes (MEGs, PHERES1 (PHE1 and ADMETOS (ADM are paternally expressed imprinted genes (PEGs. Here, we report that UPWARD CURLY LEAF1 (UCL1, a gene encoding an E3 ligase that degrades the CURLY LEAF (CLF polycomb protein, is a PEG. After fertilization, paternally inherited UCL1 is expressed in the endosperm, but not in the embryo. The expression pattern of a β-glucuronidase (GUS reporter gene driven by the UCL1 promoter suggests that the imprinting control region (ICR of UCL1 is adjacent to a transposable element in the UCL1 5'-upstream region. Polycomb Repressive Complex 2 (PRC2 silences the maternal UCL1 allele in the central cell prior to fertilization and in the endosperm after fertilization. The UCL1 imprinting pattern was not affected in paternal PRC2 mutants. We found unexpectedly that the maternal UCL1 allele is reactivated in the endosperm of Arabidopsis lines with mutations in cytosine DNA METHYLTRANSFERASE 1 (MET1 or the DNA glycosylase DEMETER (DME, which antagonistically regulate CpG methylation of DNA. By contrast, maternal UCL1 silencing was not altered in mutants with defects in non-CpG methylation. Thus, silencing of the maternal UCL1 allele is regulated by both MET1 and DME as well as by PRC2, suggesting that divergent mechanisms for the regulation of PEGs evolved in Arabidopsis.

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

    Science.gov (United States)

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

    1999-03-05

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

  19. An apicoplast localized ubiquitylation system is required for the import of nuclear-encoded plastid proteins.

    Directory of Open Access Journals (Sweden)

    Swati Agrawal

    Full Text Available Apicomplexan parasites are responsible for numerous important human diseases including toxoplasmosis, cryptosporidiosis, and most importantly malaria. There is a constant need for new antimalarials, and one of most keenly pursued drug targets is an ancient algal endosymbiont, the apicoplast. The apicoplast is essential for parasite survival, and several aspects of its metabolism and maintenance have been validated as targets of anti-parasitic drug treatment. Most apicoplast proteins are nuclear encoded and have to be imported into the organelle. Recently, a protein translocon typically required for endoplasmic reticulum associated protein degradation (ERAD has been proposed to act in apicoplast protein import. Here, we show ubiquitylation to be a conserved and essential component of this process. We identify apicoplast localized ubiquitin activating, conjugating and ligating enzymes in Toxoplasma gondii and Plasmodium falciparum and observe biochemical activity by in vitro reconstitution. Using conditional gene ablation and complementation analysis we link this activity to apicoplast protein import and parasite survival. Our studies suggest ubiquitylation to be a mechanistic requirement of apicoplast protein import independent to the proteasomal degradation pathway.

  20. Specificity of leaf mitochondrial and chloroplast processing systems for nuclear-encoded precursor proteins.

    Science.gov (United States)

    Whelan, J; Knorpp, C; Harmey, M A; Glaser, E

    1991-02-01

    The specificity of the mitochondrial and chloroplast processing enzymes for the nuclear-encoded precursor proteins was investigated. Mitochondrial precursor proteins of the Nicotiana plumbaginifolia and the Neurospora crassa beta subunits of F1-ATPase and the Neurospora Rieske FeS precursor protein were processed to the correct mature size by matrix extracts isolated from spinach leaves, yeast, rat liver and beef heart. The mitochondrial extracts failed to process chloroplast precursor proteins of the stromal small subunit of ribulose 1,5-bisphosphate carboxylase and the thylakoid 33 kDa protein of the oxygen-evolving complex. Both mitochondrial F1 beta precursors were specifically processed by a soluble stromal extract from chloroplasts. However, no processing of the Rieske FeS precursor protein was observed under the same conditions with the chloroplast extract. The cleavage of the mitochondrial F1 beta precursors by the chloroplast extract was shown to be sensitive to the metal chelators EDTA and ortho-phenanthroline. The cleavage site of the mitochondrial F1 beta precursor by the chloroplast soluble extract appears to be located at the N-terminus.

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

    Energy Technology Data Exchange (ETDEWEB)

    Nagamine, Tadashi; Nomada, Shohgo; Onouchi, Takashi; Kameshita, Isamu; Sueyoshi, Noriyuki, E-mail: sueyoshi@ag.kagawa-u.ac.jp

    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.

  2. Imbalance of different types of CD4(+) forkhead box protein 3 (FoxP3)(+) T cells in patients with new-onset systemic lupus erythematosus.

    Science.gov (United States)

    Ma, L; Zhao, P; Jiang, Z; Shan, Y; Jiang, Y

    2013-12-01

    The aim of this study was to examine the numbers of CD4(+) CD25(-) forkhead box protein 3 (FoxP3)(+) , CD4(+) CD25(+) FoxP3(+) and CD4(+) CXCR5(+) FoxP3(+) T cells in patients with new-onset systemic lupus erythematosus (SLE). The numbers of CD4(+) CD25(-) FoxP3(+) , CD4(+) CD25(+) FoxP3(+) and CD4(+) CXCR5(+) FoxP3(+) T cells and the concentrations of serum interleukin (IL)-10 in 23 patients and 20 healthy controls (HC) were measured. The potential correlations between CD4(+) FoxP3(+) T cells, serum IL-10 and clinical measures in SLE patients were analysed. In comparison with that in the HC, significantly reduced numbers of CD4(+) CD25(+) FoxP3(+) and CD4(+) CXCR5(+) FoxP3(+) T cells, but increased numbers of CD4(+) CD25(-) FoxP3(+) T cells, were detected, accompanied by significantly lower levels of serum IL-10 in the patients. Stratification analysis indicated the numbers of CD4(+) CD25(+) FoxP3(+) and CD4(+) CXCR5(+) FoxP3(+) T cells and serum IL-10 levels in the patients with seropositive anti-dsDNA were significantly less than that in those with seronegative anti-dsDNA. Treatment with the anti-SLE therapy, particularly with prednisone, leflunomide and methotrexate, significantly improved the imbalance of these types of FoxP3(+) T cells and increased the concentrations of serum IL-10 in the drug-responding patients. The numbers of CD4(+) CD25(+) FoxP3(+) T cells were correlated negatively with the values of SLE disease activity index (SLEDAI), whereas the numbers of CD4(+) CD25(-) FoxP3(+) T cells were correlated positively with the values of SLEDAI, erythrocyte sedimentation rate (ESR) and serum C3. In addition, the concentrations of serum IL-10 were correlated positively with the numbers of CD4(+) CD25(+) FoxP3(+) T cells, but negatively with the values of SLEDAI, serum C3, CRP and ESR in these patients. Our data indicate that the imbalance of different types of FoxP3(+) CD4(+) T cells may contribute to the development of SLE in Chinese patients. © 2013

  3. Forkhead box protein P3 (Foxp3) expression serves as an early chronic inflammation marker of squamous cell differentiation and aggressive pathology of urothelial carcinomas in neurological patients.

    Science.gov (United States)

    Phé, Véronique; Rouprêt, Morgan; Cussenot, Olivier; Chartier-Kastler, Emmanuel; Gamé, Xavier; Compérat, Eva

    2015-04-01

    To establish whether the expression of forkhead box protein P3 (Foxp3) provides specific diagnostic information about neurological patients with urothelial carcinoma of the bladder (UCB). UCB tissue samples from neurological patients were retrieved and compared with control samples. The expression of Foxp3 was analysed via immunohistochemistry of microarray tissue sections. The correlation between Foxp3 expression, histological parameters and tumour stage was assessed. Overall, 20 UCB tissue samples and 20 others without UCB from neurological patients, and 46 UCB tissue samples from non-neurological patients were analysed. The distribution of pT of UCB in the neurological patients was as follows: one low-grade pTa (5%), three high-grade pTa (15%), three pT1(15%), one pT2(5%), seven pT3(35%) and five pT4(25%). Squamous cell differentiation was seen in nine UCB samples (45%). Foxp3 expression was detected in tumour tissues, including one pTa high grade, one pT1, one pT2, five pT3 and five pT4 tumours. Foxp3 was expressed in 11/13 muscle-invasive tumours. All tumours displaying squamous cell differentiation expressed Foxp3. Foxp3 was not expressed in the pT3 tumours that displayed sarcomatoid and micropapillary properties. Among the bladder samples without UCB from neurological patients, no expression of Foxp3 was observed. Among the UCB samples from the non-neurological patients, only seven displayed squamous cell differentiation. All tumours that displayed squamous cell differentiation expressed Foxp3, including one pTa high grade, four pT3 and two pT4 tumours. Other tumours displaying urothelial differentiation did not express Foxp3. The expression of Foxp3 correlated to squamous cell differentiation in neurological (P = 0.004) and non-neurological UCB tissue (P differentiation. Targeting Foxp3 may represent a novel strategy to improve anti-tumour immunotherapy for UCB. © 2015 The Authors. BJU International © 2015 BJU International.

  4. An epidemiological investigation of a Forkhead box protein E3 founder mutation underlying the high frequency of sclerocornea, aphakia, and microphthalmia in a Mexican village.

    Science.gov (United States)

    Pantoja-Melendez, Carlos; Ali, Manir; Zenteno, Juan C

    2013-01-01

    To investigate the molecular epidemiological basis for the unusually high incidence of sclerocornea, aphakia, and microphthalmia in a village in the Tlaxcala province of central Mexico. A population census was performed in a village to identify all sclerocornea, aphakia, and microphthalmia cases. Molecular analysis of the previously identified Forkhead box protein E3 (FOXE3) mutation, c.292T>C (p.Y98H), was performed with PCR amplification and direct DNA sequencing. In addition, DNA from 405 randomly selected unaffected villagers was analyzed to establish the carrier frequency of the causal mutation. To identify the number of generations since the mutation arose in the village, 17 polymorphic markers distributed in a region of 6 Mb around the mutated locus were genotyped in the affected individuals, followed by DMLE software analysis to calculate mutation age. A total of 22 patients with sclerocornea, aphakia, and microphthalmia were identified in the village, rendering a disease prevalence of 2.52 cases per 1,000 habitants (1 in 397). The FOXE3 homozygous mutation was identified in all 17 affected subjects who consented to molecular analysis. Haplotype analysis indicated that the mutation arose 5.0-6.5 generations ago (approximately 106-138 years). Among the 405 unaffected villagers who were genotyped, ten heterozygote carriers were identified, yielding a population carrier frequency of approximately 1 in 40 and a predicted incidence of affected of 1 in 6,400 based on random marriages between two carriers in the village. This study demonstrates that a cluster of patients with sclerocornea, aphakia, and microphthalmia in a small Mexican village is due to a FOXE3 p.Y98H founder mutation that arose in the village just over a century ago at a time when a population migrated from a nearby village because of land disputes. The actual disease incidence is higher than the calculated predicted value and suggests non-random marriages (i.e., consanguinity) within the

  5. Nuclear envelope proteins and chromatin arrangement: a pathogenic mechanism for laminopathies

    Directory of Open Access Journals (Sweden)

    NM Maraldi

    2009-06-01

    Full Text Available The involvement of the nuclear envelope in the modulation of chromatin organization is strongly suggested by the increasing number of human diseases due to mutations of nuclear envelope proteins. A common feature of these diseases, named laminopathies, is the occurrence of major chromatin defects. Laminopathies share in some instances their clinical features, but each of them is characterized by a phenotype that involves one or multiple tissues.We previously reported that cells from laminopathic patients show an altered nuclear profile, and loss or detachment of heterochromatin from the nuclear envelope. Recent evidence indicates that processing of the lamin A precursor is altered in laminopathies featuring pre-mature aging and/or lipodystrophy phenotype. In these cases, pre-lamin A is accumulated in the nucleus and heterochromatin is severely disorganized. Moreover, altered distribution and solubility properties of heterochromatin-associated proteins such as HP1 are observed. These findings indicate that defects of chromatin remodeling are involved in the cascade of epigenetic events leading to the laminopathic phenotypes. Here we report evidence indicating that pre-lamin A is mis-localized in the nuclei of Emery-Dreifuss muscular dystrophy fibroblasts, either bearing lamin A/C or emerin mutations. Abnornal pre-lamin A-containing structures are formed following treatment with a farnesyl-transferase inhibitor, a drug that causes accumulation of non-farnesylated pre-lamin A. Pre-lamin A-labeled structures co-localize with heterochromatin clumps. These data indicate that in almost all laminopathies the expression of the mutant lamin A precursor disrupts the organization of heterochromatin domains so that affected cells are unable to maintain the silenced chromatin state capable to allow/preserve terminal differentiation. Our results further show that the absence of emerin expression alters the distribution of pre-lamin A and of heterochromatin

  6. The PCNA interaction protein box sequence in Rad54 is an integral part of its ATPase domain and is required for efficient DNA repair and recombination

    DEFF Research Database (Denmark)

    Burgess, Rebecca C; Sebesta, Marek; Sisakova, Alexandra

    2013-01-01

    was defective in primer extension at the MAT locus as well as in vitro, but additional biochemical analysis revealed that this mutant also had diminished ATPase activity and an inability to promote D-loop formation. Further mutational analysis of the putative PIP-box uncovered that other phenotypically relevant...

  7. The Nuclear Death Domain Protein p84N5; a Candidate Breast Cancer Susceptibility Gene

    Science.gov (United States)

    2005-05-01

    DATES COVERED ( Leave blank) May 2005 Annual (1 May 04 - 30 Apr 05) 4. TITLE AND SUBTITLE 5. FUNDING NUMBERS The Nuclear Death Domain Protein p84N5; a...p84N5. Cell proliferation and apoptosis (data not shown) was examined using Guava ViaCount and Nexin assays, respectively. Plotted is the number of...sequence results identical to those of breast cancer cell lines. Based on these data, we concluded that there are no somatic mutations in p84N5 in these

  8. Nuclear export and import of human hepatitis B virus capsid protein and particles.

    Directory of Open Access Journals (Sweden)

    Hung-Cheng Li

    Full Text Available It remains unclear what determines the subcellular localization of hepatitis B virus (HBV core protein (HBc and particles. To address this fundamental issue, we have identified four distinct HBc localization signals in the arginine rich domain (ARD of HBc, using immunofluorescence confocal microscopy and fractionation/Western blot analysis. ARD consists of four tight clustering arginine-rich subdomains. ARD-I and ARD-III are associated with two co-dependent nuclear localization signals (NLS, while ARD-II and ARD-IV behave like two independent nuclear export signals (NES. This conclusion is based on five independent lines of experimental evidence: i Using an HBV replication system in hepatoma cells, we demonstrated in a double-blind manner that only the HBc of mutant ARD-II+IV, among a total of 15 ARD mutants, can predominantly localize to the nucleus. ii These results were confirmed using a chimera reporter system by placing mutant or wild type HBc trafficking signals in the heterologous context of SV40 large T antigen (LT. iii By a heterokaryon or homokaryon analysis, the fusion protein of SV40 LT-HBc ARD appeared to transport from nuclei of transfected donor cells to nuclei of recipient cells, suggesting the existence of an NES in HBc ARD. This putative NES is leptomycin B resistant. iv We demonstrated by co-immunoprecipitation that HBc ARD can physically interact with a cellular factor TAP/NXF1 (Tip-associated protein/nuclear export factor-1, which is known to be important for nuclear export of mRNA and proteins. Treatment with a TAP-specific siRNA strikingly shifted cytoplasmic HBc to nucleus, and led to a near 7-fold reduction of viral replication, and a near 10-fold reduction in HBsAg secretion. v HBc of mutant ARD-II+IV was accumulated predominantly in the nucleus in a mouse model by hydrodynamic delivery. In addition to the revised map of NLS, our results suggest that HBc could shuttle rapidly between nucleus and cytoplasm via a novel

  9. AtSKIP18 and AtSKIP31, F-box subunits of the SCF E3 ubiquitin ligase complex, mediate the degradation of 14-3-3 proteins in Arabidopsis.

    Science.gov (United States)

    Hong, Jong-Pil; Adams, Eri; Yanagawa, Yuki; Matsui, Minami; Shin, Ryoung

    2017-03-25

    14-3-3 proteins regulate numerous cellular processes through interaction with their target proteins in a phosphorylation dependent manner. Although proteins that are regulated by 14-3-3s have been studied, the regulatory mechanism of 14-3-3s is poorly understood. In the present study, F-box proteins, a component of Skp1-Cullin-F-box E3 ubiquitin ligase, were identified as 14-3-3 targets using yeast two-hybrid screening. Among them, AtSKIP18 and AtSKIP31, were shown to mediate the degradation of Arabidopsis 14-3-3s. Mutational analyses of AtSKIP18 and AtSKIP31 indicated that the phosphorylation of AtSKIPs is critical for interaction and degradation of 14-3-3s. The loss-of-function mutation in AtSKIP31 resulted in enhanced primary root growth under nitrogen deficient conditions. These findings suggest that AtSKIP31 regulates the primary root growth in nitrogen deficiency via degrading 14-3-3s. Copyright © 2017 Elsevier Inc. All rights reserved.

  10. Math in the Box

    Science.gov (United States)

    DeYoung, Mary J.

    2009-01-01

    This article describes how to make an origami paper box and explores the algebra, geometry, and other mathematics that unfolds. A set of origami steps that transforms the paper into an open box can hold mathematical surprises for both students and teachers. An origami lesson can engage students in an open-ended exploration of the relationship…

  11. ALUMINUM BOX BUNDLING PRESS

    Directory of Open Access Journals (Sweden)

    Iosif DUMITRESCU

    2015-05-01

    Full Text Available In municipal solid waste, aluminum is the main nonferrous metal, approximately 80- 85% of the total nonferrous metals. The income per ton gained from aluminum recuperation is 20 times higher than from glass, steel boxes or paper recuperation. The object of this paper is the design of a 300 kN press for aluminum box bundling.

  12. Y-box-binding protein-1 (YB-1) promotes cell proliferation, adhesion and drug resistance in diffuse large B-cell lymphoma.

    Science.gov (United States)

    Miao, Xiaobing; Wu, Yaxun; Wang, Yuchan; Zhu, Xinghua; Yin, Haibing; He, Yunhua; Li, Chunsun; Liu, Yushan; Lu, Xiaoyun; Chen, Yali; Shen, Rong; Xu, Xiaohong; He, Song

    2016-08-15

    YB-1 is a multifunctional protein, which has been shown to correlate with resistance to treatment of various tumor types. This study investigated the expression and biologic function of YB-1 in diffuse large B-cell lymphoma (DLBCL). Immunohistochemical analysis showed that the expression statuses of YB-1 and pYB-1(S102) were reversely correlated with the clinical outcomes of DLBCL patients. In addition, we found that YB-1 could promote the proliferation of DLBCL cells by accelerating the G1/S transition. Ectopic expression of YB-1 could markedly increase the expression of cell cycle regulators cyclin D1 and cyclin E. Furthermore, we found that adhesion of DLBCL cells to fibronectin (FN) could increase YB-1 phosphorylation at Ser102 and pYB-1(S102) nuclear translocation. In addition, overexpression of YB-1 could increase the adhesion of DLBCL cells to FN. Intriguingly, we found that YB-1 overexpression could confer drug resistance through cell-adhesion dependent and independent mechanisms in DLBCL. Silencing of YB-1 could sensitize DLBCL cells to mitoxantrone and overcome cell adhesion-mediated drug resistance (CAM-DR) phenotype in an AKT-dependent manner. Copyright © 2016 Elsevier Inc. All rights reserved.

  13. Y-box-binding protein-1 (YB-1) promotes cell proliferation, adhesion and drug resistance in diffuse large B-cell lymphoma

    Energy Technology Data Exchange (ETDEWEB)

    Miao, Xiaobing; Wu, Yaxun [Department of Pathology, Affiliated Cancer Hospital of Nantong University, Nantong 226361, Jiangsu (China); Wang, Yuchan [Department of Pathogen, Medical College, Nantong University, Nantong 226001, Jiangsu (China); Jiangsu Province Key Laboratory for Inflammation and Molecular Drug Target, Nantong University, Nantong 226001, Jiangsu (China); Zhu, Xinghua; Yin, Haibing [Department of Pathology, Affiliated Cancer Hospital of Nantong University, Nantong 226361, Jiangsu (China); He, Yunhua [Jiangsu Province Key Laboratory for Inflammation and Molecular Drug Target, Nantong University, Nantong 226001, Jiangsu (China); Li, Chunsun; Liu, Yushan; Lu, Xiaoyun; Chen, Yali; Shen, Rong [Department of Pathology, Affiliated Cancer Hospital of Nantong University, Nantong 226361, Jiangsu (China); Xu, Xiaohong, E-mail: xuxiaohongnantong@126.com [Department of Oncology, Affiliated Cancer Hospital of Nantong University, Nantong 226361, Jiangsu (China); He, Song, E-mail: hesongnt@126.com [Department of Pathology, Affiliated Cancer Hospital of Nantong University, Nantong 226361, Jiangsu (China)

    2016-08-15

    YB-1 is a multifunctional protein, which has been shown to correlate with resistance to treatment of various tumor types. This study investigated the expression and biologic function of YB-1 in diffuse large B-cell lymphoma (DLBCL). Immunohistochemical analysis showed that the expression statuses of YB-1 and pYB-1{sup S102} were reversely correlated with the clinical outcomes of DLBCL patients. In addition, we found that YB-1 could promote the proliferation of DLBCL cells by accelerating the G1/S transition. Ectopic expression of YB-1 could markedly increase the expression of cell cycle regulators cyclin D1 and cyclin E. Furthermore, we found that adhesion of DLBCL cells to fibronectin (FN) could increase YB-1 phosphorylation at Ser102 and pYB-1{sup S102} nuclear translocation. In addition, overexpression of YB-1 could increase the adhesion of DLBCL cells to FN. Intriguingly, we found that YB-1 overexpression could confer drug resistance through cell-adhesion dependent and independent mechanisms in DLBCL. Silencing of YB-1 could sensitize DLBCL cells to mitoxantrone and overcome cell adhesion-mediated drug resistance (CAM-DR) phenotype in an AKT-dependent manner. - Highlights: • The expression statuses of YB-1 and pYB-1{sup S102} are reversely correlated with outcomes of DLBCL patients. • YB-1 promotes cell proliferation by accelerating G1/S transition in DLBCL. • YB-1 confers drug resistance to mitoxantrone in DLBCL.

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

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Chung Kwon; Lee, Sang Bae; Nguyen, Truong L.X. [Department of Molecular Cell Biology, Sungkyunkwan University School of Medicine, Suwon, 440-746 (Korea, Republic of); Lee, Kyung-Hoon [Department of Anatomy, Sungkyunkwan University School of Medicine, Suwon, 440-746 (Korea, Republic of); Center for Molecular Medicine, Samsung Biomedical Research Institute, Sungkyunkwan University School of Medicine, Suwon, 440-746 (Korea, Republic of); Um, Sung Hee [Department of Molecular Cell Biology, Sungkyunkwan University School of Medicine, Suwon, 440-746 (Korea, Republic of); Center for Molecular Medicine, Samsung Biomedical Research Institute, Sungkyunkwan University School of Medicine, Suwon, 440-746 (Korea, Republic of); Kim, Jihoe [School of Biotechnology, Yeungnam University, Gyeongsan 712-749 (Korea, Republic of); Ahn, Jee-Yin, E-mail: jeeahn@skku.edu [Department of Molecular Cell Biology, Sungkyunkwan University School of Medicine, Suwon, 440-746 (Korea, Republic of); Center for Molecular Medicine, Samsung Biomedical Research Institute, Sungkyunkwan University School of Medicine, Suwon, 440-746 (Korea, Republic of)

    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.

  15. Cross-regulation of protein stability by p53 and nuclear receptor SHP.

    Directory of Open Access Journals (Sweden)

    Zhihong Yang

    Full Text Available We report here a novel interplay between tumor suppressor p53 and nuclear receptor SHP that controls p53 and SHP stability. Overexpression of p53 causes rapid SHP protein degradation, which does not require the presence of Mdm2 and is mediated by the proteosome pathway. Overexpressing SHP alone does not affect p53 stability. However, SHP destabilizes p53 by augmentation of Mdm2 ubiquitin ligase activity toward p53. The single amino acid substitution in the SHP protein SHPK170R increases SHP binding to p53 relative to SHP wild-type, whereas SHPG171A variant shows a diminished p53 binding. As a result of the cross-regulation, the tumor suppressor function of p53 and SHP in inhibition of colon cancer growth is compromised. Our findings reveal a unique scenario for a cross-inhibition between two tumor suppressors to keep their expression and function in check.

  16. Recrystallization inhibition in ice due to ice binding protein activity detected by nuclear magnetic resonance

    Directory of Open Access Journals (Sweden)

    Jennifer R. Brown

    2014-09-01

    Full Text Available Liquid water present in polycrystalline ice at the interstices between ice crystals results in a network of liquid-filled veins and nodes within a solid ice matrix, making ice a low porosity porous media. Here we used nuclear magnetic resonance (NMR relaxation and time dependent self-diffusion measurements developed for porous media applications to monitor three dimensional changes to the vein network in ices with and without a bacterial ice binding protein (IBP. Shorter effective diffusion distances were detected as a function of increased irreversible ice binding activity, indicating inhibition of ice recrystallization and persistent small crystal structure. The modification of ice structure by the IBP demonstrates a potential mechanism for the microorganism to enhance survivability in ice. These results highlight the potential of NMR techniques in evaluation of the impact of IBPs on vein network structure and recrystallization processes; information useful for continued development of ice-interacting proteins for biotechnology applications.

  17. Iterative Development of an Application to Support Nuclear Magnetic Resonance Data Analysis of Proteins.

    Science.gov (United States)

    Ellis, Heidi J C; Nowling, Ronald J; Vyas, Jay; Martyn, Timothy O; Gryk, Michael R

    2011-04-11

    The CONNecticut Joint University Research (CONNJUR) team is a group of biochemical and software engineering researchers at multiple institutions. The vision of the team is to develop a comprehensive application that integrates a variety of existing analysis tools with workflow and data management to support the process of protein structure determination using Nuclear Magnetic Resonance (NMR). The use of multiple disparate tools and lack of data management, currently the norm in NMR data processing, provides strong motivation for such an integrated environment. This manuscript briefly describes the domain of NMR as used for protein structure determination and explains the formation of the CONNJUR team and its operation in developing the CONNJUR application. The manuscript also describes the evolution of the CONNJUR application through four prototypes and describes the challenges faced while developing the CONNJUR application and how those challenges were met.

  18. The R35 residue of the influenza A virus NS1 protein has minimal effects on nuclear localization but alters virus replication through disrupting protein dimerization

    Energy Technology Data Exchange (ETDEWEB)

    Lalime, Erin N.; Pekosz, Andrew, E-mail: apekosz@jhsph.edu

    2014-06-15

    The influenza A virus NS1 protein has a nuclear localization sequence (NLS) in the amino terminal region. This NLS overlaps sequences that are important for RNA binding as well as protein dimerization. To assess the significance of the NS1 NLS on influenza virus replication, the NLS amino acids were individually mutated to alanines and recombinant viruses encoding these mutations were rescued. Viruses containing NS1 proteins with mutations at R37, R38 and K41 displayed minimal changes in replication or NS1 protein nuclear localization. Recombinant viruses encoding NS1 R35A were not recovered but viruses containing second site mutations at position D39 in addition to the R35A mutation were isolated. The mutations at position 39 were shown to partially restore NS1 protein dimerization but had minimal effects on nuclear localization. These data indicate that the amino acids in the NS1 NLS region play a more important role in protein dimerization compared to nuclear localization. - Highlights: • Mutations were introduced into influenza NS1 NLS1. • NS1 R37A, R38A, K41A viruses had minimal changes in replication and NS1 localization. • Viruses from NS1 R35A rescue all contained additional mutations at D39. • NS1 R35A D39X mutations recover dimerization lost in NS1 R35A mutations. • These results reaffirm the importance of dimerization for NS1 protein function.

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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

  1. Variants within the SP110 nuclear body protein modify risk of canine degenerative myelopathy.

    Science.gov (United States)

    Ivansson, Emma L; Megquier, Kate; Kozyrev, Sergey V; Murén, Eva; Körberg, Izabella Baranowska; Swofford, Ross; Koltookian, Michele; Tonomura, Noriko; Zeng, Rong; Kolicheski, Ana L; Hansen, Liz; Katz, Martin L; Johnson, Gayle C; Johnson, Gary S; Coates, Joan R; Lindblad-Toh, Kerstin

    2016-05-31

    Canine degenerative myelopathy (DM) is a naturally occurring neurodegenerative disease with similarities to some forms of amyotrophic lateral sclerosis (ALS). Most dogs that develop DM are homozygous for a common superoxide dismutase 1 gene (SOD1) mutation. However, not all dogs homozygous for this mutation develop disease. We performed a genome-wide association analysis in the Pembroke Welsh Corgi (PWC) breed comparing DM-affected and -unaffected dogs homozygous for the SOD1 mutation. The analysis revealed a modifier locus on canine chromosome 25. A haplotype within the SP110 nuclear body protein (SP110) was present in 40% of affected compared with 4% of unaffected dogs (P = 1.5 × 10(-5)), and was associated with increased probability of developing DM (P = 4.8 × 10(-6)) and earlier onset of disease (P = 1.7 × 10(-5)). SP110 is a nuclear body protein involved in the regulation of gene transcription. Our findings suggest that variations in SP110-mediated gene transcription may underlie, at least in part, the variability in risk for developing DM among PWCs that are homozygous for the disease-related SOD1 mutation. Further studies are warranted to clarify the effect of this modifier across dog breeds.

  2. HTLV-1 Tax upregulates early growth response protein 1 through nuclear factor-κB signaling.

    Science.gov (United States)

    Huang, Qingsong; Niu, Zhiguo; Han, Jingxian; Liu, Xihong; Lv, Zhuangwei; Li, Huanhuan; Yuan, Lixiang; Li, Xiangping; Sun, Shuming; Wang, Hui; Huang, Xinxiang

    2017-08-01

    Human T cell leukemia virus type 1 (HTLV-1) is a complex retrovirus that causes adult T cell leukemia (ATL) in susceptible individuals. The HTLV-1-encoded oncoprotein Tax induces persistent activation of the nuclear factor-κB (NF-κB) pathway. Early growth response protein 1 (EGR1) is overexpressed in HTLV-1-infected T cell lines and ATL cells. Here, we showed that both Tax expression and HTLV-1 infection promoted EGR1 overexpression. Loss of the NF-κB binding site in the EGR1 promotor or inhibition of NF-κB activation reduced Tax-induced EGR1 upregulation. Tax mutants unable to activate NF-κB induced only slight EGR1 upregulation as compared with wild-type Tax, confirming NF-κB pathway involvement in EGR1 regulation. Tax also directly interacted with the EGR1 protein and increased endogenous EGR1 stability. Elevated EGR1 in turn promoted p65 nuclear translocation and increased NF-κB activation. These results demonstrate a positive feedback loop between EGR1 expression and NF-κB activation in HTLV-1-infected and Tax-expressing cells. Both NF-κB activation and Tax-induced EGR1 stability upregulated EGR1, which in turn enhanced constitutive NF-κB activation and facilitated ATL progression in HTLV-1-infected cells. These findings suggest EGR1 may be an effective anti-ATL therapeutic target.

  3. Yes-Associated Protein (YAP) Promotes the Nuclear Import of p73

    Science.gov (United States)

    Zhang, Heng; Wu, Shengnan

    2011-01-01

    p73 has been identified as a structural and functional homolog of the tumor suppressor p53. However, mechanisms that regulate the localization of p73 have not been fully clarified. The Yes-associated protein (YAP) is a transcriptional coactivator. As a transcriptional coactivator, YAP needs to bind transcription factors to stimulate gene expression. p73 is a reported YAP target transcription factors and YAP has been shown to positively regulate p73 in promoting apoptosis. Previous studies show that p73 interacts with YAP through its PPPY motif, and increases p73 transactivation of apoptotic genes. In this study, we focused on YAP's regulation of the localization of p73. After transient transfection into Rat pheochromocytoma (PC12) cells and Human embryonic kidney 293T cells with GFP-YAP and/or YFP-p73, and incubated for 24 hours expression. p73 was fused to YFP to allow the examination of its subcellular localization. When expressed alone, YFP-p73 was distributed throughout the cell. When coexpressed with YAP, nuclear accumulation of YFP-p73 became evident. We quantitated the effect of YAP on the redistribution of YFP-p73 by counting cells with nuclear-only YFP signal. We found that YAP can influence the subcellular distribution of p73. Altogether, coexpression with YAP affected the subcellular distribution of the p73 protein. Our studies attribute a central role to YAP in regulating p73 accumulation and YAP, at least in part, might promote the nuclear import of p73.

  4. Identification of a novel nuclear localization signal sequence in Chlamydia trachomatis-secreted hypothetical protein CT311.

    Directory of Open Access Journals (Sweden)

    Lei Lei

    Full Text Available 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 ((21AVEGKPLSRAAQLRERRKDLHVSGKPSPRYALKKRALEAKKNK(63. This sequence was sufficient for targeting a heterologous protein into mammalian cell nucleus and it contains two independent clusters of basic residues ((34RERRK(38 and (53KKRALEAKKNK(63 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.

  5. Inhibition of CRM1-mediated nuclear export of influenza A nucleoprotein and nuclear export protein as a novel target for antiviral drug development.

    Science.gov (United States)

    Chutiwitoonchai, Nopporn; Mano, Takafumi; Kakisaka, Michinori; Sato, Hirotaka; Kondoh, Yasumitsu; Osada, Hiroyuki; Kotani, Osamu; Yokoyama, Masaru; Sato, Hironori; Aida, Yoko

    2017-07-01

    An anti-influenza compound, DP2392-E10 based on inhibition of the nuclear export function of the viral nucleoprotein-nuclear export signal 3 (NP-NES3) domain was successfully identified by our previous high-throughput screening system. Here, we demonstrated that DP2392-E10 exerts its antiviral effect by inhibiting replication of a broad range of influenza A subtypes. In regard to the molecular mechanism, we revealed that DP2392-E10 inhibits nuclear export of both viral NP and nuclear export protein (NEP). More specifically, in vitro pull-down assays revealed that DP2392-E10 directly binds cellular CRM1, which mediates nuclear export of NP and NEP. In silico docking suggested that DP2392-E10 binds at a region close to the HEAT9 and HEAT10 domains of CRM1. Together, these results indicate that the CRM1-mediated nuclear export function of influenza virus represents a new potential target for antiviral drug development, and also provide a core structure for a novel class of inhibitors that target this function. Copyright © 2017 Elsevier Inc. All rights reserved.

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

    Directory of Open Access Journals (Sweden)

    Morgane eBatzenschlager

    2013-11-01

    Full Text Available During interphase, the microtubular cytoskeleton of cycling plant cells is organized in both cortical and perinuclear arrays. Perinuclear microtubules (MTs are nucleated from γ-Tubulin Complexes (γ-TuCs located at the surface of the nucleus. The molecular mechanisms of γ-TuC association to the nuclear envelope are currently unknown. The γ-TuC Protein 3 (GCP3-Interacting Protein 1 (GIP1 is the smallest γ-TuC component identified so far. AtGIP1 and its homologous protein AtGIP2 participate in the localization of active γ-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 γ-TuC core proteins and MT fibre 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 nuclear envelope.These results provide the first evidence for the involvement of a γ-TuC component in both nuclear shaping and nuclear envelope organization. Functional hypotheses are discussed in order to propose a model for a GIP-dependent nucleo-cytoplasmic continuum.

  7. Engineering light-inducible nuclear localization signals for precise spatiotemporal control of protein dynamics in living cells.

    Science.gov (United States)

    Niopek, Dominik; Benzinger, Dirk; Roensch, Julia; Draebing, Thomas; Wehler, Pierre; Eils, Roland; Di Ventura, Barbara

    2014-07-14

    The function of many eukaryotic proteins is regulated by highly dynamic changes in their nucleocytoplasmic distribution. The ability to precisely and reversibly control nuclear translocation would, therefore, allow dissecting and engineering cellular networks. Here we develop a genetically encoded, light-inducible nuclear localization signal (LINuS) based on the LOV2 domain of Avena sativa phototropin 1. LINuS is a small, versatile tag, customizable for different proteins and cell types. LINuS-mediated nuclear import is fast and reversible, and can be tuned at different levels, for instance, by introducing mutations that alter AsLOV2 domain photo-caging properties or by selecting nuclear localization signals (NLSs) of various strengths. We demonstrate the utility of LINuS in mammalian cells by controlling gene expression and entry into mitosis with blue light.

  8. Role of a nuclear localization signal on the minor capsid Proteins VP2 and VP3 in BKPyV nuclear entry

    Energy Technology Data Exchange (ETDEWEB)

    Bennett, Shauna M. [Cellular and Molecular Biology Program University of Michigan 1150W Medical Center Dr 5724 Medical Science Bldg II Ann Arbor, MI 48109 (United States); Zhao, Linbo [Doctoral Program in Cancer Biology Program University of Michigan 1150W Medical Center Dr 5724 Medical Science Bldg II Ann Arbor, MI 48109 (United States); Bosard, Catherine [Department of Microbiology and Immunology University of Michigan 1150W Medical Center Dr 5724 Medical Science Bldg II Ann Arbor, MI 48109 (United States); Imperiale, Michael J., E-mail: imperial@umich.edu [Cellular and Molecular Biology Program University of Michigan 1150W Medical Center Dr 5724 Medical Science Bldg II Ann Arbor, MI 48109 (United States); Doctoral Program in Cancer Biology Program University of Michigan 1150W Medical Center Dr 5724 Medical Science Bldg II Ann Arbor, MI 48109 (United States); Department of Microbiology and Immunology University of Michigan 1150W Medical Center Dr 5724 Medical Science Bldg II Ann Arbor, MI 48109 (United States)

    2015-01-01

    BK Polyomavirus (BKPyV) is a ubiquitous nonenveloped human virus that can cause severe disease in immunocompromised populations. After internalization into renal proximal tubule epithelial cells, BKPyV traffics through the ER and enters the cytosol. However, it is unclear how the virus enters the nucleus. In this study, we elucidate a role for the nuclear localization signal located on the minor capsid proteins VP2 and VP3 during infection. Site-directed mutagenesis of a single lysine in the basic region of the C-terminus of the minor capsid proteins abrogated their nuclear localization, and the analogous genomic mutation reduced infectivity. Additionally, through use of the inhibitor ivermectin and knockdown of importin β1, we found that the importin α/β pathway is involved during infection. Overall these data are the first to show the significance of the NLS of the BKPyV minor capsid proteins during infection in a natural host cell. - Highlights: • Polyomaviruses must deliver their genome to the nucleus to replicate. • The minor capsid proteins have a well-conserved nuclear localization signal. • Mutation of this NLS diminishes, but does not completely inhibit, infection.

  9. Intermolecular masking of the HIV-1 Rev NLS by the cellular protein HIC: novel insights into the regulation of Rev nuclear import.

    LENUS (Irish Health Repository)

    Gu, Lili

    2011-01-01

    The HIV-1 regulatory protein Rev, which is essential for viral replication, mediates the nuclear export of unspliced viral transcripts. Rev nuclear function requires active nucleocytoplasmic shuttling, and Rev nuclear import is mediated by the recognition of its Nuclear Localisation Signal (NLS) by multiple import factors, which include transportin and importin β. However, it remains unclear which nuclear import pathway(s) predominate in vivo, and the cellular environment that modulates Rev nucleocytoplasmic shuttling remains to be characterised.

  10. Importin α-importin β complex mediated nuclear translocation of insulin-like growth factor binding protein-5.

    Science.gov (United States)

    Sun, Min; Long, Juan; Yi, Yuxin; Xia, Wei

    2017-10-28

    Insulin-like growth factor-binding protein (IGFBP)-5 is a secreted protein that binds to IGFs and modulates IGF actions, as well as regulates cell proliferation, migration, and apoptosis independent of IGF. Proper cellular localization is critical for the effective function of most signaling molecules. In previous studies, we have shown that the nuclear IGFBP-5 comes from ER-cytosol retro-translocation. In this study, we further investigated the pathway mediating IGFBP-5 nuclear import after it retro-translocation. Importin-α5 was identified as an IGFBP-5-interacting protein with a yeast two-hybrid system, and its interaction with IGFBP-5 was further confirmed by GST pull down and co-immunoprecipitation. Binding affinity of IGFBP-5 and importins were determined by surface plasmon resonance (IGFBP-5/importin-β: K D =2.44e-7, IGFBP-5/importin-α5: K D =3.4e-7). Blocking the importin-α5/importin-β nuclear import pathway using SiRNA or dominant negative impotin-β dramatically inhibited IGFBP-5-EGFP nuclear import, though importin-α5 overexpress does not affect IGFBP-5 nuclear import. Furthermore, nuclear IGFBP-5 was quantified using luciferase report assay. When deleted the IGFBP-5 nuclear localization sequence (NLS), IGFBP-5 ΔNLS loss the ability to translocate into the nucleus and accumulation of IGFBP-5 ΔNLS was visualized in the cytosol. Altogether, our findings provide a substantially evidence showed that the IGFBP-5 nuclear import is mediated by importin-α/importin-β complex, and NLS is critical domain in IGFBP-5 nuclear translocation.

  11. Mutations causing Greenberg dysplasia but not Pelger anomaly uncouple enzymatic from structural functions of a nuclear membrane protein.

    OpenAIRE

    Clayton, P; Fischer, B; Mann, A.; Mansour, S.; Rossier, E; Van der Veen, M.; C. Lang; Baasanjav, S.; Kieslich, M.; Brossuleit, K.; Gravemann, S; Schnipper, N.; Karbasyian, M.; Demuth, I.; Zwerger, M.

    2010-01-01

    The lamin B receptor (LBR) is an inner nuclear membrane protein with a structural function interacting with chromatin and lamins, and an enzymatic function as a sterol reductase. Heterozygous LBR mutations cause nuclear hyposegmentation in neutrophils (Pelger anomaly), while homozygous mutations cause prenatal death with skeletal defects and abnormal sterol metabolism (Greenberg dysplasia). It has remained unclear whether the lethality in Greenberg dysplasia is due to cholesterol defects or a...

  12. BoxPlot++

    OpenAIRE

    Azmeh, Zeina; Hamoui, Fady; Huchard, Marianne

    2011-01-01

    We propose the BoxPlot++ as an extension of Tukey's boxplot. We improve the resulting data values distribution by removing the repeated values and by calculating distances between the points and the nearest median.

  13. Importin α1 is required for nuclear import of herpes simplex virus proteins and capsid assembly in fibroblasts and neurons

    Science.gov (United States)

    Anderson, Fenja; Rother, Franziska; Rudolph, Kathrin; Prank, Ute; Binz, Anne; Hügel, Stefanie; Hartmann, Enno; Bader, Michael; Bauerfeind, Rudolf; Sodeik, Beate

    2018-01-01

    Herpesviruses are large DNA viruses which depend on many nuclear functions, and therefore on host transport factors to ensure specific nuclear import of viral and host components. While some import cargoes bind directly to certain transport factors, most recruit importin β1 via importin α. We identified importin α1 in a small targeted siRNA screen to be important for herpes simplex virus (HSV-1) gene expression. Production of infectious virions was delayed in the absence of importin α1, but not in cells lacking importin α3 or importin α4. While nuclear targeting of the incoming capsids, of the HSV-1 transcription activator VP16, and of the viral genomes were not affected, the nuclear import of the HSV-1 proteins ICP4 and ICP0, required for efficient viral transcription, and of ICP8 and pUL42, necessary for DNA replication, were reduced. Furthermore, quantitative electron microscopy showed that fibroblasts lacking importin α1 contained overall fewer nuclear capsids, but an increased proportion of mature nuclear capsids indicating that capsid formation and capsid egress into the cytoplasm were impaired. In neurons, importin α1 was also not required for nuclear targeting of incoming capsids, but for nuclear import of ICP4 and for the formation of nuclear capsid assembly compartments. Our data suggest that importin α1 is specifically required for the nuclear localization of several important HSV1 proteins, capsid assembly, and capsid egress into the cytoplasm, and may become rate limiting in situ upon infection at low multiplicity or in terminally differentiated cells such as neurons. PMID:29304174

  14. Nuclear import and dimerization of tomato ASR1, a water stress-inducible protein exclusive to plants.

    Science.gov (United States)

    Ricardi, Martiniano M; Guaimas, Francisco F; González, Rodrigo M; Burrieza, Hernán P; López-Fernández, María P; Jares-Erijman, Elizabeth A; Estévez, José M; Iusem, Norberto D

    2012-01-01

    The ASR (for ABA/water stress/ripening) protein family, first described in tomato as nuclear and involved in adaptation to dry climates, is widespread in the plant kingdom, including crops of high agronomic relevance. We show both nuclear and cytosolic localization for ASR1 (the most studied member of the family) in histological plant samples by immunodetection, typically found in small proteins readily diffusing through nuclear pores. Indeed, a nuclear localization was expected based on sorting prediction software, which also highlight a monopartite nuclear localization signal (NLS) in the primary sequence. However, here we prove that such an "NLS" of ASR1 from tomato is dispensable and non-functional, being the transport of the protein to the nucleus due to simple diffusion across nuclear pores. We attribute such a targeting deficiency to the misplacing in that cryptic NLS of two conserved contiguous lysine residues. Based on previous in vitro experiments regarding quaternary structure, we also carried out live cell imaging assays through confocal microscopy to explore dimer formation in planta. We found homodimers in both the cytosol and the nucleus and demonstrated that assembly of both subunits together can occur in the cytosol, giving rise to translocation of preformed dimers. The presence of dimers was further corroborated by means of in vivo crosslinking of nuclei followed by SDS-PAGE.

  15. The inner nuclear membrane protein Src1 associates with subtelomeric genes and alters their regulated gene expression.

    Science.gov (United States)

    Grund, Stefanie E; Fischer, Tamás; Cabal, Ghislain G; Antúnez, Oreto; Pérez-Ortín, José E; Hurt, Ed

    2008-09-08

    Inner nuclear membrane proteins containing a LEM (LAP2, emerin, and MAN1) domain participate in different processes, including chromatin organization, gene expression, and nuclear envelope biogenesis. In this study, we identify a robust genetic interaction between transcription export (TREX) factors and yeast Src1, an integral inner nuclear membrane protein that is homologous to vertebrate LEM2. DNA macroarray analysis revealed that the expression of the phosphate-regulated genes PHO11, PHO12, and PHO84 is up-regulated in src1Delta cells. Notably, these PHO genes are located in subtelomeric regions of chromatin and exhibit a perinuclear location in vivo. Src1 spans the nuclear membrane twice and exposes its N and C domains with putative DNA-binding motifs to the nucleoplasm. Genome-wide chromatin immunoprecipitation-on-chip analyses indicated that Src1 is highly enriched at telomeres and subtelomeric regions of the yeast chromosomes. Our data show that the inner nuclear membrane protein Src1 functions at the interface between subtelomeric gene expression and TREX-dependent messenger RNA export through the nuclear pore complexes.

  16. Boxes and Shelves

    OpenAIRE

    Hughes, Dean

    2008-01-01

    The Boxes and Shelves series from 2008 are are all made from the backing card from discarded writing pads. Boxes and Shelves extended my investigation of quotidian materials and their relationship to the origins of creative toil. Since 1996 my research has sought to identify and locate instances where the 'unmeasurable' meets the measurable. I have consistently employed a range of utilitarian materials such as bus seats, bus tickets, puddles, A4 writing paper, to present a series of 'problem ...

  17. Freedom to box.

    Science.gov (United States)

    Warburton, N

    1998-02-01

    The british Medical Association wants to criminalise all boxing. This article examines the logic of the arguments it uses and finds them wanting. The move from medical evidence about the risk of brain damage to the conclusion that boxing should be banned is not warranted. The BMA's arguments are a combination of inconsistent paternalism and legal moralism. Consistent application of the principles implicit in the BMA's arguments would lead to absurd consequences and to severe limitations being put on individual freedom.

  18. Targeting proliferating cell nuclear antigen and its protein interactions induces apoptosis in multiple myeloma cells.

    Directory of Open Access Journals (Sweden)

    Rebekka Müller

    Full Text Available Multiple myeloma is a hematological cancer that is considered incurable despite advances in treatment strategy during the last decade. Therapies targeting single pathways are unlikely to succeed due to the heterogeneous nature of the malignancy. Proliferating cell nuclear antigen (PCNA is a multifunctional protein essential for DNA replication and repair that is often overexpressed in cancer cells. Many proteins involved in the cellular stress response interact with PCNA through the five amino acid sequence AlkB homologue 2 PCNA-interacting motif (APIM. Thus inhibiting PCNA's protein interactions may be a good strategy to target multiple pathways simultaneously. We initially found that overexpression of peptides containing the APIM sequence increases the sensitivity of cancer cells to contemporary therapeutics. Here we have designed a cell-penetrating APIM-containing peptide, ATX-101, that targets PCNA and show that it has anti-myeloma activity. We found that ATX-101 induced apoptosis in multiple myeloma cell lines and primary cancer cells, while bone marrow stromal cells and primary healthy lymphocytes were much less sensitive. ATX-101-induced apoptosis was caspase-dependent and cell cycle phase-independent. ATX-101 also increased multiple myeloma cells' sensitivity against melphalan, a DNA damaging agent commonly used for treatment of multiple myeloma. In a xenograft mouse model, ATX-101 was well tolerated and increased the anti-tumor activity of melphalan. Therefore, targeting PCNA by ATX-101 may be a novel strategy in multiple myeloma treatment.

  19. Targeting and processing of nuclear-encoded apicoplast proteins in plastid segregation mutants of Toxoplasma gondii.

    Science.gov (United States)

    He, C Y; Striepen, B; Pletcher, C H; Murray, J M; Roos, D S

    2001-07-27

    The apicoplast is a distinctive organelle associated with apicomplexan parasites, including Plasmodium sp. (which cause malaria) and Toxoplasma gondii (the causative agent of toxoplasmosis). This unusual structure (acquired by the engulfment of an ancestral alga and retention of the algal plastid) is essential for long-term parasite survival. Similar to other endosymbiotic organelles (mitochondria, chloroplasts), the apicoplast contains proteins that are encoded in the nucleus and post-translationally imported. Translocation across the four membranes surrounding the apicoplast is mediated by an N-terminal bipartite targeting sequence. Previous studies have described a recombinant "poison" that blocks plastid segregation during mitosis, producing parasites that lack an apicoplast and siblings containing a gigantic, nonsegregating plastid. To learn more about this remarkable phenomenon, we examined the localization and processing of the protein produced by this construct. Taking advantage of the ability to isolate apicoplast segregation mutants, we also demonstrated that processing of the transit peptide of nuclear-encoded apicoplast proteins requires plastid-associated activity.

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

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Cheng-Der; Cheng, Chi-Ping; Fang, Jia-Shih; Chen, Ling-Chih [Department of Life Sciences, Tzu-Chi University, 701 Chung-Yang Rd. Sec 3, Hualien 97004, Taiwan (China); Zhao, Bo; Kieff, Elliott [Department of Medicine and Microbiology and Molecular Genetics, Channing Laboratory, Brigham and Women’s Hospital and Harvard Medical School, 181 Longwood Ave., Boston 02115, MA (United States); Peng, Chih-Wen, E-mail: pengcw@mail.tcu.edu.tw [Department of Life Sciences, Tzu-Chi University, 701 Chung-Yang Rd. Sec 3, Hualien 97004, Taiwan (China)

    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.

  1. Specific interactions of three proliferating cell nuclear antigens with replication-related proteins in Aeropyrum pernix.

    Science.gov (United States)

    Imamura, Kaori; Fukunaga, Kenzo; Kawarabayasi, Yutaka; Ishino, Yoshizumi

    2007-04-01

    Proliferating cell nuclear antigen (PCNA) is a well-known multifunctional protein involved in eukaryotic and archaeal DNA transactions. The homotrimeric PCNA ring encircles double-stranded DNA within its central hole and tethers many proteins on DNA. Plural genes encoding PCNA-like proteins have been found in the genome sequence of crenarchaeal organisms. We describe here the biochemical properties of the three PCNAs, PCNA1, PCNA2 and PCNA3, from the hyperthermophilic archaeon, Aeropyrum pernix. PCNA2 can form a trimeric structure by itself, and it also forms heterotrimeric structures with PCNA1 and PCNA3. However, neither PCNA1 nor PCNA3 can form homotrimers. The DNA synthesis activity of DNA polymerase I and II, the endonuclease activity of FEN1, and the nick-sealing activity of DNA ligase were stimulated by the complex of PCNA2 and 3 or PCNA1, 2 and 3. These results suggest that the heterotrimeric PCNA at least including PCNA2 and 3 function as the clamp in the replisome. However, PCNA2 is the most abundant in the cells throughout the growth stages among the three PCNAs, and therefore, PCNA2 may perform multitasks by changing complex composition.

  2. Thinking Outside the Box

    Energy Technology Data Exchange (ETDEWEB)

    Green, Lorne [World Nuclear Transport Institute, Remo House, 310-312 Regent Street, London, W1B 3AX (United Kingdom)

    2009-06-15

    The World Nuclear Transport Institute was formed to fill a need to provide a dedicated vehicle for the radioactive transport and packaging industry sectors worldwide, to exchange information and ideas, all with a view to working toward consolidated industry positions on the key issues affecting safe, efficient and reliable transport. WNTI was also intended to be a strong voice for industry in those international and national bodies where deliberations on such transport safety issues take place. The very fact that companies, sometimes in competition with each other, were prepared to come together in this way, reflects two important points: firstly, it represents an acknowledgement on industry's part that safe, effective and reliable transport is the sine qua non, the absolute essential. And second, it is a recognition that it is enhanced to the extent that industry is able to collaborate to this end. This is thinking outside the box. Another important attribute of safety is 'stability'. Everyone likes to know where he or she stands. The radioactive materials packaging and transport industry thrives within a stable regulatory framework for safety. For a stable regulatory regime allows operators to be properly trained; it allows operators to become familiar with safety requirements, and to be at ease with them. Stability is conducive to safety and efficiency. Stability is good for business too - for stability in package and transport requirements allows sufficient time for a fair return on investment in expensive package design, manufacture, licensing and use over time. Stability should not, however, be opposed to creativity. From experience we can develop new thinking to improve efficiency as illustrated in examples of work related to the packaging and transport of Uranium Concentrates for instance.. Another example is work within WNTI on the thermal test requirements for the packaging of uranium hexafluoride. The robustness of packages is based on the

  3. The lamin B receptor of the nuclear envelope inner membrane: a polytopic protein with eight potential transmembrane domains

    OpenAIRE

    1990-01-01

    The lamin B receptor is a previously identified integral membrane protein in the nuclear envelope of turkey erythrocytes that associates with the nuclear intermediate filament protein lamin B (Worman, H. J., J. Yuan, G. Blobel, and S. D. Georgatos. 1988. Proc. Natl. Acad. Sci. USA. 85:8531-8534). In the present report, we use cell fractionation and antibodies against the lamin B receptor to localize it to an 8-M urea-extracted membrane fraction of chicken liver nuclei, supporting an inner nuc...

  4. Cable Tester Box

    Science.gov (United States)

    Lee, Jason H.

    2011-01-01

    Cables are very important electrical devices that carry power and signals across multiple instruments. Any fault in a cable can easily result in a catastrophic outcome. Therefore, verifying that all cables are built to spec is a very important part of Electrical Integration Procedures. Currently, there are two methods used in lab for verifying cable connectivity. (1) Using a Break-Out Box and an ohmmeter this method is time-consuming but effective for custom cables and (2) Commercial Automated Cable Tester Boxes this method is fast, but to test custom cables often requires pre-programmed configuration files, and cables used on spacecraft are often uniquely designed for specific purposes. The idea is to develop a semi-automatic continuity tester that reduces human effort in cable testing, speeds up the electrical integration process, and ensures system safety. The JPL-Cable Tester Box is developed to check every single possible electrical connection in a cable in parallel. This system indicates connectivity through LED (light emitting diode) circuits. Users can choose to test any pin/shell (test node) with a single push of a button, and any other nodes that are shorted to the test node, even if they are in the same connector, will light up with the test node. The JPL-Cable Tester Boxes offers the following advantages: 1. Easy to use: The architecture is simple enough that it only takes 5 minutes for anyone to learn how operate the Cable Tester Box. No pre-programming and calibration are required, since this box only checks continuity. 2. Fast: The cable tester box checks all the possible electrical connections in parallel at a push of a button. If a cable normally takes half an hour to test, using the Cable Tester Box will improve the speed to as little as 60 seconds to complete. 3. Versatile: Multiple cable tester boxes can be used together. As long as all the boxes share the same electrical potential, any number of connectors can be tested together.

  5. A nuclear envelope-associated kinase phosphorylates arginine-serine motifs and modulates interactions between the lamin B receptor and other nuclear proteins.

    Science.gov (United States)

    Nikolakaki, E; Simos, G; Georgatos, S D; Giannakouros, T

    1996-04-05

    Previous studies have identified a subassembly of nuclear envelope proteins, termed "the LBR complex." This complex includes the lamin B receptor protein (LBR or p58), a kinase which phosphorylates LBR in a constitutive fashion (LBR kinase), the nuclear lamins A and B, an 18-kDa polypeptide (p18), and a 34-kDa protein (p34/p32). The latter polypeptide has been shown to interact with the HIV-1 proteins Rev and Tat and with the splicing factor 2 (SF2). Using recombinant proteins produced in bacteria and synthetic peptides representing different regions of LBR, we now show that the LBR kinase modifies specifically arginine-serine (RS) dipeptide motifs located at the nucleoplasmic, NH2-terminal domain of LBR and in members of the SR family of splicing factors. Furthermore, we show that the NH2-terminal domain of LBR binds to p34/p32, whereas a mutated domain lacking the RS region does not. Phosphorylation of LBR by the RS kinase completely abolishes binding of p34/p32, suggesting that this enzyme regulates interactions among the components of the LBR complex.

  6. 20-Hydroxyecdysone stimulates nuclear accumulation of BmNep1, a nuclear ribosome biogenesis-related protein in the silkworm, Bombyx mori.

    Science.gov (United States)

    Ji, M-M; Liu, A-Q; Sima, Y-H; Xu, S-Q

    2016-10-01

    The pathway of communication between endocrine hormones and ribosome biogenesis critical for physiological adaptation is largely unknown. Nucleolar essential protein 1 (Nep1) is an essential gene for ribosome biogenesis and is functionally conserved in many in vertebrate and invertebrate species. In this study, we cloned Bombyx mori Nep1 (BmNep1) due to its high expression in silk glands of silkworms on day 3 of the fifth instar. We found that BmNep1 mRNA and protein levels were upregulated in silk glands during fourth-instar ecdysis and larval-pupal metamorphosis. By immunoprecipitation with the anti-BmNep1 antibody and liquid chromatography-tandem mass spectrometry analyses, it was shown that BmNep1 probably interacts with proteins related to ribosome structure formation. Immunohistochemistry, biochemical fractionation and immunocytochemistry revealed that BmNep1 is localized to the nuclei in Bombyx cells. Using BmN cells originally derived from ovaries, we demonstrated that 20-hydroxyecdysone (20E) induced BmNep1 expression and stimulated nuclear accumulation of BmNep1. Under physiological conditions, BmNep1 was also upregulated in ovaries during larval-pupal metamorphosis. Overall, our results indicate that the endocrine hormone 20E facilitates nuclear accumulation of BmNep1, which is involved in nuclear ribosome biogenesis in Bombyx. © 2016 The Royal Entomological Society.

  7. High-Mobility Group Box-1 Protein Mediates the Regulation of Signal Transducer and Activator of Transcription-3 in the Diabetic Retina and in Human Retinal Müller Cells.

    Science.gov (United States)

    Mohammad, Ghulam; Jomar, Deema; Siddiquei, Mohammad Mairaj; Alam, Kaiser; Abu El-Asrar, Ahmed M

    2017-01-01

    The expression of high-mobility group box-1 (HMGB1) and signal transducer and activator of transcription-3 (STAT-3) is upregulated in the diabetic retina. We hypothesized that the activation of STAT-3 is under the control of HMGB1. Retinas from 1-month-old diabetic rats and from normal rats intravitreally injected with HMGB1 and human retinal Müller glial cells (MIO-M1) stimulated with HMGB1 or high glucose were studied by Western blot analysis and immunofluorescence. We also studied the effect of the HMGB1 inhibitor glycyrrhizin (GA) on high-glucose-induced pSTAT-3 nuclear translocation and upregulation in Müller cells and on pSTAT-3 expression in the retinas of diabetic rats (n = 7-10 in each group). In addition, we studied the effect of STAT-3 inhibitor on the HMGB1-induced induction of vascular endothelial growth factor (VEGF) by Müller cells and human retinal microvascular endothelial cell (HRMEC) migration. Treatment of retinal Müller cells with recombinant HMGB1 induced nuclear translocation of pSTAT-3 but did not alter pSTAT-3 expression. High glucose induced a significant upregulation of HMGB1 and pSTAT-3 upregulation and nuclear translocation in retinal Müller cells. GA co-treatment normalized the high-glucose-induced upregulation of HMGB1 and pSTAT-3 upregulation and nuclear translocation in Müller cells. Intravitreal administration of HMGB1 in normal and diabetic rats upregulated pSTAT-3 expression in the retina. GA attenuated the diabetes-induced upregulation of pSTAT-3 in the retina. The STAT-3 inhibitor attenuated HMGB1-induced VEGF upregulation by Müller cells and HRMEC migration. The results suggest a role for HMGB1 in the modulation of STAT-3 expression in the diabetic retina. © 2016 S. Karger AG, Basel.

  8. LEM2 is a novel MAN1-related inner nuclear membrane protein associated with A-type lamins.

    Science.gov (United States)

    Brachner, Andreas; Reipert, Siegfried; Foisner, Roland; Gotzmann, Josef

    2005-12-15

    The LEM (lamina-associated polypeptide-emerin-MAN1) domain is a motif shared by a group of lamin-interacting proteins in the inner nuclear membrane (INM) and in the nucleoplasm. The LEM domain mediates binding to a DNA-crosslinking protein, barrier-to-autointegration factor (BAF). We describe a novel, ubiquitously expressed LEM domain protein, LEM2, which is structurally related to MAN1. LEM2 contains an N-terminal LEM motif, two predicted transmembrane domains and a MAN1-Src1p C-terminal (MSC) domain highly homologous to MAN1, but lacks the MAN1-specific C-terminal RNA-recognition motif. Immunofluorescence microscopy of digitonin-treated cells and subcellular fractionation identified LEM2 as a lamina-associated protein residing in the INM. LEM2 binds to the lamin C tail in vitro. Targeting of LEM2 to the nuclear envelope requires A-type lamins and is mediated by the N-terminal and transmembrane domains. Highly overexpressed LEM2 accumulates in patches at the nuclear envelope and forms membrane bridges between nuclei of adjacent cells. LEM2 structures recruit A-type lamins, emerin, MAN1 and BAF, whereas lamin B and lamin B receptor are excluded. Our data identify LEM2 as a novel A-type-lamin-associated INM protein involved in nuclear structure organization.

  9. Children's (Pediatric) Nuclear Medicine

    Medline Plus

    Full Text Available ... information you were looking for? Yes No Please type your comment or suggestion into the following text box: Comment: ... Images related to Children's (Pediatric) Nuclear Medicine Videos ...

  10. Induction of polyploidy by nuclear fusion mechanism upon decreased expression of the nuclear envelope protein LAP2β in the human osteosarcoma cell line U2OS.

    Science.gov (United States)

    Ben-Shoshan, Shirley Oren; Simon, Amos J; Jacob-Hirsch, Jasmine; Shaklai, Sigal; Paz-Yaacov, Nurit; Amariglio, Ninette; Rechavi, Gideon; Trakhtenbrot, Luba

    2014-01-28

    Polyploidy has been recognized for many years as an important hallmark of cancer cells. Polyploid cells can arise through cell fusion, endoreplication and abortive cell cycle. The inner nuclear membrane protein LAP2β plays key roles in nuclear envelope breakdown and reassembly during mitosis, initiation of replication and transcriptional repression. Here we studied the function of LAP2β in the maintenance of cell ploidy state, a role which has not yet been assigned to this protein. By knocking down the expression of LAP2β, using both viral and non-viral RNAi approaches in osteosarcoma derived U2OS cells, we detected enlarged nuclear size, nearly doubling of DNA content and chromosomal duplications, as analyzed by fluorescent in situ hybridization and spectral karyotyping methodologies. Spectral karyotyping analyses revealed that near-hexaploid karyotypes of LAP2β knocked down cells consisted of not only seven duplicated chromosomal markers, as could be anticipated by genome duplication mechanism, but also of four single chromosomal markers. Furthermore, spectral karyotyping analysis revealed that both of two near-triploid U2OS sub-clones contained the seven markers that were duplicated in LAP2β knocked down cells, whereas the four single chromosomal markers were detected only in one of them. Gene expression profiling of LAP2β knocked down cells revealed that up to a third of the genes exhibiting significant changes in their expression are involved in cancer progression. Our results suggest that nuclear fusion mechanism underlies the polyploidization induction upon LAP2β reduced expression. Our study implies on a novel role of LAP2β in the maintenance of cell ploidy status. LAP2β depleted U2OS cells can serve as a model to investigate polyploidy and aneuploidy formation by nuclear fusion mechanism and its involvement in cancerogenesis.

  11. LBR, a chromatin and lamin binding protein from the inner nuclear membrane, is proteolyzed at late stages of apoptosis.

    Science.gov (United States)

    Duband-Goulet, I; Courvalin, J C; Buendia, B

    1998-05-01

    Chromatin condensation and apposition to the nuclear envelope is an important feature of the execution phase of apoptosis. During this process, lamin proteins that are located between the inner nuclear membrane and heterochromatin are proteolyzed by the apoptosis-specific protease caspase 6. We have investigated the fate of nuclear membranes during apoptosis by studying the lamin B receptor (LBR), a transmembrane protein of the inner nuclear membrane. LBR interacts through its nucleoplasmic amino-terminal domain with both heterochromatin and B-type lamins, and is phosphorylated throughout the cell cycle, but on different sites in interphase and mitosis. We report here that: (i) the amino-terminal domain of LBR is specifically cleaved during apoptosis to generate an approximately 20 kDa soluble fragment; (ii) the cleavage of LBR is a late event of apoptosis and occurs subsequent to lamin B cleavage; (iii) the phosphorylation of LBR during apoptosis is similar to that occurring in interphase. As the association of condensed chromatin with the inner nuclear membrane persists until the late stages of apoptosis, we suggest that the chromatin binding protein LBR plays a major role in maintaining this association.

  12. Fast track, dynein-dependent nuclear targeting of human immunodeficiency virus Vpr protein; impaired trafficking in a clinical isolate

    Energy Technology Data Exchange (ETDEWEB)

    Caly, Leon [Department of Biochemistry and Molecular Biology, Monash University, Clayton, Vic. 3800 (Australia); Kassouf, Vicki T. [Centre for Virus Research, The Westmead Institute for Medical Research, The University of Sydney, Westmead, NSW 2145 (Australia); Moseley, Gregory W. [Department of Biochemistry and Molecular Biology, Monash University, Clayton, Vic. 3800 (Australia); Diefenbach, Russell J.; Cunningham, Anthony L. [Centre for Virus Research, The Westmead Institute for Medical Research, The University of Sydney, Westmead, NSW 2145 (Australia); Jans, David A., E-mail: david.jans@monash.edu [Department of Biochemistry and Molecular Biology, Monash University, Clayton, Vic. 3800 (Australia)

    2016-02-12

    Nuclear import of the accessory protein Vpr is central to infection by human immunodeficiency virus (HIV). We previously identified the Vpr F72L mutation in a HIV-infected, long-term non-progressor, showing that it resulted in reduced Vpr nuclear accumulation and altered cytoplasmic localisation. Here we demonstrate for the first time that the effects of nuclear accumulation of the F72L mutation are due to impairment of microtubule-dependent-enhancement of Vpr nuclear import. We use high resolution imaging approaches including fluorescence recovery after photobleaching and other approaches to document interaction between Vpr and the dynein light chain protein, DYNLT1, and impaired interaction of the F72L mutant with DYNLT1. The results implicate MTs/DYNLT1 as drivers of Vpr nuclear import and HIV infection, with important therapeutic implications. - Highlights: • HIV-1 Vpr utilizes the microtubule network to traffic towards the nucleus. • Mechanism relies on interaction between Vpr and dynein light chain protein DYNLT1. • Long-term non-progressor derived mutation (F72L) impairs this interaction. • Key residues in the vicinity of F72 contribute to interaction with DYNLT1.

  13. The BOXES Methodology Black Box Dynamic Control

    CERN Document Server

    Russell, David W

    2012-01-01

    Robust control mechanisms customarily require knowledge of the system’s describing equations which may be of the high order differential type.  In order to produce these equations, mathematical models can often be derived and correlated with measured dynamic behavior.  There are two flaws in this approach one is the level of inexactness introduced by linearizations and the other when no model is apparent.  Several years ago a new genre of control systems came to light that are much less dependent on differential models such as fuzzy logic and genetic algorithms. Both of these soft computing solutions require quite considerable a priori system knowledge to create a control scheme and sometimes complicated training program before they can be implemented in a real world dynamic system. Michie and Chambers’ BOXES methodology created a black box system that was designed to control a mechanically unstable system with very little a priori system knowledge, linearization or approximation.  All the method need...

  14. In vitro FRAP reveals the ATP-dependent nuclear mobilization of the exon junction complex protein SRm160

    OpenAIRE

    Wagner, Stefan; Chiosea, Simion; Ivshina, Maria; Nickerson, Jeffrey A.

    2004-01-01

    We present a new in vitro system for characterizing the binding and mobility of enhanced green fluorescent protein (EGFP)–labeled nuclear proteins by fluorescence recovery after photobleaching in digitonin-permeabilized cells. This assay reveals that SRm160, a splicing coactivator and component of the exon junction complex (EJC) involved in RNA export, has an adenosine triphosphate (ATP)–dependent mobility. Endogenous SRm160, lacking the EGFP moiety, could also be released from sites at splic...

  15. The insulator protein SU(HW fine-tunes nuclear lamina interactions of the Drosophila genome.

    Directory of Open Access Journals (Sweden)

    Joke G van Bemmel

    Full Text Available Specific interactions of the genome with the nuclear lamina (NL are thought to assist chromosome folding inside the nucleus and to contribute to the regulation of gene expression. High-resolution mapping has recently identified hundreds of large, sharply defined lamina-associated domains (LADs in the human genome, and suggested that the insulator protein CTCF may help to demarcate these domains. Here, we report the detailed structure of LADs in Drosophila cells, and investigate the putative roles of five insulator proteins in LAD organization. We found that the Drosophila genome is also organized in discrete LADs, which are about five times smaller than human LADs but contain on average a similar number of genes. Systematic comparison to new and published insulator binding maps shows that only SU(HW binds preferentially at LAD borders and at specific positions inside LADs, while GAF, CTCF, BEAF-32 and DWG are mostly absent from these regions. By knockdown and overexpression studies we demonstrate that SU(HW weakens genome - NL interactions through a local antagonistic effect, but we did not obtain evidence that it is essential for border formation. Our results provide insights into the evolution of LAD organization and identify SU(HW as a fine-tuner of genome - NL interactions.

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

    Energy Technology Data Exchange (ETDEWEB)

    Seo, Jae Sung; Kim, Ha Na; Kim, Sun-Jick; Bang, Jiyoung; Kim, Eun-A; Sung, Ki Sa [Department of Biological Sciences, Sungkyunkwan University, Suwon 440-746 (Korea, Republic of); Yoon, Hyun-Joo [TissueGene Inc. 9605 Medical Center Dr., Rockville, MD 20850 (United States); Yoo, Hae Yong [Department of Health Sciences and Technology, Samsung Advanced Institute for Health Sciences and Technology, Samsung Medical Center, Sungkyunkwan University, Seoul 135-710 (Korea, Republic of); Choi, Cheol Yong, E-mail: choicy@skku.ac.kr [Department of Biological Sciences, Sungkyunkwan University, Suwon 440-746 (Korea, Republic of)

    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.

  17. Using FRET to Measure the Angle at Which a Protein Bends DNA: TBP Binding a TATA Box as a Model System

    Science.gov (United States)

    Kugel, Jennifer F.

    2008-01-01

    An undergraduate biochemistry laboratory experiment that will teach the technique of fluorescence resonance energy transfer (FRET) while analyzing protein-induced DNA bending is described. The experiment uses the protein TATA binding protein (TBP), which is a general transcription factor that recognizes and binds specific DNA sequences known as…

  18. Identification of two HSP70-related Xenopus oocyte proteins that are capable of recycling across the nuclear envelope

    Energy Technology Data Exchange (ETDEWEB)

    Mandell, R.B.; Feldherr, C.M. (Univ. of Florida, Gainesville (USA))

    1990-11-01

    Two 70-kD polypeptides, B3 and B4, are present in equivalent concentrations in the nucleus and cytoplasm of Xenopus oocytes. The objectives of this study were to determine if they (a) are members of the 70-kD family of heat shock proteins, and (b) recycle between the nuclear and cytoplasmic compartments. Evidence based on high-affinity binding to ATP, cross-reactivity of B3/B4-specific antibodies with rat hsc70, and a comparison of cyanogen bromide cleavage peptide maps with hsc70, verified that B3 and B4 are members of the 70-kD family of heat-shock proteins. Nuclear uptake studies were performed by microinjecting 125I-labeled B3/B4, rat hsc70, and BSA into the cytoplasm of oocytes, and examining their subsequent intracellular distributions. By 6 h postinjection, the nuclear concentration of B3/B4 and hsc70 were approximately 24-fold greater than BSA controls. It was also found that B3/B4-coated gold particles as large as 120A in diameter were able to enter the nucleus by passing through the pores. Nuclear efflux was analyzed by microinjecting the iodinated proteins directly into the oocyte nuclei. 2 h after nuclear injection, at least 46% of the B3/B4 and 60% of the hsc70 were found in the cytoplasmic fractions, compared with less than 10% for the BSA controls. Cell fusion experiments, in which labeled, anucleate oocyte vegetal hemispheres were fused, under oil, with nucleate unlabeled animal hemispheres, demonstrated that cytoplasmic B3 and B4 could enter the nucleus after equilibration was reached, arguing against the existence of separate nuclear and cytoplasmic populations. Collectively, these results show that B3, B4, and rat hsc70 are transported across the nuclear envelope and recycle between the nucleus and cytoplasm.

  19. Tau-Induced Ca2+/Calmodulin-Dependent Protein Kinase-IV Activation Aggravates Nuclear Tau Hyperphosphorylation.

    Science.gov (United States)

    Wei, Yu-Ping; Ye, Jin-Wang; Wang, Xiong; Zhu, Li-Ping; Hu, Qing-Hua; Wang, Qun; Ke, Dan; Tian, Qing; Wang, Jian-Zhi

    2017-06-23

    Hyperphosphorylated tau is the major protein component of neurofibrillary tangles in the brains of patients with Alzheimer's disease (AD). However, the mechanism underlying tau hyperphosphorylation is not fully understood. Here, we demonstrated that exogenously expressed wild-type human tau40 was detectable in the phosphorylated form at multiple AD-associated sites in cytoplasmic and nuclear fractions from HEK293 cells. Among these sites, tau phosphorylated at Thr205 and Ser214 was almost exclusively found in the nuclear fraction at the conditions used in the present study. With the intracellular tau accumulation, the Ca 2+ concentration was significantly increased in both cytoplasmic and nuclear fractions. Further studies using site-specific mutagenesis and pharmacological treatment demonstrated that phosphorylation of tau at Thr205 increased nuclear Ca 2+ concentration with a simultaneous increase in the phosphorylation of Ca 2+ /calmodulin-dependent protein kinase IV (CaMKIV) at Ser196. On the other hand, phosphorylation of tau at Ser214 did not significantly change the nuclear Ca 2+ /CaMKIV signaling. Finally, expressing calmodulin-binding protein-4 that disrupts formation of the Ca 2+ /calmodulin complex abolished the okadaic acid-induced tau hyperphosphorylation in the nuclear fraction. We conclude that the intracellular accumulation of phosphorylated tau, as detected in the brains of AD patients, can trigger nuclear Ca 2+ /CaMKIV signaling, which in turn aggravates tau hyperphosphorylation. Our findings provide new insights for tauopathies: hyperphosphorylation of intracellular tau and an increased Ca 2+ concentration may induce a self-perpetuating harmful loop to promote neurodegeneration.

  20. Glycyrrhizin inhibits porcine epidemic diarrhea virus infection and attenuates the proinflammatory responses by inhibition of high mobility group box-1 protein.

    Science.gov (United States)

    Huan, Chang-Chao; Wang, Hua-Xia; Sheng, Xiang-Xiang; Wang, Rui; Wang, Xin; Mao, Xiang

    2017-06-01

    Porcine epidemic diarrhea (PED), caused by porcine epidemic diarrhea virus (PEDV) infection, leads to significant economic losses in the swine industry worldwide. In our studies, we found that glycyrrhizin, the major component of licorice root extracts, could moderately inhibit PEDV infection in Vero cells, when analyzed by western blot, qRT-PCR and a plaque formation assay. We also revealed that glycyrrhizin inhibited the entry and replication of PEDV. In addition, we demonstrated that glycyrrhizin decreased the mRNA levels of proinflammatory cytokines. Since glycyrrhizin is a competitive inhibitor of high mobility group box-1 (HMGB1), we confirmed that TLR4 and RAGE (£ associated with PEDV pathogenesis during the infection in Vero cells. In summary, our studies provide a molecular basis for developing novel therapeutic methods to control PEDV infection, based on glycyrrhizin and its derivatives.

  1. Steatosis-induced proteins adducts with lipid peroxidation products and nuclear electrophilic stress in hepatocytes.

    Science.gov (United States)

    Anavi, Sarit; Ni, Zhixu; Tirosh, Oren; Fedorova, Maria

    2015-01-01

    Accumulating evidence suggests that fatty livers are particularly more susceptible to several pathological conditions, including hepatic inflammation, cirrhosis and liver cancer. However the exact mechanism of such susceptibility is still largely obscure. The current study aimed to elucidate the effect of hepatocytes lipid accumulation on the nuclear electrophilic stress. Accumulation of intracellular lipids was significantly increased in HepG2 cells incubated with fatty acid (FA) complex (1mM, 2:1 oleic and palmitic acids). In FA-treated cells, lipid droplets were localized around the nucleus and seemed to induce mechanical force, leading to the disruption of the nucleus morphology. Level of reactive oxygen species (ROS) was significantly increased in FA-loaded cells and was further augmented by treatment with moderate stressor (CoCl2). Increased ROS resulted in formation of reactive carbonyls (aldehydes and ketones, derived from lipid peroxidation) with a strong perinuclear accumulation. Mass-spectroscopy analysis indicated that lipid accumulation per-se can results in modification of nuclear protein by reactive lipid peroxidation products (oxoLPP). 235 Modified proteins involved in transcription regulation, splicing, protein synthesis and degradation, DNA repair and lipid metabolism were identified uniquely in FA-treated cells. These findings suggest that steatosis can affect nuclear redox state, and induce modifications of nuclear proteins by reactive oxoLPP accumulated in the perinuclear space upon FA-treatment. Copyright © 2014 The Authors. Published by Elsevier B.V. All rights reserved.

  2. Use of spin labels to study membrane proteins by high-frequency electron nuclear double resonance spectroscopy

    NARCIS (Netherlands)

    Orlinkskii, S.B.; Borovykh, I.V.; Zielke, V.; Steinhoff, H.J.

    2007-01-01

    The applicability of spin labels to study membrane proteins by high-frequency electron nuclear double resonance spectroscopy is demonstrated. With the use of bacteriorhodopsin embedded in a lipid membrane as an example, the spectra of protons of neighboring amino acids are recorded, electric field

  3. Steatosis-induced proteins adducts with lipid peroxidation products and nuclear electrophilic stress in hepatocytes

    Directory of Open Access Journals (Sweden)

    Sarit Anavi

    2015-04-01

    Full Text Available Accumulating evidence suggests that fatty livers are particularly more susceptible to several pathological conditions, including hepatic inflammation, cirrhosis and liver cancer. However the exact mechanism of such susceptibility is still largely obscure. The current study aimed to elucidate the effect of hepatocytes lipid accumulation on the nuclear electrophilic stress. Accumulation of intracellular lipids was significantly increased in HepG2 cells incubated with fatty acid (FA complex (1 mM, 2:1 oleic and palmitic acids. In FA-treated cells, lipid droplets were localized around the nucleus and seemed to induce mechanical force, leading to the disruption of the nucleus morphology. Level of reactive oxygen species (ROS was significantly increased in FA-loaded cells and was further augmented by treatment with moderate stressor (CoCl2. Increased ROS resulted in formation of reactive carbonyls (aldehydes and ketones, derived from lipid peroxidation with a strong perinuclear accumulation. Mass-spectroscopy analysis indicated that lipid accumulation per-se can results in modification of nuclear protein by reactive lipid peroxidation products (oxoLPP. 235 Modified proteins involved in transcription regulation, splicing, protein synthesis and degradation, DNA repair and lipid metabolism were identified uniquely in FA-treated cells. These findings suggest that steatosis can affect nuclear redox state, and induce modifications of nuclear proteins by reactive oxoLPP accumulated in the perinuclear space upon FA-treatment.

  4. Protein profiles in cortical and nuclear regions of aged human donor lenses: A confocal Raman microspectroscopic and imaging study

    NARCIS (Netherlands)

    Vrensen, G.F.J.M.; Otto, Cornelis; Lenferink, Aufrid T.M.; Liszka, B.; Montenegro, G.A.; Barraquer, R.I.; Michael, R.

    2016-01-01

    A combination of Raman spectroscopy, imaging, hierarchical cluster analysis (HCA) and peak ratio analysis was used to analyze protein profiles in the superficial cortex (SC), deep cortex (DC) and nucleus of old human lenses with cortical, nuclear and mixed cataracts. No consistent differences were

  5. Hepatitis B virus X promotes hepatocellular carcinoma development via nuclear protein 1 pathway

    Energy Technology Data Exchange (ETDEWEB)

    Bak, Yesol; Shin, Hye-jun; Bak, In seon [Disease Model Research Laboratory, Aging Intervention Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejeon (Korea, Republic of); Yoon, Do-young [Department of Bioscience and Biotechnology, Bio/Molecular Informatics Center, Konkuk University, Seoul (Korea, Republic of); Yu, Dae-Yeul, E-mail: dyyu10@kribb.re.kr [Disease Model Research Laboratory, Aging Intervention Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejeon (Korea, Republic of)

    2015-10-30

    Hepatocellular carcinoma (HCC) is one of the most common malignancies and chronic hepatitis B virus (HBV) infection is a major risk factor for HCC. Hepatitis B virus X (HBx) protein relates to trigger oncogenesis. HBx has oncogenic properties with a hyperproliferative response to HCC. Nuclear protein 1 (NUPR1) is a stress-response protein, frequently upregulated in several cancers. Recent data revealed that NUPR1 is involved in tumor progression, but its function in HCC is not revealed yet. Here we report HBx can induce NUPR1 in patients, mice, and HCC cell lines. In an HBx transgenic mouse model, we found that HBx overexpression upregulates NUPR1 expression consistently with tumor progression. Further, in cultured HBV positive cells, HBx knockdown induces downregulation of NUPR1. Smad4 is a representative transcription factor, regulated by HBx, and we showed that HBx upregulates NUPR1 by Smad4 dependent way. We found that NUPR1 can inhibit cell death and induce vasculogenic mimicry in HCC cell lines. Moreover, NUPR1 silencing in HepG2-HBx showed reduced cell motility. These results suggest that HBx can modulate NUPR1 expression through the Smad4 pathway and NUPR1 has a role in hepatocellular carcinoma progression. - Highlights: • NUPR1 is overexpressed in HBx transgenic mouse and HCC patients. • NUPR1 inactivation hampers the HBx induced growth, VM formation, and migration of HepG2 cells in vitro. • NUPR1 has a role for survival of HCC and mechanistically NUPR1 is activated by HBx-Smad4 axis.

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

    Energy Technology Data Exchange (ETDEWEB)

    Sugioka-Sugiyama, Rie [Graduate School of Life and Environmental Sciences, University of Tsukuba, Tsukuba, Ibaraki 305-8577 (Japan); Initiative for the Promotion of Young Scientists' Independent Research, University of Tsukuba, Tsukuba, Ibaraki 305-8577 (Japan); Sugiyama, Tomoyasu, E-mail: sugiyamt@biol.tsukuba.ac.jp [Graduate School of Life and Environmental Sciences, University of Tsukuba, Tsukuba, Ibaraki 305-8577 (Japan); Initiative for the Promotion of Young Scientists' Independent Research, University of Tsukuba, Tsukuba, Ibaraki 305-8577 (Japan); Precursory Research for Embryonic Science and Technology (PRESTO), Japan Science and Technology Agency (JST), Kawaguchi, Saitama 332-0012 (Japan)

    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.

  7. Opto-Box

    CERN Document Server

    Bertsche, David; The ATLAS collaboration; Welch, Steven; Smith, Dale Shane; Che, Siinn; Gan, K.K.; Boyd, George Russell Jr

    2015-01-01

    The opto-box is a custom mini-crate for housing optical modules, which process and transfer optoelectronic data. The system tightly integrates electrical, mechanical, and thermal functionality into a small package of size 35x10x8 cm^3. Special attention was given to ensure proper shielding, grounding, cooling, high reliability, and environmental tolerance. The custom modules, which incorporate Application Specific Integrated Circuits (ASICs), were developed through a cycle of rigorous testing and redesign. In total, fourteen opto-boxes have been installed and loaded with modules on the ATLAS detector. They are currently in operation as part of the LHC run 2 data read-out chain.

  8. [Dead-box RNA helicases in animal gametogenesis].

    Science.gov (United States)

    Kotov, A A; Akulenko, N V; Kibanov, M V; Olenina, L V

    2014-01-01

    This review analyzes and summarizes a current knowledge about a role of RNA helicases in the development and maintenance of gametogenesis of eukaryotic organisms. Here we focused on three representatives of RNA helicase family containing the characteristic motifs in the amino acid sequence (DEAD-box) and carrying substantial and conservative functions in the germinal tissues of various species from drosophila to human. There are such proteins as Vasa/DDX4, BelIe/DDX3 and Spindle-E/TDRD9. They are involved in a wide range of activities that are associated with the regulation of transcription, splicing, nuclear export, and especially with the initiation of translation. The expression of genes required for the gametogenesis appears to be regulated mainly at the translational level. RNA helicases are involved in the formation of cytoplasmic RNP granules and in the implementation of gene RNA-silencing. "DEAD-box RNA helicases" that carry highly homologous central domain, which determines their basic biochemical activity in the ATP-dependent unwinding of short RNA duplexes, perform the essential and non-overlapping functions in the germinal tissues.

  9. A sugar beet chlorophyll a/b binding protein promoter void of G-box like elements confers strong and leaf specific reporter gene expression in transgenic sugar beet

    Directory of Open Access Journals (Sweden)

    Kloos Dorothee U

    2004-12-01

    Full Text Available Abstract Background Modification of leaf traits in sugar beet requires a strong leaf specific promoter. With such a promoter, expression in taproots can be avoided which may otherwise take away available energy resources for sugar accumulation. Results Suppression Subtractive Hybridization (SSH was utilized to generate an enriched and equalized cDNA library for leaf expressed genes from sugar beet. Fourteen cDNA fragments corresponding to thirteen different genes were isolated. Northern blot analysis indicates the desired tissue specificity of these genes. The promoters for two chlorophyll a/b binding protein genes (Bvcab11 and Bvcab12 were isolated, linked to reporter genes, and transformed into sugar beet using promoter reporter gene fusions. Transient and transgenic analysis indicate that both promoters direct leaf specific gene expression. A bioinformatic analysis revealed that the Bvcab11 promoter is void of G-box like regulatory elements with a palindromic ACGT core sequence. The data indicate that the presence of a G-box element is not a prerequisite for leaf specific and light induced gene expression in sugar beet. Conclusions This work shows that SSH can be successfully employed for the identification and subsequent isolation of tissue specific sugar beet promoters. These promoters are shown to drive strong leaf specific gene expression in transgenic sugar beet. The application of these promoters for expressing resistance improving genes against foliar diseases is discussed.

  10. Nuclear size is sensitive to NTF2 protein levels in a manner dependent on Ran binding

    Science.gov (United States)

    Vuković, Lidija D.; Jevtić, Predrag; Zhang, Zhaojie; Stohr, Bradley A.; Levy, Daniel L.

    2016-01-01

    ABSTRACT Altered nuclear size is associated with many cancers, and determining whether cancer-associated changes in nuclear size contribute to carcinogenesis necessitates an understanding of mechanisms of nuclear size regulation. Although nuclear import rates generally positively correlate with nuclear size, NTF2 levels negatively affect nuclear size, despite the role of NTF2 (also known as NUTF2) in nuclear recycling of the import factor Ran. We show that binding of Ran to NTF2 is required for NTF2 to inhibit nuclear expansion and import of large cargo molecules in Xenopus laevis egg and embryo extracts, consistent with our observation that NTF2 reduces the diameter of the nuclear pore complex (NPC) in a Ran-binding-dependent manner. Furthermore, we demonstrate that ectopic NTF2 expression in Xenopus embryos and mammalian tissue culture cells alters nuclear size. Finally, we show that increases in nuclear size during melanoma progression correlate with reduced NTF2 expression, and increasing NTF2 levels in melanoma cells is sufficient to reduce nuclear size. These results show a conserved capacity for NTF2 to impact on nuclear size, and we propose that NTF2 might be a new cancer biomarker. PMID:26823604

  11. Functional characterization of nuclear localization and export signals in hepatitis C virus proteins and their role in the membranous web.

    Directory of Open Access Journals (Sweden)

    Aviad Levin

    Full Text Available 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 α5 (IPOA5/kapα1, importin β3 (IPO5/kap β3, 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.

  12. Identification of interacting proteins of retinoid-related orphan nuclear receptor gamma in HepG2 cells

    Directory of Open Access Journals (Sweden)

    Ze-Min Huang1,#, Jun Wu2,#, Zheng-Cai Jia1, Yi Tian1, Jun Tang3, Yan Tang1, Ying Wang2, Yu-Zhang Wu1,* & Bing Ni1,*

    2012-06-01

    Full Text Available The retinoid-related orphan nuclear receptor gamma (RORγplays critical roles in regulation of development, immunity andmetabolism. As transcription factor usually forms a proteincomplex to function, thus capturing and dissecting of theRORγ protein complex will be helpful for exploring themechanisms underlying those functions. After construction ofthe recombinant tandem affinity purification (TAP plasmid,pMSCVpuro RORγ-CTAP(SG, the nuclear localization ofRORγ-CTAP(SG fusion protein was verified. Followingisolation of RORγ protein complex by TAP strategy, sevencandidate interacting proteins were identified. Finally, the heatshock protein 90 (HSP90 and receptor-interacting protein 140(RIP140 were confirmed to interplay with RORγ byco-immunoprecipitation. Interference of HSP90 or/and RIP140genes resulted in dramatically decreased expression ofCYP2C8 gene, the RORγ target gene. Data from this studydemonstrate that HSP90 and RIP140 proteins interact withRORγ protein in a complex format and function asco-activators in the RORγ-mediated regulatory processes ofHepG2 cells.

  13. Leukemia-Associated Nup214 Fusion Proteins Disturb the XPO1-Mediated Nuclear-Cytoplasmic Transport Pathway and Thereby the NF-κB Signaling Pathway.

    Science.gov (United States)

    Saito, Shoko; Cigdem, Sadik; Okuwaki, Mitsuru; Nagata, Kyosuke

    2016-07-01

    Nuclear-cytoplasmic transport through nuclear pore complexes is mediated by nuclear transport receptors. Previous reports have suggested that aberrant nuclear-cytoplasmic transport due to mutations or overexpression of nuclear pore complexes and nuclear transport receptors is closely linked to diseases. Nup214, a component of nuclear pore complexes, has been found as chimeric fusion proteins in leukemia. Among various Nup214 fusion proteins, SET-Nup214 and DEK-Nup214 have been shown to be engaged in tumorigenesis, but their oncogenic mechanisms remain unclear. In this study, we examined the functions of the Nup214 fusion proteins by focusing on their effects on nuclear-cytoplasmic transport. We found that SET-Nup214 and DEK-Nup214 interact with exportin-1 (XPO1)/CRM1 and nuclear RNA export factor 1 (NXF1)/TAP, which mediate leucine-rich nuclear export signal (NES)-dependent protein export and mRNA export, respectively. SET-Nup214 and DEK-Nup214 decreased the XPO1-mediated nuclear export of NES proteins such as cyclin B and proteins involved in the NF-κB signaling pathway by tethering XPO1 onto nuclear dots where Nup214 fusion proteins are localized. We also demonstrated that SET-Nup214 and DEK-Nup214 expression inhibited NF-κB-mediated transcription by abnormal tethering of the complex containing p65 and its inhibitor, IκB, in the nucleus. These results suggest that SET-Nup214 and DEK-Nup214 perturb the regulation of gene expression through alteration of the nuclear-cytoplasmic transport system. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

  14. Multiple protein domains contribute to nuclear import and cell toxicity of DUX4, a candidate pathogenic protein for facioscapulohumeral muscular dystrophy.

    Directory of Open Access Journals (Sweden)

    Edgardo Daniel Corona

    Full Text Available DUX4 (Double Homeobox Protein 4 is a nuclear transcription factor encoded at each D4Z4 unit of a tandem-repeat array at human chromosome 4q35. DUX4 constitutes a major candidate pathogenic protein for facioscapulohumeral muscular dystrophy (FSHD, the third most common form of inherited myopathy. A low-level expression of DUX4 compromises cell differentiation in myoblasts and its overexpression induces apoptosis in cultured cells and living organisms. In this work we explore potential molecular determinants of DUX4 mediating nuclear import and cell toxicity. Deletion of the hypothetical monopartite nuclear localization sequences RRRR(23, RRKR(98 and RRAR(148 (i.e. NLS1, NLS2 and NLS3, respectively only partially delocalizes DUX4 from the cell nuclei. Nuclear entrance guided by NLS1, NLS2 and NLS3 does not follow the classical nuclear import pathway mediated by α/β importins. NLS and homeodomain mutants from DUX4 are dramatically less cell-toxic than the wild type molecule, independently of their subcellular localization. A triple ΔNLS1-2-3 deletion mutant is still partially localized in the nuclei, indicating that additional sequences in DUX4 contribute to nuclear import. Deletion of ≥111 amino acids from the C-terminal of DUX4, on a ΔNLS1-2-3 background, almost completely re-localizes DUX4 to the cytoplasm, indicating that the C-ter tail contributes to subcellular trafficking of DUX4. Also, C-terminal deletion mutants of DUX4 on a NLS wild type background are less toxic than wild type DUX4. Results reported here indicate that DUX4 possesses redundant mechanisms to assure nuclear entrance and that its various transcription-factor associated domains play an essential role in cell toxicity.

  15. 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

    Energy Technology Data Exchange (ETDEWEB)

    Eto, Masumi, E-mail: masumi.eto@jefferson.edu [Department of Molecular Physiology and Biophysics, and Kimmel Cancer Center, Thomas Jefferson University, 1020 Locust Street, PA 19107 (United States); Kirkbride, Jason A.; Chugh, Rishika; Karikari, Nana Kofi [Department of Molecular Physiology and Biophysics, and Kimmel Cancer Center, Thomas Jefferson University, 1020 Locust Street, PA 19107 (United States); Kim, Jee In [Department of Molecular Physiology and Biophysics, and Kimmel Cancer Center, Thomas Jefferson University, 1020 Locust Street, PA 19107 (United States); Cardiovascular Research Institute, Kyungpook National University School of Medicine, Daegu 700-422 (Korea, Republic of)

    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.

  16. Characterization of p18, a component of the lamin B receptor complex and a new integral membrane protein of the avian erythrocyte nuclear envelope.

    Science.gov (United States)

    Simos, G; Maison, C; Georgatos, S D

    1996-05-24

    Employing avian erythrocytes, we have previously isolated a multimeric complex consisting of the lamin B receptor (LBR, or p58), the nuclear lamins, an LBR-specific kinase, a 34-kDa protein, and an 18-kDa polypeptide termed p18. As the LBR kinase and the 34-kDa component have been recently characterized, we now proceed in the characterization of p18. We show here that p18 is an integral membrane protein specific to the erythrocyte nuclear envelope which binds to LBR and B-type lamins. NH2-terminal sequencing indicates that p18 is distinct from other nuclear envelope components, but has similarity to the mitochondrial isoquinoline-binding protein. In situ analysis by immunoelectron microscopy and examination of digitonin-permeabilized cells by indirect immunofluorescence show that p18, unlike LBR and other lamin-binding proteins, is equally distributed between the inner and outer nuclear membrane. Furthermore, cycloheximide inhibition experiments reveal that the fraction of p18 that resides in the outer nuclear membrane does not represent nascent chains en route to the inner nuclear membrane, but rather material in equilibrium with the p18 that partitions with the inner nuclear membrane. The paradigm of p18 suggests that transmembrane complexes formed by the nuclear lamins and LBR provide potential docking sites for integral membrane proteins of the nuclear envelope that equilibrate between the rough endoplasmic reticulum and the inner nuclear membrane.

  17. HMG-box sequences from microbats homologous to the human SOX30 HMG-box.

    Science.gov (United States)

    Bullejos, M; Díaz de la Guardia, R; Barragán, M J; Sánchez, A

    2000-01-01

    SOX genes are a family of genes that encode for proteins which are characterised by the presence of a HMG-domain related to that of the mammalian sex-determining gene (SRY). By definition, the DNA binding domain of SOX genes is at least 50% identical to the 79 amino acid HMG domain of the SRY gene. We report here two HMG-box sequences from two microbat species (R. ferrumequinum and P. Pipistrellus) which were PCR amplified using a primer pair specific to the mouse Sry HMG-box. The high percentage of identity of this sequences with the human and mouse SOX30 HMG-box suggests that they are the SOX30 HMG-box for these two bat species.

  18. Development of a radioiodinated triazolopyrimidine probe for nuclear medical imaging of fatty acid binding protein 4.

    Directory of Open Access Journals (Sweden)

    Kantaro Nishigori

    Full Text Available Fatty acid binding protein 4 (FABP4 is the most well-characterized FABP isoform. FABP4 regulates inflammatory pathways in adipocytes and macrophages and is involved in both inflammatory diseases and tumor formation. FABP4 expression was recently reported for glioblastoma, where it may participate in disease malignancy. While FABP4 is a potential molecular imaging target, with the exception of a tritium labeled probe there are no reports of other nuclear imaging probes that target this protein. Here we designed and synthesized a nuclear imaging probe, [123I]TAP1, and evaluated its potential as a FABP4 targeting probe in in vitro and in vivo assays. We focused on the unique structure of a triazolopyrimidine scaffold that lacks a carboxylic acid to design the TAP1 probe that can undergo facilitated delivery across cell membranes. The affinity of synthesized TAP1 was measured using FABP4 and 8-anilino-1-naphthalene sulfonic acid. [125I]TAP1 was synthesized by iododestannylation of a precursor, followed by affinity and selectivity measurements using immobilized FABPs. Biodistributions in normal and C6 glioblastoma-bearing mice were evaluated, and excised tumors were subjected to autoradiography and immunohistochemistry. TAP1 and [125I]TAP1 showed high affinity for FABP4 (Ki = 44.5±9.8 nM, Kd = 69.1±12.3 nM. The FABP4 binding affinity of [125I]TAP1 was 11.5- and 35.5-fold higher than for FABP3 and FABP5, respectively. In an in vivo study [125I]TAP1 displayed high stability against deiodination and degradation, and moderate radioactivity accumulation in C6 tumors (1.37±0.24% dose/g 3 hr after injection. The radioactivity distribution profile in tumors partially corresponded to the FABP4 positive area and was also affected by perfusion. The results indicate that [125I]TAP1 could detect FABP4 in vitro and partly in vivo. As such, [125I]TAP1 is a promising lead compound for further refinement for use in in vivo FABP4 imaging.

  19. Teaching with Box Tops.

    Science.gov (United States)

    Raiser, Lynne; D'Zamko, Mary Elizabeth

    1984-01-01

    Using environmental materials (such as the phone book and placemats from fast food restaurants) can be a motivating way to teach learning disabled students skills and concepts, as shown in an approach to reading, math, science and nutrition, and social studies instruction using a JELL-O brand gelatin box. (CL)

  20. Many-box locality

    Science.gov (United States)

    Zhou, Yuqian; Cai, Yu; Bancal, Jean-Daniel; Gao, Fei; Scarani, Valerio

    2017-11-01

    There is an ongoing search for a physical or operational definition for quantum mechanics. Several informational principles have been proposed which are satisfied by a theory less restrictive than quantum mechanics. Here, we introduce the principle of "many-box locality," which is a refined version of the previously proposed "macroscopic locality." These principles are based on coarse graining the statistics of several copies of a given box. The set of behaviors satisfying many-box locality for N boxes is denoted LNM B. We study these sets in the bipartite scenario with two binary measurements, in relation with the sets Q and Q1 +A B of quantum and "almost quantum" correlations, respectively. We find that the LNM B sets are, in general, not convex. For unbiased marginals, by working in the Fourier space we can prove analytically that LNM B⊈Q for any finite N , while L∞M B=Q . Then, with suitably developed numerical tools, we find an example of a point that belongs to L16M B but not to Q1 +A B. Among the problems that remain open is whether Q ⊂L∞M B .

  1. IN-A-BOX

    Indian Academy of Sciences (India)

    Home; Journals; Resonance – Journal of Science Education; Volume 7; Issue 2. Claude Elwood Shannon. Priti Shankar. Article-in-a-Box Volume 7 Issue 2 February 2002 pp 2-3. Fulltext. Click here to view fulltext PDF. Permanent link: http://www.ias.ac.in/article/fulltext/reso/007/02/0002-0003. Author Affiliations.

  2. Mystery Box Marvels

    Science.gov (United States)

    Santos, Joel; Centurio, Tina

    2012-01-01

    What happens in the first week of school could very well set the stage for the rest of the school year. Setting high standards for science activities based in inquiry can start on the first day of science class and develop as the year unfolds. With the use of simple, readily available, inexpensive materials, an efficient mystery box lesson can be…

  3. Operant-Box Sociology

    Science.gov (United States)

    Dimond, Stuart J.

    1971-01-01

    Sketches a model which considers society a teacher which instructs like an operant box..." The author contends that the family, the school, the play group, the adolescent gang, the office, the prison, the mental hospital, each reward, instruct and control the individual who learns by operant means what the institution teaches." (Author/AL)

  4. Nuclear Wiskott–Aldrich syndrome protein co-regulates T cell factor 1-mediated transcription in T cells

    Directory of Open Access Journals (Sweden)

    Nikolai V. Kuznetsov

    2017-10-01

    Full Text Available Abstract Background The Wiskott–Aldrich syndrome protein (WASp family of actin-nucleating factors are present in the cytoplasm and in the nucleus. The role of nuclear WASp for T cell development remains incompletely defined. Methods We performed WASp chromatin immunoprecipitation and deep sequencing (ChIP-seq in thymocytes and spleen CD4+ T cells. Results WASp was enriched at genic and intergenic regions and associated with the transcription start sites of protein-coding genes. Thymocytes and spleen CD4+ T cells showed 15 common WASp-interacting genes, including the gene encoding T cell factor (TCF12. WASp KO thymocytes had reduced nuclear TCF12 whereas thymocytes expressing constitutively active WASpL272P and WASpI296T had increased nuclear TCF12, suggesting that regulated WASp activity controlled nuclear TCF12. We identify a putative DNA element enriched in WASp ChIP-seq samples identical to a TCF1-binding site and we show that WASp directly interacted with TCF1 in the nucleus. Conclusions These data place nuclear WASp in proximity with TCF1 and TCF12, essential factors for T cell development.

  5. The human β-globin gene promoter; nuclear protein factors and erythroid specific induction of transcription.

    NARCIS (Netherlands)

    E. de Boer (Ernie); M. Antoniou (Michael); V. Mignotte; L. Wall (Lee); F.G. Grosveld (Frank)

    1988-01-01

    textabstractWe have shown that the promoter of the human beta-globin gene contains three regions in addition to the known CAC, CAAT and TATA box regions that are important for the induction of transcription in erythroid cells. By using DNaseI footprinting and gel mobility shift assays we were able

  6. The phosphatidylserine receptor from Hydra is a nuclear protein with potential Fe(II dependent oxygenase activity

    Directory of Open Access Journals (Sweden)

    Stiening Beate

    2004-06-01

    Full Text Available Abstract Background Apoptotic cell death plays an essential part in embryogenesis, development and maintenance of tissue homeostasis in metazoan animals. The culmination of apoptosis in vivo is the phagocytosis of cellular corpses. One morphological characteristic of cells undergoing apoptosis is loss of plasma membrane phospholipid asymmetry and exposure of phosphatidylserine on the outer leaflet. Surface exposure of phosphatidylserine is recognised by a specific receptor (phosphatidylserine receptor, PSR and is required for phagocytosis of apoptotic cells by macrophages and fibroblasts. Results We have cloned the PSR receptor from Hydra in order to investigate its function in this early metazoan. Bioinformatic analysis of the Hydra PSR protein structure revealed the presence of three nuclear localisation signals, an AT-hook like DNA binding motif and a putative 2-oxoglutarate (2OG-and Fe(II-dependent oxygenase activity. All of these features are conserved from human PSR to Hydra PSR. Expression of GFP tagged Hydra PSR in hydra cells revealed clear nuclear localisation. Deletion of one of the three NLS sequences strongly diminished nuclear localisation of the protein. Membrane localisation was never detected. Conclusions Our results suggest that Hydra PSR is a nuclear 2-oxoglutarate (2OG-and Fe(II-dependent oxygenase. This is in contrast with the proposed function of Hydra PSR as a cell surface receptor involved in the recognition of apoptotic cells displaying phosphatidylserine on their surface. The conservation of the protein from Hydra to human infers that our results also apply to PSR from higher animals.

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

    Energy Technology Data Exchange (ETDEWEB)

    Guarda, Alessia, E-mail: alessiaguarda@libero.it [Department of Structural and Functional Biology, University of Insubria, via Alberto da Giussano 12, Busto Arsizio (Italy); Bolognese, Fabrizio, E-mail: fabrizio.bolognese@uninsubria.it [Department of Structural and Functional Biology, University of Insubria, via Alberto da Giussano 12, Busto Arsizio (Italy); Bonapace, Ian Marc, E-mail: ian.bonapace@uninsubria.it [Department of Structural and Functional Biology, University of Insubria, via Alberto da Giussano 12, Busto Arsizio (Italy); Badaracco, Gianfranco, E-mail: gianfranco.badaracco@uninsubria.it [Department of Structural and Functional Biology, University of Insubria, via Alberto da Giussano 12, Busto Arsizio (Italy)

    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.

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

    Science.gov (United States)

    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.

  9. Phylogeny of pteromalid parasitic wasps (Hymenoptera: Pteromalidae): initial evidence from four protein-coding nuclear genes.

    Science.gov (United States)

    Desjardins, Christopher A; Regier, Jerome C; Mitter, Charles

    2007-11-01

    Chalcidoidea (approximately 22,000 described species) is the most ecologically diverse superfamily of parasitic Hymenoptera and plays a major role in the biological control of insect pests. However, phylogenetic relationships both within and between chalcidoid families have been poorly understood, particularly for the large family Pteromalidae and relatives. Forty-two taxa, broadly representing Chalcidoidea but concentrated in the 'pteromalid lineage,' were sequenced for 4620 bp of protein-coding sequence from four nuclear genes for which we present new primers. These are: CAD (1719 bp) DDC (708 bp), enolase (1149 bp), and PEPCK (1044 bp). The combined data set was analyzed using parsimony, maximum likelihood, and Bayesian methods. Statistical significance of the apparent non-monophyly of some taxonomic groups on our trees was evaluated using the approximately unbiased test of Shimodaira [Shimodaira, H. 2002. An approximately unbiased test of phylogenetic tree selection. Syst. Biol. 51(3), 492-508]. In accord with previous studies, we find moderate to strong support for monophyly of Chalcidoidea, a sister-group relationship of Mymaridae to the remainder of Chalcidoidea, and a relatively basal placement of Encarsia (Aphelinidae) within the latter. The 'pteromalid lineage' of families is generally recovered as monophyletic, but the hypothesis of monophyly for Pteromalidae, which appear paraphyletic with respect to all other families sampled in that lineage, is decisively rejected (P Initial phylogenetic comparisons of life history traits suggest that the ancestral chalcidoid was small-bodied and parasitized insect eggs.

  10. Prominent increase in synthesis of a nuclear protein is an early signal associated with mitogenesis of B cells

    Energy Technology Data Exchange (ETDEWEB)

    Feuerstein, N.; Mond, J.J.

    1987-05-01

    Activation of murine splenic B lymphocytes with various mitogens was found to be associated with a prominent increase in synthesis and abundance of a 40 KDa/pI 5.0 nuclear protein (p40). Subcellular fractionation revealed that this protein is not found in the cytosol fraction and cannot be solubilized by DNAse or RNAse digestion, indication that the protein is mainly associated with the nuclear matrix. The increase in synthesis of p40 was detected at early G1 phase, 60 min following activation of the cells by the mitogen, reached a peak at 16h and declined to control level at 48h (during S phase). Activation of the cells with non mitogenic stimuli such as BSF-1, A23187, and PMA, induced an increase in synthesis of discrete nuclear proteins, but failed to affect the synthesis of p40, suggesting that the increase in synthesis of p40 is specifically associated with the signal of mitogenic stimuli but not with this of non mitogenic stimuli. Inhibition of the mitogenic effect of anti-Ig by PMA treatment of the cells was associated with specific inhibition in the synthesis of p40, while overcoming this inhibition by addition of 8-mercaptoguanosine was associated with restoration of the mitogenic effect of anti-Ig. These results suggest that p40 may have an important role in early induction of mitogenesis in B cells.

  11. Hermit Points on a Box

    Science.gov (United States)

    Hess, Richard; Grinstead, Charles; Grindstead, Marshall; Bergstrand, Deborah

    2008-01-01

    Suppose that we are given a rectangular box in 3-space. Given any two points on the surface of this box, we can define the surface distance between them to be the length of the shortest path between them on the surface of the box. This paper determines the pairs of points of maximum surface distance for all boxes. It is often the case that these…

  12. Microglial Amyloid-β1-40 Phagocytosis Dysfunction Is Caused by High-Mobility Group Box Protein-1: Implications for the Pathological Progression of Alzheimer’s Disease

    Directory of Open Access Journals (Sweden)

    Kazuyuki Takata

    2012-01-01

    Full Text Available In Alzheimer disease (AD patient brains, the accumulation of amyloid-β (Aβ peptides is associated with activated microglia. Aβ is derived from the amyloid precursor protein; two major forms of Aβ, that is, Aβ1-40 (Aβ40 and Aβ1-42 (Aβ42, exist. We previously reported that rat microglia phagocytose Aβ42, and high mobility group box protein 1 (HMGB1, a chromosomal protein, inhibits phagocytosis. In the present study, we investigated the effects of exogenous HMGB1 on rat microglial Aβ40 phagocytosis. In the presence of exogenous HMGB1, Aβ40 markedly increased in microglial cytoplasm, and the reduction of extracellular Aβ40 was inhibited. During this period, HMGB1 was colocalized with Aβ40 in the cytoplasm. Furthermore, exogenous HMGB1 inhibited the degradation of Aβ40 induced by the rat microglial cytosolic fraction. Thus, extracellular HMGB1 may internalize with Aβ40 in the microglial cytoplasm and inhibit Aβ40 degradation by microglia. This may subsequently delay Aβ40 clearance. We further confirmed that in AD brains, the parts of senile plaques surrounded by activated microglia are composed of Aβ40, and extracellular HMGB1 is deposited on these plaques. Taken together, microglial Aβ phagocytosis dysfunction may be caused by HMGB1 that accumulates extracellularly on Aβ plaques, and it may be critically involved in the pathological progression of AD.

  13. Virus-Like Particles Derived from HIV-1 for Delivery of Nuclear Proteins: Improvement of Production and Activity by Protein Engineering.

    Science.gov (United States)

    Robert, Marc-André; Lytvyn, Viktoria; Deforet, Francis; Gilbert, Rénald; Gaillet, Bruno

    2017-01-01

    Virus-like particles (VLPs) derived from retroviruses and lentiviruses can be used to deliver recombinant proteins without the fear of causing insertional mutagenesis to the host cell genome. In this study we evaluate the potential of an inducible lentiviral vector packaging cell line for VLP production. The Gag gene from HIV-1 was fused to a gene encoding a selected protein and it was transfected into the packaging cells. Three proteins served as model: the green fluorescent protein and two transcription factors-the cumate transactivator (cTA) of the inducible CR5 promoter and the human Krüppel-like factor 4 (KLF4). The sizes of the VLPs were 120-150 nm in diameter and they were resistant to freeze/thaw cycles. Protein delivery by the VLPs reached up to 100% efficacy in human cells and was well tolerated. Gag-cTA triggered up to 1100-fold gene activation of the reporter gene in comparison to the negative control. Protein engineering was required to detect Gag-KLF4 activity. Thus, insertion of the VP16 transactivation domain increased the activity of the VLPs by eightfold. An additional 2.4-fold enhancement was obtained by inserting nuclear export signal. In conclusion, our platform produced VLPs capable of efficient protein transfer, and it was shown that protein engineering can be used to improve the activity of the delivered proteins as well as VLP production.

  14. Nuclear LSm8 affects number of cytoplasmic processing bodies via controlling cellular distribution of Like-Sm proteins.

    Science.gov (United States)

    Novotny, Ivan; Podolská, Katerina; Blazíková, Michaela; Valásek, Leos Shivaya; Svoboda, Petr; Stanek, David

    2012-10-01

    Processing bodies (P-bodies) are dynamic cytoplasmic structures involved in mRNA degradation, but the mechanism that governs their formation is poorly understood. In this paper, we address a role of Like-Sm (LSm) proteins in formation of P-bodies and provide evidence that depletion of nuclear LSm8 increases the number of P-bodies, while LSm8 overexpression leads to P-body loss. We show that LSm8 knockdown causes relocalization of LSm4 and LSm6 proteins to the cytoplasm and suggest that LSm8 controls nuclear accumulation of all LSm2-7 proteins. We propose a model in which redistribution of LSm2-7 to the cytoplasm creates new binding sites for other P-body components and nucleates new, microscopically visible structures. The model is supported by prolonged residence of two P-body proteins, DDX6 and Ago2, in P-bodies after LSm8 depletion, which indicates stronger interactions between these proteins and P-bodies. Finally, an increased number of P-bodies has negligible effects on microRNA-mediated translation repression and nonsense mediated decay, further supporting the view that the function of proteins localized in P-bodies is independent of visible P-bodies.

  15. sel-7, a positive regulator of lin-12 activity, encodes a novel nuclear protein in Caenorhabditis elegans.

    Science.gov (United States)

    Chen, Jiabin; Li, Xiajun; Greenwald, Iva

    2004-01-01

    Suppressor genetics in C. elegans has identified key components of the LIN-12/Notch signaling pathway. Here, we describe a genetic and molecular characterization of the suppressor gene sel-7. We show that reducing or eliminating sel-7 activity suppresses the effects of constitutive lin-12 activity, enhances the effects of partially reduced lin-12 activity, and causes a synthetic Lin-12(0) phenotype when combined with a null mutation in the sel-12 presenilin gene. These observations suggest that sel-7 is a positive regulator of lin-12 activity. We also show that SEL-7 encodes a novel nuclear protein. Through yeast two-hybrid screening, we identified an apparent interaction partner, K08E3.8, that also interacts with SEL-8, a known component of the nuclear complex that forms upon LIN-12 activation. Our data suggest potential roles for SEL-7 in the assembly or function of this nuclear complex.

  16. Correlation between high mobility group box-1 protein and chronic hepatitis B infection with severe hepatitis B and acute-on-chronic liver failure: a meta-analysis.

    Science.gov (United States)

    Hu, Yi-Bing; Hu, Dan-Ping; Fu, Rong-Quan

    2017-06-01

    The aim of this study was to evaluate the correlation of High-mobility group box 1 (HMGB1) expression in the serum with chronic hepatitis B (CHB) related liver fibrosis, severe hepatitis B and acute-on-chronic liver failure (ACLF). We made a literature search in PubMed, Embase, Web of Science, Medline, Google Scholar, China National Knowledge Infrastructure, WanFang with no language restriction. Pooled data were analyzed and mean difference with corresponding 95% confidence intervals were calculated. A total of 16 relevant studies were identified. HMGB1 serum levels were higher in severe hepatitis B or ACLF patients than those in CHB patients. Pooled mean differences of HMGB1 in severe hepatitis B and ACLF patients compared with CHB patients were 4.32 (95% CI: 0.34-8.29, Z=2.13, I2=59%, P=0.03) and 15.96 (95% CI: -0.37-32.28, Z=1.92, P=0.06). Four studies showed there was a different HMGB1 expression in mild, moderate and severe CHB patients (P values were <0.05, <0.05, <0.05 and <0.01, respectively). Pooled mean difference of HMGB1 in low liver fibrosis patients compared with high liver fibrosis was -125.38 (95% CI: -539.44-288.68, Z=0.59, I2=98%, P=0.55). The results suggested that HMGB1 levels in the serum were statistically higher in severe hepatitis B and ACLF patients. Therefore, HMGB1 may be a useful therapeutic target for severe hepatitis B and ACLF diagnosis.

  17. Nuclear location of tumor suppressor protein maspin inhibits proliferation of breast cancer cells without affecting proliferation of normal epithelial cells

    Science.gov (United States)

    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

  18. Venezuelan equine Encephalitis virus capsid protein forms a tetrameric complex with CRM1 and importin alpha/beta that obstructs nuclear pore complex function.

    Science.gov (United States)

    Atasheva, Svetlana; Fish, Alexander; Fornerod, Maarten; Frolova, Elena I

    2010-05-01

    Development of the cellular antiviral response requires nuclear translocation of multiple transcription factors and activation of a wide variety of cellular genes. To counteract the antiviral response, several viruses have developed an efficient means of inhibiting nucleocytoplasmic traffic. In this study, we demonstrate that the pathogenic strain of Venezuelan equine encephalitis virus (VEEV) has developed a unique mechanism of nuclear import inhibition. Its capsid protein forms a tetrameric complex with the nuclear export receptor CRM1 and the nuclear import receptor importin alpha/beta. This unusual complex accumulates in the center channel of the nuclear pores and blocks nuclear import mediated by different karyopherins. The inhibitory function of VEEV capsid protein is determined by a short 39-amino-acid-long peptide that contains both nuclear import and supraphysiological nuclear export signals. Mutations in these signals or in the linker peptide attenuate or completely abolish capsid-specific inhibition of nuclear traffic. The less pathogenic VEEV strains contain a wide variety of mutations in this peptide that affect its inhibitory function in nuclear import. Thus, these mutations appear to be the determinants of this attenuated phenotype. This novel mechanism of inhibiting nuclear transport also shows that the nuclear pore complex is vulnerable to unusual cargo receptor complexes and sheds light on the importance of finely adjusted karyopherin-nucleoporin interactions for efficient cargo translocation.

  19. Opto-Box

    CERN Document Server

    AUTHOR|(INSPIRE)INSPIRE-00377159; The ATLAS collaboration

    2015-01-01

    The opto-box is a custom mini-crate for housing optical modules, which process and transfer optoelectronic data. Many novel solutions were developed for the custom design and manufacturing. The system tightly integrates electrical, mechanical, and thermal functionality into a small package of size 35x10x8 cm$^{3}$. Special attention was given to ensure proper shielding, grounding, cooling, high reliability, and environmental tolerance. The custom modules, which incorporate Application Specific Integrated Circuits (ASICs), were developed through a cycle of rigorous testing and redesign. In total, fourteen opto-boxes have been installed and loaded with modules on the ATLAS detector. They are currently in operation as part of the LHC run 2 data read-out chain.

  20. Multiple conformational states of DnaA protein regulate its interaction with DnaA boxes in the initiation of DNA replication.

    Science.gov (United States)

    Patel, Meera J; Bhatia, Lavesh; Yilmaz, Gulden; Biswas-Fiss, Esther E; Biswas, Subhasis B

    2017-09-01

    DnaA protein is the initiator of genomic DNA replication in prokaryotes. It binds to specific DNA sequences in the origin of DNA replication and unwinds small AT-rich sequences downstream for the assembly of the replisome. The mechanism of activation of DnaA that enables it to bind and organize the origin DNA and leads to replication initiation remains unclear. In this study, we have developed double-labeled fluorescent DnaA probes to analyze conformational states of DnaA protein upon binding DNA, nucleotide, and Soj sporulation protein using Fluorescence Resonance Energy Transfer (FRET). Our studies demonstrate that DnaA protein undergoes large conformational changes upon binding to substrates and there are multiple distinct conformational states that enable it to initiate DNA replication. DnaA protein adopted a relaxed conformation by expanding ~15Å upon binding ATP and DNA to form the ATP·DnaA·DNA complex. Hydrolysis of bound ATP to ADP led to a contraction of DnaA within the complex. The relaxed conformation of DnaA is likely required for the formation of the multi-protein ATP·DnaA·DNA complex. In the initiation of sporulation, Soj binding to DnaA prevented relaxation of its conformation. Soj·ADP appeared to block the activation of DnaA, suggesting a mechanism for Soj·ADP in switching initiation of DNA replication to sporulation. Our studies demonstrate that multiple conformational states of DnaA protein regulate its binding to DNA in the initiation of DNA replication. Copyright © 2017 Elsevier B.V. All rights reserved.

  1. Boxed Permutation Pattern Matching

    DEFF Research Database (Denmark)

    Amit, Mika; Bille, Philip; Cording, Patrick Hagge

    2016-01-01

    the goal is to only find the boxed subsequences of T that are order-isomorphic to P. This problem was introduced by Bruner and Lackner who showed that it can be solved in O(n3) time. Cho et al. [CPM 2015] gave an O(n2m) time algorithm and improved it to O(n2 logm). In this paper we present a solution...

  2. Immunoreactivity to the pre-core box antibody shows that most glycine-rich beta-proteins accumulate in lepidosaurian beta-layer and in the corneous layer of crocodilian and turtle epidermis.

    Science.gov (United States)

    Alibardi, L

    2014-02-01

    The differentiation of the corneous layers of reptilian epidermis has been analyzed by ultrastructural immunocytochemistry using specific antibodies against the conserved pre-core box region of their keratin-associated beta-proteins (KAbetaPs, formerly indicated as beta-keratins) and silver-intensification. The epitope analysis in the sequences of different reptilian KAbetaPs indicates that this antibody recognizes mainly glycine-rich beta-proteins in lizards and snakes. The immunoreactivity of the beta-layer of the tuatara to this antibody also suggests that a similar epitope is present in beta-proteins of this relict species. In crocodilians the antibody recognizes glycine-rich beta-proteins, so far representing all the known crocodilian KAbetaPs. In hard-shelled turtle the antibody labels mainly type 1 KAbetaPs that represent most types found in this turtle. The antibody does not label the corneous layer of the soft-shelled turtle that contains exclusively type 2 KAbetaPs, with a low identity to the epitope recognized by the antibody. The prevalent labeling of the beta-layers in lepidosaurian epidermis and of the corneous layer in turtle and crocodilian epidermis suggest that this antibody is mainly directed toward KAbetaPs rich in glycine. The latter are main constituents of the corneous layer in turtles and crocodilians and of the beta-layer in lizards, snakes and the tuatara. These proteins are largely responsible for the inflexibility, mechanical resistance, chromophobicity and relative hydrophobicity of the reptilian corneous layer. Copyright © 2013. Published by Elsevier Ltd.

  3. Dissociation of heterochromatin protein 1 from lamin B receptor induced by human polyomavirus agnoprotein: role in nuclear egress of viral particles.

    Science.gov (United States)

    Okada, Yuki; Suzuki, Tadaki; Sunden, Yuji; Orba, Yasuko; Kose, Shingo; Imamoto, Naoko; Takahashi, Hidehiro; Tanaka, Shinya; Hall, William W; Nagashima, Kazuo; Sawa, Hirofumi

    2005-05-01

    The nuclear envelope is one of the chief obstacles to the translocation of macromolecules that are larger than the diameter of nuclear pores. Heterochromatin protein 1 (HP1) bound to the lamin B receptor (LBR) is thought to contribute to reassembly of the nuclear envelope after cell division. Human polyomavirus agnoprotein (Agno) has been shown to bind to HP1alpha and to induce its dissociation from LBR, resulting in destabilization of the nuclear envelope. Fluorescence recovery after photobleaching showed that Agno increased the lateral mobility of LBR in the inner nuclear membrane. Biochemical and immunofluorescence analyses showed that Agno is targeted to the nuclear envelope and facilitates the nuclear egress of polyomavirus-like particles. These results indicate that dissociation of HP1alpha from LBR and consequent perturbation of the nuclear envelope induced by polyomavirus Agno promote the translocation of virions out of the nucleus.

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

    Science.gov (United States)

    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.

  5. The role of nuclear matrix proteins binding to matrix attachment regions (Mars) in prostate cancer cell differentiation.

    Science.gov (United States)

    Barboro, Paola; Repaci, Erica; D'Arrigo, Cristina; Balbi, Cecilia

    2012-01-01

    In tumor progression definite alterations in nuclear matrix (NM) protein composition as well as in chromatin structure occur. The NM interacts with chromatin via specialized DNA sequences called matrix attachment regions (MARs). In the present study, using a proteomic approach along with a two-dimensional Southwestern assay and confocal laser microscopy, we show that the differentiation of stabilized human prostate carcinoma cells is marked out by modifications both NM protein composition and bond between NM proteins and MARs. Well-differentiated androgen-responsive and slowly growing LNCaP cells are characterized by a less complex pattern and by a major number of proteins binding MAR sequences in comparison to 22Rv1 cells expressing androgen receptor but androgen-independent. Finally, in the poorly differentiated and strongly aggressive androgen-independent PC3 cells the complexity of NM pattern further increases and a minor number of proteins bind the MARs. Furthermore, in this cell line with respect to LNCaP cells, these changes are synchronous with modifications in both the nuclear distribution of the MAR sequences and in the average loop dimensions that significantly increase. Although the expression of many NM proteins changes during dedifferentiation, only a very limited group of MAR-binding proteins seem to play a key role in this process. Variations in the expression of poly (ADP-ribose) polymerase (PARP) and special AT-rich sequence-binding protein-1 (SATB1) along with an increase in the phosphorylation of lamin B represent changes that might trigger passage towards a more aggressive phenotype. These results suggest that elucidating the MAR-binding proteins that are involved in the differentiation of prostate cancer cells could be an important tool to improve our understanding of this carcinogenesis process, and they could also be novel targets for prostate cancer therapy.

  6. The role of nuclear matrix proteins binding to matrix attachment regions (Mars in prostate cancer cell differentiation.

    Directory of Open Access Journals (Sweden)

    Paola Barboro

    Full Text Available In tumor progression definite alterations in nuclear matrix (NM protein composition as well as in chromatin structure occur. The NM interacts with chromatin via specialized DNA sequences called matrix attachment regions (MARs. In the present study, using a proteomic approach along with a two-dimensional Southwestern assay and confocal laser microscopy, we show that the differentiation of stabilized human prostate carcinoma cells is marked out by modifications both NM protein composition and bond between NM proteins and MARs. Well-differentiated androgen-responsive and slowly growing LNCaP cells are characterized by a less complex pattern and by a major number of proteins binding MAR sequences in comparison to 22Rv1 cells expressing androgen receptor but androgen-independent. Finally, in the poorly differentiated and strongly aggressive androgen-independent PC3 cells the complexity of NM pattern further increases and a minor number of proteins bind the MARs. Furthermore, in this cell line with respect to LNCaP cells, these changes are synchronous with modifications in both the nuclear distribution of the MAR sequences and in the average loop dimensions that significantly increase. Although the expression of many NM proteins changes during dedifferentiation, only a very limited group of MAR-binding proteins seem to play a key role in this process. Variations in the expression of poly (ADP-ribose polymerase (PARP and special AT-rich sequence-binding protein-1 (SATB1 along with an increase in the phosphorylation of lamin B represent changes that might trigger passage towards a more aggressive phenotype. These results suggest that elucidating the MAR-binding proteins that are involved in the differentiation of prostate cancer cells could be an important tool to improve our understanding of this carcinogenesis process, and they could also be novel targets for prostate cancer therapy.

  7. A human Polycomb isoform lacking the Pc box does not participate to PRC1 complexes but forms protein assemblies and represses transcription

    DEFF Research Database (Denmark)

    Völkel, Pamela; Le Faou, Perrine; Vandamme, Julien

    2012-01-01

    Polycomb repression controls the expression of hundreds of genes involved in development and is mediated by essentially two classes of chromatin-associated protein complexes. The Polycomb repressive complex 2 (PRC2) trimethylates histone H3 at lysine 27, an epigenetic mark that serves as a docking...... site for the PRC1 protein complex. Drosophila core PRC1 is composed of four subunits: Polycomb (Pc), Posterior sex combs (Psc), Polyhomeotic (Ph) and Sex combs extra (Sce). Each of these proteins has multiple orthologs in vertebrates, thus generating an enormous scope for potential combinatorial...... diversity. In particular, mammalian genomes encode five Pc family members: CBX2, CBX4, CBX6, CBX7 and CBX8. To complicate matters further, distinct isoforms might arise from single genes. Here, we address the functional role of the two human CBX2 isoforms. Owing to different polyadenylation sites...

  8. Mutations causing Greenberg dysplasia but not Pelger anomaly uncouple enzymatic from structural functions of a nuclear membrane protein.

    Science.gov (United States)

    Clayton, Peter; Fischer, Björn; Mann, Anuska; Mansour, Sahar; Rossier, Eva; Veen, Markus; Lang, Christine; Baasanjav, Sevjidmaa; Kieslich, Moritz; Brossuleit, Katja; Gravemann, Sophia; Schnipper, Nele; Karbasyian, Mohsen; Demuth, Ilja; Zwerger, Monika; Vaya, Amparo; Utermann, Gerd; Mundlos, Stefan; Stricker, Sigmar; Sperling, Karl; Hoffmann, Katrin

    2010-01-01

    The lamin B receptor (LBR) is an inner nuclear membrane protein with a structural function interacting with chromatin and lamins, and an enzymatic function as a sterol reductase. Heterozygous LBR mutations cause nuclear hyposegmentation in neutrophils (Pelger anomaly), while homozygous mutations cause prenatal death with skeletal defects and abnormal sterol metabolism (Greenberg dysplasia). It has remained unclear whether the lethality in Greenberg dysplasia is due to cholesterol defects or altered nuclear morphology.To answer this question we characterized two LBR missense mutations and showed that they cause Greenberg dysplasia. Both mutations affect residues that are evolutionary conserved among sterol reductases. In contrast to wildtype LBR, both mutations failed to rescue C14 sterol reductase deficient yeast, indicating an enzymatic defect. We found no Pelger anomaly in the carrier parent excluding marked effects on nuclear structure. We studied Lbr in mouse embryos and demonstrate expression in skin and the developing skeletal system consistent with sites of histological changes in Greenberg dysplasia. Unexpectedly we found in disease-relevant cell types not only nuclear but also cytoplasmatic LBR localization. The cytoplasmatic LBR staining co-localized with ER-markers and is thus consistent with the sites of endogeneous sterol synthesis. We conclude that LBR missense mutations can abolish sterol reductase activity, causing lethal Greenberg dysplasia but not Pelger anomaly. The findings separate the metabolic from the structural function and indicate that the sterol reductase activity is essential for human intrauterine development.

  9. A protein interaction atlas for the nuclear receptors: properties and quality of a hub-based dimerisation network

    Directory of Open Access Journals (Sweden)

    De Graaf David

    2007-07-01

    Full Text Available Abstract Background The nuclear receptors are a large family of eukaryotic transcription factors that constitute major pharmacological targets. They exert their combinatorial control through homotypic heterodimerisation. Elucidation of this dimerisation network is vital in order to understand the complex dynamics and potential cross-talk involved. Results Phylogeny, protein-protein interactions, protein-DNA interactions and gene expression data have been integrated to provide a comprehensive and up-to-date description of the topology and properties of the nuclear receptor interaction network in humans. We discriminate between DNA-binding and non-DNA-binding dimers, and provide a comprehensive interaction map, that identifies potential cross-talk between the various pathways of nuclear receptors. Conclusion We infer that the topology of this network is hub-based, and much more connected than previously thought. The hub-based topology of the network and the wide tissue expression pattern of NRs create a highly competitive environment for the common heterodimerising partners. Furthermore, a significant number of negative feedback loops is present, with the hub protein SHP [NR0B2] playing a major role. We also compare the evolution, topology and properties of the nuclear receptor network with the hub-based dimerisation network of the bHLH transcription factors in order to identify both unique themes and ubiquitous properties in gene regulation. In terms of methodology, we conclude that such a comprehensive picture can only be assembled by semi-automated text-mining, manual curation and integration of data from various sources.

  10. Magnesium Presence Prevents Removal of Antigenic Nuclear-Associated Proteins from Bovine Pericardium for Heart Valve Engineering.

    Science.gov (United States)

    Dalgliesh, Ailsa J; Liu, Zhi Zhao; Griffiths, Leigh G

    2017-07-01

    Current heart valve prostheses are associated with significant complications, including aggressive immune response, limited valve life expectancy, and inability to grow in juvenile patients. Animal derived "tissue" valves undergo glutaraldehyde fixation to mask tissue antigenicity; however, chronic immunological responses and associated calcification still commonly occur. A heart valve formed from an unfixed bovine pericardium (BP) extracellular matrix (ECM) scaffold, in which antigenic burden has been eliminated or significantly reduced, has potential to overcome deficiencies of current bioprostheses. Decellularization and antigen removal methods frequently use sequential solutions extrapolated from analytical chemistry approaches to promote solubility and removal of tissue components from resultant ECM scaffolds. However, the extent to which such prefractionation strategies may inhibit removal of antigenic tissue components has not been explored. We hypothesize that presence of magnesium in prefractionation steps causes DNA precipitation and reduces removal of nuclear-associated antigenic proteins. Keeping all variables consistent bar the addition or absence of magnesium (2 mM magnesium chloride hexahydrate), residual BP ECM scaffold antigenicity and removed antigenicity were assessed, along with residual and removed DNA content, ECM morphology, scaffold composition, and recellularization potential. Furthermore, we used proteomic methods to determine the mechanism by which magnesium presence or absence affects scaffold residual antigenicity. This study demonstrates that absence of magnesium from antigen removal solutions enhances solubility and subsequent removal of antigenic nuclear-associated proteins from BP. We therefore conclude that the primary mechanism of action for magnesium removal during antigen removal processes is avoidance of DNA precipitation, facilitating solubilization and removal of nuclear-associated antigenic proteins. Future studies are

  11. Arthropod relationships revealed by phylogenomic analysis of nuclear protein-coding sequences.

    Science.gov (United States)

    Regier, Jerome C; Shultz, Jeffrey W; Zwick, Andreas; Hussey, April; Ball, Bernard; Wetzer, Regina; Martin, Joel W; Cunningham, Clifford W

    2010-02-25

    The remarkable antiquity, diversity and ecological significance of arthropods have inspired numerous attempts to resolve their deep phylogenetic history, but the results of two decades of intensive molecular phylogenetics have been mixed. The discovery that terrestrial insects (Hexapoda) are more closely related to aquatic Crustacea than to the terrestrial centipedes and millipedes (Myriapoda) was an early, if exceptional, success. More typically, analyses based on limited samples of taxa and genes have generated results that are inconsistent, weakly supported and highly sensitive to analytical conditions. Here we present strongly supported results from likelihood, Bayesian and parsimony analyses of over 41 kilobases of aligned DNA sequence from 62 single-copy nuclear protein-coding genes from 75 arthropod species. These species represent every major arthropod lineage, plus five species of tardigrades and onychophorans as outgroups. Our results strongly support Pancrustacea (Hexapoda plus Crustacea) but also strongly favour the traditional morphology-based Mandibulata (Myriapoda plus Pancrustacea) over the molecule-based Paradoxopoda (Myriapoda plus Chelicerata). In addition to Hexapoda, Pancrustacea includes three major extant lineages of 'crustaceans', each spanning a significant range of morphological disparity. These are Oligostraca (ostracods, mystacocarids, branchiurans and pentastomids), Vericrustacea (malacostracans, thecostracans, copepods and branchiopods) and Xenocarida (cephalocarids and remipedes). Finally, within Pancrustacea we identify Xenocarida as the long-sought sister group to the Hexapoda, a result confirming that 'crustaceans' are not monophyletic. These results provide a statistically well-supported phylogenetic framework for the largest animal phylum and represent a step towards ending the often-heated, century-long debate on arthropod relationships.

  12. Phylogenetic analysis of Myriapoda using three nuclear protein-coding genes.

    Science.gov (United States)

    Regier, Jerome C; Wilson, Heather M; Shultz, Jeffrey W

    2005-01-01

    We assessed the ability of three nuclear protein-encoding genes-elongation factor-1alpha (EF-1alpha), RNA polymerase II (Pol II), and elongation factor-2 (EF-2)-from 59 myriapod and 12 non-myriapod species to resolve phylogenetic relationships among myriapod classes and orders. In a previous study using EF-1alpha and Pol II (2134 nt combined) from 34 myriapod taxa, Regier and Shultz recovered widely accepted classes, orders, and families but failed to resolve interclass and interordinal relationships. The result was attributed to heterogenous rates of cladogenesis (specifically, the inability of the slowly evolving sequences to capture phylogenetic signal during rapid phylogenetic diversification) but the possibility of inadequate taxon sampling or limited sequence information could not be excluded. In the present study, the myriapod taxon sample was increased by 25 taxa (73%) and sequence length per taxon was effectively doubled through addition of EF-2 (4318 nt combined). Parsimony and Bayesian analyses of the expanded data set recovered a monophyletic Myriapoda, all four myriapod classes and all multiply sampled orders, often with high node support. However, except for three diplopod clades (Colobognatha, Helminothomorpha, and a subgroup of Pentazonia), few interordinal relationships and no interclass relationships were well supported. These results are similar to those of the earlier study by Regier and Shultz, which indicates that taxon sample and sequence length alone do not readily explain the weakly supported resolution in the earlier study. We review recent paleontological evidence to further develop our proposal that heterogeneity in phylogenetic signal provided by our slowly evolving sequences is due to heterogeneity in the temporal structure of myriapod diversification.

  13. Validation of the Diagnostic Value of Nuclear Matrix Protein 22 Depending on Tumoral Stage and Grade

    Directory of Open Access Journals (Sweden)

    Zoltán A. MIHÁLY

    2013-02-01

    Full Text Available Objectives: The aim of the present study was to validate the sensitivity and specificity of the NMP22® BladderChek® test in our group of patients according to the tumoral stage and grade and to identify the patient categories that might benefit from the non-invasive nature of NMP22® BladderChek® test. Methods: Voided urine samples from 266 patients with imagistic suspicion of bladder cancer were collected to perform the NMP22® BladderChek® test. The nuclear matrix protein 22 (NMP22 levels were measured by a lateral flow immunochromatographic qualitative assay, using 10 U/ml as the cut-off value. After this patients underwent transurethral resection of bladder tumors (TUR-BT follewed by histologic grading and tumor staging for a proper and optimal patient management. Sensitivity specificity and positive predictive value of the NMP22® BladderChek® test were defined for different tumoral stage and grade. Results: Two hundred thirty-eight of the 265 patients had urothelial malignancies (76 pTa, 81 pT1, 37 pT2, 32 pT3, 12 pT4, 27 pT0; 118 G1, 54 G2, 64 G3. The sensitivity was 0.629 [0.612; 0.629] for the NMP22® BladderChek® test while the specificity was equal to 1 [0.851; 1]. Positive predictive values was 1 [0.973; 1], and the negative predictive value was 0.235 [0.200; 0.235]. Conclusions: The results demonstrate that the even if the NMP22® BladderChek® is an easily applied test, giving diagnostic findings within 30 min, cannot be recommended for screening or surveillance in clinical routine use in non muscle invasive bladder cancer because of its poor sensitivity.

  14. HCC-DETECT: a combination of nuclear, cytoplasmic, and oncofetal proteins as biomarkers for hepatocellular carcinoma.

    Science.gov (United States)

    Attallah, Abdelfattah M; El-Far, Mohamed; Malak, Camelia A Abdel; Omran, Mohamed M; Shiha, Gamal E; Farid, Khaled; Barakat, Lamiaa A; Albannan, Mohamed S; Attallah, Ahmed A; Abdelrazek, Mohamed A; Elbendary, Mohamed S; Sabry, Refaat; Hamoda, Gehan A; Elshemy, Mohamed M; Ragab, Abdallah A; Foda, Basma M; Abdallah, Sanaa O

    2015-09-01

    Currently, the search for suitable hepatocellular carcinoma (HCC) biomarkers is very intensive. Besides, efficacy and cost/effectiveness of screening and surveillance of cirrhotics for the diagnosis of HCC is still debated. So, the present study is concerned with the evaluation of cytokeratin-1 (CK-1) and nuclear matrix protein-52 (NMP-52) for identifying HCC. Two-hundred and eighty individuals categorized into three groups [liver fibrosis (F1-F3), cirrhosis (F4), and HCC] constituted this study. Western blot was used for identifying CK-1 and NMP-52 in serum samples. As a result, a single immunoreactive band was shown at 67 and 52 kDa corresponding to CK-1 and NMP-52, respectively. Both CK-1 and NMP-52 bands were cut and electroeluted separately. These markers were quantified in sera using ELISA. Patients with HCC were associated with higher concentrations of CK-1 and NMP-52 than those without HCC with a significant difference (P < 0.0001). CK-1 showed an area under receiver-operating characteristic curve (AUC) of 0.83 with 75 % sensitivity and 82 % specificity while NMP-52 yielded 0.72 AUC with 62 % sensitivity and 70 % specificity for identifying HCC. HCC-DETECT comprising CK-1 and NMP-52 together with AFP was then constructed yielding 0.90 AUC for identifying HCC with 80 % sensitivity and 92 % specificity. HCC-DETECT was then tested for separating HCC from F1-F3 showing 0.94 AUC with 80 % sensitivity and 93 % specificity. In conclusion, CK-1 in conjunction with NMP-52 and AFP could have a potential role for improving the detection of HCC with a high degree of accuracy.

  15. Nuclear LSm8 affects number of cytoplasmic processing bodies via controlling cellular distribution of Like-Sm proteins

    OpenAIRE

    Novotný, Ivan; Podolská, Kateřina; Blažíková, Michaela; Valášek, Leoš Shivaya; Svoboda, Petr; Staněk, David

    2012-01-01

    Processing bodies (P-bodies) are dynamic cytoplasmic structures involved in mRNA degradation, but the mechanism that governs their formation is poorly understood. In this paper, we address a role of Like-Sm (LSm) proteins in formation of P-bodies and provide evidence that depletion of nuclear LSm8 increases the number of P-bodies, while LSm8 overexpression leads to P-body loss. We show that LSm8 knockdown causes relocalization of LSm4 and LSm6 proteins to the cytoplasm and suggest that LSm8 c...

  16. Carbon-ion beams induce production of an immune mediator protein, high mobility group box 1, at levels comparable with X-ray irradiation.

    Science.gov (United States)

    Yoshimoto, Yuya; Oike, Takahiro; Okonogi, Noriyuki; Suzuki, Yoshiyuki; Ando, Ken; Sato, Hiro; Noda, Shin-ei; Isono, Mayu; Mimura, Kousaku; Kono, Koji; Nakano, Takashi

    2015-05-01

    X-ray radiotherapy activates tumor antigen-specific T-cell responses, and increases in the serum levels of high mobility group box 1 (HMGB1) induced by X-ray irradiation play a pivotal role in activating anti-tumor immunity. Here, we examined whether carbon-ion beams, as well as X-rays, can induce HMGB1 release from human cancer cell lines. The study examined five human cancer cell lines: TE2, KYSE70, A549, NCI-H460 and WiDr. The proportion of cells surviving X- or carbon-ion beam irradiation was assessed in a clonogenic assay. The D10, the dose at which 10% of cells survive, was calculated using a linear-quadratic model. HMGB1 levels in the culture supernatants were assessed by an ELISA. The D10 dose for X-rays in TE2, KYSE70, A549, NCI-H460 and WiDr cells was 2.1, 6.7, 8.0, 4.8 and 7.1 Gy, respectively, whereas that for carbon-ion beams was 0.9, 2.5, 2.7, 1.8 and 3.5 Gy, respectively. X-rays and carbon-ion beams significantly increased HMGB1 levels in the culture supernatants of A549, NCI-H460 and WiDr cells at 72 h post-irradiation with a D10 dose. Furthermore, irradiation with X-rays or carbon-ion beams significantly increased HMGB1 levels in the culture supernatants of all five cell lines at 96 h post-irradiation. There was no significant difference in the amount of HMGB1 induced by X-rays and carbon-ion beams at any time-point (except at 96 h for NCI-H460 cells); thus we conclude that comparable levels of HMGB1 were detected after irradiation with iso-survival doses of X-rays and carbon-ion beams. © The Author 2015. Published by Oxford University Press on behalf of The Japan Radiation Research Society and Japanese Society for Radiation Oncology.

  17. Different sets of ER-resident J-proteins regulate distinct polar nuclear-membrane fusion events in Arabidopsis thaliana.

    Science.gov (United States)

    Maruyama, Daisuke; Yamamoto, Masaya; Endo, Toshiya; Nishikawa, Shuh-ichi

    2014-11-01

    Angiosperm female gametophytes contain a central cell with two polar nuclei. In many species, including Arabidopsis thaliana, the polar nuclei fuse during female gametogenesis. We previously showed that BiP, an Hsp70 in the endoplasmic reticulum (ER), was essential for membrane fusion during female gametogenesis. Hsp70 function requires partner proteins for full activity. J-domain containing proteins (J-proteins) are the major Hsp70 functional partners. A. thaliana ER contains three soluble J-proteins, AtERdj3A, AtERdj3B, and AtP58(IPK). Here, we analyzed mutants of these proteins and determined that double-mutant ovules lacking AtP58(IPK) and AtERdj3A or AtERdj3B were defective in polar nuclear fusion. Electron microscopy analysis identified that polar nuclei were in close contact, but no membrane fusion occurred in mutant ovules lacking AtP58(IPK) and AtERdj3A. The polar nuclear outer membrane appeared to be connected via the ER remaining at the inner unfused membrane in mutant ovules lacking AtP58(IPK) and AtERdj3B. These results indicate that ER-resident J-proteins, AtP58(IPK)/AtERdj3A and AtP58(IPK)/AtERdj3B, function at distinct steps of polar nuclear-membrane fusion. Similar to the bip1 bip2 double mutant female gametophytes, the aterdj3a atp58(ipk) double mutant female gametophytes defective in fusion of the outer polar nuclear membrane displayed aberrant endosperm proliferation after fertilization with wild-type pollen. However, endosperm proliferated normally after fertilization of the aterdj3b atp58(ipk) double mutant female gametophytes defective in fusion of the inner membrane. Our results indicate that the polar nuclear fusion defect itself does not cause an endosperm proliferation defect. © The Author 2014. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.

  18. Oxidation products of 3-hydroxykynurenine bind to lens proteins: relevance for nuclear cataract.

    Science.gov (United States)

    Aquilina, J A; Carver, J A; Truscott, R J

    1997-05-01

    3-Hydroxykynurenine (3OHKyn), present as a human lens UV filter, has also been implicated as a carcinogen and neurotoxin. It has been suggested that oxidation of 3OHKyn is involved in each of these effects. In the presence of oxygen, 3OHKyn has been found to react with bovine crystallins, to give brown-coloured products (Stutchbury and Truscott, 1993). In this study the roles of UV-light, pH, glutathione and oxygen were examined, with the objective of determining how these factors may affect the binding of 3OHKyn to crystallins under the conditions found within the lens itself. The presence of oxygen was found to be an important parameter for determining the extent to which 3OHKyn reacts with protein, and when it was totally excluded, little modification was observed. UV-light was not required for activation, but was found to augment the extent of modification and cross-linking, while an elevated pH, which is known to accelerate the rate of 3OHKyn oxidation, did not markedly increase the extent of reaction with the crystallins. 3OHKyn binding was accompanied by crystallin aggregation, pigmentation, and development of non-tryptophan fluorescence, all of which have been associated with cataract formation. The inclusion of glutathione, a ubiquitous antioxidant, in reaction mixtures resulted in a delayed onset of crystallin modification. This effect was apparent at concentrations of glutathione greater than 1 mM. When glutathione levels fell below 1 mM, crystallins became modified by 3OHKyn. Since lens glutathione concentrations decrease with age, and are known to be lower in the lens nucleus than the cortex, this region appears particularly vulnerable to modification by this UV filter. Thus, whilst the other human lens UV filters, kynurenine (Kyn) and 3-hydroxykynurenine glucoside (3HKG), appear to require activation by UV-light in order to react with proteins, 3OHKyn can modify crystallins in the absence of light, under conditions of low oxygen tension, and in the

  19. HIV Integration Targeting: A Pathway Involving Transportin-3 and the Nuclear Pore Protein RanBP2

    Science.gov (United States)

    Huegel, Alyssa; Roth, Shoshannah L.; Schaller, Torsten; James, Leo C.; Towers, Greg J.; Young, John A. T.; Chanda, Sumit K.; König, Renate; Malani, Nirav; Berry, Charles C.; Bushman, Frederic D.

    2011-01-01

    Genome-wide siRNA screens have identified host cell factors important for efficient HIV infection, among which are nuclear pore proteins such as RanBP2/Nup358 and the karyopherin Transportin-3/TNPO3. Analysis of the roles of these proteins in the HIV replication cycle suggested that correct trafficking through the pore may facilitate the subsequent integration step. Here we present data for coupling between these steps by demonstrating that depletion of Transportin-3 or RanBP2 altered the terminal step in early HIV replication, the selection of chromosomal sites for integration. We found that depletion of Transportin-3 and RanBP2 altered integration targeting for HIV. These knockdowns reduced HIV integration frequency in gene-dense regions and near gene-associated features, a pattern that differed from that reported for depletion of the HIV integrase binding cofactor Psip1/Ledgf/p75. MLV integration was not affected by the Transportin-3 knockdown. Using siRNA knockdowns and integration targeting analysis, we also implicated several additional nuclear proteins in proper target site selection. To map viral determinants of integration targeting, we analyzed a chimeric HIV derivative containing MLV gag, and found that the gag replacement phenocopied the Transportin-3 and RanBP2 knockdowns. Thus, our data support a model in which Gag-dependent engagement of the proper transport and nuclear pore machinery mediate trafficking of HIV complexes to sites of integration. PMID:21423673

  20. HIV integration targeting: a pathway involving Transportin-3 and the nuclear pore protein RanBP2.

    Directory of Open Access Journals (Sweden)

    Karen E Ocwieja

    2011-03-01

    Full Text Available Genome-wide siRNA screens have identified host cell factors important for efficient HIV infection, among which are nuclear pore proteins such as RanBP2/Nup358 and the karyopherin Transportin-3/TNPO3. Analysis of the roles of these proteins in the HIV replication cycle suggested that correct trafficking through the pore may facilitate the subsequent integration step. Here we present data for coupling between these steps by demonstrating that depletion of Transportin-3 or RanBP2 altered the terminal step in early HIV replication, the selection of chromosomal sites for integration. We found that depletion of Transportin-3 and RanBP2 altered integration targeting for HIV. These knockdowns reduced HIV integration frequency in gene-dense regions and near gene-associated features, a pattern that differed from that reported for depletion of the HIV integrase binding cofactor Psip1/Ledgf/p75. MLV integration was not affected by the Transportin-3 knockdown. Using siRNA knockdowns and integration targeting analysis, we also implicated several additional nuclear proteins in proper target site selection. To map viral determinants of integration targeting, we analyzed a chimeric HIV derivative containing MLV gag, and found that the gag replacement phenocopied the Transportin-3 and RanBP2 knockdowns. Thus, our data support a model in which Gag-dependent engagement of the proper transport and nuclear pore machinery mediate trafficking of HIV complexes to sites of integration.

  1. Insights into the quality of DnaA boxes and their cooperativity

    DEFF Research Database (Denmark)

    Hansen, Flemming G.; Christensen, Bjarke Bak; Nielsen, Christina Bang

    2006-01-01

    Plasmids carrying the mioC promoter region with its two DnaA boxes are as efficient in titration of DnaA protein as plasmids carrying a replicationinactivated oriC region with its five DnaA boxes. The two DnaA boxes upstream of the mioC promoter were mutated in various ways to study the cooperati......Plasmids carrying the mioC promoter region with its two DnaA boxes are as efficient in titration of DnaA protein as plasmids carrying a replicationinactivated oriC region with its five DnaA boxes. The two DnaA boxes upstream of the mioC promoter were mutated in various ways to study...... the cooperativity between the DnaA boxes, and to study in vivo the in vitrodefined 9mer DnaA box consensus sequence TTA/TTNCACA). The quality and cooperativity of the DnaA oxes were determined in two complementary ways: as titration of DnaA protein leading to derepression of the dnaA promoter, and as repression...... of the mioC promoter caused by the DnaA protein binding to the DnaA boxes. Titration of DnaA protein correlated with repression of the mioC promoter. The level of titration and repression with the normal promoter-proximal box (TTTTCCACA) depends strongly on the presence and the quality of a DnaA box...

  2. Identification of a phosphorylation-dependent nuclear localization motif in interferon regulatory factor 2 binding protein 2.

    Directory of Open Access Journals (Sweden)

    Allen C T Teng

    Full Text Available Interferon regulatory factor 2 binding protein 2 (IRF2BP2 is a muscle-enriched transcription factor required to activate vascular endothelial growth factor-A (VEGFA expression in muscle. IRF2BP2 is found in the nucleus of cardiac and skeletal muscle cells. During the process of skeletal muscle differentiation, some IRF2BP2 becomes relocated to the cytoplasm, although the functional significance of this relocation and the mechanisms that control nucleocytoplasmic localization of IRF2BP2 are not yet known.Here, by fusing IRF2BP2 to green fluorescent protein and testing a series of deletion and site-directed mutagenesis constructs, we mapped the nuclear localization signal (NLS to an evolutionarily conserved sequence (354ARKRKPSP(361 in IRF2BP2. This sequence corresponds to a classical nuclear localization motif bearing positively charged arginine and lysine residues. Substitution of arginine and lysine with negatively charged aspartic acid residues blocked nuclear localization. However, these residues were not sufficient because nuclear targeting of IRF2BP2 also required phosphorylation of serine 360 (S360. Many large-scale phosphopeptide proteomic studies had reported previously that serine 360 of IRF2BP2 is phosphorylated in numerous human cell types. Alanine substitution at this site abolished IRF2BP2 nuclear localization in C(2C(12 myoblasts and CV1 cells. In contrast, substituting serine 360 with aspartic acid forced nuclear retention and prevented cytoplasmic redistribution in differentiated C(2C(12 muscle cells. As for the effects of these mutations on VEGFA promoter activity, the S360A mutation interfered with VEGFA activation, as expected. Surprisingly, the S360D mutation also interfered with VEGFA activation, suggesting that this mutation, while enforcing nuclear entry, may disrupt an essential activation function of IRF2BP2.Nuclear localization of IRF2BP2 depends on phosphorylation near a conserved NLS. Changes in phosphorylation status

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

    Science.gov (United States)

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

    2015-11-03

    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 α-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.

  4. Molecular characterization and expression pattern of X box-binding protein-1 (XBP1) in common carp (Cyprinus carpio L.): Indications for a role of XBP1 in antibacterial and antiviral immunity.

    Science.gov (United States)

    Li, Ting; Li, Hua; Peng, Shaoqing; Zhang, Fumiao; An, Liguo; Yang, Guiwen

    2017-08-01

    X box-binding protein-1 (XBP1) is a transcription factor that is essential for the unfolded protein response (UPR) and the differentiation of plasma cells, and some findings have also uncovered its function in innate immunity. XBP1 typically has two different transcripts, un-spliced (XBP1u) and spliced forms (XBP1s), but XBP1s is an active transcription factor in the regulation of target genes. To date, there is no evidence about the identification and function of XBP1 in common carp. Moreover, no data are currently available regarding the role of fish XBP1 in innate immunity. Thus, to determine whether XBP1 is involved in innate immune response in common carp, we cloned CcXBP1s and examined the expression of XBP1s and a XBP1s stimulated gene (IL-6) after Aeromonas hydrophila (A. hydrophila) and polyinosinic-polycytidylic acid (polyI:C) challenges. The results imply that CcXBP1s, as an active transcription factor, might play regulation roles in the antibacterial and antiviral innate immune responses of common carp. This allows us to gain new insights into the immunological function of XBP1 in fish innate immunity and the evolution of this important class of genes across vertebrates. Copyright © 2017 Elsevier Ltd. All rights reserved.

  5. Phytoplasma Effector SAP54 Hijacks Plant Reproduction by Degrading MADS-box Proteins and Promotes Insect Colonization in a RAD23-Dependent Manner

    Science.gov (United States)

    MacLean, Allyson M.; Orlovskis, Zigmunds; Kowitwanich, Krissana; Zdziarska, Anna M.; Angenent, Gerco C.; Immink, Richard G. H.; Hogenhout, Saskia A.

    2014-01-01

    Pathogens that rely upon multiple hosts to complete their life cycles often modify behavior and development of these hosts to coerce them into improving pathogen fitness. However, few studies describe mechanisms underlying host coercion. In this study, we elucidate the mechanism by which an insect-transmitted pathogen of plants alters floral development to convert flowers into vegetative tissues. We find that phytoplasma produce a novel effector protein (SAP54) that interacts with members of the MADS-domain transcription factor (MTF) family, including key regulators SEPALLATA3 and APETALA1, that occupy central positions in the regulation of floral development. SAP54 mediates degradation of MTFs by interacting with proteins of the RADIATION SENSITIVE23 (RAD23) family, eukaryotic proteins that shuttle substrates to the proteasome. Arabidopsis rad23 mutants do not show conversion of flowers into leaf-like tissues in the presence of SAP54 and during phytoplasma infection, emphasizing the importance of RAD23 to the activity of SAP54. Remarkably, plants with SAP54-induced leaf-like flowers are more attractive for colonization by phytoplasma leafhopper vectors and this colonization preference is dependent on RAD23. An effector that targets and suppresses flowering while simultaneously promoting insect herbivore colonization is unprecedented. Moreover, RAD23 proteins have, to our knowledge, no known roles in flower development, nor plant defence mechanisms against insects. Thus SAP54 generates a short circuit between two key pathways of the host to alter development, resulting in sterile plants, and promotes attractiveness of these plants to leafhopper vectors helping the obligate phytoplasmas reproduce and propagate (zombie plants). PMID:24714165

  6. Phytoplasma effector SAP54 hijacks plant reproduction by degrading MADS-box proteins and promotes insect colonization in a RAD23-dependent manner.

    Directory of Open Access Journals (Sweden)

    Allyson M MacLean

    2014-04-01

    Full Text Available Pathogens that rely upon multiple hosts to complete their life cycles often modify behavior and development of these hosts to coerce them into improving pathogen fitness. However, few studies describe mechanisms underlying host coercion. In this study, we elucidate the mechanism by which an insect-transmitted pathogen of plants alters floral development to convert flowers into vegetative tissues. We find that phytoplasma produce a novel effector protein (SAP54 that interacts with members of the MADS-domain transcription factor (MTF family, including key regulators SEPALLATA3 and APETALA1, that occupy central positions in the regulation of floral development. SAP54 mediates degradation of MTFs by interacting with proteins of the RADIATION SENSITIVE23 (RAD23 family, eukaryotic proteins that shuttle substrates to the proteasome. Arabidopsis rad23 mutants do not show conversion of flowers into leaf-like tissues in the presence of SAP54 and during phytoplasma infection, emphasizing the importance of RAD23 to the activity of SAP54. Remarkably, plants with SAP54-induced leaf-like flowers are more attractive for colonization by phytoplasma leafhopper vectors and this colonization preference is dependent on RAD23. An effector that targets and suppresses flowering while simultaneously promoting insect herbivore colonization is unprecedented. Moreover, RAD23 proteins have, to our knowledge, no known roles in flower development, nor plant defence mechanisms against insects. Thus SAP54 generates a short circuit between two key pathways of the host to alter development, resulting in sterile plants, and promotes attractiveness of these plants to leafhopper vectors helping the obligate phytoplasmas reproduce and propagate (zombie plants.

  7. Phytoplasma Effector SAP54 Hijacks Plant Reproduction by Degrading MADS-box Proteins and Promotes Insect Colonization in a RAD23-Dependent Manner

    OpenAIRE

    Allyson M MacLean; Zigmunds Orlovskis; Krissana Kowitwanich; Zdziarska, Anna M.; Angenent, Gerco C.; Immink, Richard G. H.; Hogenhout, Saskia A.

    2014-01-01

    Pathogens that rely upon multiple hosts to complete their life cycles often modify behavior and development of these hosts to coerce them into improving pathogen fitness. However, few studies describe mechanisms underlying host coercion. In this study, we elucidate the mechanism by which an insect-transmitted pathogen of plants alters floral development to convert flowers into vegetative tissues. We find that phytoplasma produce a novel effector protein (SAP54) that interacts with members of ...

  8. The DEAD-box helicase eIF4A

    Science.gov (United States)

    Andreou, Alexandra Z.; Klostermeier, Dagmar

    2013-01-01

    DEAD-box helicases catalyze the ATP-dependent unwinding of RNA duplexes. They share a helicase core formed by two RecA-like domains that carries a set of conserved motifs contributing to ATP binding and hydrolysis, RNA binding and duplex unwinding. The translation initiation factor eIF4A is the founding member of the DEAD-box protein family, and one of the few examples of DEAD-box proteins that consist of a helicase core only. It is an RNA-stimulated ATPase and a non-processive helicase that unwinds short RNA duplexes. In the catalytic cycle, a series of conformational changes couples the nucleotide cycle to RNA unwinding. eIF4A has been considered a paradigm for DEAD-box proteins, and studies of its function have revealed the governing principles underlying the DEAD-box helicase mechanism. However, as an isolated helicase core, eIF4A is rather the exception, not the rule. Most helicase modules in other DEAD-box proteins are modified, some by insertions into the RecA-like domains, and the majority by N- and C-terminal appendages. While the basic catalytic function resides within the helicase core, its modulation by insertions, additional domains or a network of interaction partners generates the diversity of DEAD-box protein functions in the cell. This review summarizes the current knowledge on eIF4A and its regulation, and discusses to what extent eIF4A serves as a model DEAD-box protein. PMID:22995829

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

    Directory of Open Access Journals (Sweden)

    St-Laurent Georges

    2006-11-01

    Full Text Available Abstract 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.

  10. C. elegans PAT-9 is a nuclear zinc finger protein critical for the assembly of muscle attac