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Sample records for box nuclear protein

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

    Science.gov (United States)

    Jöhnk, Bastian; Bayram, Özgür; Heinekamp, Thorsten; Mattern, Derek J.; Brakhage, Axel A.; Jacobsen, Ilse D.; Valerius, Oliver; Braus, Gerhard H.

    2016-01-01

    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. PMID:27649508

  2. F-box proteins in flowering plants

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    In eukaryotes, the ubiquitin-mediated protein degradation pathway has been shown to control several key biological processes such as cell division, development, metabolism and immune response. F-box proteins, as a part of SCF (Skp1-Cullin (or Cdc53)-F-box) complex, functioned by interacting with substrate proteins, leading to their subsequent degradation by the 26S proteasome. To date, several F-box proteins identified in Arabidopsis and Antirrhinum have been shown to play important roles in auxin signal transduction, floral organ formation, flowering and leaf senescence. Arabidopsis genome sequence analysis revealed that it encodes over 1000 predicted F-box proteins accounting for about 5% of total predicted proteins. These results indicate that the ubiquitin-mediated protein degradation involving the F-box proteins is an important mechanism controlling plant gene expression. Here, we review the known F-box proteins and their functionsin flowering plants.

  3. Nuclear respiratory factor 1 mediates the transcription initiation of insulin-degrading enzyme in a TATA box-binding protein-independent manner.

    Directory of Open Access Journals (Sweden)

    Lang Zhang

    Full Text Available CpG island promoters often lack canonical core promoter elements such as the TATA box, and have dispersed transcription initiation sites. Despite the prevalence of CpG islands associated with mammalian genes, the mechanism of transcription initiation from CpG island promoters remains to be clarified. Here we investigate the mechanism of transcription initiation of the CpG island-associated gene, insulin-degrading enzyme (IDE. IDE is ubiquitously expressed, and has dispersed transcription initiation sites. The IDE core promoter locates within a 32-bp region, which contains three CGGCG repeats and a nuclear respiratory factor 1 (NRF-1 binding motif. Sequential mutation analysis indicates that the NRF-1 binding motif is critical for IDE transcription initiation. The NRF-1 binding motif is functional, because NRF-1 binds to this motif in vivo and this motif is required for the regulation of IDE promoter activity by NRF-1. Furthermore, the NRF-1 binding site in the IDE promoter is conserved among different species, and dominant negative NRF-1 represses endogenous IDE expression. Finally, TATA-box binding protein (TBP is not associated with the IDE promoter, and inactivation of TBP does not abolish IDE transcription, suggesting that TBP is not essential for IDE transcription initiation. Our studies indicate that NRF-1 mediates IDE transcription initiation in a TBP-independent manner, and provide insights into the potential mechanism of transcription initiation for other CpG island-associated genes.

  4. MicroRNA-96 promotes the proliferation of colorectal cancer cells and targets tumor protein p53 inducible nuclear protein 1, forkhead box protein O1 (FOXO1) and FOXO3a.

    Science.gov (United States)

    Gao, Feng; Wang, Wenhui

    2015-02-01

    MicroRNAs (miRNAs) are a conserved class of small, endogenous, non protein-coding RNA molecules that are capable of regulating gene expression at post-transcriptional levels and are involved in diverse cellular processes, including cancer pathogenesis. It has previously been reported that miRNA-96 (miR-96) is overexpressed in human colorectal cancer (CRC). However, the underlying mechanism of miR-96 regulation in CRC remains to be elucidated. In the present study, miR-96 was confirmed to be upregulated in CRC tissues by reverse transcription quantitative polymerase chain reaction. MTT assay, colony formation assay and cell cycle analysis revealed that miR-96 overexpression led to increased tumor cell viability, colony formation ability and cell cycle progression. By contrast, inhibition of miR-96 resulted in the suppression of cell proliferation. It was also demonstrated that miR-96 reduced the messenger RNA and protein expression levels of tumor protein p53 inducible nuclear protein 1 (TP53INP1), forkhead box protein O1 (FOXO1) and FOXO3a, which are closely associated with cell proliferation. A luciferase reporter assay indicated that miR-96 inhibited luciferase intensity controlled by the 3'UTRs of TP53INP1, FOXO1 and FOXO3a. In conclusion, the results of the present study demonstrated that miR-96 contributed to CRC cell growth and that TP53INP1, FOXO1 and FOXO3a were direct targets of miR-96, suggesting that miR-96 may have the potential to be used in the development of miRNA‑based therapies for CRC patients.

  5. Roles of F-box Proteins in Plant Hormone Responses

    Institute of Scientific and Technical Information of China (English)

    Haichuan YU; Jiao WU; Nanfei XU; Ming PENG

    2007-01-01

    The F-box protein is an important component of the E3 ubiquitin ligase Skpl-Cullin-F-box protein complex. It binds specific substrates for ubiquitin-mediated proteolysis. The F-box proteins contain a signature F-box motif at their amino-terminus and some protein-protein interaction motifs at their carboxyterminus, such as Trp-Asp repeats or leucine rich repeats. Many F-box proteins have been identified to be involved in plant hormone response as receptors or important medial components. These breakthrough findings shed light on our current understanding of the structure and function of the various F-box proteins,their related plant hormone signaling pathways, and their roles in regulating plant development.

  6. 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

  7. 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.

  8. High Mobility Group Box Protein-1 Correlates with Renal Function in Chronic Kidney Disease (CKD)

    OpenAIRE

    Bruchfeld, Annette; Qureshi, Abdul Rashid; Lindholm, Bengt; Barany, Peter; Yang, Lihong; Stenvinkel, Peter; Tracey, Kevin J.

    2007-01-01

    Chronic kidney disease (CKD) is associated with inflammation and malnutrition and carries a markedly increased risk of cardiovascular disease (CVD). High Mobility Group Box Protein-1 (HMGB-1) is a 30-kDa nuclear and cytosolic protein known as a transcription and growth factor, recently identified as a proinflammatory mediator of tissue injury. Recent data implicates HMGB-1 in endotoxin lethality, rheumatoid arthritis, and atherosclerosis. The aim of this post-hoc, cross-sectional study was to...

  9. Cajal body proteins differentially affect the processing of box C/D scaRNPs.

    Science.gov (United States)

    Enwerem, Isioma I; Wu, Guowei; Yu, Yi Tao; Hebert, Michael D

    2015-01-01

    Small nuclear ribonucleoproteins (snRNPs), which are required for pre-mRNA splicing, contain extensively modified snRNA. Small Cajal body-specific ribonucleoproteins (scaRNPs) mediate these modifications. It is unknown how the box C/D class of scaRNPs localizes to Cajal Bodies (CBs). The processing of box C/D scaRNA is also unclear. Here, we explore the processing of box C/D scaRNA 2 and 9 by coilin. We also broaden our investigation to include WRAP53 and SMN, which accumulate in CBs, play a role in RNP biogenesis and associate with coilin. These studies demonstrate that the processing of an ectopically expressed scaRNA2 is altered upon the reduction of coilin, WRAP53 or SMN, but the extent and direction of this change varies depending on the protein reduced. We also show that box C/D scaRNP activity is reduced in a cell line derived from coilin knockout mice. Collectively, the findings presented here further implicate coilin as being a direct participant in the formation of box C/D scaRNPs, and demonstrate that WRAP53 and SMN may also play a role, but the activity of these proteins is divergent to coilin.

  10. Cajal body proteins differentially affect the processing of box C/D scaRNPs.

    Directory of Open Access Journals (Sweden)

    Isioma I Enwerem

    Full Text Available Small nuclear ribonucleoproteins (snRNPs, which are required for pre-mRNA splicing, contain extensively modified snRNA. Small Cajal body-specific ribonucleoproteins (scaRNPs mediate these modifications. It is unknown how the box C/D class of scaRNPs localizes to Cajal Bodies (CBs. The processing of box C/D scaRNA is also unclear. Here, we explore the processing of box C/D scaRNA 2 and 9 by coilin. We also broaden our investigation to include WRAP53 and SMN, which accumulate in CBs, play a role in RNP biogenesis and associate with coilin. These studies demonstrate that the processing of an ectopically expressed scaRNA2 is altered upon the reduction of coilin, WRAP53 or SMN, but the extent and direction of this change varies depending on the protein reduced. We also show that box C/D scaRNP activity is reduced in a cell line derived from coilin knockout mice. Collectively, the findings presented here further implicate coilin as being a direct participant in the formation of box C/D scaRNPs, and demonstrate that WRAP53 and SMN may also play a role, but the activity of these proteins is divergent to coilin.

  11. Ubiquitination-mediated degradation of cell cycle-related proteins by F-box proteins.

    Science.gov (United States)

    Zheng, Nana; Wang, Zhiwei; Wei, Wenyi

    2016-04-01

    F-box proteins, subunits of SKP1-cullin 1-F-box protein (SCF) type of E3 ubiquitin ligase complexes, have been validated to play a crucial role in governing various cellular processes such as cell cycle, cell proliferation, apoptosis, migration, invasion and metastasis. Recently, a wealth of evidence has emerged that F-box proteins is critically involved in tumorigenesis in part through governing the ubiquitination and subsequent degradation of cell cycle proteins, and dysregulation of this process leads to aberrant cell cycle progression and ultimately, tumorigenesis. Therefore, in this review, we describe the critical role of F-box proteins in the timely regulation of cell cycle. Moreover, we discuss how F-box proteins involve in tumorigenesis via targeting cell cycle-related proteins using biochemistry studies, engineered mouse models, and pathological gene alternations. We conclude that inhibitors of F-box proteins could have promising therapeutic potentials in part through controlling of aberrant cell cycle progression for cancer therapies.

  12. Calmodulin-dependent nuclear import of HMG-box family nuclear factors: importance of the role of SRY in sex reversal

    Science.gov (United States)

    Kaur, Gurpreet; Delluc-Clavieres, Aurelie; Poon, Ivan K. H.; Forwood, Jade K.; Glover, Dominic J.; Jans, David A.

    2010-01-01

    The HMG (high-mobility group)-box-containing chromatin-remodelling factor SRY (sex-determining region on the Y chromosome) plays a key role in sex determination. Its role in the nucleus is critically dependent on two NLSs (nuclear localization signals) that flank its HMG domain: the C-terminally located ‘β-NLS’ that mediates nuclear transport through Impβ1 (importin β1) and the N-terminally located ‘CaM-NLS’ which is known to recognize the calcium-binding protein CaM (calmodulin). In the present study, we examined a number of missense mutations in the SRY CaM-NLS from human XY sex-reversed females for the first time, showing that they result in significantly reduced nuclear localization of GFP (green fluorescent protein)–SRY fusion proteins in transfected cells compared with wild-type. The CaM antagonist CDZ (calmidazolium chloride) was found to significantly reduce wild-type SRY nuclear accumulation, indicating dependence of SRY nuclear import on CaM. Intriguingly, the CaM-NLS mutants were all resistant to CDZ's effects, implying a loss of interaction with CaM, which was confirmed by direct binding experiments. CaM-binding/resultant nuclear accumulation was the only property of SRY found to be impaired by two of the CaM-NLS mutations, implying that inhibition of CaM-dependent nuclear import is the basis of sex reversal in these cases. Importantly, the CaM-NLS is conserved in other HMG-box-domain-containing proteins such as SOX-2, -9, -10 and HMGN1, all of which were found for the first time to rely on CaM for optimal nuclear localization. CaM-dependent nuclear translocation is thus a common mechanism for this family of important transcription factors. PMID:20528776

  13. High Mobility Group Box Protein-1 in Wound Repair

    Directory of Open Access Journals (Sweden)

    Mauro Patrone

    2012-09-01

    Full Text Available High-mobility group box 1 protein (HMGB1, a member of highly conserved non-histone DNA binding protein family, has been studied as transcription factor and growth factor. Secreted extracellularly by activated monocytes and macrophages or passively released by necrotic or damaged cells, extracellular HMGB1 is a potent mediator of inflammation. Extracellular HMGB1 has apparently contrasting biological actions: it sustains inflammation (with the possible establishment of autoimmunity or of self-maintaining tissue damage, but it also activates and recruits stem cells, boosting tissue repair. Here, we focus on the role of HMGB1 in physiological and pathological responses, the mechanisms by which it contributes to tissue repair and therapeutic strategies base on targeting HMGB1.

  14. Protein loss during nuclear isolation

    OpenAIRE

    1983-01-01

    Cryomicrodissection makes possible the measurement of the entire in vivo protein content of the amphibian oocyte nucleus and provides a heretofore missing baseline for estimating protein loss during nuclear isolation by other methods. When oocyte nuclei are isolated into an aqueous medium, they lose 95% of their protein with a half-time of 250 s. This result implies an even more rapid loss of protein from aqueously isolated nuclei of ordinary-size cells.

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

    Science.gov (United States)

    Rentetzi, Maria

    2017-03-15

    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.

  16. Binding of Y-box proteins to RNA: involvement of different protein domains.

    Science.gov (United States)

    Ladomery, M; Sommerville, J

    1994-01-01

    Eukaryotic Y-box proteins are reported to interact with a wide variety of nucleic acid structures to act as transcription factors and mRNA masking proteins. The modular structure of Y-box proteins includes a highly conserved N-terminal cold-shock domain (CSD, equivalent to the bacterial cold-shock proteins) plus four basic C-terminal domains containing arginine clusters and aromatic residues. In addition, the basic domains are separated by acidic regions which contain several potential sites for serine/threonine phosphorylation. The interaction of Y-box proteins, isolated from Xenopus oocytes (FRGY2 type), with RNA molecules has been studied by UV crosslinking and protein fragmentation. We have identified two distinct binding activities. The CSD interacts preferentially with the polypurines poly(A,G) and poly(G) but not poly(A), this activity being sensitive to 5 mM MgCl2 but not to 5 mM spermidine. In the presence of 1 mM MgCl2 or 1 mM spermidine, the basic domains interact preferentially with poly(C,U), this activity being sensitive to 0.5 M NaCl. Binding of the basic domains is also sensitive to low concentrations of heparin. The basic domains can be crosslinked individually to labelled RNA. These results are discussed with reference to the various specificities noted in the binding of Y-box proteins to RNA and DNA. Images PMID:7530842

  17. 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.

  18. 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.

  19. F-box protein FBXL2 inhibits gastric cancer proliferation by ubiquitin-mediated degradation of forkhead box M1.

    Science.gov (United States)

    Li, Liang-qing; Pan, Dun; Chen, Hui; Zhang, Lin; Xie, Wen-jun

    2016-02-01

    F-box/LRR-repeat protein 2 (FBXL2), a component of Skp-Cullin-F box (SCF) ubiquitin E3 ligase, has been shown to inhibit tumorigenesis by targeting and ubiquitinating several oncoproteins. However, its role in gastric cancer remains poorly understood. Here, by tandem mass spectrometry, we show that FBXL2 interacts with forkhead box M1 (FoxM1) transcription factor. As a result, FBXL2 promotes ubiquitination and degradation of FoxM1 in gastric cancer cells. Furthermore, overexpression of FBXL2 inhibits, while its deficiency promotes cell proliferation and invasion. Expression levels of cell-cycle regulators (Cdc25B and p27), which are down-stream target effectors of FoxM1, are also regulated by FBXL2. Therefore, our results uncover a previous unknown network involving FBXL2 and FoxM1 in the regulation of gastric cancer growth.

  20. 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

    INTRODUCTION: Sepsis is a serious condition with a significant morbidity and mortality. New insight into the immunopathogenesis of sepsis could promote the development of new strategies for diagnosis and therapy. High mobility group box-1 protein (HMGB1) has been known for many years as a nuclear......-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

  1. MicroRNA regulation of F-box proteins and its role in cancer.

    Science.gov (United States)

    Wu, Zhao-Hui; Pfeffer, Lawrence M

    2016-02-01

    MicroRNAs (miRNAs) are small endogenous non-coding RNAs, which play critical roles in cancer development by suppressing gene expression at the post-transcriptional level. In general, oncogenic miRNAs are upregulated in cancer, while miRNAs that act as tumor suppressors are downregulated, leading to decreased expression of tumor suppressors and upregulated oncogene expression, respectively. F-box proteins function as the substrate-recognition components of the SKP1-CUL1-F-box (SCF)-ubiquitin ligase complex for the degradation of their protein targets by the ubiquitin-proteasome system. Therefore F-box proteins and miRNAs both negatively regulate target gene expression post-transcriptionally. Since each miRNA is capable of fine-tuning the expression of multiple target genes, multiple F-box proteins may be suppressed by the same miRNA. Meanwhile, one F-box proteins could be regulated by several miRNAs in different cancer types. In this review, we will focus on miRNA-mediated downregulation of various F-box proteins, the resulting stabilization of F-box protein substrates and the impact of these processes on human malignancies. We provide insight into how the miRNA: F-box protein axis may regulate cancer progression and metastasis. We also consider the broader role of F-box proteins in the regulation of pathways that are independent of the ubiquitin ligase complex and how that impacts on oncogenesis. The area of miRNAs and the F-box proteins that they regulate in cancer is an emerging field and will inform new strategies in cancer treatment.

  2. 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.

  3. Analog sensitive chemical inhibition of the DEAD-box protein DDX3.

    Science.gov (United States)

    Floor, Stephen N; Barkovich, Krister J; Condon, Kendall J; Shokat, Kevan M; Doudna, Jennifer A

    2016-03-01

    Proper maintenance of RNA structure and dynamics is essential to maintain cellular health. Multiple families of RNA chaperones exist in cells to modulate RNA structure, RNA-protein complexes, and RNA granules. The largest of these families is the DEAD-box proteins, named after their catalytic Asp-Glu-Ala-Asp motif. The human DEAD-box protein DDX3 is implicated in diverse biological processes including translation initiation and is mutated in numerous cancers. Like many DEAD-box proteins, DDX3 is essential to cellular health and exhibits dosage sensitivity, such that both decreases and increases in protein levels can be lethal. Therefore, chemical inhibition would be an ideal tool to probe the function of DDX3. However, most DEAD-box protein active sites are extremely similar, complicating the design of specific inhibitors. Here, we show that a chemical genetic approach best characterized in protein kinases, known as analog-sensitive chemical inhibition, is viable for DDX3 and possibly other DEAD-box proteins. We present an expanded active-site mutant that is tolerated in vitro and in vivo, and is sensitive to chemical inhibition by a novel bulky inhibitor. Our results highlight a course towards analog sensitive chemical inhibition of DDX3 and potentially the entire DEAD-box protein family.

  4. X-box-binding protein 1-modified neural stem cells for treatment of Parkinson's disease.

    Science.gov (United States)

    Si, Lihui; Xu, Tianmin; Wang, Fengzhang; Liu, Qun; Cui, Manhua

    2012-04-01

    X-box-binding protein 1-transfected neural stem cells were transplanted into the right lateral ventricles of rats with rotenone-induced Parkinson's disease. The survival capacities and differentiation rates of cells expressing the dopaminergic marker tyrosine hydroxylase were higher in X-box-binding protein 1-transfected neural stem cells compared to non-transfected cells. Moreover, dopamine and 3,4-dihydroxyphenylacetic acid levels in the substantia nigra were significantly increased, α-synuclein expression was decreased, and neurological behaviors were significantly ameliorated in rats following transplantation of X-box-binding protein 1-transfected neural stem cells. These results indicate that transplantation of X-box-binding protein 1-transfected neural stem cells can promote stem cell survival and differentiation into dopaminergic neurons, increase dopamine and 3,4-dihydroxyphenylacetic acid levels, reduce α-synuclein aggregation in the substantia nigra, and improve the symptoms of Parkinson's disease in rats.

  5. X-box-binding protein 1-modified neural stem cells for treatment of Parkinson's disease

    Institute of Scientific and Technical Information of China (English)

    Lihui Si; Tianmin Xu; Fengzhang Wang; Qun Liu; Manhua Cui

    2012-01-01

    X-box-binding protein 1-transfected neural stem cells were transplanted into the right lateral ventricles of rats with rotenone-induced Parkinson's disease. The survival capacities and differentiation rates of cells expressing the dopaminergic marker tyrosine hydroxylase were higher in X-box-binding protein 1-transfected neural stem cells compared to non-transfected cells. Moreover, dopamine and 3,4-dihydroxyphenylacetic acid levels in the substantia nigra were significantly increased, α-synuclein expression was decreased, and neurological behaviors were significantly ameliorated in rats following transplantation of X-box-binding protein 1-transfected neural stem cells. These results indicate that transplantation of X-box-binding protein 1-transfected neural stem cells can promote stem cell survival and differentiation into dopaminergic neurons, increase dopamine and 3,4-dihydroxyphenylacetic acid levels, reduce α-synuclein aggregation in the substantia nigra, and improve the symptoms of Parkinson's disease in rats.

  6. LOV Domain-Containing F-Box Proteins:Light-Dependent Protein Degradation Modules in Arabidopsis

    Institute of Scientific and Technical Information of China (English)

    Shogo Ito; Young Hun Song; Takato Imaizumi

    2012-01-01

    Plants constantly survey the surrounding environment using several sets of photoreceptors.They can sense changes in the quantity (=intensity) and quality (=wavelength) of light and use this information to adjust their physiological responses,growth,and developmental patterns.In addition to the classical photoreceptors,such as phytochromes,cryptochromes,and phototropins,ZEITLUPE (ZTL),FLAVIN-BINDING,KELCH REPEAT,F-BOX 1 (FKF1),and LOV KELCH PROTEIN 2 (LKP2) proteins have been recently identified as blue-light photoreceptors that are important for regulation of the circadian clock and photoperiodic flowering.The ZTL/FKF1/LKP2 protein family possesses a unique combination of domains:a blue-light-absorbing LOV (Light,Oxygen,or Voltage) domain along with domains involved in protein degradation.Here,we summarize recent advances in our understanding of the function of the Arabidopsis ZTL/FKF1/LKP2 proteins.We summarize the distinct photochemical properties of their LOV domains and discuss the molecular mechanisms by which the ZTL/FKF1/LKP2 proteins regulate the circadian clock and photoperiodic flowering by controlling blue-light-dependent protein degradation.

  7. A novel F-box protein CaF-box is involved in responses to plant hormones and abiotic stress in pepper (Capsicum annuum L.).

    Science.gov (United States)

    Chen, Rugang; Guo, Weili; Yin, Yanxu; Gong, Zhen-Hui

    2014-02-10

    The F-box protein family is characterized by an F-box motif that has been shown to play an important role in regulating various developmental processes and stress responses. In this study, a novel F-box-containing gene was isolated from leaves of pepper cultivar P70 (Capsicum annuum L.) and designated CaF-box. The full-length cDNA is 2088 bp and contains an open reading frame of 1914 bp encoding a putative polypeptide of 638 amino acids with a mass of 67.8 kDa. CaF-box was expressed predominantly in stems and seeds, and the transcript was markedly upregulated in response to cold stress, abscisic acid (ABA) and salicylic acid (SA) treatment, and downregulated under osmotic and heavy metal stress. CaF-box expression was dramatically affected by salt stress, and was rapidly increased for the first hour, then sharply decreased thereafter. In order to further assess the role of CaF-box in the defense response to abiotic stress, a loss-of-function experiment in pepper plants was performed using a virus-induced gene silencing (VIGS) technique. Measurement of thiobarbituric acid reactive substances (TBARS) and electrolyte leakage revealed stronger lipid peroxidation and cell death in the CaF-box-silenced plants than in control plants, suggesting CaF-box plays an important role in regulating the defense response to abiotic stress resistance in pepper plants.

  8. Robotic and nuclear safety for an automated/teleoperated glove box system

    Energy Technology Data Exchange (ETDEWEB)

    Domning, E.E. (Lawrence Livermore National Lab., CA (United States)); McMahon, T.T.; Sievers, R.H. (Science Applications International Corp., Pleasanton, CA (United States))

    1991-09-01

    Lawrence Livermore National Laboratory (LLNL) is developing a fully automated system to handle the processing of special nuclear materials (SNM). This work is performed in response to the new goals at the Department of Energy (DOE) for hazardous waste minimization and radiation dose reduction. This fully automated system, called the automated test bed (ATB), consists of an IBM gantry robot and automated processing equipment sealed within a glove box. While the ATB is a cold system, we are designing it as a prototype of the future hot system. We recognized that identification and application of safety requirements early in the design phase will lead to timely installation and approval of the hot system. This paper identifies these safety issues as well as the general safety requirements necessary for the safe operation of the ATB. 4 refs., 2 figs.

  9. Cloning and Sequence Analysis of Y-box Binding Protein Gene in Min Pig

    Institute of Scientific and Technical Information of China (English)

    Zhang Dong-jie; Liu Di; Wang Liang; He Xin-miao; Wang Wen-tao

    2014-01-01

    In order to study the gene sequence of Min pig Y-box binding protein (YB-1) gene, the complete coding sequence of Min pig YB-1 gene was cloned by RT-PCR, the sequence features were analyzed by some software and online website. The results showed that the complete CDS of Min pig Y-box was found to be 975 bp long, encoding 324 amino acids. It contained a conserved cold shock domain and several phosphorylation sites, but had no transmembrane domains, and was consistent with a protein found in the cytoplasm. Min pig YB-1 nucleotides shared high similarity (61.37%-97.66%) with other mammals.

  10. 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.

    Science.gov (United States)

    Rodríguez-Lima, Oscar; García-Gutierrez, Ponciano; Jiménez, Lucía; Zarain-Herzberg, Ángel; Lazzarini, Roberto; Landa, Abraham

    2015-01-01

    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.

  11. Classificati,Expression Patter,and E3 Ligase Activity Assay of Rice U-Box-Containing Proteins

    Institute of Scientific and Technical Information of China (English)

    Li-Rong Zeng; Chan Ho Park; R.C.Venu; Julian Gough; Guo-Liang Wang

    2008-01-01

    Ubiquitin ligases play a central role in determining the specificity of the ubiquitination system by selecting a myriad of appropriate candidate proteins for modification.The U-box is a recently identified,ubiquitin ligase activityrelated protein domain that shows greater presence in plants than in other organisms.In this study,we identified 77 putative U-box proteins from the rice genome using a battery of whole genome analysis algorithms.Most of the U-box protein genes are expressed,as supported by the identification of their corresponding expressed sequence tags (ESTs),full-length cDNAs,or massively parallel signature sequencing(MPSS)tags.Using the same algorithms,we identified 61 U-box proteins from the Arabidopsis genome.The rice and Arabidopsis U-box proteins were classified into nine major classes based on their domain compositions.Comparison between rice and Arabidopsis U-box proteins indicates that the majority of rice and Arabidopsis U-box proteins have the same domain organizations.The inferred phylogeny established the homology between rice and Arabidopsis U-box/ARM proteins.Cell death assay using the rice protoplast system suggests that one rice U-box gene,OsPU851,might act as a negative regulator of cell death signaling.In addition,the selected U-box proteins were found to be functional E3 ubiquitin ligases.The identification and analysis of rice U-box proteins hereby at the genomic level will help functionally characterize this class of E3 ubiquitin ligase in the future.

  12. Spliced X-box binding protein 1 couples the unfolded protein response to hexosamine biosynthetic pathway.

    Science.gov (United States)

    Wang, Zhao V; Deng, Yingfeng; Gao, Ningguo; Pedrozo, Zully; Li, Dan L; Morales, Cyndi R; Criollo, Alfredo; Luo, Xiang; Tan, Wei; Jiang, Nan; Lehrman, Mark A; Rothermel, Beverly A; Lee, Ann-Hwee; Lavandero, Sergio; Mammen, Pradeep P A; Ferdous, Anwarul; Gillette, Thomas G; Scherer, Philipp E; Hill, Joseph A

    2014-03-13

    The hexosamine biosynthetic pathway (HBP) generates uridine diphosphate N-acetylglucosamine (UDP-GlcNAc) for glycan synthesis and O-linked GlcNAc (O-GlcNAc) protein modifications. Despite the established role of the HBP in metabolism and multiple diseases, regulation of the HBP remains largely undefined. Here, we show that spliced X-box binding protein 1 (Xbp1s), the most conserved signal transducer of the unfolded protein response (UPR), is a direct transcriptional activator of the HBP. We demonstrate that the UPR triggers HBP activation via Xbp1s-dependent transcription of genes coding for key, rate-limiting enzymes. We further establish that this previously unrecognized UPR-HBP axis is triggered in a variety of stress conditions. Finally, we demonstrate a physiologic role for the UPR-HBP axis by showing that acute stimulation of Xbp1s in heart by ischemia/reperfusion confers robust cardioprotection in part through induction of the HBP. Collectively, these studies reveal that Xbp1s couples the UPR to the HBP to protect cells under stress.

  13. 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.

  14. Protein dynamics from nuclear magnetic relaxation.

    Science.gov (United States)

    Charlier, Cyril; Cousin, Samuel F; Ferrage, Fabien

    2016-05-01

    Nuclear magnetic resonance is a ubiquitous spectroscopic tool to explore molecules with atomic resolution. Nuclear magnetic relaxation is intimately connected to molecular motions. Many methods and models have been developed to measure and interpret the characteristic rates of nuclear magnetic relaxation in proteins. These approaches shed light on a rich and diverse range of motions covering timescales from picoseconds to seconds. Here, we introduce some of the basic concepts upon which these approaches are built and provide a series of illustrations.

  15. 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

    bead is reduced compared to that of unbent DNA. We detected individual binding and dissocation events and derived kinetic parameters for the process. Dissociation was induced by increasing the salt concentration or by directly pulling on the tethered bead using optical tweezers. In addition to the well......The TATA-box binding protein (TBP) is required by all three eucaryotic RNA polymerases for the initiation of transcription from most promoters. TBP recognizes, binds to, and bends promoter sequences called "TATA-boxes" in the DNA. We present results from the study of individual Saccharomyces...... 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...

  16. 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.

  17. A calmodulin-binding/CGCG box DNA-binding protein family involved in multiple signaling pathways in plants

    Science.gov (United States)

    Yang, Tianbao; Poovaiah, B. W.

    2002-01-01

    We reported earlier that the tobacco early ethylene-responsive gene NtER1 encodes a calmodulin-binding protein (Yang, T., and Poovaiah, B. W. (2000) J. Biol. Chem. 275, 38467-38473). Here we demonstrate that there is one NtER1 homolog as well as five related genes in Arabidopsis. These six genes are rapidly and differentially induced by environmental signals such as temperature extremes, UVB, salt, and wounding; hormones such as ethylene and abscisic acid; and signal molecules such as methyl jasmonate, H(2)O(2), and salicylic acid. Hence, they were designated as AtSR1-6 (Arabidopsis thaliana signal-responsive genes). Ca(2+)/calmodulin binds to all AtSRs, and their calmodulin-binding regions are located on a conserved basic amphiphilic alpha-helical motif in the C terminus. AtSR1 targets the nucleus and specifically recognizes a novel 6-bp CGCG box (A/C/G)CGCG(G/T/C). The multiple CGCG cis-elements are found in promoters of genes such as those involved in ethylene signaling, abscisic acid signaling, and light signal perception. The DNA-binding domain in AtSR1 is located on the N-terminal 146 bp where all AtSR1-related proteins share high similarity but have no similarity to other known DNA-binding proteins. The calmodulin-binding nuclear proteins isolated from wounded leaves exhibit specific CGCG box DNA binding activities. These results suggest that the AtSR gene family encodes a family of calmodulin-binding/DNA-binding proteins involved in multiple signal transduction pathways in plants.

  18. High Mobility Group Box Protein-1 correlates with renal function in chronic kidney disease (CKD).

    Science.gov (United States)

    Bruchfeld, Annette; Qureshi, Abdul Rashid; Lindholm, Bengt; Barany, Peter; Yang, Lihong; Stenvinkel, Peter; Tracey, Kevin J

    2008-01-01

    Chronic kidney disease (CKD) is associated with inflammation and malnutrition and carries a markedly increased risk of cardiovascular disease (CVD). High Mobility Group Box Protein-1 (HMGB-1) is a 30-kDa nuclear and cytosolic protein known as a transcription and growth factor, recently identified as a proinflammatory mediator of tissue injury. Recent data implicates HMGB-1 in endotoxin lethality, rheumatoid arthritis, and atherosclerosis. The aim of this post-hoc, cross-sectional study was to determine whether HMGB-1 serum levels are elevated in CKD patients. The study groups were categorized as follows: 110 patients starting dialysis defined as CKD 5; 67 patients with moderately to severely reduced renal function or CKD 3-4; and 48 healthy controls. High-sensitivity C-reactive-protein (hs-CRP), interleukin-6 (IL-6), tumor necrosis factor (TNF), serum-albumin (S-albumin), hemoglobin A(1c) (HbA(1c)), hemoglobin, subjective global nutritional assessment (SGA), and glomerular filtration rate (GFR) were analyzed. Kruskal-Wallis test was used to compare groups and Spearman's rank correlation test was used for continuous variables. HMGB-1, measured by Western blot, was significantly (P < 0.001) elevated in CKD 5 (146.7 +/- 58.6 ng/mL) and CKD 3-4 (85.6 +/- 31.8) compared with controls (10.9 +/- 10.5). HMGB-1 levels were correlated positively with TNF (Rho = 0.52; P < 0.001), hs-CRP (Rho = 0.38; P < 0.001), IL-6 (Rho = 0.30; P < 0.001), HbA(1c) (Rho = 0.14; P = 0.02) and SGA (Rho = 0.21; P = 0.002) and negatively correlated with GFR (Rho = -0.69; P = 0.0001), Hb (Rho = -0.60; P < 0.001), S-albumin (Rho = -0.31; P < 0.001). The current study has revealed that HMGB-1 is elevated significantly in CKD patients and correlates with GFR as well as markers of inflammation and malnutrition. Future studies may delineate whether HMGB-1 is also a marker of disease activity and severity as well as a predictor of outcome in CKD.

  19. The DEAD box protein Mrh4 functions in the assembly of the mitochondrial large ribosomal subunit.

    Science.gov (United States)

    De Silva, Dasmanthie; Fontanesi, Flavia; Barrientos, Antoni

    2013-11-01

    Proteins in a cell are universally synthesized by ribosomes. Mitochondria contain their own ribosomes, which specialize in the synthesis of a handful of proteins required for oxidative phosphorylation. The pathway of mitoribosomal biogenesis and factors involved are poorly characterized. An example is the DEAD box proteins, widely known to participate in the biogenesis of bacterial and cytoplasmic eukaryotic ribosomes as either RNA helicases or RNA chaperones, whose mitochondrial counterparts remain completely unknown. Here, we have identified the Saccharomyces cerevisiae mitochondrial DEAD box protein Mrh4 as essential for large mitoribosome subunit biogenesis. Mrh4 interacts with the 21S rRNA, mitoribosome subassemblies, and fully assembled mitoribosomes. In the absence of Mrh4, the 21S rRNA is matured and forms part of a large on-pathway assembly intermediate missing proteins Mrpl16 and Mrpl39. We conclude that Mrh4 plays an essential role during the late stages of mitoribosome assembly by promoting remodeling of the 21S rRNA-protein interactions.

  20. Nuclear variants of bone morphogenetic proteins

    Directory of Open Access Journals (Sweden)

    Meinhart Christopher A

    2010-03-01

    Full Text Available Abstract Background Bone morphogenetic proteins (BMPs contribute to many different aspects of development including mesoderm formation, heart development, neurogenesis, skeletal development, and axis formation. They have previously been recognized only as secreted growth factors, but the present study detected Bmp2, Bmp4, and Gdf5/CDMP1 in the nuclei of cultured cells using immunocytochemistry and immunoblotting of nuclear extracts. Results In all three proteins, a bipartite nuclear localization signal (NLS was found to overlap the site at which the proproteins are cleaved to release the mature growth factors from the propeptides. Mutational analyses indicated that the nuclear variants of these three proteins are produced by initiating translation from downstream alternative start codons. The resulting proteins lack N-terminal signal peptides and are therefore translated in the cytoplasm rather than the endoplasmic reticulum, thus avoiding proteolytic processing in the secretory pathway. Instead, the uncleaved proteins (designated nBmp2, nBmp4, and nGdf5 containing the intact NLSs are translocated to the nucleus. Immunostaining of endogenous nBmp2 in cultured cells demonstrated that the amount of nBmp2 as well as its nuclear/cytoplasmic distribution differs between cells that are in M-phase versus other phases of the cell cycle. Conclusions The observation that nBmp2 localization varies throughout the cell cycle, as well as the conservation of a nuclear localization mechanism among three different BMP family members, suggests that these novel nuclear variants of BMP family proteins play an important functional role in the cell.

  1. Transcriptional regulation of fruit ripening by tomato FRUITFULL homologs and associated MADS box proteins.

    Science.gov (United States)

    Fujisawa, Masaki; Shima, Yoko; Nakagawa, Hiroyuki; Kitagawa, Mamiko; Kimbara, Junji; Nakano, Toshitsugu; Kasumi, Takafumi; Ito, Yasuhiro

    2014-01-01

    The tomato (Solanum lycopersicum) MADS box FRUITFULL homologs FUL1 and FUL2 act as key ripening regulators and interact with the master regulator MADS box protein RIPENING INHIBITOR (RIN). Here, we report the large-scale identification of direct targets of FUL1 and FUL2 by transcriptome analysis of FUL1/FUL2 suppressed fruits and chromatin immunoprecipitation coupled with microarray analysis (ChIP-chip) targeting tomato gene promoters. The ChIP-chip and transcriptome analysis identified FUL1/FUL2 target genes that contain at least one genomic region bound by FUL1 or FUL2 (regions that occur mainly in their promoters) and exhibit FUL1/FUL2-dependent expression during ripening. These analyses identified 860 direct FUL1 targets and 878 direct FUL2 targets; this set of genes includes both direct targets of RIN and nontargets of RIN. Functional classification of the FUL1/FUL2 targets revealed that these FUL homologs function in many biological processes via the regulation of ripening-related gene expression, both in cooperation with and independent of RIN. Our in vitro assay showed that the FUL homologs, RIN, and tomato AGAMOUS-LIKE1 form DNA binding complexes, suggesting that tetramer complexes of these MADS box proteins are mainly responsible for the regulation of ripening.

  2. 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.

  3. High mobility group box 1 protein as a late-acting mediator of acute lung inflammation.

    Science.gov (United States)

    Lutz, Waldemar; Stetkiewicz, Jan

    2004-01-01

    Acute inflammatory lung injury is often a delayed complication of critical illness and is associated with increased mortality. High mobility group box 1 (HMGB1) protein, in addition to its role as a transcriptional regulator factor, has been identified as a late mediator of endotoxin lethality and might be also involved in the development and progression of acute lung injury. HMGB1 protein itself can cause an acute inflammatory response manifested by increased production of proinflammatory cytokines and neutrophil accumulation. The delayed kinetics of HMGB1 protein release indicate that this protein is a distal mediator of acute inflamatory lung injury. Anti-HMGB1 protein antibodies attenuated endotoxin-induced lung injury, but not the early release of TNF-alpha and IL-1beta, indicating that HMGB1 protein is a late mediator of endotoxin-induced acute lung injury. HMGB1 protein is not released by apoptotic cells but is passively released by necrotic or damaged somatic and immune cells and it functions as a major stimulus of necrosis-induced inflammation. HMGB1 protein is also released by activated monocytes/macrophages and induces delayed and biphasic release of proinflammatory mediators from these cells. HMGB1 protein failed to stimulate cytokines release in lymphocytes, indicating that cellular stimulation is specific. We would like to suggest that HMGB1 protein may be also a primary mediator of the inflammatory responses to lung cells injury caused by toxic environmental chemicals.

  4. APC/C(Cdh1-mediated degradation of the F-box protein NIPA is regulated by its association with Skp1.

    Directory of Open Access Journals (Sweden)

    Christine von Klitzing

    Full Text Available NIPA (Nuclear Interaction Partner of Alk kinase is an F-box like protein that targets nuclear Cyclin B1 for degradation. Integrity and therefore activity of the SCF(NIPA E3 ligase is regulated by cell-cycle-dependent phosphorylation of NIPA, restricting substrate ubiquitination to interphase. Here we show that phosphorylated NIPA is degraded in late mitosis in an APC/C(Cdh1-dependent manner. Binding of the unphosphorylated form of NIPA to Skp1 interferes with binding to the APC/C-adaptor protein Cdh1 and therefore protects unphosphorylated NIPA from degradation in interphase. Our data thus define a novel mode of regulating APC/C-mediated ubiquitination.

  5. SRY interacts with ribosomal proteins S7 and L13a in nuclear speckles.

    Science.gov (United States)

    Sato, Youichi; Yano, Shojiro; Ewis, Ashraf A; Nakahori, Yutaka

    2011-05-01

    The SRY (sex-determining region on the Y chromosome) is essential for male development; however, the molecular mechanism by which the SRY induces testis development is still unclear. To elucidate the mechanism of testis development, we identified SRY-interacting proteins using a yeast two-hybrid system. We found two ribosomal proteins, RPS7 (ribosomal protein S7) and RPL13a (ribosomal protein L13a) that interact with the HMG (high-mobility group) box domain of SRY. Furthermore, we confirmed the intracellular distributions of RPS7, RPL13a and SRY and found that the three proteins were co-expressed in COS1 cells. SRY, RPS7 and RPL13a were co-localized in nuclear speckles. These findings suggest that SRY plays an important role in activities associated with nuclear speckles via an unknown mechanism.

  6. 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.

  7. Structures of genes encoding TATA box-binding proteins from Trimeresurus gramineus and T. flavoviridis snakes.

    Science.gov (United States)

    Nakashima, K; Nobuhisa, I; Deshimaru, M; Ogawa, T; Shimohigashi, Y; Fukumaki, Y; Hattori, M; Sakaki, Y; Hattori, S; Ohno, M

    1995-01-23

    A cDNA encoding the Trimeresurus gramineus (Tg; green habu snake) TATA-box-binding protein (TgTBP) was cloned and sequenced. The cDNA encodes a 33-kDa protein with an extensive sequence similarity to those derived from other organisms, except for the N-terminal domain. Genes encoding TgTBP and Trimeresurus flavoviridis (Tf; habu snake) TBP (TfTBP) were isolated using a TgTBP cDNA and their nt sequences were determined. They are the first TBP genes entirely sequenced in higher animals. Both genes span over 15 kb and are constructed from eight exons and seven introns. Comparison of the loci of introns on the aligned amino-acid sequences of TBP from six organisms (Tg, Tf, mouse, Arabidopsis thaliana, Schizosaccharomyces pombe and Acanthamoeba castellanii) indicated that there are three highly conserved loci in the C-terminal domain.

  8. Insulin Is Required to Maintain Albumin Expression by Inhibiting Forkhead Box O1 Protein.

    Science.gov (United States)

    Chen, Qing; Lu, Mingjian; Monks, Bobby R; Birnbaum, Morris J

    2016-01-29

    Diabetes is accompanied by dysregulation of glucose, lipid, and protein metabolism. In recent years, much effort has been spent on understanding how insulin regulates glucose and lipid metabolism, whereas the effect of insulin on protein metabolism has received less attention. In diabetes, hepatic production of serum albumin decreases, and it has been long established that insulin positively controls albumin gene expression. In this study, we used a genetic approach in mice to identify the mechanism by which insulin regulates albumin gene transcription. Albumin expression was decreased significantly in livers with insulin signaling disrupted by ablation of the insulin receptor or Akt. Concomitant deletion of Forkhead Box O1 (Foxo1) in these livers rescued the decreased albumin secretion. Furthermore, activation of Foxo1 in the liver is sufficient to suppress albumin expression. These results suggest that Foxo1 acts as a repressor of albumin expression.

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

    Directory of Open Access Journals (Sweden)

    Fanchi Meng

    2015-12-01

    Full Text Available 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.

  10. Conditional depletion of nuclear proteins by the Anchor Away system.

    Science.gov (United States)

    Fan, Xiaochun; Geisberg, Joseph V; Wong, Koon Ho; Jin, Yi

    2011-01-01

    Nuclear proteins play key roles in the regulation of many important cellular processes. In Saccharomyces cerevisiae, many genes encoding nuclear proteins are essential. This unit describes a method termed Anchor Away that can be used to conditionally and rapidly deplete nuclear proteins of interest. It involves conditional export of the protein of interest out of the nucleus and its subsequent sequestration in the cytoplasm. This method can be used to simultaneously deplete multiple proteins from the nucleus.

  11. High-mobility group box protein 1 promotes the survival of myeloid-derived suppressor cells by inducing autophagy.

    Science.gov (United States)

    Parker, Katherine H; Horn, Lucas A; Ostrand-Rosenberg, Suzanne

    2016-09-01

    Myeloid-derived suppressor cells are immune-suppressive cells that are elevated in most individuals with cancer, where their accumulation and suppressive activity are driven by inflammation. As myeloid-derived suppressor cells inhibit anti-tumor immunity and promote tumor progression, we are determining how their viability is regulated. Previous studies have established that the damage-associated molecular pattern molecule high-mobility group box protein 1 drives myeloid-derived suppressor cell accumulation and suppressive potency and is ubiquitously present in the tumor microenvironment. As high-mobility group box protein 1 also facilitates tumor cell survival by inducing autophagy, we sought to determine if high-mobility group box protein 1 regulates myeloid-derived suppressor cell survival through induction of autophagy. Inhibition of autophagy increased the quantity of apoptotic myeloid-derived suppressor cells, demonstrating that autophagy extends the survival and increases the viability of myeloid-derived suppressor cells. Inhibition of high-mobility group box protein 1 similarly increased the level of apoptotic myeloid-derived suppressor cells and reduced myeloid-derived suppressor cell autophagy, demonstrating that in addition to inducing the accumulation of myeloid-derived suppressor cells, high-mobility group box protein 1 sustains myeloid-derived suppressor cell viability. Circulating myeloid-derived suppressor cells have a default autophagic phenotype, and tumor-infiltrating myeloid-derived suppressor cells are more autophagic, consistent with the concept that inflammatory and hypoxic conditions within the microenvironment of solid tumors contribute to tumor progression by enhancing immune-suppressive myeloid-derived suppressor cells. Overall, these results demonstrate that in addition to previously recognized protumor effects, high-mobility group box protein 1 contributes to tumor progression by increasing myeloid-derived suppressor cell viability by

  12. 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.

  13. Rice ABERRANT PANICLE ORGANIZATION 1, encoding an F-box protein, regulates meristem fate.

    Science.gov (United States)

    Ikeda, Kyoko; Ito, Momoyo; Nagasawa, Nobuhiro; Kyozuka, Junko; Nagato, Yasuo

    2007-09-01

    Inflorescence architecture is one of the most important agronomical traits. Characterization of rice aberrant panicle organization 1 (apo1) mutants revealed that APO1 positively controls spikelet number by suppressing the precocious conversion of inflorescence meristems to spikelet meristems. In addition, APO1 is associated with the regulation of the plastchron, floral organ identity, and floral determinacy. Phenotypic analyses of apo1 and floral homeotic double mutants demonstrate that APO1 positively regulates class-C floral homeotic genes, but not class-B genes. Molecular studies revealed that APO1 encodes an F-box protein, an ortholog of Arabidopsis UNUSUAL FLORAL ORGAN (UFO), which is a positive regulator of class-B genes. Overexpression of APO1 caused an increase in inflorescence branches and spikelets. As the mutant inflorescences and flowers differed considerably between apo1 and ufo, the functions of APO1 and UFO appear to have diverged during evolution.

  14. Analysis of MADS box protein-protein interactions in living plant cells

    NARCIS (Netherlands)

    Immink, R.G.H.; Gadella, T.W.J.; Ferrario, S.I.T.; Busscher, M.; Angenent, G.C.

    2002-01-01

    Over the last decade, the yeast two-hybrid system has become the tool to use for the identification of protein-protein interactions and recently, even complete interactomes were elucidated by this method. Nevertheless, it is an artificial system that is sensitive to errors resulting in the identific

  15. 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.

  16. Plant F-box protein evolution is determined by lineage-specific timing of major gene family expansion waves.

    Directory of Open Access Journals (Sweden)

    Aura Navarro-Quezada

    Full Text Available F-box proteins (FBPs represent one of the largest and fastest evolving gene/protein families in the plant kingdom. The FBP superfamily can be divided in several subfamilies characterized by different C-terminal protein-protein interaction domains that recruit targets for proteasomal degradation. Hence, a clear picture of their phylogeny and molecular evolution is of special interest for the general understanding of evolutionary histories of multi-domain and/or large protein families in plants. In an effort to further understand the molecular evolution of F-box family proteins, we asked whether the largest subfamily in Arabidopsis thaliana, which carries a C-terminal F-box associated domain (FBA proteins shares evolutionary patterns and signatures of selection with other FBPs. To address this question, we applied phylogenetic and molecular evolution analyses in combination with the evaluation of transcriptional profiles. Based on the 2219 FBA proteins we de novo identified in 34 completely sequenced plant genomes, we compared their evolutionary patterns to a previously analyzed large subfamily carrying C-terminal kelch repeats. We found that these two large FBP subfamilies generally tend to evolve by massive waves of duplication, followed by sequence conservation of the F-box domain and sequence diversification of the target recruiting domain. We conclude that the earlier in evolutionary time a major wave of expansion occurred, the more pronounced these selection signatures are. As a consequence, when performing cross species comparisons among FBP subfamilies, significant differences will be observed in the selective signatures of protein-protein interaction domains. Depending on the species, the investigated subfamilies comprise up to 45% of the complete superfamily, indicating that other subfamilies possibly follow similar modes of evolution.

  17. Intrinsic and extrinsic negative regulators of nuclear protein transport processes

    OpenAIRE

    Sekimoto, Toshihiro; Yoneda, Yoshihiro

    2012-01-01

    The nuclear–cytoplasmic protein transport is a critical process in cellular events. The identification of transport signals (nuclear localization signal and nuclear export signal) and their receptors has facilitated our understanding of this expanding field. Nuclear transport must be appropriately regulated to deliver proteins through the nuclear pore when their functions are required in the nucleus, and to export them into the cytoplasm when they are not needed in the nucleus. Altered nuclea...

  18. Potentiation of NMDA receptor-dependent cell responses by extracellular high mobility group box 1 protein.

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    Marco Pedrazzi

    Full Text Available BACKGROUND: Extracellular high mobility group box 1 (HMGB1 protein can operate in a synergistic fashion with different signal molecules promoting an increase of cell Ca(2+ influx. However, the mechanisms responsible for this effect of HMGB1 are still unknown. PRINCIPAL FINDINGS: Here we demonstrate that, at concentrations of agonist per se ineffective, HMGB1 potentiates the activation of the ionotropic glutamate N-methyl-D-aspartate receptor (NMDAR in isolated hippocampal nerve terminals and in a neuroblastoma cell line. This effect was abolished by the NMDA channel blocker MK-801. The HMGB1-facilitated NMDAR opening was followed by activation of the Ca(2+-dependent enzymes calpain and nitric oxide synthase in neuroblastoma cells, resulting in an increased production of NO, a consequent enhanced cell motility, and onset of morphological differentiation. We have also identified NMDAR as the mediator of HMGB1-stimulated murine erythroleukemia cell differentiation, induced by hexamethylenebisacetamide. The potentiation of NMDAR activation involved a peptide of HMGB1 located in the B box at the amino acids 130-139. This HMGB1 fragment did not overlap with binding sites for other cell surface receptors of HMGB1, such as the advanced glycation end products or the Toll-like receptor 4. Moreover, in a competition assay, the HMGB1((130-139 peptide displaced the NMDAR/HMGB1 interaction, suggesting that it comprised the molecular and functional site of HMGB1 regulating the NMDA receptor complex. CONCLUSION: We propose that the multifunctional cytokine-like molecule HMGB1 released by activated, stressed, and damaged or necrotic cells can facilitate NMDAR-mediated cell responses, both in the central nervous system and in peripheral tissues, independently of other known cell surface receptors for HMGB1.

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

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    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.

  20. Identification and characterization of GIP1, an Arabidopsis thaliana protein that enhances the DNA binding affinity and reduces the oligomeric state of G-box binding factors

    Institute of Scientific and Technical Information of China (English)

    Paul C. SEHNKE; Beth J. LAUGHNER; Carla R. LYERLY LINEBARGER; William B. GURLEY; Robert J.FERL

    2005-01-01

    Environmental control of the alcohol dehydrogenase (Adh) and other stress response genes in plants is in part brought about by transcriptional regulation involving the G-box cis-acting DNA element and bZIP G-box Binding Factors (GBFs).The mechanisms of GBF regulation and requirements for additional factors in this control process are not well understood.In an effort to identify potential GBF binding and control partners, maize GBF1 was used as bait in a yeast two-hybrid screen of an A. thaliana cDNA library. GBF Interacting Protein 1 (GIP1) arose from the screen as a 496 amino acid protein with a predicted molecular weight of 53,748 kDa that strongly interacts with GBFs. Northern analysis of A.thaliana tissue suggests a 1.8-1.9 kb GIP1 transcript, predominantly in roots. Immunolocalization studies indicate that GIP1 protein is mainly localized to the nucleus. In vitro electrophoretic mobility shift assays using an Adh G-box DNA probe and recombinant A. thaliana GBF3 or maize GBF1, showed that the presence of GIP1 resulted in a tenfold increase in GBF DNA binding activity without altering the migration, suggesting a transient association between GIP1 and GBF. Addition of GIP1 to intentionally aggregated GBF converted GBF to lower molecular weight macromolecular complexes and GIP1 also refolded denatured rhodanese in the absence of ATP. These data suggest GIP1 functions to enhance GBF DNA binding activity by acting as a potent nuclear chaperone or crowbar, and potentially regulates the multimeric state of GBFs, thereby contributing to bZIP-mediated gene regulation.

  1. Nuclear accumulation of β-catenin and forkhead box O3a in colon cancer:Dangerous liaison

    Institute of Scientific and Technical Information of China (English)

    Wolfgang; Link

    2012-01-01

    The WNT/-catenin and phosphoinositide 3-kinase(PI3K/AKT) signaling cascades both have been implicated in the formation and progression of colorectal cancer.Oncogenic PI3K/AKT signaling suppresses the activity of forkhead box O3a(FOXO3a) transcription factor through phosphorylation leading to its nuclear exclusion.Inhibition of the PI3K/AKT signaling by PI3K or AKT inhibitors results in the translocation of FOXO3a to the nucleus,and is considered to be a promising therapeutic strategy for many cancers including colon cancer.Now,however,a new study in Nature Medicine has revealed a nuclear interaction of-catenin with FOXO3a as a promoter of metastatic progression in colon cancer.The work has important implications for the treatment of colon cancers,suggests a companion biomarker strategy to enable a personalized medicine approach,and offers an alternative therapeutic strategy to overcome resistance to PI3K and AKT inhibitors.

  2. Clinical significance of serum high mobility group box 1 protein in patients with severe traumatic brain injury

    Institute of Scientific and Technical Information of China (English)

    濮雪华

    2014-01-01

    Objective To detect the levels of high mobility group box 1 protein(HMGB1),tumor necrosis factor-α(TNF-α),interleukin-6(IL-6),C-reactive protein(CRP)in order to explore the clinical significance of HMGB1 in patients with severely traumatic brain injury.Methods A total of 75 patients composed of 40 male and35 female with severely traumatic brain

  3. Molecular characterization and expression analysis of Triticum aestivum squamosa-promoter binding protein-box genes involved in ear development

    Institute of Scientific and Technical Information of China (English)

    Bin Zhang; a Xia Liu; a Guangyao Zhao; Xinguo Mao; Ang Li; Ruilian Jing

    2014-01-01

    Wheat (Triticum aestivum L.) is one of the most important crops in the world. Squamosa-promoter binding protein (SBP)-box genes play a critical role in regulating flower and fruit development. In this study, 10 novel SBP-box genes (TaSPL genes) were isolated from wheat ((Triticum aestivum L.) cultivar Yanzhan 4110). Phylogenetic analysis classified the TaSPL genes into five groups (G1-G5). The motif combinations and expression patterns of the TaSPL genes varied among the five groups with each having own distinctive characteristics: TaSPL20/21 in G1 and TaSPL17 in G2 mainly expressed in the shoot apical meristem and the young ear, and their expression levels responded to development of the ear; TaSPL6/15 belonging to G3 were upregulated and TaSPL1/23 in G4 were downregulated during grain development; the gene in G5 (TaSPL3) expressed constitutively. Thus, the consistency of the phylogenetic analysis, motif compositions, and expression patterns of the TaSPL genes revealed specific gene structures and functions. On the other hand, the diverse gene structures and different expression patterns suggested that wheat SBP-box genes have a wide range of functions. The results also suggest a potential role for wheat SBP-box genes in ear development. This study provides a significant beginning of functional analysis of SBP-box genes in wheat.

  4. The implication and potential applications of high-mobility group box 1 protein in breast cancer.

    Science.gov (United States)

    Sohun, Moonindranath; Shen, Huiling

    2016-06-01

    High-mobility group box 1 protein (HMGB1) is a highly conserved, non-histone and ubiquitous chromosomal protein found enriched in active chromatin forming part of the high mobility group family of proteins and is encoded by the HMGB1 gene (13q12) in human beings. It has various intranuclear and extracellular functions. It plays an important role in the pathogenesis of many diseases including cancer. In 2012, there was approximately 1.67 million new breast cancer cases diagnosed which makes it the second most frequent cancer in the world after lung cancer (25% of all cancers) and the commonest cancer among women. Both pre-clinical and clinical studies have suggested that HMGB1 might be a useful target in the management of breast cancer. This review summarises the structure and functions of HMGB1 and its dual role in carcinogenesis both as a pro-tumorigenic and anti-tumorigenic factor. It also sums up evidence from in vitro and in vivo studies using breast cancer cell lines and samples which demonstrate its influence in radiotherapy, chemotherapy and hormonal therapy in breast cancer. It may have particular importance in HER2 positive and metastatic breast cancer. It might pave the way for new breast cancer treatments through development of novel drugs, use of microRNAs (miRNAs), targeting breast cancer stem cells (CSCs) and breast cancer immunotherapy. It may also play a role in determining breast cancer prognosis. Thus HMGB1 may open up novel avenues in breast cancer management.

  5. 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.

  6. Differential distribution of Y-box-binding protein 1 and cold shock domain protein A in developing and adult human brain.

    Science.gov (United States)

    Bernstein, Hans-Gert; Lindquist, Jonathan A; Keilhoff, Gerburg; Dobrowolny, Henrik; Brandt, Sabine; Steiner, Johann; Bogerts, Bernhard; Mertens, Peter R

    2015-07-01

    The two cold shock domain containing proteins, Y-box-binding protein-1 and cold shock domain protein A were immunolocalized in developing and adult human brain. With the exception of a small population of hypothalamic astrocytes, brain Y-box-binding protein-1 was predominantly found in multiple neurons in the mature human CNS, which might be related to its involvement in neurotransmission and other neuron-associated functions. Cold shock domain protein A was typically observed in astrocytes, oligodendrocytes, choroid plexus epithelia and nerve fibers. However, in circumscribed brain regions as hypothalamus, habenula, and cerebellum, this protein was also expressed in neurons. In the prenatal brain, both proteins were found to be abundantly expressed in radial glial cells, neuroblasts and neurons, which might be an anatomical correlate of the proposed roles of both proteins in cell proliferation and differentiation. In addition, Y-box-binding protein-1 was identified in cultured, lipopolysaccharide-stimulated microglial cells, which underscores its putative role as a mediator in immune and inflammatory processes.

  7. High mobility group box 1 protein: possible pathogenic link to atrial fibrillation

    Institute of Scientific and Technical Information of China (English)

    HU Xiao-rong; WANG Xiao-hong; LIU Hue-fen; ZHOU Wen-jie; JIANG Hong

    2012-01-01

    Atrial fibrillation (AF) is the most common sustained dysrhythmia in clinical practice.The bulk of evidence suggests that inflammatory processes,oxidative stress and matrix metalloproteinase are associated with development of AF.However,these agents may be involved in high mobility group box 1 protein (HMGB1).We hypothesized that HMGB1 may be a possible pathogenic link to AF.A growing body of evidence supports these hypotheses.First,the level of serum HMGB1 is significantly increased in patients with AF including paroxysmal and persistent AF.Second,HMGB1 has been identified as a new pro-inflammatory cytokine in cardiovascular diseases,along with tumor necrosis factor (TNF)-α,interleukin (IL)-6,and C-reactive protein,and there is cross-talk between HMGB1 and inflammatory cytokines.Third,oxidative stress is involved in the release of the pro-inflammatory cytokine,HMGB1,indicating there is cross-talk between oxidative stress and inflammation,and oxidative stress may reinforce the effect of inflammation on the pathogenesis of AF and inflammation may play a more important role in the pathogenesis of AF.Fourth,HMGB1 can promote matrix metalloproteinase-9 upregulation and activation.Fifth,HMGB1 receptors (receptor for advanced glycation end products,Toll-like receptor-2,4) may mediate the atrial structural remodeling or be up-regulated in patients with non-valvular AF.These results suggest that HMGB1 may participate in the pathogenesis of AF and provide a potential target for pharmacological interruption of AF.

  8. Characterization of differential ripening pattern in association with ethylene biosynthesis in the fruits of five naturally occurring banana cultivars and detection of a GCC-box-specific DNA-binding protein.

    Science.gov (United States)

    Choudhury, Swarup Roy; Roy, Sujit; Saha, Progya Paramita; Singh, Sanjay Kumar; Sengupta, Dibyendu N

    2008-07-01

    MA-ACS1 and MA-ACO1 are the two major ripening genes in banana and play crucial role in the regulation of ethylene production during ripening. Here, we report a comparative ripening pattern in five different naturally occurring banana cultivars namely Cavendish (AAA), Rasthali (AAB), Kanthali (AB), Poovan (AAB) and Monthan (ABB), which have distinct genome composition. We found a distinct variation in the climacteric ethylene production and in-vivo ACC oxidase activity level during the ripening stages in the five cultivars. We identified the cDNAs for MA-ACS1 and MA-ACO1 from the five cultivars and studied the transcript accumulation patterns of the two genes, which correlated well with the differential timing in the expression of these two genes during ripening. The GCC-box is one of the ethylene-responsive elements (EREs) found in the promoters of many ethylene-inducible genes. We have identified a GCC-box motif (putative ERE) in the promoters of MA-ACS1 and MA-ACO1 in banana cultivars. DNA-protein interaction studies revealed the presence of a GCC-box-specific DNA-binding activity in the fruit nuclear extract and such DNA-binding activity was enhanced following ethylene treatment. South-Western blotting revealed a 25-kDa nuclear protein that binds specifically to GCC-box DNA in the climacteric banana fruit. Together, these results indicate the probable involvement of the GCC-box motif as the cis-acting ERE in the regulation of MA-ACS1 and MA-ACO1 during ripening in banana fruits via binding of specific ERE-binding protein.

  9. A novel family of plant nuclear envelope-associated proteins.

    Science.gov (United States)

    Pawar, Vidya; Poulet, Axel; Détourné, Gwénaëlle; Tatout, Christophe; Vanrobays, Emmanuel; Evans, David E; Graumann, Katja

    2016-10-01

    This paper describes the characterisation of a new family of higher plant nuclear envelope-associated proteins (NEAPs) that interact with other proteins of the nuclear envelope. In the model plant Arabidopsis thaliana, the family consists of three genes expressed ubiquitously (AtNEAP1-3) and a pseudogene (AtNEAP4). NEAPs consist of extensive coiled-coil domains, followed by a nuclear localisation signal and a C-terminal predicted transmembrane domain. Domain deletion mutants confirm the presence of a functional nuclear localisation signal and transmembrane domain. AtNEAP proteins localise to the nuclear periphery as part of stable protein complexes, are able to form homo- and heteromers, and interact with the SUN domain proteins AtSUN1 and AtSUN2, involved in the linker of nucleoskeleton and cytoskeleton (LINC) complex. An A. thaliana cDNA library screen identified a putative transcription factor called AtbZIP18 as a novel interactor of AtNEAP1, which suggest a connection between NEAP and chromatin. An Atneap1 Atneap3 double-knockout mutant showed reduced root growth, and altered nuclear morphology and chromatin structure. Thus AtNEAPs are suggested as inner nuclear membrane-anchored coiled-coil proteins with roles in maintaining nuclear morphology and chromatin structure.

  10. 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.

    Directory of Open Access Journals (Sweden)

    Rebecca C Burgess

    Full Text Available Rad54 is an ATP-driven translocase involved in the genome maintenance pathway of homologous recombination (HR. Although its activity has been implicated in several steps of HR, its exact role(s at each step are still not fully understood. We have identified a new interaction between Rad54 and the replicative DNA clamp, proliferating cell nuclear antigen (PCNA. This interaction was only mildly weakened by the mutation of two key hydrophobic residues in the highly-conserved PCNA interaction motif (PIP-box of Rad54 (Rad54-AA. Intriguingly, the rad54-AA mutant cells displayed sensitivity to DNA damage and showed HR defects similar to the null mutant, despite retaining its ability to interact with HR proteins and to be recruited to HR foci in vivo. We therefore surmised that the PCNA interaction might be impaired in vivo and was unable to promote repair synthesis during HR. Indeed, the Rad54-AA mutant 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 mutants in this domain also resulted in a loss of ATPase activity. Therefore, we have found that although Rad54 interacts with PCNA, the PIP-box motif likely plays only a minor role in stabilizing the PCNA interaction, and rather, this conserved domain is probably an extension of the ATPase domain III.

  11. Peroxiredoxins, thioredoxin, and Y-box-binding protein-1 are involved in the pathogenesis and progression of dialysis-associated renal cell carcinoma.

    Science.gov (United States)

    Fushimi, Fumiyoshi; Taguchi, Kenichi; Izumi, Hiroto; Kohno, Kimitoshi; Kuwano, Michihiko; Ono, Mayumi; Nakashima, Yutaka; Takesue, Tetsuro; Naito, Seiji; Oda, Yoshinao

    2013-10-01

    Patients with end-stage renal disease are exposed to increased oxidative stress and impairment of antioxidant mechanisms. We focused on dialysis renal cell carcinoma (RCC), including epithelial hyperplasia in acquired cystic disease of the kidney (ACDK). We attempted to obtain insight into the carcinogenesis and tumor progression in terms of cellular defense mechanisms associated with oxidative stress by investigating the expression of antioxidant proteins by immunohistochemistry. We evaluated retrospectively 43 cases of dialysis RCC and, as a control group, 49 cases of sporadic RCC. Peroxiredoxin (Prx) 1, 3, 4, 5, and 6 expression in dialysis RCC was positively correlated with the duration of dialysis. In epithelial hyperplasia, in 17 cases of acquired cystic disease of the kidney, Prxs and thioredoxin were highly expressed. Moreover, in dialysis RCC, Prx 3, 4, and 5 immunoreactivity and nuclear expression of Y-box-binding protein-1 were higher than in sporadic RCC. In dialysis RCC, Prx 3, 4, and 5 immunoreactivity positively correlated with the Fuhrman nuclear grade. These data suggest that oxidative stress during dialysis enhances antioxidant activity, with an inhibiting effect on carcinogenesis. Once cancer has developed, antioxidant activity might have a stimulating effect on the progression of dialysis RCC.

  12. Generation of a Monoclonal Antibody Specifically Reacting with Neuron-specific TATA-Box Binding Protein-Associated Factor 1 (N-TAF1

    Directory of Open Access Journals (Sweden)

    Gen Tamiya

    2012-12-01

    Full Text Available TATA-box binding protein-associated factor 1 (TAF1, the largest subunit of the transcription factor IID complex, plays an important role in the RNA polymerase II-mediated gene transcription pathway regulating the transcription of a large number of genes related to cell division. The neuron-specific isoform of the TAF1 gene (N-TAF1 may have an essential role in neurons through transcriptional regulation of many neuron-specific genes. The present study reports the preparation and properties of a monoclonal antibody directed against N-TAF1. The monoclonal antibody, 3A-11F, specifically recognized N-TAF1 protein with no reactivity to TAF1 protein, as evidenced by immunocytochemistry and immunoprecipitation using cultured cells expressing recombinant N-TAF1 or TAF1 protein. Immunohistochemistry using 3A-11F showed that N-TAF1-imunoreactivity was detected in the nuclear region of neurons in the rat brain. The 3A-11F monoclonal antibody promises to be a useful tool for determining the expression pattern and biological function of N-TAF1 in the brain.

  13. 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...... manner. Demographic data, comorbidity, routine biochemistry, microbiological data, infection focus, severity score and mortality on day 28 were recorded. Plasma and serum were sampled within 24 hours after admission. Levels of all studied markers (HMGB1, LBP, PCT, IL-6, C-reactive protein, white blood...... patients compared with nonbacteraemic patients (P white blood cell count and neutrophils (P

  14. 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

  15. NSs Virulence Factor of Rift Valley Fever Virus Engages the F-Box Proteins FBXW11 and β-TRCP1 To Degrade the Antiviral Protein Kinase PKR

    Science.gov (United States)

    Kainulainen, Markus; Lau, Simone; Samuel, Charles E.; Hornung, Veit

    2016-01-01

    ABSTRACT Rift Valley fever virus (RVFV, family Bunyaviridae, genus Phlebovirus) is a relevant pathogen of both humans and livestock in Africa. The nonstructural protein NSs is a major virulence factor known to suppress the type I interferon (IFN) response by inhibiting host cell transcription and by proteasomal degradation of a major antiviral IFN effector, the translation-inhibiting protein kinase PKR. Here, we identified components of the modular SCF (Skp1, Cul1, F-box protein)-type E3 ubiquitin ligases as mediators of PKR destruction by NSs. Small interfering RNAs (siRNAs) against the conserved SCF subunit Skp1 protected PKR from NSs-mediated degradation. Consequently, RVFV replication was severely reduced in Skp1-depleted cells when PKR was present. SCF complexes have a variable F-box protein subunit that determines substrate specificity for ubiquitination. We performed an siRNA screen for all (about 70) human F-box proteins and found FBXW11 to be involved in PKR degradation. The partial stabilization of PKR by FBXW11 depletion upregulated PKR autophosphorylation and phosphorylation of the PKR substrate eIF2α and caused a shutoff of host cell protein synthesis in RVFV-infected cells. To maximally protect PKR from the action of NSs, knockdown of structurally and functionally related FBXW1 (also known as β-TRCP1), in addition to FBXW11 deletion, was necessary. Consequently, NSs was found to interact with both FBXW11 and β-TRCP1. Thus, NSs eliminates the antiviral kinase PKR by recruitment of SCF-type E3 ubiquitin ligases containing FBXW11 and β-TRCP1 as substrate recognition subunits. This antagonism of PKR by NSs is essential for efficient RVFV replication in mammalian cells. IMPORTANCE Rift Valley fever virus is a pathogen of humans and animals that has the potential to spread from Africa and the Arabian Peninsula to other regions. A major virulence mechanism is the proteasomal degradation of the antiviral kinase PKR by the viral protein NSs. Here, we

  16. Nonredundant roles of mitochondria-associated F-box proteins Mfb1 and Mdm30 in maintenance of mitochondrial morphology in yeast.

    Science.gov (United States)

    Dürr, Mark; Escobar-Henriques, Mafalda; Merz, Sandra; Geimer, Stefan; Langer, Thomas; Westermann, Benedikt

    2006-09-01

    Mitochondria constantly fuse and divide to adapt organellar morphology to the cell's ever-changing physiological conditions. Little is known about the molecular mechanisms regulating mitochondrial dynamics. F-box proteins are subunits of both Skp1-Cullin-F-box (SCF) ubiquitin ligases and non-SCF complexes that regulate a large number of cellular processes. Here, we analyzed the roles of two yeast F-box proteins, Mfb1 and Mdm30, in mitochondrial dynamics. Mfb1 is a novel mitochondria-associated F-box protein. Mitochondria in mutants lacking Mfb1 are fusion competent, but they form aberrant aggregates of interconnected tubules. In contrast, mitochondria in mutants lacking Mdm30 are highly fragmented due to a defect in mitochondrial fusion. Fragmented mitochondria are docked but nonfused in Deltamdm30 cells. Mitochondrial fusion is also blocked during sporulation of homozygous diploid mutants lacking Mdm30, leading to a mitochondrial inheritance defect in ascospores. Mfb1 and Mdm30 exert nonredundant functions and likely have different target proteins. Because defects in F-box protein mutants could not be mimicked by depletion of SCF complex and proteasome core subunits, additional yet unknown factors are likely involved in regulating mitochondrial dynamics. We propose that mitochondria-associated F-box proteins Mfb1 and Mdm30 are key components of a complex machinery that regulates mitochondrial dynamics throughout yeast's entire life cycle.

  17. Regulation of neuronal differentiation by proteins associated with nuclear bodies.

    Directory of Open Access Journals (Sweden)

    Benjamin Förthmann

    Full Text Available Nuclear bodies are large sub-nuclear structures composed of RNA and protein molecules. The Survival of Motor Neuron (SMN protein localizes to Cajal bodies (CBs and nuclear gems. Diminished cellular concentration of SMN is associated with the neurodegenerative disease Spinal Muscular Atrophy (SMA. How nuclear body architecture and its structural components influence neuronal differentiation remains elusive. In this study, we analyzed the effects of SMN and two of its interaction partners in cellular models of neuronal differentiation. The nuclear 23 kDa isoform of Fibroblast Growth Factor - 2 (FGF-2(23 is one of these interacting proteins - and was previously observed to influence nuclear bodies by destabilizing nuclear gems and mobilizing SMN from Cajal bodies (CBs. Here we demonstrate that FGF-2(23 blocks SMN-promoted neurite outgrowth, and also show that SMN disrupts FGF-2(23-dependent transcription. Our results indicate that FGF-2(23 and SMN form an inactive complex that interferes with neuronal differentiation by mutually antagonizing nuclear functions. Coilin is another nuclear SMN binding partner and a marker protein for Cajal bodies (CBs. In addition, coilin is essential for CB function in maturation of small nuclear ribonucleoprotein particles (snRNPs. The role of coilin outside of Cajal bodies and its putative impacts in tissue differentiation are poorly defined. The present study shows that protein levels of nucleoplasmic coilin outside of CBs decrease during neuronal differentiation. Overexpression of coilin has an inhibitory effect on neurite outgrowth. Furthermore, we find that nucleoplasmic coilin inhibits neurite outgrowth independent of SMN binding revealing a new function for coilin in neuronal differentiation.

  18. Two Prp19-like U-box proteins in the MOS4-associated complex play redundant roles in plant innate immunity.

    Directory of Open Access Journals (Sweden)

    Jacqueline Monaghan

    2009-07-01

    Full Text Available Plant Resistance (R proteins play an integral role in defense against pathogen infection. A unique gain-of-function mutation in the R gene SNC1, snc1, results in constitutive activation of plant immune pathways and enhanced resistance against pathogen infection. We previously found that mutations in MOS4 suppress the autoimmune phenotypes of snc1, and that MOS4 is part of a nuclear complex called the MOS4-Associated Complex (MAC along with the transcription factor AtCDC5 and the WD-40 protein PRL1. Here we report the immuno-affinity purification of the MAC using HA-tagged MOS4 followed by protein sequence analysis by mass spectrometry. A total of 24 MAC proteins were identified, 19 of which have predicted roles in RNA processing based on their homology to proteins in the Prp19-Complex, an evolutionarily conserved spliceosome-associated complex containing homologs of MOS4, AtCDC5, and PRL1. Among these were two highly similar U-box proteins with homology to the yeast and human E3 ubiquitin ligase Prp19, which we named MAC3A and MAC3B. MAC3B was recently shown to exhibit E3 ligase activity in vitro. Through reverse genetics analysis we show that MAC3A and MAC3B are functionally redundant and are required for basal and R protein-mediated resistance in Arabidopsis. Like mos4-1 and Atcdc5-1, mac3a mac3b suppresses snc1-mediated autoimmunity. MAC3 localizes to the nucleus and interacts with AtCDC5 in planta. Our results suggest that MAC3A and MAC3B are members of the MAC that function redundantly in the regulation of plant innate immunity.

  19. 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.

  20. Autoinhibitory Interdomain Interactions and Subfamily-specific Extensions Redefine the Catalytic Core of the Human DEAD-box Protein DDX3.

    Science.gov (United States)

    Floor, Stephen N; Condon, Kendall J; Sharma, Deepak; Jankowsky, Eckhard; Doudna, Jennifer A

    2016-01-29

    DEAD-box proteins utilize ATP to bind and remodel RNA and RNA-protein complexes. All DEAD-box proteins share a conserved core that consists of two RecA-like domains. The core is flanked by subfamily-specific extensions of idiosyncratic function. The Ded1/DDX3 subfamily of DEAD-box proteins is of particular interest as members function during protein translation, are essential for viability, and are frequently altered in human malignancies. Here, we define the function of the subfamily-specific extensions of the human DEAD-box protein DDX3. We describe the crystal structure of the subfamily-specific core of wild-type DDX3 at 2.2 Å resolution, alone and in the presence of AMP or nonhydrolyzable ATP. These structures illustrate a unique interdomain interaction between the two ATPase domains in which the C-terminal domain clashes with the RNA-binding surface. Destabilizing this interaction accelerates RNA duplex unwinding, suggesting that it is present in solution and inhibitory for catalysis. We use this core fragment of DDX3 to test the function of two recurrent medulloblastoma variants of DDX3 and find that both inactivate the protein in vitro and in vivo. Taken together, these results redefine the structural and functional core of the DDX3 subfamily of DEAD-box proteins.

  1. The involvement of wheat F-box protein gene TaFBA1 in the oxidative stress tolerance of plants.

    Directory of Open Access Journals (Sweden)

    Shu-Mei Zhou

    Full Text Available As one of the largest gene families, F-box domain proteins have been found to play important roles in abiotic stress responses via the ubiquitin pathway. TaFBA1 encodes a homologous F-box protein contained in E3 ubiquitin ligases. In our previous study, we found that the overexpression of TaFBA1 enhanced drought tolerance in transgenic plants. To investigate the mechanisms involved, in this study, we investigated the tolerance of the transgenic plants to oxidative stress. Methyl viologen was used to induce oxidative stress conditions. Real-time PCR and western blot analysis revealed that TaFBA1 expression was up-regulated by oxidative stress treatments. Under oxidative stress conditions, the transgenic tobacco plants showed a higher germination rate, higher root length and less growth inhibition than wild type (WT. The enhanced oxidative stress tolerance of the transgenic plants was also indicated by lower reactive oxygen species (ROS accumulation, malondialdehyde (MDA content and cell membrane damage under oxidative stress compared with WT. Higher activities of antioxidant enzymes, including superoxide dismutase (SOD, catalase (CAT, ascorbate peroxidase (APX and peroxidase (POD, were observed in the transgenic plants than those in WT, which may be related to the upregulated expression of some antioxidant genes via the overexpression of TaFBA1. In others, some stress responsive elements were found in the promoter region of TaFBA1, and TaFBA1 was located in the nucleus, cytoplasm and plasma membrane. These results suggest that TaFBA1 plays an important role in the oxidative stress tolerance of plants. This is important for understanding the functions of F-box proteins in plants' tolerance to multiple stress conditions.

  2. The involvement of wheat F-box protein gene TaFBA1 in the oxidative stress tolerance of plants.

    Science.gov (United States)

    Zhou, Shu-Mei; Kong, Xiang-Zhu; Kang, Han-Han; Sun, Xiu-Dong; Wang, Wei

    2015-01-01

    As one of the largest gene families, F-box domain proteins have been found to play important roles in abiotic stress responses via the ubiquitin pathway. TaFBA1 encodes a homologous F-box protein contained in E3 ubiquitin ligases. In our previous study, we found that the overexpression of TaFBA1 enhanced drought tolerance in transgenic plants. To investigate the mechanisms involved, in this study, we investigated the tolerance of the transgenic plants to oxidative stress. Methyl viologen was used to induce oxidative stress conditions. Real-time PCR and western blot analysis revealed that TaFBA1 expression was up-regulated by oxidative stress treatments. Under oxidative stress conditions, the transgenic tobacco plants showed a higher germination rate, higher root length and less growth inhibition than wild type (WT). The enhanced oxidative stress tolerance of the transgenic plants was also indicated by lower reactive oxygen species (ROS) accumulation, malondialdehyde (MDA) content and cell membrane damage under oxidative stress compared with WT. Higher activities of antioxidant enzymes, including superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX) and peroxidase (POD), were observed in the transgenic plants than those in WT, which may be related to the upregulated expression of some antioxidant genes via the overexpression of TaFBA1. In others, some stress responsive elements were found in the promoter region of TaFBA1, and TaFBA1 was located in the nucleus, cytoplasm and plasma membrane. These results suggest that TaFBA1 plays an important role in the oxidative stress tolerance of plants. This is important for understanding the functions of F-box proteins in plants' tolerance to multiple stress conditions.

  3. Mutation analysis of the TATA box-binding protein (TBP) gene in Chinese Han patients with spinocerebellar ataxia.

    Science.gov (United States)

    Xu, Q; Li, X H; Wang, J L; Jiang, H; Zhang, S; Lei, L F; Shen, L; Xia, K; Pan, Q; Long, Z G; Tang, B S

    2009-10-01

    Spinocerebellar ataxia type 17 (SCA17) is a rare autosomal dominant progressive neurodegenerative disease caused by the CAG/CAA expansion in the TATA box-binding protein (TBP) gene. This study aimed to assess the frequency of SCA17 in patients from mainland China. Analysis of CAG/CAA expansion in this gene was performed in 263 patients consisting of 100 probands with dominantly inherited ataxias and 163 patients with sporadic ataxias. Abnormal expansion of CAG/CAA repeats in the SCA17 locus was found in a proband and her younger sister. To our knowledge, we are providing the first kindred analysis of SCA17 in mainland China.

  4. 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 ...

  5. 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.

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

    Science.gov (United States)

    Pfisterer, Karin; Jayo, Asier; Parsons, Maddy

    2017-01-06

    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.

  7. 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.

  8. 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.

  9. The RING finger/B-Box factor TAM-1 and a retinoblastoma-like protein LIN-35 modulate context-dependent gene silencing in Caenorhabditis elegans

    Science.gov (United States)

    Hsieh, Jenny; Liu, Jing; Kostas, Stephen A.; Chang, Chieh; Sternberg, Paul W.; Fire, Andrew

    1999-01-01

    Context-dependent gene silencing is used by many organisms to stably modulate gene activity for large chromosomal regions. We have used tandem array transgenes as a model substrate in a screen for Caenorhabditis elegans mutants that affect context-dependent gene silencing in somatic tissues. This screen yielded multiple alleles of a previously uncharacterized gene, designated tam-1 (for tandem-array-modifier). Loss-of-function mutations in tam-1 led to a dramatic reduction in the activity of numerous highly repeated transgenes. These effects were apparently context dependent, as nonrepetitive transgenes retained activity in a tam-1 mutant background. In addition to the dramatic alterations in transgene activity, tam-1 mutants showed modest alterations in expression of a subset of endogenous cellular genes. These effects include genetic interactions that place tam-1 into a group called the class B synMuv genes (for a Synthetic Multivulva phenotype); this family plays a negative role in the regulation of RAS pathway activity in C. elegans. Loss-of-function mutants in other members of the class-B synMuv family, including lin-35, which encodes a protein similar to the tumor suppressor Rb, exhibit a hypersilencing in somatic transgenes similar to that of tam-1 mutants. Molecular analysis reveals that tam-1 encodes a broadly expressed nuclear protein with RING finger and B-box motifs. PMID:10580003

  10. Role of SKP1-CUL1-F-Box-Protein (SCF) E3 Ubiquitin Ligases in Skin Cancer

    Institute of Scientific and Technical Information of China (English)

    Chuan-Ming Xie; Wenyi Wei; Yi Sun

    2013-01-01

    Many biological processes such as cell proliferation,differentiation,and cell death depend precisely on the timely synthesis and degradation of key regulatory proteins.While protein synthesis can be regulated at multiple levels,protein degradation is mainly controlled by the ubiquitin-proteasome system (UPS),which consists of two distinct steps:(1) ubiquitylation of targeted protein by E1 ubiquitin-activating enzyme,E2 ubiquitin-conjugating enzyme and E3 ubiquitin ligase,and (2) subsequent degradation by the 26S proteasome.Among all E3 ubiquitin ligases,the SCF (SKP1-CUL1-F-box protein) E3 ligases are the largest family and are responsible for the turnover of many key regulatory proteins.Aberrant regulation of SCF E3 ligases is associated with various human diseases,such as cancers,including skin cancer.In this review,we provide a comprehensive overview of all currently published data to define a promoting role of SCF E3 ligases in the development of skin cancer.The future directions in this area of research are also discussed with an ultimate goal to develop small molecule inhibitors of SCF E3 ligases as a novel approach for the treatment of human skin cancer.Furthermore,altered components or substrates of SCF E3 ligases may also be developed as the biomarkers for early diagnosis or predicting prognosis.

  11. Plasma membrane-association of SAUL1-type plant U-box armadillo repeat proteins is conserved in land plants

    Directory of Open Access Journals (Sweden)

    Katja eVogelmann

    2014-02-01

    Full Text Available Post-translational protein modification plays a pivotal role in the regulation and specific turnover of proteins. One of these important modifications is the ubiquitination of target proteins, which can occur at distinct cellular compartments. At the plasma membrane, ubiquitination regulates the internalization and thus trafficking of membrane proteins such as receptors and channels. The Arabidopsis plant U-box armadillo repeat (PUB-ARM ubiquitin ligase SAUL1 (SENESCENCE-ASSOCIATED UBIQUITIN LIGASE1 is part of the ubiquitination machinery at the plasma membrane. In contrast to most other PUB-ARM proteins, SAUL1 carries additional C-terminal ARM repeats responsible for plasma membrane-association. Here, we demonstrated that the C-terminal ARM repeat domain is also essential and sufficient to mediate plasma membrane-association of the closest Arabidopis paralog AtPUB43. We investigated targeting of PUB-ARM ubiquitin ligases of different plant species to find out whether plasma membrane-association of SAUL1-type PUB-ARM proteins is conserved. Phylogenetic analysis identified orthologs of SAUL1 in these plant species. Intracellular localization of transiently expressed GFP fusion proteins revealed that indeed plasma membrane-association due to additional C-terminal ARM repeats represents a conserved feature of SAUL1-type proteins. Analyses of transgenic Arabidopsis plants overexpressing N-terminally masked or truncated proteins revealed that interfering with the function of SAUL1-type proteins resulted in severe growth defects. Our results suggest an ancient origin of ubiquitination at the plasma membrane in the evolution of land plants.

  12. DNA polymerases BI and D from the hyperthermophilic archaeon Pyrococcus furiosus both bind to proliferating cell nuclear antigen with their C-terminal PIP-box motifs.

    Science.gov (United States)

    Tori, Kazuo; Kimizu, Megumi; Ishino, Sonoko; Ishino, Yoshizumi

    2007-08-01

    Proliferating cell nuclear antigen (PCNA) is the sliding clamp that is essential for the high processivity of DNA synthesis during DNA replication. Pyrococcus furiosus, a hyperthermophilic archaeon, has at least two DNA polymerases, polymerase BI (PolBI) and PolD. Both of the two DNA polymerases interact with the archaeal P. furiosus PCNA (PfuPCNA) and perform processive DNA synthesis in vitro. This phenomenon, in addition to the fact that both enzymes display 3'-5' exonuclease activity, suggests that both DNA polymerases work in replication fork progression. We demonstrated here that both PolBI and PolD functionally interact with PfuPCNA at their C-terminal PIP boxes. The mutant PolBI and PolD enzymes lacking the PIP-box sequence do not respond to the PfuPCNA at all in an in vitro primer extension reaction. This is the first experimental evidence that the PIP-box motif, located at the C termini of the archaeal DNA polymerases, is actually critical for PCNA binding to form a processive DNA-synthesizing complex.

  13. 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...

  14. 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

  15. 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

  16. 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.

  17. Poxvirus host range protein CP77 contains an F-box-like domain that is necessary to suppress NF-kappaB activation by tumor necrosis factor alpha but is independent of its host range function.

    Science.gov (United States)

    Chang, Shu-Jung; Hsiao, Jye-Chian; Sonnberg, Stephanie; Chiang, Cheng-Ting; Yang, Min-Hsiang; Tzou, Der-Lii; Mercer, Andrew A; Chang, Wen

    2009-05-01

    Tumor necrosis factor alpha (TNF-alpha) activates the nuclear factor kappaB (NF-kappaB) signaling pathway that regulates expression of many cellular factors playing important roles in innate immune responses and inflammation in infected hosts. Poxviruses employ many strategies to inhibit NF-kappaB activation in cells. In this report, we describe a poxvirus host range protein, CP77, which blocked NF-kappaB activation by TNF-alpha. Immunofluorescence analyses revealed that nuclear translocation of NF-kappaB subunit p65 protein in TNF-alpha-treated HeLa cells was blocked by CP77. CP77 did so without blocking IkappaBalpha phosphorylation, suggesting that upstream kinase activation was not affected by CP77. Using GST pull-down, we showed that CP77 bound to the NF-kappaB subunit p65 through the N-terminal six-ankyrin-repeat region in vitro. CP77 also bound to Cullin-1 and Skp1 of the SCF complex through a C-terminal 13-amino-acid F-box-like sequence. Both regions of CP77 are required to block NF-kappaB activation. We thus propose a model in which poxvirus CP77 suppresses NF-kappaB activation by two interactions: the C-terminal F-box of CP77 binding to the SCF complex and the N-terminal six ankyrins binding to the NF-kappaB subunit p65. In this way, CP77 attenuates innate immune response signaling in cells. Finally, we expressed CP77 or a CP77 F-box deletion protein from a vaccinia virus host range mutant (VV-hr-GFP) and showed that either protein was able to rescue the host range defect, illustrating that the F-box region, which is important for NF-kappaB modulation and binding to SCF complex, is not required for CP77's host range function. Consistently, knocking down the protein level of NF-kappaB did not relieve the growth restriction of VV-hr-GFP in HeLa cells.

  18. The F-box protein Fbp1 functions in the invasive growth and cell wall integrity mitogen-activated protein kinase (MAPK) pathways in Fusarium oxysporum.

    Science.gov (United States)

    Miguel-Rojas, Cristina; Hera, Concepcion

    2016-01-01

    F-box proteins determine substrate specificity of the ubiquitin-proteasome system. Previous work has demonstrated that the F-box protein Fbp1, a component of the SCF(Fbp1) E3 ligase complex, is essential for invasive growth and virulence of the fungal plant pathogen Fusarium oxysporum. Here, we show that, in addition to invasive growth, Fbp1 also contributes to vegetative hyphal fusion and fungal adhesion to tomato roots. All of these functions have been shown previously to require the mitogen-activated protein kinase (MAPK) Fmk1. We found that Fbp1 is required for full phosphorylation of Fmk1, indicating that Fbp1 regulates virulence and invasive growth via the Fmk1 pathway. Moreover, the Δfbp1 mutant is hypersensitive to sodium dodecylsulfate (SDS) and calcofluor white (CFW) and shows reduced phosphorylation levels of the cell wall integrity MAPK Mpk1 after SDS treatment. Collectively, these results suggest that Fbp1 contributes to both the invasive growth and cell wall integrity MAPK pathways of F. oxysporum.

  19. A bifunctional archaeal protein that is a component of 30S ribosomal subunits and interacts with C/D box small RNAs

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    Andrea Ciammaruconi

    2008-01-01

    Full Text Available We have identified a novel archaeal protein that apparently plays two distinct roles in ribosome metabolism. It is a polypeptide of about 18 kDa (termed Rbp18 that binds free cytosolic C/D box sRNAs in vivo and in vitro and behaves as a structural ribosomal protein, specifically a component of the 30S ribosomal subunit. As Rbp18 is selectively present in Crenarcheota and highly thermophilic Euryarchaeota, we propose that it serves to protect C/D box sRNAs from degradation and perhaps to stabilize thermophilic 30S subunits.

  20. F-box protein AFB4 plays a crucial role in plant growth,development and innate immunity

    Institute of Scientific and Technical Information of China (English)

    Zhubing Hu; Mehmet Ali Keceli; Maria Piisil(a); JingF Li; Mantas Survila; Pekka Heino; Günter Brader; E Tapio Palva; Jing Li

    2012-01-01

    Dear Editor,Auxin-signaling F-box protein 4 (AFB4) encoded by At4g24390 shares a significant sequence similarity to auxin receptor TIR1.In this study,we used a combination of physiological,molecular,and genetic approaches to characterize a T-DNA insertion line (GABI-KAT accession no.:068E01;henceforth designated afb4-1) as a knockout allele.Complete loss-of-function of the AFB4gene confers defects in many aspects of the plant life cycle including lateral root development,hypocotyl elongation,leaf organogenesis,flowering time,seed formation,and disease resistance to specific phytopathogens.The results presented here argue against the previously proposed mechanism of AFB4 action in negatively controlling auxin sensitivity [1].

  1. DEAD-box protein Ddx46 is required for the development of the digestive organs and brain in zebrafish.

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    Shunya Hozumi

    Full Text Available Spatially and temporally controlled gene expression, including transcription, several mRNA processing steps, and the export of mature mRNA to the cytoplasm, is essential for developmental processes. It is well known that RNA helicases of the DExD/H-box protein family are involved in these gene expression processes, including transcription, pre-mRNA splicing, and rRNA biogenesis. Although one DExD/H-box protein, Prp5, a homologue of vertebrate Ddx46, has been shown to play important roles in pre-mRNA splicing in yeast, the in vivo function of Ddx46 remains to be fully elucidated in metazoans. In this study, we isolated zebrafish morendo (mor, a mutant that shows developmental defects in the digestive organs and brain, and found that it encodes Ddx46. The Ddx46 transcript is maternally supplied, and as development proceeds in zebrafish larvae, its ubiquitous expression gradually becomes restricted to those organs. The results of whole-mount in situ hybridization showed that the expression of various molecular markers in these organs is considerably reduced in the Ddx46 mutant. Furthermore, splicing status analysis with RT-PCR revealed unspliced forms of mRNAs in the digestive organ and brain tissues of the Ddx46 mutant, suggesting that Ddx46 may be required for pre-mRNA splicing during zebrafish development. Therefore, our results suggest a model in which zebrafish Ddx46 is required for the development of the digestive organs and brain, possibly through the control of pre-mRNA splicing.

  2. A modified box model including charge regulation for protein adsorption in a spherical polyelectrolyte brush

    NARCIS (Netherlands)

    Biesheuvel, P.M.; Wittemann, A.

    2005-01-01

    Recent experiments showed significant adsorption of bovine serum albumin (BSA) in spherical polyelectrolyte brushes (SPB) consisting of polyacrylic acid, even for pH values above the isoelectric point of the protein, when both protein and polyion are negatively charged. To describe these experimenta

  3. Blockade of high mobility group box-1 protein attenuates experimental severe acute pancreatitis

    Institute of Scientific and Technical Information of China (English)

    Hidehiro Sawa; Takashi Ueda; Yoshifumi Takeyama; Takeo Yasuda; Makoto Shinzeki; Takahiro Nakajima; Yoshikazu Kuroda

    2006-01-01

    AIM: To examine the effects of anti-high mobility group box 1 (HMGB1) neutralizing antibody in experimental severe acute pancreatitis (SAP).METHODS: SAP was induced by creating closed duodenal loop in C3H/HeN mice. SAP was induced immediately after intraperitoneal injection of anti-HMGB1 neutralizing antibody (200 μg). Severity of pancreatitis, organ injury (liver, kidney and lung), and bacterial translocation to pancreas was examined 12 h after induction of SAP.RESULTS: Anti-HMGB1 neutralizing antibody significantly improved the elevation of the serum amylase level and the histological alterations of pancreas and lung in SAP.Anti-HMGB1 antibody also significantly ameliorated the elevations of serum alanine aminotransferase and creatinine in SAP. However, anti-HMGB1 antibody worsened the bacterial translocation to pancreas.CONCLUSION: Blockade of HMGB1 attenuated the development of SAP and associated organ dysfunction,suggesting that HMGB1 may act as a key mediator for inflammatory response and organ injury in SAP.

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

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    Margaret Rohrbaugh

    Full Text Available BACKGROUND: 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. METHODOLOGY/PRINCIPAL FINDINGS: 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. CONCLUSIONS/SIGNIFICANCE: 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

  5. 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.

  6. The Arabidopsis B-box protein BZS1/BBX20 interacts with HY5 and mediates strigolactone regulation of photomorphogenesis.

    Science.gov (United States)

    Wei, Chuang-Qi; Chien, Chih-Wei; Ai, Lian-Feng; Zhao, Jun; Zhang, Zhenzhen; Li, Kathy H; Burlingame, Alma L; Sun, Yu; Wang, Zhi-Yong

    2016-09-20

    Plant growth is controlled by integration of hormonal and light-signaling pathways. BZS1 is a B-box zinc finger protein previously characterized as a negative regulator in the brassinosteroid (BR)-signaling pathway and a positive regulator in the light-signaling pathway. However, the mechanisms by which BZS1/BBX20 integrates light and hormonal pathways are not fully understood. Here, using a quantitative proteomic workflow, we identified several BZS1-associated proteins, including light-signaling components COP1 and HY5. Direct interactions of BZS1 with COP1 and HY5 were verified by yeast two-hybrid and co-immunoprecipitation assays. Overexpression of BZS1 causes a dwarf phenotype that is suppressed by the hy5 mutation, while overexpression of BZS1 fused with the SRDX transcription repressor domain (BZS1-SRDX) causes a long-hypocotyl phenotype similar to hy5, indicating that BZS1's function requires HY5. BZS1 positively regulates HY5 expression, whereas HY5 negatively regulates BZS1 protein level, forming a feedback loop that potentially contributes to signaling dynamics. In contrast to BR, strigolactone (SL) increases BZS1 level, whereas the SL responses of hypocotyl elongation, chlorophyll and HY5 accumulation are diminished in the BZS1-SRDX seedlings, indicating that BZS1 is involved in these SL responses. These results demonstrate that BZS1 interacts with HY5 and plays a central role in integrating light and multiple hormone signals for photomorphogenesis in Arabidopsis.

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

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    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.

  8. High mobility group box 1 protein (HMGB1) as an immune-modulating factor for polarization of human T lymphocytes

    Institute of Scientific and Technical Information of China (English)

    Lifeng Huang; Yongming Yao; Haidong Meng; Xiaodong Zhao; Ning Dong; Yan Yu

    2008-01-01

    Objective This study was performed to investigate the effect of high mobility group box-1 protein (HMGB 1) on immune function of human T lymphocytes in vitro and explore its potential role in cell-mediated immune dysfunction.Methods Fresh blood was obtained from healthy adult volunteers and peripheral blood mononuclear cells (PBMCs) were isolated,then rhHMGB 1 was added to PBMCs.Four-color flow cytometric (FCM) analysis was used for the measurement of intracellular cytokine including interleukin Results (1) Different stimulating time and dosage of rhHMGB 1 did not alter the number of IFN-a positive cells (Th 1).rhHMGB 1 stimulation provoked a dose-dependent and time-dependent increase in Th2 subset and decrease in ratio of Th 1 to Th2.(2) Compared with the untreated cells,when the cells were coincubated with rhHMGB 1 (10-100ng/ml) for 12 hrs,protein release of IL-2 and sIL-2R were significantly up-regulated.At 48 hrs,in contrast,protein production was relatively lower in cells after exposure to 100-1000 ng/ml rhHMGBI.Conclusions These findings demonstrated that HMGB1 has a dual influence on immune functions of human T lymphocytes.

  9. A DEAD box protein is required for formation of a hidden break in Arabidopsis chloroplast 23S rRNA.

    Science.gov (United States)

    Nishimura, Kenji; Ashida, Hiroki; Ogawa, Taro; Yokota, Akiho

    2010-09-01

    In plant chloroplasts, the ribosomal RNA (rRNA) of the large subunit of the ribosome undergoes post-maturation fragmentation processing. This processing consists of site-specific cleavage that generates gapped, discontinuous rRNA molecules. However, the molecular mechanism underlying introduction of the gap structure (the 'hidden break') is poorly understood. Here, we found that the DEAD box protein RH39 plays a key role in introduction of the hidden break into the 23S rRNA in Arabidopsis chloroplasts. Genetic screening for an Arabidopsis plant with a drastically reduced level of ribulose-1,5-bisphosphate carboxylase/oxygenase identified an RH39 mutant. The levels of other chloroplast-encoded photosynthetic proteins were also severely reduced. The reductions were not due to a failure of transcription, but rather inefficiency in translation. RNA gel blotting revealed incomplete fragmentation of 23S rRNA in chloroplasts during maturation. In vitro analysis with recombinant RH39 suggested that the protein binds to the adjacent sequence upstream of the hidden break site to exert its function. We propose a molecular mechanism for the RH39-mediated fragmentation processing of 23S rRNA in chloroplasts.

  10. The UNUSUAL FLORAL ORGANS gene of Arabidopsis thaliana is an F-box protein required for normal patterning and growth in the floral meristem.

    Science.gov (United States)

    Samach, A; Klenz, J E; Kohalmi, S E; Risseeuw, E; Haughn, G W; Crosby, W L

    1999-11-01

    Genetic and molecular studies have suggested that the UNUSUAL FLORAL ORGANS (UFO) gene, from Arabidopsis thaliana, is expressed in all shoot apical meristems, and is involved in the regulation of a complex set of developmental events during floral development, including floral meristem and floral organ identity. Results from in situ hybridization using genes expressed early in floral development as probes indicate that UFO controls growth of young floral primordia. Transgenic constructs were used to provide evidence that UFO regulates floral organ identity by activating or maintaining transcription of the class B organ-identity gene APETALA 3, but not PISTILLATA. In an attempt to understand the biochemical mode of action of the UFO gene product, we show here that UFO is an F-box protein that interacts with Arabidopsis SKP1-like proteins, both in the yeast two-hybrid system and in vitro. In yeast and other organisms both F-box proteins and SKP1 homologues are subunits of specific ubiquitin E3 enzyme complexes that target specific proteins for degradation. The protein selected for degradation by the complex is specified by the F-box proteins. It is therefore possible that the role of UFO is to target for degradation specific proteins controlling normal growth patterns in the floral primordia, as well as proteins that negatively regulate APETALA 3 transcription.

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

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    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.

  12. C-terminal binding protein (CtBP activates the expression of E-box clock genes with CLOCK/CYCLE in Drosophila.

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    Taichi Q Itoh

    Full Text Available In Drosophila, CLOCK/CYCLE heterodimer (CLK/CYC is the primary activator of circadian clock genes that contain the E-box sequence in their promoter regions (hereafter referred to as "E-box clock genes". Although extensive studies have investigated the feedback regulation of clock genes, little is known regarding other factors acting with CLK/CYC. Here we show that Drosophila C-terminal binding protein (dCtBP, a transcriptional co-factor, is involved in the regulation of the E-box clock genes. In vivo overexpression of dCtBP in clock cells lengthened or abolished circadian locomotor rhythm with up-regulation of a subset of the E-box clock genes, period (per, vrille (vri, and PAR domain protein 1ε (Pdp1ε. Co-expression of dCtBP with CLK in vitro also increased the promoter activity of per, vri, Pdp1ε and cwo depending on the amount of dCtBP expression, whereas no effect was observed without CLK. The activation of these clock genes in vitro was not observed when we used mutated dCtBP which carries amino acid substitutions in NAD+ domain. These results suggest that dCtBP generally acts as a putative co-activator of CLK/CYC through the E-box sequence.

  13. 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.

  14. Identification of two Y-box binding proteins that interact with the promoters of columbid annexin I genes.

    Science.gov (United States)

    Pratt, S L; Horseman, N D

    1998-07-01

    Two annexin I (anxI) genes, called cp35 and cp37, are expressed from the pigeon (Columba livia) genome, but they are regulated differently at both the transcriptional and post-transcriptional levels. The proximal promoter elements of these two genes are very similar. A conserved sequence from the cp35 and cp37 promoters bound specifically with proteins present in cropsac cell extracts. This sequence of DNA was used to screen a lambdagt11 cDNA expression library. Clones encoding two pigeon Y-box binding proteins (YB) were isolated. One of the pigeon YB cDNAs was found to be most similar to YB1 from other species, and the other was most similar to chicken YB2. Each YB is encoded by a single-copy gene in the pigeon, and their mRNAs are expressed in many tissues. On Northern blots, the sizes of the mRNAs encoding pigeon YB1 (pYB1) and pigeon YB2 (pYB2) were 1.8 and 1.7kb, respectively. The sequences of both pYB1 and pYB2 diverge from their previously identified relatives in the N-terminal domain 'A'. Antisera were developed to unique peptide epitopes in YB1 or 2. Affinity-purified anti-YB1 and anti-YB2 detected immunoreactive proteins in extracts from a variety of pigeon tissues, including the cropsac. To confirm that pYB1 and pYB2 interact with the cp35 promoter, electrophoretic gel mobility shift reactions were carried out in the presence or absence of YB antibodies. Binding to the cp35 promoter was specifically neutralized by either anti-pYB1 or anti-pYB2. These results are the first evidence that two YB proteins simultaneously bind to a promoter element, and thereby may interact during regulation of gene expression.

  15. 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…

  16. The role of high mobility group box chromosomal protein 1 expression in the differential diagnosis of hepatic actinomycosis: a case report

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    Wu Chuan-Xin

    2013-01-01

    Full Text Available Abstract Introduction Primary hepatic actinomycosis is a rare disease, but is important in the differential diagnosis of hepatoma in endemic areas. As high mobility group box chromosomal protein 1 plays an important role in the pathogenesis of both acute and chronic inflammatory conditions, we postulate that high mobility group box chromosomal protein 1 may have a possible pathogenic role in hepatic actinomycosis. To the best of our knowledge, our report is the first to detect an association between highly elevated high mobility group box chromosomal protein 1 expression and hepatic actinomycosis. Case presentation A 67-year-old Chinese man was admitted to our hospital with a three-month history of epigastric pain, anorexia, and subjective weight loss. Ultrasonography and computed tomography of the patient’s abdomen confirmed a hypodense mass measuring seven cm in diameter in the left lateral segment of his liver. A hepatic tumor was suspected and surgical resection was scheduled. Histopathologic examination revealed that the overall features of the hepatic tissues were consistent with hepatic actinomycosis. Whole blood and hepatic tissue samples of the patient, of patients who had hepatocellular carcinoma and of healthy donors were collected. Serum high mobility group box chromosomal protein 1 concentration in actinomycosis was 8.5ng/mL, which was higher than the hepatocellular carcinoma level of 5.2ng/mL and the normal level of Conclusion High mobility group box chromosomal protein 1 may have a potent biological effect on the pathogenesis of hepatic actinomycosis as a novel cytokine and may be a useful marker in the differential diagnosis of hepatic actinomycosis.

  17. STUDY ON NUCLEAR MATRIX PROTEINS FROM HUMAN BREAST CARCINOMA

    Institute of Scientific and Technical Information of China (English)

    HE Qian; ZHANG Shu-qun; CHU Yong-lie; JIA Xiao-li; JIANG Jian-tao

    2009-01-01

    Objective To investigate the marker protein of human breast carcinoma from nuclear matrix proteins (NMPs).Methods NMPs were injected subcutaneously into rabbit to get antiserum, which was used to detect the NMPs specificity for breast carcinoma.Results There was an apparent positive band (100kD) in the NMPs of breast carcinoma, which did not exist in normal breast and other tumors that were detected.Conclusion One or one group of 100kD NMPs were found to be related to human breast carcinoma, which may be involved in the carcinogenesis and development of human breast carcinoma and valuable for breast carcinoma diagnosis.

  18. 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

  19. 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.

  20. 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

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

    Science.gov (United States)

    Blavet, Nicolas; Uřinovská, Jana; Jeřábková, Hana; Chamrád, Ivo; Vrána, Jan; Lenobel, René; Beinhauer, Jana; Šebela, Marek; Doležel, Jaroslav; Petrovská, Beáta

    2017-01-02

    Proteins are the most abundant component of the cell nucleus, where they perform a plethora of functions, including the assembly of long DNA molecules into condensed chromatin, DNA replication and repair, regulation of gene expression, synthesis of RNA molecules and their modification. Proteins are important components of nuclear bodies and are involved in the maintenance of the nuclear architecture, transport across the nuclear envelope and cell division. Given their importance, the current poor knowledge of plant nuclear proteins and their dynamics during the cell's life and division is striking. Several factors hamper the analysis of the plant nuclear proteome, but the most critical seems to be the contamination of nuclei by cytosolic material during their isolation. With the availability of an efficient protocol for the purification of plant nuclei, based on flow cytometric sorting, contamination by cytoplasmic remnants can be minimized. Moreover, flow cytometry allows the separation of nuclei in different stages of the cell cycle (G1, S, and G2). This strategy has led to the identification of large number of nuclear proteins from barley (Hordeum vulgare), thus triggering the creation of a dedicated database called UNcleProt, http://barley.gambrinus.ueb.cas.cz/ .

  2. The human YB-1 cold shock domain : structural, dynamical and binding properties of the central nucleic acid binding domain of the human Y-box protein YB-1, a transcription and translation regulating protein

    NARCIS (Netherlands)

    Kloks, Cathelijne Petra Anne Marie

    2003-01-01

    Y-box proteins are a highly conserved group of proteins present in bacteria, plants and animals. They are essential in regulating transcription and translation and the coupling between theses two processes. Their central domain, the so-called cold shock domain (CSD), is responsible for the nucleic a

  3. Forkhead box protein A1 inhibits the expression of uncoupling protein 2 in hydrogen peroxide-induced A549 cell line.

    Science.gov (United States)

    Song, Lan; Xu, Zhaojun; Li, Ling; Hu, Mei; Cheng, Lijuan; Chen, Lingli; Zhang, Bo

    2014-01-01

    Forkhead box protein A1 (FoxA1) is a transcription factor that is involved in embryonic development and cell differentiation. In this study, we show that hydrogen peroxide (H2O2) treatment upregulated expression of FoxA1 and UCP2 in the A549 cell line. Overexpression of FoxA1 by full-length complementary DNA reduced UCP2 expression, while silencing of FoxA1 expression by small interfering RNA significantly increased UCP2 levels. FoxA1 binds to a site from -919 to -913 bp relative to the UCP2 transcription start site. The overexpression of FoxA1 promoted the DNA binding activity and attenuated the transcription of UCP2 promoter as shown by electromobility shift, chromatin immunoprecipitation assays, and luciferase reporter assay. These data indicate an important role of FoxA1 in regulating expression of UCP2.

  4. 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.

  5. High Mobility Group Box-1 Protein and Outcomes in Critically Ill Surgical Patients Requiring Open Abdominal Management

    Directory of Open Access Journals (Sweden)

    Michelle S. Malig

    2017-01-01

    Full Text Available Background. Previous studies assessing various cytokines in the critically ill/injured have been uninformative in terms of translating to clinical care management. Animal abdominal sepsis work suggests that enhanced intraperitoneal (IP clearance of Damage-Associated Molecular Patterns (DAMPs improves outcome. Thus measuring the responses of DAMPs offers alternate potential insights and a representative DAMP, High Mobility Group Box-1 protein (HMGB-1, was considered. While IP biomediators are being recognized in critical illness/trauma, HMGB-1 behaviour has not been examined in open abdomen (OA management. Methods. A modified protocol for HMGB-1 detection was used to examine plasma/IP fluid samples from 44 critically ill/injured OA patients enrolled in a randomized controlled trial comparing two negative pressure peritoneal therapies (NPPT: Active NPPT (ANPPT and Barker’s Vacuum Pack NPPT (BVP. Samples were collected and analyzed at the time of laparotomy and at 24 and 48 hours after. Results. There were no statistically significant differences in survivor versus nonsurvivor HMGB-1 plasma or IP concentrations at baseline, 24 hours, or 48 hours. However, plasma HMGB-1 levels tended to increase continuously in the BVP cohort. Conclusions. HMGB-1 appeared to behave differently between NPPT cohorts. Further studies are needed to elucidate the relationship of HMGB-1 and outcomes in septic/injured patients.

  6. High Mobility Group Box Protein 1 Boosts Endothelial Albumin Transcytosis through the RAGE/Src/Caveolin-1 Pathway

    Science.gov (United States)

    Shang, Dan; Peng, Tao; Gou, Shanmiao; Li, Yiqing; Wu, Heshui; Wang, Chunyou; Yang, Zhiyong

    2016-01-01

    High-mobility group box protein 1 (HMGB1), an inflammatory mediator, has been reported to destroy cell-cell junctions, resulting in vascular endothelial hyperpermeability. Here, we report that HMGB1 increases the endothelial transcytosis of albumin. In mouse lung vascular endothelial cells (MLVECs), HMGB1 at a concentration of 500 ng/ml or less did not harm cell-cell junctions but rapidly induced endothelial hyperpermeability to 125I-albumin. HMGB1 induced an increase in 125I-albumin and AlexaFluor 488-labeled albumin internalization in endocytosis assays. Depletion of receptor for advanced glycation end products (RAGE), but not TLR2 or TLR4, suppressed HMGB1-induced albumin transcytosis and endocytosis. Genetic and pharmacological destruction of lipid rafts significantly inhibited HMGB1-induced albumin endocytosis and transcytosis. HMGB1 induced the rapid phosphorylation of caveolin (Cav)-1 and Src. Either RAGE gene silencing or soluble RAGE suppressed Cav-1 Tyr14 phosphorylation and Src Tyr418 phosphorylation. The Src inhibitor 4-amino-5-(4-chlorophenyl)-7-(t-butyl) pyrazolo[3,4-d] pyrimidine (PP2) blocked HMGB1-induced Cav-1 Tyr14 phosphorylation. PP2 and overexpression of Cav-1 with a T14F mutation significantly inhibited HMGB1-induced transcytosis and albumin endocytosis. Our findings suggest that HMGB1 induces the transcytosis of albumin via RAGE-dependent Src phosphorylation and Cav-1 phosphorylation. These studies revealed a new mechanism of HMGB1-induced endothelial hyperpermeability. PMID:27572515

  7. Ethyl pyruvate reduces myocardial ischemia and reperfusion injury by inhibiting high mobility group box 1 protein in rats.

    Science.gov (United States)

    Hu, Xiaorong; Cui, Bo; Zhou, Xiaoya; Xu, Changwu; Lu, Zhibing; Jiang, Hong

    2012-01-01

    High mobility group box 1 protein (HMGB1) plays an important role in myocardial ischemia and reperfusion (I/R) injury. Ethyl pyruvate (EP), a potent reactive oxygen species scavenger, has been reported to inhibit myocardial apoptosis and reduce myocardial I/R injury. The aim of this study was to investigate the mechanism by which EP reduces myocardial I/R injury in rats. Anesthetized male rats were once treated with EP (50 mg/kg, i.p.) before ischemia, and then subjected to ischemia for 30 min followed by reperfusion for 4 h. Lactate dehydrogenase (LDH), creatine kinase (CK), malondialdehyde (MDA), superoxide dismutase (SOD) activity and infarct size were measured. HMGB1 expression was assessed by immunoblotting. The results showed that pretreatment of EP (50 mg/kg) could significantly reduce the infarct size and the levels of LDH and CK after 4 h reperfusion (all PR. The present study suggested that ethyl pyruvate could attenuate myocardial I/R injury by inhibiting HMGB1 expression.

  8. The DEAD-Box Protein Dhh1p Couples mRNA Decay and Translation by Monitoring Codon Optimality.

    Science.gov (United States)

    Radhakrishnan, Aditya; Chen, Ying-Hsin; Martin, Sophie; Alhusaini, Najwa; Green, Rachel; Coller, Jeff

    2016-09-22

    A major determinant of mRNA half-life is the codon-dependent rate of translational elongation. How the processes of translational elongation and mRNA decay communicate is unclear. Here, we establish that the DEAD-box protein Dhh1p is a sensor of codon optimality that targets an mRNA for decay. First, we find mRNAs whose translation elongation rate is slowed by inclusion of non-optimal codons are specifically degraded in a Dhh1p-dependent manner. Biochemical experiments show Dhh1p is preferentially associated with mRNAs with suboptimal codon choice. We find these effects on mRNA decay are sensitive to the number of slow-moving ribosomes on an mRNA. Moreover, we find Dhh1p overexpression leads to the accumulation of ribosomes specifically on mRNAs (and even codons) of low codon optimality. Lastly, Dhh1p physically interacts with ribosomes in vivo. Together, these data argue that Dhh1p is a sensor for ribosome speed, targeting an mRNA for repression and subsequent decay.

  9. 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.

  10. 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.

  11. Related polymorphic F-box protein genes between haplotypes clustering in the BAC contig sequences around the S-RNase of Japanese pear.

    Science.gov (United States)

    Okada, Kazuma; Tonaka, Nozomi; Taguchi, Tomio; Ichikawa, Takehiko; Sawamura, Yutaka; Nakanishi, Tetsu; Takasaki-Yasuda, Takeshi

    2011-03-01

    Most fruit trees in the Rosaceae exhibit self-incompatibility, which is controlled by the pistil S gene, encoding a ribonuclease (S-RNase), and the pollen S gene at the S-locus. The pollen S in Prunus is an F-box protein gene (SLF/SFB) located near the S-RNase, but it has not been identified in Pyrus and Malus. In the Japanese pear, various F-box protein genes (PpSFBB(-α-γ)) linked to the S-RNase are proposed as the pollen S candidate. Two bacterial artificial chromosome (BAC) contigs around the S-RNase genes of Japanese pear were constructed, and 649 kb around S(4)-RNase and 378 kb around S(2)-RNase were sequenced. Six and 10 pollen-specific F-box protein genes (designated as PpSFBB(4-u1-u4, 4-d1-d2) and PpSFBB(2-u1-u5,) (2-d1-d5), respectively) were found, but PpSFBB(4-α-γ) and PpSFBB(2-γ) were absent. The PpSFBB(4) genes showed 66.2-93.1% amino acid identity with the PpSFBB(2) genes, which indicated clustering of related polymorphic F-box protein genes between haplotypes near the S-RNase of the Japanese pear. Phylogenetic analysis classified 36 F-box protein genes of Pyrus and Malus into two major groups (I and II), and also generated gene pairs of PpSFBB genes and PpSFBB/Malus F-box protein genes. Group I consisted of gene pairs with 76.3-94.9% identity, while group II consisted of gene pairs with higher identities (>92%) than group I. This grouping suggests that less polymorphic PpSFBB genes in group II are non-S pollen genes and that the pollen S candidates are included in the group I PpSFBB genes.

  12. Defective chloroplast development inhibits maintenance of normal levels of abscisic acid in a mutant of the Arabidopsis RH3 DEAD-box protein during early post-germination growth.

    Science.gov (United States)

    Lee, Kwang-Hee; Park, Jiyoung; Williams, Donna S; Xiong, Yuqing; Hwang, Inhwan; Kang, Byung-Ho

    2013-03-01

    The plastid has its own translation system, and its ribosomes are assembled through a complex process in which rRNA precursors are processed and ribosomal proteins are inserted into the rRNA backbone. DEAD-box proteins have been shown to play roles in multiple steps in ribosome biogenesis. To investigate the cellular and physiological roles of an Arabidopsis DEAD-box protein, RH3, we examined its expression and localization and the phenotypes of rh3-4, a T-DNA insertion mutant allele of RH3. The promoter activity of RH3 is strongest in the greening tissues of 3-day and 1-week-old seedlings but reduced afterwards. Cotyledons were pale and seedling growth was retarded in the mutant. The most obvious abnormality in the mutant chloroplasts was their lack of normal ribosomes. Electron tomography analysis indicated that ribosome density in the 3-day-old mutant chloroplasts is only 20% that of wild-type chloroplasts, and the ribosomes in the mutant are smaller. These chloroplast defects in rh3-4 were alleviated in 2-week-old cotyledons and true leaves. Interestingly, rh3-4 seedlings have lower amounts of abscisic acid prior to recovery of their chloroplasts, and were more sensitive to abiotic stresses. Transcriptomic analysis indicated that nuclear genes for chloroplast proteins are down-regulated, and proteins mediating chloroplast-localized steps of abscisic acid biosynthesis are expressed to a lower extent in 1-week-old rh3-4 seedlings. Taken together, these results suggest that conversion of eoplasts into chloroplasts in young seedlings is critical for the seedlings to start carbon fixation as well as for maintenance of abscisic acid levels for responding to environmental challenges.

  13. BZS1, a B-box Protein, Promotes Photomorphogenesis Downstream of Both Brassinosteroid and Light Signaling Pathways

    Institute of Scientific and Technical Information of China (English)

    Xi-Ying Fan; Kang Chong; Zhi-Yong Wang; Yu Sun; Dong-Mei Cao; Ming-Yi Bai; Xiao-Min Luo; Hong-Juan Yang; Chuang-Qi Wei; Sheng-Wei Zhu; Ying Sun

    2012-01-01

    Photomorphogenesis is controlled by multiple signaling pathways,including the light and brassinosteroid (BR) pathways.BR signaling activates the BZR1 transcription factor,which is required for suppressing photomorphogenesis in the dark.We identified a suppressor of the BR hypersensitive mutant bzr1-1D and named it bzr1-1D suppressor1-Dominant (bzs1-D).The bzs1-D mutation was caused by overexpression of a B-box zinc finger protein BZS1,which is transcriptionally repressed by BZR1.Overexpression of BZS1 causes de-etiolation in the dark,short hypocotyls in the light,reduced sensitivity to BR treatment,and repression of many BR-activated genes.Knockdown of BZS1 by co-suppression partly suppressed the short hypocotyl phenotypes of BR-deficient or insensitive mutants.These results support that BZS1 is a negative regulator of BR response.BZS1 overexpressors are hypersensitive to different wavelengths of light and loss of function of BZS1 reduces plant sensitivity to light and partly suppresses the constitutive photomorphogenesis 1 (cop1) mutant in the dark,suggesting a positive role in light response.BZS1 protein accumulates at an increased level after light treatment of dark-grown BZS1-OX plants and in the cop1 mutants,and BZS1 interacts with COP1 in vitro,suggesting that light regulates BZS1 through COP1-mediated ubiquitination and proteasomal degradation.These results demonstrate that BZS1 mediates the crosstalk between BR and light pathways.

  14. Evaluation of high mobility group box 1 protein as a presurgical diagnostic marker reflecting the severity of acute appendicitis

    Directory of Open Access Journals (Sweden)

    Wu Chuanxin

    2012-09-01

    Full Text Available Abstract Objectives To validate the role of high mobility group box-1(HMGB1 in diagnosis of acute appendicitis (AA with different pathological severity. Methods According to the pathologically diagnosis, 150 patients underwent appendectomies between Jan. 2007 and Dec, 2010 were divided into acute simple, acute suppurative and acute gangrenous appendicitis as group 1, 2 and 3, respectively. Each patient group contains 50 sex and age matched cases to make comparison with 50 healthy volunteers. The mRNA and protein expression levels of serum HMGB1 were determined by real-time quantitative PCR and enzyme linked immunosorbent assay (ELISA. Serum High-sensitivity C-reactive protein (hs-CRP levels were determined by rate nephelometric immunoassay. Results In comparison with health volunteers, relative HMGB1 mRNA levels in group 1, 2 and 3 were significantly increased 3.05 ± 0.51,8.33 ± 0.75 and 13.74 ± 1.09 folds, reflecting a tendency of augmented severity. In accordance, serum protein levels of HMGB1 were 10.97 ± 1.64, 14.42 ± 1.56 and 18.08 ± 2.41 ng/ml in 3 patient groups, which are significantly higher than that of healthy volunteers’ 5.47 ± 0.73 ng/ml. hs-CRP levels were 12.85 ± 3.41, 21.04 ± 1.98 and 31.07 ± 5.46 ng/ml in 3 patients groups compared with 2.06 ± 0.77 ng/ml in controls. The concentrations of HMGB1 and hs-CRP were both positively correlated with disease severity. Conclusion Serum HMGB1 constitutes as a valuable marker in diagnosis of AA. Positively correlated with hs-CRP level, mRNA and protein expression of HMGB1 to a certain extent reflected the severity of AA.

  15. Membrane proteins structure and dynamics by nuclear magnetic resonance.

    Science.gov (United States)

    Maltsev, Sergey; Lorigan, Gary A

    2011-10-01

    Membrane proteins represent a challenging class of biological systems to study. They are extremely difficult to crystallize and in most cases they retain their structure and functions only in membrane environments. Therefore, commonly used diffraction methods fail to give detailed molecular structure and other approaches have to be utilized to obtain biologically relevant information. Nuclear magnetic resonance (NMR) spectroscopy, however, can provide powerful structural and dynamical constraints on these complicated systems. Solution- and solid-state NMR are powerful methods for investigating membrane proteins studies. In this work, we briefly review both solution and solid-state NMR techniques for membrane protein studies and illustrate the applications of these methods to elucidate proteins structure, conformation, topology, dynamics, and function. Recent advances in electronics, biological sample preparation, and spectral processing provided opportunities for complex biological systems, such as membrane proteins inside lipid vesicles, to be studied faster and with outstanding quality. New analysis methods therefore have emerged, that benefit from the combination of sample preparation and corresponding specific high-end NMR techniques, which give access to more structural and dynamic information.

  16. Airway epithelial inflammation-induced endoplasmic reticulum Ca2+ store expansion is mediated by X-box binding protein-1.

    Science.gov (United States)

    Martino, Mary E B; Olsen, John C; Fulcher, Nanette B; Wolfgang, Matthew C; O'Neal, Wanda K; Ribeiro, Carla M P

    2009-05-29

    Inflamed cystic fibrosis (CF) human bronchial epithelia (HBE), or normal HBE exposed to supernatant from mucopurulent material (SMM) from CF airways, exhibit endoplasmic reticulum (ER)/Ca(2+) store expansion and amplified Ca(2+)-mediated inflammation. HBE inflammation triggers an unfolded protein response (UPR) coupled to mRNA splicing of X-box binding protein-1 (XBP-1). Because spliced XBP-1 (XBP-1s) promotes ER expansion in other cellular models, we hypothesized that XBP-1s is responsible for the ER/Ca(2+) store expansion in inflamed HBE. XBP-1s was increased in freshly isolated infected/inflamed CF in comparison with normal HBE. The link between airway epithelial inflammation, XBP-1s, and ER/Ca(2+) store expansion was then addressed in murine airways challenged with phosphate-buffered saline or Pseudomonas aeruginosa. P. aeruginosa-challenged mice exhibited airway epithelial ER/Ca(2+) store expansion, which correlated with airway inflammation. P. aeruginosa-induced airway inflammation triggered XBP-1s in ER stress-activated indicator (ERAI) mice. To evaluate the functional role of XBP-1s in airway inflammation linked to ER/Ca(2+) store expansion, control, XBP-1s, or dominant negative XBP-1 (DN-XBP-1) stably expressing 16HBE14o(-) cell lines were used. Studies with cells transfected with an unfolded protein response element (UPRE) luciferase reporter plasmid confirmed that the UPRE was activated or inhibited by expression of XBP-1s or DN-XBP-1, respectively. Expression of XBP-1s induced ER/Ca(2+) store expansion and potentiated bradykinin-increased interleukin (IL)-8 secretion, whereas expression of DN-XBP-1 inhibited bradykinin-dependent IL-8 secretion. In addition, expression of DN-XBP-1 blunted SMM-induced ER/Ca(2+) store expansion and SMM-induced IL-8 secretion. These findings suggest that, in inflamed HBE, XBP-1s is responsible for the ER/Ca(2+) store expansion that confers amplification of Ca(2+)-dependent inflammatory responses.

  17. Early localization of NPA58, a rat nuclear pore-associated protein, to the reforming nuclear envelope during mitosis

    Indian Academy of Sciences (India)

    Radhika Ganeshan; Nandini Rangaraj; Veena K Parnaik

    2001-03-01

    We have studied the mitotic reassembly of the nuclear envelope, using antibodies to nuclear marker proteins and NPA58 in F-111 rat fibroblast cells. In earlier studies we have proposed that NPA58, a 58 kDa rat nuclear protein, is involved in nuclear protein import. In this report, NPA58 is shown to be localized on the cytoplasmic face of the envelope in interphase cells, in close association with nuclear pores. In mitotic cells NPA58 is dispersed in the cytoplasm till anaphase. 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 localization of NPA58 in the newly-formed envelope. The early targeting of NPA58 is consistent with its proposed role in nuclear transport.

  18. A pollen-expressed gene for a novel protein with an F-box motif that is very tightly linked to a gene for S-RNase in two species of cherry, Prunus cerasus and P. avium.

    Science.gov (United States)

    Yamane, Hisayo; Ikeda, Kazuo; Ushijima, Koichiro; Sassa, Hidenori; Tao, Ryutaro

    2003-07-01

    This study describes a novel F-box protein gene in the S-locus of sour cherry (Prunus cerasus) and sweet cherry (P. avium). The gene showed an S-haplotype-specific sequence polymorphism and the expression was specific to pollen. Genomic DNA blot analysis of eight sweet cherry cultivars with the probe for the F-box protein gene under low stringency conditions yielded RFLP bands specific to the S-haplotypes of each cultivar. We discuss the possibility of the gene for the F-box protein being a candidate for the male determinant of gametophytic self-incompatibility in PRUNUS:

  19. Differential response of multiple zebrafish hepatic F-box protein genes to 17α-ethinylestradiol treatment

    Institute of Scientific and Technical Information of China (English)

    Hongping Zheng; Shifeng Li; Zhili Wu; Yunbin Zhang; Shengnan Hu; Yuanchang Yan; Yiping Li

    2011-01-01

    Estrogens are accumulating in environment and their effects on a variety of reproductive processes and tumorigenesis were reported by previous study, but the mechanism of estrogen promoting neoplasia was still not clear. F-box protein (FBP) is the component of E3 ubiquitin ligase which takes part in a variety of key biological processes. In this study, using mature male zebrafish, which are more sensitive to estrogen treatment, we examined influence of 17α-ethinylestradiol (EE2) exposure on the expression of a series of hepatic FBP genes, which take part in a variety of biological processes, including tumorigenesis. The influence of EE2 on the expression of hepatic mRNA concentrations of FBP genes were quantified based on the expression of the optimal internal control gene in male zebrafish after 7-day exposure to EE2, from a low-dose concentration (1 ng/L) to environmentally relevant concentrations (10, 100 ng/L). Our results showed that EE2 exposure reduced the expression offbxll4a, fbxl14b, fbxo25 and β-TRCP2b, but enchanced the expression of skp2. While the alterations infbxl2, fbxw7,fbxo9, β-TRCP2a, fbxll8 andfbxo45 mRNA levels were not observed after EE2 exposure. Thus, our results showed that the expression of hepatic FBP genes exhibited differentially in male zebrafish exposed EE2. The changes of the expression level of FBP genes induced by EE2 may be an important clue to elucidate the correlations of estrogen and hepatic tumors.

  20. 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.

  1. 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

    Rad54 is an ATP-driven translocase involved in the genome maintenance pathway of homologous recombination (HR). Although its activity has been implicated in several steps of HR, its exact role(s) at each step are still not fully understood. We have identified a new interaction between Rad54...... and the replicative DNA clamp, proliferating cell nuclear antigen (PCNA). This interaction was only mildly weakened by the mutation of two key hydrophobic residues in the highly-conserved PCNA interaction motif (PIP-box) of Rad54 (Rad54-AA). Intriguingly, the rad54-AA mutant cells displayed sensitivity to DNA damage...

  2. High-mobility group box-1 protein (HMGB1) is increased in antineutrophilic cytoplasmatic antibody (ANCA)-associated vasculitis with renal manifestations.

    Science.gov (United States)

    Bruchfeld, Annette; Wendt, Mårten; Bratt, Johan; Qureshi, Abdul R; Chavan, Sangeeta; Tracey, Kevin J; Palmblad, Karin; Gunnarsson, Iva

    2011-01-01

    High-mobility group box 1 (HMGB1) is a nuclear and cytosolic protein that is increasingly recognized as an important proinflammatory mediator actively secreted from monocytes and macrophages and passively released from necrotic cells. In antineutrophilic cytoplasmatic antibody (ANCA)-associated vasculitis (AAV), the kidneys are commonly affected vital organs, characterized by focal necrotizing and/or crescentic pauci-immune glomerulonephritis. The aim of the study was to determine whether HMGB1 serum levels are elevated in AAV with renal manifestations. A total of 30 AAV patients (16 female and 14 male; median age 59 years, range 17-82) with Wegener granulomatosis, microscopic polyangiitis and Churg-Strauss syndrome with available renal biopsies and serum samples were included. In seven cases, serum was also obtained at rebiopsy in remission. HMGB1 was analyzed with Western blot. Birmingham Vasculitis Activity Score (BVAS, version 2003), C-reactive protein (CRP), erythrocyte sedimentation rate (ESR), urinanalysis, creatinine, estimated glomerular filtration rate, sex and age were included in the analysis. Twenty-five episodes of biopsy-proven active disease with BVAS 17.9 ± 4.6 and 13 cases with inactive biopsies and BVAS 2.3 ± 3.7 (P = 0.0001) were identified. CRP, ESR, hematuria and proteinuria were significantly higher in active cases. HMGB1 was significantly elevated (P = 0.01) comparing active with inactive cases (120 ± 48 versus 78 ± 46 ng/mL) and significantly lower in the seven control patients (P = 0.03) at rebiopsy in remission. HMGB1 remained higher in inactive cases compared with historic healthy controls (10.9 ± 10.5 ng/mL). HMGB1 levels did not differ significantly between AAV subgroups. CRP and ESR did not correlate with HMGB1. HMGB1 is significantly increased in AAV with renal involvement. Residual HMGB1 elevation in remission could possibly reflect low-grade inflammatory activity or tissue damage. Future studies may further reveal whether HMGB

  3. Identifying Protein-Protein Associations at the Nuclear Envelope with BioID.

    Science.gov (United States)

    Kim, Dae In; Jensen, Samuel C; Roux, Kyle J

    2016-01-01

    The nuclear envelope (NE) is a critical cellular structure whose constituents and roles in a myriad of cellular processes seem ever expanding. To determine the underlying mechanisms by which the NE constituents participate in various cellular events, it is necessary to understand the nature of their protein-protein associations. BioID (proximity-dependent biotin identification) is a recently established method to generate a history of protein-protein associations as they occur over time in living cells. BioID is based on fusion of a bait protein to a promiscuous biotin ligase. Expression of the BioID fusion protein in a relevant cellular environment enables biotinylation of vicinal and interacting proteins of the bait protein, permitting isolation and identification by conventional biotin-affinity capture and mass-spec analysis. In this way, BioID provides unique capabilities to identify protein-protein associations at the NE. In this chapter we provide a detailed protocol for the application of BioID to the study of NE proteins.

  4. Identification of four nuclear transport signal-binding proteins that interact with diverse transport signals.

    Science.gov (United States)

    Yamasaki, L; Kanda, P; Lanford, R E

    1989-07-01

    The transport of proteins into the nucleus requires not only the presence of a nuclear transport signal on the targeted protein but also the signal recognition proteins and the nuclear pore translocation apparatus. Complicating the search for the signal recognition proteins is the fact that the nuclear transport signals identified share little obvious homology. In this study, synthetic peptides homologous to the nuclear transport signals from the simian virus 40 large T antigen, Xenopus oocyte nucleoplasmin, adenovirus E1A, and Saccharomyces cerevisiae MAT alpha 2 proteins were coupled to a UV-photoactivable cross-linker and iodinated for use in an in vitro cross-linking reaction with cellular lysates. Four proteins, p140, p100, p70, and p55, which specifically interacted with the nuclear transport signal peptides were identified. Unique patterns of reactivity were observed with closely related pairs of nuclear transport signal peptides. Competition experiments with labeled and unlabeled peptides demonstrated that heterologous signals were able to bind the same protein and suggested that diverse signals use a common transport pathway. The subcellular distribution of the four nuclear transport signal-binding proteins suggested that nuclear transport involves both cytoplasmic and nuclear receptors. The four proteins were not bound by wheat germ agglutinin and were not associated tightly with the nuclear pore complex.

  5. Identification of a Ubiquitin-Binding Structure in the S-Locus F-Box Protein Controlling S-RNase-Based Self-Incompatibility

    Institute of Scientific and Technical Information of China (English)

    Guang Chen; Bin Zhang; Lijing Liu; Qun Li; Yu'e Zhang; Qi Xie; Yongbiao Xue

    2012-01-01

    In flowering plants,self-incompatibility (SI) serves as an important intraspecific reproductive barrier to promote outbreeding.In species from the Solanaceae,Plantaginaceae and Rosaceae,S-RNase and SLF (S-locus F-box) proteins have been shown to control the female and male specificity of SI,respectively.However,little is known about structure features of the SLF protein apart from its conserved F-box domain.Here we show that the SLF C-terminal region possesses a novel ubiquitin-binding domain (UBD) structure conserved among the SLF protein family.By using an ex vivo system of Nicotiana benthamiana,we found that the UBD mediates the SLF protein turnover by the ubiquitin-proteasome pathway.Furthermore,we detected that the SLF protein was directly involved in S-RNase degradation.Taken together,our results provide a novel insight into the SLF structure and highlight a potential role of SLF protein stability and degradation in S-RNase-based self-incompatibility.

  6. 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.

  7. Aberrant expression of nuclear matrix proteins during HMBA-induced differentiation of gastric cancer cells

    Institute of Scientific and Technical Information of China (English)

    2010-01-01

    AIM: To investigate the aberrant expression of nuclear matrix proteins in human gastric cancer cells before and after hexamethylene bisacetamide (HMBA) treatment.METHODS: Proteomics analysis of differential nuclear matrix proteins was performed by two dimensional electrophoresis polyacrylamide gel electrophoresis and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.The expression levels of three nuclear matrix proteins were further confirmed by Western blotting and their location...

  8. Identification of an E-box DNA binding protein, activated protein 4, and its function in regulating the expression of the gene encoding diapause hormone and pheromone biosynthesis-activating neuropeptide in Helicoverpa armigera.

    Science.gov (United States)

    Hu, C-H; Hong, B; Xu, W-H

    2010-04-01

    Activated protein 4 (AP-4), an E-box DNA-binding protein, was cloned from the cotton bollworm, Helicoverpa armigera (Har). The expression of Har-AP-4 mRNA and the protein that it encodes are significantly higher in nondiapause pupae than in diapause pupae. In vitro-translated Har-AP-4 can bind specifically to the E-box motif on the promoter of the diapause hormone and pheromone biosynthesis-activating neuropeptide (DH-PBAN). Har-AP-4, fused with the green fluorescent protein (GFP), is localized to the nucleus, and overexpression of Har-AP-4 can significantly activate the promoter of the DH-PBAN gene that is involved in nondiapause pupal development in H. armigera. These results suggest that Har-AP-4, which binds to the promoter of DH-PBAN, may play a role in regulating pupal development in H. armigera.

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

    Science.gov (United States)

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

    2014-01-01

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

  10. 压水堆核电站循环冷却水泵齿轮箱传动设计研究%Design of Circulating Pump Gear Box for Pressurized Water Reator Nuclear Power Plant

    Institute of Scientific and Technical Information of China (English)

    邢志伟

    2011-01-01

    The development of nuclear power is our inevitable choice for sustainable economic development, but also is it a very arduous and difficult task. Based on pressurized water reactor nuclear power plant, the basic characteristics, domestic difficulties and development of the circulating pump gear box are introduced. Then, the transmission scheme of the gear box is discussed, involving the determination of basic parameters, design of contained bodies, selection and heat treatment of major components and so on. Finally, the future of the gear box for nuclear power is prospected.%基于压水堆核电站,介绍了循环水泵用齿轮箱减速器的基本特点、国产化难点及国内发展概况.讨论了核电用齿轮箱的传动方案设计,涉及基本参数的确定、均载机构的设计、主要零件的选材与热处理等.最后展望核电用齿轮箱的发展前景.

  11. Subcellular Localization of the S Locus F-box Protein AhSLF-S2 in Pollen and Pollen Tubes of Self-Incompatible Antirrhinum

    Institute of Scientific and Technical Information of China (English)

    Hong-Yun WANG; Yong-Biao XUE

    2005-01-01

    The distribution of the S locus F-box (SLF) protein was examined by immunocytochemistry and Western blot techniques using an antibody against the C-terminal part of AhSLF-S2 in self-incompatible lines of Antirrhinum. Abundant gold particles were found where pollen tubes emerge in vitro. With the elongation of pollen tubes, binding sites for the antibody were found in the cytoplasm of the pollen tubes,including the peripheral part of the endoplasmic reticulum. After germination in vitro for 16 h, the product of AhSLF-S2 and possibly its allelic products could still be detectable, implying that the SLF protein has a role in the elongating process of pollen tubes. The present study provides evidence at the protein level that the SLF protein is present in pollen cytoplasm during pollen tube growth. These findings are discussed, as is their potential role in the self-incompatible response in Antirrhinum.

  12. 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

    -protein interaction sites. Of these, one specifically interacts with NRs bound to their agonistic ligand and not with NR mutants that are defective in the AF-2 activity. Immediately adjacent to this 'NR box', TIF1alpha contains an interaction site for members of the chromatin organization modifier (chromo) family, HP...

  13. The centromeric/nucleolar chromatin protein ZFP-37 may function to specify neuronal nuclear domains

    NARCIS (Netherlands)

    E. Payen; T. Verkerk (Ton); D. Michalovich (David); S.D. Dreyer; A. Winterpacht; B. Lee (Brendan); C.I. de Zeeuw (Chris); N.J. Galjart (Niels); F.G. Grosveld (Frank)

    1998-01-01

    textabstractMurine ZFP-37 is a member of the large family of C2H2 type zinc finger proteins. It is characterized by a truncated NH2-terminal Kruppel-associated box and is thought to play a role in transcriptional regulation. During development Zfp-37 mRNA is most abunda

  14. Einstein's Boxes

    CERN Document Server

    Norsen, T

    2005-01-01

    At the 1927 Solvay conference, Einstein presented a thought experiment intended to demonstrate the incompleteness of the quantum mechanical description of reality. In the following years, the thought experiment was picked up and modified by Einstein, de Broglie, and several other commentators into a simple scenario involving the splitting in half of the wave function of a single particle in a box. In this paper we collect together several formulations of this thought experiment from the existing literature; analyze and assess it from the point of view of the Einstein-Bohr debates, the EPR dilemma, and Bell's theorem; and generally lobby for Einstein's Boxes taking its rightful place alongside similar but historically better-known quantum mechanical thought experiments such as EPR and Schroedinger's Cat.

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

    Science.gov (United States)

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

    2015-03-01

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

  16. Genetically engineered mouse models for functional studies of SKP1-CUL1-F-box-protein (SCF) E3 ubiquitin ligases

    Institute of Scientific and Technical Information of China (English)

    Weihua Zhou; Wenyi Wei; Yi Sun

    2013-01-01

    The SCF (SKP1 (S-phase-kinase-associated protein 1),Cullin-1,F-box protein) E3 ubiquitin ligases,the founding member of Cullin-RING ligases (CRLs),are the largest family of E3 ubiquitin ligases in mammals.Each individual SCF E3 ligase consists of one adaptor protein SKP1,one scaffold protein cullin-1 (the first family member of the eight cullins),one F-box protein out of 69 family members,and one out of two RING (Really Interesting New Gene) family proteins RBX1/ROC1 or RBX2/ROC2/SAG/RNF7.Various combinations of these four components construct a large number of SCF E3s that promote the degradation of many key regulatory proteins in cell-context,temporally,and spatially dependent manners,thus controlling precisely numerous important cellular processes,including cell cycle progression,apoptosis,gene transcription,signal transduction,DNA replication,maintenance of genome integrity,and tumorigenesis.To understand how the SCF E3 ligases regulate these cellular processes and embryonic development under in vivo physiological conditions,a number of mouse models with transgenic (Tg) expression or targeted deletion of components of SCF have been established and characterized.In this review,we will provide a brief introduction to the ubiquitin-proteasome system (UPS) and the SCF E3 ubiquitin ligases,followed by a comprehensive overview on the existing Tg and knockout (KO) mouse models of the SCF E3s,and discuss the role of each component in mouse embryogenesis,cell proliferation,apoptosis,carcinogenesis,as well as other pathogenic processes associated with human diseases.We will end with a brief discussion on the future directions of this research area and the potential applications of the knowledge gained to more effective therapeutic interventions of human diseases.

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

    OpenAIRE

    2015-01-01

    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 e...

  18. A motif unique to the human DEAD-box protein DDX3 is important for nucleic acid binding, ATP hydrolysis, RNA/DNA unwinding and HIV-1 replication.

    Directory of Open Access Journals (Sweden)

    Anna Garbelli

    Full Text Available DEAD-box proteins are enzymes endowed with nucleic acid-dependent ATPase, RNA translocase and unwinding activities. The human DEAD-box protein DDX3 has been shown to play important roles in tumor proliferation and viral infections. In particular, DDX3 has been identified as an essential cofactor for HIV-1 replication. Here we characterized a set of DDX3 mutants biochemically with respect to nucleic acid binding, ATPase and helicase activity. In particular, we addressed the functional role of a unique insertion between motifs I and Ia of DDX3 and provide evidence for its implication in nucleic acid binding and HIV-1 replication. We show that human DDX3 lacking this domain binds HIV-1 RNA with lower affinity. Furthermore, a specific peptide ligand for this insertion selected by phage display interferes with HIV-1 replication after transduction into HelaP4 cells. Besides broadening our understanding of the structure-function relationships of this important protein, our results identify a specific domain of DDX3 which may be suited as target for antiviral drugs designed to inhibit cellular cofactors for HIV-1 replication.

  19. A motif unique to the human DEAD-box protein DDX3 is important for nucleic acid binding, ATP hydrolysis, RNA/DNA unwinding and HIV-1 replication.

    Science.gov (United States)

    Garbelli, Anna; Beermann, Sandra; Di Cicco, Giulia; Dietrich, Ursula; Maga, Giovanni

    2011-05-12

    DEAD-box proteins are enzymes endowed with nucleic acid-dependent ATPase, RNA translocase and unwinding activities. The human DEAD-box protein DDX3 has been shown to play important roles in tumor proliferation and viral infections. In particular, DDX3 has been identified as an essential cofactor for HIV-1 replication. Here we characterized a set of DDX3 mutants biochemically with respect to nucleic acid binding, ATPase and helicase activity. In particular, we addressed the functional role of a unique insertion between motifs I and Ia of DDX3 and provide evidence for its implication in nucleic acid binding and HIV-1 replication. We show that human DDX3 lacking this domain binds HIV-1 RNA with lower affinity. Furthermore, a specific peptide ligand for this insertion selected by phage display interferes with HIV-1 replication after transduction into HelaP4 cells. Besides broadening our understanding of the structure-function relationships of this important protein, our results identify a specific domain of DDX3 which may be suited as target for antiviral drugs designed to inhibit cellular cofactors for HIV-1 replication.

  20. 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.

  1. 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.

  2. 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

    2017-01-30

    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.

  3. Preparation of the Human Cytomegalovirus Nuclear Egress Complex and Associated Proteins.

    Science.gov (United States)

    Sharma, Mayuri; Kamil, Jeremy P; Coen, Donald M

    2016-01-01

    Herpesviruses, like most DNA viruses, replicate their genomes in the host cell nucleus. Their DNA is then packaged and assembled into viral nucleocapsids, which, in most cases, are too large to pass through the nuclear pore complex. Instead, herpesviruses use a complex multistep pathway, termed nuclear egress, to exit the nucleus. Key players in this process include two conserved viral proteins that form the nuclear egress complex (NEC). In human cytomegalovirus, these NEC proteins are UL50, embedded in the inner nuclear membrane, and its nucleoplasmic partner UL53. Both are essential for viral nuclear egress. However, other viral components as well as host nuclear envelope proteins may also participate in nuclear egress. Identifying these viral and cellular factors may provide important insight into the herpesvirus lifecycle and its relationship to the underlying, yet still-mysterious, host nuclear egress pathway. We developed an immunoprecipitation-based protocol, described herein, to identify protein-protein interactions involving the NEC from the nuclear fraction of infected cells that express an epitope-tagged version of NEC subunit UL53.

  4. PABPN1 overexpression leads to upregulation of genes encoding nuclear proteins that are sequestered in oculopharyngeal muscular dystrophy nuclear inclusions.

    Science.gov (United States)

    Corbeil-Girard, Louis-Philippe; Klein, Arnaud F; Sasseville, A Marie-Josée; Lavoie, Hugo; Dicaire, Marie-Josée; Saint-Denis, Anik; Pagé, Martin; Duranceau, André; Codère, François; Bouchard, Jean-Pierre; Karpati, George; Rouleau, Guy A; Massie, Bernard; Langelier, Yves; Brais, Bernard

    2005-04-01

    Oculopharyngeal muscular dystrophy (OPMD) is an adult-onset disease caused by expanded (GCN)12-17 stretches encoding the N-terminal polyalanine domain of the poly(A) binding protein nuclear 1 (PABPN1). OPMD is characterized by intranuclear inclusions (INIs) in skeletal muscle fibers, which contain PABPN1, molecular chaperones, ubiquitin, proteasome subunits, and poly(A)-mRNA. We describe an adenoviral model of PABPN1 expression that produces INIs in most cells. Microarray analysis revealed that PABPN1 overexpression reproducibly changed the expression of 202 genes. Sixty percent of upregulated genes encode nuclear proteins, including many RNA and DNA binding proteins. Immunofluorescence microscopy revealed that all tested nuclear proteins encoded by eight upregulated genes colocalize with PABPN1 within the INIs: CUGBP1, SFRS3, FKBP1A, HMG2, HNRPA1, PRC1, S100P, and HSP70. In addition, CUGBP1, SFRS3, and FKBP1A were also found in OPMD muscle INIs. This study demonstrates that a large number of nuclear proteins are sequestered in OPMD INIs, which may compromise cellular function.

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

    Energy Technology Data Exchange (ETDEWEB)

    Sakamoto, Hikaru [Department of Bioproduction, Faculty of Bioindustry, Tokyo University of Agriculture, 196 Yasaka, Abashiri-shi, Hokkaido 093-2422 (Japan); Sakata, Keiko; Kusumi, Kensuke [Department of Biology, Faculty of Sciences, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581 (Japan); Kojima, Mikiko; Sakakibara, Hitoshi [RIKEN Plant Science Center, 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama 230-0045 (Japan); Iba, Koh, E-mail: koibascb@kyushu-u.org [Department of Biology, Faculty of Sciences, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581 (Japan)

    2012-06-29

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

  6. Tandem affinity purification to identify cytosolic and nuclear gβγ-interacting proteins.

    Science.gov (United States)

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

    2015-01-01

    It has become clear in recent years that the Gβγ subunits of heterotrimeric proteins serve broad roles in the regulation of cellular activity and interact with many proteins in different subcellular locations including the nucleus. Protein affinity purification is a common method to identify and confirm protein interactions. When used in conjugation with mass spectrometry it can be used to identify novel protein interactions with a given bait protein. The tandem affinity purification (TAP) technique identifies partner proteins bound to tagged protein bait. Combined with protocols to enrich the nuclear fraction of whole cell lysate through sucrose cushions, TAP allows for purification of interacting proteins found specifically in the nucleus. Here we describe the use of the TAP technique on cytosolic and nuclear lysates to identify candidate proteins, through mass spectrometry, that bind to Gβ1 subunits.

  7. Alteration of nuclear matrix-intermediate filament system and differential expression of nuclear matrix proteins during human hepatocarcinoma cell differentiation

    Institute of Scientific and Technical Information of China (English)

    Jian Tang; Jing-Wen Niu; Dong-Hui Xu; Zhi-Xing Li; Qi-Fu Li; Jin-An Chen

    2007-01-01

    AIM:To investigate the association between the configurational and compositional changes of nuclear matrix and the differentiation of carcinoma cells.METHODS: Cells cultured with or without 5 × 10-3mmol/L of hexamethylene bisacetamide (HMBA) on Nickel grids were treated by selective extraction and prepared for whole mount observation under electron microscopy. The samples were examined under transmission electron microscope. Nuclear matrix proteins were selectively extracted and subjected to subcellular proteomics study. The protein expression patterns were analyzed by PDQuest software. Spots of differentially expressed nuclear matrix proteins were excised and subjected to in situ digestion with trypsin.The peptides were analyzed by matrix-assisted laserdesorption/ionization time of flight mass spectrometry (MALDI-TOF-MS). Data were submitted for database searching using Mascot tool (www. Matrixscience.com).RESULTS: The nuclear matrix (NM) and intermediate filament (IF) in SMMC-7721 hepatocarcinoma cells were found relatively sparse and arranged irregularly.The nuclear lamina was non-uniform, and two kinds of filaments were not tightly connected. After induction for differentiation by HMBA, the NM-IF filaments were concentrated and distributed uniformly. The heterogeneous population of filaments, including highly branched utrathin filaments could also be seen in the regular meshwork. The connection between the two kinds of filaments and the relatively thin, condensed and sharply demarcated lamina composed of intermediatesized filaments was relatively fastened. Meanwhile, 21NM proteins changed remarkably during SMMC-7721cell differentiation. Four proteins, I.e. Mutant Pyst1,hypothetical protein, nucleophosmin1, and LBP were downregulated, whereas four other proteins, eIF6, p44subunit, β-tubulin, and SIN3B were upregulated with the last one, SR2/ASF found only in the differentiated SMMC-7721 cells.CONCLUSION: The induced differentiation of SMMC-7721cells by HMBA is

  8. 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...

  9. Main: BOX1PSGS2 [PLACE

    Lifescience Database Archive (English)

    Full Text Available BOX1PSGS2 S000222 19-August-2004 (last modified) kehi Box 1 element in pea (P.s.) glutamine synthetase (GS...2) gene; An element in a 33-bp AT-rich sequence (box 1) of the 5' end of a GS2 promot...er; Located at -837 to -827 of pea GS2; Multimer of box 1 element was used to isolate a cDNA encoding an AT-...rich DNA binding protein (ATBP-1) (Tjaden & Coruzzi, 1994); Box 1; glutamine synthetase; GS2; ATBp; ATBP-1; pea (Pisum sativum) ATAGAAATCAA ...

  10. Altered expression of nuclear matrix proteins in etoposide induced apoptosis in HL-60 cells

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    The events of cell death and the expression of nuclear matrix protein(NMP)have been investigated in a promyelocytic leukemic cell line HL-60 induced with etoposide.By means of TUNEL assay,the nuclei displayed a characteristic morphology change,and the amount of apoptotic cells increased early and reached maximun about 39% after treatment with etoposide for 2 h.Nucleosomal DNA fragmentation was observed after treatment for 4 h.The morphological change of HL-60 cells,thus,occurred earlier than the appearance of DNA ladder.Total nuclear matrix proteins were analyzed by 2-dimensional gel electrophoresis.Differential expression of 59 nuclear matrix proteins was found in 4 h etoposide treated cells.Western blotting was then performed on three nuclear matrix acssociated proteins,PML,HSC70 and NuMA.The expression of the suppressor PML protein and heat shock protein HSC70 were significantly upregulated after etoposide treatment,while NuMA,a nuclear mitotic apparatus protein,was down regulated.These results demonstrate that significant biochemical alterations in nuclear matrix proteins take place during the apoptotic process.

  11. Poly(ADP-ribosyl)ation of proteins associated with nuclear matrix in rat testis

    Energy Technology Data Exchange (ETDEWEB)

    Quesada, P.; Atorino, L.; Faraone-Mennella, M.R.; Farina, B. [Naples Univ. (Italy); Caiafa, P. [Rome Univ. (Italy)

    1995-12-31

    We have previously demonstrated that a significant percentage of poly(ADPR)polymerase is present, as a tightly-bound form, at the third level of chromatin organization defined by chromosomal loops and nuclear matrix. The present work is focused on the study of poly(ADP-ribosyl)ation of proteins present in these nuclear subfractions. It has been shown that, due to the action of poly(ADPR) polymerase, the ADP-ribose moiety of [{sup 14}C]NAD is transferred to both loosely-bound and tightly-bound chromosomal proteins, which in consequence are modified by chain polymers of ADP-ribose of different lengths. Moreover, histone-like proteins seem to be ADP-ribosylated in chromosomal loops and nuclear matrix associated regions of DNA loops (MARS). A hypothesis can be put forward that the ADP-ribosylation system is functionally related to the nuclear processes, actively coordinated by the nuclear matrix. (author). 34 refs, 4 figs.

  12. The proteins of intra-nuclear bodies: a data-driven analysis of sequence, interaction and expression

    Directory of Open Access Journals (Sweden)

    Bodén Mikael

    2010-04-01

    Full Text Available Abstract Background Cajal bodies, nucleoli, PML nuclear bodies, and nuclear speckles are morpohologically distinct intra-nuclear structures that dynamically respond to cellular cues. Such nuclear bodies are hypothesized to play important regulatory roles, e.g. by sequestering and releasing transcription factors in a timely manner. While the nucleolus and nuclear speckles have received more attention experimentally, the PML nuclear body and the Cajal body are still incompletely characterized in terms of their roles and protein complement. Results By collating recent experimentally verified data, we find that almost 1000 proteins in the mouse nuclear proteome are known to associate with one or more of the nuclear bodies. Their gene ontology terms highlight their regulatory roles: splicing is confirmed to be a core activity of speckles and PML nuclear bodies house a range of proteins involved in DNA repair. We train support-vector machines to show that nuclear proteins contain discriminative sequence features that can be used to identify their intra-nuclear body associations. Prediction accuracy is highest for nucleoli and nuclear speckles. The trained models are also used to estimate the full protein complement of each nuclear body. Protein interactions are found primarily to link proteins in the nuclear speckles with proteins from other compartments. Cell cycle expression data provide support for increased activity in nucleoli, nuclear speckles and PML nuclear bodies especially during S and G2 phases. Conclusions The large-scale analysis of the mouse nuclear proteome sheds light on the functional organization of physically embodied intra-nuclear compartments. We observe partial support for the hypothesis that the physical organization of the nucleus mirrors functional modularity. However, we are unable to unambiguously identify proteins' intra-nuclear destination, suggesting that critical drivers behind of intra-nuclear translocation are yet to

  13. Identification of nuclear phosphatidylinositol 4,5-bisphosphate-interacting proteins by neomycin extraction.

    Science.gov (United States)

    Lewis, Aurélia E; Sommer, Lilly; Arntzen, Magnus Ø; Strahm, Yvan; Morrice, Nicholas A; Divecha, Nullin; D'Santos, Clive S

    2011-02-01

    Considerable insight into phosphoinositide-regulated cytoplasmic functions has been gained by identifying phosphoinositide-effector proteins. Phosphoinositide-regulated nuclear functions however are fewer and less clear. To address this, we established a proteomic method based on neomycin extraction of intact nuclei to enrich for nuclear phosphoinositide-effector proteins. We identified 168 proteins harboring phosphoinositide-binding domains. Although the vast majority of these contained lysine/arginine-rich patches with the following motif, K/R-(X(n= 3-7)-K-X-K/R-K/R, we also identified a smaller subset of known phosphoinositide-binding proteins containing pleckstrin homology or plant homeodomain modules. Proteins with no prior history of phosphoinositide interaction were identified, some of which have functional roles in RNA splicing and processing and chromatin assembly. The remaining proteins represent potentially other novel nuclear phosphoinositide-effector proteins and as such strengthen our appreciation of phosphoinositide-regulated nuclear functions. DNA topology was exemplar among these: Biochemical assays validated our proteomic data supporting a direct interaction between phosphatidylinositol 4,5-bisphosphate and DNA Topoisomerase IIα. In addition, a subset of neomycin extracted proteins were further validated as phosphatidyl 4,5-bisphosphate-interacting proteins by quantitative lipid pull downs. In summary, data sets such as this serve as a resource for a global view of phosphoinositide-regulated nuclear functions.

  14. DEAD-box protein DDX3 associates with eIF4F to promote translation of selected mRNAs.

    Science.gov (United States)

    Soto-Rifo, Ricardo; Rubilar, Paulina S; Limousin, Taran; de Breyne, Sylvain; Décimo, Didier; Ohlmann, Théophile

    2012-09-12

    Here, we have characterized a step in translation initiation of viral and cellular mRNAs that contain RNA secondary structures immediately at the vicinity of their m(7)GTP cap. This is mediated by the DEAD-box helicase DDX3 which can directly bind to the 5' of the target mRNA where it clamps the entry of eIF4F through an eIF4G and Poly A-binding protein cytoplasmic 1 (PABP) double interaction. This could induce limited local strand separation of the secondary structure to allow 43S pre-initiation complex attachment to the 5' free extremity of the mRNA. We further demonstrate that the requirement for DDX3 is highly specific to some selected transcripts, cannot be replaced or substituted by eIF4A and is only needed in the very early steps of ribosome binding and prior to 43S ribosomal scanning. Altogether, these data define an unprecedented role for a DEAD-box RNA helicase in translation initiation.

  15. Fludarabine nucleoside modulates nuclear "survival and death" proteins in resistant chronic lymphocytic leukemia cells.

    Science.gov (United States)

    Henrich, Silke; Mactier, Swetlana; Best, Giles; Mulligan, Stephen P; Crossett, Ben; Christopherson, Richard Ian

    2011-12-01

    The nuclear mechanisms by which fludarabine nucleoside (F-ara-A) induces apoptosis have been investigated in human MEC1 cells derived from B-cell chronic lymphocytic leukemia. Upon treatment of cells with F-ara-A (100 μM, 72 hours), 15 nuclear proteins changed in abundance by more than 2-fold. Nuclear proteins up-regulated included calmodulin (4.3-fold), prohibitin (3.9-fold), β-actin variant (3.7-fold), and structure-specific recognition protein 1 (3.7-fold); those down-regulated included 60S ribosomal protein P2B (0.12-fold), fumarate hydratase (0.19-fold), splicing factor arginine/serine-rich 3 (0.35-fold), and replication protein A2 (0.42-fold). These changes in the levels of specific proteins promote survival or apoptosis; because the end result is apoptosis of MEC1 cells, apoptotic effects predominate.

  16. 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.

  17. Higher-order nuclear organization in growth arrest of human mammary epithelial cells: a novel role for telomere-associated protein TIN2

    DEFF Research Database (Denmark)

    Kaminker, P.; Plachot, C.; Kim, SH.;

    2005-01-01

    Nuclear structure, Three-dimensional culture, Breast, Morphogenesis, Quiescence, Heterochromatin protein 1......Nuclear structure, Three-dimensional culture, Breast, Morphogenesis, Quiescence, Heterochromatin protein 1...

  18. Dimerization and nuclear entry of mPER proteins in mammalian cells

    NARCIS (Netherlands)

    K. Yagita (Kazuhiro); S. Yamaguchi; F. Tamanini (Filippo); A. Yasui (Akira); J.J. Loros; J.C. Dunlap; H. Okamura (Hitoshi); J.H.J. Hoeijmakers (Jan); G.T.J. van der Horst (Gijsbertus)

    2000-01-01

    textabstractNuclear entry of circadian oscillatory gene products is a key step for the generation of a 24-hr cycle of the biological clock. We have examined nuclear import of clock proteins of the mammalian period gene family and the effect of serum shock, which induces a synchrono

  19. Manufacture Process of Steel Box Girder for RP Gantry in Some Nuclear Power Station%某核电站RP龙门架箱形钢结构梁制作工艺

    Institute of Scientific and Technical Information of China (English)

    展之芳

    2014-01-01

    In steel structure project, fabrication process of steel box girder is complex, so it is difficult to control welding deformation. This paper introduced the fabrication process of steel box girder for RP gantry in nuclear power station from the material, assembly, welding and correction. The fabrication process ensures the overall geometry of box girder, and guarantees the effective control of welding deformation and welding quality, achieves a certain economic benefits.%钢结构工程中,箱形钢梁制作工艺复杂,焊接变形控制困难。通过从下料、组装、焊接、矫正等方面详细介绍了核电站RP龙门架箱形钢结构梁的制作工艺过程,该过程保证了箱形钢梁的整体几何尺寸,同时又保证了焊接质量并有效控制了焊接变形,取得了一定经济效益。

  20. Sumoylation of Human Translationally Controlled Tumor Protein Is Important for Its Nuclear Transport

    OpenAIRE

    Gnanasekar Munirathinam; Kalyanasundaram Ramaswamy

    2012-01-01

    Translationally controlled tumor protein (TCTP) lacks nuclear bipartite localization signal sequence; yet TCTP is present abundantly in the nucleus. At present it is not known how TCTP gets transported to the nucleus. Sequence analyses showed that all TCTPs described to date have putative small ubiquitin-like modifier (SUMO) motifs. Since SUMO modification plays an important role in the nuclear transport of proteins, we evaluated whether SUMO motifs are important for transport of TCTP into th...

  1. Karyopherin α 3 and karyopherin α 4 proteins mediate the nuclear import of methyl-CpG binding protein 2.

    Science.gov (United States)

    Baker, Steven Andrew; Lombardi, Laura Marie; Zoghbi, Huda Yahya

    2015-09-11

    Methyl-CpG binding protein 2 (MeCP2) is a nuclear protein with important roles in regulating chromatin structure and gene expression, and mutations in MECP2 cause Rett syndrome (RTT). Within the MeCP2 protein sequence, the nuclear localization signal (NLS) is reported to reside between amino acids 255-271, and certain RTT-causing mutations overlap with the MeCP2 NLS, suggesting that they may alter nuclear localization. One such mutation, R270X, is predicted to interfere with the localization of MeCP2, but recent in vivo studies have demonstrated that this mutant remains entirely nuclear. To clarify the mechanism of MeCP2 nuclear import, we isolated proteins that interact with the NLS and identified karyopherin α 3 (KPNA3 or Kap-α3) and karyopherin α 4 (KPNA4 or Kap-α4) as key binding partners of MeCP2. MeCP2-R270X did not interact with KPNA4, consistent with a requirement for an intact NLS in this interaction. However, this mutant retains binding to KPNA3, accounting for the normal localization of MeCP2-R270X to the nucleus. These data provide a mechanism for MeCP2 nuclear import and have implications for the design of therapeutics aimed at modulating the function of MeCP2 in RTT patients.

  2. 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

  3. FoxO proteins' nuclear retention and BH3-only protein Bim induction evoke mitochondrial dysfunction-mediated apoptosis in berberine-treated HepG2 cells.

    Science.gov (United States)

    Shukla, Shatrunajay; Rizvi, Fatima; Raisuddin, Sheikh; Kakkar, Poonam

    2014-11-01

    Mammalian forkhead-box family members belonging to the 'O' category (FoxO) manipulate a plethora of genes modulating a wide array of cellular functions including cell cycle regulation, apoptosis, DNA damage repair, and energy metabolism. FoxO overexpression and nuclear accumulation have been reported to show correlation with hindered tumor growth in vitro and size in vivo, while FoxO's downregulation via phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) pathway has been linked with tumor promotion. In this study, we have explored for the first time intervention of berberine, a plant-derived isoquinoline alkaloid, with FoxO family proteins in hepatoma cells. We observed that berberine significantly upregulated the mRNA expression of both FoxO1 and FoxO3a. Their phosphorylation-mediated cytoplasmic sequestration followed by degradation was prevented by berberine-induced downmodulation of the PI3K/Akt/mTOR pathway which promoted FoxO nuclear retention. PTEN, a tumor suppressor gene and negative regulator of the PI3K/Akt axis, was upregulated while phosphorylation of its Ser380 residue (possible mechanism of PTEN degradation) was significantly decreased in treated HepG2 cells. Exposure to berberine induced a significant increase in transcriptional activity of FoxO, as shown by GFP reporter assay. FoxO transcription factors effectively heightened BH3-only protein Bim expression, which in turn, being a direct activator of proapoptotic protein Bax, altered Bax/Bcl-2 ratio, culminating into mitochondrial dysfunction, caspases activation, and DNA fragmentation. The pivotal role of Bim in berberine-mediated cytotoxicity was further corroborated by knockdown experiments where Bim-silencing partially restored HepG2 cell viability during berberine exposure. In addition, a correlation between oxidative overload and FoxO's nuclear accumulation via JNK activation was evident as berberine treatment led to a pronounced increase in JNK phosphorylation together with enhanced

  4. Ternary complex formation between the MADS-box proteins SQUAMOSA, DEFICIENS and GLOBOSA is involved in the control of floral architecture in Antirrhinum majus.

    Science.gov (United States)

    Egea-Cortines, M; Saedler, H; Sommer, H

    1999-10-01

    In Antirrhinum, floral meristems are established by meristem identity genes. Floral meristems give rise to floral organs in whorls, with their identity established by combinatorial activities of organ identity genes. Double mutants of the floral meristem identity gene SQUAMOSA and organ identity genes DEFICIENS or GLOBOSA produce flowers in which whorled patterning is partially lost. In yeast, SQUA, DEF and GLO proteins form ternary complexes via their C-termini, which in gel-shift assays show increased DNA binding to CArG motifs compared with DEF/GLO heterodimers or SQUA/SQUA homodimers. Formation of ternary complexes by plant MADS-box factors increases the complexity of their regulatory functions and might be the molecular basis for establishment of whorled phyllotaxis and combinatorial interactions of floral organ identity genes.

  5. 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.

  6. Laminopathies: involvement of structural nuclear proteins in the pathogenesis of an increasing number of human diseases.

    Science.gov (United States)

    Maraldi, Nadir M; Squarzoni, Stefano; Sabatelli, Patrizia; Capanni, Cristina; Mattioli, Elisabetta; Ognibene, Andrea; Lattanzi, Giovanna

    2005-05-01

    Just at the beginning of the millennium the neologism laminopathies has been introduced in the scientific vocabulary. An exponential increase of interest on the subject started concomitantly, so that a formerly quite neglected group of rare human diseases is now widely investigated. This review will cover the history of the identification of the molecular basis for fourteen (since now) hereditary diseases arising from defects in genes that encode nuclear envelope and nuclear lamina-associated proteins and will also consider the hypotheses that can account for the role of structural nuclear proteins in the pathogenesis of diseases affecting a wide spectrum of tissues.

  7. Dual localized mitochondrial and nuclear proteins as gene expression regulators in plants?

    Directory of Open Access Journals (Sweden)

    Philippe eGiegé

    2012-09-01

    Full Text Available Mitochondria heavily depend on the coordinated expression of both mitochondrial and nuclear genomes because some of their most significant activities are held by multi-subunit complexes composed of both mitochondrial and nuclear encoded proteins. Thus, precise communication and signaling pathways are believed to exist between the two compartments. Proteins dual localized to both mitochondria and the nucleus make excellent candidates for a potential involvement in the envisaged communication. Here, we review the identified instances of dual localized nucleo-mitochondrial proteins with an emphasis on plant proteins and discuss their functions, which are seemingly mostly related to gene expression regulation. We discuss whether dual localization could be achieved by dual targeting and / or by re-localization and try to apprehend the signals required for the respective processes. Finally, we propose that in some instances, dual localized mitochondrial and nuclear proteins might act as retrograde signaling molecules for mitochondrial biogenesis.

  8. 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.

  9. Dynamic SPR monitoring of yeast nuclear protein binding to a cis-regulatory element.

    Science.gov (United States)

    Mao, Grace; Brody, James P

    2007-11-09

    Gene expression is controlled by protein complexes binding to short specific sequences of DNA, called cis-regulatory elements. Expression of most eukaryotic genes is controlled by dozens of these elements. Comprehensive identification and monitoring of these elements is a major goal of genomics. In pursuit of this goal, we are developing a surface plasmon resonance (SPR) based assay to identify and monitor cis-regulatory elements. To test whether we could reliably monitor protein binding to a regulatory element, we immobilized a 16bp region of Saccharomyces cerevisiae chromosome 5 onto a gold surface. This 16bp region of DNA is known to bind several proteins and thought to control expression of the gene RNR1, which varies through the cell cycle. We synchronized yeast cell cultures, and then sampled these cultures at a regular interval. These samples were processed to purify nuclear lysate, which was then exposed to the sensor. We found that nuclear protein binds this particular element of DNA at a significantly higher rate (as compared to unsynchronized cells) during G1 phase. Other time points show levels of DNA-nuclear protein binding similar to the unsynchronized control. We also measured the apparent association complex of the binding to be 0.014s(-1). We conclude that (1) SPR-based assays can monitor DNA-nuclear protein binding and that (2) for this particular cis-regulatory element, maximum DNA-nuclear protein binding occurs during G1 phase.

  10. Nuclear export signal-interacting protein forms complexes with lamin A/C-Nups to mediate the CRM1-independent nuclear export of large hepatitis delta antigen.

    Science.gov (United States)

    Huang, Cheng; Jiang, Jia-Yin; Chang, Shin C; Tsay, Yeou-Guang; Chen, Mei-Ru; Chang, Ming-Fu

    2013-02-01

    Nuclear export is an important process that not only regulates the functions of cellular factors but also facilitates the assembly of viral nucleoprotein complexes. Chromosome region maintenance 1 (CRM1) that mediates the transport of proteins bearing the classical leucine-rich nuclear export signal (NES) is the best-characterized nuclear export receptor. Recently, several CRM1-independent nuclear export pathways were also identified. The nuclear export of the large form of hepatitis delta antigen (HDAg-L), a nucleocapsid protein of hepatitis delta virus (HDV), which contains a CRM1-independent proline-rich NES, is mediated by the host NES-interacting protein (NESI). The mechanism of the NESI protein in mediating nuclear export is still unknown. In this study, NESI was characterized as a highly glycosylated membrane protein. It interacted and colocalized well in the nuclear envelope with lamin A/C and nucleoporins. Importantly, HDAg-L could be coimmunoprecipitated with lamin A/C and nucleoporins. In addition, binding of the cargo HDAg-L to the C terminus of NESI was detected for the wild-type protein but not for the nuclear export-defective HDAg-L carrying a P205A mutation [HDAg-L(P205A)]. Knockdown of lamin A/C effectively reduced the nuclear export of HDAg-L and the assembly of HDV. These data indicate that by forming complexes with lamin A/C and nucleoporins, NESI facilitates the CRM1-independent nuclear export of HDAg-L.

  11. The DEAD-box RNA helicase DDX3 associates with export messenger ribonucleoproteins as well as tip-associated protein and participates in translational control.

    Science.gov (United States)

    Lai, Ming-Chih; Lee, Yan-Hwa Wu; Tarn, Woan-Yuh

    2008-09-01

    Nuclear export of mRNA is tightly linked to transcription, nuclear mRNA processing, and subsequent maturation in the cytoplasm. Tip-associated protein (TAP) is the major nuclear mRNA export receptor, and it acts coordinately with various factors involved in mRNA expression. We screened for protein factors that associate with TAP and identified several candidates, including RNA helicase DDX3. We demonstrate that DDX3 directly interacts with TAP and that its association with TAP as well as mRNA ribonucleoprotein complexes may occur in the nucleus. Depletion of TAP resulted in nuclear accumulation of DDX3, suggesting that DDX3 is, at least in part, exported along with messenger ribonucleoproteins to the cytoplasm via the TAP-mediated pathway. Moreover, the observation that DDX3 localizes transiently in cytoplasmic stress granules under cell stress conditions suggests a role for DDX3 in translational control. Indeed, DDX3 associates with translation initiation complexes. However, DDX3 is probably not critical for general mRNA translation but may instead promote efficient translation of mRNAs containing a long or structured 5' untranslated region. Given that the DDX3 RNA helicase activity is essential for its involvement in translation, we suggest that DDX3 facilitates translation by resolving secondary structures of the 5'-untranslated region in mRNAs during ribosome scanning.

  12. American Society of Nuclear Cardiology

    Science.gov (United States)

    ... much more! class="box-li"> Journal of Nuclear Cardiology Official publication of the American Society of Nuclear Cardiology Clinical Guidelines Procedures, Appropriate Use Criteria, Information Statements ...

  13. Ezrin Binds to DEAD-Box RNA Helicase DDX3 and Regulates Its Function and Protein Level.

    Science.gov (United States)

    Çelik, Haydar; Sajwan, Kamal P; Selvanathan, Saravana P; Marsh, Benjamin J; Pai, Amrita V; Kont, Yasemin Saygideger; Han, Jenny; Minas, Tsion Z; Rahim, Said; Erkizan, Hayriye Verda; Toretsky, Jeffrey A; Üren, Aykut

    2015-09-01

    Ezrin is a key regulator of cancer metastasis that links the extracellular matrix to the actin cytoskeleton and regulates cell morphology and motility. We discovered a small-molecule inhibitor, NSC305787, that directly binds to ezrin and inhibits its function. In this study, we used a nano-liquid chromatography-tandem mass spectrometry (nano-LC-MS-MS)-based proteomic approach to identify ezrin-interacting proteins that are competed away by NSC305787. A large number of the proteins that interact with ezrin were implicated in protein translation and stress granule dynamics. We validated direct interaction between ezrin and the RNA helicase DDX3, and NSC305787 blocked this interaction. Downregulation or long-term pharmacological inhibition of ezrin led to reduced DDX3 protein levels without changes in DDX3 mRNA. Ectopic overexpression of ezrin in low-ezrin-expressing osteosarcoma cells caused a notable increase in DDX3 protein levels. Ezrin inhibited the RNA helicase activity of DDX3 but increased its ATPase activity. Our data suggest that ezrin controls the translation of mRNAs preferentially with a structured 5' untranslated region, at least in part, by sustaining the protein level of DDX3 and/or regulating its function. Therefore, our findings suggest a novel function for ezrin in regulation of gene translation that is distinct from its canonical role as a cytoskeletal scaffold at the cell membrane.

  14. 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.

  15. 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.

  16. The complete amino acid sequence of the basic nuclear protein of bull spermatozoa

    NARCIS (Netherlands)

    Coelingh, J.P.; Monfoort, Cornelis H.; Rozijn, Thomas H.; Gevers Leuven, Jan A.; Schiphof, R.; Steyn-Parvé, Elizabeth P.; Braunitzer, Gerhard; Schrank, Barbara; Ruhfus, Annette

    1972-01-01

    The complete amino acid sequence of the basic nuclear protein of bull spermatozoa has been established. The sequence was partially deduced by characterization of peptides isolated from thermolysine and chymotryptic digests of the reduced and S-aminoethylated protein. The complete sequence of the fir

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

    Energy Technology Data Exchange (ETDEWEB)

    Yan Chunlan; Wu Wei [Department of Toxicology, Zhejiang University School of Public Health, 388 Yu-Hang-Tang Road, Hangzhou, Zhejiang 310058 (China); Li Haiyan [Department of Toxicology, Zhejiang University School of Public Health, 388 Yu-Hang-Tang Road, Hangzhou, Zhejiang 310058 (China); Huzhou Maternity and Child Care Hospital, Huzhou, Zhejiang 313000 (China); Zhang Guanglin [Department of Toxicology, Zhejiang University School of Public Health, 388 Yu-Hang-Tang Road, Hangzhou, Zhejiang 310058 (China); Duerksen-Hughes, Penelope J. [Department of Basic Sciences, Loma Linda University School of Medicine, Loma Linda, CA 92354 (United States); Zhu Xinqiang, E-mail: zhuxq@zju.edu.cn [Department of Toxicology, Zhejiang University School of Public Health, 388 Yu-Hang-Tang Road, Hangzhou, Zhejiang 310058 (China); Yang Jun, E-mail: gastate@zju.edu.cn [Department of Toxicology, Zhejiang University School of Public Health, 388 Yu-Hang-Tang Road, Hangzhou, Zhejiang 310058 (China); Zhejiang-California International Nanosystems Institute, Hangzhou, Zhejiang 310029 (China)

    2010-04-01

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

  18. Alterations in oxidant/antioxidant balance, high-mobility group box 1 protein and acute phase response in cross-bred suckling piglets suffering from rotaviral enteritis.

    Science.gov (United States)

    Kumar De, Ujjwal; Mukherjee, Reena; Nandi, Sukdeb; Patel, Bhimnere Hanumatnagouda Manjunatha; Dimri, Umesh; Ravishankar, Chintu; Verma, Ashok Kumar

    2014-10-01

    Rotaviral enteritis has emerged as a major cause of morbidity and mortality in piglets during their post-natal life. The present study was carried out to examine high-mobility group box 1 (HMGB1) protein, acute phase response and oxidative stress indices in the serum of suckling piglets suffering from enteritis with or without association of porcine group A rotavirus infection. The present investigation utilized 23 clinical cases with signs of acute enteritis and 12 more healthy piglets of a similar age group as control animals. Out of 23 enteritis cases, 12 cases were found to be positive for porcine group A rotavirus infection as confirmed by reverse transcription-polymerase chain reaction (RT-PCR) using specific primers for group A rotavirus, and the rest were found negative. The acute enteritis cases in piglets were associated with an elevated level of HMGB1 protein and serum haptoglobin and ceruloplasmin suggestive of an acute phase response. Among the oxidative stress indices, the concentrations of malondialdehyde (MDA) and nitric oxide (NO) in serum were significantly increased. A pronounced drop of total antioxidant capacity and the activity of antioxidant enzymes such as catalase and superoxide dismutase in the serum of piglets suffering from acute enteritis compared to healthy ones were also noticed. The alterations in HMGB1 protein, acute phase response and oxidative stress indices were more pronounced in cases with the involvement of porcine rotavirus as compared to rotavirus-negative cases. It is concluded that HMGB1 protein, markers of oxidative stress and acute phase proteins might play an important role in the aetiopathogenesis of porcine diarrhoea caused by rotavirus and might be true markers in diagnosing the conditions leading to the extension of the prompt and effective therapeutic care.

  19. Mechanistic Insights from Structural Analyses of Ran-GTPase-Driven Nuclear Export of Proteins and RNAs.

    Science.gov (United States)

    Matsuura, Yoshiyuki

    2016-05-22

    Understanding how macromolecules are rapidly exchanged between the nucleus and the cytoplasm through nuclear pore complexes is a fundamental problem in biology. Exportins are Ran-GTPase-dependent nuclear transport factors that belong to the karyopherin-β family and mediate nuclear export of a plethora of proteins and RNAs, except for bulk mRNA nuclear export. Exportins bind cargo macromolecules in a Ran-GTP-dependent manner in the nucleus, forming exportin-cargo-Ran-GTP complexes (nuclear export complexes). Transient weak interactions between exportins and nucleoporins containing characteristic FG (phenylalanine-glycine) repeat motifs facilitate nuclear pore complex passage of nuclear export complexes. In the cytoplasm, nuclear export complexes are disassembled, thereby releasing the cargo. GTP hydrolysis by Ran promoted in the cytoplasm makes the disassembly reaction virtually irreversible and provides thermodynamic driving force for the overall export reaction. In the past decade, X-ray crystallography of some of the exportins in various functional states coupled with functional analyses, single-particle electron microscopy, molecular dynamics simulations, and small-angle solution X-ray scattering has provided rich insights into the mechanism of cargo binding and release and also begins to elucidate how exportins interact with the FG repeat motifs. The knowledge gained from structural analyses of nuclear export is being translated into development of clinically useful inhibitors of nuclear export to treat human diseases such as cancer and influenza.

  20. 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.

  1. 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.

  2. The Effect of Tripterygium Wilfordii Monomer T4 on Rat Spermatid Nuclear Protein Transition

    Institute of Scientific and Technical Information of China (English)

    戴文平; 刘平; 陈啸梅; 薛社普

    1996-01-01

    Rat testis elongating spermatids and epididymal sperms were collected after 7 weeks of treatment with Tripterygium wilfordii monomer T4. Total nuclear basic protein (TNBP) was extracted from the elongating spermatid nuclei and the sperm nuclei isolated by sonication. Polyacrylamide gel electrophoresis has beep used to separate the TNBP and individual proteins were quantified by scanning microdensitometry. It was found that the content of protamine was reduced and the TH (Total Histones) /RP (Rat Protamine) ratios were increased following treatment in the testis elongating spermatids, and same result was found in the epididymal sperms. These results suggest that the interruption of nuclear protein transition of testis spermatids induced by T4 might cause aberrant epididymal sperm nuclear protein and lead to infertility. The relationship between protamine and fertility was discussed.

  3. Chronic cyclophosphamide exposure alters the profile of rat sperm nuclear matrix proteins.

    Science.gov (United States)

    Codrington, Alexis M; Hales, Barbara F; Robaire, Bernard

    2007-08-01

    Chronic exposure of male rats to the alkylating agent cyclophosphamide, a well-known male-mediated developmental toxicant, alters gene expression in male germ cells as well as in early preimplantation embryos sired by cyclophosphamide-exposed males. Sperm DNA is organized by the nuclear matrix into loop-domains in a sequence-specific manner. In somatic cells, loop-domain organization is involved in gene regulation. Various structural and functional components of the nuclear matrix are targets for chemotherapeutic agents. Consequently, we hypothesized that cyclophosphamide treatment would alter the expression of sperm nuclear matrix proteins. Adult male rats were treated for 4 wk with saline or cyclophosphamide (6.0 mg kg(-1) day(-1)), and the nuclear matrix was extracted from cauda epididymal sperm. Proteins were analyzed by two-dimensional gel electrophoresis. Identified proteins within the nuclear matrix proteome were mainly involved in cell structure, transcription, translation, DNA binding, protein processing, signal transduction, metabolism, cell defense, or detoxification. Interestingly, cyclophosphamide selectively induced numerous changes in cell defense and detoxification proteins, most notably, in all known forms of the antioxidant enzyme glutathione peroxidase 4, in addition to an uncharacterized 54-kDa form; an overall increase in glutathione peroxidase 4 immunoreactivity was observed in the nuclear matrix extracts from cyclophosphamide-exposed spermatozoa. An increase in glutathione peroxidase 4 expression suggests a role for this enzyme in maintaining nuclear matrix stability and function. These results led us to propose that a change in composition of the nuclear matrix in response to drug exposure was a factor in altered sperm function and embryo development.

  4. F-Box Protein FBXO22 Mediates Polyubiquitination and Degradation of CD147 to Reverse Cisplatin Resistance of Tumor Cells

    OpenAIRE

    Bo Wu; Zhen-Yu Liu; Jian Cui; Xiang-Min Yang; Lin Jing; Yang Zhou; Zhi-Nan Chen; Jian-Li Jiang

    2017-01-01

    Drug resistance remains a major clinical obstacle to successful treatment of cancer. As posttranslational modification is becoming widely recognized to affect the function of oncoproteins, targeting specific posttranslational protein modification provides an attractive strategy for anticancer drug development. CD147 is a transmembrane glycoprotein contributing to chemo-resistance of cancer cells in a variety of human malignancies. Ubiquitination is an important posttranslational modification ...

  5. Down-expression of tumor protein p53-induced nuclear protein 1 in human gastric cancer

    Institute of Scientific and Technical Information of China (English)

    Pei-Hong Jiang; Yoshiharu Motoo; Stéphane Garcia; Juan Lucio Iovanna; Marie-Josèphe Pébusque; Norio Sawabu

    2006-01-01

    AIM: Overexpression of tumor protein p53-induced nuclear protein 1 (TP53INP1) induces G1 cell cycle arrest and increases p53-mediated apoptosis. To clarify the clinical importance of TP53INP1, we analyzed TP53INP1and p53 expression in gastric cancer.METHODS: TP53INP1 and p53 expression were examined using immunohistochemistry in 142 cases of gastric cancer. The apoptosis of gastric cancer cells was analyzed using the TUNEL method. The relationship between the expression of TP53INP1 and clinicopathological factors was statistically analyzed.RESULTS: TP53INP1 was expressed in 98% (139/142cases) of non-cancerous gastric tissues and was downexpressed in 64% (91/142 cases) of gastric cancer lesions from the same patients. TP53INP1 expression was significantly decreased (43.9%) in poorly differentiated adenocarcinoma compared with well or moderately differentiated adenocarcinoma (81.6%).Cancers invading the submucosa or deeper showed lower positively (59.1%) compared with mucosal cancers (85.2%). Decrease or loss of TP53INP1 expression was significantly correlated with lymphatic invasion (54.3%vs 82.0% without lymphatic invasion) and node-positive patients (31.3% vs 68.3% in node-negative patients).P53 was expressed in 68 (47.9%) patients of gastric cancer, whereas it was absent in normal gastric tissues.A significant association was also observed between TP53INP1 status and the level of apoptosis in tumor cells: the apoptotic index in TP53INP1-positive tissues was significantly higher than that in TP53INP1-negative portions. Finally, when survival data were analyzed,loss of TP53INP1 expression had a significant effect in predicting a poor prognosis (P= 0.0006).CONCLUSION: TP53INP1-positive rate decreases with the progression of gastric cancer. TP53INP1 protein negativity is significantly associated with aggressive pathological phenotypes of gastric cancer. TP53INP1is related to the apoptosis of gastric cancer cells. The decreased expression of the TP53INP1 protein may

  6. Electrostatic potentials of the S-locus F-box proteins contribute to the pollen S specificity in self-incompatibility in Petunia hybrida.

    Science.gov (United States)

    Li, Junhui; Zhang, Yue; Song, Yanzhai; Zhang, Hui; Fan, Jiangbo; Li, Qun; Zhang, Dongfen; Xue, Yongbiao

    2017-01-01

    Self-incompatibility (SI) is a self/non-self discrimination system found widely in angiosperms and, in many species, is controlled by a single polymorphic S-locus. In the Solanaceae, Rosaceae and Plantaginaceae, the S-locus encodes a single S-RNase and a cluster of S-locus F-box (SLF) proteins to control the pistil and pollen expression of SI, respectively. Previous studies have shown that their cytosolic interactions determine their recognition specificity, but the physical force between their interactions remains unclear. In this study, we show that the electrostatic potentials of SLF contribute to the pollen S specificity through a physical mechanism of 'like charges repel and unlike charges attract' between SLFs and S-RNases in Petunia hybrida. Strikingly, the alteration of a single C-terminal amino acid of SLF reversed its surface electrostatic potentials and subsequently the pollen S specificity. Collectively, our results reveal that the electrostatic potentials act as a major physical force between cytosolic SLFs and S-RNases, providing a mechanistic insight into the self/non-self discrimination between cytosolic proteins in angiosperms.

  7. 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.

  8. 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.

  9. A high mobility group box 1 (HMGB1) gene from Chlamys farreri and the DNA-binding ability and pro-inflammatory activity of its recombinant protein.

    Science.gov (United States)

    Wang, Mengqiang; Wang, Lingling; Guo, Ying; Zhou, Zhi; Yi, Qilin; Zhang, Daoxiang; Zhang, Huan; Liu, Rui; Song, Linsheng

    2014-02-01

    High-mobility group box 1 (HMGB1) protein, a highly conserved DNA binding protein, plays an important role in maintaining nucleosome structures, transcription, and inflammation. In the present research, a cDNA of 1268 bp for the Zhikong scallop Chlamys farreri HMGB1 (designed as CfHMGB1) was cloned via rapid amplification of cDNA ends (RACE) technique and expression sequence tag (EST) analysis. The complete cDNA sequence of CfHMGB1 contained an open reading frame (ORF) of 648 bp, which encoded a protein of 215 amino acids. The amino acid sequence of CfHMGB1 shared 53-57% similarity with other identified HMGB1s. There were two HMG domains, two low complexity regions and a conserved acidic tail in the amino acid sequence of CfHMGB1. The mRNA transcripts of CfHMGB1 were constitutively expressed in all the tested tissues, including haemocytes, muscle, mantle, gill, hepatopancreas, kidney and gonad, with the highest expression level in hepatopancreas. The mRNA expression profiles of CfHMGB1 in haemocytes after the stimulation with different pathogen-associated molecular patterns (PAMPs), including lipopolysaccharide (LPS), peptidoglycan (PGN) and glucan (Glu), were similar with an up-regulation in the early stage and then recovered to the original level. The recombinant CfHMGB1 protein could bind double-stranded DNA and induce the release of TNF-α activity in mixed primary culture of scallop haemocytes. These results collectively indicated that CfHMGB1, with DNA-binding ability and pro-inflammatory activity, could play an important role in the immune response of scallops.

  10. Ets-1 facilitates nuclear entry of NFAT proteins and their recruitment to the IL-2 promoter.

    Science.gov (United States)

    Tsao, Hsiao-Wei; Tai, Tzong-Shyuan; Tseng, William; Chang, Hui-Hsin; Grenningloh, Roland; Miaw, Shi-Chuen; Ho, I-Cheng

    2013-09-24

    E26 transformation-specific sequence 1 (Ets-1), the prototype of the ETS family of transcription factors, is critical for the expression of IL-2 by murine Th cells; however, its mechanism of action is still unclear. Here we show that Ets-1 is also essential for optimal production of IL-2 by primary human Th cells. Although Ets-1 negatively regulates the expression of Blimp1, a known suppressor of IL-2 expression, ablation of B lymphocyte-induced maturation protein 1 (Blimp1) does not rescue the expression of IL-2 by Ets-1-deficient Th cells. Instead, Ets-1 physically and functionally interacts with the nuclear factor of activated T-cells (NFAT) and is required for the recruitment of NFAT to the IL-2 promoter. In addition, Ets-1 is located in both the nucleus and cytoplasm of resting Th cells. Nuclear Ets-1 quickly exits the nucleus in response to calcium-dependent signals and competes with NFAT proteins for binding to protein components of noncoding RNA repressor of NFAT complex (NRON), which serves as a cytoplasmic trap for phosphorylated NFAT proteins. This nuclear exit of Ets-1 precedes rapid nuclear entry of NFAT and Ets-1 deficiency results in impaired nuclear entry, but not dephosphorylation, of NFAT proteins. Thus, Ets-1 promotes the expression of IL-2 by modulating the activity of NFAT.

  11. Protein Tpr is required for establishing nuclear pore-associated zones of heterochromatin exclusion.

    Science.gov (United States)

    Krull, Sandra; Dörries, Julia; Boysen, Björn; Reidenbach, Sonja; Magnius, Lars; Norder, Helene; Thyberg, Johan; Cordes, Volker C

    2010-05-19

    Amassments of heterochromatin in somatic cells occur in close contact with the nuclear envelope (NE) but are gapped by channel- and cone-like zones that appear largely free of heterochromatin and associated with the nuclear pore complexes (NPCs). To identify proteins involved in forming such heterochromatin exclusion zones (HEZs), we used a cell culture model in which chromatin condensation induced by poliovirus (PV) infection revealed HEZs resembling those in normal tissue cells. HEZ occurrence depended on the NPC-associated protein Tpr and its large coiled coil-forming domain. RNAi-mediated loss of Tpr allowed condensing chromatin to occur all along the NE's nuclear surface, resulting in HEZs no longer being established and NPCs covered by heterochromatin. These results assign a central function to Tpr as a determinant of perinuclear organization, with a direct role in forming a morphologically distinct nuclear sub-compartment and delimiting heterochromatin distribution.

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

    DEFF Research Database (Denmark)

    Bennetzen, Martin; Andersen, J.S.; Lasen, D.H.

    2013-01-01

    -dependent posttranslational modifications (PT Ms). 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......-based proteomic approaches. Apart from cataloging acetylation sites through SILAC proteomic analyses before IR and at 5 and 60 min after IR exposure of U2OS cells, we report that: (1) key components of the transcriptional machinery, such as EP 300 and CREBBP, are dynamically acetylated; (2) that nuclear...... to assess lysine acetylation status and thereby validate the mass spectrometry data. We thus present evidence that nuclear proteins, including those known to regulate cellular functions via epigenetic modifications of histones, are regulated by (de)acetylation in a timely manner upon cell's exposure...

  13. Nuclear localization of Sindbis virus nonstructural protein nsP2

    Institute of Scientific and Technical Information of China (English)

    WANGXIAOZHONG; MINGXIAODING

    1993-01-01

    In early infection, approximately 10% of nonstructural protein nsP2 of Sindbis virus was transported into the nuclei of virus-infected BHK-21 cells. Nuclear asP2 was dominantly associated with nuclear matrix. During the course of infection, increasing amounts of nsP2 accumulated in the nuclear fraction. A prominent accumulation of nuclear nsP2 occurred early in infection, from 1 h to 3 h postinfection. Meanwhile. a weak NTPase activity was found to be associated with the immunocomplexed nsP2. Nuclear localization of nsP2 and its possible role were diseussed in relation to the inhibition of host macromolecular synthesis.

  14. Quantitative Proteomics Identifies Vasopressin-Responsive Nuclear Proteins in Collecting Duct Cells

    OpenAIRE

    Schenk, Laura K.; Bolger, Steven J.; Luginbuhl, Kelli; Gonzales, Patricia A.; Rinschen, Markus M.; Yu, Ming-Jiun; Hoffert, Jason D.; Pisitkun, Trairak; Knepper, Mark A.

    2012-01-01

    Vasopressin controls transport in the renal collecting duct, in part, by regulating transcription. This complex process, which can involve translocation and/or modification of transcriptional regulators, is not completely understood. Here, we applied a method for large-scale profiling of nuclear proteins to quantify vasopressin-induced changes in the nuclear proteome of cortical collecting duct (mpkCCD) cells. Using stable isotope labeling and tandem mass spectrometry, we quantified 3987 nucl...

  15. 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.

  16. The nuclear export protein of H5N1 influenza A viruses recruits Matrix 1 (M1) protein to the viral ribonucleoprotein to mediate nuclear export.

    Science.gov (United States)

    Brunotte, Linda; Flies, Joe; Bolte, Hardin; Reuther, Peter; Vreede, Frank; Schwemmle, Martin

    2014-07-18

    In influenza A virus-infected cells, replication and transcription of the viral genome occurs in the nucleus. To be packaged into viral particles at the plasma membrane, encapsidated viral genomes must be exported from the nucleus. Intriguingly, the nuclear export protein (NEP) is involved in both processes. Although NEP stimulates viral RNA synthesis by binding to the viral polymerase, its function during nuclear export implicates interaction with viral ribonucleoprotein (vRNP)-associated M1. The observation that both interactions are mediated by the C-terminal moiety of NEP raised the question whether these two features of NEP are linked functionally. Here we provide evidence that the interaction between M1 and the vRNP depends on the NEP C terminus and its polymerase activity-enhancing property for the nuclear export of vRNPs. This suggests that these features of NEP are linked functionally. Furthermore, our data suggest that the N-terminal domain of NEP interferes with the stability of the vRNP-M1-NEP nuclear export complex, probably mediated by its highly flexible intramolecular interaction with the NEP C terminus. On the basis of our data, we propose a new model for the assembly of the nuclear export complex of Influenza A vRNPs.

  17. 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.

  18. F-Box Protein FBXO22 Mediates Polyubiquitination and Degradation of CD147 to Reverse Cisplatin Resistance of Tumor Cells

    Directory of Open Access Journals (Sweden)

    Bo Wu

    2017-01-01

    Full Text Available Drug resistance remains a major clinical obstacle to successful treatment of cancer. As posttranslational modification is becoming widely recognized to affect the function of oncoproteins, targeting specific posttranslational protein modification provides an attractive strategy for anticancer drug development. CD147 is a transmembrane glycoprotein contributing to chemo-resistance of cancer cells in a variety of human malignancies. Ubiquitination is an important posttranslational modification mediating protein degradation. Degradation of oncoproteins, CD147 included, emerges as an attractive alternative for tumor inhibition. However, the ubiquitination of CD147 remains elusive. Here in this study, we found that deletion of the CD147 intracellular domain (CD147-ICD prolonged the half-life of CD147 in HEK293T cells, and we identified that CD147-ICD interacts with FBXO22 using mass spectrometry and Western blot. Then, we demonstrated that FBXO22 mediates the polyubiquitination and degradation of CD147 by recognizing CD147-ICD. While knocking down of FBXO22 prolonged the half-life of CD147 in HEK293T cells, we found that FBXO22 regulates CD147 protein turnover in SMMC-7721, Huh-7 and A549 cells. Moreover, we found that the low level of FBXO22 contributes to the accumulation of CD147 and thereafter the cisplatin resistance of A549/DDP cells. To conclude, our study demonstrated that FBXO22 mediated the polyubiquitination and degradation of CD147 by interacting with CD147-ICD, and CD147 polyubiquitination by FBXO22 reversed cisplatin resistance of tumor cells.

  19. F-Box Protein FBXO22 Mediates Polyubiquitination and Degradation of CD147 to Reverse Cisplatin Resistance of Tumor Cells

    Science.gov (United States)

    Wu, Bo; Liu, Zhen-Yu; Cui, Jian; Yang, Xiang-Min; Jing, Lin; Zhou, Yang; Chen, Zhi-Nan; Jiang, Jian-Li

    2017-01-01

    Drug resistance remains a major clinical obstacle to successful treatment of cancer. As posttranslational modification is becoming widely recognized to affect the function of oncoproteins, targeting specific posttranslational protein modification provides an attractive strategy for anticancer drug development. CD147 is a transmembrane glycoprotein contributing to chemo-resistance of cancer cells in a variety of human malignancies. Ubiquitination is an important posttranslational modification mediating protein degradation. Degradation of oncoproteins, CD147 included, emerges as an attractive alternative for tumor inhibition. However, the ubiquitination of CD147 remains elusive. Here in this study, we found that deletion of the CD147 intracellular domain (CD147-ICD) prolonged the half-life of CD147 in HEK293T cells, and we identified that CD147-ICD interacts with FBXO22 using mass spectrometry and Western blot. Then, we demonstrated that FBXO22 mediates the polyubiquitination and degradation of CD147 by recognizing CD147-ICD. While knocking down of FBXO22 prolonged the half-life of CD147 in HEK293T cells, we found that FBXO22 regulates CD147 protein turnover in SMMC-7721, Huh-7 and A549 cells. Moreover, we found that the low level of FBXO22 contributes to the accumulation of CD147 and thereafter the cisplatin resistance of A549/DDP cells. To conclude, our study demonstrated that FBXO22 mediated the polyubiquitination and degradation of CD147 by interacting with CD147-ICD, and CD147 polyubiquitination by FBXO22 reversed cisplatin resistance of tumor cells. PMID:28117675

  20. Structural mechanism of nuclear transport mediated by importin β and flexible amphiphilic proteins.

    Science.gov (United States)

    Yoshimura, Shige H; Kumeta, Masahiro; Takeyasu, Kunio

    2014-12-02

    Karyopherin β family proteins mediate the nuclear/cytoplasmic transport of various proteins through the nuclear pore complex (NPC), although they are substantially larger than the size limit of the NPC.To elucidate the molecular mechanism underlying this paradoxical function, we focused on the unique structures called HEAT repeats, which consist of repetitive amphiphilic α helices. An in vitro transport assay and FRAP analyses demonstrated that not only karyopherin β family proteins but also other proteins with HEAT repeats could pass through the NPC by themselves, and serve as transport mediators for their binding partners. Biochemical and spectroscopic analyses and molecular dynamics simulations of purified HEAT-rich proteins revealed that they interact with hydrophobic groups, including phenyl and alkyl groups, and undergo reversible conformational changes in tertiary structures, but not in secondary structures. These results show that conformational changes in the flexible amphiphilic motifs play a critical role in translocation through the NPC.

  1. 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.

  2. 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.

  3. Tus, an E. coli protein, contains mammalian nuclear targeting and exporting signals.

    Science.gov (United States)

    Kaczmarczyk, Stanislaw J; Sitaraman, Kalavathy; Hill, Thomas; Hartley, James L; Chatterjee, Deb K

    2010-01-26

    Shuttling of proteins between nucleus and cytoplasm in mammalian cells is facilitated by the presence of nuclear localization signals (NLS) and nuclear export signals (NES), respectively. However, we have found that Tus, an E. coli replication fork arresting protein, contains separate sequences that function efficiently as NLS and NES in mammalian cell lines, as judged by cellular location of GFP-fusion proteins. The NLS was localized to a short stretch of 9 amino acids in the carboxy-terminus of Tus protein. Alterations of any of these basic amino acids almost completely abolished the nuclear targeting. The NES comprises a cluster of leucine/hydrophobic residues located within 21 amino acids at the amino terminus of Tus. Finally, we have shown that purified GFP-Tus fusion protein or GFP-Tus NLS fusion protein, when added to the culture media, was internalized very efficiently into mammalian cells. Thus, Tus is perhaps the first reported bacterial protein to possess both NLS and NES, and has the capability to transduce protein into mammalian cells.

  4. Proneural proteins Achaete and Scute associate with nuclear actin to promote formation of external sensory organs.

    Science.gov (United States)

    Hsiao, Yun-Ling; Chen, Yu-Ju; Chang, Yi-Jie; Yeh, Hsiao-Fong; Huang, Yi-Chun; Pi, Haiwei

    2014-01-01

    Basic helix-loop-helix (bHLH) proneural proteins promote neurogenesis through transcriptional regulation. Although much is known about the tissue-specific regulation of proneural gene expression, how proneural proteins interact with transcriptional machinery to activate downstream target genes is less clear. Drosophila proneural proteins Achaete (Ac) and Scute (Sc) induce external sensory organ formation by activating neural precursor gene expression. Through co-immunoprecipitation and mass spectrometric analyses, we found that nuclear but not cytoplasmic actin associated with the Ac and Sc proteins in Drosophila S2 cells. Daughterless (Da), the common heterodimeric partner of Drosophila bHLH proteins, was observed to associate with nuclear actin through proneural proteins. A yeast two-hybrid assay revealed that the binding specificity between actin and Ac or Sc was conserved in yeast nuclei without the presence of additional Drosophila factors. We further show that actin is required in external sensory organ formation. Reduction in actin gene activity impaired proneural-protein-dependent expression of the neural precursor genes, as well as formation of neural precursors. Furthermore, increased nuclear actin levels, obtained by expression of nucleus-localized actin, elevated Ac-Da-dependent gene transcription as well as Ac-mediated external sensory organ formation. Taken together, our in vivo and in vitro observations suggest a novel link for actin in proneural-protein-mediated transcriptional activation and neural precursor differentiation.

  5. 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.

  6. DEAD-box RNA helicase DDX3 connects CRM1-dependent nuclear export and translation of the HIV-1 unspliced mRNA through its N-terminal domain.

    Science.gov (United States)

    Fröhlich, Alvaro; Rojas-Araya, Bárbara; Pereira-Montecinos, Camila; Dellarossa, Alessandra; Toro-Ascuy, Daniela; Prades-Pérez, Yara; García-de-Gracia, Francisco; Garcés-Alday, Andrea; Rubilar, Paulina S; Valiente-Echeverría, Fernando; Ohlmann, Théophile; Soto-Rifo, Ricardo

    2016-05-01

    DEAD-box RNA helicase DDX3 is a host factor essential for HIV-1 replication and thus, a potential target for novel therapies aimed to overcome viral resistance. Previous studies have shown that DDX3 promotes nuclear export and translation of the HIV-1 unspliced mRNA. Although the function of DDX3 during both processes requires its catalytic activity, it is unknown whether other domains surrounding the helicase core are involved. Here, we show the involvement of the N- and C-terminal domains of DDX3 in the regulation of HIV-1 unspliced mRNA translation. Our results suggest that the intrinsically disordered N-terminal domain of DDX3 regulates its functions in translation by acting prior to the recruitment of the 43S pre-initiation complex onto the viral 5'-UTR. Interestingly, this regulation was conserved in HIV-2 and was dependent on the CRM1-dependent nuclear export pathway suggesting a role of the RNA helicase in interconnecting nuclear export with ribosome recruitment of the viral unspliced mRNA. This specific function of DDX3 during HIV gene expression could be exploited as an alternative target for pharmaceutical intervention.

  7. Transcription of the human beta enolase gene (ENO-3) is regulated by an intronic muscle-specific enhancer that binds myocyte-specific enhancer factor 2 proteins and ubiquitous G-rich-box binding factors.

    Science.gov (United States)

    Feo, S; Antona, V; Barbieri, G; Passantino, R; Calì, L; Giallongo, A

    1995-01-01

    To provide evidence for the cis-regulatory DNA sequences and trans-acting factors involved in the complex pattern of tissue- and stage-specific expression of the beta enolase gene, constructs containing fragments of the gene fused to the chloramphenicol acetyltransferase gene were used in transient-transfection assays of C2C12 myogenic cells. Deletion analysis revealed the presence of four major regions: two negative regions in the 5'-flanking sequence, a basal promoter region which directs expression at low levels in proliferating and differentiated muscle cells, and a positive region within the first intron that confers cell-type-specific and differentiation-induced expression. This positive regulatory element is located in the 3'-proximal portion of the first intron (nucleotides +504 to +637) and acts as an enhancer irrespective of orientation and position from the homologous beta enolase promoter or the heterologous thymidine kinase promoter, conferring in both cases muscle-specific expression to the linked reporter gene. Deletion of a putative myocyte-specific enhancer factor 1 (MEF-1) binding site, containing a canonical E-box motif, had no effects on muscle-specific transcription, indicating that this site is not required for the activity of the enhancer. Gel mobility shift assays, competition analysis, DNase I footprinting, and mutagenesis studies indicated that this element interacts through an A/T-rich box with a MEF-2 protein(s) and through a G-rich box with a novel ubiquitous factor(s). Mutation of either the G-rich box or the A/T-rich box resulted in a significantly reduced activity of the enhancer in transient-transfection assays. These data indicate that MEF-2 and G-rich-box binding factors are each necessary for tissue-specific expression of the beta enolase gene in skeletal muscle cells. PMID:7565752

  8. Nuclear translocation of EGF receptor regulated by Epstein-Barr virus encoded latent membrane protein 1

    Institute of Scientific and Technical Information of China (English)

    TAO; Yongguang; SONG; Xin; TAN; Yunnian; LIN; Xiaofeng; ZH

    2004-01-01

    Epstein-Barr virus (EBV) encoded latent membrane protein 1 (LMP1) is considered to be the major oncogenic protein of EBV encoded proteins, and also it has always been the core of the oncogenic mechanism of EBV. Traditional receptor theory demonstrates that cell surface receptors exert biological functions on the membrane, which neither enter into the nucleus nor directly affect the transcription of the target genes. But, advanced studies on nuclear translocation of the epidermal growth factor receptor (EGFR) family have greatly developed our knowledge of the biological function of cell surface receptors. In this study, we used Tet-on LMP1 HNE2 cell line as a cell model, which is a dual-stable LMP1 integrated NPC cell line and the expression of LMP1 in which could be regulated by Tet system. We found that LMP1 could regulate the nuclear translocation of EGFR in a dose-dependent manner from both quantitative and qualitative levels through the Western blot analysis and the immunofluorescent analysis with a laser scanning confocal microscope. We further demonstrated that the nuclear localization sequence of EGFR played some roles in the location of the protein within the nucleus under LMP1 regulation, and the nuclear accumulation of EGFR regulated by LMP1 was in a ligand-independent manner. These findings provide a novel view that the regulation of LMP1 on the nuclear translocation of EGFR is critical for the process of nasopharyngeal carcinoma.

  9. Characterization of a nuclear localization signal of canine parvovirus capsid proteins.

    Science.gov (United States)

    Vihinen-Ranta, M; Kakkola, L; Kalela, A; Vilja, P; Vuento, M

    1997-12-01

    We investigated the abilities of synthetic peptides mimicking the potential nuclear localization signal of canine parvovirus (CPV) capsid proteins to translocate a carrier protein to the nucleus following microinjection into the cytoplasm of A72 cells. Possible nuclear localization sequences were chosen for synthesis from CPV capsid protein sequences (VP1, VP2) on the basis of the presence of clustered basic residues, which is a common theme in most of the previously identified targeting peptides. Nuclear targeting activity was found within the N-terminal residues 4-13 (PAKRARRGYK) of the VP1 capsid protein. While replacement of Arg10 with glycine did not affect the activity, replacement of Lys6, Arg7, or Arg9 with glycine abolished it. The targeting activity was found to residue in a cluster of basic residues, Lys5, Arg7, and Arg9. Nuclear import was saturated by excess of unlabelled peptide conjugates (showing that it was a receptor-mediated process). Transport into the nucleus was an energy-dependent and temperature-dependent process actively mediated by the nuclear pores and inhibited by wheat germ agglutinin.

  10. Laryngeal (Voice Box) Cancer

    Science.gov (United States)

    ... ENTCareers Marketplace Find an ENT Doctor Near You Voice Box (Laryngeal) Cancer Voice Box (Laryngeal) Cancer Patient Health Information News media ... laryngeal cancer can be severe with respect to voice, breathing, or swallowing. It is fundamentally a preventable ...

  11. Nuclear localization and secretion competence is conserved amongst 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

    2017-01-05

    Viruses of the genus Henipavirus of the family Paramyxoviridae are zoonotic pathogens, which have emerged in South East Asia, Australia and Africa. Nipah virus (NiV) and Hendra virus (HeV) are highly virulent pathogens transmitted from bats to animals and humans, whilst the henipavirus Cedar virus (CedV) 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, KV) and Mojiang virus (MojV). 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, CedV-, KV- and MojV-M proteins were mutated in a bipartite nuclear localization signal indicating that a key lysine residue is essential for nuclear import, export and for the 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.

  12. Altered profiles of nuclear matrix proteins during the differentiation of human gastric mucous adenocarcinoma MGc80-3 cells

    Institute of Scientific and Technical Information of China (English)

    Chun-Hong Zhao; Qi-Fu Li

    2005-01-01

    AIM: To find and identify specific nuclear matrix proteins associated with proliferation and differentiation of carcinoma cells, which will be potential markers for cancer diagnosis and targets in cancer therapy.METHODS: Nuclear matrix proteins were selectively extracted from MGc80-3 cells treated with or without hexamethylamine bisacetamide (HMBA), and subjected to 2-D gel electrophoresis. The resulted protein patterns were analyzed by Melanie software. Spots of nuclear matrix proteins differentially expressed were excised and subjected to in situ digestion with trypsin. Peptide masses were obtained by matrix-assisted laser-desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS) analysis and submitted for database searching using Mascot tool.RESULTS: The MGc80-3 cells were induced into differentiation by HMBA. There were 22 protein spots which changed remarkably in the nuclear matrix, from differentiation of MGc80-3 cells compared to control.Eleven of which were identified. Seven proteins -actin, prohibitin, porin 31HL, heterogeneous nuclear ribonucleoprotein A2/B1, vimentin, ATP synthase, and heatshock protein 60 were downregulated, whereas three proteins - heat shock protein gp96, heat shock protein 90-beta, and valosin-containing protein were upregulated,and the oxygen-regulated protein was only found in the differentiated MGc80-3 cells.CONCLUSION: The induced differentiation of carcinoma cells is accompanied by the changes of nuclear matrix proteins. Further characterization of those proteins will show the mechanism of cellular proliferation and differentiation, as well as cancer differentiation.

  13. High mobility group box protein-1 promotes cerebral edema after traumatic brain injury via activation of toll-like receptor 4.

    Science.gov (United States)

    Laird, Melissa D; Shields, Jessica S; Sukumari-Ramesh, Sangeetha; Kimbler, Donald E; Fessler, R David; Shakir, Basheer; Youssef, Patrick; Yanasak, Nathan; Vender, John R; Dhandapani, Krishnan M

    2014-01-01

    Traumatic brain injury (TBI) is a major cause of mortality and morbidity worldwide. Cerebral edema, a life-threatening medical complication, contributes to elevated intracranial pressure (ICP) and a poor clinical prognosis after TBI. Unfortunately, treatment options to reduce post-traumatic edema remain suboptimal, due in part, to a dearth of viable therapeutic targets. Herein, we tested the hypothesis that cerebral innate immune responses contribute to edema development after TBI. Our results demonstrate that high-mobility group box protein 1 (HMGB1) was released from necrotic neurons via a NR2B-mediated mechanism. HMGB1 was clinically associated with elevated ICP in patients and functionally promoted cerebral edema after TBI in mice. The detrimental effects of HMGB1 were mediated, at least in part, via activation of microglial toll-like receptor 4 (TLR4) and the subsequent expression of the astrocytic water channel, aquaporin-4 (AQP4). Genetic or pharmacological (VGX-1027) TLR4 inhibition attenuated the neuroinflammatory response and limited post-traumatic edema with a delayed, clinically implementable therapeutic window. Human and rodent tissue culture studies further defined the cellular mechanisms demonstrating neuronal HMGB1 initiates the microglial release of interleukin-6 (IL-6) in a TLR4 dependent mechanism. In turn, microglial IL-6 increased the astrocytic expression of AQP4. Taken together, these data implicate microglia as key mediators of post-traumatic brain edema and suggest HMGB1-TLR4 signaling promotes neurovascular dysfunction after TBI.

  14. X-box binding protein 1 (XBP1s is a critical determinant of Pseudomonas aeruginosa homoserine lactone-mediated apoptosis.

    Directory of Open Access Journals (Sweden)

    Cathleen D Valentine

    Full Text Available Pseudomonas aeruginosa infections are associated with high mortality rates and occur in diverse conditions including pneumonias, cystic fibrosis and neutropenia. Quorum sensing, mediated by small molecules including N-(3-oxo-dodecanoyl homoserine lactone (C12, regulates P. aeruginosa growth and virulence. In addition, host cell recognition of C12 initiates multiple signalling responses including cell death. To gain insight into mechanisms of C12-mediated cytotoxicity, we studied the role of endoplasmic reticulum stress in host cell responses to C12. Dramatic protection against C12-mediated cell death was observed in cells that do not produce the X-box binding protein 1 transcription factor (XBP1s. The leucine zipper and transcriptional activation motifs of XBP1s were sufficient to restore C12-induced caspase activation in XBP1s-deficient cells, although this polypeptide was not transcriptionally active. The XBP1s polypeptide also regulated caspase activation in cells stimulated with N-(3-oxo-tetradecanoyl homoserine lactone (C14, produced by Yersinia enterolitica and Burkholderia pseudomallei, and enhanced homoserine lactone-mediated caspase activation in the presence of endogenous XBP1s. In C12-tolerant cells, responses to C12 including phosphorylation of p38 MAPK and eukaryotic initiation factor 2α were conserved, suggesting that C12 cytotoxicity is not heavily dependent on these pathways. In summary, this study reveals a novel and unconventional role for XBP1s in regulating host cell cytotoxic responses to bacterial acyl homoserine lactones.

  15. Polymorphism - 116C/G of the human X box binding protein 1 gene is associated with risk of type 2 diabetes in a Chinese Han population.

    Science.gov (United States)

    Liu, Shengyuan; Ma, Guoda; Yao, Songpo; Chen, Zhongwei; Wang, Changyi; Zhao, Bin; Li, Keshen

    2016-01-01

    Evidence has been obtained showing that endoplasmic reticulum (ER) stress is closely associated with the development of type 2 diabetes (T2D) and that the human X box binding protein 1 (XBP1) is an important transcription factor involved in the development of ER stress. The study aimed to analyze the potential association between polymorphism -116C/G of XBP1 and the risk of T2D. The association between XBP1 polymorphism -116C/G and T2D risk was assessed among 1058 consecutive unrelated subjects, including 523 T2D patients and 535 healthy controls, in a case control study. The -116GG genotype and -116G allele were more frequent in T2D subjects compared with control subjects by statistical analysis, showing that the -116GG homozygote polymorphism of XBP1 might lead to increased susceptibility to T2D in a Chinese Han population. T2D subjects with the -116GG genotype had higher fasting plasma glucose levels, fasting insulin levels, and HbA1c and worse HOMA-IR than the T2D subjects with -116CG and -116CC genotypes (PG polymorphism of the XBP1 promoter in the pathogenesis of T2D in a Chinese Han population, and more studies are needed to further evaluate our results.

  16. 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.

  17. High mobility group box-1 protein inhibits regulatory T cell immune activity in liver failure in patients with chronic hepatitis B

    Institute of Scientific and Technical Information of China (English)

    Lu-WenWang; Hui Chen; Zuo-Jiong Gong

    2010-01-01

    BACKGROUND: Liver failure in chronic hepatitis B (CHB) patients is a severe, life-threatening condition. Intestinal endotoxemia plays a significant role in the progress to liver failure. High mobility group box-1 (HMGB1) protein is involved in the process of endotoxemia. Regulatory T (Treg) cells maintain immune tolerance and contribute to the immunological hyporesponsiveness against HBV infection. However, the roles of HMGB1 and Treg cells in the pathogenesis of liver failure in CHB patients, and whether HMGB1 affects the immune activity of Treg cells are poorly known at present, and so were explored in this study. METHODS: The levels of HMGB1 expression were detected by ELISA, real-time RT-PCR, and Western blotting, and the percentage of CD4+CD25+CD127low Treg cells among CD4+cells was detected by flow cytometry in liver failure patients with chronic HBV infection, CHB patients, and healthy controls. Then, CD4+CD25+CD127low Treg cells isolated from the peripheral blood mononuclear cells from CHB patients were stimulated with HMGB1 at different concentrations or at various intervals. The effect of HMGB1 on the immune activity of Treg cells was assessed by a suppression assay of the allogeneic mixed lymphocyte response. The levels of forkhead box P3 (Foxp3) expression in Treg cells treated with HMGB1 were detected by RT-PCR and Western blotting. RESULTS: A higher level of HMGB1 expression and a lower percentage of Treg cells within the population of CD4+ cells were found in liver failure patients than in CHB patients (82.6±20.1 μg/L vs. 34.2±13.7 μg/L; 4.55±1.34% vs. 9.52± 3.89%, respectively). The immune activity of Treg cells was significantly weakened and the levels of Foxp3 expression were reduced in a dose- or time-dependent manner when Treg cells were stimulated with HMGB1 in vitro. CONCLUSIONS: The high level of HMGB1 and the low percentage of Treg cells play an important role in the pathogenesis of liver failure in patients with chronic HBV infection

  18. Nuclear localization signal in a cancer-related transcriptional regulator protein NAC1.

    Science.gov (United States)

    Okazaki, Kosuke; Nakayama, Naomi; Nariai, Yuko; Nakayama, Kentaro; Miyazaki, Kohji; Maruyama, Riruke; Kato, Hiroaki; Kosugi, Shunichi; Urano, Takeshi; Sakashita, Gyosuke

    2012-10-01

    Nucleus accumbens-associated protein 1 (NAC1) might have potential oncogenic properties and participate in regulatory networks for pluripotency. Although NAC1 is described as a transcriptional regulator, the nuclear import machinery of NAC1 remains unclear. We found, using a point mutant, that dimer formation was not committed to the nuclear localization of NAC1 and, using deletion mutants, that the amino-terminal half of NAC1 harbored a potential nuclear localization signal (NLS). Wild type, but not mutants of this region, alone was sufficient to drive the importation of green fluorescent protein (GFP) into the nucleus. Bimax1, a synthetic peptide that blocks the importin α/β pathway, impaired nuclear localization of NAC1 in cells. We also used the binding properties of importin to demonstrate that this region is an NLS. Furthermore, the transcriptional regulator function of NAC1 was dependent on its nuclear localization activity in cells. Taken together, these results show that the region with a bipartite motif constitutes a functional nuclear import sequence in NAC1 that is independent of NAC1 dimer formation. The identification of an NAC1 NLS thus clarifies the mechanism through which NAC1 translocates to the nucleus to regulate the transcription of genes involved in oncogenicity and pluripotency.

  19. Expression of ankyrin repeat and suppressor of cytokine signaling box protein 4 (Asb-4) in proopiomelanocortin neurons of the arcuate nucleus of mice produces a hyperphagic, lean phenotype.

    Science.gov (United States)

    Li, Ji-Yao; Chai, Biao-Xin; Zhang, Weizhen; Wang, Hui; Mulholland, Michael W

    2010-01-01

    Ankyrin repeat and suppressor of cytokine signaling box-containing protein 4 (Asb-4) is specifically expressed in the energy homeostasis-related brain areas and colocalizes with proopiomelanocortin (POMC) neurons of the arcuate nucleus (ARC). Injection of insulin into the third ventricle of the rat brain increased Asb-4 mRNA expression in the paraventricular nucleus but not in the ARC of the hypothalamus, whereas injection of leptin (ip) increased Asb-4 expression in both mouse paraventricular nucleus and ARC. A transgenic mouse in which Myc-tagged Asb-4 is specifically expressed in POMC neurons of the ARC was made and used to study the effects of Asb-4 on ingestive behavior and metabolic rate. Animals with overexpression of Asb-4 in POMC neurons demonstrated an increase in food intake. However, POMC-Asb-4 transgenic animals gained significantly less weight from 6-30 wk of age. The POMC-Asb-4 mice had reduced fat mass and increased lean mass and lower levels of blood leptin. The transgenic animals were resistant to high-fat diet-induced obesity. Transgenic mice had significantly higher rates of oxygen consumption and carbon dioxide production than wild-type mice during both light and dark periods. The locomotive activity of transgenic mice was increased. The overexpression of Asb-4 in POMC neurons increased POMC mRNA expression in the ARC. The transgenic animals had no observed effect on peripheral glucose metabolism and the activity of the autonomic nervous system. These results indicate that Asb-4 is a key regulatory protein in the central nervous system, involved in the control of feeding behavior and metabolic rate.

  20. A rapid screening system evaluates novel inhibitors of DNA methylation and suggests F-box proteins as potential therapeutic targets for high-risk neuroblastoma.

    Science.gov (United States)

    Penter, Livius; Maier, Bert; Frede, Ute; Hackner, Benjamin; Carell, Thomas; Hagemeier, Christian; Truss, Matthias

    2015-12-01

    After extensive research on radiochemotherapy, 5-year survival rates of children with high risk neuroblastoma still do not exceed 50%, owing to adverse side-effects exemplified by doxorubicin-induced cardiomyopathy. A promising new approach is the combination of conventional therapies with specific modulation of cell signaling pathways promoting therapeutic resistance, such as inhibition of aberrant kinase activity or re-expression of silenced tumor suppressor genes by means of chromatin remodeling. In this regard, we established a system that allows to identify potential drug targets as well as to validate respective candidate inhibitors in high-risk neuroblastoma model cell lines. Cell culture, drug exposure, shRNA-mediated knockdown and phenotype analysis are integrated into an efficient and versatile single well-based protocol. By utilizing this system, we assessed RG108, SGI-1027 and nanaomycin A, three novel DNA methyltransferase inhibitors that have not been tested in neuroblastoma cell lines so far, for their potential of synergistic anti-tumor activity in combination with doxorubicin. We found that, similarly to azacytidine, SGI-1027 and nanaomycin A mediate synergistic growth inhibition with doxorubicin independently of N-Myc status. However, they display high cytotoxicity but lack global DNA demethylation activity. Secondly, we conducted a lentiviral shRNA screen of F-box proteins, key regulators of protein stability, and identified Fbxw11/β-TrCP2 as well as Fbxo5/Emi1 as potential therapeutic targets in neuroblastoma. These results complement existing studies and underline the reliability and versatility of our single well-based protocol.

  1. 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

    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.

  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. Ephemeral Protein Binding to DNA Shapes Stable Nuclear Bodies and Chromatin Domains.

    Science.gov (United States)

    Brackley, Chris A; Liebchen, Benno; Michieletto, Davide; Mouvet, Francois; Cook, Peter R; Marenduzzo, Davide

    2017-03-28

    Fluorescence microscopy reveals that the contents of many (membrane-free) nuclear bodies exchange rapidly with the soluble pool while the underlying structure persists; such observations await a satisfactory biophysical explanation. To shed light on this, we perform large-scale Brownian dynamics simulations of a chromatin fiber interacting with an ensemble of (multivalent) DNA-binding proteins able to switch between an "on" (binding) and an "off" (nonbinding) state. This system provides a model for any DNA-binding protein that can be posttranslationally modified to change its affinity for DNA (e.g., through phosphorylation). Protein switching is a nonequilibrium process, and it leads to the formation of clusters of self-limiting size, where individual proteins in a cluster exchange with the soluble pool with kinetics similar to those seen in photobleaching experiments. This behavior contrasts sharply with that exhibited by nonswitching proteins, which are permanently in the on-state; when these bind to DNA nonspecifically, they form clusters that grow indefinitely in size. To explain these findings, we propose a mean-field theory from which we obtain a scaling relation between the typical cluster size and the protein switching rate. Protein switching also reshapes intrachromatin contacts to give networks resembling those seen in topologically associating domains, as switching markedly favors local (short-range) contacts over distant ones. Our results point to posttranslational modification of chromatin-bridging proteins as a generic mechanism driving the self-assembly of highly dynamic, nonequilibrium, protein clusters with the properties of nuclear bodies.

  4. GTP-dependent binding and nuclear transport of RNA polymerase II by Npa3 protein

    DEFF Research Database (Denmark)

    Staresincic, Lidija; Walker, Jane; Dirac-Svejstrup, A Barbara;

    2011-01-01

    in yeast extracts. Indeed, Npa3 depletion in vivo affects nuclear localization of RNAPII; the polymerase accumulates in the cytoplasm. Npa3 is a member of the GPN-LOOP family of GTPases. Npa3 mutants that either cannot bind GTP or that bind but cannot hydrolyze it are inviable and unable to support nuclear...... transport of RNAPII. Surprisingly, we were unable to detect interactions between Npa3 and proteins in the classical importin a/ß pathway for nuclear import. Interestingly, Npa3-RNAPII binding is significantly increased by the addition of GTP or its slowly hydrolyzable analogue guanosine 5'-3-O......-(thio)triphosphate (GTP¿S). Moreover, the Npa3 mutant that binds GTP, but cannot hydrolyze it, binds RNAPII even in the absence of added GTP, whereas the mutant that cannot bind GTP is unable to bind the polymerase. Together, our data suggest that Npa3 defines an unconventional pathway for nuclear import of RNAPII, which...

  5. Mx1 GTPase accumulates in distinct nuclear domains and inhibits influenza A virus in cells that lack promyelocytic leukaemia protein nuclear bodies.

    Science.gov (United States)

    Engelhardt, Othmar G; Sirma, Hüseyin; Pandolfi, Pier-Paolo; Haller, Otto

    2004-08-01

    The interferon-induced murine Mx1 GTPase is a nuclear protein. It specifically inhibits influenza A viruses at the step of primary transcription, a process known to occur in the nucleus of infected cells. However, the exact mechanism of inhibition is still poorly understood. The Mx1 GTPase has previously been shown to accumulate in distinct nuclear dots that are spatially associated with promyelocytic leukaemia protein (PML) nuclear bodies (NBs), but the significance of this association is not known. Here it is reported that, in cells lacking PML and, as a consequence, PML NBs, Mx1 still formed nuclear dots. These dots were indistinguishable from the dots observed in wild-type cells, indicating that intact PML NBs are not required for Mx1 dot formation. Furthermore, Mx1 retained its antiviral activity against influenza A virus in these PML-deficient cells, which were fully permissive for influenza A virus. Nuclear Mx proteins from other species showed a similar subnuclear distribution. This was also the case for the human MxA GTPase when this otherwise cytoplasmic protein was translocated into the nucleus by virtue of a foreign nuclear localization signal. Human MxA and mouse Mx1 do not interact or form heterooligomers. Yet, they co-localized to a large degree when co-expressed in the nucleus. Taken together, these findings suggest that Mx1 dots represent distinct nuclear domains ('Mx nuclear domains') that are frequently associated with, but functionally independent of, PML NBs.

  6. Box-Behnken试验设计法提取玉米胚芽蛋白工艺的研究%Study on Proteins Extraction of Corn Germ By Box-Behnken

    Institute of Scientific and Technical Information of China (English)

    李秀娟; 鲁曾

    2010-01-01

    采用Osboren分类法对玉米胚芽蛋白进行了分类,并采用Box-Behnken试验设计确定了玉米胚芽蛋白质的最佳提取条件:缓冲溶液的pH值10.17、提取时间1.85h、提取温度52.4℃、料液比1∶9.5814,蛋白质提取率75.2%,提取出的玉米胚芽蛋白中以谷氨酸含量最高.

  7. Nuclear magnetic resonance detection and spectroscopy of single proteins using quantum logic.

    Science.gov (United States)

    Lovchinsky, I; Sushkov, A O; Urbach, E; de Leon, N P; Choi, S; De Greve, K; Evans, R; Gertner, R; Bersin, E; Müller, C; McGuinness, L; Jelezko, F; Walsworth, R L; Park, H; Lukin, M D

    2016-02-19

    Nuclear magnetic resonance spectroscopy is a powerful tool for the structural analysis of organic compounds and biomolecules but typically requires macroscopic sample quantities. We use a sensor, which consists of two quantum bits corresponding to an electronic spin and an ancillary nuclear spin, to demonstrate room temperature magnetic resonance detection and spectroscopy of multiple nuclear species within individual ubiquitin proteins attached to the diamond surface. Using quantum logic to improve readout fidelity and a surface-treatment technique to extend the spin coherence time of shallow nitrogen-vacancy centers, we demonstrate magnetic field sensitivity sufficient to detect individual proton spins within 1 second of integration. This gain in sensitivity enables high-confidence detection of individual proteins and allows us to observe spectral features that reveal information about their chemical composition.

  8. 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.

  9. FGFR2 protein expression in breast cancer: nuclear localisation and correlation with patient genotype

    Directory of Open Access Journals (Sweden)

    Thompson Alastair M

    2011-03-01

    Full Text Available Abstract Background Single Nucleotide Polymorphisms (SNPs in intron 2 of the Fibroblast Growth Factor Receptor Type 2 (FGFR2 gene, including rs2981582, contribute to multifactorial breast cancer susceptibility. The high risk polymorphism haplotype in the FGFR2 gene has been associated with increased mRNA transcription and altered transcription factor binding but the effect on FGFR2 protein expression is unknown. 40 breast tumours were identified from individuals with known rs2981582 genotype. Tumour sections were stained for FGFR2 protein expression, and scored for nuclear and cytoplasmic staining in tumour and surrounding normal tissue. Findings FGFR2 immunohistochemistry demonstrated variable nuclear staining in normal tissue and tumour tissue, as well as consistent cytoplasmic staining. We did not find an association between nuclear staining for FGFR2 and genotype, and there was no association between FGFR2 staining and estrogen or progestogen receptor status. There was an association between presence of nuclear staining for FGFR2 in normal tissue and presence of nuclear staining in the adjacent tumour (Fishers exact test, p = 0.002. Conclusions Variable nuclear staining for FGFR2 in breast cancer, but an absence of correlation with rs2981582 genotype suggests that the mechanism of action of polymorphisms at the FGFR2 locus may be more complex than a direct effect on mRNA expression levels in the final cancer. The effect may relate to FGFR2 function or localisation during breast development or tumourigenesis. Nuclear localisation of FGFR2 suggests an important additional role for this protein in breast development and breast cancer, in addition to its function as a classical cell surface receptor.

  10. 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.

    Directory of Open Access Journals (Sweden)

    Sohail Khoshnevis

    2016-06-01

    Full Text Available 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.

  11. 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.

  12. Sumoylation of Human Translationally Controlled Tumor Protein Is Important for Its Nuclear Transport

    Directory of Open Access Journals (Sweden)

    Gnanasekar Munirathinam

    2012-01-01

    Full Text Available Translationally controlled tumor protein (TCTP lacks nuclear bipartite localization signal sequence; yet TCTP is present abundantly in the nucleus. At present it is not known how TCTP gets transported to the nucleus. Sequence analyses showed that all TCTPs described to date have putative small ubiquitin-like modifier (SUMO motifs. Since SUMO modification plays an important role in the nuclear transport of proteins, we evaluated whether SUMO motifs are important for transport of TCTP into the nucleus. We show that TCTP exists in sumoylated form in cytoplasm and nucleus of mammalian cells. Point mutation of lysine residue in the SUMO motif compromised the ability of TCTP to get sumoylated in vitro. When cells were transfected with FLAG-tagged mutated TCTP, nuclear transport of TCTP was inhibited confirming that sumoylation is critical for the nuclear transport of TCTP. Our previous studies demonstrated that TCTP can function as an antioxidant protein in the nucleus. When we mutated TCTP at the SUMO motif the antioxidant function of TCTP was compromised. Results presented in this study thus show that sumoylation plays an important role in the transport of TCTP into the nucleus where they function as antioxidant protein.

  13. Nuclear actin-related protein is required for chromosome segregation in Toxoplasma gondii.

    Science.gov (United States)

    Suvorova, Elena S; Lehmann, Margaret M; Kratzer, Stella; White, Michael W

    2012-01-01

    Apicomplexa parasites use complex cell cycles to replicate that are not well understood mechanistically. We have established a robust forward genetic strategy to identify the essential components of parasite cell division. Here we describe a novel temperature sensitive Toxoplasma strain, mutant 13-20C2, which growth arrests due to a defect in mitosis. The primary phenotype is the mis-segregation of duplicated chromosomes with chromosome loss during nuclear division. This defect is conditional-lethal with respect to temperature, although relatively mild in regard to the preservation of the major microtubule organizing centers. Despite severe DNA loss many of the physical structures associated with daughter budding and the assembly of invasion structures formed and operated normally at the non-permissive temperature before completely arresting. These results suggest there are coordinating mechanisms that govern the timing of these events in the parasite cell cycle. The defect in mutant 13-20C2 was mapped by genetic complementation to Toxoplasma chromosome III and to a specific mutation in the gene encoding an ortholog of nuclear actin-related protein 4. A change in a conserved isoleucine to threonine in the helical structure of this nuclear actin related protein leads to protein instability and cellular mis-localization at the higher temperature. Given the age of this protist family, the results indicate a key role for nuclear actin-related proteins in chromosome segregation was established very early in the evolution of eukaryotes.

  14. 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...

  15. Quantitative proteomics identifies vasopressin-responsive nuclear proteins in collecting duct cells.

    Science.gov (United States)

    Schenk, Laura K; Bolger, Steven J; Luginbuhl, Kelli; Gonzales, Patricia A; Rinschen, Markus M; Yu, Ming-Jiun; Hoffert, Jason D; Pisitkun, Trairak; Knepper, Mark A

    2012-06-01

    Vasopressin controls transport in the renal collecting duct, in part, by regulating transcription. This complex process, which can involve translocation and/or modification of transcriptional regulators, is not completely understood. Here, we applied a method for large-scale profiling of nuclear proteins to quantify vasopressin-induced changes in the nuclear proteome of cortical collecting duct (mpkCCD) cells. Using stable isotope labeling and tandem mass spectrometry, we quantified 3987 nuclear proteins and identified significant changes in the abundance of 65, including previously established targets of vasopressin signaling in the collecting duct. Vasopressin-induced changes in the abundance of the transcription factors JunB, Elf3, Gatad2b, and Hmbox1; transcriptional co-regulators Ctnnb1 (β-catenin) and Crebbp; subunits of the Mediator complex; E3 ubiquitin ligase Nedd4; nuclear transport regulator RanGap1; and several proteins associated with tight junctions and adherens junctions. Bioinformatic analysis showed that many of the quantified transcription factors have putative binding sites in the 5'-flanking regions of genes coding for the channel proteins Aqp2, Aqp3, Scnn1b (ENaCβ), and Scnn1g (ENaCγ), which are known targets of vasopressin. Immunoblotting demonstrated that the increase in β-catenin in nuclear fractions was accompanied by an even larger increase in its phosphorylated form (pSer552). The findings provide a new online database resource for nuclear proteomics (http://helixweb.nih.gov/ESBL/Database/mNPD/) and generate new hypotheses regarding vasopressin-mediated transcriptional regulation in the collecting duct.

  16. Proteomic analysis of nuclear matrix proteins during arsenic trioxide induced apoptosis in leukemia K562 cells

    Institute of Scientific and Technical Information of China (English)

    WANG Zi-hui; YU Ding; CHEN Yan; HAO Jian-zhong

    2005-01-01

    Background Arsenic trioxide (As2O3) has been identified as a very potent anti-acute leukemic agent. However its role in apoptosis needs to be elucidated. As2O3 interferes with the proliferation and survival of tumor cells via a variety of mechanisms. Drug-target interactions at the level of nuclear matrix (NM) may be critical events in the induction of cell death by As2O3. This study dealt with As2O3-target interactions at the level of NM in chronic myelogenous leukemia cell line K562 by proteomics. Methods K562 cells were cultured in MEM and treated with different concentrations of As2O3. The nuclear matrix proteins were analyzed by high-resolution two-dimensional gel electrophoresis and computer-assisted image analysis. Results As2O3 significantly inhibited the growth of chronic myelogenous leukemia cell line K562 at low concentrations. While more than 200 protein spots were shared among the nuclear matrices, about 18 distinct spots in the nuclear matrices were found characteristic for As2O3 treated cells. Conclusions: As2O3 induces apoptosis in K562 cells in a dose and time-dependent manner. Our results demonstrated that for the detection of the onset of apoptosis, the alteration in the composition of nuclear matrix proteins was a more sensitive indicator than nucleosomal DNA fragmentation test. These results indicated that As2O3 might be clinically useful in the treatment of chronic myelogenous leukemia. The changes of nuclear matrix proteins in the treated cells can be used as a useful indicator for this treatment.

  17. Legionella pneumophila infection induces programmed cell death, caspase activation, and release of high-mobility group box 1 protein in A549 alveolar epithelial cells: inhibition by methyl prednisolone

    Directory of Open Access Journals (Sweden)

    Koide Michio

    2008-05-01

    Full Text Available Abstract Background Legionella pneumophila pneumonia often exacerbates acute lung injury (ALI and acute respiratory distress syndrome (ARDS. Apoptosis of alveolar epithelial cells is considered to play an important role in the pathogenesis of ALI and ARDS. In this study, we investigated the precise mechanism by which A549 alveolar epithelial cells induced by L. pneumophila undergo apoptosis. We also studied the effect of methyl prednisolone on apoptosis in these cells. Methods Nuclear deoxyribonucleic acid (DNA fragmentation and caspase activation in L. pneumophila-infected A549 alveolar epithelial cells were assessed using the terminal deoxyribonucleotidyl transferase-mediated triphosphate (dUTP-biotin nick end labeling method (TUNEL method and colorimetric caspase activity assays. The virulent L. pneumophila strain AA100jm and the avirulent dotO mutant were used and compared in this study. In addition, we investigated whether methyl prednisolone has any influence on nuclear DNA fragmentation and caspase activation in A549 alveolar epithelial cells infected with L. pneumophila. Results The virulent strain of L. pneumophila grew within A549 alveolar epithelial cells and induced subsequent cell death in a dose-dependent manner. The avirulent strain dotO mutant showed no such effect. The virulent strains of L. pneumophila induced DNA fragmentation (shown by TUNEL staining and activation of caspases 3, 8, 9, and 1 in A549 cells, while the avirulent strain did not. High-mobility group box 1 (HMGB1 protein was released from A549 cells infected with virulent Legionella. Methyl prednisolone (53.4 μM did not influence the intracellular growth of L. pneumophila within alveolar epithelial cells, but affected DNA fragmentation and caspase activation of infected A549 cells. Conclusion Infection of A549 alveolar epithelial cells with L. pneumophila caused programmed cell death, activation of various caspases, and release of HMGB1. The dot/icm system, a

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

    Science.gov (United States)

    Kanda, Kojun; Pflug, James M; Sproul, John S; Dasenko, Mark A; Maddison, David R

    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

  19. Poly(A) binding proteins located at the inner surface of resealed nuclear envelopes.

    Science.gov (United States)

    Prochnow, D; Riedel, N; Agutter, P S; Fasold, H

    1990-04-25

    We have used a photoreactive cross-linking reagent, poly(A/8-N3-A) (a poly(A) of average molecular mass of 100 kDa in which 5-10% of the A residues are replaced by 8-N3-A), to label poly(A) binding proteins of rat liver nuclear envelopes. This reagent was prepared by polymerizing a mixture of ADP and 8-N3-ADP with polynucleotide phosphorylase. The purified poly(A) was labeled in the 5'-position with a 32P group. In nuclear envelopes prepared by a low salt DNase I procedure, the poly(A/8-N3-A) labeled a protein-nucleic acid complex of approximately 270 kDa, which on degradation with RNase U2 or NaOH at pH 10 yielded two polypeptides of approximately 50 and 30 kDa. These photoreaction products were markedly decreased when resealed nuclear envelopes or non-nuclear envelope proteins were irradiated in the presence of poly(A/8-N3-A). The affinity labeling was intensified when resealed vesicles were made leaky by freezing or ultrasonication, suggesting that the poly(A) binding proteins are accessible from the nucleoplasmic but not the cytoplasmic face of the envelope. Moreover binding was specific for poly(A). Alternative reagents, random poly(A/8-N3-A,C,G,U) of about 100 kDa and poly(dA) (molecular mass between 350 and 515 kDa), showed a very low affinity for poly(A) recognition proteins in the low salt DNase I-treated nuclear envelopes; the 270-kDa band was labeled only weakly. The binding site was not protected by poly(A,C,G,U), weakly by poly(dA), and distinctly by poly(A).

  20. Ephemeral protein binding to DNA shapes stable nuclear bodies and chromatin domains

    CERN Document Server

    Brackley, C A; Michieletto, D; Mouvet, F; Cook, P R; Marenduzzo, D

    2016-01-01

    Fluorescence microscopy reveals that the contents of many (membrane-free) nuclear "bodies" exchange rapidly with the soluble pool whilst the underlying structure persists; such observations await a satisfactory biophysical explanation. To shed light on this, we perform large-scale Brownian dynamics simulations of a chromatin fiber interacting with an ensemble of (multivalent) DNA-binding proteins; these proteins switch between two states -- active (binding) and inactive (non-binding). This system provides a model for any DNA-binding protein that can be modified post-translationally to change its affinity for DNA (e.g., like the phosphorylation of a transcription factor). Due to this out-of-equilibrium process, proteins spontaneously assemble into clusters of self-limiting size, as individual proteins in a cluster exchange with the soluble pool with kinetics like those seen in photo-bleaching experiments. This behavior contrasts sharply with that exhibited by "equilibrium", or non-switching, proteins that exis...

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

    Directory of Open Access Journals (Sweden)

    Takashi Shibano

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

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

    Science.gov (United States)

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

    2015-01-01

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

  3. Human Cytomegalovirus Nuclear Egress Proteins Ectopically Expressed in the Heterologous Environment of Plant Cells are Strictly Targeted to the Nuclear Envelope.

    Science.gov (United States)

    Lamm, Christian E; Link, Katrin; Wagner, Sabrina; Milbradt, Jens; Marschall, Manfred; Sonnewald, Uwe

    2016-03-10

    In all eukaryotic cells, the nucleus forms a prominent cellular compartment containing the cell's nuclear genome. Although structurally similar, animal and plant nuclei differ substantially in details of their architecture. One example is the nuclear lamina, a layer of tightly interconnected filament proteins (lamins) underlying the nuclear envelope of metazoans. So far no orthologous lamin genes could be detected in plant genomes and putative lamin-like proteins are only poorly described in plants. To probe for potentially conserved features of metazoan and plant nuclear envelopes, we ectopically expressed the core nuclear egress proteins of human cytomegalovirus pUL50 and pUL53 in plant cells. pUL50 localizes to the inner envelope of metazoan nuclei and recruits the nuclear localized pUL53 to it, forming heterodimers. Upon expression in plant cells, a very similar localization pattern of both proteins could be determined. Notably, pUL50 is specifically targeted to the plant nuclear envelope in a rim-like fashion, a location to which coexpressed pUL53 becomes strictly corecruited from its initial nucleoplasmic distribution. Using pUL50 as bait in a yeast two-hybrid screening, the cytoplasmic re-initiation supporting protein RISP could be identified. Interaction of pUL50 and RISP could be confirmed by coexpression and coimmunoprecipitation in mammalian cells and by confocal laser scanning microscopy in plant cells, demonstrating partial pUL50-RISP colocalization in areas of the nuclear rim and other intracellular compartments. Thus, our study provides strong evidence for conserved structural features of plant and metazoan nuclear envelops and identifies RISP as a potential pUL50-interacting plant protein.

  4. 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.

  5. Mitochondria and forkhead box protein O 3a%线粒体和叉头框蛋白O类3a

    Institute of Scientific and Technical Information of China (English)

    赵琳; 戴琼艳; 张露; 段满林

    2014-01-01

    Background Forkhead box O (FOXO) 3a transcription factors are regulators of cell-type specific apoptosis and cell cycle arrest,but also control cell survival and production of reactive oxygen species(ROS).Objective To review the FOXO3a self-reactivating loop and novel functions of FOXO3a in controlling mitochondrial respiration of cells,which further supports the current view that FOXO3a transcription factors are information-integrating sentinels of cellular stress and critical modulators of cell homeostasis.Content In this article,we will discuss the current knowledge on the involvement of FOXO3a transcription factors in the regulation of cellular homestasis with specific emphasis on mitochondrial integrity,morphology and activity.In neuronal tumor cells,FOXO3a triggers ROS-accumulation as a consequence of transient mitochondrial outer membrane permeabilization,which is essential for FOXO3a-induced apoptosis in these cells.Cellular levels of reactive oxygen species are affected by the FOXO3a-targets including Bim,BclxL,and Survivin.All three proteins localize to mitochondria and affect mitochondrial membrane potential and respiration,as well as cellular levels of reactive oxygen species.Trend FOXO3a controls a delicate balance between mitochondrial reactive oxygeu species-generation and levels of reactive oxygen species-preventing or detoxifying processes,which is critical for cell death decision in neuronal cells.%背景 叉头框蛋白O类(forkhead box protein O,FOXO)3a转录因子是细胞凋亡和细胞周期的调节者,也调控细胞生存或活性氧簇(reactive oxygen species,ROS)生成.目的 阐述FOXO3a激活对细胞线粒体呼吸作用的调控,进一步说明FOXO3a是细胞应激的信息整合因子和细胞稳态的主要调控因子.内容 讨论FOXO3a转录因子在调节细胞稳态中的作用,重点在线粒体完整性、形态和活性.在神经肿瘤细胞中,FOXO3a诱发ROS积聚,短暂性增加线粒体外膜通透性,这对FOXO3a诱

  6. 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.

  7. A subset of FG-nucleoporins is necessary for efficient Msn5-mediated nuclear protein export.

    Science.gov (United States)

    Finn, Erin M; DeRoo, Elise P; Clement, George W; Rao, Sheila; Kruse, Sarah E; Kokanovich, Kate M; Belanger, Kenneth D

    2013-05-01

    The transport of proteins between the cytoplasm and nucleus requires interactions between soluble transport receptors (karyopherins) and phenylalanine-glycine (FG) repeat domains on nuclear pore complex proteins (nucleoporins). However, the role of specific FG repeat-containing nucleoporins in nuclear protein export has not been carefully investigated. We have developed a novel kinetic assay to investigate the relative export kinetics mediated by the karyopherin Msn5/Kap142 in yeast containing specific FG-Nup mutations. Using the Msn5 substrate Crz1 as a marker for Msn5-mediated protein export, we observe that deletions of NUP100 or NUP2 result in decreased rates of Crz1 export, while nup60Δ and nup42Δ mutants do not vary significantly from wild type. The decreased Msn5 export rate in nup100Δ was confirmed using Mig1-GFP as a transport substrate. A nup100ΔGLFG mutant shows defects in nuclear export kinetics similar to a nup100Δ deletion. Removal of FG-repeats from Nsp1 also decreases export kinetics, while a loss of Nup1 FXFGs does not. To confirm that our export data reflected functional differences in protein localization, we performed Crz1 transcription activation assays using a CDRE::LacZ reporter gene that is upregulated upon increased transcription activation by Crz1 in vivo. We observe that expression from this reporter increases in nup100ΔGLFG and nsp1ΔFGΔFXFG strains that exhibit decreased Crz1 export kinetics but resembles wild-type levels in nup1ΔFXFG strains that do not exhibit export defects. These data provide evidence that the export of Msn5 is likely mediated by a specific subset of FG-Nups and that the GLFG repeat domain of Nup100 is important for Msn5-mediated nuclear protein export.

  8. The S haplotype-specific F-box protein gene, SFB, is defective in self-compatible haplotypes of Prunus avium and P. mume.

    Science.gov (United States)

    Ushijima, Koichiro; Yamane, Hisayo; Watari, Akiko; Kakehi, Eiko; Ikeda, Kazuo; Hauck, Nathanael R; Iezzoni, Amy F; Tao, Ryutaro

    2004-08-01

    Many Prunus species, including sweet cherry and Japanese apricot, of the Rosaceae, display an S-RNase-based gametophytic self-incompatibility (GSI). The specificity of this outcrossing mechanism is determined by a minimum of two genes that are located in a multigene complex, termed the S locus, which controls the pistil and pollen specificities. SFB, a gene located in the S locus region, encodes an F-box protein that has appropriate S haplotype-specific variation to be the pollen determinant in the self-incompatibility reaction. This study characterizes SFBs of two self-compatible (SC) haplotypes, S(4') and S(f), of Prunus. S(4') of sweet cherry is a pollen-part mutant (PPM) that was produced by X-ray irradiation, while S(f) of Japanese apricot is a naturally occurring SC haplotype that is considered to be a PPM. DNA sequence analysis revealed defects in both SFB(4') and SFB(f). A 4 bp deletion upstream from the HVa coding region of SFB(4') causes a frame-shift that produces transcripts of a defective SFB lacking the two hypervariable regions, HVa and HVb. Similarly, the presence of a 6.8 kbp insertion in the middle of the SFB(f) coding region leads to transcripts for a defective SFB lacking the C-terminal half that contains HVa and HVb. As all reported SFBs of functional S haplotypes encode intact SFB, the fact that the partial loss-of-function mutations in SFB are present in SC mutant haplotypes of Prunus provides additional evidence that SFB is the pollen S gene in GSI in Prunus.

  9. Update of studies on DEAD-box protein family and spermatogenesis%DEAD box家族蛋白与精子生成的研究进展

    Institute of Scientific and Technical Information of China (English)

    胡秀玉; 彭弋峰

    2011-01-01

    The normal development and maturation of sperm need not only a variety of related gene expressions, but also the participation of a variety of RNAs.These RNAs are required to have a better stability to maintain their functions.DEAD-box protein family is an ATP-dependent RNA helicase family that is found to be involved in a variety of RNA metabolic processes such as RNA secondary structure transformation, transcription initiation, mitochondrial RNA splicing, ribosome and spliceosome assembly,mRNA degradation, and the maintenance of mRNA stability.Recent studies have shown that some members of this family including DDX3, DDX4, DDX25 and others, are closely related to spermatogenesis.%精子的发生和成熟过程不但需要多种相关基因正确表达,而且还需要多种RNA的参与.因此,这些RNA需要较好的稳定性以保证其功能的正确执行.DEAD-box家族蛋白是一个ATP依赖的RNA解旋酶家族,参与RNA的各种代谢过程如RNA二级结构变换,转录起始,线粒体RNA剪接,核糖体和剪接体装配、mRNA降解以及维持mRNA的稳定性等.最近的一些研究显示这个家族的一些成员如DDX3,DDX4,DDX25等与精子生成有着密切的关系.

  10. 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.

  11. X-box binding protein 1 is essential for the anti-oxidant defense and cell survival in the retinal pigment epithelium.

    Directory of Open Access Journals (Sweden)

    Yimin Zhong

    Full Text Available Damage to the retinal pigment epithelium (RPE is an early event in the pathogenesis of age-related macular degeneration (AMD. X-box binding protein 1 (XBP1 is a key transcription factor that regulates endoplasmic reticulum (ER homeostasis and cell survival. This study aimed to delineate the role of endogenous XBP1 in the RPE. Our results show that in a rat model of light-induced retinal degeneration, XBP1 activation was suppressed in the RPE/choroid complex, accompanied by decreased anti-oxidant genes and increased oxidative stress. Knockdown of XBP1 by siRNA resulted in reduced expression of SOD1, SOD2, catalase, and glutathione synthase and sensitized RPE cells to oxidative damage. Using Cre/LoxP system, we generated a mouse line that lacks XBP1 only in RPE cells. Compared to wildtype littermates, RPE-XBP1 KO mice expressed less SOD1, SOD2, and catalase in the RPE, and had increased oxidative stress. At age 3 months and older, these mice exhibited apoptosis of RPE cells, decreased number of cone photoreceptors, shortened photoreceptor outer segment, reduced ONL thickness, and deficit in retinal function. Electron microscopy showed abnormal ultrastructure, Bruch's membrane thickening, and disrupted basal membrane infolding in XBP1-deficient RPE. These results indicate that XBP1 is an important gene involved in regulation of the anti-oxidant defense in the RPE, and that impaired activation of XBP1 may contribute to RPE dysfunction and cell death during retinal degeneration and AMD.

  12. The pro-inflammatory role of high-mobility group box 1 protein (HMGB-1) in photoreceptors and retinal explants exposed to elevated pressure.

    Science.gov (United States)

    Böhm, Michael R R; Schallenberg, Maurice; Brockhaus, Katrin; Melkonyan, Harutyun; Thanos, Solon

    2016-04-01

    To determine the role of high-mobility group box 1 protein (HMGB-1) in cellular and tissue models of elevated pressure-induced neurodegeneration, regeneration, and inflammation. Mouse retinal photoreceptor-derived cells (661W) and retinal explants were incubated either under elevated pressure or in the presence of recombinant HMGB-1 (rHMGB-1) to investigate the mechanisms of response of photoreceptors. Immunohistochemistry, western blotting, and the quantitative real-time PCR were used to examine the expression levels of immunological factors (eg, HMGB-1, receptor for advanced glycation end products (RAGE)), Toll-like receptors 2 and 4 (TLR-2, TLR-4), apoptosis-related factors (eg, B-cell lymphoma 2 (Bcl-2), Bcl-2-associated death promoter (Bad)) as well as cytokine expression (eg, tumor necrosis factor alpha (TNF-α), interleukin (IL)-4, IL-6, and vascular endothelial growth factor (VEGF)). The data revealed increased the expression of HMGB-1 and its receptors RAGE, TLR-2, and TLR-4, and TNF-α as well as pro-apoptotic factors (eg, Bad) as well as apoptosis in 661W cells exposed to elevated pressure. Co-cultivation of 661W cells with rHMGB-1 increased the expression levels of pro-apoptotic Bad and cleaved Caspase-3 resulting in apoptosis. Cytokine array studies revealed an increased release of TNF-α, IL-4, IL-6, and VEGF after incubation of 661W cells with rHMGB-1. Upregulation of HMGB-1, TLR-2, and RAGE as well as anti-apoptotic Bcl-2 expression levels was found in the retinal explants exposed to rHMGB-1 or elevated pressure. The results suggest that HMGB-1 promotes an inflammatory response and mediates apoptosis in the pathology of photoreceptors and retinal homeostasis. HMGB-1 may have a key role in ongoing damage of retinal cells under conditions of elevated intraocular pressure.

  13. 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

    localization signals (NLSs) in hEXO1. Using fluorescent fusion proteins, we show that the sequence 418KRPR421, which exhibit strong homology to other monopartite NLS sequences, is responsible for correct nuclear localization of hEXO1. This NLS sequence is located in a region that is also required for hEXO1......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...... interaction with hMLH1 and we show that defective nuclear localization of hEXO1 mutant proteins could be rescued by hMLH1 or hMSH2. Both hEXO1 and hMLH1 form complexes with the nuclear import factors importin beta/alpha1,3,7 whereas hMSH2 specifically recognizes importin beta/alpha3. Taken together, we infer...

  14. 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.

  15. 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.

  16. The human nuclear poly(a-binding protein promotes RNA hyperadenylation and decay.

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    Stefan M Bresson

    Full Text Available Control of nuclear RNA stability is essential for proper gene expression, but the mechanisms governing RNA degradation in mammalian nuclei are poorly defined. In this study, we uncover a mammalian RNA decay pathway that depends on the nuclear poly(A-binding protein (PABPN1, the poly(A polymerases (PAPs, PAPα and PAPγ, and the exosome subunits RRP6 and DIS3. Using a targeted knockdown approach and nuclear RNA reporters, we show that PABPN1 and PAPα, redundantly with PAPγ, generate hyperadenylated decay substrates that are recognized by the exosome and degraded. Poly(A tail extension appears to be necessary for decay, as cordycepin treatment or point mutations in the PAP-stimulating domain of PABPN1 leads to the accumulation of stable transcripts with shorter poly(A tails than controls. Mechanistically, these data suggest that PABPN1-dependent promotion of PAP activity can stimulate nuclear RNA decay. Importantly, efficiently exported RNAs are unaffected by this decay pathway, supporting an mRNA quality control function for this pathway. Finally, analyses of both bulk poly(A tails and specific endogenous transcripts reveals that a subset of nuclear RNAs are hyperadenylated in a PABPN1-dependent fashion, and this hyperadenylation can be either uncoupled or coupled with decay. Our results highlight a complex relationship between PABPN1, PAPα/γ, and nuclear RNA decay, and we suggest that these activities may play broader roles in the regulation of human gene expression.

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

    Directory of Open Access Journals (Sweden)

    Chieko Sugita

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

  18. Comparison of Nuclear Accumulation of p53 Protein with Mutations in the p53 Gene of Human Breast Cancer Tissues

    Institute of Scientific and Technical Information of China (English)

    王萱仪; 查小明; 武正炎; 范萍

    2001-01-01

    Objective The objective was to compare nuclear accumulation of p53 protein with mutations in the p53 gene on the tissues of human breast cancer. Methods Fifty-four invasive ductal carcinomas of breast were analyzed by the method of polymerase chain reaction-single strand conformational polymorphism (PCR-SSCP) silver stain and strep-avidin-biotin-peroxidase complex (SABC) immunohistochemistry. Results A statistically significant association between the presence of p53 gene mutation and nuclear accumulation of p53 protein was found (P<0.01). 22 tumors that demonstrated p53 gene mutations showed nuclear accumulation of p53 protein, while only 9 (28%) showed nuclear accumulation of p53 protein in 32 tumors without p53 gene mutations. Both p53 mutation protein and p53 gene mutations were prevalent in steroid and progesterone receptors negative tumors (P<0.05). A statistically significant association was found between the nuclear accumulation of p53 protein and lymph node invasion (P<0.05), and between p53 gene mutations and lymph node invasion (P<0.05). p53 abnormalities might be associated with an aggressive phenotype in breast cancer. Conclusion The immunohistochemical detection of nuclear p53 protein accumulation is highly associated with p53 gene mutations in breast cancer tissues, and that this method is useful for rapid screening of p53 abnormalities. However, in order to avoid false positive reaction, the p53 gene mutations should be determined in cases slightly positive for p53 nuclear protein.

  19. Lipophilic proteins encoded by mitochondrial and nuclear genes in Neurospora crassa.

    Science.gov (United States)

    Küntzel, H; Pieniaźek, N J; Pieniaźek, D; Leister, D E

    1975-06-01

    Mitochondrial proteins soluble in neutral chloroform-methanol (2:1) were separated from lipids by ether precipitation and resolved by Sephadex G-200 filtration in the presence of dodecylsulfate into two major fractions eluting in the excluded region (peak I) and in a region of an apparent molecular weight 8000 (peak II). Residual phospholipids are found only in peak II. Peak I consists of several aggregated small polypeptides of molecular weights around 8000, which can be disaggregated by mild oxidation with performic acid. Cycloheximide stimulates almost two-fold incorporation of radioactive phenylalanine into peak I proteins but inhibits labelling of peak II proteins by 95%. Chloramphenicol and ethidium bromide inhibit the synthesis of peak I proteins by 70% and 95% respectively, but do not affect labelling of peak II proteins. At least 30% of the translation products of mitochondrial DNA in vitro behave like peak I proteins: they are soluble in organic solvents, they aggregate in dodecylsulfate buffer after removal of phospholipids and they contain species of molecular weights around 8000 that disaggregate upon oxidation. The data strongly suggest that the proteins of peak I are encoded by mitochondrial genes and synthesized on mitochondrial ribosomes, whereas the proteins of peak II are encoded by nuclear genes and synthesized on cytoplasmic ribosomes. Both groups of lipophilic proteins are very similar in their molecular weights, but the mitochondrially coded peak I proteins have the unique property of forming large heat-stable aggregates in the presence of dodecylsulfate.

  20. A G-Box-Binding Protein from Soybean Binds to the E1 Auxin-Response Element in the Soybean GH3 Promoter and Contains a Proline-Rich Repression Domain.

    Science.gov (United States)

    Liu, Z. B.; Hagen, G.; Guilfoyle, T. J.

    1997-10-01

    The E1 promoter fragment (-249 to -203) is one of three auxin-response elements (AuxREs) in the soybean (Glycine max L.) GH3 promoter (Z.-B. Liu, T. Ulmasov, X. Shi, G. Hagen, T.J. Guilfoyle [1994] Plant Cell 6: 645-657). Results presented here further characterize and delimit the AuxRE within the E1 fragment. The E1 fragment functioned as an AuxRE in transgenic tobacco (Nicotiana tabacum L.) plants, as well as in transfected protoplasts. The AuxRE within E1 contains a G-box, and this G-box was used to clone a G-box-binding factor (GBF) from soybean (SGBF-2). This 45-kD GBF contains an N-terminal proline-rich domain and a C-terminal basic/leucine zipper DNA-binding domain. Gel-mobility shift assays were used to characterize the binding specificity of SGBF-2. Antiserum raised against recombinant SGBF-2 was used to further characterize SGBF-2 and antigenically related GBFs in soybean nuclear extracts. Co-transfection assays with effector and reporter plasmids in carrot (Daucus carota L.) protoplasts indicated that the N-terminal proline-rich domain of SGBF-2 functioned as a repression domain in both basal and auxin-inducible transcription.

  1. Presence of a functional but dispensable Nuclear Export Signal in the HTLV-2 Tax protein

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    Kiemer Lars

    2005-11-01

    Full Text Available Abstract Background Human T-cell leukemia virus type 1 and type 2 are related human retroviruses. HTLV-1 is the etiological agent of the Adult T-cell Leukemia/Lymphoma and of the Tropical Spastic Paraparesis/HTLV-1 Associated Myelopathy, whereas, HTLV-2 infection has not been formally associated with any T-cell malignancy. HTLV-1 and 2 genomes encode, respectively, the Tax1 and Tax2 proteins whose role is to transactivate the viral promoter. HTLV-1 and HTLV-2 Tax sequences display 28% divergence at the amino acid level. Tax1 is a shuttling protein that possesses both a non canonical nuclear import (NLS and a nuclear export (NES signal. We have recently demonstrated that Tax1 and Tax2 display different subcellular localization and that residues 90–100 are critical for this process. We investigate in the present report, whether Tax2 also possesses a functional NES. Results We first used a NES prediction method to determine whether the Tax2 protein might contain a NES and the results do suggest the presence of a NES sequence in Tax2. Using Green Fluorescent Protein-NES (GFP-NES fusion proteins, we demonstrate that the Tax2 sequence encompasses a functional NES (NES2. As shown by microscope imaging, NES2 is able to mediate translocation of GFP from the nucleus, without the context of a full length Tax protein. Furthermore, point mutations or leptomycin B treatment abrogate NES2 function. However, within the context of full length Tax2, similar point mutations in the NES2 leucine rich stretch do not modify Tax2 localization. Finally, we also show that Tax1 NES function is dependent upon the positioning of the nuclear export signal "vis-à-vis" GFP. Conclusion HTLV-2 Tax NES is functional but dispensable for the protein localization in vitro.

  2. CHANGES OF NUCLEAR MATRIX PROTEIN AND ITS RELATIONSHIP WITH c-erbB-2 IN HUMAN COLON ADENOCARCINOMA

    Institute of Scientific and Technical Information of China (English)

    WANG Ya-lan; GAO Jing; LI Yuan-yuan

    2005-01-01

    Objective: Nuclear matrix protein is tissue, cell-type specific, and tumor-relative. It plays an important role in the regulation of intranuclear processes. Some researches also showed that a c-erbB-2 promoter-specific DNA-binding nuclear matrix protein is present only in malignant human breast tissues and induces mitogenesis and cell surface expression of the c-erbB-2 protein in resting NIH/3T3 cells. But it is not clear that how it in colon adenocarcinomas. Methods:Two-dimensional gel electrophoretic method was used for NMP identification and immunohistochemistry was used for c-erbB-2 detection in 12 cases of colon adenocarcinomas and matched adjacent normal colon tissues. Results: 5 different nuclear matrix proteins (named C1-C5) were identified in 12 colon adenocarcinoma specimens, but not in the matched adjacent normal colon tissues; 3 nuclear matrix proteins (named N1-N3) were identified in all 12 matched adjacent normal colon tissues, but not in colon adenocarcinoma specimens. A nuclear matrix protein (named N4) was detected in all of 9moderated-well differentiated adenocarcinomas and all 12 matched adjacent normal colon tissues, but not in 3poor-differentiated adenocarcinomas. All of the 10 colon adenocarcinomas which had the nuclear matrix protein C4 were c-erbB-2 expression positive. Conclusion: The data suggest that there are specific nuclear matrix proteins in colon adenocarcinomas and its subtypes, which maybe valuable to serve as markers of colon adenocarcinomas in future. Nuclear matrix protein C4 probably is a c-erbB-2 promotor-specific nuclear matrix protein in colon adenocarcinomas, and may induce the expression of c-erbB-2.

  3. 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.

  4. Phenotype Clustering of Breast Epithelial Cells in Confocal Imagesbased on Nuclear Protein Distribution Analysis

    Energy Technology Data Exchange (ETDEWEB)

    Long, Fuhui; Peng, Hanchuan; Sudar, Damir; Levievre, Sophie A.; Knowles, David W.

    2006-09-05

    Background: The distribution of the chromatin-associatedproteins plays a key role in directing nuclear function. Previously, wedeveloped an image-based method to quantify the nuclear distributions ofproteins and showed that these distributions depended on the phenotype ofhuman mammary epithelial cells. Here we describe a method that creates ahierarchical tree of the given cell phenotypes and calculates thestatistical significance between them, based on the clustering analysisof nuclear protein distributions. Results: Nuclear distributions ofnuclear mitotic apparatus protein were previously obtained fornon-neoplastic S1 and malignant T4-2 human mammary epithelial cellscultured for up to 12 days. Cell phenotype was defined as S1 or T4-2 andthe number of days in cultured. A probabilistic ensemble approach wasused to define a set of consensus clusters from the results of multipletraditional cluster analysis techniques applied to the nucleardistribution data. Cluster histograms were constructed to show how cellsin any one phenotype were distributed across the consensus clusters.Grouping various phenotypes allowed us to build phenotype trees andcalculate the statistical difference between each group. The resultsshowed that non-neoplastic S1 cells could be distinguished from malignantT4-2 cells with 94.19 percent accuracy; that proliferating S1 cells couldbe distinguished from differentiated S1 cells with 92.86 percentaccuracy; and showed no significant difference between the variousphenotypes of T4-2 cells corresponding to increasing tumor sizes.Conclusion: This work presents a cluster analysis method that canidentify significant cell phenotypes, based on the nuclear distributionof specific proteins, with high accuracy.

  5. Abnormal expressions of proliferating cell nuclear antigen and P27 protein in brain glioma

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    BACKGROUND: Both proliferating cell nuclear antigen and P27 protein are important factors to regulate cell cycle. While, the combination of them can provide exactly objective markers to evaluate prognosis of patients with brain glioma needs to be further studied based on pathological level.OBJECTIVE: To observe the expressions of proliferating cell nuclear antigen and P27 protein in both injured and normal brain glioma tissues and analyze the effect of them on onset and development of brain glioma.DESIGN: Case contrast observation.SETTING: Department of Neurosurgery, the Second Affiliated Hospital of Xi'an Jiaotong University.PARTICIPANTS: A total of 63 patients with brain glioma were selected from Department of Neurosurgery,the Second Affiliated Hospital of Xi'an Jiaotong University from July 1996 to June 2000. There were 38 males and 25 females and their ages ranged from 23 to 71 years. Based on pathological classification and grading standards of brain glioma, patients were divided into grade Ⅰ - tⅡ (n =30) and grade Ⅲ - Ⅳ (n =33). All cases received one operation but no radiotherapy and chemiotherapy before operation. Sample tissues were collected from tumor parenchyma. Non-neoplastic brain tissues were collected from another 12 non-tumor subjects who received craniocerebral trauma infra-decompression and regarded as the control group. There were 10 males and 2 females and their ages ranged from 16 to 54 years. The experiment had got confirmed consent from local ethic committee and the collection was provided confirmed consent from patients and their relatives. All samples were restained with HE staining so as to diagnose as the brain glioma.While, all patients with brain glioma received radiotherapy after operation and their survival periods were followed up.METHODS: Primary lesion wax of brain glioma was cut into serial sections and stained with S-P immunohistochemical staining. Brown substance which was observed in tumor nucleus was regarded as the

  6. A Herpesvirus Protein Selectively Inhibits Cellular mRNA Nuclear Export.

    Science.gov (United States)

    Gong, Danyang; Kim, Yong Hoon; Xiao, Yuchen; Du, Yushen; Xie, Yafang; Lee, Kevin K; Feng, Jun; Farhat, Nisar; Zhao, Dawei; Shu, Sara; Dai, Xinghong; Chanda, Sumit K; Rana, Tariq M; Krogan, Nevan J; Sun, Ren; Wu, Ting-Ting

    2016-11-09

    Nuclear mRNA export is highly regulated to ensure accurate cellular gene expression. Viral inhibition of cellular mRNA export can enhance viral access to the cellular translation machinery and prevent anti-viral protein production but is generally thought to be nonselective. We report that ORF10 of Kaposi's sarcoma-associated herpesvirus (KSHV), a nuclear DNA virus, inhibits mRNA export in a transcript-selective manner to control cellular gene expression. Nuclear export inhibition by ORF10 requires an interaction with an RNA export factor, Rae1. Genome-wide analysis reveals a subset of cellular mRNAs whose nuclear export is blocked by ORF10 with the 3' UTRs of ORF10-targeted transcripts conferring sensitivity to export inhibition. The ORF10-Rae1 interaction is important for the virus to express viral genes and produce infectious virions. These results suggest that a nuclear DNA virus can selectively interfere with RNA export to restrict host gene expression for optimal replication.

  7. Nuclear localization of phosphorylated c-Myc protein in human tumor cells.

    Directory of Open Access Journals (Sweden)

    C. Soldani

    2010-05-01

    Full Text Available Using immunocytochemical techniques at light and electron microscopy, we analysed the distribution of phosphorylated c-Myc in actively proliferating human HeLa cells. The distribution pattern of c-Myc was also compared with those of other ribonucleoprotein (RNP-containing components (PANA, hnRNP-core proteins, fibrillarin or RNP-associated nuclear proteins (SC-35 splicing factor. Our results provide the first evidence that phosphorylated c-Myc accumulates in the nucleus of tumor cells, where it colocalizes with fibrillarin, both in the nucleolus and in extranucleolar structures.

  8. Small molecule inhibitors of PCNA/PIP-box interaction suppress translesion DNA synthesis.

    Science.gov (United States)

    Actis, Marcelo; Inoue, Akira; Evison, Benjamin; Perry, Scott; Punchihewa, Chandanamali; Fujii, Naoaki

    2013-04-01

    Proliferating cell nuclear antigen (PCNA) is an essential component for DNA replication and DNA damage response. Numerous proteins interact with PCNA through their short sequence called the PIP-box to be promoted to their respective functions. PCNA supports translesion DNA synthesis (TLS) by interacting with TLS polymerases through PIP-box interaction. Previously, we found a novel small molecule inhibitor of the PCNA/PIP-box interaction, T2AA, which inhibits DNA replication in cells. In this study, we created T2AA analogues and characterized them extensively for TLS inhibition. Compounds that inhibited biochemical PCNA/PIP-box interaction at an IC50 <5 μM inhibited cellular DNA replication at 10 μM as measured by BrdU incorporation. In cells lacking nucleotide-excision repair activity, PCNA inhibitors inhibited reactivation of a reporter plasmid that was globally damaged by cisplatin, suggesting that the inhibitors blocked the TLS that allows replication of the plasmid. PCNA inhibitors increased γH2AX induction and cell viability reduction mediated by cisplatin. Taken together, these findings suggest that inhibitors of PCNA/PIP-box interaction could chemosensitize cells to cisplatin by inhibiting TLS.

  9. Nucleophosmin/B23 is a proliferate shuttle protein associated with nuclear matrix.

    Science.gov (United States)

    Yun, Jing-Ping; Chew, Eng Ching; Liew, Choong-Tsek; Chan, John Y H; Jin, Mei-Lin; Ding, Ming-Xiao; Fai, Yam Hin; Li, H K Richard; Liang, Xiao-Man; Wu, Qiu-Liang

    2003-12-15

    It has become obvious that a better understanding and potential elucidation of the nucleolar phosphoprotein B23 involving in functional interrelationship between nuclear organization and gene expression. In present study, protein B23 expression were investigated in the regenerative hepatocytes at different periods (at days 0, 1, 2, 3, 4, 7) during liver regeneration after partial hepatectomy on the rats with immunohistochemistry and Western blot analysis. Another experiment was done with immunolabeling methods and two-dimensional (2-D) gel electrophoresis for identification of B23 in the regenerating hepatocytes and HepG2 cells (hepatoblastoma cell line) after sequential extraction with detergents, nuclease, and salt. The results showed that its expression in the hepatocytes had a locative move and quantitative change during the process of liver regeneration post-operation. Its immunochemical localization in the hepatocytes during the process showed that it moved from nucleoli of the hepatocytes in the stationary stage to nucleoplasm, cytoplasm, mitotic spindles, and mitotic chromosomes of the hepatocytes in the regenerating livers. It was quantitatively increased progressively to peak level at day 3 post-operation and declined gradually to normal level at day 7. It was detected in nuclear matrix protein (NMP) composition extracted from the regenerating hepatocytes and HepG2 cells and identified with isoelectric point (pI) value of 5.1 and molecular weight of 40 kDa. These results indicated that B23 was a proliferate shuttle protein involving in cell cycle and cell proliferation associated with nuclear matrix.

  10. The nuclear protein Waharan is required for endosomal-lysosomal trafficking in Drosophila.

    Science.gov (United States)

    Lone, Mohiddin; Kungl, Theresa; Koper, Andre; Bottenberg, Wolfgang; Kammerer, Richard; Klein, Melanie; Sweeney, Sean T; Auburn, Richard P; O'Kane, Cahir J; Prokop, Andreas

    2010-07-15

    Here we report Drosophila Waharan (Wah), a 170-kD predominantly nuclear protein with two potential human homologues, as a newly identified regulator of endosomal trafficking. Wah is required for neuromuscular-junction development and muscle integrity. In muscles, knockdown of Wah caused novel accumulations of tightly packed electron-dense tubules, which we termed 'sausage bodies'. Our data suggest that sausage bodies coincide with sites at which ubiquitylated proteins and a number of endosomal and lysosomal markers co-accumulate. Furthermore, loss of Wah function generated loss of the acidic LysoTracker compartment. Together with data demonstrating that Wah acts earlier in the trafficking pathway than the Escrt-III component Drosophila Shrb (snf7 in Schizosaccharomyces pombe), our results indicate that Wah is essential for endocytic trafficking at the late endosome. Highly unexpected phenotypes result from Wah knockdown, in that the distribution of ubiquitylated cargos and endolysosomal morphologies are affected despite Wah being a predominant nuclear protein. This finding suggests the existence of a relationship between nuclear functions and endolysosomal trafficking. Future studies of Wah function will give us insights into this interesting phenomenon.

  11. Nuclear Targeting of Methyl-Recycling Enzymes in Arabidopsis thaliana Is Mediated by Specific Protein Interactions

    Institute of Scientific and Technical Information of China (English)

    Sanghyun Lee; Andrew C. Doxey; Brendan J. McConkey; Barbara A. Moffatt

    2012-01-01

    Numerous transmethylation reactions are required for normal plant growth and development.S-adenosylhomocysteine hydrolase (SAHH) and adenosine kinase (ADK) act coordinately to recycle the by-product of these reactions,S-adenosylhomocysteine (SAH) that would otherwise competitively inhibit methyltransferase (MT) activities.Here,we report on investigations to understand how the SAH produced in the nucleus is metabolized by SAHH and ADK.Localization analyses using green fluorescent fusion proteins demonstrated that both enzymes are capable of localizing to the cytoplasm and the nucleus,although no obvious nuclear localization signal was found in their sequences.Deletion analysis revealed that a 41-amino-acid segment of SAHH (Gly1 50-Lys190) is required for nuclear targeting of this enzyme.This segment is surface exposed,shows unique sequence conservation patterns in plant SAHHs,and possesses additional features of protein-protein interaction motifs.ADK and SAHH interact in Arabidopsis via this segment and also interact with an mRNA cap MT.We propose that the targeting of this complex is directed by the nuclear localization signal of the MT; other MTs may similarly target SAHH/ADK to other subcellular compartments to ensure uninterrupted transmethylation.

  12. Differentially expressed nuclear proteins in human CCRF-CEM, HL-60, MEC-1 and Raji cells correlate with cellular properties.

    Science.gov (United States)

    Henrich, Silke; Crossett, Ben; Christopherson, Richard I

    2007-10-01

    The human cell lines CCRF-CEM (T-cell acute lymphocytic leukemia), HL-60 (acute myeloid leukemia), MEC-1 (B-cell chronic lymphocytic leukemia) and Raji (Burkitt's B-cell lymphoma) have been analysed for differences in their nuclear proteomes. Using 2-D DIGE, 55 nuclear proteins have been identified that are differentially expressed (p<0.025) between the four cell lines, including proteins associated with transcription, proliferation, DNA repair and apoptosis. Of these 55 proteins, 22 were over-expressed in just one cell line, and four were down-regulated in one cell line. Proteins uniquely over-expressed between myeloid and lymphoid cell lines include those that may have use as markers for diagnosis, disease progression and B-cell maturation and differentiation. Expression of various proliferation-associated nuclear proteins correlated with relative growth rates of the cell lines, giving these proteins potential diagnostic applications for distinction of chronic versus acute subtypes of haematological malignancies. Identification of these differentially expressed nuclear proteins should facilitate elucidation of the molecular mechanisms underlying leukocyte differentiation and transformation to leukemias and lymphomas. The nuclear expression profiles should enable classification of subtypes of leukemia, and identify potential nuclear protein targets for development of diagnostic and therapeutic strategies.

  13. 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.

  14. Nuclear matrix associated protein PML: an arsenic trioxide apoptosis therapeutic target protein in HepG2 cells

    Institute of Scientific and Technical Information of China (English)

    于鼎; 王子慧; 朱立元; 邱殷庆

    2003-01-01

    Objective To investigate arsenic trioxide (As2O3)-induced apoptosis and the effects on cell nuclear matrix related protein promyelocytic leukaemia (PML). Methods HepG2 cells were cultured in MEM medium and treated with 0.5, 2, 5 and 10 μmol/L As2O3 for either 24 h or 96 h at each concentration. In situ terminal deoxynucleotidyl transferase (TdT) labeling (TUNEL) and DNA ladders were used to detect apoptosis. Confocal microscopy and Western blotting were used to observe the expression of PML. Results The growth rates of HepG2 cells were slower in the As2O3 treated than the untreated control group. DNA ladder and TUNEL positive apoptotic cells could be detected in As2O3 treated groups. The expression of PML decreased in HepG2 cells with 2 μmol/L As2O3 treatment. Confocal images demonstrated that the expression of PML protein in HepG2 cell nuclei decreased after treatment with 2 μmol/L As2O3, and micropunctates characteristic of PML protein in HepG2 cell nuclei disappeared after treatment with 5 μmol/L As2O3.Conclusions Our results show that arsenic trioxide can significantly inhibit the growth of HepG2 cells in vitro. As2O3 induces apoptosis in HepG2 tumor cells in a time and concentration dependent manner. As2O3 may degrade the PML protein in HepG2 cell nuclei. The decreased expression of PML in As2O3 treated tumor cells is most likely to be caused by apoptosis. Nuclear matrix associated protein PML could be the target of As2O3 therapy.

  15. Straw in a Box

    Science.gov (United States)

    Jerrard, Richard; Schneider, Joel; Smallberg, Ralph; Wetzel, John

    2006-01-01

    A problem on a state's high school exit exam asked for the longest straw that would fit in a box. The examiners apparently wanted the length of a diagonal of the box, but the figure accompanying the question suggested otherwise--that the radius of the straw be considered. This article explores that more general problem.

  16. 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.

  17. 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...

  18. The mirror box

    Science.gov (United States)

    Thompson, Gene; Mathieson, Don

    2001-11-01

    The mirror box is an old standby in magic shows and an impressive demonstration of the law of reflection for the physics instructor. The box creates the illusion of an object floating in space by the use of a plane mirror.

  19. Boxing with neutrino oscillations

    Science.gov (United States)

    Wagner, D. J.; Weiler, Thomas J.

    1999-06-01

    We develop a characterization of neutrino oscillations based on the coefficients of the oscillating terms. These coefficients are individually observable; although they are quartic in the elements of the unitary mixing matrix, they are independent of the conventions chosen for the angle and phase parametrization of the mixing matrix. We call these reparametrization-invariant observables ``boxes'' because of their geometric relation to the mixing matrix, and because of their association with the Feynman box diagram that describes oscillations in field theory. The real parts of the boxes are the coefficients for the CP- or T-even oscillation modes, while the imaginary parts are the coefficients for the CP- or T-odd oscillation modes. Oscillation probabilities are linear in the boxes, so measurements can straightforwardly determine values for the boxes (which can then be manipulated to yield magnitudes of mixing matrix elements). We examine the effects of unitarity on the boxes and discuss the reduction of the number of boxes to a minimum basis set. For the three-generation case, we explicitly construct the basis. Using the box algebra, we show that CP violation may be inferred from measurements of neutrino flavor mixing even when the oscillatory factors have averaged. The framework presented here will facilitate general analyses of neutrino oscillations among n>=3 flavors.

  20. Autographa californica Multiple Nucleopolyhedrovirus Ac34 Protein Retains Cellular Actin-Related Protein 2/3 Complex in the Nucleus by Subversion of CRM1-Dependent Nuclear Export

    NARCIS (Netherlands)

    Mu, Jingfang; Zhang, Yongli; Hu, Yangyang; Hu, Xue; Zhou, Yuan; Zhao, He; Pei, Rongjuan; Wu, Chunchen; Chen, Jizheng; Zhao, Han; Yang, Kai; Oers, van Monique; Chen, Xinwen; Wang, Yun

    2016-01-01

    Actin, nucleation-promoting factors (NPFs), and the actin-related protein 2/3 complex (Arp2/3) are key elements of the cellular actin polymerization machinery. With nuclear actin polymerization implicated in ever-expanding biological processes and the discovery of the nuclear import mechanisms of ac

  1. Interactions of cullin3/KCTD5 complexes with both cytoplasmic and nuclear proteins: Evidence for a role in protein stabilization

    Energy Technology Data Exchange (ETDEWEB)

    Rutz, Natalja; Heilbronn, Regine; Weger, Stefan, E-mail: stefan.weger@charite.de

    2015-08-28

    Based on its specific interaction with cullin3 mediated by an N-terminal BTB/POZ homologous domain, KCTD5 has been proposed to function as substrate adapter for cullin3 based ubiquitin E3 ligases. In the present study we tried to validate this hypothesis through identification and characterization of additional KCTD5 interaction partners. For the replication protein MCM7, the zinc finger protein ZNF711 and FAM193B, a yet poorly characterized cytoplasmic protein, we could demonstrate specific interaction with KCTD5 both in yeast two-hybrid and co-precipitation studies in mammalian cells. Whereas trimeric complexes of cullin3 and KCTD5 with the respective KCTD5 binding partner were formed, KCTD5/cullin3 induced polyubiquitylation and/or proteasome-dependent degradation of these binding partners could not be demonstrated. On the contrary, KCTD5 or Cullin3 overexpression increased ZNF711 protein stability. - Highlights: • KCTD5 nuclear translocation depends upon M phase and protein oligomerization. • Identification of MCM7, ZNF711 and FAM193 as KCTD5 interaction partners. • Formation of trimeric complexes of KCTD5/cullin3 with MCM7, ZNF711 and FAM193B. • KCTD5 is not involved in polyubiquitylation of MCM7 replication factor. • The KCTD5/cullin3 complex stabilizes ZNF711 transcription factor.

  2. 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

  3. A second CRM1-dependent nuclear export signal in the influenza A virus NS2 protein contributes to the nuclear export of viral ribonucleoproteins.

    Science.gov (United States)

    Huang, Shengping; Chen, Jingjing; Chen, Quanjiao; Wang, Huadong; Yao, Yanfeng; Chen, Jianjun; Chen, Ze

    2013-01-01

    Influenza A virus NS2 protein, also called nuclear export protein (NEP), is crucial for the nuclear export of viral ribonucleoproteins. However, the molecular mechanisms of NEP mediation in this process remain incompletely understood. A leucine-rich nuclear export signal (NES2) in NEP, located at the predicted N2 helix of the N-terminal domain, was identified in the present study. NES2 was demonstrated to be a transferable NES, with its nuclear export activity depending on the nuclear export receptor chromosome region maintenance 1 (CRM1)-mediated pathway. The interaction between NEP and CRM1 is coordinately regulated by both the previously reported NES (NES1) and now the new NES2. Deletion of the NES1 enhances the interaction between NEP and CRM1, and deletion of the NES1 and NES2 motifs completely abolishes this interaction. Moreover, NES2 interacts with CRM1 in the mammalian two-hybrid system. Mutant viruses containing NES2 alterations generated by reversed genetics exhibit reduced viral growth and delay in the nuclear export of viral ribonucleoproteins (vRNPs). The NES2 motif is highly conserved in the influenza A and B viruses. The results demonstrate that leucine-rich NES2 is involved in the nuclear export of vRNPs and contributes to the understanding of nucleocytoplasmic transport of influenza virus vRNPs.

  4. A helminth cestode parasite express an estrogen-binding protein resembling a classic nuclear estrogen receptor.

    Science.gov (United States)

    Ibarra-Coronado, Elizabeth Guadalupe; Escobedo, Galileo; Nava-Castro, Karen; Jesús Ramses, Chávez-Rios; Hernández-Bello, Romel; García-Varela, Martìn; Ambrosio, Javier R; Reynoso-Ducoing, Olivia; Fonseca-Liñán, Rocío; Ortega-Pierres, Guadalupe; Pavón, Lenin; Hernández, María Eugenia; Morales-Montor, Jorge

    2011-01-01

    The role of an estrogen-binding protein similar to a known mammalian estrogen receptor (ER) is described in the estradiol-dependent reproduction of the helminth parasite Taenia crassiceps. Previous results have shown that 17-β-estradiol induces a concentration-dependent increase in bud number of in vitro cultured cysticerci. This effect is inhibited when parasites are also incubated in the presence of an ER binding-inhibitor (tamoxifen). RT-PCR assays using specific oligonucleotides of the most conserved ER sequences, showed expression by the parasite of a mRNA band of molecular weight and sequence corresponding to an ER. Western blot assays revealed reactivity with a 66 kDa protein corresponding to the parasite ER protein. Tamoxifen treatment strongly reduced the production of the T. crassiceps ER-like protein. Antibody specificity was demonstrated by immunoprecipitating the total parasite protein extract with anti-ER-antibodies. Cross-contamination by host cells was discarded by flow cytometry analysis. ER was specifically detected on cells expressing paramyosin, a specific helminth cell marker. Parasite cells expressing the ER-like protein were located by confocal microscopy in the subtegumental tissue exclusively. Analysis of the ER-like protein by bidimensional electrophoresis and immunoblot identified a specific protein of molecular weight and isoelectric point similar to a vertebrates ER. Sequencing of the spot produced a small fragment of protein similar to the mammalian nuclear ER. Together these results show that T. crassiceps expresses an ER-like protein which activates the budding of T. crassiceps cysticerci in vitro. To the best of our knowledge, this is the first report of an ER-like protein in parasites. This finding may have strong implications in the fields of host-parasite co-evolution as well as in sex-associated susceptibility to this infection, and could be an important target for the design of new drugs.

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

    Science.gov (United States)

    Rodríguez-Muñoz, Rafael; Cárdenas-Aguayo, María Del Carmen; Alemán, Víctor; Osorio, Beatriz; Chávez-González, Oscar; Rendon, Alvaro; Martínez-Rojas, Dalila; Meraz-Ríos, Marco Antonio

    2015-01-01

    The precise functional role of the dystrophin 71 in neurons is still elusive. Previously, we reported that dystrophin 71d and dystrophin 71f are present in nuclei from cultured neurons. In the present work, we performed a detailed analysis of the intranuclear distribution of dystrophin 71 isoforms (Dp71d and Dp71f), during the temporal course of 7-day postnatal rats hippocampal neurons culture for 1h, 2, 4, 10, 15 and 21 days in vitro (DIV). By immunofluorescence assays, we detected the highest level of nuclear expression of both dystrophin Dp71 isoforms at 10 DIV, during the temporal course of primary culture. Dp71d and Dp71f were detected mainly in bipolar GABAergic (≥60%) and multipolar Glutamatergic (≤40%) neurons, respectively. We also characterized the existence of two nuclear dystrophin-associated protein complexes (DAPC): dystrophin 71d or dystrophin 71f bound to 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.

  6. 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.

  7. Genome-wide screen of three herpesviruses for protein subcellular localization and alteration of PML nuclear bodies.

    Directory of Open Access Journals (Sweden)

    Jayme Salsman

    2008-07-01

    Full Text Available Herpesviruses are large, ubiquitous DNA viruses with complex host interactions, yet many of the proteins encoded by these viruses have not been functionally characterized. As a first step in functional characterization, we determined the subcellular localization of 234 epitope-tagged proteins from herpes simplex virus, cytomegalovirus, and Epstein-Barr virus. Twenty-four of the 93 proteins with nuclear localization formed subnuclear structures. Twelve of these localized to the nucleolus, and five at least partially localized with promyelocytic leukemia (PML bodies, which are known to suppress viral lytic infection. In addition, two proteins disrupted Cajal bodies, and 19 of the nuclear proteins significantly decreased the number of PML bodies per cell, including six that were shown to be SUMO-modified. These results have provided the first functional insights into over 120 previously unstudied proteins and suggest that herpesviruses employ multiple strategies for manipulating nuclear bodies that control key cellular processes.

  8. 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.

  9. 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.

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

    Directory of Open Access Journals (Sweden)

    Sébastien Gingras

    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.

  11. Production and Characterization of Monoclonal Antibodies against Human Nuclear Protein FAM76B.

    Directory of Open Access Journals (Sweden)

    Xiaojing Zheng

    Full Text Available Human FAM76B (hFAM76B is a 39 kDa protein that contains homopolymeric histidine tracts, a targeting signal for nuclear speckles. FAM76B is highly conserved among different species, suggesting that it may play an important physiological role in normal cellular functions. However, a lack of appropriate tools has hampered study of this potentially important protein. To facilitate research into the biological function(s of FAM76B, murine monoclonal antibodies (MAbs against hFAM76B were generated by using purified, prokaryotically expressed hFAM76B protein. Six strains of MAbs specific for hFAM76B were obtained and characterized. The specificity of MAbs was validated by using FAM76B-/- HEK 293 cell line. Double immunofluorescence followed by laser confocal microscopy confirmed the nuclear speckle localization of hFAM76B, and the specific domains recognized by different MAbs were further elucidated by Western blot. Due to the high conservation of protein sequences between mouse and human FAM76B, MAbs against hFAM76B were shown to react with mouse FAM76B (mFAM76B specifically. Lastly, FAM76B was found to be expressed in the normal tissues of most human organs, though to different extents. The MAbs produced in this study should provide a useful tool for investigating the biological function(s of FAM76B.

  12. Protein Kinase C-{delta} mediates down-regulation of heterogeneous nuclear ribonucleoprotein K protein: involvement in apoptosis induction

    Energy Technology Data Exchange (ETDEWEB)

    Gao, Feng-Hou [NO.3 People' s Hospital affiliated to Shanghai Jiao-Tong University School of Medicine (SJTU-SM), Shanghai 201900 (China); The Department of Pathophysiology, Key Laboratory of Cell Differentiation and Apoptosis of National Ministry of Education, Shanghai Jiao-Tong University School of Medicine (SJTU-SM), Shanghai 200025 (China); Wu, Ying-Li [The Department of Pathophysiology, Key Laboratory of Cell Differentiation and Apoptosis of National Ministry of Education, Shanghai Jiao-Tong University School of Medicine (SJTU-SM), Shanghai 200025 (China); Zhao, Meng [Institute of Health Science, SJTU-SM/Shanghai Institutes for Biological Science, Chinese Academy of Sciences, Shanghai (China); Liu, Chuan-Xu; Wang, Li-Shun [The Department of Pathophysiology, Key Laboratory of Cell Differentiation and Apoptosis of National Ministry of Education, Shanghai Jiao-Tong University School of Medicine (SJTU-SM), Shanghai 200025 (China); Chen, Guo-Qiang, E-mail: chengq@shsmu.edu.cn [The Department of Pathophysiology, Key Laboratory of Cell Differentiation and Apoptosis of National Ministry of Education, Shanghai Jiao-Tong University School of Medicine (SJTU-SM), Shanghai 200025 (China); Institute of Health Science, SJTU-SM/Shanghai Institutes for Biological Science, Chinese Academy of Sciences, Shanghai (China)

    2009-11-15

    We reported previously that NSC606985, a camptothecin analogue, induces apoptosis of acute myeloid leukemia (AML) cells through proteolytic activation of protein kinase C delta ({Delta}PKC-{delta}). By subcellular proteome analysis, heterogeneous nuclear ribonucleoprotein K (hnRNP K) was identified as being significantly down-regulated in NSC606985-treated leukemic NB4 cells. HnRNP K, a docking protein for DNA, RNA, and transcriptional or translational molecules, is implicated in a host of processes involving the regulation of gene expression. However, the molecular mechanisms of hnRNP K reduction and its roles during apoptosis are still not understood. In the present study, we found that, following the appearance of the {Delta}PKC-{delta}, hnRNP K protein was significantly down-regulated in NSC606985, doxorubicin, arsenic trioxide and ultraviolet-induced apoptosis. We further provided evidence that {Delta}PKC-{delta} mediated the down-regulation of hnRNP K protein during apoptosis: PKC-{delta} inhibitor could rescue the reduction of hnRNP K; hnRNP K failed to be decreased in PKC-{delta}-deficient apoptotic KG1a cells; conditional induction of {Delta}PKC-{delta} in U937T cells directly down-regulated hnRNP K protein. Moreover, the proteasome inhibitor also inhibited the down-regulation of hnRNP K protein by apoptosis inducer and the conditional expression of {Delta}PKC-{delta}. More intriguingly, the suppression of hnRNP K with siRNA transfection significantly induced apoptosis. To our knowledge, this is the first demonstration that proteolytically activated PKC-{delta} down-regulates hnRNP K protein in a proteasome-dependent manner, which plays an important role in apoptosis induction.

  13. Identification of potential nuclear reprogramming and differentiation factors by a novel selection method for cloning chromatin-binding proteins

    Institute of Scientific and Technical Information of China (English)

    LiuWang; AihuaZheng; LingYi; ChongrenXu; MingxiaoDing; HongkuiDeng

    2005-01-01

    Nuclear reprogramming is critical for animal cloning and stem cell creation through nuclear transfer, which requires extensive remodeling of chromosomal architecture involving dramatic changes in chromatin-binding proteins. To understand the mechanism of nuclear reprogramming, it is critical to identify chromatin-binding factors specify the reprogramming process. In this report, we have developed a high-throughput selection method, based on T7 phage display and chromatin immunoprecipitation, to isolate chromatin-binding factors expressed in mouse embryonic stem cells using primary mouse embryonic fibroblast chromatin. Seven chromatin-binding proteins have been isolated by this method. We have also isolated several chromatin-binding proteins involved in hepatocyte differentiation. Our method provides a powerful tool to rapidly and selectively identify chromatin-binding proteins. The method can be used to study epigenetic modification of chromatin during nuclear reprogramming, cell differentiation, and transdifferentiation.

  14. 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.

  15. Changes of Nuclear Matrix Proteins Following the Differentiation of Human Osteosarcoma MG-63 Cells

    Institute of Scientific and Technical Information of China (English)

    Chun-Hong Zhao; Qi-Fu Li; Yan Zhao; Jing-Wen Niu; Zhi-Xing Li; Jin-An Chen

    2006-01-01

    Human osteosarcoma MG-63 cells were induced into differentiation by 5 mmol/L hexamethylene bisacetamide (HMBA). Their nuclear matrix proteins (NMPs) were selectively extracted and subjected to two-dimensional gel electrophoresis analysis.The results of protein patterns were analyzed by Melanie software. The spots of differentially expressed NMPs were excised and subjected to in situ digestion with trypsin. The maps of peptide mass fingerprinting were obtained by MALDI-TOFMS analysis, and were submitted for NCBI database searches by Mascot tool.There were twelve spots changed remarkably during the differentiation induced by HMBA, nine of which were identified. The roles of the regulated proteins during the MG-63 differentiation were analyzed. This study suggests that the induced differentiation of cancer cells is accompanied by the changes of NMPs, and confirms the presence of some specific NMPs related to the cancer cell proliferation and differentiation. The changed NMPs are potential markers for cancer diagnosis or targets for cancer therapy.

  16. 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.

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

    Energy Technology Data Exchange (ETDEWEB)

    Paterson, Carolyn P.; Ayalew, Lisanework E. [Vaccine and Infectious Disease Organization-International Vaccine Center (VIDO-InterVac), University of Saskatchewan, Saskatoon, SK S7N 5E3 Canada (Canada); Veterinary Microbiology, University of Saskatchewan, Saskatoon, SK S7N 5E3 S7N 5B4 Canada (Canada); Tikoo, Suresh K., E-mail: suresh.tik@usask.ca [Vaccine and Infectious Disease Organization-International Vaccine Center (VIDO-InterVac), University of Saskatchewan, Saskatoon, SK S7N 5E3 Canada (Canada); Veterinary Microbiology, University of Saskatchewan, Saskatoon, SK S7N 5E3 S7N 5B4 Canada (Canada); School of Public Health, University of Saskatchewan, Saskatoon, SK S7N 5E5 Canada (Canada)

    2012-10-10

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

  18. 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.

  19. Timing of human protein evolution as revealed by massively parallel capture of Neandertal nuclear DNA sequences

    Science.gov (United States)

    Burbano, Hernán A.; Hodges, Emily; Green, Richard E.; Briggs, Adrian W.; Krause, Johannes; Meyer, Matthias; Good, Jeffrey M.; Maricic, Tomislav; Johnson, Philipp L.F.; Xuan, Zhenyu; Rooks, Michelle; Bhattacharjee, Arindam; Brizuela, Leonardo; Albert, Frank W.; de la Rasilla, Marco; Fortea, Javier; Rosas, Antonio; Lachmann, Michael; Hannon, Gregory J.; Pääbo, Svante

    2010-01-01

    Whole genome shotgun sequencing is now possible for extinct organisms, as well as the targeted capture of specific regions. However, targeted resequencing of megabase sized parts of nuclear genomes has yet to be demonstrated for ancient DNA. Here we show that hybridization capture on microarrays can be used to generate large scale targeted data from Neandertal DNA even in the presence of ~99.8% microbial DNA. It is thus now possible to generate high quality data from large regions of the nuclear genome from Neandertals and other extinct organisms. Using this approach we have sequenced ~14,000 protein coding positions that have been inferred to have changed on the human lineage since the last common ancestor shared with chimpanzees. We identify 88 amino acid substitutions that have become fixed in all humans since the divergence from the Neandertals. PMID:20448179

  20. 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.

  1. 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.

  2. Generation of transgenic Wuzhishan miniature pigs expressing monomeric red fluorescent protein by somatic cell nuclear transfer.

    Science.gov (United States)

    Lu, Yue; Kang, Jin-Dan; Li, Suo; Wang, Wei; Jin, Jun-Xue; Hong, Yu; Cui, Cheng-du; Yan, Chang-Guo; Yin, Xi-Jun

    2013-08-01

    Red fluorescent protein and its variants enable researchers to study gene expression, localization, and protein-protein interactions in vitro in real-time. Fluorophores with higher wavelengths are usually preferred since they efficiently penetrate tissues and produce less toxic emissions. A recently developed fluorescent protein marker, monomeric red fluorescent protein (mRFP1), is particularly useful because of its rapid maturation and minimal interference with green fluorescent protein (GFP) and GFP-derived markers. We generated a pCX-mRFP1-pgk-neoR construct and evaluated the ability of mRFP1 to function as a fluorescent marker in transgenic Wuzhishan miniature pigs. Transgenic embryos were generated by somatic cell nuclear transfer (SCNT) of nuclei isolated from ear fibroblasts expressing mRFP1. Embryos generated by SCNT developed into blastocysts in vitro (11.65%; 31/266). Thereafter, a total of 685 transgenic embryos were transferred into the oviducts of three recipients, two of which became pregnant. Of these, one recipient had six aborted fetuses, whereas the other recipient gave birth to four offspring. All offspring expressed the pCX-mRFP1-pgk-neoR gene as shown by PCR and fluorescence in situ hybridization analysis. The transgenic pigs expressed mRFP1 in all organs and tissues at high levels. These results demonstrate that Wuzhishan miniature pigs can express mRFP1. To conclude, this transgenic animal represents an excellent model with widespread applications in medicine and agriculture.

  3. 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.

  4. Silencing of Y-box binding protein-1 by RNA interference inhibits proliferation, invasion, and metastasis, and enhances sensitivity to cisplatin through NF-κB signaling pathway in human neuroblastoma SH-SY5Y cells.

    Science.gov (United States)

    Wang, Hong; Sun, Ruowen; Chi, Zuofei; Li, Shuang; Hao, Liangchun

    2017-04-05

    Y-box binding protein-1 (YB-1), a member of Y-box protein family binding DNA and RNA, has been proposed as a novel marker in multiple malignant tumors and found to be associated with tumor malignancy. Neuroblastoma is an embryonal tumor arising from neuroblast cells of the autonomic nervous system, which is the most common cancer diagnosed in infants. It has been reported that YB-1 is highly expressing in various human tumors including nasopharynx, thyroid, lung, breast, colon, ovary, and prostate cancers. This study aimed to investigate the functional role of YB-1 in neuroblastoma by silencing YB-1 using RNA interference (shRNA) in neuroblastoma SH-SY5Y cells. We found that silencing of YB-1 decreased the proliferation, migration, and invasion of SH-SY5Y cells. At molecular level, inhibition of YB-1 decreased the expression level of PCNA as well as MMP-2 in neuroblastoma SH-SY5Y cells. Also, we discovered that YB-1 silencing sensitized SH-SY5Y cells to cisplatin and promoted the apoptosis induced by cisplatin due to down-regulation of multidrug resistance (MDR) 1 protein via NF-κB signaling pathway. Therefore, we consider that targeting YB-1 is promising for neuroblastoma treatment and for overcoming its cisplatin resistance in the development of new neuroblastoma therapeutic strategies.

  5. Analysis of origin and protein-protein interaction maps suggests distinct oncogenic role of nuclear EGFR during cancer evolution

    Science.gov (United States)

    Sharip, Ainur; Abdukhakimova, Diyora; Wang, Xiao; Kim, Alexey; Kim, Yevgeniy; Sharip, Aigul; Orakov, Askarbek; Miao, Lixia; Sun, Qinglei; Chen, Yue; Chen, Zhenbang; Xie, Yingqiu

    2017-01-01

    Receptor tyrosine kinase EGFR usually is localized on plasma membrane to induce progression of many cancers including cancers in children (Bodey et al. In Vivo. 2005, 19:931-41), but it contains a nuclear localization signal (NLS) that mediates EGFR nuclear translocation (Lin et al. Nat Cell Biol. 2001, 3:802-8). Here we report that NLS of EGFR has its old evolutionary origin. Protein-protein interaction maps suggests that nEGFR pathways are different from membrane EGFR and EGF is not found in nEGFR network while androgen receptor (AR) is found, which suggests the evolution of prostate cancer, a well-known AR driven cancer, through changes in androgen- or EGF-dependence. Database analysis suggests that nEGFR correlates with the tumor grades especially in prostate cancer patients. Structural predication analysis suggests that NLS can compromise the differential protein binding to EGFR through stretch linkers with evolutionary mutation from N to V. In experiment, elevation of nEGFR but not membrane EGFR was found in castration resistant prostate cancer cells. Finally, systems analysis of NLS and transmembrane domain (TM) suggests that NLS has old origin while NLS neighboring domain of TM has been undergone accelerated evolution. Thus nEGFR has an old origin resembling the cancer evolution but TM may interfere with NLS driven signaling for natural selection of survival to evade NLS induced aggressive cancers. Our data suggest NLS is a dynamic inducer of EGFR oncogenesis during evolution for advanced cancers. Our model provides novel insights into the evolutionary role of NLS of oncogenic kinases in cancers.

  6. Pion in a Box

    CERN Document Server

    Bietenholz, W; Horsley, R; Nakamura, Y; Pleiter, D; Rakow, P E L; Schierholz, G; Zanotti, J M

    2010-01-01

    The residual mass of the pion in a finite spatial box at vanishing quark masses is computed with two flavors of dynamical clover fermions. The result is compared with predictions of chiral perturbation theory in the delta regime.

  7. Voice box (image)

    Science.gov (United States)

    The larynx, or voice box, is located in the neck and performs several important functions in the body. The larynx is involved in swallowing, breathing, and voice production. Sound is produced when the air which ...

  8. 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 ...

  9. [Boxing: traumatology and prevention].

    Science.gov (United States)

    Cabanis, Emmanuel-Alain; Iba-Zizen, Marie-Thérèse; Perez, Georges; Senegas, Xavier; Furgoni, Julien; Pineau, Jean-Claude; Louquet, Jean-Louis; Henrion, Roger

    2010-10-01

    In 1986, a surgeon who, as an amateur boxer himself was concerned with boxers' health, approached a pioneering Parisian neuroimaging unit. Thus began a study in close cooperation with the French Boxing Federation, spanning 25 years. In a first series of 52 volunteer boxers (13 amateurs and 39 professionals), during which MRI gradually replaced computed tomography, ten risk factors were identified, which notably included boxing style: only one of 40 "stylists" with a good boxing technique had cortical atrophy (4.5 %), compared to 15 % of "sloggers". Changes to the French Boxing Federation rules placed the accent on medical prevention. The second series, of 247 boxers (81 amateurs and 266 professionals), showed a clear improvement, as lesions were suspected in 14 individuals, of which only 4 (1.35 %) were probably due to boxing. The third and fourth series were part of a protocol called "Brain-Boxing-Ageing", which included 76 boxers (11 having suffered KOs) and 120 MRI scans, with reproducible CT and MRI acquisitions (9 sequences with 1.5 T then 3 T, and CT). MRI anomalies secondary to boxing were found in 11 % of amateurs and 38 % of professionals (atrophy, high vascular T2 signal areas, 2 cases of post-KO subdural bleeding). CT revealed sinus damage in 13 % of the amateurs and 19 % of the professionals. The risk of acute and chronic facial and brain damage was underline, along with detailed precautionary measures (organization of bouts, role of the referee and ringside doctor, and application of French Boxing Federation rules).

  10. Infectious disease and boxing.

    Science.gov (United States)

    King, Osric S

    2009-10-01

    There are no unique boxing diseases but certain factors contributing to the spread of illnesses apply strongly to the boxer, coach, and the training facility. This article examines the nature of the sport of boxing and its surrounding environment, and the likelihood of spread of infection through airborne, contact, or blood-borne routes of transmission. Evidence from other sports such as running, wrestling, and martial arts is included to help elucidate the pathophysiologic elements that could be identified in boxers.

  11. Nonneurologic emergencies in boxing.

    Science.gov (United States)

    Coletta, Domenic F

    2009-10-01

    Professional boxing has done an admirable job in promoting safety standards in its particular sport. However, injuries occur during the normal course of competition and, unfortunately, an occasional life-threatening emergency may arise. Although most common medical emergencies in boxing are injuries from closed head trauma, in this article those infrequent but potentially catastrophic nonneurologic conditions are reviewed along with some less serious emergencies that the physician must be prepared to address.

  12. Characterizing RNA-protein interaction using cross-linking and metabolite supplemented nuclear RNA-immunoprecipitation.

    Science.gov (United States)

    Au, Phil Chi Khang; Helliwell, Chris; Wang, Ming-Bo

    2014-05-01

    RNA-immunoprecipitation (RNA-IP) is a method used to isolate and identify RNA molecules specifically associated with an RNA-binding protein. Non-coding RNAs are emerging as key regulators of many biological and developmental pathways and RNA-IP has become an important tool in studying their function(s). While RNA-IP is successfully used to determine protein-RNA interaction, specific details regarding the level of this association and the metabolic requirement of this interaction which can influence the success of RNA-IP remain unclear. Here, we investigate the conditions required for efficient nuclear RNA-IP using Arabidopsis AGO4 (Argonaute 4) and siRNA binding as the study model. We showed that formaldehyde cross-linking, but not UV cross-linking, allowed for efficient pull-down of 24-nt siRNAs, suggesting that AGO4-siRNA interaction involves other protein(s). We also showed that, while formaldehyde cross-linking could also be performed on purified nuclei, ATP supplementation to the nuclei isolation buffer was needed to efficiently pull down 24-nt siRNAs. This result indicates that ATP is required for efficient siRNA loading onto AGO4. As most of the known RNA-mediated regulatory processes occur in the nucleus, our findings on cross-linking conditions and metabolite requirement for successful AGO4 nuclear RNA-IP provide a valuable insight and future consideration when studying the function of protein-RNA interactions in plants.

  13. 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

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

    Energy Technology Data Exchange (ETDEWEB)

    Iwahara, Naotoshi [Department of Pharmacology, Sapporo Medical University, Sapporo 060-8556 (Japan); Hisahara, Shin; Hayashi, Takashi [Department of Pharmacology, Sapporo Medical University, Sapporo 060-8556 (Japan); Department of Neurology, Sapporo Medical University, Sapporo 060-8556 (Japan); Horio, Yoshiyuki, E-mail: horio@sapmed.ac.jp [Department of Pharmacology, Sapporo Medical University, Sapporo 060-8556 (Japan)

    2009-09-04

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

  15. Liver-type fatty acid binding protein interacts with hepatocyte nuclear factor 4α

    OpenAIRE

    McIntosh, Avery L.; Petrescu, Anca D.; Hostetler, Heather A.; Kier, Ann B.; Schroeder, Friedhelm

    2013-01-01

    Hepatocyte nuclear factor 4α (HNF4α) regulates liver type fatty acid binding protein (L-FABP) gene expression. Conversely as shown herein, L-FABP structurally and functionally also interacts with HNF4α. Fluorescence resonance energy transfer (FRET) between Cy3-HNF4α (donor) and Cy5-L-FABP (acceptor) as well as FRET microscopy detected L-FABP in close proximity (~80 Å) to HNF4α, binding with high affinity Kd ~250–300 nM. Circular dichroism (CD) determined that the HNF4α/L-FABP interaction alte...

  16. DMPD: Manipulation of mitogen-activated protein kinase/nuclear factor-kappaB-signalingcascades during intracellular Toxoplasma gondii infection. [Dynamic Macrophage Pathway CSML Database

    Lifescience Database Archive (English)

    Full Text Available 15361242 Manipulation of mitogen-activated protein kinase/nuclear factor-kappaB-signalingcas...in kinase/nuclear factor-kappaB-signalingcascades during intracellular Toxoplasma... gondii infection. PubmedID 15361242 Title Manipulation of mitogen-activated protein kinase/nuclear factor-kappaB-signalingcas

  17. Mapping functional group free energy patterns at protein occluded sites: nuclear receptors and G-protein coupled receptors.

    Science.gov (United States)

    Lakkaraju, Sirish Kaushik; Yu, Wenbo; Raman, E Prabhu; Hershfeld, Alena V; Fang, Lei; Deshpande, Deepak A; MacKerell, Alexander D

    2015-03-23

    Occluded ligand-binding pockets (LBP) such as those found in nuclear receptors (NR) and G-protein coupled receptors (GPCR) represent a significant opportunity and challenge for computer-aided drug design. To determine free energies maps of functional groups of these LBPs, a Grand-Canonical Monte Carlo/Molecular Dynamics (GCMC/MD) strategy is combined with the Site Identification by Ligand Competitive Saturation (SILCS) methodology. SILCS-GCMC/MD is shown to map functional group affinity patterns that recapitulate locations of functional groups across diverse classes of ligands in the LBPs of the androgen (AR) and peroxisome proliferator-activated-γ (PPARγ) NRs and the metabotropic glutamate (mGluR) and β2-adreneric (β2AR) GPCRs. Inclusion of protein flexibility identifies regions of the binding pockets not accessible in crystal conformations and allows for better quantitative estimates of relative ligand binding affinities in all the proteins tested. Differences in functional group requirements of the active and inactive states of the β2AR LBP were used in virtual screening to identify high efficacy agonists targeting β2AR in Airway Smooth Muscle (ASM) cells. Seven of the 15 selected ligands were found to effect ASM relaxation representing a 46% hit rate. Hence, the method will be of use for the rational design of ligands in the context of chemical biology and the development of therapeutic agents.

  18. Sequential expression, activity and nuclear localization of prolyl oligopeptidase protein in the developing rat brain.

    Science.gov (United States)

    Hannula, Mirva J; Männistö, Pekka T; Myöhänen, Timo T

    2011-01-01

    Prolyl oligopeptidase (POP) is a serine protease that hydrolyzes peptides shorter than 30-mer. Some evidence has recently been obtained that POP can generate protein-protein interactions and therefore participate in various physiological and pathological events. Several studies have reported that POP may be involved in neurogenesis since its activity increases during development and can be found in the nucleus of proliferating tissues. In cell cultures, POP has been shown to be localized in the nucleus, but only early in the development, since during maturation it is moved to the cytosol. We have now studied for the first time the expression of POP protein, its enzymatic activity and nuclear localization in vivo in the developing rat brain. We observed that enzymatic activity of POP is highest on embryonic day 18 while the protein amounts reach their peak at birth. Furthermore, POP is located in the nucleus only early in the development but is transferred to the cytosol already before parturition. Our in vivo results confirm the previous cell culture results supporting the role of POP in neurogenesis. A discordance of antenatal protein amounts and enzymatic activities is suggesting a tight regulation of POP activity and possibly even a nonhydrolytic role at that stage.

  19. Predicting the Nuclear Localization Signals of 107 Types of HPV L1 Proteins by Bioinformatic Analysis

    Institute of Scientific and Technical Information of China (English)

    Jun Yang; Yi-Li Wang; Lü-Sheng Si

    2006-01-01

    In this study, 107 types of human papillomavirus (HPV) L1 protein sequences were obtained from available databases, and the nuclear localization signals (NLSs) of these HPV L1 proteins were analyzed and predicted by bioinformatic analysis.Out of the 107 types, the NLSs of 39 types were predicted by PredictNLS software (35 types of bipartite NLSs and 4 types of monopartite NLSs). The NLSs of the remaining HPV types were predicted according to the characteristics and the homology of the already predicted NLSs as well as the general rule of NLSs.According to the result, the NLSs of 107 types of HPV L1 proteins were classified into 15 categories. The different types of HPV L1 proteins in the same NLS category could share the similar or the same nucleocytoplasmic transport pathway.They might be used as the same target to prevent and treat different types of HPV infection. The results also showed that bioinformatic technology could be used to analyze and predict NLSs of proteins.

  20. 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.

  1. 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.

  2. EXPRESSION OF P53 PROTEIN AND PROLIFERATING CELL NUCLEAR ANTIGEN IN HUMAN GESTATION TROPHOBLASTIC DISEASE

    Institute of Scientific and Technical Information of China (English)

    黄铁军; 王志忠; 方光光; 刘志恒

    2004-01-01

    Objective: To study the relationship between p53 protein, proliferating cell nuclear antigen (PCNA) expression and benign or malignant gestational trophoblastic disease (MGTD). Methods: The histotomic sections of 48 patients with gestational trophoblastic disease and 24 patients of normal chorionic villi were stained using immunohistochemistry. The monoclonal antibodies were used to determine p53 protein and PCNA. Results: The frequency of p53 and PCNA positive expression were significantly different among the chorionic villi of normal pregnancy, hydratidiform mole (HM) and MGTD. But neither p53 nor PCNA has any relation with the clinical staging or metastasis of MGTD. Conclusion: Both P53 and PCNA are valuable in diagnosis of human gestational trophoblastic disease.

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

    Science.gov (United States)

    Pastor, Nina; Amero, Carlos

    2015-01-01

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

  4. Information Flow and Protein Dynamics: the Interplay Between Nuclear Magnetic Resonance Spectroscopy and Molecular Dynamics Simulations

    Directory of Open Access Journals (Sweden)

    Nina ePastor

    2015-05-01

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

  5. Data in support of DPF2 regulates OCT4 protein level and nuclear distribution

    Directory of Open Access Journals (Sweden)

    Chao Liu

    2015-12-01

    Full Text Available DPF2, also named ubi-d4/requiem (REQU, interacts with a protein complex containing OCT4. This paper provides data in support of the research article entitled “DPF2 regulates OCT4 protein level and nuclear distribution”. The highlights include: (1 Denature-immunoprecipitation assay revealed ubiquitination of OCT4 in pluripotent H9 cells, which was enhancedby MG132, a proteasome inhibitor. (2 Well colocalization of ectopic OCT4 and FLAG-Ub was found in HeLa cells, which was also increased by MG132. (3 MG132 treatment decreased DPF2 cytoplasmic expression in vivo. These data give insights into how proteasome inhibition contributes to studying ubiquitnation of OCT4.

  6. Nuclear TAR DNA-binding protein 43 A new target for amyotrophic lateral sclerosis treatment

    Institute of Scientific and Technical Information of China (English)

    Mei Zheng; Yujie Shi; Dongsheng Fan

    2013-01-01

    Abnormal TAR DNA-binding protein 43 (TDP-43) inclusion bodies can be detected in the degener-ative neurons of amyotrophic lateral sclerosis. In this study, we induced chronic oxidative stress in-jury by applying malonate to cultured mouse cortical motor neurons. In the later stages of the ma-lonate insult, TDP-43 expression reduced in the nuclei and transferred to the cytoplasm. This was accompanied by neuronal death, mimicking the pathological changes in TDP-43 that are seen in patients with amyotrophic lateral sclerosis. Interestingly, in the early stages of the response to ma-lonate treatment, nuclear TDP-43 expression increased, and neurons remained relatively intact, without inclusion bodies or fragmentation. Therefore, we hypothesized that the increase of nuclear TDP-43 expression might be a pro-survival factor against oxidative stress injury. This hypothesis was confirmed by an in vitro transgenic experiment, in which overexpression of wild type mouse TDP-43 in cultured cortical motor neurons significantly reduced malonate-induced neuronal death. Our findings suggest that the loss of function of TDP-43 is an important cause of neuronal dege-neration, and upregulation of nuclear TDP-43 expression might be neuroprotective in amyotrophic lateral sclerosis.

  7. A redox-sensitive yellow fluorescent protein sensor for monitoring nuclear glutathione redox dynamics.

    Science.gov (United States)

    Banach-Latapy, Agata; Dardalhon, Michèle; Huang, Meng-Er

    2015-01-01

    Intracellular redox homeostasis is crucial for many cellular functions, but accurate measurements of cellular compartment-specific redox states remain technically challenging. Genetically encoded biosensors, including the glutathione-specific redox-sensitive yellow fluorescent protein (rxYFP), provide an alternative approach to overcome the limitations of conventional glutathione/glutathione disulfide (GSH/GSSG) redox measurements. In this chapter we describe methods to measure the nuclear rxYFP redox state in human cells by a redox Western blot technique. A nucleus-targeted rxYFP sensor can be used to sense nuclear steady-state and dynamic redox changes in response to oxidative stress. Complementary to existing redox sensors and conventional redox measurements, nucleus-targeted rxYFP sensors provide a novel tool for examining nuclear redox homeostasis in mammalian cells, permitting high-resolution readout of steady glutathione state and dynamics of redox changes. The technique described may be used with minimal variations to study the effects of stress conditions which lead to glutathione redox changes.

  8. Src1 is a Protein of the Inner Nuclear Membrane Interacting with the Dictyostelium Lamin NE81

    Directory of Open Access Journals (Sweden)

    Petros Batsios

    2016-03-01

    Full Text Available The nuclear envelope (NE consists of the outer and inner nuclear membrane (INM, whereby the latter is bound to the nuclear lamina. Src1 is a Dictyostelium homologue of the helix-extension-helix family of proteins, which also includes the human lamin-binding protein MAN1. Both endogenous Src1 and GFP-Src1 are localized to the NE during the entire cell cycle. Immuno-electron microscopy and light microscopy after differential detergent treatment indicated that Src1 resides in the INM. FRAP experiments with GFP-Src1 cells suggested that at least a fraction of the protein could be stably engaged in forming the nuclear lamina together with the Dictyostelium lamin NE81. Both a BioID proximity assay and mis-localization of soluble, truncated mRFP-Src1 at cytosolic clusters consisting of an intentionally mis-localized mutant of GFP-NE81 confirmed an interaction of Src1 and NE81. Expression GFP-Src11–646, a fragment C-terminally truncated after the first transmembrane domain, disrupted interaction of nuclear membranes with the nuclear lamina, as cells formed protrusions of the NE that were dependent on cytoskeletal pulling forces. Protrusions were dependent on intact microtubules but not actin filaments. Our results indicate that Src1 is required for integrity of the NE and highlight Dictyostelium as a promising model for the evolution of nuclear architecture.

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

    Energy Technology Data Exchange (ETDEWEB)

    Cambier, Linda [CNRS UMR5237, Universite Montpellier 1, Universite Montpellier 2, Centre de Recherche en Biochimie Macromoleculaire, Montpellier (France); Pomies, Pascal, E-mail: pascal.pomies@crbm.cnrs.fr [CNRS UMR5237, Universite Montpellier 1, Universite Montpellier 2, Centre de Recherche en Biochimie Macromoleculaire, Montpellier (France)

    2011-06-17

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

  10. Yes-Associated Protein (YAP) Promotes the Nuclear Import of p73

    Energy Technology Data Exchange (ETDEWEB)

    Zhang Heng; Wu Shengnan, E-mail: wushn@scnu.edu.cn [MOE Key Laboratory of Laser Life Science and Institute of Laser Life Science, College of Biophotonics, South China Normal University, Guangzhou 510631 (China)

    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.

  11. 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.

  12. Identification of a nuclear transport inhibitory signal (NTIS) in the basic domain of HIV-1 Vif protein.

    Science.gov (United States)

    Friedler, A; Zakai, N; Karni, O; Friedler, D; Gilon, C; Loyter, A

    1999-06-11

    The HIV-1 auxiliary protein Vif contains a basic domain within its sequence. This basic region,90RKKR93, is similar to the prototypic nuclear localization signal (NLS). However, Vif is not a nuclear protein and does not function in the nucleus. Here we have studied the karyophilic properties of this basic region. We have synthesized peptides corresponding to this positively charged NLS-like region and observed that these peptides inhibited nuclear transport via the importin pathway in vitro with IC50values in the micromolar range. Inhibition was observed only with peptides derived from the positively charged region, but not from other regions of the Vif protein, showing sequence specificity. On the other hand, the Vif inhibitory peptide Vif88-98 did not confer karyophilic properties when conjugated to BSA. The inactive Vif conjugate and the active SV40-NLS-BSA conjugate both contained a similar number of peptides conjugated to each BSA molecule, as was determined by amino acid analysis of the peptide-BSA conjugates. Thus, the lack of nuclear import of the Vif peptide-BSA conjugate cannot be attributed to insufficient number of conjugated peptide molecules per BSA molecule. Our results suggest that the HIV-1 Vif protein carries an NLS-like sequence that inhibits, but does not mediate, nuclear import via the importin pathway. We have termed such signals as nuclear transport inhibitory signals (NTIS). The possible role of NTIS in controlling nuclear uptake, and specifically during virus infection, is discussed herein. Our results raise the possibility that NLS-like sequences of certain low molecular weight viral proteins may serve as regulators of nucleocytoplasmic trafficking and not neccessarily as mediators of nuclear import.

  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. [The nuclear matrix proteins (mol. mass 38 and 50 kDa) are transported by chromosomes in mitosis].

    Science.gov (United States)

    Murasheva, M I; Chentsov, Iu S

    2010-01-01

    It was shown by immunofluorescence method that serum M68 and serum K43 from patients with autoimmune disease stain interphase nuclei and periphery of mitotic chromosomes of pig kidney cells. Western blotting reveals the polypeptide with mol. mass of 50 kDa in serum M68, and the polypeptide with mol. mass of 38 kDa in serum K43. In the nuclear protein matrix, the antibodies to protein with mol. mass of 38 kDa stained only nucleolar periphery, while the antibodies to the protein with mol. mass of 50 kDa stained both the nucleolar periphery and all the interphase nucleus. It shows that among all components of nuclear protein matrix (lamina, internuclear network, residual nucleoli) only nucleolar periphery contains the 38 kDa protein, while the 50 kDa protein is a part of residual nucleolar periphery and takes part in nuclear protein network formation. In the interphase cells, both proteins were in situ localized in the nuclei, but one of them with mol. mass of 50 kDa was in the form of small clearly outlined granules, while the other (38 kDa) was in the form of small bright granules against the background of diffusely stained nuclei. Both proteins were also revealed as continuous ring around nucleolar periphery. During all mitotic stages, the 50 kDa protein was seen on the chromosomal periphery as a cover, and the 38 kDa protein formed separate fragments and granules around them. After nuclear and chromosome decondensation induced by hypotonic treatment, both antibodies stain interphase nuclei in diffuse manner, but in mitotic cells they stained the surface of the swollen chromosomes. The polypeptide with mol. mass of 50 kDa maintained strong connection with chromosome periphery both in norm and under condition of decondensation induced by hypotonic treatment and at subsequent recondensation in isotonic medium. In contrast, the protein with mol. mass of 38 kDa partially lost the contact with a chromosome during recondensation appearing also in the form of granules in

  15. Interaction between composite elements in the napA promoter: both the B-box ABA-responsive complex and the RY/G complex are necessary for seed-specific expression.

    Science.gov (United States)

    Ezcurra, I; Ellerström, M; Wycliffe, P; Stålberg, K; Rask, L

    1999-07-01

    During seed maturation, the transcriptional activity of napin genes is regulated by developmental signals involving the transcriptional activator ABI3 and abscisic acid (ABA). To localize cis elements involved in the seed-specific activity of the napin napA promoter, a systematic analysis was performed focusing on two major element complexes, the B-box and RY/G. Substitution mutation analysis using promoter-reporter gene fusions in stable transgenic tobacco showed synergistic interactions between elements within these complexes. The distal part of the B-box shows similarities to abscisic acid response elements and the proximal portion contains a CA-rich element. In vitro studies involving Exonuclease III protection and electrophoretic mobility shift assays revealed binding by nuclear proteins to elements within the B-box. The distal and proximal parts of the B-box were found to bind distinct nuclear protein complexes. By gain-of-function analysis with a tetramer of the B-box fused to a truncated (-46) cauliflower mosaic virus (CaMV) 35S minimal promoter, it was demonstrated that the B-box mediates strong activity in seeds. Further, it was shown that the elements in the B-box constitute an ABA-responsive complex, since the B-box tetramer mediates ABA-responsiveness in vegetative tissues to a construct containing the CaMV virus 35S enhancer (-343 to -90). Thus, the seed-specific activity of the napA promoter relies on the combinatorial interaction between the RY/G complex and the B-box ABA-responsive complex during the ABA response in seed development.

  16. 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

    remain largely unexplored. To increase our understanding of the ASB proteins function, we conducted a family-wide SILAC (Stable Isotope Labeling by Amino acids in Cell Culture)-based protein-protein interaction analysis. This investigation led to the identification of novel as well as known ASB...... in vivo. In summary, we provide a comprehensive protein-protein interaction data resource that can aid the biological and functional characterization of ASB ubiquitin ligases....

  17. Search for Conditions to Detect Epigenetic Marks and Nuclear Proteins in Immunostaining of the Testis and Cartilage

    Directory of Open Access Journals (Sweden)

    Hisashi Ideno

    2014-01-01

    Full Text Available The localization of nuclear proteins and modified histone tails changes during cell differentiation at the tissue as well as at the cellular level. Immunostaining in paraffin sections is the most powerful approach available to evaluate protein localization. Since nuclear proteins are sensitive to fixation, immunohistochemical conditions should be optimized in light of the particular antibodies and tissues employed. In this study, we searched for optimal conditions to detect histone modification at histone H3 lysine 9 (H3K9 and H3K9 methyltransferase G9a in the testis and cartilage in paraffin sections. In the testis, antigen retrieval (AR was indispensable for detecting H3K9me1 and me3, G9a, and nuclear protein proliferating cell nuclear antigen (PCNA. With AR, shorter fixation times yielded better results for the detection of G9a and PCNA. Without AR, H3K9me2 and H3K9ac could be detected at shorter fixation times in primary spermatocytes of the testis. In contrast to the testis, all antibodies tested could detect their epitopes irrespective of AR application in the growth plate cartilage. Thus, conditions for the detection of epigenetic marks and nuclear proteins should be optimized in consideration of fixation time and AR application in different tissues and antibodies.

  18. Transfection of influenza A virus nuclear export protein induces the expression of tumor necrosis factor alpha.

    Science.gov (United States)

    Lara-Sampablo, Alejandra; Flores-Alonso, Juan Carlos; De Jesús-Ortega, Nereyda; Santos-López, Gerardo; Vallejo-Ruiz, Verónica; Rosas-Murrieta, Nora; Reyes-Carmona, Sandra; Herrera-Camacho, Irma; Reyes-Leyva, Julio

    2014-06-24

    Influenza A virus genomic segments eight codes for non-structural 1 (NS1) protein that is involved in evasion of innate antiviral response, and nuclear export protein (NEP) that participates in the export of viral ribonucleoprotein (RNP) complexes, transcription and replication. Tumor necrosis factor alpha (TNF-α) is highly expressed during influenza virus infections and is considered an anti-infective cytokine. NS1 and NEP proteins were overexpressed and their role on TNF-α expression was evaluated. Both TNF-α mRNA and protein increased in cells transfected with NEP but not with NS1. We further investigate if NS1 or NEP regulates the activity of TNF-α promoter. In the presence of NEP the activity of TNF-α promoter increased significantly compared with the control (83.5±2.9 vs. 30.9±2.8, respectively; p=0.001). This effect decreased 15-fold when the TNF-α promoter distal region was deleted, suggesting the involvement of mitogen-activated protein kinases (MAPK) and NF-kB response elements. This was corroborated by testing the effect produced on TNF-α promoter by the treatment with Raf/MEK/ERK (U0126), NF-kB (Bay-11-7082) and PI3K (Ly294-002) cell signaling inhibitors. Treatment with U0126 and Bay-117082 reduced the activity of TNF-α promoter mediated by NEP (41.5±3.2, 70% inhibition; and 80.6±7.4, 35% inhibition, respectively) compared to mock-treated control. The results suggest a new role for NEP protein that participates in the transcriptional regulation of human TNF-α expression.

  19. Novel Nuclear Protein ALC-INTERACTING PROTEIN1 is Expressed in Vascular and Mesocarp Cells in Arabidopsis

    Institute of Scientific and Technical Information of China (English)

    Fang Wang; Dong-Qiao Shi; Jie Liu; Wei-Cai Yang

    2008-01-01

    Pod shattering is an agronomical trait that is a result of the coordinated action of cell differentiation and separation. In Arabidopsis, pod shattering is controlled by a complex genetic network in which ALCATRAZ (ALC), a member of the basic helix-loop-helix family, is critical for cell separation during fruit dehiscence. Herein, we report the identification of ALC-INTERACTiNG PROTEIN1 (ACI1) via the yeast two-hybrid screen. ACI1 encodes a nuclear protein with a lysine-rich domain and a C-terminal serine-rich domain. ACI1 is mainly expressed in the vascular system throughout the plant and mesocarp of the valve in siliques. Our data showed that ACI1 interacts strongly with the N-terminal portion of ALC in yeast cells and in plant cells in the nucleus as demonstrated by bimolecular fluorescence complementation assay. Both ACl1 and ALC share an overlapping expression pattern, suggesting that they likely function together in planta. However, no detectable phenotype was found in plants with reduced ACI1 expression by RNA interference technology, suggesting that ACI1 may be redundant. Taken together, these data indicate that ALC may interact with ACll and its homologs to control cell separation during fruit dehiscence in Arabidopsis.

  20. Epstein - Barr virus latent membrane protein 1 suppresses reporter activity through modulation of promyelocytic leukemia protein-nuclear bodies

    Directory of Open Access Journals (Sweden)

    Flemington Erik K

    2011-10-01

    Full Text Available Abstract The Epstein-Barr virus (EBV encoded Latent Membrane Protein 1 (LMP1 has been shown to increase the expression of promyelocytic leukemia protein (PML and the immunofluorescent intensity of promyelocytic leukemia nuclear bodies (PML NBs. PML NBs have been implicated in the modulation of transcription and the association of reporter plasmids with PML NBs has been implicated in repression of reporter activity. Additionally, repression of various reporters in the presence of LMP1 has been noted. This study demonstrates that LMP1 suppresses expression of reporter activity in a dose responsive manner and corresponds with the LMP1 induced increase in PML NB intensity. Disruption of PML NBs with arsenic trioxide or a PML siRNA restores reporter activity. These data offer an explanation for previously conflicting data on LMP1 signaling and calls attention to the possibility of false-positives and false-negatives when using reporter assays as a research tool in cells expressing LMP1.

  1. Depth in box spaces.

    Science.gov (United States)

    Pont, Sylvia C; Nefs, Harold T; van Doorn, Andrea J; Wijntjes, Maarten W A; Te Pas, Susan F; de Ridder, Huib; Koenderink, Jan J

    2012-01-01

    Human observers adjust the frontal view of a wireframe box on a computer screen so as to look equally deep and wide, so that in the intended setting the box looks like a cube. Perspective cues are limited to the size-distance effect, since all angles are fixed. Both the size on the screen, and the viewing distance from the observer to the screen were varied. All observers prefer a template view of a cube over a veridical rendering, independent of picture size and viewing distance. If the rendering shows greater or lesser foreshortening than the template, the box appears like a long corridor or a shallow slab, that is, like a 'deformed' cube. Thus observers ignore 'veridicality'. This does not fit an 'inverse optics' model. We discuss a model of 'vision as optical user interface'.

  2. Homeostatic restitution of cell membranes. Nuclear membrane lipid biogenesis and transport of protein from cytosol to intranuclear spaces.

    Directory of Open Access Journals (Sweden)

    Amalia Slomiany, Maria Grabska, Bronislaw L. Slomiany

    2006-01-01

    Full Text Available Our studies on homeostatic restitution of cellular and subcellular membranes showed that vesicular intracellular transport is engaged in systematic and coordinated replacement of lipids and proteins in the membranes of the secretory, non-dividing epithelial cells (Slomiany et al., J. Physiol. Pharmacol. 2004; 55: 837-860. In this report, we present evidence on the homeostatic restitution of lipids in the biomembranes that constitute nuclear envelopes. We investigated nuclear membranes lipid synthesis by employing purified intact nuclei (IN, the outer nuclear membrane (ONM, the inner nuclear membrane (INM and the cell cytosol (CC. In contrast to Endoplasmic Reticulum (ER which in the presence of CC generates new biomembrane that forms ER vesicles transporting ER products to Golgi, the IN, ONM and INM are not producing transport vesicles. Instead, the newly synthesized lipids remain in the nuclear membranes. The membranes (INM, ONM of IN incubated with CC become enriched with newly synthesized phosphatidylcholine (PC, phosphatidylinositol (PI, phosphatidylinositol phosphates (PIPs and phosphatidic acid (PA. The incubation of separated ONM and INM with CC also enriched the membranes with IN specific lipids identified above. Moreover, the incubation of IN or its membranes with CC afforded retention of numerous CC proteins on the nuclear membrane. Here, we concentrated on 30kDa CC protein that displayed affinity to nuclear membrane PIP2. The 30kDa CC protein bound to PIP2 of IN, INM, and ONM. With IN, initially the PIP2-30kDa CC protein complex was detected on ONM, after 30-120 min of incubation, was found on INM and in nuclear contents. At the same time when the 30 kDa protein was released from INM and found in nuclear contents, the PIP2 of INM and ONM became undetectable, while the lipid extract from the membrane displaced from IN contained labeled PI only. Since ONM is an uninterrupted continuum of ER and INM, we speculate that the synthesis of

  3. 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.

  4. 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.

  5. 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.

  6. 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.

  7. Eye trauma in boxing.

    Science.gov (United States)

    Corrales, Gustavo; Curreri, Anthony

    2009-10-01

    In boxing, along with a few other sports, trauma is inherent to the nature of the sport; therefore it is considered a high-risk sport for ocular injuries. The long-term morbidity of ocular injuries suffered by boxers is difficult to estimate due to the lack of structured long-term follow-up of these athletes. Complications of blunt ocular trauma may develop years after the athlete has retired from the ring and is no longer considered to be at risk for boxing-related injuries. This article describes the wide range of eye injuries a boxer can sustain, and their immediate and long-term clinical management.

  8. The Nuclear PolyA-Binding Protein Nab2p Is Essential for mRNA Production

    DEFF Research Database (Denmark)

    Schmid, Manfred; Olszewski, Pawel; Pelechano, Vicent;

    2015-01-01

    Polyadenylation of mRNA is a key step in eukaryotic gene expression. However, despite the major impact of poly(A) tails on mRNA metabolism, the precise roles of poly(A)-binding proteins (PABPs) in nuclear mRNA biogenesis remain elusive. Here, we demonstrate that rapid nuclear depletion of the S....... cerevisiae PABP Nab2p leads to a global loss of cellular mRNA, but not of RNA lacking poly(A) tails. Disappearance of mRNA is a nuclear event, but not due to decreased transcription. Instead, the absence of Nab2p results in robust nuclear mRNA decay by the ribonucleolytic RNA exosome in a polyadenylation......-dependent process. We conclude that Nab2p is required to protect early mRNA and therefore constitutes a crucial nuclear mRNA biogenesis factor....

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

    Science.gov (United States)

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

    2015-12-01

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

  10. 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.

  11. 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.; 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. PMID:22899993

  12. Nuclear import and dimerization of tomato ASR1, a water stress-inducible protein exclusive to plants.

    Directory of Open Access Journals (Sweden)

    Martiniano M Ricardi

    Full Text Available 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.

  13. 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.

  14. Nuclear Fragile X Mental Retardation Protein is localized to Cajal bodies.

    Science.gov (United States)

    Dury, Alain Y; El Fatimy, Rachid; Tremblay, Sandra; Rose, Timothy M; Côté, Jocelyn; De Koninck, Paul; Khandjian, Edouard W

    2013-10-01

    Fragile X syndrome is caused by loss of function of a single gene encoding the Fragile X Mental Retardation Protein (FMRP). This RNA-binding protein, widely expressed in mammalian tissues, is particularly abundant in neurons and is a component of messenger ribonucleoprotein (mRNP) complexes present within the translational apparatus. The absence of FMRP in neurons is believed to cause translation dysregulation and defects in mRNA transport essential for local protein synthesis and for synaptic development and maturation. A prevalent model posits that FMRP is a nucleocytoplasmic shuttling protein that transports its mRNA targets from the nucleus to the translation machinery. However, it is not known which of the multiple FMRP isoforms, resulting from the numerous alternatively spliced FMR1 transcripts variants, would be involved in such a process. Using a new generation of anti-FMRP antibodies and recombinant expression, we show here that the most commonly expressed human FMRP isoforms (ISO1 and 7) do not localize to the nucleus. Instead, specific FMRP isoforms 6 and 12 (ISO6 and 12), containing a novel C-terminal domain, were the only isoforms that localized to the nuclei in cultured human cells. These isoforms localized to specific p80-coilin and SMN positive structures that were identified as Cajal bodies. The Cajal body localization signal was confined to a 17 amino acid stretch in the C-terminus of human ISO6 and is lacking in a mouse Iso6 variant. As FMRP is an RNA-binding protein, its presence in Cajal bodies suggests additional functions in nuclear post-transcriptional RNA metabolism. Supporting this hypothesis, a missense mutation (I304N), known to alter the KH2-mediated RNA binding properties of FMRP, abolishes the localization of human FMRP ISO6 to Cajal bodies. These findings open unexplored avenues in search for new insights into the pathophysiology of Fragile X Syndrome.

  15. Nuclear Fragile X Mental Retardation Protein is localized to Cajal bodies.

    Directory of Open Access Journals (Sweden)

    Alain Y Dury

    2013-10-01

    Full Text Available Fragile X syndrome is caused by loss of function of a single gene encoding the Fragile X Mental Retardation Protein (FMRP. This RNA-binding protein, widely expressed in mammalian tissues, is particularly abundant in neurons and is a component of messenger ribonucleoprotein (mRNP complexes present within the translational apparatus. The absence of FMRP in neurons is believed to cause translation dysregulation and defects in mRNA transport essential for local protein synthesis and for synaptic development and maturation. A prevalent model posits that FMRP is a nucleocytoplasmic shuttling protein that transports its mRNA targets from the nucleus to the translation machinery. However, it is not known which of the multiple FMRP isoforms, resulting from the numerous alternatively spliced FMR1 transcripts variants, would be involved in such a process. Using a new generation of anti-FMRP antibodies and recombinant expression, we show here that the most commonly expressed human FMRP isoforms (ISO1 and 7 do not localize to the nucleus. Instead, specific FMRP isoforms 6 and 12 (ISO6 and 12, containing a novel C-terminal domain, were the only isoforms that localized to the nuclei in cultured human cells. These isoforms localized to specific p80-coilin and SMN positive structures that were identified as Cajal bodies. The Cajal body localization signal was confined to a 17 amino acid stretch in the C-terminus of human ISO6 and is lacking in a mouse Iso6 variant. As FMRP is an RNA-binding protein, its presence in Cajal bodies suggests additional functions in nuclear post-transcriptional RNA metabolism. Supporting this hypothesis, a missense mutation (I304N, known to alter the KH2-mediated RNA binding properties of FMRP, abolishes the localization of human FMRP ISO6 to Cajal bodies. These findings open unexplored avenues in search for new insights into the pathophysiology of Fragile X Syndrome.

  16. Deuterated protein folds obtained directly from unassigned nuclear overhauser effect data.

    Science.gov (United States)

    Bermejo, Guillermo A; Llinás, Miguel

    2008-03-26

    We demonstrate the feasibility of determining the global fold of a highly deuterated protein from unassigned experimental NMR nuclear Overhauser effect (NOE) data only. The method relies on the calculation of a spatial configuration of covalently unconnected protons-a "cloud"-directly from unassigned distance restraints derived from 13C- and 15N-edited NOESY spectra. Each proton in the cloud, labeled by its chemical shift and that of the directly bound 13C or 15N, is subsequently mapped to specific atoms in the protein. This is achieved via graph-theoretical protocols that search for connectivities in graphs that encode the structural information within the cloud. The peptidyl HN chain is traced by seeking for all possible routes and selecting the one that yields the minimal sum of sequential distances. Complete proton identification in the cloud is achieved by linking the side-chain protons to proximal main-chain HNs via bipartite graph matching. The identified protons automatically yield the NOE assignments, which in turn are used for structure calculation with RosettaNMR, a protocol that incorporates structural bias derived from protein databases. The method, named Sparse-Constraint CLOUDS, was applied to experimental NOESY data on the 58-residue Z domain of staphylococcal protein A. The generated structures are of similar accuracy to those previously reported, which were derived via a conventional approach involving a larger NMR data set. Additional tests were performed on seven reported protein structures of various folds, using restraint lists simulated from the known atomic coordinates.

  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. 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. Tissue factor pathway inhibitor-2 may interact with nuclear protein RASSF1C

    Institute of Scientific and Technical Information of China (English)

    Xudong Chen; Zhenwu Li; Jin Zhang; Zuohua Mao; Duan Ma; Huijun Wang

    2012-01-01

    Tissue factor pathway inhibitor-2 (TFPI-2) is a 32 kDa matrix-associated Kunitz-type serine proteinase inhibitor consisting of a short amino-terminal region,three tandem Kunitz-type domains,and a positively charged carboxyterminal tail.Human TFPI-2 (hTFPI-2) inhibits a broad spectrum of serine proteinases (including trypsin,plasmin,plasma kallikrein,XIa,and chymotrypsin) almost exclusively via its first Kunitz-type domain,and potentially plays an important role in the regulation of extracellular matrix digestion and remodeling [1].Reduced TFPI-2 synthesis has been related to numerous pathophysiological processes such as inflammation,angiogenesis,atherosclerosis [2,3],retinal degeneration,and tumor growth/metastasis [4-6].It has been suggested that TFPI-2 is a tumor suppressor gene in some cancers [7,8].However,the specific physiological functions of hTFPI-2 in humans are unclear,particularly its interactions with other proteins.To better understand the physiological function of hTFPI-2,we used yeast two-hybrid system screening and bioinformatics analysis to identify its interacting proteins and confirm its interactions with nuclear protein RASSF1C using confocal microscopy and co-immunoprecipitation.

  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. Role of fatty acid binding proteins and long chain fatty acids in modulating nuclear receptors and gene transcription.

    Science.gov (United States)

    Schroeder, Friedhelm; Petrescu, Anca D; Huang, Huan; Atshaves, Barbara P; McIntosh, Avery L; Martin, Gregory G; Hostetler, Heather A; Vespa, Aude; Landrock, Danilo; Landrock, Kerstin K; Payne, H Ross; Kier, Ann B

    2008-01-01

    Abnormal energy regulation may significantly contribute to the pathogenesis of obesity, diabetes mellitus, cardiovascular disease, and cancer. For rapid control of energy homeostasis, allosteric and posttranslational events activate or alter activity of key metabolic enzymes. For longer impact, transcriptional regulation is more effective, especially in response to nutrients such as long chain fatty acids (LCFA). Recent advances provide insights into how poorly water-soluble lipid nutrients [LCFA; retinoic acid (RA)] and their metabolites (long chain fatty acyl Coenzyme A, LCFA-CoA) reach nuclei, bind their cognate ligand-activated receptors, and regulate transcription for signaling lipid and glucose catabolism or storage: (i) while serum and cytoplasmic LCFA levels are in the 200 mircroM-mM range, real-time imaging recently revealed that LCFA and LCFA-CoA are also located within nuclei (nM range); (ii) sensitive fluorescence binding assays show that LCFA-activated nuclear receptors [peroxisome proliferator-activated receptor-alpha (PPARalpha) and hepatocyte nuclear factor 4alpha (HNF4alpha)] exhibit high affinity (low nM KdS) for LCFA (PPARalpha) and/or LCFA-CoA (PPARalpha, HNF4alpha)-in the same range as nuclear levels of these ligands; (iii) live and fixed cell immunolabeling and imaging revealed that some cytoplasmic lipid binding proteins [liver fatty acid binding protein (L-FABP), acyl CoA binding protein (ACBP), cellular retinoic acid binding protein-2 (CRABP-2)] enter nuclei, bind nuclear receptors (PPARalpha, HNF4alpha, CRABP-2), and activate transcription of genes in fatty acid and glucose metabolism; and (iv) studies with gene ablated mice provided physiological relevance of LCFA and LCFA-CoA binding proteins in nuclear signaling. This led to the hypothesis that cytoplasmic lipid binding proteins transfer and channel lipidic ligands into nuclei for initiating nuclear receptor transcriptional activity to provide new lipid nutrient signaling pathways that

  2. Inhibition of host protein synthesis by Sindbis virus: correlation with viral RNA replication and release of nuclear proteins to the cytoplasm.

    Science.gov (United States)

    Sanz, Miguel A; García-Moreno, Manuel; Carrasco, Luis

    2015-04-01

    Infection of mammalian cells by Sindbis virus (SINV) profoundly blocks cellular mRNA translation. Experimental evidence points to viral non-structural proteins (nsPs), in particular nsP2, as the mediator of this inhibition. However, individual expression of nsP1, nsP2, nsP3 or nsP1-4 does not block cellular protein synthesis in BHK cells. Trans-complementation of a defective SINV replicon lacking most of the coding region for nsPs by the co-expression of nsP1-4 propitiates viral RNA replication at low levels, and inhibition of cellular translation is not observed. Exit of nuclear proteins including T-cell intracellular antigen and polypyrimidine tract-binding protein is clearly detected in SINV-infected cells, but not upon the expression of nsPs, even when the defective replicon was complemented. Analysis of a SINV variant with a point mutation in nsP2, exhibiting defects in the shut-off of host protein synthesis, indicates that both viral RNA replication and the release of nuclear proteins to the cytoplasm are greatly inhibited. Furthermore, nucleoside analogues that inhibit cellular and viral RNA synthesis impede the blockade of host mRNA translation, in addition to the release of nuclear proteins. Prevention of the shut-off of host mRNA translation by nucleoside analogues is not due to the inhibition of eIF2α phosphorylation, as this prevention is also observed in PKR(-/-) mouse embryonic fibroblasts that do not phosphorylate eIF2α after SINV infection. Collectively, our observations are consistent with the concept that for the inhibition of cellular protein synthesis to occur, viral RNA replication must take place at control levels, leading to the release of nuclear proteins to the cytoplasm.

  3. Hydrophobic, Porous Battery Boxes

    Science.gov (United States)

    Bragg, Bobby J.; Casey, John E., Jr.

    1995-01-01

    Boxes made of porous, hydrophobic polymers developed to contain aqueous potassium hydroxide electrolyte solutions of zinc/air batteries while allowing air to diffuse in as needed for operation. Used on other types of batteries for in-cabin use in which electrolytes aqueous and from which gases generated during operation must be vented without allowing electrolytes to leak out.

  4. 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…

  5. 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.

  6. Intranuclear targeting and nuclear export of the adenovirus E1B-55K protein are regulated by SUMO1 conjugation

    OpenAIRE

    Kindsmüller, Kathrin; Groitl, Peter; Härtl, Barbara; Blanchette, Paola; Hauber, Joachim; Dobner, Thomas

    2007-01-01

    We have investigated the requirements for CRM1-mediated nuclear export and SUMO1 conjugation of the adenovirus E1B-55K protein during productive infection. Our data show that CRM1 is the major export receptor for E1B-55K in infected cells. Functional inactivation of the E1B-55K CRM1-dependent nuclear export signal (NES) or leptomycin B treatment causes an almost complete redistribution of the viral protein from the cytoplasm to the nucleus and its accumulation at the periphery of the viral re...

  7. Green fluorescent protein (GFP) transgenic pig produced by somatic cell nuclear transfer

    Institute of Scientific and Technical Information of China (English)

    LIU ZhongHua; SUN Shuang; LI YuTian; WANG HongBin; R S PRATHER; SONG Jun; WANG ZhenKun; TIAN JiangTian; KONG QingRan; ZHENG Zhong; YIN Zhi; GAO Li; MA HaiKun

    2008-01-01

    Transgenic somatic cell nuclear transfer is a very promising route for producing transgenic farm ani-mals. Research on GFP transgenic pigs can provide useful information for breeding transgenic pigs, human disease models and human organ xenotransplantation. In this study, a liposomal transfection system was screened and transgenic embryos were reconstructed by nuclear transfer of GFP positive cells into enucleated in vitro matured oocytes. The development of reconstructed embryos both in vitro and in vivo was observed, and GFP expression was determined. The results showed that porcine fe-tal-derived fibroblast cells cultured with 4.0 plJmL liposome and 1.6 pg/mL plasmid DNA for 6 h re-sulted in the highest transfection rate (3.6%). The percentage of GFP reconstructed embryos that de-veloped in vitro to the blastocyst stage was 10%. Of those the GFP positive percentage was 48%. Re-constructed transgenic embryos were transferred to 10 recipients. 5 of them were pregnant, and 3 de-livered 6 cloned piglets in which 4 piglets were transgenic for the GFP as verified by both GFP protein expression and GFP DNA sequence analysis. The percentage of reconstructed embryos that resulted in cloned piglets was 1.0%; while the percentage of piglets that were transgenic was 0.7%. This is the first group of transgenic cloned pigs born in China, marking a great progress in Chinese transgenic cloned pig research.

  8. Comparison of nuclear matrix proteins between gastric cancer and normal gastric tissue

    Institute of Scientific and Technical Information of China (English)

    Qin-Xian Zhang; Yi Ding; Zhuo Li; Xiao-Ping Le; Wei Zhang; Ling Sun; Hui-Rong Shi

    2004-01-01

    AIM: To study the alteration of nuclear matrix proteins (NMPs) in gastric cancer.METHODS: The NMPs extracted from 22 cases of gastric cancer and normal gastric tissues were investigated by SDS-PAGE technique and the data were analyzed using Genetools analysis software.RESULTS: Compared with normal gastric tissue, the expression of 30 ku and 28 ku NMPs in gastric cancer decreased significantly (P=0.002, P=0.001, P<0.05). No significant difference was found in the expression of the two NMPs between the various differentiated grades (P=0.947, P=0.356) and clinical stages of gastric cancer (P=0.920, P=0.243, P>0.05).CONCLUSION: The results suggested that the alteration of NMPs in gastric cancer occurred at the early stage of gastric cancer development.

  9. Nuclear inclusions mimicking poly(A)-binding protein nuclear 1 inclusions in a case of inclusion body myopathy associated with Paget disease of bone and frontotemporal dementia with a novel mutation in the valosin-containing protein gene.

    Science.gov (United States)

    Matsubara, Shiro; Shimizu, Toshio; Komori, Takashi; Mori-Yoshimura, Madoka; Minami, Narihiro; Hayashi, Yukiko K

    2016-07-01

    A middle-aged Japanese man presented with slowly progressive asymmetric weakness of legs and arm but had neither ptosis nor dysphagia. He had a family history of similar condition suggestive of autosomal dominant inheritance. A muscle biopsy showed mixture of neurogenic atrophy and myopathy with rimmed vacuoles. Furthermore we found intranuclear inclusions that had a fine structure mimicking that of inclusions reported in oculopharyngeal muscular dystrophy (OPMD). Immunohistochemical staining for polyadenylate-binding nuclear protein 1, which is identified within the nuclear inclusions of OPMD, demonstrated nuclear positivity in this case. However, OPMD was thought unlikely based on the clinical features and results of genetic analyses. Instead, a novel mutation in valosin-containing protein, c.376A>T (p.Ile126Phe), was revealed. A diagnosis of inclusion body myopathy associated with Paget disease of bone and frontotemporal dementia was made. This is the first report of polyadenylate-binding nuclear protein 1-positive nuclear inclusions in the muscle of this condition.

  10. Structure and biochemical function of a prototypical Arabidopsis U-box domain

    DEFF Research Database (Denmark)

    Andersen, Pernille; Kragelund, Birthe B; Olsen, Addie N;

    2004-01-01

    U-box proteins, as well as other proteins involved in regulated protein degradation, are apparently over-represented in Arabidopsis compared with other model eukaryotes. The Arabidopsis protein AtPUB14 contains a typical U-box domain followed by an Armadillo repeat region, a domain organization t...

  11. Role of NF-Kappa B Signaling in X-Box Binding Protein 1 (XBP1)-Mediated Antiestrogen Resistance in Breast Cancer

    Science.gov (United States)

    2013-10-01

    the cell death response to antiestrogen therapy. ERα knockdown, but not ICI treatment, reduced nuclear Nrf2 (a UPR-induced antioxidant signaling...Cancer Center, 09/2007-09/2009 Skp2 regulation of melanoma cell proliferation: mechanism and role in a skin -like microenvironment Post-doctoral

  12. Mapping of the nuclear localization signals in open reading frame 2 protein from porcine circovirus type 1

    Institute of Scientific and Technical Information of China (English)

    Jiangbing Shuai; Wei Wei; Lingli Jiang; Xiaoliang Li; Ning Chen; Weihuan Fang

    2008-01-01

    Porcine circovirus type 1 (PCV1) contains two major open reading frames encoding the replication-associated proteins and the major structural capsid (Cap) protein.PCV1 Cap has an N-terminus carrying several potential monopartite or bipartite nuclear localization signals (NLS).The contribution of these partially overlapping motifs to nuclear importing was identified by expression of mutated PCV1 Cap versions fused to enhanced green fluorescent protein (EGFP).The Cterminus truncated PCV1 Cap-EGFP was localized in nuclei of PK-15 cells similar to the wild-type PCV1 Cap-EGFP,whereas truncation of the N-terminus rendered the fusion protein distributed into cytoplasm,indicating that the nuclear import of PCV1 Cap was efficiently mediated by its N-terminal region.Substitutions of basic residues in stretches 9RRRR12 or the right part of 25RRPYLAHPAFRNRYRWRRK43 resulted in a diffused distribution of the fusion protein in both nuclei and cytoplasm,indicating that the two NLSs were responsible for restricted nuclear targeting of PCV1 Cap.

  13. Identification of a nuclear localization motif in the serine/arginine protein kinase PSRPK of physarum polycephalum

    Directory of Open Access Journals (Sweden)

    Tian Shengli

    2009-08-01

    Full Text Available Abstract Background Serine/arginine (SR protein-specific kinases (SRPKs are conserved in a wide range of organisms, from humans to yeast. Studies showed that SRPKs can regulate the nuclear import of SR proteins in cytoplasm, and regulate the sub-localization of SR proteins in the nucleus. But no nuclear localization signal (NLS of SRPKs was found. We isolated an SRPK-like protein PSRPK (GenBank accession No. DQ140379 from Physarum polycephalum previously, and identified a NLS of PSRPK in this study. Results We carried out a thorough molecular dissection of the different domains of the PSRPK protein involved in its nuclear localization. By truncation of PSRPK protein, deletion of and single amino acid substitution in a putative NLS and transfection of mammalian cells, we observed the distribution of PSRPK fluorescent fusion protein in mammalian cells using confocal microscopy and found that the protein was mainly accumulated in the nucleus; this indicated that the motif contained a nuclear localization signal (NLS. Further investigation with truncated PSPRK peptides showed that the NLS (318PKKGDKYDKTD328 was localized in the alkaline Ω-loop of a helix-loop-helix motif (HLHM of the C-terminal conserved domain. If the 318PKKGDK322 sequence was deleted from the loop or K320 was mutated to T320, the PSRPK fluorescent fusion protein could not enter and accumulate in the nucleus. Conclusion This study demonstrated that the 318PKKGDKYDKTD328 peptides localized in the C-terminal conserved domain of PSRPK with the Ω-loop structure could play a crucial role in the NLS function of PSRPK.

  14. Protein kinase C α regulates nuclear pri-microRNA 15a release as part of endothelin signaling.

    Science.gov (United States)

    von Brandenstein, Melanie; Depping, Reinhard; Schäfer, Ekaterine; Dienes, Hans-Peter; Fries, Jochen W U

    2011-10-01

    Endothelin-1 induced signaling is characterized by an early induction of a nuclear factor-kappa B p65/mitogen-activated phosphokinase p38 transcription complex via its A-receptor versus a late induction via diacylglycerol, and protein kinase C. A possible interaction between these two pathways and a potential function for protein kinase C in this context has not previously been elucidated. Here we report that in Caki-1 tumor cells, protein kinase C α is a part of the transcription complex. With importin α4 and α5 as chaperones, the transcription complex transmigrates into the nucleus. Protein kinase C α blocks the nuclear release of pri-microRNA 15a by direct binding shown by electrophoretic mobility shift assay and Duolink immune histology. The expression levels of miRNA 15a can be further manipulated by transfection of si-protein kinase C α, or an expression vector containing protein kinase C α or miRNA 15. The miRNA 15a regulation by protein kinase C α is detectable in different malignant human tumor cell lines (renal cell carcinoma, breast carcinoma, and melanoma). Furthermore, all three cell lines harbor both endothelin receptors (ETAR/ETBR). Specific blockage of each receptor leads to major reduction of miRNA 15a expression due to increased nuclear protein kinase C α translocation. We conclude that the nuclear binding of pri-microRNA 15a is a novel function of protein kinase C α, which plays an important role in endothelin-1 mediated signaling. Since several endothelin-sensitive, malignant tumor cell lines harbor this regulation, it could indicate a more general role in tumor biology.

  15. lin-8, which antagonizes Caenorhabditis elegans Ras-mediated vulval induction, encodes a novel nuclear protein that interacts with the LIN-35 Rb protein.

    Science.gov (United States)

    Davison, Ewa M; Harrison, Melissa M; Walhout, Albertha J M; Vidal, Marc; Horvitz, H Robert

    2005-11-01

    Ras-mediated vulval development in C. elegans is inhibited by the functionally redundant sets of class A, B, and C synthetic Multivulva (synMuv) genes. Three of the class B synMuv genes encode an Rb/DP/E2F complex that, by analogy with its mammalian and Drosophila counterparts, has been proposed to silence genes required for vulval specification through chromatin modification and remodeling. Two class A synMuv genes, lin-15A and lin-56, encode novel nuclear proteins that appear to function as a complex. We show that a third class A synMuv gene, lin-8, is the defining member of a novel C. elegans gene family. The LIN-8 protein is nuclear and can interact physically with the product of the class B synMuv gene lin-35, the C. elegans homolog of mammalian Rb. LIN-8 likely acts with the synMuv A proteins LIN-15A and LIN-56 in the nucleus, possibly in a protein complex with the synMuv B protein LIN-35 Rb. Other LIN-8 family members may function in similar complexes in different cells or at different stages. The nuclear localization of LIN-15A, LIN-56, and LIN-8, as well as our observation of a direct physical interaction between class A and class B synMuv proteins, supports the hypothesis that the class A synMuv genes control vulval induction through the transcriptional regulation of gene expression.

  16. Autoantibodies from patients with primary biliary cirrhosis recognize a region within the nucleoplasmic domain of inner nuclear membrane protein LBR.

    Science.gov (United States)

    Lin, F; Noyer, C M; Ye, Q; Courvalin, J C; Worman, H J

    1996-01-01

    Autoantibodies from rare patients with primary biliary cirrhosis (PBC) recognize LBR, or lamin B receptor, an integral membrane protein of the inner nuclear membrane. Human LBR has a nucleoplasmic, amino-terminal domain of 208 amino acids followed by a carboxyl-terminal domain with eight putative transmembrane segments. Autoantibodies against LBR from four patients with PBC recognized the nucleoplasmic, amino-terminal domain but not the carboxyl-terminal domain. Immunoblotting of smaller fusion proteins demonstrated that these autoantibodies recognized a conformational epitope(s) contained within the stretch of amino acids from 1 to 60. These results, combined with those of previous studies, show that autoepitopes of nuclear membrane proteins are located within their nucleocytoplasmic domains and that autoantibodies from patients with PBC predominantly react with one domain of a protein antigen. This work also provides further characterization of anti-LBR antibodies that have found utility as reagents in cell biology research.

  17. Progress in relationship between high mobility group box 1 protein and cardiovascular diseases%高迁移率族蛋白B1与心血管疾病相关性的研究进展

    Institute of Scientific and Technical Information of China (English)

    刘宇; 曹清心

    2012-01-01

    High mobility group box 1 protein (HMGB1) is a kind of nucleoprotein. It is secreted by necrosis cells and activated immunocytes into the extracellular environment, which has the activity of a cytokine, and thereby initiates immune responses and cause inflammatory reactions. It has been confirmed by numerous studies that HMGB1 is closely related to heart failure, myocardial ischemia reperfusion injury, atherosclerosis, heart infarction, etc. Studying the mechanism of HMGB1 in the development of angiocardiopathy will find a new target for curing cardiovascular diseases clinically.%高迁移率族蛋白B1(high mobility group box1 protein,HMGB1)是一种核蛋白质,其可由坏死细胞以及活化的免疫细胞释放至细胞外而具有细胞因子的活性,继之启动免疫反应和引发炎性反应.近年来,大量研究证实HMGB1与心力衰竭( heart failure,HF)、心肌缺血-再灌注(ischemia/reperfusion,I/R)损伤、动脉粥样硬化(atherosclerosis,AS)、心肌梗死(myocardial infarction,MI)等疾病有密切关系,通过研究HMGB1在心血管疾病发生发展过程中的机制,可为心血管疾病的临床治疗找到新靶点.

  18. 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

    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...

  19. Making Connections with Memory Boxes.

    Science.gov (United States)

    Whatley, April

    2000-01-01

    Addresses the use of children's literature within the social studies classroom on the topic of memory boxes. Includes discussions of four books: (1) "The Littlest Angel" (Charles Tazewell); (2) "The Hundred Penny Box" (Sharon Bell Mathis); (3) "Wilfrid Gordon McDonald Partridge" (Mem Fox); and (4) "The Memory Box" (Mary Bahr). (CMK)

  20. Liver X receptor regulates hepatic nuclear O-GlcNAc signaling and carbohydrate responsive element-binding protein activity

    DEFF Research Database (Denmark)

    Bindesbøll, Christian; Fan, Qiong; Nørgaard, Rikke C;

    2015-01-01

    Liver X receptor (LXR)α and LXRβ play key roles in hepatic de novo lipogenesis through their regulation of lipogenic genes, including sterol regulatory element-binding protein (SREBP)-1c and carbohydrate responsive element-binding protein (ChREBP). LXRs activate lipogenic gene transcription...... metabolic sensors upstream of ChREBP by modulating GK expression, nuclear O-GlcNAc signaling, and ChREBP expression and activity....

  1. Dissociation of the dorsal-cactus complex and phosphorylation of the dorsal protein correlate with the nuclear localization of dorsal

    OpenAIRE

    1993-01-01

    The formation of dorsal-ventral polarity in Drosophila requires the asymmetric nuclear localization of the dorsal protein along the D/V axis. This process is regulated by the action of the dorsal group genes and cactus. We show that dorsal and cactus are both phosphoproteins that form a stable cytoplasmic complex, and that the cactus protein is stabilized by its interaction with dorsal. The dorsal-cactus complex dissociates when dorsal is targeted to the nucleus. While the phosphorylation of ...

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

    Energy Technology Data Exchange (ETDEWEB)

    Cunningham, J; Gatenby, R [Moffitt Cancer Research Institute, Tampa, FL (United States)

    2014-06-01

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

  3. 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.

  4. Tim50a, a nuclear isoform of the mitochondrial Tim50, interacts with proteins involved in snRNP biogenesis

    Directory of Open Access Journals (Sweden)

    Robinson Melvin L

    2005-07-01

    Full Text Available Abstract Background The Cajal body (CB is a nuclear suborganelle involved in the biogenesis of small nuclear ribonucleoproteins (snRNPs, which are vital for pre-mRNA splicing. Newly imported Sm-class snRNPs traffic through CBs, where the snRNA component of the snRNP is modified, and then target to other nuclear domains such as speckles and perichromatin fibrils. It is not known how nascent snRNPs localize to the CB and are released from this structure after modification. The marker protein for CBs, coilin, may play a role in snRNP biogenesis given that it can interact with snRNPs and SMN, the protein mutated in Spinal Muscular Atrophy. Loss of coilin function in mice leads to significant viability and fertility problems and altered CB formation. Results In this report, we identify a minor isoform of the mitochondrial Tim50, Tim50a, as a coilin interacting protein. The Tim50a transcript can be detected in some cancer cell lines and normal brain tissue. The Tim50a protein differs only from Tim50 in that it contains an additional 103 aa N-terminal to the translation start of Tim50. Importantly, a putative nuclear localization signal is found within these 103 residues. In contrast to Tim50, which localizes to the cytoplasm and mitochondria, Tim50a is strictly nuclear and is enriched in speckles with snRNPs. In addition to coilin, Tim50a interacts with snRNPs and SMN. Competition binding experiments demonstrate that coilin competes with Sm proteins of snRNPs and SMN for binding sites on Tim50a. Conclusion Tim50a may play a role in snRNP biogenesis given its cellular localization and protein interaction characteristics. We hypothesize that Tim50a takes part in the release of snRNPs and SMN from the CB.

  5. 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.

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

    Science.gov (United States)

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

    2015-10-15

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

  7. 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.

  8. Heterogeneous nuclear expression of the promyelocytic leukemia (PML) protein in normal and neoplastic human tissues.

    Science.gov (United States)

    Gambacorta, M.; Flenghi, L.; Fagioli, M.; Pileri, S.; Leoncini, L.; Bigerna, B.; Pacini, R.; Tanci, L. N.; Pasqualucci, L.; Ascani, S.; Mencarelli, A.; Liso, A.; Pelicci, P. G.; Falini, B.

    1996-01-01

    The RING-finger promyelocytic leukemia (PML) protein is the product of the PML gene that fuses with the retinoic acid receptor-alpha gene in the t(15; 17) translocation of acute promyelocytic leukemia. Wild-type PML localizes in the nucleus with a typical speckled pattern that is a consequence of the concentration of the protein within discrete subnuclear domains known as nuclear bodies. Delocalization of PML from nuclear bodies has been documented in acute promyelocytic leukemia cells and suggested to contribute to leukemogenesis. In an attempt to get new insights into the function of the wild-type PML protein and to investigate whether it displays an altered expression pattern in neoplasms other than acute promyelocytic leukemia, we stained a large number of normal and neoplastic human tissues with a new murine monoclonal antibody (PG-M3) directed against the amino-terminal region of PML. As the PG-M3 epitope is partially resistant to fixatives, only cells that overexpress PML are detected by the antibody in microwave-heated paraffin sections. Among normal tissues, PML was characteristically up-regulated in activated epithelioid histiocytes and fibroblasts in a variety of pathological conditions, columnar epithelium in small active thyroid follicles, well differentiated foamy cells in the center of sebaceous glands, and hypersecretory endometria (Arias-Stella). Interferons, the PML of which is a primary target gene, and estrogens are likely to represent some of the cytokines and/or hormones that may be involved in the up-regulation of PML under these circumstances. In keeping with this concept, we found that PML is frequently overexpressed in Hodgkin and Reed-Sternberg cells of Hodgkin's disease, a tumor of cytokine-producing cells. Among solid tumors, overexpression of PML was frequently found in carcinomas of larynx and thyroid (papillary), epithelial thymomas, and Kaposi's sarcoma, whereas carcinomas of the lung, thyroid (follicular), breast, and colon were

  9. Opto-Box

    CERN Document Server

    Bertsche, David; 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.

  10. Identification and Characterization of Nuclear Localization Signals within the Nucleocapsid Protein VP15 of White Spot Syndrome Virus

    Institute of Scientific and Technical Information of China (English)

    Li-juan LI; Hua-jun ZHANG; Cong ZHANG; Zheng-li SHI

    2009-01-01

    The nucleocapsid protein VP15 of white spot syndrome virus (WSSV) is a basic DNA-binding protein. Three canonical bipartite nuclear localization signals (NLSs), called NLS1 (aa 11-27), NLS2 (aa 33-49) and NLS3 (44-60), have been detected in this protein, using the ScanProsite computer program. To determine the nuclear localization sequence of VP15, the full-length open reading frame, or the sequence of one of the three NLSs, was fused to the green fluorescent protein (GFP) gene, and transiently expressed in insect Sf9 cells. Transfection with full-length VP15 resulted in GFP fluorescence being distributed exclusively in the nucleus. NLS 1 alone could also direct GFP to the nucleus, but less efficiently. Neither of the other two NLSs (NLS2 and 3) was functional when expressed alone, but exhibited similar activity to NLS1 when they were expressed as a fusion peptide. Furthermore, a mutated VP15, in which the two basic amino acids (11RR12) of NLSI were changed to two alanines (11AA12), caused GFP to be localized only in the cytoplasm of Sf9 cells. These results demonstrated that VP15, as a nuclear localization protein, needs cooperation between its three NLSs, and that the two residues (11RR12) of NLS1 play a key role in transporting the protein to the nucleus.

  11. Nuclear export of the influenza virus ribonucleoprotein complex: Interaction of Hsc70 with viral proteins M1 and NS2.

    Science.gov (United States)

    Watanabe, Ken; Shimizu, Teppei; Noda, Saiko; Tsukahara, Fujiko; Maru, Yoshiro; Kobayashi, Nobuyuki

    2014-01-01

    The influenza virus replicates in the host cell nucleus, and the progeny viral ribonucleoprotein complex (vRNP) is exported to the cytoplasm prior to maturation. NS2 has a nuclear export signal that mediates the nuclear export of vRNP by the vRNP-M1-NS2 complex. We previously reported that the heat shock cognate 70 (Hsc70) protein binds to M1 protein and mediates vRNP export. However, the interactions among M1, NS2, and Hsc70 are poorly understood. In the present study, we demonstrate that Hsc70 interacts with M1 more strongly than with NS2 and competes with NS2 for M1 binding, suggesting an important role of Hsc70 in the nuclear export of vRNP.

  12. Decreased activity and enhanced nuclear export of CCAAT-enhancer-binding protein beta during inhibition of adipogenesis by ceramide.

    Science.gov (United States)

    Sprott, Kam M; Chumley, Michael J; Hanson, Janean M; Dobrowsky, Rick T

    2002-07-01

    To identify novel molecular mechanisms by which ceramide regulates cell differentiation, we examined its effect on adipogenesis of 3T3-L1 preadipocytes. Hormonal stimulation of 3T3-L1 preadipocytes induced formation of triacylglycerol-laden adipocytes over 7 days; in part, via the co-ordinated action of CCAAT-enhancer-binding proteins alpha, beta and delta (C/EBP-alpha, -beta and -delta) and peroxisome-proliferator-activated receptor gamma (PPARgamma). The addition of exogenous N-acetylsphingosine (C2-ceramide) or increasing endogenous ceramide levels inhibited the expression of C/EBPalpha and PPARgamma, and blocked adipocyte development. C2-ceramide did not decrease the cellular expression of C/EBPbeta, which is required for expression of C/EBPalpha and PPARgamma, but significantly blocked its transcriptional activity from a promoter construct after 24 h. The ceramide-induced decrease in the transcriptional activity of C/EBPbeta correlated with a strong decrease in its phosphorylation, DNA-binding ability and nuclear localization at 24 h. However, ceramide did not change the nuclear level of C/EBPbeta after a period of 4 or 16 h, suggesting that it was not affecting nuclear import. CRM1 (more recently named 'exportin-1') is a nuclear membrane protein that regulates protein export from the nucleus by binding to a specific nuclear export sequence. Leptomycin B is an inhibitor of CRM1/exportin-1, and reversed the ceramide-induced decrease in nuclear C/EBPbeta at 24 h. Taken together, these data support the hypothesis that ceramide may inhibit adipogenesis, at least in part, by enhancing dephosphorylation and premature nuclear export of C/EBPbeta at a time when its maximal transcriptional activity is required to drive adipogenesis.

  13. 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...

  14. The Box Method

    DEFF Research Database (Denmark)

    Nielsen, Peter Vilhelm

    The velocity level in a room ventilated by jet ventilation is strongly influenced by the supply conditions. The momentum flow in the supply jets controls the air movement in the room and, therefore, it is very important that the inlet conditions and the numerical method can generate a satisfactory...... description of this momentum flow. The Box Method is a practical method for the description of an Air Terminal Device which will save grid points and ensure the right level of the momentum flow....

  15. A Dual Mechanism Controls Nuclear Localization in the Atypical Basic-Helix-Loop-Helix Protein PAR1 of Arabidopsis thaliana

    Institute of Scientific and Technical Information of China (English)

    Anahit Galstyan; Jordi Bou-Torrent; Irma Roig-Villanova; Jaime F. Martínez-García

    2012-01-01

    PAR1 is an atypical basic-helix-loop-helix (bHLH) protein that negatively regulates the shade avoidance syndrome in Arabidopsis thaliana acting as a transcriptional cofactor.Consistently with this function,PAR1 has to be in the nucleus to display biological activity.Previous structure-function analyses revealed that the N-terminal region of PAR1 drives the protein to the nucleus.However,truncated forms of PAR1 lacking this region still display biological activity,implying that PAR1 has additional mechanisms to localize into the nucleus.In this work,we compared the primary structure of PAR1 and various related and unrelated plant bHLH proteins,which led us to suggest that PAR1 contains a non-canonical nuclear localization signal (NLS) in the N-terminal region.By overexpressing truncated and mutated derivatives of PAR1,we have also investigated the importance of other regions of PAR1,such as the acidic and the extended HLH dimerization domains,for its nuclear localization.We found that,in the absence of the N-terminal region,a functional HLH domain is required for nuclear localization.Our results suggest the existence of a dual mechanism for PAR1 nuclear localization:(1) one mediated by the N-terminal non-consensus NLS and (2) a second one that involves interaction with other proteins via the dimerization domain.

  16. 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.

  17. Protein (Viridiplantae): 225463823 [PGDBj - Ortholog DB

    Lifescience Database Archive (English)

    Full Text Available S-box protein SVP Vitis vinifera MARQKIQIKKIDNTAARQVTFSKRRRGLFKKAQELSILCDAEIALIVFSAAGKLFEYSSSSVSQVIGRHNQHPQT...PGKPEPPSLELQLENSTCAALSKEIAQQTQRLRQMKGEELQVLKIEELTELEELLEAGLCNVVEEKEERIRTEISDLQRKGDLLQEENERLRKEMENIFEAQPLL ...

  18. The Electronic Battle Box

    Science.gov (United States)

    Gouin, Denis; Turcotte, Guy; Lebel, Eric; Gilbert, Annie

    2000-08-01

    The Electronic Battle Box is an integrated suite of planning and decision-aid tools specially designed to facilitate Canadian Armed Force Officers during their training and during their tasks of preparing and conducting military operations. It is the result of a collaborative effort between the Defence Research Establishment Valcartier, the Directorate of Army Doctrine (DAD), the Directorate of Land Requirements (DLR), the G4 staff of 1Cdn Div HQ and CGI Information and Management Consultants Inc. Distributed on CD-ROM, the Electronic Battle Box contains efficient and user-friendly tools that significantly reduce the planning time for military operations and ensure staff officers a better focus on significant tasks. Among the tools are an OrBat Browser and an Equipment Browser allowing to view and edit military organizations, a Task Browser providing facilities to prepare plans using Gantt charts, a Logistic Planner allowing to estimate supply requirements applying complex calculations, and Road, Air and Rail Movement Planners. EBB also provides staff officers with a large set of doctrinal documents in an electronic format. This paper provides an overview of the various tools of the Electronic Battle Box.

  19. 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.

  20. 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.

  1. Preformed beading and boxing appliance.

    Science.gov (United States)

    Reddy, J Sashi Deepth; Padmanabhan, T V; Veerareddy, Chandrika; Chandrasekhar, M; Narendra, R

    2013-03-01

    Conventional beading and boxing procedure is time consuming and involves application of heat that might distort green stick compound used for border molding. Earlier studies regarding beading and boxing methods have shown usage of various materials that were disposable and that cannot be recycled. To reduce the time consumed for beading and boxing procedure and to make this procedure cost-effective by using recyclable beading material, "Preformed boxing appliance" with moldable clay meant for beading the secondary impression was used. Secondary impression was supported by 3 studs provided on the floor of the boxing appliance. The cast was poured. The duration for the entire procedure was much less than the conventional procedure.

  2. 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.

  3. Cdc20 mediates D-box-dependent degradation of Sp100

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Ran; Li, Ke-min; Zhou, Cai-hong; Xue, Jing-lun [State Key Laboratory of Genetic Engineering, Institute of Genetics, School of Life Science, Fudan University, Shanghai (China); Ji, Chao-neng, E-mail: Chnji@fudan.edu.cn [State Key Laboratory of Genetic Engineering, Institute of Genetics, School of Life Science, Fudan University, Shanghai (China); Chen, Jin-zhong, E-mail: kingbellchen@fudan.edu.cn [State Key Laboratory of Genetic Engineering, Institute of Genetics, School of Life Science, Fudan University, Shanghai (China)

    2011-12-02

    Highlights: Black-Right-Pointing-Pointer Cdc20 is a co-activator of APC/C complex. Black-Right-Pointing-Pointer Cdc20 recruits Sp100 and mediates its degradation. Black-Right-Pointing-Pointer The D-box of Sp100 is required for Cdc20-mediated degradation. Black-Right-Pointing-Pointer Sp100 expresses consistently at both the mRNA and protein levels in cell cycle. -- Abstract: Cdc20 is a co-activator of the anaphase-promoting complex/cyclosome (APC/C complex), which recruits substrates at particular phases of the cell cycle and mediates their degradation. Sp100 is a PML-NB scaffold protein, which localizes to nuclear particles during interphase and disperses from them during mitosis, participates in viral resistance, transcriptional regulation, and apoptosis. However, its metabolism during the cell cycle has not yet been fully characterized. We found a putative D-box in Sp100 using the Eukaryotic Linear Motif (ELM) predictor database. The putative D-box of Sp100 was verified by mutational analysis. Overexpression of Cdc20 resulted in decreased levels of both endogenous Sp100 protein and overexpressed Sp100 mRNA in HEK 293 cells. Only an overexpressed D-box deletion mutant of Sp100 accumulated in HEK293 cells that also overexpressed Cdc20. Cdc20 knockdown by cdc20 specific siRNA resulted in increased Sp100 protein levels in cells. Furthermore, we discovered that the Cdc20 mediated degradation of Sp100 is diminished by the proteasome inhibitor MG132, which suggests that the ubiquitination pathway is involved in this process. However, unlike the other Cdc20 substrates, which display oscillating protein levels, the level of Sp100 protein remains constant throughout the cell cycle. Additionally, both overexpression and knockdown of endogenous Sp100 had no effect on the cell cycle. Our results suggested that sp100 is a novel substrate of Cdc20 and it is degraded by the ubiquitination pathway. The intact D-box of Sp100 was necessary for this process. These findings expand

  4. Serine 249 phosphorylation by ATM protein kinase regulates hepatocyte nuclear factor-1α transactivation.

    Science.gov (United States)

    Zhao, Long; Chen, Hui; Zhan, Yi-Qun; Li, Chang-Yan; Ge, Chang-Hui; Zhang, Jian-Hong; Wang, Xiao-Hui; Yu, Miao; Yang, Xiao-Ming

    2014-07-01

    Hepatocyte nuclear factor-1 alpha (HNF1α) exerts important effects on gene expression in multiple tissues. Several studies have directly or indirectly supported the role of phosphorylation processes in the activity of HNF1α. However, the molecular mechanism of this phosphorylation remains largely unknown. Using microcapillary liquid chromatography MS/MS and biochemical assays, we identified a novel phosphorylation site in HNF1α at Ser249. We also found that the ATM protein kinase phosphorylated HNF1α at Ser249 in vitro in an ATM-dependent manner and that ATM inhibitor KU55933 treatment inhibited phosphorylation of HNF1α at Ser249 in vivo. Coimmunoprecipitation assays confirmed the association between HNF1α and ATM. Moreover, ATM enhanced HNF1α transcriptional activity in a dose-dependent manner, whereas the ATM kinase-inactive mutant did not. The use of KU55933 confirmed our observation. Compared with wild-type HNF1α, a mutation in Ser249 resulted in a pronounced decrease in HNF1α transactivation, whereas no dominant-negative effect was observed. The HNF1αSer249 mutant also exhibited normal nuclear localization but decreased DNA-binding activity. Accordingly, the functional studies of HNF1αSer249 mutant revealed a defect in glucose metabolism. Our results suggested that ATM regulates the activity of HNF1α by phosphorylation of serine 249, particularly in glucose metabolism, which provides valuable insights into the undiscovered mechanisms of ATM in the regulation of glucose homeostasis.

  5. 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.

  6. KIFC1-like motor protein associates with the cephalopod manchette and participates in sperm nuclear morphogenesis in Octopus tankahkeei.

    Directory of Open Access Journals (Sweden)

    Wei Wang

    Full Text Available BACKGROUND: Nuclear morphogenesis is one of the most fundamental cellular transformations taking place during spermatogenesis. In rodents, a microtubule-based perinuclear structure, the manchette, and a C-terminal kinesin motor KIFC1 are believed to play crucial roles in this process. Spermatogenesis in Octopus tankahkeei is a good model system to explore whether evolution has created a cephalopod prototype of mammalian manchette-based and KIFC1-dependent sperm nuclear shaping machinery. METHODOLOGY/PRINCIPAL FINDINGS: We detected the presence of a KIFC1-like protein in the testis, muscle, and liver of O. tankahkeei by Western Blot. Then we tracked its dynamic localization in spermatic cells at various stages using Immunofluorescence and Immunogold Electron Microscopy. The KIFC1-like protein was not expressed at early stages of spermatogenesis when no significant morphological changes occur, began to be present in early spermatid, localized around and in the nucleus of intermediate and late spermatids where the nucleus was dramatically elongated and compressed, and concentrated at one end of final spermatid. Furthermore, distribution of the motor protein during nuclear elongation and condensation overlapped with that of the cephalopod counterpart of manchette at a significant level. CONCLUSIONS/SIGNIFICANCE: The results support the assumption that the protein is actively involved in sperm nuclear morphogenesis in O. tankahkeei possibly through bridging the manchette-like perinuclear microtubules to the nucleus and assisting in the nucleocytoplasmic trafficking of specific cargoes. This study represents the first description of the role of a motor protein in sperm nuclear shaping in cephalopod.

  7. Structure–function–folding relationships and native energy landscape of dynein light chain protein: nuclear magnetic resonance insights

    Indian Academy of Sciences (India)

    P M Krishna Mohan; Ramakrishna V Hosur

    2009-09-01

    The detailed characterization of the structure, dynamics and folding process of a protein is crucial for understanding the biological functions it performs. Modern biophysical and nuclear magnetic resonance (NMR) techniques have provided a way to obtain accurate structural and thermodynamic information on various species populated on the energy landscape of a given protein. In this context, we review here the structure–function–folding relationship of an important protein, namely, dynein light chain protein (DLC8). DLC8, the smallest subunit of the dynein motor complex, acts as a cargo adaptor. The protein exists as a dimer under physiological conditions and dissociates into a pure monomer below pH 4. Cargo binding occurs at the dimer interface. Dimer stability and relay of perturbations through the dimer interface are anticipated to be playing crucial roles in the variety of functions the protein performs. NMR investigations have provided great insights into these aspects of DLC8 in recent years.

  8. All 17 S-locus F-box proteins of the S2 - and S3 -haplotypes of Petunia inflata are assembled into similar SCF complexes with a specific function in self-incompatibility.

    Science.gov (United States)

    Li, Shu; Williams, Justin S; Sun, Penglin; Kao, Teh-Hui

    2016-09-01

    The collaborative non-self-recognition model for S-RNase-based self-incompatibility predicts that multiple S-locus F-box proteins (SLFs) produced by pollen of a given S-haplotype collectively mediate ubiquitination and degradation of all non-self S-RNases, but not self S-RNases, in the pollen tube, thereby resulting in cross-compatible pollination but self-incompatible pollination. We had previously used pollen extracts containing GFP-fused S2 -SLF1 (SLF1 with an S2 -haplotype) of Petunia inflata for co-immunoprecipitation (Co-IP) and mass spectrometry (MS), and identified PiCUL1-P (a pollen-specific Cullin1), PiSSK1 (a pollen-specific Skp1-like protein) and PiRBX1 (a conventional Rbx1) as components of the SCF(S) (2-) (SLF) (1) complex. Using pollen extracts containing PiSSK1:FLAG:GFP for Co-IP/MS, we identified two additional SLFs (SLF4 and SLF13) that were assembled into SCF(SLF) complexes. As 17 SLF genes (SLF1 to SLF17) have been identified in S2 and S3 pollen, here we examined whether all 17 SLFs are assembled into similar complexes and, if so, whether these complexes are unique to SLFs. We modified the previous Co-IP/MS procedure, including the addition of style extracts from four different S-genotypes to pollen extracts containing PiSSK1:FLAG:GFP, to perform four separate experiments. The results taken together show that all 17 SLFs and an SLF-like protein, SLFLike1 (encoded by an S-locus-linked gene), co-immunoprecipitated with PiSSK1:FLAG:GFP. Moreover, of the 179 other F-box proteins predicted by S2 and S3 pollen transcriptomes, only a pair with 94.9% identity and another pair with 99.7% identity co-immunoprecipitated with PiSSK1:FLAG:GFP. These results suggest that SCF(SLF) complexes have evolved specifically to function in self-incompatibility.

  9. 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.

  10. 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...

  11. The 5‘—flanking cis—acting elements of the human ε—globin gene associates with the nuclear matrix and binds to the nuclear matrix proteins

    Institute of Scientific and Technical Information of China (English)

    YANZHIJIANG; RUOLANQIAN

    1998-01-01

    The nuclear matrix attachment regions(MARs) and the binding nuclear matrix proteins in the 5'-flanking cisacting elements of the human ε-globin gene have been examined.Using in vitro DNA-matrix binding assay,it has been shown that the positive stage-specific regulatory element (ε-PREII,-446bp- -419bp) upstream of this gene could specifically associate with the nuclear matrix from K562 cells,indicating that ε-PREII may be an erythroidspecific facultative MAR.In gel mobility shift assay and Southwestern blotting assay,an erythroid-specific nuclear matrix protein (ε-NMPk) in K562 cells has been revealed to bind to this positive regulatory element (ε-PREII).Furthermore,we demonstrated that the silencer (-392bp- -177bp) upstream of the human ε-globin gene could associate with the nuclear matrices from K562,HEL and Raji cells.In addition,the nuclear matrix proteins prepared from these three cell lines could also bind to this silencer,suggesting that this silencer element might be a constitutive nuclear matrix attachment region(constitutive MAR).Our results demonstrated that the nuclear matrix and nuclear matrix proteins might play an important role in the regulation of the human ε-globin gene expression.

  12. Key motifs in EBV (Epstein-Barr virus)-encoded protein kinase for phosphorylation activity and nuclear localization.

    Science.gov (United States)

    Gershburg, Svetlana; Murphy, Leann; Marschall, Manfred; Gershburg, Edward

    2010-10-15

    A sole EBV (Epstein-Barr virus)-encoded protein kinase (EBV-PK) (the BGLF4 gene product) plays important roles in viral infection. Although a number of targets of this protein have been identified, the kinase itself remains largely unstudied with regard to its enzymology and structure. In the present study, site-directed mutagenesis has been employed to generate mutations targeting residues involved in nuclear localization of the EBV-PK, core residues in subdomain III of the protein kinase domain conserved in most protein kinases or residues in subdomain VIa conserved only within the HPK (herpesvirus-encoded protein kinase) group. Deletion of amino acids 389-391 resulted in exclusive cytoplasmic localization of the protein, indicating the involvement of this region in nuclear translocation of the EBV-PK. Mutations at the amino acids Glu113 (core component), Phe175, Leu178, Phe184, Leu185 and Asn186 (conserved in HPKs) resulted in loss of EBV-PK autophosphorylation, protein substrate [EBV EA-D (early antigen diffused)] phosphorylation, and ability to facilitate ganciclovir phosphorylation. These results reiterate the unique features of this group of kinases and present an opportunity for designing more specific antiviral compounds.

  13. Nuclear envelope proteins modulate proliferation of vascular smooth muscle cells during cyclic stretch application.

    Science.gov (United States)

    Qi, Ying-Xin; Yao, Qing-Ping; Huang, Kai; Shi, Qian; Zhang, Ping; Wang, Guo-Liang; Han, Yue; Bao, Han; Wang, Lu; Li, Hai-Peng; Shen, Bao-Rong; Wang, Yingxiao; Chien, Shu; Jiang, Zong-Lai

    2016-05-10

    Cyclic stretch is an important inducer of vascular smooth muscle cell (VSMC) proliferation, which is crucial in vascular remodeling during hypertension. However, the molecular mechanism remains unclear. We studied the effects of emerin and lamin A/C, two important nuclear envelope proteins, on VSMC proliferation in hypertension and the underlying mechano-mechanisms. In common carotid artery of hypertensive rats in vivo and in cultured cells subjected to high (15%) cyclic stretch in vitro, VSMC proliferation was increased significantly, and the expression of emerin and lamin A/C was repressed compared with normotensive or normal (5%) cyclic stretch controls. Using targeted siRNA to mimic the repressed expression of emerin or lamin A/C induced by 15% stretch, we found that VSMC proliferation was enhanced under static and 5%-stretch conditions. Overexpression of emerin or lamin A/C reversed VSMC proliferation induced by 15% stretch. Hence, emerin and lamin A/C play critical roles in suppressing VSMC hyperproliferation induced by hyperstretch. ChIP-on-chip and MOTIF analyses showed that the DNAs binding with emerin contain three transcription factor motifs: CCNGGA, CCMGCC, and ABTTCCG; DNAs binding with lamin A/C contain the motifs CVGGAA, GCCGCYGC, and DAAGAAA. Protein/DNA array proved that altered emerin or lamin A/C expression modulated the activation of various transcription factors. Furthermore, accelerating local expression of emerin or lamin A/C reversed cell proliferation in the carotid artery of hypertensive rats in vivo. Our findings establish the pathogenetic role of emerin and lamin A/C repression in stretch-induced VSMC proliferation and suggest mechanobiological mechanism underlying this process that involves the sequence-specific binding of emerin and lamin A/C to specific transcription factor motifs.

  14. Impaired protein stability and nuclear localization of NOBOX variants associated with premature ovarian insufficiency.

    Science.gov (United States)

    Ferrari, Ilaria; Bouilly, Justine; Beau, Isabelle; Guizzardi, Fabiana; Ferlin, Alberto; Pollazzon, Marzia; Salerno, Mariacarolina; Binart, Nadine; Persani, Luca; Rossetti, Raffaella

    2016-10-23

    Premature ovarian insufficiency (POI) is a clinical syndrome defined by a loss of ovarian activity before the age of 40. Its pathogenesis is still largely unknown, but increasing evidences support a genetic basis in most cases. Among these, heterozygous mutations in NOBOX, a homeobox gene encoding a transcription factor expressed specifically by oocyte and granulosa cells within the ovary, have been reported in ∼6% of women with sporadic POI. The pivotal role of NOBOX in early folliculogenesis is supported by findings in knock-out mice. Here, we report the genetic screening of 107 European women with idiopathic POI, recruited in various settings, and the molecular and functional characterization of the identified variants to evaluate their involvement in POI onset. Specifically, we report the identification of two novel and two recurrent heterozygous NOBOX variants in 7 out of 107 patients, with a prevalence of 6.5% (upper 95% confidence limit of 11.17%). Furthermore, immunolocalization, Western Blot and transcriptional assays conducted in either HEK293T or CHO cells revealed that all the studied variants (p.R44L, p.G91W, p.G111R, p.G152R, p.K273*, p.R449* and p.D452N) display variable degrees of functional impairment, including defects in transcriptional activity, autophagosomal degradation, nuclear localization or protein instability. Several variants conserve the ability to interact with FOXL2 in intracellular aggregates. Their inability to sustain gene expression, together with their likely aberrant effects on protein stability and degradation, make the identified NOBOX mutations a plausible cause of POI onset.

  15. Distribution of Pico- and Nanosecond Motions in Disordered Proteins from Nuclear Spin Relaxation.

    Science.gov (United States)

    Khan, Shahid N; Charlier, Cyril; Augustyniak, Rafal; Salvi, Nicola; Déjean, Victoire; Bodenhausen, Geoffrey; Lequin, Olivier; Pelupessy, Philippe; Ferrage, Fabien

    2015-09-01

    Intrinsically disordered proteins and intrinsically disordered regions (IDRs) are ubiquitous in the eukaryotic proteome. The description and understanding of their conformational properties require the development of new experimental, computational, and theoretical approaches. Here, we use nuclear spin relaxation to investigate the distribution of timescales of motions in an IDR from picoseconds to nanoseconds. Nitrogen-15 relaxation rates have been measured at five magnetic fields, ranging from 9.4 to 23.5 T (400-1000 MHz for protons). This exceptional wealth of data allowed us to map the spectral density function for the motions of backbone NH pairs in the partially disordered transcription factor Engrailed at 11 different frequencies. We introduce an approach called interpretation of motions by a projection onto an array of correlation times (IMPACT), which focuses on an array of six correlation times with intervals that are equidistant on a logarithmic scale between 21 ps and 21 ns. The distribution of motions in Engrailed varies smoothly along the protein sequence and is multimodal for most residues, with a prevalence of motions around 1 ns in the IDR. We show that IMPACT often provides better quantitative agreement with experimental data than conventional model-free or extended model-free analyses with two or three correlation times. We introduce a graphical representation that offers a convenient platform for a qualitative discussion of dynamics. Even when relaxation data are only acquired at three magnetic fields that are readily accessible, the IMPACT analysis gives a satisfactory characterization of spectral density functions, thus opening the way to a broad use of this approach.

  16. Construction of Yeast One-Hybrid Library for Screening of G-box Binding Proteins%筛选G-box结合蛋白的酵母单杂交文库的构建

    Institute of Scientific and Technical Information of China (English)

    杨鹏程; 周波; 李玉花

    2012-01-01

    目的:筛选花青素合成中的关键基因查尔酮合成酶基因CHS启动子中G-box的结合蛋白,从而找到调节CHS表达的转录因子.方法:采用Matchmaker Gold Yeast One-Hybrid Library Screening System,将CHS启动子G-box序列串联后整合入酵母染色体,构建诱饵菌株;采用SMART技术合成芜菁幼苗下胚轴cDNA,将该cDNA与pGA DT7-Rec表达载体共同转化诱饵菌株,通过同源重组在酵母细胞内同步进行cDNA文库的构建和筛选;用酵母菌落PCR法获得阳性克隆中的cDNA插入片段,测序后在NCBI网站进行Blast分析.结果:共筛选了2.52×106个酵母克隆,得到94个阳性克隆,菌落PCR获得了长度为0.4~2.0 kb的cDNA插入片段,并通过Blast推测了其编码蛋白.结论:实验结果证明酵母单杂交文库构建成功,初步筛选获得了G-box结合蛋白的候选蛋白,为研究CHS的表达调控奠定了基础.%Objective: In order to screen binding proteins of G-box, an important element in chalcone synthase (CHS) promoter region, and find transcriptional regulators of CHS gene. Methods: Matchmaker Gold Yeast One-Hybrid Library Screening System was employed in this study. Bait yeast strain was constructed by synthesizing oligonucleotides containing three tandem copies of G-box core sequences and integrating it into the genome of yeast. The cDNA for hypocotyls of turnip(Brassica rapa L. subsp. rapa Tsuda) was synthesized via SMART technology and co-transformed into bait yeast strain with pGADT7-Rec vector, one-hybrid cDNA library was simultaneously constructed and screened directly in yeast as a result of in vivo plasmid recombination. cDNA inserts in positive clones was amplified by yeast colony PCR and analyzed through NCBI Blast after sequencing. Results: Based on the experiments, we screened 2.52×106 yeast clones and got 94 positive clones. Colony PCR amplification products were 0.4~2.0 kb in length and proteins encoded by them were inferred by NCBI Blast analysis

  17. Localization and Differential Expression of the Krüppel-Associated Box Zinc Finger Proteins 1 and 54 in Early Mouse Development

    DEFF Research Database (Denmark)

    Albertsen, Maria; Teperek, Marta; Elholm, Grethe;

    2010-01-01

    -fused reporter gene into zygotes demonstrated the intracellular distribution of ZFP1-green fluorescent protein (GFP) and ZFP54-GFP colocalized with a DNA marker in the two-cell embryo. The KRAB domain was essential to colocalize with DNA, and deletion of the KRAB domain in ZFP1-GFP and ZFP54-GFP localized...

  18. Functional Significance of the Interaction between the mRNA-binding Protein, Nab2, and the Nuclear Pore-associated Protein, Mlp1, in mRNA Export* S⃞

    OpenAIRE

    Fasken, Milo B.; Stewart, Murray; Corbett, Anita H.

    2008-01-01

    Nuclear export of mRNA requires several key mRNA-binding proteins that recognize and remodel the mRNA and target it for export via interactions with the nuclear pore complex. In Saccharomyces cerevisiae, the shuttling heterogeneous nuclear ribonucleoprotein, Nab2, which is essential for mRNA export, specifically recognizes poly(A) RNA and binds to the nuclear pore-associated protein, myosin-like protein 1 (Mlp1), which functions in mRNA export and quality control. Specifically, the N-terminal...

  19. The folate-coupled enzyme MTHFD2 is a nuclear protein and promotes cell proliferation.

    Science.gov (United States)

    Gustafsson Sheppard, Nina; Jarl, Lisa; Mahadessian, Diana; Strittmatter, Laura; Schmidt, Angelika; Madhusudan, Nikhil; Tegnér, Jesper; Lundberg, Emma K; Asplund, Anna; Jain, Mohit; Nilsson, Roland

    2015-01-01

    Folate metabolism is central to cell proliferation and a target of commonly used cancer chemotherapeutics. In particular, the mitochondrial folate-coupled metabolism is thought to be important for proliferating cancer cells. The enzyme MTHFD2 in this pathway is highly expressed in human tumors and broadly required for survival of cancer cells. Although the enzymatic activity of the MTHFD2 protein is well understood, little is known about its larger role in cancer cell biology. We here report that MTHFD2 is co-expressed with two distinct gene sets, representing amino acid metabolism and cell proliferation, respectively. Consistent with a role for MTHFD2 in cell proliferation, MTHFD2 expression was repressed in cells rendered quiescent by deprivation of growth signals (serum) and rapidly re-induced by serum stimulation. Overexpression of MTHFD2 alone was sufficient to promote cell proliferation independent of its dehydrogenase activity, even during growth restriction. In addition to its known mitochondrial localization, we found MTHFD2 to have a nuclear localization and co-localize with DNA replication sites. These findings suggest a previously unknown role for MTHFD2 in cancer cell proliferation, adding to its known function in mitochondrial folate metabolism.

  20. A case of anti-nuclear matrix protein 2 antibody positive myopathy associated with lung cancer.

    Science.gov (United States)

    Ohta, Shin; Unoda, Ki-Ichi; Nakajima, Hideto; Ikeda, Soichiro; Hamaguchi, Yasuhito; Kimura, Fumiharu

    2016-08-31

    Myositis-specific autoantibodies (MSAs) are associated with myositis. Anti-nuclear matrix protein 2 (NXP-2) antibody was recently identified as a major MSA and was observed mostly in juvenile dermatomyositis. We report the case of a 44-year-old man who presented with myopathy with anti-NXP-2 antibody and large cell carcinoma of the lung. He was hospitalized because of myalgia and edema of limbs. Neurological examination revealed mild proximal-dominant weakness in all four extremities, and laboratory studies showed elevated creatine kinase level (6,432 IU/l). Needle electromyography showed myogenic patterns. MRI of the lower limbs demonstrated inflammatory lesions in the thighs. Biopsied specimen from the left quadriceps femoris muscle showed mild mononuclear inflammatory infiltrate surrounding muscle fibres but no fiber necrosis. He was diagnosed with myopathy based on neurological examinations and clinical symptoms. His chest X-ray and CT showed tumor shadow on the right upper lung field, but CT didn't indicate the findings of interstitial lung disease. This was surgically removed, and a histological diagnosis of non-small cell lung cancer was suspected. He was also treated with definitive chemoradiotherapy before and after operation. His symptoms of myopathy promptly remitted with the preoperative chemotherapy. His serum analysis was positive for the anti-NXP-2. Further investigation and experience of MSAs are necessary to evaluate the therapeutic strategy against cancer-associated myopathy/myositis.

  1. The prenylation status of a novel plant calmodulin directs plasma membrane or nuclear localization of the protein.

    Science.gov (United States)

    Rodríguez-Concepción, M; Yalovsky, S; Zik, M; Fromm, H; Gruissem, W

    1999-04-01

    Post-translational attachment of isoprenyl groups to conserved cysteine residues at the C-terminus of a number of regulatory proteins is important for their function and subcellular localization. We have identified a novel calmodulin, CaM53, with an extended C-terminal basic domain and a CTIL CaaX-box motif which are required for efficient prenylation of the protein in vitro and in vivo. Ectopic expression of wild-type CaM53 or a non-prenylated mutant protein in plants causes distinct morphological changes. Prenylated CaM53 associates with the plasma membrane, but the non-prenylated mutant protein localizes to the nucleus, indicating a dual role for the C-terminal domain. The subcellular localization of CaM53 can be altered by a block in isoprenoid biosynthesis or sugar depletion, suggesting that CaM53 activates different targets in response to metabolic changes. Thus, prenylation of CaM53 appears to be a novel mechanism by which plant cells can coordinate Ca2+ signaling with changes in metabolic activities.

  2. Observation of Transcription Regulation in the Mouse Heart Nuclear DNA Fragments and the Specific-protein Interaction by AFM

    Institute of Scientific and Technical Information of China (English)

    2003-01-01

    Using atom force microscopy (AFM), in vitro transcription, PAGE and other experimental technologies, it is observed that, in active genes of mice (Balb/c) nuclear DNA fragments of non-transcriptional state, only regulation sequences at both ends are associated with scaffold proteins (indissociable proteins) and some transcriptional factors such as complexes (dissociable proteins) made of gene-coding proteins and specific auxiliary small molecules, while there are no combining proteins in intermediate coding sequences. However, in active genes of transcriptional state, both regulation sequences and intermediate coding sequences are associated with active transcriptional factors by non-covalent bonds.This paper shows the prospective application of AFM observation and in vitro transcription in the research on gene expression and regulation. It also offers some theoretical basis for localization of specific genes in human genomes.

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

    Science.gov (United States)

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

    2009-12-16

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

  4. 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.

  5. Forkhead Box Protein 1 (Foxa1) and the Sumoylation Pathway that Regulates Foxa1 Stability are Potential Targets for Breast Cancer Treatment

    Science.gov (United States)

    2007-09-01

    98% aminoacid identity between mature SUMO-2 and SUMO-3) (3), antibodies that react with both SUMO-2 and SUMO- 3 were used for IPs. IP reactions...Foxa1 protein are shown. The numbers above the diagram correspond to aminoacid coordinates of human Foxa1. Potential sumoylation sites (denoted by...arrowheads) and sequences are shown below the diagram. Numbers in parenthesis next to the aminoacid sequences correspond to the potential sumoylation

  6. 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.

  7. Projection optics box

    Science.gov (United States)

    Hale, Layton C.; Malsbury, Terry; Hudyma, Russell M.; Parker, John M.

    2000-01-01

    A projection optics box or assembly for use in an optical assembly, such as in an extreme ultraviolet lithography (EUVL) system using 10-14 nm soft x-ray photons. The projection optics box utilizes a plurality of highly reflective optics or mirrors, each mounted on a precision actuator, and which reflects an optical image, such as from a mask, in the EUVL system onto a point of use, such as a target or silicon wafer, the mask, for example, receiving an optical signal from a source assembly, such as a developed from laser system, via a series of highly reflective mirrors of the EUVL system. The plurality of highly reflective optics or mirrors are mounted in a housing assembly comprised of a series of bulkheads having wall members secured together to form a unit construction of maximum rigidity. Due to the precision actuators, the mirrors must be positioned precisely and remotely in tip, tilt, and piston (three degrees of freedom), while also providing exact constraint.

  8. Chromatin-bound NLS proteins recruit membrane vesicles and nucleoporins for nuclear envelope assembly via importin-α/β

    Institute of Scientific and Technical Information of China (English)

    Quanlong Lu; Zhigang Lu; Qinying Liu; Li Guo; He Ren; Jingyan Fu; Qing Jiang; Paul R Clarke; Chuanmao Zhang

    2012-01-01

    The mechanism for nuclear envelope (NE) assembly is not fully understood.Importin-β and the small GTPase Ran have been implicated in the spatial regulation of NE assembly process.Here we report that chromatin-bound NLS (nuclear localization sequence) proteins provide docking sites for the NE precursor membrane vesicles and nucleoporins via importin-α and -β during NE assembly in Xenopus egg extracts.We show that along with the fast recruitment of the abundant NLS proteins such as nucleoplasmin and histones to the demembranated sperm chromatin in the extracts,importin-α binds the chromatin NLS proteins rapidly.Meanwhile,importin-β binds cytoplasmic NE precursor membrane vesicles and nucleoporins.Through interacting with importin-α on the chromatin NLS proteins,importin-β targets the membrane vesicles and nucleoporins to the chromatin surface.Once encountering RanGTP on the chromatin generated by RCC1,importin-β preferentially binds Ran-GTP and releases the membrane vesicles and nucleoporins for NE assembly.NE assembly is disrupted by blocking the interaction between importin-α and NLS proteins with excess soluble NLS proteins or by depletion of importin-β from the extract.Our findings reveal a novel molecular mechanism for NE assembly in Xenopus egg extracts.

  9. Structural basis of nucleic acid recognition by FK506-binding protein 25 (FKBP25), a nuclear immunophilin.

    Science.gov (United States)

    Prakash, Ajit; Shin, Joon; Rajan, Sreekanth; Yoon, Ho Sup

    2016-04-01

    The nuclear immunophilin FKBP25 interacts with chromatin-related proteins and transcription factors and is suggested to interact with nucleic acids. Currently the structural basis of nucleic acid binding by FKBP25 is unknown. Here we determined the nuclear magnetic resonance (NMR) solution structure of full-length human FKBP25 and studied its interaction with DNA. The FKBP25 structure revealed that the N-terminal helix-loop-helix (HLH) domain and C-terminal FK506-binding domain (FKBD) interact with each other and that both of the domains are involved in DNA binding. The HLH domain forms major-groove interactions and the basic FKBD loop cooperates to form interactions with an adjacent minor-groove of DNA. The FKBP25-DNA complex model, supported by NMR and mutational studies, provides structural and mechanistic insights into the nuclear immunophilin-mediated nucleic acid recognition.

  10. The Classroom Animal: Box Turtles.

    Science.gov (United States)

    Kramer, David C.

    1986-01-01

    Provides basic information on the anatomy, physiology, behaviors, and distribution patterns of the box turtle. Offers suggestions for the turtle's care and maintenance in a classroom environment. (ML)

  11. Boxing-related head injuries.

    Science.gov (United States)

    Jayarao, Mayur; Chin, Lawrence S; Cantu, Robert C

    2010-10-01

    Fatalities in boxing are most often due to traumatic brain injury that occurs in the ring. In the past 30 years, significant improvements in ringside and medical equipment, safety, and regulations have resulted in a dramatic reduction in the fatality rate. Nonetheless, the rate of boxing-related head injuries, particularly concussions, remains unknown, due in large part to its variability in clinical presentation. Furthermore, the significance of repeat concussions sustained when boxing is just now being understood. In this article, we identify the clinical manifestations, pathophysiology, and management of boxing-related head injuries, and discuss preventive strategies to reduce head injuries sustained by boxers.

  12. Ultrastructural study of nuclear inclusions immunohistochemically positive for surfactant protein A in pulmonary adenocarcinoma with special reference to their morphogenesis.

    Science.gov (United States)

    Lu, Shu-Hui; Ohtsuki, Yuji; Nonami, Yoshiki; Sasaguri, Shiro; Fujita, Jiro; Uomoto, Masashi; Tao, Fu-Shan; Kobayashi, Makoto; Furihata, Mutsuo

    2006-12-01

    To investigate the fine-structural nature of nuclear inclusions immunopositive for surfactant protein A (SP-A) antibody staining, a detailed ultrastructural study was performed, as well as immunohistochemical examination of pulmonary adenocarcinomas. Surgically resected tumor specimens from 31 patients were examined by immunohistochemistry focused on reactivity to SP-A and thyroid transcription factor 1 (TTF-1) antibodies. Only cases with >5% positive nuclear inclusions in cancer cells were considered positive, some of which were examined by electron microscopy. Immunohistochemically, 6 of 31 cases were doubly positive for SP-A and TTF-1 antibodies. On electron microscopy, SP-A-positive nuclei contained diffuse or globular fine granular substance as inclusions. Both types of globular and diffuse inclusions were sometimes connected to the inner nuclear membrane, in association with fragmented or stacked membranous structures. The findings of this study suggested that nuclear inclusions positive for SP-A antibody staining in adenocarcinomas of the lung were derived from accumulated content in the perinuclear cistern resembling pseudoinclusion processes and composed of proteins antigenically cross-reactive with SP-A.

  13. Localization of a bacterial group II intron-encoded protein in eukaryotic nuclear splicing-related cell compartments.

    Directory of Open Access Journals (Sweden)

    Rafael Nisa-Martínez

    Full Text Available Some bacterial group II introns are widely used for genetic engineering in bacteria, because they can be reprogrammed to insert into the desired DNA target sites. There is considerable interest in developing this group II intron gene targeting technology for use in eukaryotes, but nuclear genomes present several obstacles to the use of this approach. The nuclear genomes of eukaryotes do not contain group II introns, but these introns are thought to have been the progenitors of nuclear spliceosomal introns. We investigated the expression and subcellular localization of the bacterial RmInt1 group II intron-encoded protein (IEP in Arabidopsis thaliana protoplasts. Following the expression of translational fusions of the wild-type protein and several mutant variants with EGFP, the full-length IEP was found exclusively in the nucleolus, whereas the maturase domain alone targeted EGFP to nuclear speckles. The distribution of the bacterial RmInt1 IEP in plant cell protoplasts suggests that the compartmentalization of eukaryotic cells into nucleus and cytoplasm does not prevent group II introns from invading the host genome. Furthermore, the trafficking of the IEP between the nucleolus and the speckles upon maturase inactivation is consistent with the hypothesis that the spliceosomal machinery evolved from group II introns.

  14. PROTEIN L-ISOASPARTYL METHYLTRANSFERASE2 is differentially expressed in chickpea and enhances seed vigor and longevity by reducing abnormal isoaspartyl accumulation predominantly in seed nuclear proteins.

    Science.gov (United States)

    Verma, Pooja; Kaur, Harmeet; Petla, Bhanu Prakash; Rao, Venkateswara; Saxena, Saurabh C; Majee, Manoj

    2013-03-01

    PROTEIN l-ISOASPARTYL METHYLTRANSFERASE (PIMT) is a widely distributed protein-repairing enzyme that catalyzes the conversion of abnormal l-isoaspartyl residues in spontaneously damaged proteins to normal aspartyl residues. This enzyme is encoded by two divergent genes (PIMT1 and PIMT2) in plants, unlike many other organisms. While the biological role of PIMT1 has been elucidated, the role and significance of the PIMT2 gene in plants is not well defined. Here, we isolated the PIMT2 gene (CaPIMT2) from chickpea (Cicer arietinum), which exhibits a significant increase in isoaspartyl residues in seed proteins coupled with reduced germination vigor under artificial aging conditions. The CaPIMT2 gene is found to be highly divergent and encodes two possible isoforms (CaPIMT2 and CaPIMT2') differing by two amino acids in the region I catalytic domain through alternative splicing. Unlike CaPIMT1, both isoforms possess a unique 56-amino acid amino terminus and exhibit similar yet distinct enzymatic properties. Expression analysis revealed that CaPIMT2 is differentially regulated by stresses and abscisic acid. Confocal visualization of stably expressed green fluorescent protein-fused PIMT proteins and cell fractionation-immunoblot analysis revealed that apart from the plasma membrane, both CaPIMT2 isoforms localize predominantly in the nucleus, while CaPIMT1 localizes in the cytosol. Remarkably, CaPIMT2 enhances seed vigor and longevity by repairing abnormal isoaspartyl residues predominantly in nuclear proteins upon seed-specific expression in Arabidopsis (Arabidopsis thaliana), while CaPIMT1 enhances seed vigor and longevity by repairing such abnormal proteins mainly in the cytosolic fraction. Together, our data suggest that CaPIMT2 has most likely evolved through gene duplication, followed by subfunctionalization to specialize in repairing the nuclear proteome.

  15. The role of mitochondria and the CIA machinery in the maturation of cytosolic and nuclear iron-sulfur proteins.

    Science.gov (United States)

    Lill, Roland; Dutkiewicz, Rafal; Freibert, Sven A; Heidenreich, Torsten; Mascarenhas, Judita; Netz, Daili J; Paul, Viktoria D; Pierik, Antonio J; Richter, Nadine; Stümpfig, Martin; Srinivasan, Vasundara; Stehling, Oliver; Mühlenhoff, Ulrich

    2015-01-01

    Mitochondria have been derived from alpha-bacterial endosymbionts during the evolution of eukaryotes. Numerous bacterial functions have been maintained inside the organelles including fatty acid degradation, citric acid cycle, oxidative phosphorylation, and the synthesis of heme or lipoic acid cofactors. Additionally, mitochondria have inherited the bacterial iron-sulfur cluster assembly (ISC) machinery. Many of the ISC components are essential for cell viability because they generate a still unknown, sulfur-containing compound for the assembly of cytosolic and nuclear Fe/S proteins that perform important functions in, e.g., protein translation, DNA synthesis and repair, and chromosome segregation. The sulfur-containing compound is exported by the mitochondrial ABC transporter Atm1 (human ABCB7) and utilized by components of the cytosolic iron-sulfur protein assembly (CIA) machinery. An appealing minimal model for the striking compartmentation of eukaryotic Fe/S protein biogenesis is provided by organisms that contain mitosomes instead of mitochondria. Mitosomes have been derived from mitochondria by reductive evolution, during which they have lost virtually all classical mitochondrial tasks. Nevertheless, mitosomes harbor all core ISC components which presumably have been maintained for assisting the maturation of cytosolic-nuclear Fe/S proteins. The current review is centered around the Atm1 export process. We present an overview on the mitochondrial requirements for the export reaction, summarize recent insights into the 3D structure and potential mechanism of Atm1, and explain how the CIA machinery uses the mitochondrial export product for the assembly of cytosolic and nuclear Fe/S proteins.

  16. Epstein-Barr virus encoded nuclear protein EBNA-3 binds a novel human uridine kinase/uracil phosphoribosyltransferase

    Directory of Open Access Journals (Sweden)

    Klein George

    2002-08-01

    Full Text Available Abstract Background Epstein-Barr virus (EBV infects resting B-lymphocytes and transforms them into immortal proliferating lymphoblastoid cell lines (LCLs in vitro. The transformed immunoblasts may grow up as immunoblastic lymphomas in immuno-suppressed hosts. Results In order to identify cellular protein targets that may be involved in Epstein-Barr virus mediated B-cell transformation, human LCL cDNA library was screened with one of the transformation associated nuclear antigens, EBNA-3 (also called EBNA-3A, using the yeast two-hybrid system. A clone encoding a fragment of a novel human protein was isolated (clone 538. The interaction was confirmed using in vitro binding assays. A full-length cDNA clone (F538 was isolated. Sequence alignment with known proteins and 3D structure predictions suggest that F538 is a novel human uridine kinase/uracil phosphoribosyltransferase. The GFP-F538 fluorescent fusion protein showed a preferentially cytoplasmic distribution but translocated to the nucleus upon co-expression of EBNA-3. A naturally occurring splice variant of F538, that lacks the C-terminal uracil phosphoribosyltransferase part but maintain uridine kinase domain, did not translocate to the nucleus in the presence of EBNA3. Antibody that was raised against the bacterially produced GST-538 protein showed cytoplasmic staining in EBV negative Burkitt lymphomas but gave a predominantly nuclear staining in EBV positive LCL-s and stable transfected cells expressing EBNA-3. Conclusion We suggest that EBNA-3 by direct protein-potein interaction induces the nuclear accumulation of a novel enzyme, that is part of the ribonucleotide salvage pathway. Increased intranuclear levels of UK/UPRT may contribute to the metabolic build-up that is needed for blast transformation and rapid proliferation.

  17. Orphan Nuclear Receptor NR4A1 Binds a Novel Protein Interaction Site on Anti-apoptotic B Cell Lymphoma Gene 2 Family Proteins.

    Science.gov (United States)

    Godoi, Paulo H C; Wilkie-Grantham, Rachel P; Hishiki, Asami; Sano, Renata; Matsuzawa, Yasuko; Yanagi, Hiroko; Munte, Claudia E; Chen, Ya; Yao, Yong; Marassi, Francesca M; Kalbitzer, Hans R; Matsuzawa, Shu-Ichi; Reed, John C

    2016-07-01

    B cell lymphoma gene 2 (Bcl-2) family proteins are key regulators of programmed cell death and important targets for drug discovery. Pro-apoptotic and anti-apoptotic Bcl-2 family proteins reciprocally modulate their activities in large part through protein interactions involving a motif known as BH3 (Bcl-2 homology 3). Nur77 is an orphan member of the nuclear receptor family that lacks a BH3 domain but nevertheless binds certain anti-apoptotic Bcl-2 family proteins (Bcl-2, Bfl-1, and Bcl-B), modulating their effects on apoptosis and autophagy. We used a combination of NMR spectroscopy-based methods, mutagenesis, and functional studies to define the interaction site of a Nur77 peptide on anti-apoptotic Bcl-2 family proteins and reveal a novel interaction surface. Nur77 binds adjacent to the BH3 peptide-binding crevice, suggesting the possibility of cross-talk between these discrete binding sites. Mutagenesis of residues lining the identified interaction site on Bcl-B negated the interaction with Nur77 protein in cells and prevented Nur77-mediated modulation of apoptosis and autophagy. The findings establish a new protein interaction site with the potential to modulate the apoptosis and autophagy mechanisms governed by Bcl-2 family proteins.

  18. Position-dependent interactions of Y-box protein 2 (YBX2) with mRNA enable mRNA storage in round spermatids by repressing mRNA translation and blocking translation-dependent mRNA decay.

    Science.gov (United States)

    Kleene, Kenneth C

    2016-03-01

    Many mRNAs encoding proteins needed for the construction of the specialized organelles of spermatozoa are stored as translationally repressed, free messenger ribonucleoproteins in round spermatids, to be actively translated in elongating and elongated spermatids. The factors that repress translation in round spermatids, however, have been elusive. Two lines of evidence implicate the highly abundant and well-known translational repressor, Y-box protein 2 (YBX2), as a critical factor: First, protamine 1 (Prm1) and sperm-mitochondria cysteine-rich protein (Smcp) mRNAs are prematurely recruited onto polysomes in Ybx2-knockout mouse round spermatids. Second, mutations in 3' untranslated region (3'UTR) cis-elements that abrogate YBX2 binding activate translation of Prm1 and Smcp mRNAs in round spermatids of transgenic mice. The abundance of YBX2 and its affinity for variable sequences, however, raise questions of how YBX2 targets specific mRNAs for repression. Mutations to the Prm1 and Smcp mRNAs in transgenic mice reveal that strong repression in round spermatids requires YBX2 binding sites located near the 3' ends of their 3'UTRs as locating the same sites in upstream positions produce negligible repression. This location-dependence implies that the assembly of repressive complexes is nucleated by adjacent cis-elements that enable cooperative interactions of YBX2 with co-factors. The available data suggest that, in vertebrates, YBX2 has the important role of coordinating the storage of translationally repressed mRNAs in round spermatids by inhibiting translational activity and the degradation of transcripts via translation-dependent deadenylation. These insights should facilitiate future experiments designed to unravel how YBX2 targets mRNAs for repression in round spermatids and how mutations in the YBX2 gene cause infertility in humans. Mol. Reprod. Dev. 83: 190-207, 2016. © 2016 Wiley Periodicals, Inc.

  19. Arcuate nucleus transcriptome profiling identifies ankyrin repeat and suppressor of cytokine signalling box-containing protein 4 as a gene regulated by fasting in central nervous system feeding circuits.

    Science.gov (United States)

    Li, J-Y; Kuick, R; Thompson, R C; Misek, D E; Lai, Y-M; Liu, Y-Q; Chai, B-X; Hanash, S M; Gantz, I

    2005-06-01

    The arcuate nucleus of the hypothalamus is a primary site for sensing blood borne nutrients and hormonal messengers that reflect caloric status. To identify novel energy homeostatic genes, we examined RNA extracts from the microdissected arcuate nucleus of fed and 48-h fasted rats using oligonucleotide microarrays. The relative abundance of 118 mRNA transcripts was increased and 203 mRNA transcripts was decreased during fasting. One of the down-regulated mRNAs was ankyrin-repeat and suppressor of cytokine signalling box-containing protein 4 (Asb-4). The predicted structure of Asb-4 protein suggested that it might encode an intracellular regulatory protein, and therefore its mRNA expression was investigated further. Reverse transcription quantitative polymerase chain reaction was used to validate down-regulation of Asb-4 mRNA in the arcuate nucleus of the fasted Sprague-Dawley rat (relative expression of Asb-4 mRNA: fed = 4.66 +/- 0.26; fasted = 3.96 +/- 0.23; n = 4, P regulation was also demonstrated in the obese fa/fa Zucker rat, another model of energy disequilibrium (relative expression of Asb-4 mRNA: lean Zucker = 3.91 +/- 0.32; fa/fa = 2.93 +/- 0.26; n = 5, P fasted state, the percentage of POMC neurones expressing Asb-4 mRNA drops to 73.2% (P fasted POMC neurone is markedly decreased. Conversely, expression of Asb-4 mRNA by NPY neurones in the fasted state is modestly increased to 52.7% (P < 0.05). Based on its differential expression, neuroanatomical distribution and colocalisation, we hypothesise that Asb-4 is a gene involved in energy homeostasis.

  20. Distinct domains of the spinal muscular atrophy protein SMN are required for targeting to Cajal bodies in mammalian cells.

    OpenAIRE

    Renvoisé, Benoît; Khoobarry, Kevinee; Gendron, Marie-Claude; Cibert, Christian; Viollet, Louis; Lefebvre, Suzie

    2006-01-01

    Mutations of the survival motor neuron gene SMN1 cause the inherited disease spinal muscular atrophy (SMA). The ubiquitous SMN protein facilitates the biogenesis of spliceosomal small nuclear ribonucleoproteins (snRNPs). The protein is detected in the cytoplasm, nucleoplasm and enriched with snRNPs in nuclear Cajal bodies. It is structurally divided into at least an amino-terminal region rich in basic amino acid residues, a central Tudor domain, a self-association tyrosine-glycine-box and an ...