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Sample records for important cellular protein

  1. Cellular Promyelocytic Leukemia Protein Is an Important Dengue Virus Restriction Factor

    OpenAIRE

    Giovannoni, Federico; Damonte, Elsa B.; Garc?a, Cybele C.

    2015-01-01

    The intrinsic antiviral defense is based on cellular restriction factors that are constitutively expressed and, thus, active even before a pathogen enters the cell. The promyelocytic leukemia (PML) nuclear bodies (NBs) are discrete nuclear foci that contain several cellular proteins involved in intrinsic antiviral responses against a number of viruses. Accumulating reports have shown the importance of PML as a DNA virus restriction factor and how these pathogens evade this antiviral activity....

  2. 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.

  3. 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

    2011-03-14

    Abstract Background 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. Results In our study, we have identified the cellular protein HIC (Human I-mfa domain-Containing protein) as a novel interactor of HIV-1 Rev. We demonstrate that HIC selectively interferes with Rev NLS interaction with importin β and impedes its nuclear import and function, but does not affect Rev nuclear import mediated by transportin. Hence, the molecular determinants mediating Rev-NLS recognition by importin β and transportin appear to be distinct. Furthermore, we have employed HIC and M9 M, a peptide specifically designed to inhibit the transportin-mediated nuclear import pathway, to characterise Rev nuclear import pathways within different cellular environments. Remarkably, we could show that in 293T, HeLa, COS7, Jurkat, U937, THP-1 and CEM cells, Rev nuclear import is cell type specific and alternatively mediated by transportin or importin β, in a mutually exclusive fashion. Conclusions Rev cytoplasmic sequestration by HIC may represent a novel mechanism for the control of Rev function. These studies highlight that the multivalent nature of the Rev NLS for different import receptors enables Rev to adapt its nuclear trafficking strategy.

  4. Intermolecular masking of the HIV-1 Rev NLS by the cellular protein HIC: Novel insights into the regulation of Rev nuclear import

    Directory of Open Access Journals (Sweden)

    Sheehy Noreen

    2011-03-01

    Full Text Available Abstract Background 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. Results In our study, we have identified the cellular protein HIC (Human I-mfa domain-Containing protein as a novel interactor of HIV-1 Rev. We demonstrate that HIC selectively interferes with Rev NLS interaction with importin β and impedes its nuclear import and function, but does not affect Rev nuclear import mediated by transportin. Hence, the molecular determinants mediating Rev-NLS recognition by importin β and transportin appear to be distinct. Furthermore, we have employed HIC and M9 M, a peptide specifically designed to inhibit the transportin-mediated nuclear import pathway, to characterise Rev nuclear import pathways within different cellular environments. Remarkably, we could show that in 293T, HeLa, COS7, Jurkat, U937, THP-1 and CEM cells, Rev nuclear import is cell type specific and alternatively mediated by transportin or importin β, in a mutually exclusive fashion. Conclusions Rev cytoplasmic sequestration by HIC may represent a novel mechanism for the control of Rev function. These studies highlight that the multivalent nature of the Rev NLS for different import receptors enables Rev to adapt its nuclear trafficking strategy.

  5. Cellular strategies to cope with protein aggregation

    NARCIS (Netherlands)

    Scior, Annika; Juenemann, Katrin; Kirstein, Janine

    2016-01-01

    Nature has evolved several mechanisms to detoxify intracellular protein aggregates that arise upon proteotoxic challenges. These include the controlled deposition of misfolded proteins at distinct cellular sites, the protein disaggregation and refolding by molecular chaperones and/or degradation of

  6. Role of Crk Adaptor Proteins in Cellular Migration and Invasion in Human Breast Cancer

    National Research Council Canada - National Science Library

    Fathers, Kelly E

    2007-01-01

    The Crk adaptor proteins (CrkI, CrkII and CrkL) play an important role during cellular signalling by mediating the formation of protein-protein complexes and are involved in cellular migration, invasion, and adhesion...

  7. Role of Crk Adaptor Proteins in Cellular Migration and Invasion in Human Breast Cancer

    National Research Council Canada - National Science Library

    Fathers, Kelly E

    2008-01-01

    The Crk adaptor proteins (CrkI, CrkII and CrkL) play an important role during cellular signalling by mediating the formation of protein-protein complexes and are involved in cellular migration, invasion, and adhesion...

  8. Piezo proteins: regulators of mechanosensation and other cellular processes.

    Science.gov (United States)

    Bagriantsev, Sviatoslav N; Gracheva, Elena O; Gallagher, Patrick G

    2014-11-14

    Piezo proteins have recently been identified as ion channels mediating mechanosensory transduction in mammalian cells. Characterization of these channels has yielded important insights into mechanisms of somatosensation, as well as other mechano-associated biologic processes such as sensing of shear stress, particularly in the vasculature, and regulation of urine flow and bladder distention. Other roles for Piezo proteins have emerged, some unexpected, including participation in cellular development, volume regulation, cellular migration, proliferation, and elongation. Mutations in human Piezo proteins have been associated with a variety of disorders including hereditary xerocytosis and several syndromes with muscular contracture as a prominent feature. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  9. Piezo Proteins: Regulators of Mechanosensation and Other Cellular Processes*

    Science.gov (United States)

    Bagriantsev, Sviatoslav N.; Gracheva, Elena O.; Gallagher, Patrick G.

    2014-01-01

    Piezo proteins have recently been identified as ion channels mediating mechanosensory transduction in mammalian cells. Characterization of these channels has yielded important insights into mechanisms of somatosensation, as well as other mechano-associated biologic processes such as sensing of shear stress, particularly in the vasculature, and regulation of urine flow and bladder distention. Other roles for Piezo proteins have emerged, some unexpected, including participation in cellular development, volume regulation, cellular migration, proliferation, and elongation. Mutations in human Piezo proteins have been associated with a variety of disorders including hereditary xerocytosis and several syndromes with muscular contracture as a prominent feature. PMID:25305018

  10. Chatty Mitochondria: Keeping Balance in Cellular Protein Homeostasis.

    Science.gov (United States)

    Topf, Ulrike; Wrobel, Lidia; Chacinska, Agnieszka

    2016-08-01

    Mitochondria are multifunctional cellular organelles that host many biochemical pathways including oxidative phosphorylation (OXPHOS). Defective mitochondria pose a threat to cellular homeostasis and compensatory responses exist to curtail the source of stress and/or its consequences. The mitochondrial proteome comprises proteins encoded by the nuclear and mitochondrial genomes. Disturbances in protein homeostasis may originate from mistargeting of nuclear encoded mitochondrial proteins. Defective protein import and accumulation of mistargeted proteins leads to stress that triggers translation alterations and proteasomal activation. These cytosolic pathways are complementary to the mitochondrial unfolded protein response (UPRmt) that aims to increase the capacity of protein quality control mechanisms inside mitochondria. They constitute putative targets for interventions aimed at increasing the fitness, stress resistance, and longevity of cells and organisms. Copyright © 2016 Elsevier Ltd. All rights reserved.

  11. Cellular proteostasis: degradation of misfolded proteins by lysosomes

    Science.gov (United States)

    Jackson, Matthew P.

    2016-01-01

    Proteostasis refers to the regulation of the cellular concentration, folding, interactions and localization of each of the proteins that comprise the proteome. One essential element of proteostasis is the disposal of misfolded proteins by the cellular pathways of protein degradation. Lysosomes are an important site for the degradation of misfolded proteins, which are trafficked to this organelle by the pathways of macroautophagy, chaperone-mediated autophagy and endocytosis. Conversely, amyloid diseases represent a failure in proteostasis, in which proteins misfold, forming amyloid deposits that are not degraded effectively by cells. Amyloid may then exacerbate this failure by disrupting autophagy and lysosomal proteolysis. However, targeting the pathways that regulate autophagy and the biogenesis of lysosomes may present approaches that can rescue cells from the deleterious effects of amyloidogenic proteins. PMID:27744333

  12. Piezo Proteins: Regulators of Mechanosensation and Other Cellular Processes*

    OpenAIRE

    Bagriantsev, Sviatoslav N.; Gracheva, Elena O.; Gallagher, Patrick G.

    2014-01-01

    Piezo proteins have recently been identified as ion channels mediating mechanosensory transduction in mammalian cells. Characterization of these channels has yielded important insights into mechanisms of somatosensation, as well as other mechano-associated biologic processes such as sensing of shear stress, particularly in the vasculature, and regulation of urine flow and bladder distention. Other roles for Piezo proteins have emerged, some unexpected, including participation in cellular deve...

  13. When is protein binding important?

    Science.gov (United States)

    Heuberger, Jules; Schmidt, Stephan; Derendorf, Hartmut

    2013-09-01

    The present paper is an ode to a classic citation by Benet and Hoener (2002. Clin Pharm Ther 71(3):115-121). The now classic paper had a huge impact on drug development and the way the issue of protein binding is perceived and interpreted. Although the authors very clearly pointed out the limitations and underlying assumptions for their delineations, these are too often overlooked and the classic paper's message is misinterpreted by broadening to cases that were not intended. Some members of the scientific community concluded from the paper that protein binding is not important. This was clearly not intended by the authors, as they finished their paper with a paragraph entitled: "When is protein binding important?" Misinterpretation of the underlying assumptions in the classic work can result in major pitfalls in drug development. Therefore, we revisit the topic of protein binding with the intention of clarifying when clinically relevant changes should be considered during drug development. Copyright © 2013 Wiley Periodicals, Inc.

  14. Cellular recycling of proteins in seed dormancy alleviation and germination

    Directory of Open Access Journals (Sweden)

    Krystyna Oracz

    2016-07-01

    Full Text Available Each step of the seed-to-seed cycle of plant development including seed germination is characterized by a specific set of proteins. The continual renewal and/or replacement of these biomolecules are crucial for optimal plant adaptation. As proteins are the main effectors inside the cells, their levels need to be tightly regulated. This is partially achieved by specific proteolytic pathways via multicatalytic protease complexes defined as 20S and 26S proteasomes. In plants, the 20S proteasome is responsible for degradation of carbonylated proteins, while the 26S being a part of ubiquitin-proteasome pathway (UPP is known to be involved in proteolysis of phytohormone signaling regulators. On the other hand, the role of translational control of plant development is also well documented, especially in the context of pollen tube growth and light signaling. Despite the current progress that has been made in seed biology, the sequence of cellular events that determine if the seed can germinate or not are still far from complete understanding. The role and mechanisms of regulation of proteome composition during processes occurring in the plant’s photosynthetic tissues have been well characterized since many years, but in nonphotosynthetic seeds it has emerged as a tempting research task only since the last decade. This review discusses the recent discoveries providing insights into the role of protein turnover in seed dormancy alleviation, and germination, with a focus on the control of translation and proteasomal proteolysis. The presented novel data of translatome profiling in seeds highlighted that post-transcriptional regulation of germination results from a timely regulated initiation of translation. In addition, the importance of 26S proteasome in the degradation of regulatory elements of cellular signaling and that of the 20S complex in proteolysis of specific carbonylated proteins in hormonal- and light-dependent processes occurring in seeds is

  15. Poly-ADP-ribosylation of proteins responds to cellular perturbations

    International Nuclear Information System (INIS)

    Schneeweiss, F.H.A.; Sharan, R.N.

    1999-01-01

    From the results presented above it is quite obvious that poly-ADP-ribosylation reaction is a sensitive parameter to monitor cellular responses to a wide variety of perturbations. Having developed a monolayer assay system using 32 P-NAD + as a marker, it has become possible to measure levels of cellular ADP-ribosylation more precisely. It has been demonstrated that the trigger of poly-ADP-ribosylation reaction may involve different cellular components for different perturbations. In this, membrane has been found to be important. The study has been particularly informative in the realm of DNA damage and repair following qualitatively different radiation assaults. As poly-ADP-ribosylation in eukaryotic cells primarily affects chromosomal proteins, notably histones, the reaction is strongly triggered in response to single and double strand breaks in DNA. Therefore, level of cellular poly-ADP-ribosylation can potentially be used as a biosensor of radiation induced strand breaks and can be specially useful in clinical monitoring of progress of radiotherapy. The assay of poly-ADP-ribosylation, however, requires use of radiolabelled tracer, e.g. 32 P-NAD + . Due to this, study of poly-ADP-ribosylation can not be extended to monitor effects of incorporated radionuclides. In order to overcome this shortcoming and to make the assay more sensitive and quick, a Western blot immunoassay has been developed. The preliminary indications are that the immunoassay of poly-ADP-ribosylation will fulfil the requirements to use poly-ADP-ribosylation as a sensitive, convenient and clinically applicable biosensor of cell response not only to radiations but also to different perturbations. (orig.)

  16. In vivo cellular imaging using fluorescent proteins - Methods and Protocols

    Directory of Open Access Journals (Sweden)

    M. Monti

    2012-12-01

    Full Text Available The discovery and genetic engineering of fluorescent proteins has revolutionized cell biology. What was previously invisible to the cell often can be made visible with the use of fluorescent proteins. With this words, Robert M. Hoffman introduces In vivo Cellular Imaging Using Fluorescent proteins, the eighteen chapters book dedicated to the description of how fluorescence proteins have changed the way to analyze cellular processes in vivo. Modern researches aim to study new and less invasive methods able to follow the behavior of different cell types in different biological contexts: for example, how cancer cells migrate or how they respond to different therapies. Also, in vivo systems can help researchers to better understand animal embryonic development so as how fluorescence proteins may be used to monitor different processes in living organisms at the molecular and cellular level.

  17. Cellular Reprogramming Employing Recombinant Sox2 Protein

    Directory of Open Access Journals (Sweden)

    Marc Thier

    2012-01-01

    Full Text Available Induced pluripotent stem (iPS cells represent an attractive option for the derivation of patient-specific pluripotent cells for cell replacement therapies as well as disease modeling. To become clinically meaningful, safe iPS cells need to be generated exhibiting no permanent genetic modifications that are caused by viral integrations of the reprogramming transgenes. Recently, various experimental strategies have been applied to accomplish transgene-free derivation of iPS cells, including the use of nonintegrating viruses, episomal expression, or excision of transgenes after reprogramming by site-specific recombinases or transposases. A straightforward approach to induce reprogramming factors is the direct delivery of either synthetic mRNA or biologically active proteins. We previously reported the generation of cell-permeant versions of Oct4 (Oct4-TAT and Sox2 (Sox2-TAT proteins and showed that Oct4-TAT is reprogramming-competent, that is, it can substitute for Oct4-encoding virus. Here, we explore conditions for enhanced Sox2-TAT protein stabilization and functional delivery into somatic cells. We show that cell-permeant Sox2 protein can be stabilized by lipid-rich albumin supplements in serum replacement or low-serum-supplemented media. Employing optimized conditions for protein delivery, we demonstrate that Sox2-TAT protein is able to substitute for viral Sox2. Sox2-piPS cells express pluripotency-associated markers and differentiate into all three germ layers.

  18. Imaging the lipidome: omega-alkynyl fatty acids for detection and cellular visualization of lipid-modified proteins.

    Science.gov (United States)

    Hannoush, Rami N; Arenas-Ramirez, Natalia

    2009-07-17

    Fatty acylation or lipid modification of proteins controls their cellular activation and diverse roles in physiology. It mediates protein-protein and protein-membrane interactions and plays an important role in regulating cellular signaling pathways. Currently, there is need for visualizing lipid modifications of proteins in cells. Herein we report novel chemical probes based on omega-alkynyl fatty acids for biochemical detection and cellular imaging of lipid-modified proteins. Our study shows that omega-alkynyl fatty acids of varying chain length are metabolically incorporated onto cellular proteins. Using fluorescence imaging, we describe the subcellular distribution of lipid-modified proteins across a panel of different mammalian cell lines and during cell division. Our results demonstrate that this methodology is a useful diagnostic tool for analyzing the lipid content of cellular proteins and for studying the dynamic behavior of lipid-modified proteins in various disease or physiological states.

  19. Cellular Handling of Protein Aggregates by Disaggregation Machines.

    Science.gov (United States)

    Mogk, Axel; Bukau, Bernd; Kampinga, Harm H

    2018-01-18

    Both acute proteotoxic stresses that unfold proteins and expression of disease-causing mutant proteins that expose aggregation-prone regions can promote protein aggregation. Protein aggregates can interfere with cellular processes and deplete factors crucial for protein homeostasis. To cope with these challenges, cells are equipped with diverse folding and degradation activities to rescue or eliminate aggregated proteins. Here, we review the different chaperone disaggregation machines and their mechanisms of action. In all these machines, the coating of protein aggregates by Hsp70 chaperones represents the conserved, initializing step. In bacteria, fungi, and plants, Hsp70 recruits and activates Hsp100 disaggregases to extract aggregated proteins. In the cytosol of metazoa, Hsp70 is empowered by a specific cast of J-protein and Hsp110 co-chaperones allowing for standalone disaggregation activity. Both types of disaggregation machines are supported by small Hsps that sequester misfolded proteins. Copyright © 2018 Elsevier Inc. All rights reserved.

  20. Cellular Chaperones As Therapeutic Targets in ALS to Restore Protein Homeostasis and Improve Cellular Function

    Directory of Open Access Journals (Sweden)

    Bernadett Kalmar

    2017-09-01

    Full Text Available Heat shock proteins (Hsps are ubiquitously expressed chaperone proteins that enable cells to cope with environmental stresses that cause misfolding and denaturation of proteins. With aging this protein quality control machinery becomes less effective, reducing the ability of cells to cope with damaging environmental stresses and disease-causing mutations. In neurodegenerative disorders such as Amyotrophic Lateral Sclerosis (ALS, such mutations are known to result in protein misfolding, which in turn results in the formation of intracellular aggregates cellular dysfunction and eventual neuronal death. The exact cellular pathology of ALS and other neurodegenerative diseases has been elusive and thus, hindering the development of effective therapies. However, a common scheme has emerged across these “protein misfolding” disorders, in that the mechanism of disease involves one or more aspects of proteostasis; from DNA transcription, RNA translation, to protein folding, transport and degradation via proteosomal and autophagic pathways. Interestingly, members of the Hsp family are involved in each of these steps facilitating normal protein folding, regulating the rate of protein synthesis and degradation. In this short review we summarize the evidence that suggests that ALS is a disease of protein dyshomeostasis in which Hsps may play a key role. Overwhelming evidence now indicates that enabling protein homeostasis to cope with disease-causing mutations might be a successful therapeutic strategy in ALS, as well as other neurodegenerative diseases. Novel small molecule co-inducers of Hsps appear to be able to achieve this aim. Arimoclomol, a hydroxylamine derivative, has shown promising results in cellular and animal models of ALS, as well as other protein misfolding diseases such as Inclusion Body Myositis (IBM. Initial clinical investigations of Arimoclomol have shown promising results. Therefore, it is possible that the long series of

  1. Peroxisome protein import: a complex journey.

    Science.gov (United States)

    Baker, Alison; Lanyon-Hogg, Thomas; Warriner, Stuart L

    2016-06-15

    The import of proteins into peroxisomes possesses many unusual features such as the ability to import folded proteins, and a surprising diversity of targeting signals with differing affinities that can be recognized by the same receptor. As understanding of the structure and function of many components of the protein import machinery has grown, an increasingly complex network of factors affecting each step of the import pathway has emerged. Structural studies have revealed the presence of additional interactions between cargo proteins and the PEX5 receptor that affect import potential, with a subtle network of cargo-induced conformational changes in PEX5 being involved in the import process. Biochemical studies have also indicated an interdependence of receptor-cargo import with release of unloaded receptor from the peroxisome. Here, we provide an update on recent literature concerning mechanisms of protein import into peroxisomes. © 2016 The Author(s).

  2. Protein import into isolated pea root leucoplasts

    OpenAIRE

    Chu, Chiung-Chih; Li, Hsou-min

    2015-01-01

    Leucoplasts are important organelles for the synthesis and storage of starch, lipids and proteins. However, molecular mechanism of protein import into leucoplasts and how it differs from that of import into chloroplasts remain unknown. We used pea seedlings for both chloroplast and leucoplast isolations to compare within the same species. We further optimized the isolation and import conditions to improve import efficiency and to permit a quantitative comparison between the two plastid types....

  3. STAT proteins: from normal control of cellular events to tumorigenesis.

    Science.gov (United States)

    Calò, Valentina; Migliavacca, Manuela; Bazan, Viviana; Macaluso, Marcella; Buscemi, Maria; Gebbia, Nicola; Russo, Antonio

    2003-11-01

    Signal transducers and activators of transcription (STAT) proteins comprise a family of transcription factors latent in the cytoplasm that participate in normal cellular events, such as differentiation, proliferation, cell survival, apoptosis, and angiogenesis following cytokine, growth factor, and hormone signaling. STATs are activated by tyrosine phosphorylation, which is normally a transient and tightly regulates process. Nevertheless, several constitutively activated STATs have been observed in a wide number of human cancer cell lines and primary tumors, including blood malignancies and solid neoplasias. STATs can be divided into two groups according to their specific functions. One is made up of STAT2, STAT4, and STAT6, which are activated by a small number of cytokines and play a distinct role in the development of T-cells and in IFNgamma signaling. The other group includes STAT1, STAT3, and STAT5, activated in different tissues by means of a series of ligands and involved in IFN signaling, development of the mammary gland, response to GH, and embriogenesis. This latter group of STATS plays an important role in controlling cell-cycle progression and apoptosis and thus contributes to oncogenesis. Although an increased expression of STAT1 has been observed in many human neoplasias, this molecule can be considered a potential tumor suppressor, since it plays an important role in growth arrest and in promoting apoptosis. On the other hand, STAT3 and 5 are considered as oncogenes, since they bring about the activation of cyclin D1, c-Myc, and bcl-xl expression, and are involved in promoting cell-cycle progression, cellular transformation, and in preventing apoptosis.

  4. Protein import into isolated pea root leucoplasts

    Directory of Open Access Journals (Sweden)

    Chiung-Chih eChu

    2015-09-01

    Full Text Available Leucoplasts are important organelles for the synthesis and storage of starch, lipids and proteins. However, molecular mechanism of protein import into leucoplasts and how it differs from that of import into chloroplasts remain unknown. We used pea seedlings for both chloroplast and leucoplast isolations to compare within the same species. We further optimized the isolation and import conditions to improve import efficiency and to permit a quantitative comparison between the two plastid types. The authenticity of the import was verified using a mitochondrial precursor protein. Our results show that, when normalized to Toc75, most translocon proteins are less abundant in leucoplasts than in chloroplasts. A precursor shown to prefer the receptor Toc132 indeed had relatively more similar import efficiencies between chloroplasts and leucoplasts compared to precursors that preferred Toc159. Furthermore we found two precursors that exhibited very high import efficiency into leucoplasts. Their transit peptides may be candidates for delivering transgenic proteins into leucoplasts and for analyzing motifs important for leucoplast import.

  5. Multidrug and toxin extrusion proteins mediate cellular transport of cadmium

    International Nuclear Information System (INIS)

    Yang, Hong; Guo, Dong; Obianom, Obinna N.; Su, Tong; Polli, James E.; Shu, Yan

    2017-01-01

    Cadmium (Cd) is an environmentally prevalent toxicant posing increasing risk to human health worldwide. As compared to the extensive research in Cd tissue accumulation, little was known about the elimination of Cd, particularly its toxic form, Cd ion (Cd 2+ ). In this study, we aimed to examine whether Cd 2+ is a substrate of multidrug and toxin extrusion proteins (MATEs) that are important in renal xenobiotic elimination. HEK-293 cells overexpressing the human MATE1 (HEK-hMATE1), human MATE2-K (HEK-hMATE2-K) and mouse Mate1 (HEK-mMate1) were used to study the cellular transport and toxicity of Cd 2+ . The cells overexpressing MATEs showed a 2–4 fold increase of Cd 2+ uptake that could be blocked by the MATE inhibitor cimetidine. A saturable transport profile was observed with the Michaelis-Menten constant (K m ) of 130 ± 15.8 μM for HEK-hMATE1; 139 ± 21.3 μM for HEK-hMATE2-K; and 88.7 ± 13.5 μM for HEK-mMate1, respectively. Cd 2+ could inhibit the uptake of metformin, a substrate of MATE transporters, with the half maximal inhibitory concentration (IC 50 ) of 97.5 ± 6.0 μM, 20.2 ± 2.6 μM, and 49.9 ± 6.9 μM in HEK-hMATE1, HEK-hMATE2-K, and HEK-mMate1 cells, respectively. In addition, hMATE1 could transport preloaded Cd 2+ out of the HEK-hMATE1 cells, thus resulting in a significant decrease of Cd 2+ -induced cytotoxicity. The present study has provided the first evidence supporting that MATEs transport Cd 2+ and may function as cellular elimination machinery in Cd intoxication. - Highlights: • Cadmium is an environmentally prevalent toxicant. • Little was known regarding the elimination and detoxification of cadmium. • Cadmium ion is here demonstrated as a substrate of MATE transporters. • MATEs may function as cellular elimination machinery in cadmium detoxification.

  6. Multidrug and toxin extrusion proteins mediate cellular transport of cadmium

    Energy Technology Data Exchange (ETDEWEB)

    Yang, Hong; Guo, Dong; Obianom, Obinna N. [Department of Pharmaceutical Sciences, School of Pharmacy, University of Maryland at Baltimore, MD (United States); Su, Tong [Department of Oral Maxillofacial Surgery, the First Affiliated Hospital, Xiangya Medical School, Central South University, Hunan 410007 (China); Polli, James E. [Department of Pharmaceutical Sciences, School of Pharmacy, University of Maryland at Baltimore, MD (United States); Shu, Yan, E-mail: yshu@rx.umaryland.edu [Department of Pharmaceutical Sciences, School of Pharmacy, University of Maryland at Baltimore, MD (United States)

    2017-01-01

    Cadmium (Cd) is an environmentally prevalent toxicant posing increasing risk to human health worldwide. As compared to the extensive research in Cd tissue accumulation, little was known about the elimination of Cd, particularly its toxic form, Cd ion (Cd{sup 2+}). In this study, we aimed to examine whether Cd{sup 2+} is a substrate of multidrug and toxin extrusion proteins (MATEs) that are important in renal xenobiotic elimination. HEK-293 cells overexpressing the human MATE1 (HEK-hMATE1), human MATE2-K (HEK-hMATE2-K) and mouse Mate1 (HEK-mMate1) were used to study the cellular transport and toxicity of Cd{sup 2+}. The cells overexpressing MATEs showed a 2–4 fold increase of Cd{sup 2+} uptake that could be blocked by the MATE inhibitor cimetidine. A saturable transport profile was observed with the Michaelis-Menten constant (K{sub m}) of 130 ± 15.8 μM for HEK-hMATE1; 139 ± 21.3 μM for HEK-hMATE2-K; and 88.7 ± 13.5 μM for HEK-mMate1, respectively. Cd{sup 2+} could inhibit the uptake of metformin, a substrate of MATE transporters, with the half maximal inhibitory concentration (IC{sub 50}) of 97.5 ± 6.0 μM, 20.2 ± 2.6 μM, and 49.9 ± 6.9 μM in HEK-hMATE1, HEK-hMATE2-K, and HEK-mMate1 cells, respectively. In addition, hMATE1 could transport preloaded Cd{sup 2+} out of the HEK-hMATE1 cells, thus resulting in a significant decrease of Cd{sup 2+}-induced cytotoxicity. The present study has provided the first evidence supporting that MATEs transport Cd{sup 2+} and may function as cellular elimination machinery in Cd intoxication. - Highlights: • Cadmium is an environmentally prevalent toxicant. • Little was known regarding the elimination and detoxification of cadmium. • Cadmium ion is here demonstrated as a substrate of MATE transporters. • MATEs may function as cellular elimination machinery in cadmium detoxification.

  7. Cellular reprogramming through mitogen-activated protein kinases

    Directory of Open Access Journals (Sweden)

    Justin eLee

    2015-10-01

    Full Text Available Mitogen-activated protein kinase (MAPK cascades are conserved eukaryote signaling modules where MAPKs, as the final kinases in the cascade, phosphorylate protein substrates to regulate cellular processes. While some progress in the identification of MAPK substrates has been made in plants, the knowledge on the spectrum of substrates and their mechanistic action is still fragmentary. In this focused review, we discuss the biological implications of the data in our original paper (Sustained mitogen-activated protein kinase activation reprograms defense metabolism and phosphoprotein profile in Arabidopsis thaliana; Frontiers in Plant Science 5: 554 in the context of related research. In our work, we mimicked in vivo activation of two stress-activated MAPKs, MPK3 and MPK6, through transgenic manipulation of Arabidopsis thaliana and used phosphoproteomics analysis to identify potential novel MAPK substrates. Here, we plotted the identified putative MAPK substrates (and downstream phosphoproteins as a global protein clustering network. Based on a highly stringent selection confidence level, the core networks highlighted a MAPK-induced cellular reprogramming at multiple levels of gene and protein expression – including transcriptional, post-transcriptional, translational, post-translational (such as protein modification, folding and degradation steps, and also protein re-compartmentalization. Additionally, the increase in putative substrates/phosphoproteins of energy metabolism and various secondary metabolite biosynthesis pathways coincides with the observed accumulation of defense antimicrobial substances as detected by metabolome analysis. Furthermore, detection of protein networks in phospholipid or redox elements suggests activation of downstream signaling events. Taken in context with other studies, MAPKs are key regulators that reprogram cellular events to orchestrate defense signaling in eukaryotes.

  8. Acylation of cellular proteins with endogenously synthesized fatty acids

    International Nuclear Information System (INIS)

    Towler, D.; Glaser, L.

    1986-01-01

    A number of cellular proteins contain covalently bound fatty acids. Previous studies have identified myristic acid and palmitic acid covalently linked to protein, the former usually attached to proteins by an amide linkage and the latter by ester or thio ester linkages. While in a few instances specific proteins have been isolated from cells and their fatty acid composition has been determined, the most frequent approach to the identification of protein-linked fatty acids is to biosynthetically label proteins with fatty acids added to intact cells. This procedure introduces possible bias in that only a selected fraction of proteins may be labeled, and it is not known whether the radioactive fatty acid linked to the protein is identical with that which is attached to the protein when the fatty acid is derived from endogenous sources. We have examined the distribution of protein-bound fatty acid following labeling with [ 3 H]acetate, a general precursor of all fatty acids, using BC 3 H1 cells (a mouse muscle cell line) and A431 cells (a human epidermoid carcinoma). Myristate, palmitate, and stearate account for essentially all of the fatty acids linked to protein following labeling with [ 3 H]acetate, but at least 30% of the protein-bound palmitate in these cells was present in amide linkage. In BC3H1 cells, exogenous palmitate becomes covalently bound to protein such that less than 10% of the fatty acid is present in amide linkage. These data are compatible with multiple protein acylating activities specific for acceptor protein fatty acid chain length and linkage

  9. Insights into the physiological function of cellular prion protein

    Directory of Open Access Journals (Sweden)

    Martins V.R.

    2001-01-01

    Full Text Available Prions have been extensively studied since they represent a new class of infectious agents in which a protein, PrPsc (prion scrapie, appears to be the sole component of the infectious particle. They are responsible for transmissible spongiform encephalopathies, which affect both humans and animals. The mechanism of disease propagation is well understood and involves the interaction of PrPsc with its cellular isoform (PrPc and subsequently abnormal structural conversion of the latter. PrPc is a glycoprotein anchored on the cell surface by a glycosylphosphatidylinositol moiety and expressed in most cell types but mainly in neurons. Prion diseases have been associated with the accumulation of the abnormally folded protein and its neurotoxic effects; however, it is not known if PrPc loss of function is an important component. New efforts are addressing this question and trying to characterize the physiological function of PrPc. At least four different mouse strains in which the PrP gene was ablated were generated and the results regarding their phenotype are controversial. Localization of PrPc on the cell membrane makes it a potential candidate for a ligand uptake, cell adhesion and recognition molecule or a membrane signaling molecule. Recent data have shown a potential role for PrPc in the metabolism of copper and moreover that this metal stimulates PrPc endocytosis. Our group has recently demonstrated that PrPc is a high affinity laminin ligand and that this interaction mediates neuronal cell adhesion and neurite extension and maintenance. Moreover, PrPc-caveolin-1 dependent coupling seems to trigger the tyrosine kinase Fyn activation. These data provide the first evidence for PrPc involvement in signal transduction.

  10. Cellular oxido-reductive proteins of Chlamydomonas reinhardtii control the biosynthesis of silver nanoparticles

    Directory of Open Access Journals (Sweden)

    Barwal Indu

    2011-12-01

    Full Text Available Abstract Background Elucidation of molecular mechanism of silver nanoparticles (SNPs biosynthesis is important to control its size, shape and monodispersity. The evaluation of molecular mechanism of biosynthesis of SNPs is of prime importance for the commercialization and methodology development for controlling the shape and size (uniform distribution of SNPs. The unicellular algae Chlamydomonas reinhardtii was exploited as a model system to elucidate the role of cellular proteins in SNPs biosynthesis. Results The C. reinhardtii cell free extract (in vitro and in vivo cells mediated synthesis of silver nanoparticles reveals SNPs of size range 5 ± 1 to 15 ± 2 nm and 5 ± 1 to 35 ± 5 nm respectively. In vivo biosynthesized SNPs were localized in the peripheral cytoplasm and at one side of flagella root, the site of pathway of ATP transport and its synthesis related enzymes. This provides an evidence for the involvement of oxidoreductive proteins in biosynthesis and stabilization of SNPs. Alteration in size distribution and decrease of synthesis rate of SNPs in protein-depleted fractions confirmed the involvement of cellular proteins in SNPs biosynthesis. Spectroscopic and SDS-PAGE analysis indicate the association of various proteins on C. reinhardtii mediated in vivo and in vitro biosynthesized SNPs. We have identified various cellular proteins associated with biosynthesized (in vivo and in vitro SNPs by using MALDI-MS-MS, like ATP synthase, superoxide dismutase, carbonic anhydrase, ferredoxin-NADP+ reductase, histone etc. However, these proteins were not associated on the incubation of pre-synthesized silver nanoparticles in vitro. Conclusion Present study provides the indication of involvement of molecular machinery and various cellular proteins in the biosynthesis of silver nanoparticles. In this report, the study is mainly focused towards understanding the role of diverse cellular protein in the synthesis and capping of silver

  11. Generic framework for mining cellular automata models on protein-folding simulations.

    Science.gov (United States)

    Diaz, N; Tischer, I

    2016-05-13

    Cellular automata model identification is an important way of building simplified simulation models. In this study, we describe a generic architectural framework to ease the development process of new metaheuristic-based algorithms for cellular automata model identification in protein-folding trajectories. Our framework was developed by a methodology based on design patterns that allow an improved experience for new algorithms development. The usefulness of the proposed framework is demonstrated by the implementation of four algorithms, able to obtain extremely precise cellular automata models of the protein-folding process with a protein contact map representation. Dynamic rules obtained by the proposed approach are discussed, and future use for the new tool is outlined.

  12. Direct Cellular Lysis/Protein Extraction Protocol for Soil Metaproteomics

    Energy Technology Data Exchange (ETDEWEB)

    Chourey, Karuna [ORNL; Jansson, Janet [Lawrence Berkeley National Laboratory (LBNL); Verberkmoes, Nathan C [ORNL; Shah, Manesh B [ORNL; Chavarria, Krystle L. [Lawrence Berkeley National Laboratory (LBNL); Tom, Lauren M [Lawrence Berkeley National Laboratory (LBNL); Brodie, Eoin L. [Lawrence Berkeley National Laboratory (LBNL); Hettich, Robert {Bob} L [ORNL

    2010-01-01

    We present a novel direct protocol for deep proteome characterization of microorganisms in soil. The method employs thermally assisted detergent-based cellular lysis (SDS) of soil samples, followed by TCA precipitation for proteome extraction/cleanup prior to liquid chromatography-mass spectrometric characterization. This approach was developed and optimized using different soils inoculated with genome-sequenced bacteria (Gram-negative Pseudomonas putida or Gram-positive Arthrobacter chlorophenolicus). Direct soil protein extraction was compared to protein extraction from cells isolated from the soil matrix prior to lysis (indirect method). Each approach resulted in identification of greater than 500 unique proteins, with a wide range in molecular mass and functional categories. To our knowledge, this SDS-TCA approach enables the deepest proteome characterizations of microbes in soil to date, without significant biases in protein size, localization, or functional category compared to pure cultures. This protocol should provide a powerful tool for ecological studies of soil microbial communities.

  13. Structural classification of proteins using texture descriptors extracted from the cellular automata image.

    Science.gov (United States)

    Kavianpour, Hamidreza; Vasighi, Mahdi

    2017-02-01

    Nowadays, having knowledge about cellular attributes of proteins has an important role in pharmacy, medical science and molecular biology. These attributes are closely correlated with the function and three-dimensional structure of proteins. Knowledge of protein structural class is used by various methods for better understanding the protein functionality and folding patterns. Computational methods and intelligence systems can have an important role in performing structural classification of proteins. Most of protein sequences are saved in databanks as characters and strings and a numerical representation is essential for applying machine learning methods. In this work, a binary representation of protein sequences is introduced based on reduced amino acids alphabets according to surrounding hydrophobicity index. Many important features which are hidden in these long binary sequences can be clearly displayed through their cellular automata images. The extracted features from these images are used to build a classification model by support vector machine. Comparing to previous studies on the several benchmark datasets, the promising classification rates obtained by tenfold cross-validation imply that the current approach can help in revealing some inherent features deeply hidden in protein sequences and improve the quality of predicting protein structural class.

  14. Cellular adaptation as an important response during chemical carcinogenesis

    International Nuclear Information System (INIS)

    Farber, E.

    1992-01-01

    Since disease processes are largely expressions of how living organisms react and respond to perturbations in the external and internal environments, adaptive or protective responses and their modulations and mechanisms are of the greatest concern in fundamental studies of disease pathogenesis. Such considerations are also of the greatest relevance in toxicology, including how living organisms respond to low levels of single and multiple xenobiotics and radiations. As the steps and mechanisms during cancer development are studied in greater depth, phenomena become apparent that suggest that adaptive reactions and responses may play important or even critical roles in the process of carcinogenesis. The question becomes whether the process of carcinogenesis is fundamentally an adversarial one (i.e., an abnormal cell in a vulnerable host), or is it more in the nature of a physiological selection or differentiation, which has survival value for the host as an adaptive phenomena? The very early initial interactions of mutagenic chemical carcinogens, radiations and viruses with DNA prejudice most to consider the adversarial 'abnormal' view as the appropriate one. Yet, the unusually common nature of the earliest altered rare cells that appear during carcinogenesis, their unusually bland nature, and their spontaneous differentiation to normal-appearing adult liver should be carefully considered

  15. Mitochondrial Protein Synthesis, Import, and Assembly

    Science.gov (United States)

    Fox, Thomas D.

    2012-01-01

    The mitochondrion is arguably the most complex organelle in the budding yeast cell cytoplasm. It is essential for viability as well as respiratory growth. Its innermost aqueous compartment, the matrix, is bounded by the highly structured inner membrane, which in turn is bounded by the intermembrane space and the outer membrane. Approximately 1000 proteins are present in these organelles, of which eight major constituents are coded and synthesized in the matrix. The import of mitochondrial proteins synthesized in the cytoplasm, and their direction to the correct soluble compartments, correct membranes, and correct membrane surfaces/topologies, involves multiple pathways and macromolecular machines. The targeting of some, but not all, cytoplasmically synthesized mitochondrial proteins begins with translation of messenger RNAs localized to the organelle. Most proteins then pass through the translocase of the outer membrane to the intermembrane space, where divergent pathways sort them to the outer membrane, inner membrane, and matrix or trap them in the intermembrane space. Roughly 25% of mitochondrial proteins participate in maintenance or expression of the organellar genome at the inner surface of the inner membrane, providing 7 membrane proteins whose synthesis nucleates the assembly of three respiratory complexes. PMID:23212899

  16. Protein carbonylation and metal-catalyzed protein oxidation in a cellular perspective

    DEFF Research Database (Denmark)

    Møller, Ian Max; Rogowska-Wrzesinska, Adelina; Rao, R S P

    2011-01-01

    Proteins can become oxidatively modified in many different ways, either by direct oxidation of amino acid side chains and protein backbone or indirectly by conjugation with oxidation products of polyunsaturated fatty acids and carbohydrates. While reversible oxidative modifications are thought...... to be relevant in physiological processes, irreversible oxidative modifications are known to contribute to cellular damage and disease. The most well-studied irreversible protein oxidation is carbonylation. In this work we first examine how protein carbonylation occurs via metal-catalyzed oxidation (MCO) in vivo...... and in vitro with an emphasis on cellular metal ion homeostasis and metal binding. We then review proteomic methods currently used for identifying carbonylated proteins and their sites of modification. Finally, we discuss the identified carbonylated proteins and the pattern of carbonylation sites in relation...

  17. Efficient cellular solid-state NMR of membrane proteins by targeted protein labeling

    Energy Technology Data Exchange (ETDEWEB)

    Baker, Lindsay A. [University of Oxford, Oxford Particle Imaging Centre, The Wellcome Trust Centre for Human Genetics, Division of Structural Biology, Nuffield Department of Medicine (United Kingdom); Daniëls, Mark; Cruijsen, Elwin A. W. van der; Folkers, Gert E.; Baldus, Marc, E-mail: m.baldus@uu.nl [Utrecht University, NMR Spectroscopy, Department of Chemistry, Faculty of Science, Bijvoet Center for Biomolecular Research (Netherlands)

    2015-06-15

    Solid-state NMR spectroscopy (ssNMR) has made significant progress towards the study of membrane proteins in their native cellular membranes. However, reduced spectroscopic sensitivity and high background signal levels can complicate these experiments. Here, we describe a method for ssNMR to specifically label a single protein by repressing endogenous protein expression with rifampicin. Our results demonstrate that treatment of E. coli with rifampicin during induction of recombinant membrane protein expression reduces background signals for different expression levels and improves sensitivity in cellular membrane samples. Further, the method reduces the amount of time and resources needed to produce membrane protein samples, enabling new strategies for studying challenging membrane proteins by ssNMR.

  18. Efficient cellular solid-state NMR of membrane proteins by targeted protein labeling

    International Nuclear Information System (INIS)

    Baker, Lindsay A.; Daniëls, Mark; Cruijsen, Elwin A. W. van der; Folkers, Gert E.; Baldus, Marc

    2015-01-01

    Solid-state NMR spectroscopy (ssNMR) has made significant progress towards the study of membrane proteins in their native cellular membranes. However, reduced spectroscopic sensitivity and high background signal levels can complicate these experiments. Here, we describe a method for ssNMR to specifically label a single protein by repressing endogenous protein expression with rifampicin. Our results demonstrate that treatment of E. coli with rifampicin during induction of recombinant membrane protein expression reduces background signals for different expression levels and improves sensitivity in cellular membrane samples. Further, the method reduces the amount of time and resources needed to produce membrane protein samples, enabling new strategies for studying challenging membrane proteins by ssNMR

  19. The Role of the Multifunctional BAG3 Protein in Cellular Protein Quality Control and in Disease.

    Science.gov (United States)

    Stürner, Elisabeth; Behl, Christian

    2017-01-01

    In neurons, but also in all other cells the complex proteostasis network is monitored and tightly regulated by the cellular protein quality control (PQC) system. Beyond folding of newly synthesized polypeptides and their refolding upon misfolding the PQC also manages the disposal of aberrant proteins either by the ubiquitin-proteasome machinery or by the autophagic-lysosomal system. Aggregated proteins are primarily degraded by a process termed selective macroautophagy (or aggrephagy). One such recently discovered selective macroautophagy pathway is mediated by the multifunctional HSP70 co-chaperone BAG3 ( BCL-2-associated athanogene 3 ). Under acute stress and during cellular aging, BAG3 in concert with the molecular chaperones HSP70 and HSPB8 as well as the ubiquitin receptor p62/SQSTM1 specifically targets aggregation-prone proteins to autophagic degradation. Thereby, BAG3-mediated selective macroautophagy represents a pivotal adaptive safeguarding and emergency system of the PQC which is activated under pathophysiological conditions to ensure cellular proteostasis. Interestingly, BAG3-mediated selective macroautophagy is also involved in the clearance of aggregated proteins associated with age-related neurodegenerative disorders, like Alzheimer's disease (tau-protein), Huntington's disease (mutated huntingtin/polyQ proteins), and amyotrophic lateral sclerosis (mutated SOD1). In addition, based on its initial description BAG3 is an anti-apoptotic protein that plays a decisive role in other widespread diseases, including cancer and myopathies. Therefore, in the search for novel therapeutic intervention avenues in neurodegeneration, myopathies and cancer BAG3 is a promising candidate.

  20. Stimulation of Cellular Proliferation by Hepatitis B Virus X Protein

    Directory of Open Access Journals (Sweden)

    Charles R. Madden

    2001-01-01

    Full Text Available Chronic infection with the hepatitis B virus (HBV is a known risk factor in the development of human hepatocellular carcinoma (HCC. The HBV-encoded X protein, HBx, has been investigated for properties that may explain its cancer cofactor role in transgenic mouse lines. We discuss here recent data showing that HBx is able to induce hepatocellular proliferation in vitro and in vivo. This property of HBx is predicted to sensitize hepatocytes to other HCC cofactors, including exposure to carcinogens and to other hepatitis viruses. Cellular proliferation is intimately linked to the mechanism(s by which most tumor-associated viruses transform virus-infected cells. The HBx alteration of the cell cycle provides an additional mechanism by which chronic HBV infection may contribute to HCC.

  1. Integrating protein structures and precomputed genealogies in the Magnum database: Examples with cellular retinoid binding proteins

    Directory of Open Access Journals (Sweden)

    Bradley Michael E

    2006-02-01

    Full Text Available Abstract Background When accurate models for the divergent evolution of protein sequences are integrated with complementary biological information, such as folded protein structures, analyses of the combined data often lead to new hypotheses about molecular physiology. This represents an excellent example of how bioinformatics can be used to guide experimental research. However, progress in this direction has been slowed by the lack of a publicly available resource suitable for general use. Results The precomputed Magnum database offers a solution to this problem for ca. 1,800 full-length protein families with at least one crystal structure. The Magnum deliverables include 1 multiple sequence alignments, 2 mapping of alignment sites to crystal structure sites, 3 phylogenetic trees, 4 inferred ancestral sequences at internal tree nodes, and 5 amino acid replacements along tree branches. Comprehensive evaluations revealed that the automated procedures used to construct Magnum produced accurate models of how proteins divergently evolve, or genealogies, and correctly integrated these with the structural data. To demonstrate Magnum's capabilities, we asked for amino acid replacements requiring three nucleotide substitutions, located at internal protein structure sites, and occurring on short phylogenetic tree branches. In the cellular retinoid binding protein family a site that potentially modulates ligand binding affinity was discovered. Recruitment of cellular retinol binding protein to function as a lens crystallin in the diurnal gecko afforded another opportunity to showcase the predictive value of a browsable database containing branch replacement patterns integrated with protein structures. Conclusion We integrated two areas of protein science, evolution and structure, on a large scale and created a precomputed database, known as Magnum, which is the first freely available resource of its kind. Magnum provides evolutionary and structural

  2. Nuclear import and export signals are essential for proper cellular trafficking and function of ZIC3.

    Science.gov (United States)

    Bedard, James E J; Purnell, Jennifer D; Ware, Stephanie M

    2007-01-15

    Missense, frameshift and nonsense mutations in the zinc finger transcription factor ZIC3 cause heterotaxy as well as isolated congenital heart disease. Previously, we developed transactivation and subcellular localization assays to test the function of ZIC3 point mutations. Aberrant cytoplasmic localization suggested that the pathogenesis of ZIC3 mutations results, at least in part, from failure of appropriate cellular trafficking. To further investigate this hypothesis, the nucleocytoplasmic shuttling properties of ZIC3 have been examined. Subcellular localization assays designed to span the entire open-reading frame of wild-type and mutant ZIC3 proteins identified the presence of nucleocytoplasmic transport signals. ZIC3 domain mapping indicates that a relatively large region containing the zinc finger binding sites and a known GLI interacting domain is required for transport to the nucleus. Site-directed mutagenesis of critical residues within two putative nuclear localization signals (NLSs) leads to loss of nuclear localization. No further decrease was observed when both NLS sites were mutated, suggesting that mutation of either NLS site is sufficient for loss of importin-mediated nuclear localization. Additionally, we identify a cryptic CRM-1-dependent nuclear export signal (NES) within ZIC3, and identify a mutation within this region in a patient with heterotaxy. These results provide the first evidence that control of cellular trafficking of ZIC3 is critical for function and suggest a possible mechanism for transcriptional control during left-right patterning. Identification of mutations in mapped NLS or NES domains in heterotaxy patients demonstrates the functional importance of these domains in cardiac morphogenesis and allows for integration of structural analysis with developmental function.

  3. Vitamin D binding protein: a multifunctional protein of clinical importance.

    Science.gov (United States)

    Speeckaert, Marijn M; Speeckaert, Reinhart; van Geel, Nanja; Delanghe, Joris R

    2014-01-01

    Since the discovery of group-specific component and its polymorphism by Hirschfeld in 1959, research has put spotlight on this multifunctional transport protein (vitamin D binding protein, DBP). Besides the transport of vitamin D metabolites, DBP is a plasma glycoprotein with many important functions, including sequestration of actin, modulation of immune and inflammatory responses, binding of fatty acids, and control of bone development. A considerable DBP polymorphism has been described with a specific allele distribution in different geographic area. Multiple studies have shed light on the interesting relationship between polymorphisms of the DBP gene and the susceptibility to diseases. In this review, we give an overview of the multifunctional character of DBP and describe the clinical importance of DBP and its polymorphisms. Finally, we discuss the possibilities to use DBP as a novel therapeutic agent.

  4. Cell-Specific Establishment of Poliovirus Resistance to an Inhibitor Targeting a Cellular Protein

    Science.gov (United States)

    Viktorova, Ekaterina G.; Nchoutmboube, Jules; Ford-Siltz, Lauren A.

    2015-01-01

    ABSTRACT It is hypothesized that targeting stable cellular factors involved in viral replication instead of virus-specific proteins may raise the barrier for development of resistant mutants, which is especially important for highly adaptable small (+)RNA viruses. However, contrary to this assumption, the accumulated evidence shows that these viruses easily generate mutants resistant to the inhibitors of cellular proteins at least in some systems. We investigated here the development of poliovirus resistance to brefeldin A (BFA), an inhibitor of the cellular protein GBF1, a guanine nucleotide exchange factor for the small cellular GTPase Arf1. We found that while resistant viruses can be easily selected in HeLa cells, they do not emerge in Vero cells, in spite that in the absence of the drug both cultures support robust virus replication. Our data show that the viral replication is much more resilient to BFA than functioning of the cellular secretory pathway, suggesting that the role of GBF1 in the viral replication is independent of its Arf activating function. We demonstrate that the level of recruitment of GBF1 to the replication complexes limits the establishment and expression of a BFA resistance phenotype in both HeLa and Vero cells. Moreover, the BFA resistance phenotype of poliovirus mutants is also cell type dependent in different cells of human origin and results in a fitness loss in the form of reduced efficiency of RNA replication in the absence of the drug. Thus, a rational approach to the development of host-targeting antivirals may overcome the superior adaptability of (+)RNA viruses. IMPORTANCE Compared to the number of viral diseases, the number of available vaccines is miniscule. For some viruses vaccine development has not been successful after multiple attempts, and for many others vaccination is not a viable option. Antiviral drugs are needed for clinical practice and public health emergencies. However, viruses are highly adaptable and can

  5. Protein-protein interactions within the ensemble, eukaryotic V-ATPase, and its concerted interactions with cellular machineries.

    Science.gov (United States)

    Balakrishna, Asha Manikkoth; Manimekalai, Malathy Sony Subramanian; Grüber, Gerhard

    2015-10-01

    The V1VO-ATPase (V-ATPase) is the important proton-pump in eukaryotic cells, responsible for pH-homeostasis, pH-sensing and amino acid sensing, and therefore essential for cell growths and metabolism. ATP-cleavage in the catalytic A3B3-hexamer of V1 has to be communicated via several so-called central and peripheral stalk units to the proton-pumping VO-part, which is membrane-embedded. A unique feature of V1VO-ATPase regulation is its reversible disassembly of the V1 and VO domain. Actin provides a network to hold the V1 in proximity to the VO, enabling effective V1VO-assembly to occur. Besides binding to actin, the 14-subunit V-ATPase interacts with multi-subunit machineries to form cellular sensors, which regulate the pH in cellular compartments or amino acid signaling in lysosomes. Here we describe a variety of subunit-subunit interactions within the V-ATPase enzyme during catalysis and its protein-protein assembling with key cellular machineries, essential for cellular function. Copyright © 2015 Elsevier Ltd. All rights reserved.

  6. The Role of the Multifunctional BAG3 Protein in Cellular Protein Quality Control and in Disease

    Directory of Open Access Journals (Sweden)

    Elisabeth Stürner

    2017-06-01

    Full Text Available In neurons, but also in all other cells the complex proteostasis network is monitored and tightly regulated by the cellular protein quality control (PQC system. Beyond folding of newly synthesized polypeptides and their refolding upon misfolding the PQC also manages the disposal of aberrant proteins either by the ubiquitin-proteasome machinery or by the autophagic-lysosomal system. Aggregated proteins are primarily degraded by a process termed selective macroautophagy (or aggrephagy. One such recently discovered selective macroautophagy pathway is mediated by the multifunctional HSP70 co-chaperone BAG3 (BCL-2-associated athanogene 3. Under acute stress and during cellular aging, BAG3 in concert with the molecular chaperones HSP70 and HSPB8 as well as the ubiquitin receptor p62/SQSTM1 specifically targets aggregation-prone proteins to autophagic degradation. Thereby, BAG3-mediated selective macroautophagy represents a pivotal adaptive safeguarding and emergency system of the PQC which is activated under pathophysiological conditions to ensure cellular proteostasis. Interestingly, BAG3-mediated selective macroautophagy is also involved in the clearance of aggregated proteins associated with age-related neurodegenerative disorders, like Alzheimer’s disease (tau-protein, Huntington’s disease (mutated huntingtin/polyQ proteins, and amyotrophic lateral sclerosis (mutated SOD1. In addition, based on its initial description BAG3 is an anti-apoptotic protein that plays a decisive role in other widespread diseases, including cancer and myopathies. Therefore, in the search for novel therapeutic intervention avenues in neurodegeneration, myopathies and cancer BAG3 is a promising candidate.

  7. Prion search and cellular prion protein expression in stranded dolphins.

    Science.gov (United States)

    Di Guardo, G; Cocumelli, C; Meoli, R; Barbaro, K; Terracciano, G; Di Francesco, C E; Mazzariol, S; Eleni, C

    2012-01-01

    The recent description of a prion disease (PD) case in a free-ranging bottlenose dolphin (Tursiops truncatus) prompted us to carry out an extensive search for the disease-associated isoform (PrPSc) of the cellular prion protein (PrPC) in the brain and in a range of lymphoid tissues from 23 striped dolphins (Stenella coeruleoalba), 5 bottlenose dolphins and 2 Risso s dolphins (Grampus griseus) found stranded between 2007 and 2012 along the Italian coastline. Three striped dolphins and one bottlenose dolphin showed microscopic lesions of encephalitis, with no evidence of spongiform brain lesions being detected in any of the 30 free-ranging cetaceans investigated herein. Nevertheless, we could still observe a prominent PrPC immunoreactivity in the brain as well as in lymphoid tissues from these dolphins. Although immunohistochemical and Western blot investigations yielded negative results for PrPSc deposition in all tissues from the dolphins under study, the reported occurrence of a spontaneous PD case in a wild dolphin is an intriguing issue and a matter of concern for both prion biology and intra/inter-species transmissibility, as well as for cetacean conservation medicine.

  8. Dimer monomer transition and dimer re-formation play important role for ATM cellular function during DNA repair

    International Nuclear Information System (INIS)

    Du, Fengxia; Zhang, Minjie; Li, Xiaohua; Yang, Caiyun; Meng, Hao; Wang, Dong; Chang, Shuang; Xu, Ye; Price, Brendan; Sun, Yingli

    2014-01-01

    Highlights: • ATM phosphorylates the opposite strand of the dimer in response to DNA damage. • The PETPVFRLT box of ATM plays a key role in its dimer dissociation in DNA repair. • The dephosphorylation of ATM is critical for dimer re-formation after DNA repair. - Abstract: The ATM protein kinase, is a serine/threonine protein kinase that is recruited and activated by DNA double-strand breaks, mediates responses to ionizing radiation in mammalian cells. Here we show that ATM is held inactive in unirradiated cells as a dimer and phosphorylates the opposite strand of the dimer in response to DNA damage. Cellular irradiation induces rapid intermolecular autophosphorylation of serine 1981 that causes dimer dissociation and initiates cellular ATM kinase activity. ATM cannot phosphorylate the substrates when it could not undergo dimer monomer transition. After DNA repair, the active monomer will undergo dephosphorylation to form dimer again and dephosphorylation is critical for dimer re-formation. Our work reveals novel function of ATM dimer monomer transition and explains why ATM dimer monomer transition plays such important role for ATM cellular activity during DNA repair

  9. Ethanol cellular defense induce unfolded protein response in yeast

    Directory of Open Access Journals (Sweden)

    Elisabet eNavarro-Tapia

    2016-02-01

    Full Text Available Ethanol is a valuable industrial product and a common metabolite used by many cell types. However, this molecule produces high levels of cytotoxicity affecting cellular performance at several levels. In the presence of ethanol, cells must adjust some of their components, such as the membrane lipids to maintain homeostasis. In the case of microorganism as Saccharomyces cerevisiae, ethanol is one of the principal products of their metabolism and is the main stress factor during fermentation. Although many efforts have been made, mechanisms of ethanol tolerance are not fully understood and very little evidence is available to date for specific signaling by ethanol in the cell. This work studied two Saccharomyces cerevisiae strains, CECT10094 and Temohaya-MI26, isolated from flor wine and agave fermentation (a traditional fermentation from Mexico respectively, which differ in ethanol tolerance, in order to understand the molecular mechanisms underlying the ethanol stress response and the reasons for different ethanol tolerance. The transcriptome was analyzed after ethanol stress and, among others, an increased activation of genes related with the unfolded protein response (UPR and its transcription factor, Hac1p, was observed in the tolerant strain CECT10094. We observed that this strain also resist more UPR agents than Temohaya-MI26 and the UPR-ethanol stress correlation was corroborated observing growth of 15 more strains and discarding UPR correlation with other stresses as thermal or oxidative stress. Furthermore, higher activation of UPR pathway in the tolerant strain CECT10094 was observed using a UPR mCherry reporter. Finally, we observed UPR activation in response to ethanol stress in other S. cerevisiae ethanol tolerant strains as the wine strains T73 and EC1118. This work demonstrates that the UPR pathway is activated under ethanol stress occurring in a standard fermentation and links this response to an enhanced ethanol tolerance. Thus

  10. Keeping the LINC: the importance of nucleocytoskeletal coupling in intracellular force transmission and cellular function.

    Science.gov (United States)

    Lombardi, Maria L; Lammerding, Jan

    2011-12-01

    Providing a stable physical connection between the nucleus and the cytoskeleton is essential for a wide range of cellular functions and it could also participate in mechanosensing by transmitting intra- and extra-cellular mechanical stimuli via the cytoskeleton to the nucleus. Nesprins and SUN proteins, located at the nuclear envelope, form the LINC (linker of nucleoskeleton and cytoskeleton) complex that connects the nucleus to the cytoskeleton; underlying nuclear lamins contribute to anchoring LINC complex components at the nuclear envelope. Disruption of the LINC complex or loss of lamins can result in disturbed perinuclear actin and intermediate filament networks and causes severe functional defects, including impaired nuclear positioning, cell polarization and cell motility. Recent studies have identified the LINC complex as the major force-transmitting element at the nuclear envelope and suggest that many of the aforementioned defects can be attributed to disturbed force transmission between the nucleus and the cytoskeleton. Thus mutations in nesprins, SUN proteins or lamins, which have been linked to muscular dystrophies and cardiomyopathies, may weaken or completely eliminate LINC complex function at the nuclear envelope and result in impaired intracellular force transmission, thereby disrupting critical cellular functions.

  11. Depletion of cellular poly (A) binding protein prevents protein synthesis and leads to apoptosis in HeLa cells

    Energy Technology Data Exchange (ETDEWEB)

    Thangima Zannat, Mst.; Bhattacharjee, Rumpa B. [Department of Molecular and Cellular Biology, University of Guelph, Guelph, Ontario, Canada N1G2W1 (Canada); Bag, Jnanankur, E-mail: jbag@uoguelph.ca [Department of Molecular and Cellular Biology, University of Guelph, Guelph, Ontario, Canada N1G2W1 (Canada)

    2011-05-13

    Highlights: {yields} Depletion of cellular PABP level arrests mRNA translation in HeLa cells. {yields} PABP knock down leads to apoptotic cell death. {yields} PABP depletion does not affect transcription. {yields} PABP depletion does not lead to nuclear accumulation of mRNA. -- Abstract: The cytoplasmic poly (A) binding protein (PABP) is important in mRNA translation and stability. In yeast, depletion of PABP leads to translation arrest. Similarly, the PABP gene in Drosophila is important for proper development. It is however uncertain, whether mammalian PABP is essential for mRNA translation. Here we showed the effect of PABP depletion on mRNA metabolism in HeLa cells by using a small interfering RNA. Our results suggest that depletion of PABP prevents protein synthesis and consequently leads to cell death through apoptosis. Interestingly, no detectable effect of PABP depletion on transcription, transport and stability of mRNA was observed.

  12. Depletion of cellular poly (A) binding protein prevents protein synthesis and leads to apoptosis in HeLa cells

    International Nuclear Information System (INIS)

    Thangima Zannat, Mst.; Bhattacharjee, Rumpa B.; Bag, Jnanankur

    2011-01-01

    Highlights: → Depletion of cellular PABP level arrests mRNA translation in HeLa cells. → PABP knock down leads to apoptotic cell death. → PABP depletion does not affect transcription. → PABP depletion does not lead to nuclear accumulation of mRNA. -- Abstract: The cytoplasmic poly (A) binding protein (PABP) is important in mRNA translation and stability. In yeast, depletion of PABP leads to translation arrest. Similarly, the PABP gene in Drosophila is important for proper development. It is however uncertain, whether mammalian PABP is essential for mRNA translation. Here we showed the effect of PABP depletion on mRNA metabolism in HeLa cells by using a small interfering RNA. Our results suggest that depletion of PABP prevents protein synthesis and consequently leads to cell death through apoptosis. Interestingly, no detectable effect of PABP depletion on transcription, transport and stability of mRNA was observed.

  13. S100 Proteins As an Important Regulator of Macrophage Inflammation

    Directory of Open Access Journals (Sweden)

    Chang Xia

    2018-01-01

    Full Text Available The S100 proteins, a family of calcium-binding cytosolic proteins, have a broad range of intracellular and extracellular functions through regulating calcium balance, cell apoptosis, migration, proliferation, differentiation, energy metabolism, and inflammation. The intracellular functions of S100 proteins involve interaction with intracellular receptors, membrane protein recruitment/transportation, transcriptional regulation and integrating with enzymes or nucleic acids, and DNA repair. The S100 proteins could also be released from the cytoplasm, induced by tissue/cell damage and cellular stress. The extracellular S100 proteins, serving as a danger signal, are crucial in regulating immune homeostasis, post-traumatic injury, and inflammation. Extracellular S100 proteins are also considered biomarkers for some specific diseases. In this review, we will discuss the multi-functional roles of S100 proteins, especially their potential roles associated with cell migration, differentiation, tissue repair, and inflammation.

  14. Glis family proteins are differentially implicated in the cellular reprogramming of human somatic cells.

    Science.gov (United States)

    Lee, Seo-Young; Noh, Hye Bin; Kim, Hyeong-Taek; Lee, Kang-In; Hwang, Dong-Youn

    2017-09-29

    The ground-breaking discovery of the reprogramming of somatic cells into pluripotent cells, termed induced pluripotent stem cells (iPSCs), was accomplished by delivering 4 transcription factors, Oct4, Sox2, Klf4, and c-Myc, into fibroblasts. Since then, several efforts have attempted to unveil other factors that are directly implicated in or might enhance reprogramming. Importantly, a number of transcription factors are reported to retain reprogramming activity. A previous study suggested Gli-similar 1 (Glis1) as a factor that enhances the reprogramming of fibroblasts during iPSC generation. However, the implication of other Glis members, including Glis2 and Glis3 (variants 1 and 2), in cellular reprogramming remains unknown. In this study, we investigated the potential involvement of human Glis family proteins, including hGlis1-3, in cellular reprogramming. Our results demonstrate that hGlis1, which is reported to reprogram human fibroblasts, promotes the reprogramming of human adipose-derived stromal cells (hADSCs), indicating that the reprogramming activity of Glis1 is not cell type-specific. Strikingly, hGlis3 promoted the reprogramming of hADSCs as efficiently as hGlis1. On the contrary, hGlis2 showed a strong negative effect on reprogramming. Together, our results reveal clear differences in the cellular reprogramming activity among Glis family members and provide valuable insight into the development of a new reprogramming strategy using Glis family proteins.

  15. Cellular and functional specificity among ferritin-like proteins in the multicellular cyanobacterium Nostoc punctiforme.

    Science.gov (United States)

    Ekman, Martin; Sandh, Gustaf; Nenninger, Anja; Oliveira, Paulo; Stensjö, Karin

    2014-03-01

    Ferritin-like proteins constitute a remarkably heterogeneous protein family, including ferritins, bacterioferritins and Dps proteins. The genome of the filamentous heterocyst-forming cyanobacterium Nostoc punctiforme encodes five ferritin-like proteins. In the present paper, we report a multidimensional characterization of these proteins. Our phylogenetic and bioinformatics analyses suggest both structural and physiological differences among the ferritin-like proteins. The expression of these five genes responded differently to hydrogen peroxide treatment, with a significantly higher rise in transcript level for Npun_F3730 as compared with the other four genes. A specific role for Npun_F3730 in the cells tolerance against hydrogen peroxide was also supported by the inactivation of Npun_F3730, Npun_R5701 and Npun_R6212; among these, only the ΔNpun_F3730 strain showed an increased sensitivity to hydrogen peroxide compared with wild type. Analysis of promoter-GFP reporter fusions of the ferritin-like genes indicated that Npun_F3730 and Npun_R5701 were expressed in all cell types of a diazotrophic culture, while Npun_F6212 was expressed specifically in heterocysts. Our study provides the first comprehensive analysis combining functional differentiation and cellular specificity within this important group of proteins in a multicellular cyanobacterium. © 2013 John Wiley & Sons Ltd and Society for Applied Microbiology.

  16. The cell cycle regulator protein P16 and the cellular senescence of dental follicle cells.

    Science.gov (United States)

    Morsczeck, Christian; Hullmann, Markus; Reck, Anja; Reichert, Torsten E

    2018-02-01

    Cellular senescence is a restricting factor for regenerative therapies with somatic stem cells. We showed previously that the onset of cellular senescence inhibits the osteogenic differentiation in stem cells of the dental follicle (DFCs), although the mechanism remains elusive. Two different pathways are involved in the induction of the cellular senescence, which are driven either by the cell cycle protein P21 or by the cell cycle protein P16. In this study, we investigated the expression of cell cycle proteins in DFCs after the induction of cellular senescence. The induction of cellular senescence was proved by an increased expression of β-galactosidase and an increased population doubling time after a prolonged cell culture. Cellular senescence regulated the expression of cell cycle proteins. The expression of cell cycle protein P16 was up-regulated, which correlates with the induction of cellular senescence markers in DFCs. However, the expression of cyclin-dependent kinases (CDK)2 and 4 and the expression of the cell cycle protein P21 were successively decreased in DFCs. In conclusion, our data suggest that a P16-dependent pathway drives the induction of cellular senescence in DFCs.

  17. A new method of high-speed cellular protein separation and insight into subcellular compartmentalization of proteins.

    Science.gov (United States)

    Png, Evelyn; Lan, WanWen; Lazaroo, Melisa; Chen, Silin; Zhou, Lei; Tong, Louis

    2011-05-01

    Transglutaminase (TGM)-2 is a ubiquitous protein with important cellular functions such as regulation of cytoskeleton, cell adhesion, apoptosis, energy metabolism, and stress signaling. We identified several proteins that may interact with TGM-2 through a discovery-based proteomics method via pull down of flag-tagged TGM-2 peptide fragments. The distribution of these potential binding partners of TGM-2 was studied in subcellular fractions separated by density using novel high-speed centricollation technology. Centricollation is a compressed air-driven, low-temperature stepwise ultracentrifugation procedure where low extraction volumes can be processed in a relatively short time in non-denaturing separation conditions with high recovery yield. The fractions were characterized by immunoblots against known organelle markers. The changes in the concentrations of the binding partners were studied in cells expressing short hairpin RNA against TGM-2 (shTG). Desmin, mitochondrial intramembrane cleaving protease (PARL), protein tyrosine kinase (NTRK3), and serine protease (PRSS3) were found to be less concentrated in the 8.5%, 10%, 15%, and 20% sucrose fractions (SFs) from the lysate of shTG cells. The Golgi-associated protein (GOLGA2) was predominantly localized in 15% SF fraction, and in shTG, this shifted to predominantly in the 8.5% SF and showed larger aggregations in the cytosol of cells on immunofluorescent staining compared to control. Based on the relative concentrations of these proteins, we propose how trafficking of such proteins between cellular compartments can occur to regulate cell function. Centricollation is useful for elucidating biological function at the molecular level, especially when combined with traditional cell biology techniques.

  18. Import of peroxisomal matrix proteins in the yeast Hansenula polymorpha

    NARCIS (Netherlands)

    Gunkel, Katja

    2005-01-01

    Archaea, prokaryotes and eukaryotes form the three kingdoms of life. The smallest unit of life, which can exist independently, is a cell. Archaea and prokaryotes have a relatively very simple architecture. The cytoplasm (cellulars pace), containing all metabolites, proteins and genetic material

  19. Aspirin acetylates multiple cellular proteins in HCT-116 colon cancer cells: Identification of novel targets.

    Science.gov (United States)

    Marimuthu, Srinivasan; Chivukula, Raghavender S V; Alfonso, Lloyd F; Moridani, Majid; Hagen, Fred K; Bhat, G Jayarama

    2011-11-01

    Epidemiological and clinical observations provide consistent evidence that regular intake of aspirin may effectively inhibit the occurrence of epithelial tumors; however, the molecular mechanisms are not completely understood. In the present study, we determined the ability of aspirin to acetylate and post-translationally modify cellular proteins in HCT-116 human colon cancer cells to understand the potential mechanisms by which it may exerts anti-cancer effects. Using anti-acetyl lysine antibodies, here we demonstrate that aspirin causes the acetylation of multiple proteins whose molecular weight ranged from 20 to 200 kDa. The identity of these proteins was determined, using immuno-affinity purification, mass spectrometry and immuno-blotting. A total of 33 cellular proteins were potential targets of aspirin-mediated acetylation, while 16 were identified as common to both the control and aspirin-treated samples. These include enzymes of glycolytic pathway, cytoskeleton proteins, histones, ribosomal and mitochondrial proteins. The glycolytic enzymes which were identified include aldolase, glyceraldehyde-3-phosphate dehydrogenase, enolase, pyruvate kinase M2, and lactate dehydrogenase A and B chains. Immunoblotting experiment showed that aspirin also acetylated glucose-6-phosphate dehydrogenase and transketolase, both enzymes of pentose phosphate pathway involved in ribonucleotide biosynthesis. In vitro assays of these enzymes revealed that aspirin did not affect pyruvate kinase and lactate dehydrogenase activity; however, it decreased glucose 6 phosphate dehydrogenase activity. Similar results were also observed in HT-29 human colon cancer cells. Selective inhibition of glucose-6-phosphate dehydrogenase may represent an important mechanism by which aspirin may exert its anti-cancer effects through inhibition of ribonucleotide synthesis.

  20. Profiling cellular protein complexes by proximity ligation with dual tag microarray readout.

    Science.gov (United States)

    Hammond, Maria; Nong, Rachel Yuan; Ericsson, Olle; Pardali, Katerina; Landegren, Ulf

    2012-01-01

    Patterns of protein interactions provide important insights in basic biology, and their analysis plays an increasing role in drug development and diagnostics of disease. We have established a scalable technique to compare two biological samples for the levels of all pairwise interactions among a set of targeted protein molecules. The technique is a combination of the proximity ligation assay with readout via dual tag microarrays. In the proximity ligation assay protein identities are encoded as DNA sequences by attaching DNA oligonucleotides to antibodies directed against the proteins of interest. Upon binding by pairs of antibodies to proteins present in the same molecular complexes, ligation reactions give rise to reporter DNA molecules that contain the combined sequence information from the two DNA strands. The ligation reactions also serve to incorporate a sample barcode in the reporter molecules to allow for direct comparison between pairs of samples. The samples are evaluated using a dual tag microarray where information is decoded, revealing which pairs of tags that have become joined. As a proof-of-concept we demonstrate that this approach can be used to detect a set of five proteins and their pairwise interactions both in cellular lysates and in fixed tissue culture cells. This paper provides a general strategy to analyze the extent of any pairwise interactions in large sets of molecules by decoding reporter DNA strands that identify the interacting molecules.

  1. AMP-activated protein kinase reduces inflammatory responses and cellular senescence in pulmonary emphysema.

    Science.gov (United States)

    Cheng, Xiao-Yu; Li, Yang-Yang; Huang, Cheng; Li, Jun; Yao, Hong-Wei

    2017-04-04

    Current drug therapy fails to reduce lung destruction of chronic obstructive pulmonary disease (COPD). AMP-activated protein kinase (AMPK) has emerged as an important integrator of signals that control energy balance and lipid metabolism. However, there are no studies regarding the role of AMPK in reducing inflammatory responses and cellular senescence during the development of emphysema. Therefore, we hypothesize that AMPK reduces inflammatroy responses, senescence, and lung injury. To test this hypothesis, human bronchial epithelial cells (BEAS-2B) and small airway epithelial cells (SAECs) were treated with cigarette smoke extract (CSE) in the presence of a specific AMPK activator (AICAR, 1 mM) and inhibitor (Compound C, 5 μM). Elastase injection was performed to induce mouse emphysema, and these mice were treated with a specific AMPK activator metformin as well as Compound C. AICAR reduced, whereas Compound C increased CSE-induced increase in IL-8 and IL-6 release and expression of genes involved in cellular senescence. Knockdown of AMPKα1/α2 increased expression of pro-senescent genes (e.g., p16, p21, and p66shc) in BEAS-2B cells. Prophylactic administration of an AMPK activator metformin (50 and 250 mg/kg) reduced while Compound C (4 and 20 mg/kg) aggravated elastase-induced airspace enlargement, inflammatory responses and cellular senescence in mice. This is in agreement with therapeutic effect of metformin (50 mg/kg) on airspace enlargement. Furthermore, metformin prophylactically protected against but Compound C further reduced mitochondrial proteins SOD2 and SIRT3 in emphysematous lungs. In conclusion, AMPK reduces abnormal inflammatory responses and cellular senescence, which implicates as a potential therapeutic target for COPD/emphysema.

  2. Methods for the Analysis of Protein Phosphorylation-Mediated Cellular Signaling Networks

    Science.gov (United States)

    White, Forest M.; Wolf-Yadlin, Alejandro

    2016-06-01

    Protein phosphorylation-mediated cellular signaling networks regulate almost all aspects of cell biology, including the responses to cellular stimulation and environmental alterations. These networks are highly complex and comprise hundreds of proteins and potentially thousands of phosphorylation sites. Multiple analytical methods have been developed over the past several decades to identify proteins and protein phosphorylation sites regulating cellular signaling, and to quantify the dynamic response of these sites to different cellular stimulation. Here we provide an overview of these methods, including the fundamental principles governing each method, their relative strengths and weaknesses, and some examples of how each method has been applied to the analysis of complex signaling networks. When applied correctly, each of these techniques can provide insight into the topology, dynamics, and regulation of protein phosphorylation signaling networks.

  3. Protein Secondary Structures (α-helix and β-sheet) at a Cellular Level and Protein Fractions in Relation to Rumen Degradation Behaviours of Protein: A New Approach

    International Nuclear Information System (INIS)

    Yu, P.

    2007-01-01

    percentage of β-sheets (from 37.2% to 49.8%: S-FTIR absorption intensity) and reduced the α-helix to β-sheet ratio (from 0.3 to 0.7) in the golden flaxseeds, which indicated a negative effect of the roasting on protein values, utilisation and bioavailability. These results were proved by the Cornell Net Carbohydrate Protein System in situ animal trial, which also revealed that roasting increased the amount of protein bound to lignin, and well as of the Maillard reaction protein (both of which are poorly used by ruminants), and increased the level of indigestible and undegradable protein in ruminants. The present results demonstrate the potential of highly spatially resolved synchrotron-based infrared microspectroscopy to locate 'pure' protein in feed tissues, and reveal protein secondary structures and digestive behaviour, making a significant step forward in and an important contribution to protein nutritional research. Further study is needed to determine the sensitivities of protein secondary structures to various heat-processing conditions, and to quantify the relationship between protein secondary structures and the nutrient availability and digestive behaviour of various protein sources. Information from the present study arising from the synchrotron-based IR probing of the protein secondary structures of protein sources at the cellular level will be valuable as a guide to maintaining protein quality and predicting digestive behaviours

  4. Protein Secondary Structures (alpha-helix and beta-sheet) at a Cellular Levle and Protein Fractions in Relation to Rumen Degradation Behaviours of Protein: A New Approach

    Energy Technology Data Exchange (ETDEWEB)

    Yu,P.

    2007-01-01

    -FTIR absorption intensity), increased the percentage of {beta}-sheets (from 37.2% to 49.8%: S-FTIR absorption intensity) and reduced the {alpha}-helix to {beta}-sheet ratio (from 0.3 to 0.7) in the golden flaxseeds, which indicated a negative effect of the roasting on protein values, utilisation and bioavailability. These results were proved by the Cornell Net Carbohydrate Protein System in situ animal trial, which also revealed that roasting increased the amount of protein bound to lignin, and well as of the Maillard reaction protein (both of which are poorly used by ruminants), and increased the level of indigestible and undegradable protein in ruminants. The present results demonstrate the potential of highly spatially resolved synchrotron-based infrared microspectroscopy to locate 'pure' protein in feed tissues, and reveal protein secondary structures and digestive behaviour, making a significant step forward in and an important contribution to protein nutritional research. Further study is needed to determine the sensitivities of protein secondary structures to various heat-processing conditions, and to quantify the relationship between protein secondary structures and the nutrient availability and digestive behaviour of various protein sources. Information from the present study arising from the synchrotron-based IR probing of the protein secondary structures of protein sources at the cellular level will be valuable as a guide to maintaining protein quality and predicting digestive behaviours.

  5. Protein-lipid interactions: from membrane domains to cellular networks

    National Research Council Canada - National Science Library

    Tamm, Lukas K

    2005-01-01

    ... membranes is the lipid bilayer. Embedded in the fluid lipid bilayer are proteins of various shapes and traits. This volume illuminates from physical, chemical and biological angles the numerous - mostly quite weak - interactions between lipids, proteins, and proteins and lipids that define the delicate, highly dynamic and yet so stable fabri...

  6. Two Outer Membrane Proteins Contribute to Caulobacter crescentus Cellular Fitness by Preventing Intracellular S-Layer Protein Accumulation

    Energy Technology Data Exchange (ETDEWEB)

    Overton, K. Wesley; Park, Dan M.; Yung, Mimi C.; Dohnalkova, Alice; Smit, John; Jiao, Yongqin

    2016-09-23

    -type='genus-species'>C. crescentus.

    IMPORTANCEDecreased growth rate and reduced cell fitness are common side effects of protein production in overexpression systems. Inclusion bodies typically form inside the cell, largely due to a lack of sufficient export machinery to transport the overexpressed proteins to the extracellular environment. This phenomenon can conceivably also occur in natural systems. As one example of a system evolved to prevent intracellular protein accumulation, our study demonstrates thatCaulobacter crescentushas two homologous outer membrane transporter proteins that are involved in S-layer export. This is an interesting case study that demonstrates how bacteria can evolve redundancy to ensure adequate protein export functionality and maintain high cellular fitness. Moreover, we provide evidence that these two outer membrane proteins, although being the closestC. crescentushomologs to TolC inE. coli, do not process TolC functionality inC. crescentus.

  7. A sentinel protein assay for simultaneously quantifying cellular processes

    Czech Academy of Sciences Publication Activity Database

    Soste, M.; Hrabáková, Rita; Wanka, S.; Melnik, A.; Boersema, P.; Maiolica, A.; Wernas, T.; Tognetti, M.; von Mering, Ch.; Picotti, P.

    2014-01-01

    Roč. 11, č. 10 (2014), s. 1045-1048 ISSN 1548-7091 R&D Projects: GA MŠk ED2.1.00/03.0124 Institutional support: RVO:67985904 Keywords : targeted proteomics * selected reaction monitoring * cellular signaling Subject RIV: CE - Biochemistry Impact factor: 32.072, year: 2014

  8. Cellular prion protein expression is not regulated by the Alzheimer's amyloid precursor protein intracellular domain.

    Directory of Open Access Journals (Sweden)

    Victoria Lewis

    Full Text Available There is increasing evidence of molecular and cellular links between Alzheimer's disease (AD and prion diseases. The cellular prion protein, PrP(C, modulates the post-translational processing of the AD amyloid precursor protein (APP, through its inhibition of the β-secretase BACE1, and oligomers of amyloid-β bind to PrP(C which may mediate amyloid-β neurotoxicity. In addition, the APP intracellular domain (AICD, which acts as a transcriptional regulator, has been reported to control the expression of PrP(C. Through the use of transgenic mice, cell culture models and manipulation of APP expression and processing, this study aimed to clarify the role of AICD in regulating PrP(C. Over-expression of the three major isoforms of human APP (APP(695, APP(751 and APP(770 in cultured neuronal and non-neuronal cells had no effect on the level of endogenous PrP(C. Furthermore, analysis of brain tissue from transgenic mice over-expressing either wild type or familial AD associated mutant human APP revealed unaltered PrP(C levels. Knockdown of endogenous APP expression in cells by siRNA or inhibition of γ-secretase activity also had no effect on PrP(C levels. Overall, we did not detect any significant difference in the expression of PrP(C in any of the cell or animal-based paradigms considered, indicating that the control of cellular PrP(C levels by AICD is not as straightforward as previously suggested.

  9. Endocytic vesicle rupture is a conserved mechanism of cellular invasion by amyloid proteins.

    Science.gov (United States)

    Flavin, William P; Bousset, Luc; Green, Zachary C; Chu, Yaping; Skarpathiotis, Stratos; Chaney, Michael J; Kordower, Jeffrey H; Melki, Ronald; Campbell, Edward M

    2017-10-01

    Numerous pathological amyloid proteins spread from cell to cell during neurodegenerative disease, facilitating the propagation of cellular pathology and disease progression. Understanding the mechanism by which disease-associated amyloid protein assemblies enter target cells and induce cellular dysfunction is, therefore, key to understanding the progressive nature of such neurodegenerative diseases. In this study, we utilized an imaging-based assay to monitor the ability of disease-associated amyloid assemblies to rupture intracellular vesicles following endocytosis. We observe that the ability to induce vesicle rupture is a common feature of α-synuclein (α-syn) assemblies, as assemblies derived from WT or familial disease-associated mutant α-syn all exhibited the ability to induce vesicle rupture. Similarly, different conformational strains of WT α-syn assemblies, but not monomeric or oligomeric forms, efficiently induced vesicle rupture following endocytosis. The ability to induce vesicle rupture was not specific to α-syn, as amyloid assemblies of tau and huntingtin Exon1 with pathologic polyglutamine repeats also exhibited the ability to induce vesicle rupture. We also observe that vesicles ruptured by α-syn are positive for the autophagic marker LC3 and can accumulate and fuse into large, intracellular structures resembling Lewy bodies in vitro. Finally, we show that the same markers of vesicle rupture surround Lewy bodies in brain sections from PD patients. These data underscore the importance of this conserved endocytic vesicle rupture event as a damaging mechanism of cellular invasion by amyloid assemblies of multiple neurodegenerative disease-associated proteins, and suggest that proteinaceous inclusions such as Lewy bodies form as a consequence of continued fusion of autophagic vesicles in cells unable to degrade ruptured vesicles and their amyloid contents.

  10. An antiviral disulfide compound blocks interaction between arenavirus Z protein and cellular promyelocytic leukemia protein

    International Nuclear Information System (INIS)

    Garcia, C.C.; Topisirovic, I.; Djavani, M.; Borden, K.L.B.; Damonte, E.B.; Salvato, M.S.

    2010-01-01

    The promyelocytic leukemia protein (PML) forms nuclear bodies (NB) that can be redistributed by virus infection. In particular, lymphocytic choriomeningitis virus (LCMV) influences disruption of PML NB through the interaction of PML with the arenaviral Z protein. In a previous report, we have shown that the disulfide compound NSC20625 has antiviral and virucidal properties against arenaviruses, inducing unfolding and oligomerization of Z without affecting cellular RING-containing proteins such as the PML. Here, we further studied the effect of the zinc-finger-reactive disulfide NSC20625 on PML-Z interaction. In HepG2 cells infected with LCMV or transiently transfected with Z protein constructs, treatment with NSC20625 restored PML distribution from a diffuse-cytoplasmic pattern to punctate, discrete NB which appeared identical to NB found in control, uninfected cells. Similar results were obtained in cells transfected with a construct expressing a Z mutant in zinc-binding site 2 of the RING domain, confirming that this Z-PML interaction requires the integrity of only one zinc-binding site. Altogether, these results show that the compound NSC20625 suppressed Z-mediated PML NB disruption and may be used as a tool for designing novel antiviral strategies against arenavirus infection.

  11. Importance of bestrophin-4 protein for vasomotion

    DEFF Research Database (Denmark)

    Matchkov, Vladimir; Larsen, Per; Bouzinova, Elena V.

    2008-01-01

    We have recently characterized a cGMP-dependent Ca2+-activated Cl- current (ICl,cGMP) with unique characteristics in smooth muscle cells (SMCs) and suggested that this could be important for vasomotion. In SMCs this current co-exists with the "classical" Ca2+-activated Cl- current. We hypothesized......). In this study we tested the hypotheses that ICl,cGMP is mediated by bestrophin-4 and that bestrophin-4 is important for vasomotion. Bestrophin-4 was downregulated with small interference (siRNA) in A7r5 cells and in rat mesenteric small arteries in vivo. siRNAs targeting different exons of bestrophin-4 were...... that at least one bestrophin family member - bestrophin-4 - is essential for ICl,cGMP and for generation of vasomotion in rat mesenteric small arteries....

  12. N-terminal modifications of cellular proteins: The enzymes involved, their substrate specificities and biological effects

    Science.gov (United States)

    Varland, Sylvia; Osberg, Camilla; Arnesen, Thomas

    2015-01-01

    The vast majority of eukaryotic proteins are N-terminally modified by one or more processing enzymes. Enzymes acting on the very first amino acid of a polypeptide include different peptidases, transferases, and ligases. Methionine aminopeptidases excise the initiator methionine leaving the nascent polypeptide with a newly exposed amino acid that may be further modified. N-terminal acetyl-, methyl-, myristoyl-, and palmitoyltransferases may attach an acetyl, methyl, myristoyl, or palmitoyl group, respectively, to the α-amino group of the target protein N-terminus. With the action of ubiquitin ligases, one or several ubiquitin molecules are transferred, and hence, constitute the N-terminal modification. Modifications at protein N-termini represent an important contribution to proteomic diversity and complexity, and are essential for protein regulation and cellular signaling. Consequently, dysregulation of the N-terminal modifying enzymes is implicated in human diseases. We here review the different protein N-terminal modifications occurring co- or post-translationally with emphasis on the responsible enzymes and their substrate specificities. PMID:25914051

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

    Science.gov (United States)

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

    2015-01-01

    ABSTRACT Prion diseases involve the conversion of the endogenous cellular prion protein, PrPC, into a misfolded infectious isoform, PrPSc. Several functions have been attributed to PrPC, and its role has also been investigated in the olfactory system. PrPC is expressed in both the olfactory bulb (OB) and olfactory epithelium (OE) and the nasal cavity is an important route of transmission of diseases caused by prions. Moreover, Prnp−/− mice showed impaired behavior in olfactory tests. Given the high PrPC expression in OE and its putative role in olfaction, we screened a mouse OE cDNA library to identify novel PrPC-binding partners. Ten different putative PrPC ligands were identified, which were involved in functions such as cellular proliferation and apoptosis, cytoskeleton and vesicle transport, ubiquitination of proteins, stress response, and other physiological processes. In vitro binding assays confirmed the interaction of PrPC with STIP1 homology and U-Box containing protein 1 (Stub1) and are reported here for the first time. Stub1 is a co-chaperone with ubiquitin E3-ligase activity, which is associated with neurodegenerative diseases characterized by protein misfolding and aggregation. Physiological and pathological implications of PrPC-Stub1 interaction are under investigation. The PrPC-binding proteins identified here are not exclusive to the OE, suggesting that these interactions may occur in other tissues and play general biological roles. These data corroborate the proposal that PrPC is part of a multiprotein complex that modulates several cellular functions and provide a platform for further studies on the physiological and pathological roles of prion protein. PMID:26237451

  14. Membrane topology and cellular dynamics of foot-and-mouth disease virus 3A protein.

    Directory of Open Access Journals (Sweden)

    Mónica González-Magaldi

    Full Text Available Foot-and-mouth disease virus non-structural protein 3A plays important roles in virus replication, virulence and host-range; nevertheless little is known on the interactions that this protein can establish with different cell components. In this work, we have performed in vivo dynamic studies from cells transiently expressing the green fluorescent protein (GFP fused to the complete 3A (GFP3A and versions including different 3A mutations. The results revealed the presence of a mobile fraction of GFP3A, which was found increased in most of the mutants analyzed, and the location of 3A in a continuous compartment in the cytoplasm. A dual behavior was also observed for GFP3A upon cell fractionation, being the protein equally recovered from the cytosolic and membrane fractions, a ratio that was also observed when the insoluble fraction was further fractioned, even in the presence of detergent. Similar results were observed in the fractionation of GFP3ABBB, a 3A protein precursor required for initiating RNA replication. A nonintegral membrane protein topology of FMDV 3A was supported by the lack of glycosylation of versions of 3A in which each of the protein termini was fused to a glycosylation acceptor tag, as well as by their accessibility to degradation by proteases. According to this model 3A would interact with membranes through its central hydrophobic region exposing its N- and C- termini to the cytosol, where interactions between viral and cellular proteins required for virus replication are expected to occur.

  15. Transient Expression and Cellular Localization of Recombinant Proteins in Cultured Insect Cells.

    Science.gov (United States)

    Fabrick, Jeffrey A; Hull, J Joe

    2017-04-20

    Heterologous protein expression systems are used for the production of recombinant proteins, the interpretation of cellular trafficking/localization, and the determination of the biochemical function of proteins at the sub-organismal level. Although baculovirus expression systems are increasingly used for protein production in numerous biotechnological, pharmaceutical, and industrial applications, nonlytic systems that do not involve viral infection have clear benefits but are often overlooked and underutilized. Here, we describe a method for generating nonlytic expression vectors and transient recombinant protein expression. This protocol allows for the efficient cellular localization of recombinant proteins and can be used to rapidly discern protein trafficking within the cell. We show the expression of four recombinant proteins in a commercially available insect cell line, including two aquaporin proteins from the insect Bemisia tabaci, as well as subcellular marker proteins specific for the cell plasma membrane and for intracellular lysosomes. All recombinant proteins were produced as chimeras with fluorescent protein markers at their carboxyl termini, which allows for the direct detection of the recombinant proteins. The double transfection of cells with plasmids harboring constructs for the genes of interest and a known subcellular marker allows for live cell imaging and improved validation of cellular protein localization.

  16. Regulation of Cellular and Molecular Functions by Protein ...

    Indian Academy of Sciences (India)

    ... a high-energy linkage. The free energy of hydrolysis 1 of protein bound tyrosine phosphate ... protein kinases, cdc2 kinase (which regulates cell division cycle) and related cdc ... residues in response to extracellular signals such as hormones or growth factors. ... involved in regulating glycogen metabolism. The activity of.

  17. [Serological and cellular reactivity to mycobacterial proteins in Hansen's disease].

    Science.gov (United States)

    Rada, Elsa; Aranzazu, Nacarid; Rodríguez, Vestalia; Borges, Rafael; Convit, Jacinto

    2010-09-01

    The study was designed for evaluating immunological reactivity to various mycobacterial protein preparations using serological and cell-mediated immunological tests in patients with clinical leprosy signs, predominantly, with the multibacillary forms. All patients were adults with ages between 20 and 30 years. Fifty eight (n = 81) percent corresponded to Lepromatous Leprosy (LL), 29% (n = 41) to Borderline Lepromatous Leprosy (BL) and 10% (n = 41) to Borderline Borderline Leprosy (BB); only 3% were Borderline Tuberculoid (BT) patients: 74% males and 26% females. The most frequent reactional phenomenon was of the Erythema Nodosum (ENL) type. The mycobacterial proteins tested were: total crude Mycobacterium leprae antigens (MISA); Mycobacterium bovis (MbSA and excretion MbSA); partially purified excretion protein antigen, with a 30 kDa relative movility (Ml30); and recombinant M. leprae proteins (Mt70, Mb 65, Ml 36, 28, 18 and 10 kDa). Two of the recombinant proteins (Ml10 and Ml 36 kDa) presented a statiscally significant higher serological reactivity, directly related with a larger bacillary load (p = 0.0051 and 0.050 respectively). The 30 kDa protein was predominantly recognized by antibodies from multibacillary patients. Results show that mean antibody values were higher in non reactional patients when tested against complete proteins (MbSA and ex MbSA) when compared with the group of patients who presented reactional phenomena (p = 0.000567 and 0.000061, respectively). Comparing reactional with non reactional patients, it was seen that mean antibody values against complete proteins (MbSA and ex MbSA) were higher in non reactional individuals (p = 0.000567 and 0.000061, respectively). This same behavior occurred towards individual mycobacterial proteins (30, 10 and 36 kDa). The T lymphocyte prolypherative response in reactional and non reactional patients towards mycobacterial proteins (MlSA, Ml 10 kDa, MbSA, ex MbSA) was negative.

  18. Cellular localization of the Escherichia coli SpoT protein.

    OpenAIRE

    Gentry, D R; Cashel, M

    1995-01-01

    The SpoT protein of Escherichia coli serves as a source of degradation as well as an apparent source of synthesis of (p)ppGpp. Since the subcellular localization of SpoT might be a clue to its function, we have used SpoT-specific antisera to analyze cell extracts fractionated on sucrose gradients. We find that the SpoT protein is not bound to ribosomes or to either inner or outer membrane fractions. Although the SpoT protein is found in large aggregates, its localization is probably cytosolic.

  19. Megalin binds and mediates cellular internalization of folate binding protein

    DEFF Research Database (Denmark)

    Birn, Henrik; Zhai, Xiaoyue; Holm, Jan

    2005-01-01

    Folate is an essential vitamin involved in a number of biological processes. High affinity folate binding proteins (FBPs) exist both as glycosylphosphatidylinositol-linked, membrane associated folate binding proteins and as soluble FBPs in plasma and some secretory fluids such as milk, saliva...... to express high levels of megalin, is inhibitable by excess unlabeled FBP and by receptor associated protein, a known inhibitor of binding to megalin. Immortalized rat yolk sac cells, representing an established model for studying megalin-mediated uptake, reveal (125)I-labeled FBP uptake which is inhibited...

  20. Regulation of Cellular Redox Signaling by Matricellular Proteins in Vascular Biology, Immunology, and Cancer.

    Science.gov (United States)

    Roberts, David D; Kaur, Sukhbir; Isenberg, Jeffrey S

    2017-10-20

    In contrast to structural elements of the extracellular matrix, matricellular proteins appear transiently during development and injury responses, but their sustained expression can contribute to chronic disease. Through interactions with other matrix components and specific cell surface receptors, matricellular proteins regulate multiple signaling pathways, including those mediated by reactive oxygen and nitrogen species and H 2 S. Dysregulation of matricellular proteins contributes to the pathogenesis of vascular diseases and cancer. Defining the molecular mechanisms and receptors involved is revealing new therapeutic opportunities. Recent Advances: Thrombospondin-1 (TSP1) regulates NO, H 2 S, and superoxide production and signaling in several cell types. The TSP1 receptor CD47 plays a central role in inhibition of NO signaling, but other TSP1 receptors also modulate redox signaling. The matricellular protein CCN1 engages some of the same receptors to regulate redox signaling, and ADAMTS1 regulates NO signaling in Marfan syndrome. In addition to mediating matricellular protein signaling, redox signaling is emerging as an important pathway that controls the expression of several matricellular proteins. Redox signaling remains unexplored for many matricellular proteins. Their interactions with multiple cellular receptors remains an obstacle to defining signaling mechanisms, but improved transgenic models could overcome this barrier. Therapeutics targeting the TSP1 receptor CD47 may have beneficial effects for treating cardiovascular disease and cancer and have recently entered clinical trials. Biomarkers are needed to assess their effects on redox signaling in patients and to evaluate how these contribute to their therapeutic efficacy and potential side effects. Antioxid. Redox Signal. 27, 874-911.

  1. The Intracellular Destiny of the Protein Corona: A Study on its Cellular Internalization and Evolution.

    Science.gov (United States)

    Bertoli, Filippo; Garry, David; Monopoli, Marco P; Salvati, Anna; Dawson, Kenneth A

    2016-11-22

    It has been well established that the early stages of nanoparticle-cell interactions are governed, at least in part, by the layer of proteins and other biomolecules adsorbed and slowly exchanged with the surrounding biological media (biomolecular corona). Subsequent to membrane interactions, nanoparticles are typically internalized into the cell and trafficked along defined pathways such as, in many cases, the endolysosomal pathway. Indeed, if the original corona is partially retained on the nanoparticle surface, the biomolecules in this layer may play an important role in determining subsequent cellular processing. In this work, using a combination of organelle separation and fluorescence labeling of the initial extracellular corona, we clarify its intracellular evolution as nanoparticles travel within the cell. We show that specific proteins present in the original protein corona are retained on the nanoparticles until they accumulate in lysosomes, and, once there, they are degraded. We also report on how different bare surfaces (amino and carboxyl modified) affect the details of this evolution. One overarching discovery is that the same serum proteins can exhibit different intracellular processing when carried inside cells by nanoparticles, as components of their corona, compared to what is observed when they are transported freely from the extracellular medium.

  2. Determining the sub-cellular localization of proteins within Caenorhabditis elegans body wall muscle.

    Science.gov (United States)

    Meissner, Barbara; Rogalski, Teresa; Viveiros, Ryan; Warner, Adam; Plastino, Lorena; Lorch, Adam; Granger, Laure; Segalat, Laurent; Moerman, Donald G

    2011-01-01

    Determining the sub-cellular localization of a protein within a cell is often an essential step towards understanding its function. In Caenorhabditis elegans, the relatively large size of the body wall muscle cells and the exquisite organization of their sarcomeres offer an opportunity to identify the precise position of proteins within cell substructures. Our goal in this study is to generate a comprehensive "localizome" for C. elegans body wall muscle by GFP-tagging proteins expressed in muscle and determining their location within the cell. For this project, we focused on proteins that we know are expressed in muscle and are orthologs or at least homologs of human proteins. To date we have analyzed the expression of about 227 GFP-tagged proteins that show localized expression in the body wall muscle of this nematode (e.g. dense bodies, M-lines, myofilaments, mitochondria, cell membrane, nucleus or nucleolus). For most proteins analyzed in this study no prior data on sub-cellular localization was available. In addition to discrete sub-cellular localization we observe overlapping patterns of localization including the presence of a protein in the dense body and the nucleus, or the dense body and the M-lines. In total we discern more than 14 sub-cellular localization patterns within nematode body wall muscle. The localization of this large set of proteins within a muscle cell will serve as an invaluable resource in our investigation of muscle sarcomere assembly and function.

  3. Protein-protein interaction networks identify targets which rescue the MPP+ cellular model of Parkinson’s disease

    Science.gov (United States)

    Keane, Harriet; Ryan, Brent J.; Jackson, Brendan; Whitmore, Alan; Wade-Martins, Richard

    2015-11-01

    Neurodegenerative diseases are complex multifactorial disorders characterised by the interplay of many dysregulated physiological processes. As an exemplar, Parkinson’s disease (PD) involves multiple perturbed cellular functions, including mitochondrial dysfunction and autophagic dysregulation in preferentially-sensitive dopamine neurons, a selective pathophysiology recapitulated in vitro using the neurotoxin MPP+. Here we explore a network science approach for the selection of therapeutic protein targets in the cellular MPP+ model. We hypothesised that analysis of protein-protein interaction networks modelling MPP+ toxicity could identify proteins critical for mediating MPP+ toxicity. Analysis of protein-protein interaction networks constructed to model the interplay of mitochondrial dysfunction and autophagic dysregulation (key aspects of MPP+ toxicity) enabled us to identify four proteins predicted to be key for MPP+ toxicity (P62, GABARAP, GBRL1 and GBRL2). Combined, but not individual, knockdown of these proteins increased cellular susceptibility to MPP+ toxicity. Conversely, combined, but not individual, over-expression of the network targets provided rescue of MPP+ toxicity associated with the formation of autophagosome-like structures. We also found that modulation of two distinct proteins in the protein-protein interaction network was necessary and sufficient to mitigate neurotoxicity. Together, these findings validate our network science approach to multi-target identification in complex neurological diseases.

  4. Importance of carbon dioxide in the critical patient: Implications at the cellular and clinical levels.

    Science.gov (United States)

    Morales Quinteros, Luis; Bringué Roque, Josep; Kaufman, David; Artigas Raventós, Antonio

    2018-02-24

    Important recent insights have emerged regarding the cellular and molecular role of carbon dioxide (CO 2 ) and the effects of hypercapnia. The latter may have beneficial effects in patients with acute lung injury, affording reductions in pulmonary inflammation, lessened oxidative alveolar damage, and the regulation of innate immunity and host defenses by inhibiting the expression of inflammatory cytokines. However, other studies suggest that CO 2 can have deleterious effects upon the lung, reducing alveolar wound repair in lung injury, decreasing the rate of reabsorption of alveolar fluid, and inhibiting alveolar cell proliferation. Clearly, hypercapnia has both beneficial and harmful consequences, and it is important to determine the net effect under specific conditions. The purpose of this review is to describe the immunological and physiological effects of carbon dioxide, considering their potential consequences in patients with acute respiratory failure. Copyright © 2018 Elsevier España, S.L.U. y SEMICYUC. All rights reserved.

  5. Human Adenovirus Infection Causes Cellular E3 Ubiquitin Ligase MKRN1 Degradation Involving the Viral Core Protein pVII.

    Science.gov (United States)

    Inturi, Raviteja; Mun, Kwangchol; Singethan, Katrin; Schreiner, Sabrina; Punga, Tanel

    2018-02-01

    Human adenoviruses (HAdVs) are common human pathogens encoding a highly abundant histone-like core protein, VII, which is involved in nuclear delivery and protection of viral DNA as well as in sequestering immune danger signals in infected cells. The molecular details of how protein VII acts as a multifunctional protein have remained to a large extent enigmatic. Here we report the identification of several cellular proteins interacting with the precursor pVII protein. We show that the cellular E3 ubiquitin ligase MKRN1 is a novel precursor pVII-interacting protein in HAdV-C5-infected cells. Surprisingly, the endogenous MKRN1 protein underwent proteasomal degradation during the late phase of HAdV-C5 infection in various human cell lines. MKRN1 protein degradation occurred independently of the HAdV E1B55K and E4orf6 proteins. We provide experimental evidence that the precursor pVII protein binding enhances MKRN1 self-ubiquitination, whereas the processed mature VII protein is deficient in this function. Based on these data, we propose that the pVII protein binding promotes MKRN1 self-ubiquitination, followed by proteasomal degradation of the MKRN1 protein, in HAdV-C5-infected cells. In addition, we show that measles virus and vesicular stomatitis virus infections reduce the MKRN1 protein accumulation in the recipient cells. Taken together, our results expand the functional repertoire of the HAdV-C5 precursor pVII protein in lytic virus infection and highlight MKRN1 as a potential common target during different virus infections. IMPORTANCE Human adenoviruses (HAdVs) are common pathogens causing a wide range of diseases. To achieve pathogenicity, HAdVs have to counteract a variety of host cell antiviral defense systems, which would otherwise hamper virus replication. In this study, we show that the HAdV-C5 histone-like core protein pVII binds to and promotes self-ubiquitination of a cellular E3 ubiquitin ligase named MKRN1. This mutual interaction between the pVII and

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

    OpenAIRE

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

    2015-01-01

    Prion diseases involve the conversion of the endogenous cellular prion protein, PrPC, into a misfolded infectious isoform, PrPSc. Several functions have been attributed to PrPC, and its role has also been investigated in the olfactory system. PrPC is expressed in both the olfactory bulb (OB) and olfactory epithelium (OE) and the nasal cavity is an important route of transmission of diseases caused by prions. Moreover, Prnp−/− mice showed impaired behavior in olfactory tests. Given the high Pr...

  7. Screening of cellular proteins that interact with the classical swine ...

    Indian Academy of Sciences (India)

    In the current study, aiming to find more clues in understanding the molecular mechanisms of CSFV NS5A's function, the yeast two-hybrid (Y2H) system was adopted to screen for CSFV NS5A interactive proteins in the cDNA library of the swine umbilical vein endothelial cell (SUVEC). Alignment with the NCBI database ...

  8. Screening of cellular proteins that interact with the classical swine ...

    Indian Academy of Sciences (India)

    2014-01-27

    Jan 27, 2014 ... to screen for CSFV NS5A interactive proteins in the cDNA library of the swine umbilical vein endothelial cell. (SUVEC). Alignment ... development. The finding of ..... were unknown, the results of the BLAST against the human.

  9. The importance of the cellular stress response in the pathogenesis and treatment of type 2 diabetes.

    Science.gov (United States)

    Hooper, Philip L; Balogh, Gabor; Rivas, Eric; Kavanagh, Kylie; Vigh, Laszlo

    2014-07-01

    Organisms have evolved to survive rigorous environments and are not prepared to thrive in a world of caloric excess and sedentary behavior. A realization that physical exercise (or lack of it) plays a pivotal role in both the pathogenesis and therapy of type 2 diabetes mellitus (t2DM) has led to the provocative concept of therapeutic exercise mimetics. A decade ago, we attempted to simulate the beneficial effects of exercise by treating t2DM patients with 3 weeks of daily hyperthermia, induced by hot tub immersion. The short-term intervention had remarkable success, with a 1 % drop in HbA1, a trend toward weight loss, and improvement in diabetic neuropathic symptoms. An explanation for the beneficial effects of exercise and hyperthermia centers upon their ability to induce the cellular stress response (the heat shock response) and restore cellular homeostasis. Impaired stress response precedes major metabolic defects associated with t2DM and may be a near seminal event in the pathogenesis of the disease, tipping the balance from health into disease. Heat shock protein inducers share metabolic pathways associated with exercise with activation of AMPK, PGC1-a, and sirtuins. Diabetic therapies that induce the stress response, whether via heat, bioactive compounds, or genetic manipulation, improve or prevent all of the morbidities and comorbidities associated with the disease. The agents reduce insulin resistance, inflammatory cytokines, visceral adiposity, and body weight while increasing mitochondrial activity, normalizing membrane structure and lipid composition, and preserving organ function. Therapies restoring the stress response can re-tip the balance from disease into health and address the multifaceted defects associated with the disease.

  10. HSV-1 nucleocapsid egress mediated by UL31 in association with UL34 is impeded by cellular transmembrane protein 140

    Energy Technology Data Exchange (ETDEWEB)

    Guan, Ying [Department of Viral Immunology, Institute of Medical Biology, Chinese Academy of Medicine Science, Peking Union Medical College, Kunming 650118 (China); Yunnan Academy of Tobacco Science, Kunming, Yunnan 650106 (China); Guo, Lei; Yang, Erxia; Liao, Yun; Liu, Longding; Che, Yanchun; Zhang, Ying; Wang, Lichun; Wang, Jingjing [Department of Viral Immunology, Institute of Medical Biology, Chinese Academy of Medicine Science, Peking Union Medical College, Kunming 650118 (China); Li, Qihan, E-mail: imbcams.lq@gmail.com [Department of Viral Immunology, Institute of Medical Biology, Chinese Academy of Medicine Science, Peking Union Medical College, Kunming 650118 (China)

    2014-09-15

    During HSV-1 infection, the viral UL31 protein forms a complex with the UL34 protein at the cellular nuclear membrane, where both proteins play important roles in the envelopment of viral nucleocapsids and their egress into the cytoplasm. To characterize the mechanism of HSV-1 nucleocapsid egress, we screened host proteins to identify proteins that interacted with UL31 via yeast two-hybrid analysis. Transmembrane protein 140 (TMEM140), was identified and confirmed to bind to and co-localize with UL31 during viral infection. Further studies indicated that TMEM140 inhibits HSV-1 proliferation through selectively blocking viral nucleocapsid egress during the viral assembly process. The blockage function of TMEM140 is mediated by impeding the formation of the UL31–UL34 complex due to competitive binding to UL31. Collectively, these data suggest the essentiality of the UL31–UL34 interaction in the viral nucleocapsid egress process and provide a new anti-HSV-1 strategy in viral assembly process of nucleocapsid egress. - Highlights: • Cellular TMEM140 protein interacts with HSV-1 UL31 protein during viral infection. • Increasing expression of TMEM140 leads to inhibition of HSV-1 proliferation. • Increasing expression of TMEM140 blocks HSV-1 nucleocapsid egress process. • Binding to UL31 of TMEM140 impedes formation of HSV-1 UL31–UL34 complex.

  11. Cellular Reprogramming Using Protein and Cell-Penetrating Peptides

    Directory of Open Access Journals (Sweden)

    Bong Jong Seo

    2017-03-01

    Full Text Available Recently, stem cells have been suggested as invaluable tools for cell therapy because of their self-renewal and multilineage differentiation potential. Thus, scientists have developed a variety of methods to generate pluripotent stem cells, from nuclear transfer technology to direct reprogramming using defined factors, or induced pluripotent stem cells (iPSCs. Considering the ethical issues and efficiency, iPSCs are thought to be one of the most promising stem cells for cell therapy. Induced pluripotent stem cells can be generated by transduction with a virus, plasmid, RNA, or protein. Herein, we provide an overview of the current technology for iPSC generation and describe protein-based transduction technology in detail.

  12. Mechanism of protein import across the chloroplast envelope.

    Science.gov (United States)

    Chen, K; Chen, X; Schnell, D J

    2000-01-01

    The development and maintenance of chloroplasts relies on the contribution of protein subunits from both plastid and nuclear genomes. Most chloroplast proteins are encoded by nuclear genes and are post-translationally imported into the organelle across the double membrane of the chloroplast envelope. Protein import into the chloroplast consists of two essential elements: the specific recognition of the targeting signals (transit sequences) of cytoplasmic preproteins by receptors at the outer envelope membrane and the subsequent translocation of preproteins simultaneously across the double membrane of the envelope. These processes are mediated via the co-ordinate action of protein translocon complexes in the outer (Toc apparatus) and inner (Tic apparatus) envelope membranes.

  13. The PTEN protein: cellular localization and post-translational regulation.

    Science.gov (United States)

    Leslie, Nick R; Kriplani, Nisha; Hermida, Miguel A; Alvarez-Garcia, Virginia; Wise, Helen M

    2016-02-01

    The phosphatase and tensin homologue deleted on chromosome 10 (PTEN) phosphatase dephosphorylates PIP3, the lipid product of the class I PI 3-kinases, and suppresses the growth and proliferation of many cell types. It has been heavily studied, in large part due to its status as a tumour suppressor, the loss of function of which is observed through diverse mechanisms in many tumour types. Here we present a concise review of our understanding of the PTEN protein and highlight recent advances, particularly in our understanding of its localization and regulation by ubiquitination and SUMOylation. © 2016 Authors; published by Portland Press Limited.

  14. Intracellular Localization and Cellular Factors Interaction of HTLV-1 and HTLV-2 Tax Proteins: Similarities and Functional Differences

    Directory of Open Access Journals (Sweden)

    Maria Grazia Romanelli

    2011-05-01

    Full Text Available Human T-lymphotropic viruses type 1 (HTLV-1 and type 2 (HTLV-2 present very similar genomic structures but HTLV-1 is more pathogenic than HTLV-2. Is this difference due to their transactivating Tax proteins, Tax-1 and Tax-2, which are responsible for viral and cellular gene activation? Do Tax-1 and Tax-2 differ in their cellular localization and in their interaction pattern with cellular factors? In this review, we summarize Tax-1 and Tax-2 structural and phenotypic properties, their interaction with factors involved in signal transduction and their localization-related behavior within the cell. Special attention will be given to the distinctions between Tax-1 and Tax-2 that likely play an important role in their transactivation activity.

  15. The G-protein-coupled receptor, GPR84, is important for eye development in Xenopus laevis

    OpenAIRE

    Perry, Kimberly J.; Johnson, Verity R.; Malloch, Erica L.; Fukui, Lisa; Wever, Jason; Thomas, Alvin G.; Hamilton, Paul W.; Henry, Jonathan J.

    2010-01-01

    G-protein-coupled receptors (GPCRs) represent diverse, multifamily groups of cell signaling receptors involved in many cellular processes. We identified Xenopus laevis GPR84 as a member of the A18 subfamily of GPCRs. During development, GPR84 is detected in the embryonic lens placode, differentiating lens fiber cells, retina and cornea. Anti-sense morpholino oligonucleotide-mediated knockdown and RNA rescue experiments demonstrate GPR84’s importance in lens, cornea and retinal development. Ex...

  16. Protein Corona Analysis of Silver Nanoparticles Links to Their Cellular Effects.

    Science.gov (United States)

    Juling, Sabine; Niedzwiecka, Alicia; Böhmert, Linda; Lichtenstein, Dajana; Selve, Sören; Braeuning, Albert; Thünemann, Andreas F; Krause, Eberhard; Lampen, Alfonso

    2017-11-03

    The breadth of applications of nanoparticles and the access to food-associated consumer products containing nanosized materials lead to oral human exposure to such particles. In biological fluids nanoparticles dynamically interact with biomolecules and form a protein corona. Knowledge about the protein corona is of great interest for understanding the molecular effects of particles as well as their fate inside the human body. We used a mass spectrometry-based toxicoproteomics approach to elucidate mechanisms of toxicity of silver nanoparticles and to comprehensively characterize the protein corona formed around silver nanoparticles in Caco-2 human intestinal epithelial cells. Results were compared with respect to the cellular function of proteins either affected by exposure to nanoparticles or present in the protein corona. A transcriptomic data set was included in the analyses in order to obtain a combined multiomics view of nanoparticle-affected cellular processes. A relationship between corona proteins and the proteomic or transcriptomic responses was revealed, showing that differentially regulated proteins or transcripts were engaged in the same cellular signaling pathways. Protein corona analyses of nanoparticles in cells might therefore help in obtaining information about the molecular consequences of nanoparticle treatment.

  17. Adipocyte size and cellular expression of caveolar proteins analyzed by confocal microscopy

    DEFF Research Database (Denmark)

    Hulstrøm, Veronica; Prats Gavalda, Clara; Vinten, Jørgen

    2013-01-01

    Caveolae are abundant in adipocytes and are involved in the regulation of lipid accumulation, which is the main volume determinant of these cells. We have developed and applied a confocal microscopic technique for measuring individual cellular expression of the caveolar proteins cavin-1 and caveo......Caveolae are abundant in adipocytes and are involved in the regulation of lipid accumulation, which is the main volume determinant of these cells. We have developed and applied a confocal microscopic technique for measuring individual cellular expression of the caveolar proteins cavin-1...

  18. Rapid directed evolution of stabilized proteins with cellular high-throughput encapsulation solubilization and screening (CHESS).

    Science.gov (United States)

    Yong, K J; Scott, D J

    2015-03-01

    Directed evolution is a powerful method for engineering proteins towards user-defined goals and has been used to generate novel proteins for industrial processes, biological research and drug discovery. Typical directed evolution techniques include cellular display, phage display, ribosome display and water-in-oil compartmentalization, all of which physically link individual members of diverse gene libraries to their translated proteins. This allows the screening or selection for a desired protein function and subsequent isolation of the encoding gene from diverse populations. For biotechnological and industrial applications there is a need to engineer proteins that are functional under conditions that are not compatible with these techniques, such as high temperatures and harsh detergents. Cellular High-throughput Encapsulation Solubilization and Screening (CHESS), is a directed evolution method originally developed to engineer detergent-stable G proteins-coupled receptors (GPCRs) for structural biology. With CHESS, library-transformed bacterial cells are encapsulated in detergent-resistant polymers to form capsules, which serve to contain mutant genes and their encoded proteins upon detergent mediated solubilization of cell membranes. Populations of capsules can be screened like single cells to enable rapid isolation of genes encoding detergent-stable protein mutants. To demonstrate the general applicability of CHESS to other proteins, we have characterized the stability and permeability of CHESS microcapsules and employed CHESS to generate thermostable, sodium dodecyl sulfate (SDS) resistant green fluorescent protein (GFP) mutants, the first soluble proteins to be engineered using CHESS. © 2014 Wiley Periodicals, Inc.

  19. Solubilization of proteins: the importance of lysis buffer choice.

    Science.gov (United States)

    Peach, Mandy; Marsh, Noelle; Miskiewicz, Ewa I; MacPhee, Daniel J

    2015-01-01

    The efficient extraction of proteins of interest from cells and tissues is not always straightforward. Here we demonstrate the differences in extraction of the focal adhesion protein Kindlin-2 from choriocarcinoma cells using NP-40 and RIPA lysis buffer. Furthermore, we demonstrate the use of a more denaturing urea/thiourea lysis buffer for solubilization, by comparing its effectiveness for solubilization of small heat-shock proteins from smooth muscle with the often utilized RIPA lysis buffer. Overall, the results demonstrate the importance of establishing the optimal lysis buffer for specific protein solubilization within the experimental workflow.

  20. Systematic Characterisation of Cellular Localisation and Expression Profiles of Proteins Containing MHC Ligands

    DEFF Research Database (Denmark)

    Juncker, Agnieszka; Larsen, Mette Voldby; Weinhold, Nils

    2009-01-01

    Background: Presentation of peptides on Major Histocompatibility Complex (MHC) molecules is the cornerstone in immune system activation and increased knowledge of the characteristics of MHC ligands and their source proteins is highly desirable. Methodology/Principal Finding: In the present large......-scale study, we used a large data set of proteins containing experimentally identified MHC class I or II ligands and examined the proteins according to their expression profiles at the mRNA level and their Gene Ontology (GO) classification within the cellular component ontology. Proteins encoded by highly...

  1. Nickel decreases cellular iron level and converts cytosolic aconitase to iron-regulatory protein 1 in A549 cells

    International Nuclear Information System (INIS)

    Chen Haobin; Davidson, Todd; Singleton, Steven; Garrick, Michael D.; Costa, Max

    2005-01-01

    Nickel (Ni) compounds are well-established carcinogens and are known to initiate a hypoxic response in cells via the stabilization and transactivation of hypoxia-inducible factor-1 alpha (HIF-1α). This change may be the consequence of nickel's interference with the function of several Fe(II)-dependent enzymes. In this study, the effects of soluble nickel exposure on cellular iron homeostasis were investigated. Nickel treatment decreased both mitochondrial and cytosolic aconitase (c-aconitase) activity in A549 cells. Cytosolic aconitase was converted to iron-regulatory protein 1, a form critical for the regulation of cellular iron homeostasis. The increased activity of iron-regulatory protein 1 after nickel exposure stabilized and increased transferrin receptor (Tfr) mRNA and antagonized the iron-induced ferritin light chain protein synthesis. The decrease of aconitase activity after nickel treatment reflected neither direct interference with aconitase function nor obstruction of [4Fe-4S] cluster reconstitution by nickel. Exposure of A549 cells to soluble nickel decreased total cellular iron by about 40%, a decrease that likely caused the observed decrease in aconitase activity and the increase of iron-regulatory protein 1 activity. Iron treatment reversed the effect of nickel on cytosolic aconitase and iron-regulatory protein 1. To assess the mechanism for the observed effects, human embryonic kidney (HEK) cells over expressing divalent metal transporter-1 (DMT1) were compared to A549 cells expressing only endogenous transporters for inhibition of iron uptake by nickel. The inhibition data suggest that nickel can enter via DMT1 and compete with iron for entry into the cell. This disturbance of cellular iron homeostasis by nickel may have a great impact on the ability of the cell to regulate a variety of cell functions, as well as create a state of hypoxia in cells under normal oxygen tension. These effects may be very important in how nickel exerts phenotypic

  2. Contribution of cellular retinol-binding protein type 1 to retinol metabolism during mouse development.

    Science.gov (United States)

    Matt, Nicolas; Schmidt, Carsten K; Dupé, Valérie; Dennefeld, Christine; Nau, Heinz; Chambon, Pierre; Mark, Manuel; Ghyselinck, Norbert B

    2005-05-01

    Within cells, retinol (ROL) is bound to cytoplasmic proteins (cellular retinol-binding proteins [CRBPs]), whose proposed function is to protect it from unspecific enzymes through channeling to retinoid-metabolizing pathways. We show that, during development, ROL and retinyl ester levels are decreased in CRBP type 1 (CRBP1) -deficient embryos and fetuses by 50% and 80%, respectively. The steady state level of retinoic acid (RA) is also decreased but to a lesser extent. However, CRBP1-null fetuses do not exhibit the abnormalities characteristic of a vitamin A-deficiency syndrome. Neither CRBP1 deficiency alters the expression patterns of RA-responding genes during development, nor does CRBP1 availability modify the expression of an RA-dependent gene in primary embryonic fibroblasts treated with ROL. Therefore, CRBP1 is required in prenatal life to maintain normal amounts of ROL and to ensure its efficient storage but seems of secondary importance for RA synthesis, at least under conditions of maternal vitamin A sufficiency. Copyright 2005 Wiley-Liss, Inc.

  3. Hepatitis C virus NS2 protein activates cellular cyclic AMP-dependent pathways

    International Nuclear Information System (INIS)

    Kim, Kyoung Mi; Kwon, Shi-Nae; Kang, Ju-Il; Lee, Song Hee; Jang, Sung Key; Ahn, Byung-Yoon; Kim, Yoon Ki

    2007-01-01

    Chronic infection of the hepatitis C virus (HCV) leads to liver cirrhosis and cancer. The mechanism leading to viral persistence and hepatocellular carcinoma, however, has not been fully understood. In this study, we show that the HCV infection activates cellular cAMP-dependent pathways. Expression of a luciferase reporter gene controlled by a basic promoter with the cAMP response element (CRE) was significantly elevated in human hepatoma Huh-7 cells infected with the HCV JFH1. Analysis with viral subgenomic replicons indicated that the HCV NS2 protein is responsible for the effect. Furthermore, the level of cellular transcripts whose stability is known to be regulated by cAMP was specifically reduced in cells harboring NS2-expressing replicons. These results allude to the HCV NS2 protein having a novel function of regulating cellular gene expression and proliferation through the cAMP-dependent pathway

  4. Humoral and cellular immune responses to synthetic peptides of the Leishmania donovani kinetoplastid membrane protein-11

    DEFF Research Database (Denmark)

    Jensen, A T; Gasim, S; Ismail, A

    1998-01-01

    as solid-phase ligands in enzyme-linked immunosorbent assays (ELISAs) and as stimulating antigens in lymphoproliferative assays in order to evaluate humoral and cellular immune responses to well-defined sequences of the protein. Antibody reactivity against the three peptides was measured in plasma from 63...

  5. Cellular protein quality control and the evolution of aggregates in spinocerebellar ataxia type 3 (SCA3)

    NARCIS (Netherlands)

    Seidel, K.; Meister, M.; Dugbartey, G. J.; Zijlstra, M. P.; Vinet, J.; Brunt, E. R. P.; van Leeuwen, F. W.; Rueb, U.; Kampinga, H. H.; den Dunnen, W. F. A.

    2012-01-01

    K. Seidel, M. Meister, G. J. Dugbartey, M. P. Zijlstra, J. Vinet, E. R. P. Brunt, F. W. van Leeuwen, U. Rub, H. H. Kampinga and W. F. A. den Dunnen (2012) Neuropathology and Applied Neurobiology38, 548558 Cellular protein quality control and the evolution of aggregates in spinocerebellar ataxia type

  6. Resistance to degradation and cellular distribution are important features for the antitumor activity of gomesin.

    Directory of Open Access Journals (Sweden)

    Marcus V Buri

    Full Text Available Many reports have shown that antimicrobial peptides exhibit anticancer abilities. Gomesin (Gm exhibits potent cytotoxic activity against cancer cells by a membrane pore formation induced after well-orchestrated intracellular mechanisms. In this report, the replacements of the Cys by Ser or Thr, and the use D-amino acids in the Gm structure were done to investigate the importance of the resistance to degradation of the molecule with its cytotoxicity. [Thr(2,6,11,15]-Gm, and [Ser(2,6,11,15]-Gm exhibits low cytotoxicity, and low resistance to degradation, and after 24 h are present in localized area near to the membrane. Conversely, the use of D-amino acids in the analogue [D-Thr(2,6,11,15]-D-Gm confers resistance to degradation, increases its potency, and maintained this peptide spread in the cytosol similarly to what happens with Gm. Replacements of Cys by Thr and Gln by L- or D-Pro ([D-Thr(2,6,11,15, Pro(9]-D-Gm, and [Thr(2,6,11,15, D-Pro(9]-Gm, which induced a similar β-hairpin conformation, also increase their resistance to degradation, and cytotoxicity, but after 24 h they are not present spread in the cytosol, exhibiting lower cytotoxicity in comparison to Gm. Additionally, chloroquine, a lysosomal enzyme inhibitor potentiated the effect of the peptides. Furthermore, the binding and internalization of peptides was determined, but a direct correlation among these factors was not observed. However, cholesterol ablation, which increase fluidity of cellular membrane, also increase cytotoxicity and internalization of peptides. β-hairpin spatial conformation, and intracellular localization/target, and the capability of entry are important properties of gomesin cytotoxicity.

  7. Resistance to degradation and cellular distribution are important features for the antitumor activity of gomesin.

    Science.gov (United States)

    Buri, Marcus V; Domingues, Tatiana M; Paredes-Gamero, Edgar J; Casaes-Rodrigues, Rafael L; Rodrigues, Elaine Guadelupe; Miranda, Antonio

    2013-01-01

    Many reports have shown that antimicrobial peptides exhibit anticancer abilities. Gomesin (Gm) exhibits potent cytotoxic activity against cancer cells by a membrane pore formation induced after well-orchestrated intracellular mechanisms. In this report, the replacements of the Cys by Ser or Thr, and the use D-amino acids in the Gm structure were done to investigate the importance of the resistance to degradation of the molecule with its cytotoxicity. [Thr(2,6,11,15)]-Gm, and [Ser(2,6,11,15)]-Gm exhibits low cytotoxicity, and low resistance to degradation, and after 24 h are present in localized area near to the membrane. Conversely, the use of D-amino acids in the analogue [D-Thr(2,6,11,15)]-D-Gm confers resistance to degradation, increases its potency, and maintained this peptide spread in the cytosol similarly to what happens with Gm. Replacements of Cys by Thr and Gln by L- or D-Pro ([D-Thr(2,6,11,15), Pro(9)]-D-Gm, and [Thr(2,6,11,15), D-Pro(9)]-Gm), which induced a similar β-hairpin conformation, also increase their resistance to degradation, and cytotoxicity, but after 24 h they are not present spread in the cytosol, exhibiting lower cytotoxicity in comparison to Gm. Additionally, chloroquine, a lysosomal enzyme inhibitor potentiated the effect of the peptides. Furthermore, the binding and internalization of peptides was determined, but a direct correlation among these factors was not observed. However, cholesterol ablation, which increase fluidity of cellular membrane, also increase cytotoxicity and internalization of peptides. β-hairpin spatial conformation, and intracellular localization/target, and the capability of entry are important properties of gomesin cytotoxicity.

  8. Resistance to Degradation and Cellular Distribution are Important Features for the Antitumor Activity of Gomesin

    Science.gov (United States)

    Buri, Marcus V.; Domingues, Tatiana M.; Paredes-Gamero, Edgar J.; Casaes-Rodrigues, Rafael L.; Rodrigues, Elaine Guadelupe; Miranda, Antonio

    2013-01-01

    Many reports have shown that antimicrobial peptides exhibit anticancer abilities. Gomesin (Gm) exhibits potent cytotoxic activity against cancer cells by a membrane pore formation induced after well-orchestrated intracellular mechanisms. In this report, the replacements of the Cys by Ser or Thr, and the use D-amino acids in the Gm structure were done to investigate the importance of the resistance to degradation of the molecule with its cytotoxicity. [Thr2,6,11,15]-Gm, and [Ser2,6,11,15]-Gm exhibits low cytotoxicity, and low resistance to degradation, and after 24 h are present in localized area near to the membrane. Conversely, the use of D-amino acids in the analogue [D-Thr2,6,11,15]-D-Gm confers resistance to degradation, increases its potency, and maintained this peptide spread in the cytosol similarly to what happens with Gm. Replacements of Cys by Thr and Gln by L- or D-Pro ([D-Thr2,6,11,15, Pro9]-D-Gm, and [Thr2,6,11,15, D-Pro9]-Gm), which induced a similar β-hairpin conformation, also increase their resistance to degradation, and cytotoxicity, but after 24 h they are not present spread in the cytosol, exhibiting lower cytotoxicity in comparison to Gm. Additionally, chloroquine, a lysosomal enzyme inhibitor potentiated the effect of the peptides. Furthermore, the binding and internalization of peptides was determined, but a direct correlation among these factors was not observed. However, cholesterol ablation, which increase fluidity of cellular membrane, also increase cytotoxicity and internalization of peptides. β-hairpin spatial conformation, and intracellular localization/target, and the capability of entry are important properties of gomesin cytotoxicity. PMID:24312251

  9. Protein import into chloroplasts requires a chloroplast ATPase

    International Nuclear Information System (INIS)

    Pain, D.; Blobel, G.

    1987-01-01

    The authors have transcribed mRNA from a cDNA clone coding for pea ribulose-1,5-bisphosphate carboxylase, translated the mRNA in a wheat germ cell-free system, and studied the energy requirement for posttranslational import of the [ 35 S]methionine-labeled protein into the stroma of pea chloroplasts. They found that import depends on ATP hydrolysis within the stroma. Import is not inhibited when H + , K + , Na + , or divalent cation gradients across the chloroplast membranes are dissipated by ionophores, as long as exogenously added ATP is also present during the import reaction. The data suggest that protein import into the chloroplast stroma requires a chloroplast ATPase that does not function to generate a membrane potential for driving the import reaction but that exerts its effect in another, yet-to-be-determined, mode. They have carried out a preliminary characterization of this ATPase regarding its nucleotide specificity and the effects of various ATPase inhibitors

  10. The role of mitochondria in cellular iron-sulfur protein biogenesis and iron metabolism.

    Science.gov (United States)

    Lill, Roland; Hoffmann, Bastian; Molik, Sabine; Pierik, Antonio J; Rietzschel, Nicole; Stehling, Oliver; Uzarska, Marta A; Webert, Holger; Wilbrecht, Claudia; Mühlenhoff, Ulrich

    2012-09-01

    Mitochondria play a key role in iron metabolism in that they synthesize heme, assemble iron-sulfur (Fe/S) proteins, and participate in cellular iron regulation. Here, we review the latter two topics and their intimate connection. The mitochondrial Fe/S cluster (ISC) assembly machinery consists of 17 proteins that operate in three major steps of the maturation process. First, the cysteine desulfurase complex Nfs1-Isd11 as the sulfur donor cooperates with ferredoxin-ferredoxin reductase acting as an electron transfer chain, and frataxin to synthesize an [2Fe-2S] cluster on the scaffold protein Isu1. Second, the cluster is released from Isu1 and transferred toward apoproteins with the help of a dedicated Hsp70 chaperone system and the glutaredoxin Grx5. Finally, various specialized ISC components assist in the generation of [4Fe-4S] clusters and cluster insertion into specific target apoproteins. Functional defects of the core ISC assembly machinery are signaled to cytosolic or nuclear iron regulatory systems resulting in increased cellular iron acquisition and mitochondrial iron accumulation. In fungi, regulation is achieved by iron-responsive transcription factors controlling the expression of genes involved in iron uptake and intracellular distribution. They are assisted by cytosolic multidomain glutaredoxins which use a bound Fe/S cluster as iron sensor and additionally perform an essential role in intracellular iron delivery to target metalloproteins. In mammalian cells, the iron regulatory proteins IRP1, an Fe/S protein, and IRP2 act in a post-transcriptional fashion to adjust the cellular needs for iron. Thus, Fe/S protein biogenesis and cellular iron metabolism are tightly linked to coordinate iron supply and utilization. This article is part of a Special Issue entitled: Cell Biology of Metals. Copyright © 2012 Elsevier B.V. All rights reserved.

  11. Searching for cellular partners of hantaviral nonstructural protein NSs: Y2H screening of mouse cDNA library and analysis of cellular interactome.

    Science.gov (United States)

    Rönnberg, Tuomas; Jääskeläinen, Kirsi; Blot, Guillaume; Parviainen, Ville; Vaheri, Antti; Renkonen, Risto; Bouloy, Michele; Plyusnin, Alexander

    2012-01-01

    Hantaviruses (Bunyaviridae) are negative-strand RNA viruses with a tripartite genome. The small (S) segment encodes the nucleocapsid protein and, in some hantaviruses, also the nonstructural protein (NSs). The aim of this study was to find potential cellular partners for the hantaviral NSs protein. Toward this aim, yeast two-hybrid (Y2H) screening of mouse cDNA library was performed followed by a search for potential NSs protein counterparts via analyzing a cellular interactome. The resulting interaction network was shown to form logical, clustered structures. Furthermore, several potential binding partners for the NSs protein, for instance ACBD3, were identified and, to prove the principle, interaction between NSs and ACBD3 proteins was demonstrated biochemically.

  12. Molecular design and nanoparticle-mediated intracellular delivery of functional proteins to target cellular pathways

    Science.gov (United States)

    Shah, Dhiral Ashwin

    Intracellular delivery of specific proteins and peptides represents a novel method to influence stem cells for gain-of-function and loss-of-function. Signaling control is vital in stem cells, wherein intricate control of and interplay among critical pathways directs the fate of these cells into either self-renewal or differentiation. The most common route to manipulate cellular function involves the introduction of genetic material such as full-length genes and shRNA into the cell to generate (or prevent formation of) the target protein, and thereby ultimately alter cell function. However, viral-mediated gene delivery may result in relatively slow expression of proteins and prevalence of oncogene insertion into the cell, which can alter cell function in an unpredictable fashion, and non-viral delivery may lead to low efficiency of genetic delivery. For example, the latter case plagues the generation of induced pluripotent stem cells (iPSCs) and hinders their use for in vivo applications. Alternatively, introducing proteins into cells that specifically recognize and influence target proteins, can result in immediate deactivation or activation of key signaling pathways within the cell. In this work, we demonstrate the cellular delivery of functional proteins attached to hydrophobically modified silica (SiNP) nanoparticles to manipulate specifically targeted cell signaling proteins. In the Wnt signaling pathway, we have targeted the phosphorylation activity of glycogen synthase kinase-3beta (GSK-3beta) by designing a chimeric protein and delivering it in neural stem cells. Confocal imaging indicates that the SiNP-chimeric protein conjugates were efficiently delivered to the cytosol of human embryonic kidney cells and rat neural stem cells, presumably via endocytosis. This uptake impacted the Wnt signaling cascade, indicated by the elevation of beta-catenin levels, and increased transcription of Wnt target genes, such as c-MYC. The results presented here suggest that

  13. Animal Proteins as Important Contributors to a Healthy Human Diet.

    Science.gov (United States)

    Elmadfa, Ibrahim; Meyer, Alexa L

    2017-02-08

    Adequate protein intake is critical for health and development. Generally, protein of animal origin is of higher quality for humans owing to its amino acid pattern and good digestibility. When administered in mixtures it can enhance the quality of plant proteins, but its availability is often low in low-income communities, especially in young children, the elderly, and pregnant and lactating women, who have increased requirements and in whom high-quality protein also stimulates (bone) growth and maintenance. Although high protein intake was associated with increased type 2 diabetes mellitus risk, milk and seafood are good sources of branched chain amino acids and taurine, which act beneficially on glucose metabolism and blood pressure. However, high consumption of protein-rich animal food is also associated with adverse health effects and higher risk for noncommunicable diseases, partly related to other components of these foods, like saturated fatty acids and potential carcinogens in processed meat but also the atherogenic methionine metabolite homocysteine. In moderation, however, animal proteins are especially important for health maintenance in vulnerable persons.

  14. Cellular Stoichiometry of Methyl-Accepting Chemotaxis Proteins in Sinorhizobium meliloti.

    Science.gov (United States)

    Zatakia, Hardik M; Arapov, Timofey D; Meier, Veronika M; Scharf, Birgit E

    2018-03-15

    The chemosensory system in Sinorhizobium meliloti has several important deviations from the widely studied enterobacterial paradigm. To better understand the differences between the two systems and how they are optimally tuned, we determined the cellular stoichiometry of the methyl-accepting chemotaxis proteins (MCPs) and the histidine kinase CheA in S. meliloti Quantitative immunoblotting was used to determine the total amount of MCPs and CheA per cell in S. meliloti The MCPs are present in the cell in high abundance (McpV), low abundance (IcpA, McpU, McpX, and McpW), and very low abundance (McpY and McpZ), whereas McpT was below the detection limit. The approximate cellular ratio of these three receptor groups is 300:30:1. The chemoreceptor-to-CheA ratio is 23.5:1, highly similar to that seen in Bacillus subtilis (23:1) and about 10 times higher than that in Escherichia coli (3.4:1). Different from E. coli , the high-abundance receptors in S. meliloti are lacking the carboxy-terminal NWETF pentapeptide that binds the CheR methyltransferase and CheB methylesterase. Using transcriptional lacZ fusions, we showed that chemoreceptors are positively controlled by the master regulators of motility, VisNR and Rem. In addition, FlbT, a class IIA transcriptional regulator of flagellins, also positively regulates the expression of most chemoreceptors except for McpT and McpY, identifying chemoreceptors as class III genes. Taken together, these results demonstrate that the chemosensory complex and the adaptation system in S. meliloti deviates significantly from the established enterobacterial paradigm but shares some similarities with B. subtilis IMPORTANCE The symbiotic soil bacterium Sinorhizobium meliloti is of great agricultural importance because of its nitrogen-fixing properties, which enhances growth of its plant symbiont, alfalfa. Chemotaxis provides a competitive advantage for bacteria to sense their environment and interact with their eukaryotic hosts. For a better

  15. Mechanical and mechanobiological influences on bone fracture repair : identifying important cellular characteristics

    NARCIS (Netherlands)

    Isaksson, H.E.

    2007-01-01

    Fracture repair is a complex and multifactorial process, which involves a well-programmed series of cellular and molecular events that result in a combination of intramembranous and endochondral bone formation. The vast majority of fractures is treated successfully. They heal through ‘secondary

  16. Topology and cellular localization of the small hydrophobic protein of avian metapneumovirus.

    Science.gov (United States)

    Deng, Qiji; Weng, Yuejin; Lu, Wuxun; Demers, Andrew; Song, Minxun; Wang, Dan; Yu, Qingzhong; Li, Feng

    2011-09-01

    The small hydrophobic protein (SH) is a type II integral membrane protein that is packaged into virions and is only present in certain paramyxoviruses including metapneumovirus. In addition to a highly divergent primary sequence, SH proteins vary significantly in size amongst the different viruses. Human respiratory syncytial virus (HRSV) encodes the smallest SH protein consisting of only 64 amino acids, while metapneumoviruses have the longest SH protein ranging from 174 to 179 amino acids in length. Little is currently known about the cellular localization and topology of the metapneumovirus SH protein. Here we characterize for the first time metapneumovirus SH protein with respect to topology, subcellular localization, and transport using avian metapneumovirus subgroup C (AMPV-C) as a model system. We show that AMPV-C SH is an integral membrane protein with N(in)C(out) orientation located in both the plasma membrane as well as within intracellular compartments, which is similar to what has been described previously for SH proteins of other paramyxoviruses. Furthermore, we demonstrate that AMPV-C SH protein localizes in the endoplasmic reticulum (ER), Golgi, and cell surface, and is transported through ER-Golgi secretory pathway. Copyright © 2011 Elsevier B.V. All rights reserved.

  17. Fibrillarin, a nucleolar protein, is required for normal nuclear morphology and cellular growth in HeLa cells

    International Nuclear Information System (INIS)

    Amin, Mohammed Abdullahel; Matsunaga, Sachihiro; Ma, Nan; Takata, Hideaki; Yokoyama, Masami; Uchiyama, Susumu; Fukui, Kiichi

    2007-01-01

    Fibrillarin is a key small nucleolar protein in eukaryotes, which has an important role in pre-rRNA processing during ribosomal biogenesis. Though several functions of fibrillarin are known, its function during the cell cycle is still unknown. In this study, we confirmed the dynamic localization of fibrillarin during the cell cycle of HeLa cells and also performed functional studies by using a combination of immunofluorescence microscopy and RNAi technique. We observed that depletion of fibrillarin has almost no effect on the nucleolar structure. However, fibrillarin-depleted cells showed abnormal nuclear morphology. Moreover, fibrillarin depletion resulted in the reduction of the cellular growth and modest accumulation of cells with 4n DNA content. Our data suggest that fibrillarin would play a critical role in the maintenance of nuclear shape and cellular growth

  18. The effects of perception of risk and importance of answering and initiating a cellular phone call while driving.

    Science.gov (United States)

    Nelson, Erik; Atchley, Paul; Little, Todd D

    2009-05-01

    Recent data suggest that laws banning cellular phone use while driving may not change use patterns, especially among young drivers with high rates of mobile phone adoption. We examined reasons younger drivers choose or do not choose to talk on a phone while driving among a sample of young drivers (n=276) with very high ownership of cellular phones (over 99%) and a very high use of cellular phones while driving (100% for those that were primary operators of an automobile). Respondents were surveyed for patterns of use, types of call, perceived risk, and motivations for use. The data were analyzed using structural equation modeling (SEM) to explore the relationships between perceived risk of the behavior, emotionality of the call, perceived importance of the call, and how often calls were initiated versus answered. The model suggests that even though people believe that talking on a cellular phone while driving is dangerous, they will tend to initiate a cellular conversation if they believe that the call is important.

  19. Super-resolution imaging and tracking of protein-protein interactions in sub-diffraction cellular space

    Science.gov (United States)

    Liu, Zhen; Xing, Dong; Su, Qian Peter; Zhu, Yun; Zhang, Jiamei; Kong, Xinyu; Xue, Boxin; Wang, Sheng; Sun, Hao; Tao, Yile; Sun, Yujie

    2014-07-01

    Imaging the location and dynamics of individual interacting protein pairs is essential but often difficult because of the fluorescent background from other paired and non-paired molecules, particularly in the sub-diffraction cellular space. Here we develop a new method combining bimolecular fluorescence complementation and photoactivated localization microscopy for super-resolution imaging and single-molecule tracking of specific protein-protein interactions. The method is used to study the interaction of two abundant proteins, MreB and EF-Tu, in Escherichia coli cells. The super-resolution imaging shows interesting distribution and domain sizes of interacting MreB-EF-Tu pairs as a subpopulation of total EF-Tu. The single-molecule tracking of MreB, EF-Tu and MreB-EF-Tu pairs reveals intriguing localization-dependent heterogonous dynamics and provides valuable insights to understanding the roles of MreB-EF-Tu interactions.

  20. Matrix rigidity regulates cancer cell growth by modulating cellular metabolism and protein synthesis.

    Directory of Open Access Journals (Sweden)

    Robert W Tilghman

    Full Text Available Tumor cells in vivo encounter diverse types of microenvironments both at the site of the primary tumor and at sites of distant metastases. Understanding how the various mechanical properties of these microenvironments affect the biology of tumor cells during disease progression is critical in identifying molecular targets for cancer therapy.This study uses flexible polyacrylamide gels as substrates for cell growth in conjunction with a novel proteomic approach to identify the properties of rigidity-dependent cancer cell lines that contribute to their differential growth on soft and rigid substrates. Compared to cells growing on more rigid/stiff substrates (>10,000 Pa, cells on soft substrates (150-300 Pa exhibited a longer cell cycle, due predominantly to an extension of the G1 phase of the cell cycle, and were metabolically less active, showing decreased levels of intracellular ATP and a marked reduction in protein synthesis. Using stable isotope labeling of amino acids in culture (SILAC and mass spectrometry, we measured the rates of protein synthesis of over 1200 cellular proteins under growth conditions on soft and rigid/stiff substrates. We identified cellular proteins whose syntheses were either preferentially inhibited or preserved on soft matrices. The former category included proteins that regulate cytoskeletal structures (e.g., tubulins and glycolysis (e.g., phosphofructokinase-1, whereas the latter category included proteins that regulate key metabolic pathways required for survival, e.g., nicotinamide phosphoribosyltransferase, a regulator of the NAD salvage pathway.The cellular properties of rigidity-dependent cancer cells growing on soft matrices are reminiscent of the properties of dormant cancer cells, e.g., slow growth rate and reduced metabolism. We suggest that the use of relatively soft gels as cell culture substrates would allow molecular pathways to be studied under conditions that reflect the different mechanical

  1. Interacting factors and cellular localization of SR protein-specific kinase Dsk1

    International Nuclear Information System (INIS)

    Tang, Zhaohua; Luca, Maria; Taggart-Murphy, Laura; Portillio, Jessica; Chang, Cathey; Guven, Ayse; Lin, Ren-Jang; Murray, Johanne; Carr, Antony

    2012-01-01

    Schizosaccharomyces pombe Dsk1 is an SR protein-specific kinase (SRPK), whose homologs have been identified in every eukaryotic organism examined. Although discovered as a mitotic regulator with protein kinase activity toward SR splicing factors, it remains largely unknown about what and how Dsk1 contributes to cell cycle and pre-mRNA splicing. In this study, we investigated the Dsk1 function by determining interacting factors and cellular localization of the kinase. Consistent with its reported functions, we found that pre-mRNA processing and cell cycle factors are prominent among the proteins co-purified with Dsk1. The identification of these factors led us to find Rsd1 as a novel Dsk1 substrate, as well as the involvement of Dsk1 in cellular distribution of poly(A) + RNA. In agreement with its role in nuclear events, we also found that Dsk1 is mainly localized in the nucleus during G 2 phase and at mitosis. Furthermore, we revealed the oscillation of Dsk1 protein in a cell cycle-dependent manner. This paper marks the first comprehensive analysis of in vivo Dsk1-associated proteins in fission yeast. Our results reflect the conserved role of SRPK family in eukaryotic organisms, and provide information about how Dsk1 functions in pre-mRNA processing and cell-division cycle.

  2. Interacting factors and cellular localization of SR protein-specific kinase Dsk1

    Energy Technology Data Exchange (ETDEWEB)

    Tang, Zhaohua, E-mail: ztang@jsd.claremont.edu [W.M. Keck Science Center, The Claremont Colleges, Claremont, CA 91711 (United States); Luca, Maria; Taggart-Murphy, Laura; Portillio, Jessica; Chang, Cathey; Guven, Ayse [W.M. Keck Science Center, The Claremont Colleges, Claremont, CA 91711 (United States); Lin, Ren-Jang [Department of Molecular and Cellular Biology, Beckman Research Institute of the City of Hope, Duarte, CA 91010 (United States); Murray, Johanne; Carr, Antony [Genome Damage and Stability Center, University of Sussex, Falmer, BN1 9RQ (United Kingdom)

    2012-10-01

    Schizosaccharomyces pombe Dsk1 is an SR protein-specific kinase (SRPK), whose homologs have been identified in every eukaryotic organism examined. Although discovered as a mitotic regulator with protein kinase activity toward SR splicing factors, it remains largely unknown about what and how Dsk1 contributes to cell cycle and pre-mRNA splicing. In this study, we investigated the Dsk1 function by determining interacting factors and cellular localization of the kinase. Consistent with its reported functions, we found that pre-mRNA processing and cell cycle factors are prominent among the proteins co-purified with Dsk1. The identification of these factors led us to find Rsd1 as a novel Dsk1 substrate, as well as the involvement of Dsk1 in cellular distribution of poly(A){sup +} RNA. In agreement with its role in nuclear events, we also found that Dsk1 is mainly localized in the nucleus during G{sub 2} phase and at mitosis. Furthermore, we revealed the oscillation of Dsk1 protein in a cell cycle-dependent manner. This paper marks the first comprehensive analysis of in vivo Dsk1-associated proteins in fission yeast. Our results reflect the conserved role of SRPK family in eukaryotic organisms, and provide information about how Dsk1 functions in pre-mRNA processing and cell-division cycle.

  3. Computational Modeling of Proteins based on Cellular Automata: A Method of HP Folding Approximation.

    Science.gov (United States)

    Madain, Alia; Abu Dalhoum, Abdel Latif; Sleit, Azzam

    2018-06-01

    The design of a protein folding approximation algorithm is not straightforward even when a simplified model is used. The folding problem is a combinatorial problem, where approximation and heuristic algorithms are usually used to find near optimal folds of proteins primary structures. Approximation algorithms provide guarantees on the distance to the optimal solution. The folding approximation approach proposed here depends on two-dimensional cellular automata to fold proteins presented in a well-studied simplified model called the hydrophobic-hydrophilic model. Cellular automata are discrete computational models that rely on local rules to produce some overall global behavior. One-third and one-fourth approximation algorithms choose a subset of the hydrophobic amino acids to form H-H contacts. Those algorithms start with finding a point to fold the protein sequence into two sides where one side ignores H's at even positions and the other side ignores H's at odd positions. In addition, blocks or groups of amino acids fold the same way according to a predefined normal form. We intend to improve approximation algorithms by considering all hydrophobic amino acids and folding based on the local neighborhood instead of using normal forms. The CA does not assume a fixed folding point. The proposed approach guarantees one half approximation minus the H-H endpoints. This lower bound guaranteed applies to short sequences only. This is proved as the core and the folds of the protein will have two identical sides for all short sequences.

  4. Characterization of Silk Fibroin Modified Surface: A Proteomic View of Cellular Response Proteins Induced by Biomaterials

    Directory of Open Access Journals (Sweden)

    Ming-Hui Yang

    2014-01-01

    Full Text Available The purpose of this study was to develop the pathway of silk fibroin (SF biopolymer surface induced cell membrane protein activation. Fibroblasts were used as an experimental model to evaluate the responses of cellular proteins induced by biopolymer material using a mass spectrometry-based profiling system. The surface was covered by multiwalled carbon nanotubes (CNTs and SF to increase the surface area, enhance the adhesion of biopolymer, and promote the rate of cell proliferation. The amount of adhered fibroblasts on CNTs/SF electrodes of quartz crystal microbalance (QCM greatly exceeded those on other surfaces. Moreover, analyzing differential protein expressions of adhered fibroblasts on the biopolymer surface by proteomic approaches indicated that CD44 may be a key protein. Through this study, utilization of mass spectrometry-based proteomics in evaluation of cell adhesion on biopolymer was proposed.

  5. Molecular modeling of the conformational dynamics of the cellular prion protein

    Science.gov (United States)

    Nguyen, Charles; Colling, Ian; Bartz, Jason; Soto, Patricia

    2014-03-01

    Prions are infectious agents responsible for transmissible spongiform encephalopathies (TSEs), a type of fatal neurodegenerative disease in mammals. Prions propagate biological information by conversion of the non-pathological version of the prion protein to the infectious conformation, PrPSc. A wealth of knowledge has shed light on the nature and mechanism of prion protein conversion. In spite of the significance of this problem, we are far from fully understanding the conformational dynamics of the cellular isoform. To remedy this situation we employ multiple biomolecular modeling techniques such as docking and molecular dynamics simulations to map the free energy landscape and determine what specific regions of the prion protein are most conductive to binding. The overall goal is to characterize the conformational dynamics of the cell form of the prion protein, PrPc, to gain insight into inhibition pathways against misfolding. NE EPSCoR FIRST Award to Patricia Soto.

  6. Protein import into chloroplasts requires a chloroplast ATPase

    Energy Technology Data Exchange (ETDEWEB)

    Pain, D.; Blobel, G.

    1987-05-01

    The authors have transcribed mRNA from a cDNA clone coding for pea ribulose-1,5-bisphosphate carboxylase, translated the mRNA in a wheat germ cell-free system, and studied the energy requirement for posttranslational import of the (/sup 35/S)methionine-labeled protein into the stroma of pea chloroplasts. They found that import depends on ATP hydrolysis within the stroma. Import is not inhibited when H/sup +/, K/sup +/, Na/sup +/, or divalent cation gradients across the chloroplast membranes are dissipated by ionophores, as long as exogenously added ATP is also present during the import reaction. The data suggest that protein import into the chloroplast stroma requires a chloroplast ATPase that does not function to generate a membrane potential for driving the import reaction but that exerts its effect in another, yet-to-be-determined, mode. They have carried out a preliminary characterization of this ATPase regarding its nucleotide specificity and the effects of various ATPase inhibitors.

  7. Cellular fatty acid transport in heart and skeletal muscle as facilitated by proteins

    NARCIS (Netherlands)

    Luiken, J. J.; Schaap, F. G.; van Nieuwenhoven, F. A.; van der Vusse, G. J.; Bonen, A.; Glatz, J. F.

    1999-01-01

    Despite the importance of long-chain fatty acids (FA) as fuels for heart and skeletal muscles, the mechanism of their cellular uptake has not yet been clarified. There is dispute as to whether FA are taken up by the muscle cells via passive diffusion and/or carrier-mediated transport. Kinetic

  8. Intracellular localisation of proteins to specific cellular areas by nanocapsule mediated delivery.

    Science.gov (United States)

    Wang, Huabin; Chen, Ligang; Sun, Xianchao; Fu, Ailing

    2017-09-01

    Nanocapsules are promising carriers with great potential for intracellular protein transport. Although many studies have intended to improve cell uptake efficacy, there is an increasing interest in understanding of subcellular distribution of cargoes inside cells, which is essential for purposeful delivery of biomolecules into specific sites within cells. Herein, we interrogate the intracellular localisation of exogenous proteins, including fluorescein isothiocyanate (FITC)-labelled bovine serum albumin (BSA) and green fluorescent protein (GFP), mediated by specially designed nanocapsules. The results show that the designed nanocapsules can deliver the two types of fluorescent proteins into different cellular destinations (cytosol, nucleus or the whole cell), depending on the composition of nanocapsules. Meanwhile, several impact factors that influence the distribution of proteins in cells have also been investigated, and the results suggest that the localisation of capsule-mediated proteins in cells is strongly affected by the surface properties of nanocapsules, the types of stabilisers and proteins, and environmental temperatures. The rational control of intracellular localised delivery of exogenous proteins as we demonstrated in this study might open new avenues to obtain desired magnitude of drug effects for modulating cell activity.

  9. Poliovirus infection induces the co-localization of cellular protein SRp20 with TIA-1, a cytoplasmic stress granule protein.

    Science.gov (United States)

    Fitzgerald, Kerry D; Semler, Bert L

    2013-09-01

    Different types of environmental stress cause mammalian cells to form cytoplasmic foci, termed stress granules, which contain mRNPs that are translationally silenced. These foci are transient and dynamic, and contain components of the cellular translation machinery as well as certain mRNAs and RNA binding proteins. Stress granules are known to be induced by conditions such as hypoxia, nutrient deprivation, and oxidative stress, and a number of cellular factors have been identified that are commonly associated with these foci. More recently it was discovered that poliovirus infection also induces the formation of stress granules, although these cytoplasmic foci appear to be somewhat compositionally unique. Work described here examined the punctate pattern of SRp20 (a host cell mRNA splicing protein) localization in the cytoplasm of poliovirus-infected cells, demonstrating the partial co-localization of SRp20 with the stress granule marker protein TIA-1. We determined that SRp20 does not co-localize with TIA-1, however, under conditions of oxidative stress, indicating that the close association of these two proteins during poliovirus infection is not representative of a general response to cellular stress. We confirmed that the expression of a dominant negative version of TIA-1 (TIA-1-PRD) results in the dissociation of stress granules. Finally, we demonstrated that expression of wild type TIA-1 or dominant negative TIA-1-PRD in cells during poliovirus infection does not dramatically affect viral translation. Taken together, these studies provide a new example of the unique cytoplasmic foci that form during poliovirus infection. Copyright © 2013 Elsevier B.V. All rights reserved.

  10. Poliovirus infection induces the co-localization of cellular protein SRp20 with TIA-1, a cytoplasmic stress granule protein

    Science.gov (United States)

    Fitzgerald, Kerry D.; Semler, Bert L.

    2013-01-01

    Different types of environmental stress cause mammalian cells to form cytoplasmic foci, termed stress granules, which contain mRNPs that are translationally silenced. These foci are transient and dynamic, and contain components of the cellular translation machinery as well as certain mRNAs and RNA binding proteins. Stress granules are known to be induced by conditions such as hypoxia, nutrient deprivation, and oxidative stress, and a number of cellular factors have been identified that are commonly associated with these foci. More recently it was discovered that poliovirus infection also induces the formation of stress granules, although these cytoplasmic foci appear to be somewhat compositionally unique. Work described here examined the punctate pattern of SRp20 (a host cell mRNA splicing protein) localization in the cytoplasm of poliovirus-infected cells, demonstrating the partial co-localization of SRp20 with the stress granule marker protein TIA-1. We determined that SRp20 does not co-localize with TIA-1, however, under conditions of oxidative stress, indicating that the close association of these two proteins during poliovirus infection is not representative of a general response to cellular stress. We confirmed that the expression of a dominant negative version of TIA-1 (TIA-1-PRD) results in the dissociation of stress granules. Finally, we demonstrated that expression of wild type TIA-1 or dominant negative TIA-1-PRD in cells during poliovirus infection does not dramatically affect viral translation. Taken together, these studies provide a new example of the unique cytoplasmic foci that form during poliovirus infection. PMID:23830997

  11. Redox modification of caveolar proteins in the cardiovascular system- role in cellular signalling and disease.

    Science.gov (United States)

    Bubb, Kristen J; Birgisdottir, Asa Birna; Tang, Owen; Hansen, Thomas; Figtree, Gemma A

    2017-08-01

    Rapid and coordinated release of a variety of reactive oxygen species (ROS) such as superoxide (O 2 .- ), hydrogen peroxide (H 2 O 2 ) and peroxynitrite, in specific microdomains, play a crucial role in cell signalling in the cardiovascular system. These reactions are mediated by reversible and functional modifications of a wide variety of key proteins. Dysregulation of this oxidative signalling occurs in almost all forms of cardiovascular disease (CVD), including at the very early phases. Despite the heavily publicized failure of "antioxidants" to improve CVD progression, pharmacotherapies such as those targeting the renin-angiotensin system, or statins, exert at least part of their large clinical benefit via modulating cellular redox signalling. Over 250 proteins, including receptors, ion channels and pumps, and signalling proteins are found in the caveolae. An increasing proportion of these are being recognized as redox regulated-proteins, that reside in the immediate vicinity of the two major cellular sources of ROS, nicotinamide adenine dinucleotide phosphate oxidase (Nox) and uncoupled endothelial nitric oxide synthase (eNOS). This review focuses on what is known about redox signalling within the caveolae, as well as endogenous protective mechanisms utilized by the cell, and new approaches to targeting dysregulated redox signalling in the caveolae as a therapeutic strategy in CVD. Copyright © 2017. Published by Elsevier Inc.

  12. Cellular Immunity State of Protein-deficient Rats with the Toxic Liver Injury

    Directory of Open Access Journals (Sweden)

    O.N. Voloshchuk

    2017-05-01

    Full Text Available Studies on the role of immunity mechanisms in the emergence and maintenance of inflammatory and destructive processes in the liver under toxic hepatitis and nutrient deficiency are topical. The aim of research – to study the quantitative content and functional activity of leukocytes under the conditions of acetaminophen-induced hepatitis on the background of nutritional protein deficiency. The most pronounced changes in cell-mediated immunity are observed in protein-deficient animals with toxic hepatitis. The pronounced defects of both specific and non-specific cellular immunity were manifested by the leukocytosis, increase number of segmented neutrophils in blood serum against decrease their phagocytic index and phagocytic number, reduction of total lymphocyte number, and simultaneously lowering of T- and B-lymphocytes was established under the conditions of acetaminophen-induced hepatotoxicity on the background of protein deficiency. Installed changes indicate the defective formation of functional immunity state which can manifest by decrease the body’s ability to carry out the reaction of cellular and humoral immunity. Research results may be used for the rationale of therapeutic approaches to the elimination and correction of the consequences of immunological status disturbances under the conditions of acetaminophen-induced hepatitis, aggravated by the alimentary protein deprivation.

  13. Characterization of a Fasciola gigantica protein carrying two DM9 domains reveals cellular relocalization property.

    Science.gov (United States)

    Phadungsil, Wansika; Smooker, Peter M; Vichasri-Grams, Suksiri; Grams, Rudi

    2016-01-01

    Even at the present age of whole-organism analysis, e.g., genomics, transcriptomics, and proteomics, the biological roles of many proteins remain unresolved. Classified among the proteins of unknown function is a family of proteins harboring repeats of the DM9 domain, a 60-75 amino acids motif first described in a small number of Drosophila melanogaster proteins. Proteins may carry two or more DM9 domains either in combination with other domains or as their sole constituent. Here we have characterized a 16.8 kDa Fasciola gigantica protein comprising two tandem repeated DM9 domains (FgDM9-1). The protein was located in the parenchyma of the immature and mature parasite and consequently it was not detected in the ES product of the parasite but only in the whole worm extract. Interestingly, extraction with SDS yielded a substantially higher amount of the protein suggesting association with insoluble cell components. In Sf9 insect cells a heterologously expressed EGFP-FgDM9-1 chimera showed cell-wide distribution but relocated to vesicle-like structures in the cytoplasm after stimulating cellular stress by bacteria, heat shock or chloroquine. These structures did not colocalize with the markers of endocytosis/phagocytosis ubiquitin, RAB7, GABARAP. The same behavior was noted for Aedes aegypti PRS1, a homologous mosquito DM9 protein as a positive control while EGFP did not exhibit such relocation in the insect cells. Cross-linking experiments on soluble recombinant FgDM9-1 indicated that the protein can undergo specific oligomerization. It is speculated that proteins carrying the DM9 domain have a role in vesicular transport in flatworms and insects. Copyright © 2016 Elsevier B.V. All rights reserved.

  14. Negative Regulation of STAT3 Protein-mediated Cellular Respiration by SIRT1 Protein

    DEFF Research Database (Denmark)

    Bernier, Michel; Paul, Rajib K; Martin-Montalvo, Alejandro

    2011-01-01

    those of wild-type controls. Comparison of profiles of phospho-antibody array data indicated that the deletion of SirT1 was accompanied by constitutive activation of the pro-inflammatory NF-¿B pathway, which is key for STAT3 induction and increased cellular respiration in Sirt1-KO cells. Thus, SIRT1...... cells exhibited higher mitochondrial respiration as compared with wild-type MEFs. Two independent approaches, including ectopic expression of SIRT1 and siRNA-mediated knockdown of STAT3, led to reduction in intracellular ATP levels and increased lactate production in Sirt1-KO cells that were approaching...

  15. Cellular Protein WDR11 Interacts with Specific Herpes Simplex Virus Proteins at the trans-Golgi Network To Promote Virus Replication

    Science.gov (United States)

    Taylor, Kathryne E.

    2015-01-01

    ABSTRACT It has recently been proposed that the herpes simplex virus (HSV) protein ICP0 has cytoplasmic roles in blocking antiviral signaling and in promoting viral replication in addition to its well-known proteasome-dependent functions in the nucleus. However, the mechanisms through which it produces these effects remain unclear. While investigating this further, we identified a novel cytoplasmic interaction between ICP0 and the poorly characterized cellular protein WDR11. During an HSV infection, WDR11 undergoes a dramatic change in localization at late times in the viral replication cycle, moving from defined perinuclear structures to a dispersed cytoplasmic distribution. While this relocation was not observed during infection with viruses other than HSV-1 and correlated with efficient HSV-1 replication, the redistribution was found to occur independently of ICP0 expression, instead requiring viral late gene expression. We demonstrate for the first time that WDR11 is localized to the trans-Golgi network (TGN), where it interacts specifically with some, but not all, HSV virion components, in addition to ICP0. Knockdown of WDR11 in cultured human cells resulted in a modest but consistent decrease in yields of both wild-type and ICP0-null viruses, in the supernatant and cell-associated fractions, without affecting viral gene expression. Although further study is required, we propose that WDR11 participates in viral assembly and/or secondary envelopment. IMPORTANCE While the TGN has been proposed to be the major site of HSV-1 secondary envelopment, this process is incompletely understood, and in particular, the role of cellular TGN components in this pathway is unknown. Additionally, little is known about the cellular functions of WDR11, although the disruption of this protein has been implicated in multiple human diseases. Therefore, our finding that WDR11 is a TGN-resident protein that interacts with specific viral proteins to enhance viral yields improves both

  16. The MCM-associated protein MCM-BP is important for human nuclear morphology.

    Science.gov (United States)

    Jagannathan, Madhav; Sakwe, Amos M; Nguyen, Tin; Frappier, Lori

    2012-01-01

    Mini-chromosome maintenance complex-binding protein (MCM-BP) was discovered as a protein that is strongly associated with human MCM proteins, known to be crucial for DNA replication in providing DNA helicase activity. The Xenopus MCM-BP homologue appears to play a role in unloading MCM complexes from chromatin after DNA synthesis; however, the importance of MCM-BP and its functional contribution to human cells has been unclear. Here we show that depletion of MCM-BP by sustained expression of short hairpin RNA (shRNA) results in highly abnormal nuclear morphology and centrosome amplification. The abnormal nuclear morphology was not seen with depletion of other MCM proteins and was rescued with shRNA-resistant MCM-BP. MCM-BP depletion was also found to result in transient activation of the G2 checkpoint, slowed progression through G2 and increased replication protein A foci, indicative of replication stress. In addition, MCM-BP depletion led to increased cellular levels of MCM proteins throughout the cell cycle including soluble MCM pools. The results suggest that MCM-BP makes multiple contributions to human cells that are not limited to unloading of the MCM complex.

  17. Modification of an acetone-sodium dodecyl sulfate disruption method for cellular protein extraction from neuropathogenic Clostridium botulinum

    Science.gov (United States)

    An acetone-sodium dodecyl sulfate (SDS) disruption method was used for the extraction of cellular proteins from neurotoxigenic Clostridium botulinum. The amount of protein extracted per gram of dry weight and the protein profile as revealed by polyacrylamide gel electrophoresis (PAGE) was comparabl...

  18. Functional interactions of nucleocapsid protein of feline immunodeficiency virus and cellular prion protein with the viral RNA.

    Science.gov (United States)

    Moscardini, Mila; Pistello, Mauro; Bendinelli, M; Ficheux, Damien; Miller, Jennifer T; Gabus, Caroline; Le Grice, Stuart F J; Surewicz, Witold K; Darlix, Jean-Luc

    2002-04-19

    All lentiviruses and oncoretroviruses examined so far encode a major nucleic-acid binding protein (nucleocapsid or NC* protein), approximately 2500 molecules of which coat the dimeric RNA genome. Studies on HIV-1 and MoMuLV using in vitro model systems and in vivo have shown that NC protein is required to chaperone viral RNA dimerization and packaging during virus assembly, and proviral DNA synthesis by reverse transcriptase (RT) during infection. The human cellular prion protein (PrP), thought to be the major component of the agent causing transmissible spongiform encephalopathies (TSE), was recently found to possess a strong affinity for nucleic acids and to exhibit chaperone properties very similar to HIV-1 NC protein in the HIV-1 context in vitro. Tight binding of PrP to nucleic acids is proposed to participate directly in the prion disease process. To extend our understanding of lentiviruses and of the unexpected nucleic acid chaperone properties of the human prion protein, we set up an in vitro system to investigate replication of the feline immunodeficiency virus (FIV), which is functionally and phylogenetically distant from HIV-1. The results show that in the FIV model system, NC protein chaperones viral RNA dimerization, primer tRNA(Lys,3) annealing to the genomic primer-binding site (PBS) and minus strand DNA synthesis by the homologous FIV RT. FIV NC protein is able to trigger specific viral DNA synthesis by inhibiting self-priming of reverse transcription. The human prion protein was found to mimic the properties of FIV NC with respect to primer tRNA annealing to the viral RNA and chaperoning minus strand DNA synthesis. Copyright 2002 Elsevier Science Ltd.

  19. Fast kinase domain-containing protein 3 is a mitochondrial protein essential for cellular respiration

    Energy Technology Data Exchange (ETDEWEB)

    Simarro, Maria [Division of Rheumatology, Immunology and Allergy, Brigham and Women' s Hospital, and Department of Medicine, Harvard Medical School, Boston, MA 02115 (United States); Gimenez-Cassina, Alfredo [Department of Cancer Biology at Dana Farber Institute, Boston, MA 02115 (United States); Kedersha, Nancy [Division of Rheumatology, Immunology and Allergy, Brigham and Women' s Hospital, and Department of Medicine, Harvard Medical School, Boston, MA 02115 (United States); Lazaro, Jean-Bernard; Adelmant, Guillaume O.; Marto, Jarrod A. [Department of Cancer Biology at Dana Farber Institute, Boston, MA 02115 (United States); Rhee, Kirsten [Division of Rheumatology, Immunology and Allergy, Brigham and Women' s Hospital, and Department of Medicine, Harvard Medical School, Boston, MA 02115 (United States); Tisdale, Sarah; Danial, Nika [Department of Cancer Biology at Dana Farber Institute, Boston, MA 02115 (United States); Benarafa, Charaf [Theodor Kocher Institute, University of Bern, 3012 Bern (Switzerland); Orduna, Anonio [Unidad de Investigacion, Hospital Clinico Universitario de Valladolid, 47005 Valladolid (Spain); Anderson, Paul, E-mail: panderson@rics.bwh.harvard.edu [Division of Rheumatology, Immunology and Allergy, Brigham and Women' s Hospital, and Department of Medicine, Harvard Medical School, Boston, MA 02115 (United States)

    2010-10-22

    Research highlights: {yields} Five members of the FAST kinase domain-containing proteins are localized to mitochondria in mammalian cells. {yields} The FASTKD3 interactome includes proteins involved in various aspects of mitochondrial metabolism. {yields} Targeted knockdown of FASTKD3 significantly reduces basal and maximal mitochondrial oxygen consumption. -- Abstract: Fas-activated serine/threonine phosphoprotein (FAST) is the founding member of the FAST kinase domain-containing protein (FASTKD) family that includes FASTKD1-5. FAST is a sensor of mitochondrial stress that modulates protein translation to promote the survival of cells exposed to adverse conditions. Mutations in FASTKD2 have been linked to a mitochondrial encephalomyopathy that is associated with reduced cytochrome c oxidase activity, an essential component of the mitochondrial electron transport chain. We have confirmed the mitochondrial localization of FASTKD2 and shown that all FASTKD family members are found in mitochondria. Although human and mouse FASTKD1-5 genes are expressed ubiquitously, some of them are most abundantly expressed in mitochondria-enriched tissues. We have found that RNA interference-mediated knockdown of FASTKD3 severely blunts basal and stress-induced mitochondrial oxygen consumption without disrupting the assembly of respiratory chain complexes. Tandem affinity purification reveals that FASTKD3 interacts with components of mitochondrial respiratory and translation machineries. Our results introduce FASTKD3 as an essential component of mitochondrial respiration that may modulate energy balance in cells exposed to adverse conditions by functionally coupling mitochondrial protein synthesis to respiration.

  20. Fast kinase domain-containing protein 3 is a mitochondrial protein essential for cellular respiration

    International Nuclear Information System (INIS)

    Simarro, Maria; Gimenez-Cassina, Alfredo; Kedersha, Nancy; Lazaro, Jean-Bernard; Adelmant, Guillaume O.; Marto, Jarrod A.; Rhee, Kirsten; Tisdale, Sarah; Danial, Nika; Benarafa, Charaf; Orduna, Anonio; Anderson, Paul

    2010-01-01

    Research highlights: → Five members of the FAST kinase domain-containing proteins are localized to mitochondria in mammalian cells. → The FASTKD3 interactome includes proteins involved in various aspects of mitochondrial metabolism. → Targeted knockdown of FASTKD3 significantly reduces basal and maximal mitochondrial oxygen consumption. -- Abstract: Fas-activated serine/threonine phosphoprotein (FAST) is the founding member of the FAST kinase domain-containing protein (FASTKD) family that includes FASTKD1-5. FAST is a sensor of mitochondrial stress that modulates protein translation to promote the survival of cells exposed to adverse conditions. Mutations in FASTKD2 have been linked to a mitochondrial encephalomyopathy that is associated with reduced cytochrome c oxidase activity, an essential component of the mitochondrial electron transport chain. We have confirmed the mitochondrial localization of FASTKD2 and shown that all FASTKD family members are found in mitochondria. Although human and mouse FASTKD1-5 genes are expressed ubiquitously, some of them are most abundantly expressed in mitochondria-enriched tissues. We have found that RNA interference-mediated knockdown of FASTKD3 severely blunts basal and stress-induced mitochondrial oxygen consumption without disrupting the assembly of respiratory chain complexes. Tandem affinity purification reveals that FASTKD3 interacts with components of mitochondrial respiratory and translation machineries. Our results introduce FASTKD3 as an essential component of mitochondrial respiration that may modulate energy balance in cells exposed to adverse conditions by functionally coupling mitochondrial protein synthesis to respiration.

  1. IN-MACA-MCC: Integrated Multiple Attractor Cellular Automata with Modified Clonal Classifier for Human Protein Coding and Promoter Prediction

    Directory of Open Access Journals (Sweden)

    Kiran Sree Pokkuluri

    2014-01-01

    Full Text Available Protein coding and promoter region predictions are very important challenges of bioinformatics (Attwood and Teresa, 2000. The identification of these regions plays a crucial role in understanding the genes. Many novel computational and mathematical methods are introduced as well as existing methods that are getting refined for predicting both of the regions separately; still there is a scope for improvement. We propose a classifier that is built with MACA (multiple attractor cellular automata and MCC (modified clonal classifier to predict both regions with a single classifier. The proposed classifier is trained and tested with Fickett and Tung (1992 datasets for protein coding region prediction for DNA sequences of lengths 54, 108, and 162. This classifier is trained and tested with MMCRI datasets for protein coding region prediction for DNA sequences of lengths 252 and 354. The proposed classifier is trained and tested with promoter sequences from DBTSS (Yamashita et al., 2006 dataset and nonpromoters from EID (Saxonov et al., 2000 and UTRdb (Pesole et al., 2002 datasets. The proposed model can predict both regions with an average accuracy of 90.5% for promoter and 89.6% for protein coding region predictions. The specificity and sensitivity values of promoter and protein coding region predictions are 0.89 and 0.92, respectively.

  2. Cellular Assays for Ferredoxins: A Strategy for Understanding Electron Flow through Protein Carriers That Link Metabolic Pathways.

    Science.gov (United States)

    Atkinson, Joshua T; Campbell, Ian; Bennett, George N; Silberg, Jonathan J

    2016-12-27

    The ferredoxin (Fd) protein family is a structurally diverse group of iron-sulfur proteins that function as electron carriers, linking biochemical pathways important for energy transduction, nutrient assimilation, and primary metabolism. While considerable biochemical information about individual Fd protein electron carriers and their reactions has been acquired, we cannot yet anticipate the proportion of electrons shuttled between different Fd-partner proteins within cells using biochemical parameters that govern electron flow, such as holo-Fd concentration, midpoint potential (driving force), molecular interactions (affinity and kinetics), conformational changes (allostery), and off-pathway electron leakage (chemical oxidation). Herein, we describe functional and structural gaps in our Fd knowledge within the context of a sequence similarity network and phylogenetic tree, and we propose a strategy for improving our understanding of Fd sequence-function relationships. We suggest comparing the functions of divergent Fds within cells whose growth, or other measurable output, requires electron transfer between defined electron donor and acceptor proteins. By comparing Fd-mediated electron transfer with biochemical parameters that govern electron flow, we posit that models that anticipate energy flow across Fd interactomes can be built. This approach is expected to transform our ability to anticipate Fd control over electron flow in cellular settings, an obstacle to the construction of synthetic electron transfer pathways and rational optimization of existing energy-conserving pathways.

  3. Cellular localization of steroid hormone-regulated proteins during sexual development in achlya

    International Nuclear Information System (INIS)

    Brunt, S.A.; Silver, J.C.

    1986-01-01

    In the fungus Achlya ambisexualis sexual development in the male strain E87 is controlled by the steroid hormone antheridiol. To investigate the effects of antheridiol on the synthesis and/or accumulation of specific cellular proteins we have analyzed [ 35 S]methionine-labeled proteins from control and hormone-treated cells using both one-dimensional (1D) and two-dimensional (2D) PAGE. The addition of the hormone antheridiol to vegetatively growing cells of Achlya E87 was found to result in changes in the synthesis and/or accumulation of at least 16 specific proteins, which could be localized to the cytoplasmic, nuclear or cell was/cell membrane fractions. The most prominent changes observed in the hormone-treated cells included the appearance in the cytoplasmic fraction of labeled proteins at 28.4 and 24.3kD which were not detectable in control cells, and a significant enrichment in the labeling of a 24.3kD protein in the cell wall/cell membrane fraction. Quantitative changes in the [ 35 S]methionine labeling of several other proteins were noted in all three cell fractions

  4. Beyond voltage-gated ion channels: Voltage-operated membrane proteins and cellular processes.

    Science.gov (United States)

    Zhang, Jianping; Chen, Xingjuan; Xue, Yucong; Gamper, Nikita; Zhang, Xuan

    2018-04-18

    Voltage-gated ion channels were believed to be the only voltage-sensitive proteins in excitable (and some non-excitable) cells for a long time. Emerging evidence indicates that the voltage-operated model is shared by some other transmembrane proteins expressed in both excitable and non-excitable cells. In this review, we summarize current knowledge about voltage-operated proteins, which are not classic voltage-gated ion channels as well as the voltage-dependent processes in cells for which single voltage-sensitive proteins have yet to be identified. Particularly, we will focus on the following. (1) Voltage-sensitive phosphoinositide phosphatases (VSP) with four transmembrane segments homologous to the voltage sensor domain (VSD) of voltage-gated ion channels; VSPs are the first family of proteins, other than the voltage-gated ion channels, for which there is sufficient evidence for the existence of the VSD domain; (2) Voltage-gated proton channels comprising of a single voltage-sensing domain and lacking an identified pore domain; (3) G protein coupled receptors (GPCRs) that mediate the depolarization-evoked potentiation of Ca 2+ mobilization; (4) Plasma membrane (PM) depolarization-induced but Ca 2+ -independent exocytosis in neurons. (5) Voltage-dependent metabolism of phosphatidylinositol 4,5-bisphosphate (PtdIns[4,5]P 2 , PIP 2 ) in the PM. These recent discoveries expand our understanding of voltage-operated processes within cellular membranes. © 2018 Wiley Periodicals, Inc.

  5. A critical role of a cellular membrane traffic protein in poliovirus RNA replication.

    Directory of Open Access Journals (Sweden)

    George A Belov

    2008-11-01

    Full Text Available Replication of many RNA viruses is accompanied by extensive remodeling of intracellular membranes. In poliovirus-infected cells, ER and Golgi stacks disappear, while new clusters of vesicle-like structures form sites for viral RNA synthesis. Virus replication is inhibited by brefeldin A (BFA, implicating some components(s of the cellular secretory pathway in virus growth. Formation of characteristic vesicles induced by expression of viral proteins was not inhibited by BFA, but they were functionally deficient. GBF1, a guanine nucleotide exchange factor for the small cellular GTPases, Arf, is responsible for the sensitivity of virus infection to BFA, and is required for virus replication. Knockdown of GBF1 expression inhibited virus replication, which was rescued by catalytically active protein with an intact N-terminal sequence. We identified a mutation in GBF1 that allows growth of poliovirus in the presence of BFA. Interaction between GBF1 and viral protein 3A determined the outcome of infection in the presence of BFA.

  6. Factors of importance for a successful delivery system for proteins

    DEFF Research Database (Denmark)

    van de Weert, Marco; Jorgensen, Lene; Horn Moeller, Eva

    2005-01-01

    Protein pharmaceuticals have matured into an important class of drugs, now comprising one in three novel drugs introduced on the market. However, significant gains are still to be made in reducing the costs of production, ensuring proper pharmacokinetics and efficacy, increasing patient compliance...... and convenience, and reducing side effects such as immunogenicity. This review summarises these issues and provides recent examples of methods to reduce costs, alter pharmacokinetics and increase patient compliance. It also discusses the increasing interest in understanding immunogenicity in order to prevent...

  7. The importance of ADAM family proteins in malignant tumors

    Directory of Open Access Journals (Sweden)

    Katarzyna Walkiewicz

    2016-02-01

    Full Text Available Increasing numbers of reports about the role of adamalysins (ADAM in malignant tumors are being published. To date, more than 30 representatives of this group, out of which about 20 occur in humans, have been described. The ADAM family is a homogeneous group of proteins which regulate, from the stage of embryogenesis, a series of processes such as cell migration, adhesion, and cell fusion. Half of them have proteolytic activity and are involved in the degradation of the extracellular matrix and the disintegration of certain protein complexes, thereby regulating the bioavailability of various growth factors. Many of these functions have a direct role in the processes of carcinogenesis and promoting the growth of tumor, which affect some signaling pathways, including those related to insulin-like growth factors (IGF1, IGF2, vascular growth factor (VEGF, tumor necrosis factor α (TNFα and the EGFR/HER pathway. Another branch of studies is the evaluation of the possibility of using members of ADAM family proteins in the diagnosis, especially in breast, colon and non- small cell lung cancer. The detection of concentrations of adamalysin in serum, urine and pleural aspirates might contribute to the development of methods of early diagnosis of cancer and monitoring the therapy. However, both the role of adamalysins in the development and progression of tumors and their importance as a diagnostic and predictive further research still need to be checked on large groups of patients.

  8. [The importance of ADAM family proteins in malignant tumors].

    Science.gov (United States)

    Walkiewicz, Katarzyna; Gętek, Monika; Muc-Wierzgoń, Małgorzata; Kokot, Teresa; Nowakowska-Zajdel, Ewa

    2016-02-11

    Increasing numbers of reports about the role of adamalysins (ADAM) in malignant tumors are being published. To date, more than 30 representatives of this group, out of which about 20 occur in humans, have been described. The ADAM family is a homogeneous group of proteins which regulate, from the stage of embryogenesis, a series of processes such as cell migration, adhesion, and cell fusion. Half of them have proteolytic activity and are involved in the degradation of the extracellular matrix and the disintegration of certain protein complexes, thereby regulating the bioavailability of various growth factors. Many of these functions have a direct role in the processes of carcinogenesis and promoting the growth of tumor, which affect some signaling pathways, including those related to insulin-like growth factors (IGF1, IGF2), vascular growth factor (VEGF), tumor necrosis factor α (TNFα) and the EGFR/HER pathway. Another branch of studies is the evaluation of the possibility of using members of ADAM family proteins in the diagnosis, especially in breast, colon and non- small cell lung cancer. The detection of concentrations of adamalysin in serum, urine and pleural aspirates might contribute to the development of methods of early diagnosis of cancer and monitoring the therapy. However, both the role of adamalysins in the development and progression of tumors and their importance as a diagnostic and predictive further research still need to be checked on large groups of patients.

  9. Partial dispensability of Djp1's J domain in peroxisomal protein import in Saccharomyces cerevisiae results from genetic redundancy with another class II J protein, Caj1.

    Science.gov (United States)

    Dobriyal, Neha; Tripathi, Prerna; Sarkar, Susrita; Tak, Yogesh; Verma, Amit K; Sahi, Chandan

    2017-05-01

    J proteins are obligate co-chaperones of Hsp70s. Via their signature J domain, all J proteins interact with their partner Hsp70s and stimulate their weak ATPase activity, which is vital for Hsp70 functions. The dependency of J proteins on their J domain is such that mutations in critical amino acids in the J domain often results into a null phenotype for a particular J protein. Here, we show that the J domain of Djp1, a cytosolic J protein important for peroxisomal protein import in Saccharomyces cerevisiae, is partially dispensable. A complete deletion of Djp1 J domain resulted into only partial loss in peroxisomal protein import function. Instead, the C-terminal domain of Djp1 was found to be essential for proper localization of the peroxisomal targeted GFP-PTS1. Furthermore, we show that Caj1, another cytosolic J protein, also has some role in peroxisomal protein import. Caj1 was found to be partially redundant with Djp1 as cells lacking both Djp1 and Caj1 resulted into a much more severe defect in GFP-PTS1 localization. Based on these results, we propose that dispensability of J domains could be attributed to genetic redundancy between different J proteins sharing common structural topology and cellular localization.

  10. Lysine acetylation targets protein complexes and co-regulates major cellular functions

    DEFF Research Database (Denmark)

    Choudhary, Chuna Ram; Kumar, Chanchal; Gnad, Florian

    2009-01-01

    Lysine acetylation is a reversible posttranslational modification of proteins and plays a key role in regulating gene expression. Technological limitations have so far prevented a global analysis of lysine acetylation's cellular roles. We used high-resolution mass spectrometry to identify 3600......, cell cycle, splicing, nuclear transport, and actin nucleation. Acetylation impaired phosphorylation-dependent interactions of 14-3-3 and regulated the yeast cyclin-dependent kinase Cdc28. Our data demonstrate that the regulatory scope of lysine acetylation is broad and comparable with that of other...

  11. Interaction between cellular retinoic acid-binding protein II and histone hypoacetylation in renal cell carcinoma

    OpenAIRE

    Viroj Wiwanitkit

    2008-01-01

    Renal cell carcinoma is a rare but serious malignancy. Since a reduction in the level of retinoic acid receptor beta 2 (RARbeta2) expression in cancer cells due in part to histone hypoacetylation which is controlled by histone deacetylase (HD), the study on the interaction between cellular retinoic acid-binding proteins II (CRABP II), which is proposed to have its potential influence on retinoic acid (RA) response, and HD can be useful. Comparing to CARBP II and HD, the CARBP II-HD poses the ...

  12. Protein kinase Cepsilon is important for migration of neuroblastoma cells

    International Nuclear Information System (INIS)

    Stensman, Helena; Larsson, Christer

    2008-01-01

    Migration is important for the metastatic capacity and thus for the malignancy of cancer cells. There is limited knowledge on regulatory factors that promote the migration of neuroblastoma cells. This study investigates the hypothesis that protein kinase C (PKC) isoforms regulate neuroblastoma cell motility. PKC isoforms were downregulated with siRNA or modulated with activators and inhibitors. Migration was analyzed with scratch and transwell assays. Protein phosphorylation and expression levels were measured with Western blot. Stimulation with 12-O-tetradecanoylphorbol-13-acetate (TPA) induced migration of SK-N-BE(2)C neuroblastoma cells. Treatment with the general protein kinase C (PKC) inhibitor GF109203X and the inhibitor of classical isoforms Gö6976 inhibited migration while an inhibitor of PKCβ isoforms did not have an effect. Downregulation of PKCε, but not of PKCα or PKCδ, with siRNA led to a suppression of both basal and TPA-stimulated migration. Experiments using PD98059 and LY294002, inhibitors of the Erk and phosphatidylinositol 3-kinase (PI3K) pathways, respectively, showed that PI3K is not necessary for TPA-induced migration. The Erk pathway might be involved in TPA-induced migration but not in migration driven by PKCε. TPA induced phosphorylation of the PKC substrate myristoylated alanine-rich C kinase substrate (MARCKS) which was suppressed by the PKC inhibitors. Treatment with siRNA oligonucleotides against different PKC isoforms before stimulation with TPA did not influence the phosphorylation of MARCKS. PKCε is important for migration of SK-N-BE(2)C neuroblastoma cells. Neither the Erk pathway nor MARCKS are critical downstream targets of PKCε but they may be involved in TPA-mediated migration

  13. Anterior gradient protein-2 is a regulator of cellular adhesion in prostate cancer.

    Directory of Open Access Journals (Sweden)

    Diptiman Chanda

    Full Text Available Anterior Gradient Protein (AGR-2 is reported to be over-expressed in many epithelial cancers and promotes metastasis. A clear-cut mechanism for its observed function(s has not been previously identified. We found significant upregulation of AGR-2 expression in a bone metastatic prostate cancer cell line, PC3, following culturing in bone marrow-conditioned medium. Substantial AGR-2 expression was also confirmed in prostate cancer tissue specimens in patients with bone lesions. By developing stable clones of PC3 cells with varying levels of AGR-2 expression, we identified that abrogation of AGR-2 significantly reduced cellular attachment to fibronectin, collagen I, collagen IV, laminin I and fibrinogen. Loss of cellular adhesion was associated with sharp decrease in the expression of α4, α5, αV, β3 and β4 integrins. Failure to undergo apoptosis following detachment is a hallmark of epithelial cancer metastasis. The AGR-2-silenced PC3 cells showed higher resistance to Tumor necrosis factor-related apoptosis- inducing ligand (TRAIL induced apoptosis in vitro. This observation was also supported by significantly reduced Caspase-3 expression in AGR-2-silenced PC3 cells, which is a key effector of both extrinsic and intrinsic death signaling pathways. These data suggest that AGR-2 influence prostate cancer metastasis by regulation of cellular adhesion and apoptosis.

  14. Transient expression of protein tyrosine phosphatases encoded in Cotesia plutellae bracovirus inhibits insect cellular immune responses

    Science.gov (United States)

    Ibrahim, Ahmed M. A.; Kim, Yonggyun

    2008-01-01

    Several immunosuppressive factors are associated with parasitism of an endoparasitoid wasp, Cotesia plutellae, on the diamondback moth, Plutella xylostella. C. plutellae bracovirus (CpBV) encodes a large number of putative protein tyrosine phosphatases (PTPs), which may play a role in inhibiting host cellular immunity. To address this inhibitory hypothesis of CpBV-PTPs, we performed transient expression of individual CpBV-PTPs in hemocytes of the beet armyworm, Spodoptera exigua, and analyzed their cellular immune responses. Two different forms of CpBV-PTPs were chosen and cloned into a eukaryotic expression vector under the control of the p10 promoter of baculovirus: one with the normal cysteine active site (CpBV-PTP1) and the other with a mutated active site (CpBV-PTP5). The hemocytes transfected with CpBV-PTP1 significantly increased in PTP activity compared to control hemocytes, but those with CpBV-PTP5 exhibited a significant decrease in the PTP activity. All transfected hemocytes exhibited a significant reduction in both cell spreading and encapsulation activities compared to control hemocytes. Co-transfection of CpBV-PTP1 together with its double-stranded RNA reduced the messenger RNA (mRNA) level of CpBV-PTP1 and resulted in recovery of both hemocyte behaviors. This is the first report demonstrating that the polydnaviral PTPs can manipulate PTP activity of the hemocytes to interrupt cellular immune responses.

  15. Molecular basis of cellular localization of poly C binding protein 1 in neuronal cells

    International Nuclear Information System (INIS)

    Berry, Andrea M.; Flock, Kelly E.; Loh, Horace H.; Ko, Jane L.

    2006-01-01

    Poly C binding protein 1 (PCBP) is involved in the transcriptional regulation of neuronal mu-opioid receptor gene. In this study, we examined the molecular basis of PCBP cellular/nuclear localization in neuronal cells using EGFP fusion protein. PCBP, containing three KH domains and a variable domain, distributed in cytoplasm and nucleus with a preferential nuclear expression. Domain-deletional analyses suggested the requirement of variable and KH3 domains for strong PCBP nuclear expression. Within the nucleus, a low nucleolar PCBP expression was observed, and PCBP variable domain contributed to this restricted nucleolar expression. Furthermore, the punctate nuclear pattern of PCBP was correlated to its single-stranded (ss) DNA binding ability, with both requiring cooperativity of at least three sequential domains. Collectively, certain PCBP domains thus govern its nuclear distribution and transcriptional regulatory activity in the nucleus of neurons, whereas the low nucleolar expression implicates the disengagement of PCBP in the ribosomal RNA synthesis

  16. Detection of interferon alpha protein reveals differential levels and cellular sources in disease.

    Science.gov (United States)

    Rodero, Mathieu P; Decalf, Jérémie; Bondet, Vincent; Hunt, David; Rice, Gillian I; Werneke, Scott; McGlasson, Sarah L; Alyanakian, Marie-Alexandra; Bader-Meunier, Brigitte; Barnerias, Christine; Bellon, Nathalia; Belot, Alexandre; Bodemer, Christine; Briggs, Tracy A; Desguerre, Isabelle; Frémond, Marie-Louise; Hully, Marie; van den Maagdenberg, Arn M J M; Melki, Isabelle; Meyts, Isabelle; Musset, Lucile; Pelzer, Nadine; Quartier, Pierre; Terwindt, Gisela M; Wardlaw, Joanna; Wiseman, Stewart; Rieux-Laucat, Frédéric; Rose, Yoann; Neven, Bénédicte; Hertel, Christina; Hayday, Adrian; Albert, Matthew L; Rozenberg, Flore; Crow, Yanick J; Duffy, Darragh

    2017-05-01

    Type I interferons (IFNs) are essential mediators of antiviral responses. These cytokines have been implicated in the pathogenesis of autoimmunity, most notably systemic lupus erythematosus (SLE), diabetes mellitus, and dermatomyositis, as well as monogenic type I interferonopathies. Despite a fundamental role in health and disease, the direct quantification of type I IFNs has been challenging. Using single-molecule array (Simoa) digital ELISA technology, we recorded attomolar concentrations of IFNα in healthy donors, viral infection, and complex and monogenic interferonopathies. IFNα protein correlated well with functional activity and IFN-stimulated gene expression. High circulating IFNα levels were associated with increased clinical severity in SLE patients, and a study of the cellular source of IFNα protein indicated disease-specific mechanisms. Measurement of IFNα attomolar concentrations by digital ELISA will enhance our understanding of IFN biology and potentially improve the diagnosis and stratification of pathologies associated with IFN dysregulation. © 2017 Rodero et al.

  17. Inactivation of cellular enzymes by carbonyls and protein-bound glycation/glycoxidation products

    DEFF Research Database (Denmark)

    Morgan, Philip E; Dean, Roger T; Davies, Michael Jonathan

    2002-01-01

    products. In this study, we have examined the effect of glucose and carbonyl compounds (methylglyoxal, glyoxal, glycolaldehyde, and hydroxyacetone), and glycation products arising from reaction of these materials with model proteins, on the activity of three key cellular enzymes: glyceraldehyde-3-phosphate...... dehydrogenase (GAPDH), glutathione reductase, and lactate dehydrogenase, both in isolation and in cell lysates. In contrast to glucose (1M, both fresh and aged for 8 weeks), which had no effect, marked inhibition of all three enzymes was observed with methylglyoxal and glyoxal. GAPDH was also inhibited...... by glycolaldehyde and hydroxyacetone. Incubation of these enzymes with proteins that had been preglycated with methylglyoxal, but not glucose, also resulted in significant time- and concentration-dependent inhibition with both isolated enzymes and cell lysates. This inhibition was not metal ion, oxygen, superoxide...

  18. Dynamic shaping of cellular membranes by phospholipids and membrane-deforming proteins.

    Science.gov (United States)

    Suetsugu, Shiro; Kurisu, Shusaku; Takenawa, Tadaomi

    2014-10-01

    All cellular compartments are separated from the external environment by a membrane, which consists of a lipid bilayer. Subcellular structures, including clathrin-coated pits, caveolae, filopodia, lamellipodia, podosomes, and other intracellular membrane systems, are molded into their specific submicron-scale shapes through various mechanisms. Cells construct their micro-structures on plasma membrane and execute vital functions for life, such as cell migration, cell division, endocytosis, exocytosis, and cytoskeletal regulation. The plasma membrane, rich in anionic phospholipids, utilizes the electrostatic nature of the lipids, specifically the phosphoinositides, to form interactions with cytosolic proteins. These cytosolic proteins have three modes of interaction: 1) electrostatic interaction through unstructured polycationic regions, 2) through structured phosphoinositide-specific binding domains, and 3) through structured domains that bind the membrane without specificity for particular phospholipid. Among the structured domains, there are several that have membrane-deforming activity, which is essential for the formation of concave or convex membrane curvature. These domains include the amphipathic helix, which deforms the membrane by hemi-insertion of the helix with both hydrophobic and electrostatic interactions, and/or the BAR domain superfamily, known to use their positively charged, curved structural surface to deform membranes. Below the membrane, actin filaments support the micro-structures through interactions with several BAR proteins as well as other scaffold proteins, resulting in outward and inward membrane micro-structure formation. Here, we describe the characteristics of phospholipids, and the mechanisms utilized by phosphoinositides to regulate cellular events. We then summarize the precise mechanisms underlying the construction of membrane micro-structures and their involvements in physiological and pathological processes. Copyright © 2014 the

  19. Expression Profiles of Cellular Retinol-binding Protein, Type II (CRBP II in Erlang Mountainous Chickens

    Directory of Open Access Journals (Sweden)

    H. D. Yin

    2014-03-01

    Full Text Available Cellular retinol-binding protein II (CRBP II belongs to the family of cellular retinol-binding proteins and plays a major role in absorption, transport, and metabolism of vitamin A. In addition, because vitamin A is correlated with reproductive performance, we measured CRBP II mRNA abundance in erlang mountainous chickens by real-time PCR using the relative quantification method. The expression of CRBP II showed a tissue-specific pattern and egg production rate-dependent changes. The expression was very high (p<0.05 in jejunum and liver, intermediate in kidney, ovary, and oviduct, and lowest (p<0.05 in heart, hypothalamus, and pituitary. In the hypothalamus, oviduct, ovary, and pituitary, CRBP II mRNA abundance were correlated to egg production rate, which increased from 12 wk to 32 wk, peaked at 32 wk relative to the other time points, and then decreased from 32 wk to 45 wk. In contrast, the expression of CRBP II mRNA in heart, jejunum, kidney, and liver was not different at any of the ages evaluated in this study. These data may help to understand the genetic basis of vitamin A metabolism, and suggest that CRBP II may be a candidate gene to affect egg production traits in chickens.

  20. Activation of human natural killer cells by the soluble form of cellular prion protein

    Energy Technology Data Exchange (ETDEWEB)

    Seong, Yeon-Jae [Laboratory of Immunology and Infectious Diseases, Graduate School of Medical Science and Engineering, KAIST, Daejeon (Korea, Republic of); Hafis Clinic, Seoul (Korea, Republic of); Sung, Pil Soo; Jang, Young-Soon; Choi, Young Joon [Laboratory of Immunology and Infectious Diseases, Graduate School of Medical Science and Engineering, KAIST, Daejeon (Korea, Republic of); Park, Bum-Chan [Aging Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon (Korea, Republic of); Park, Su-Hyung [Laboratory of Translational Immunology and Vaccinology, Graduate School of Medical Science and Engineering, KAIST, Daejeon (Korea, Republic of); Park, Young Woo [Aging Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon (Korea, Republic of); Shin, Eui-Cheol, E-mail: ecshin@kaist.ac.kr [Laboratory of Immunology and Infectious Diseases, Graduate School of Medical Science and Engineering, KAIST, Daejeon (Korea, Republic of)

    2015-08-21

    Cellular prion protein (PrP{sup C}) is widely expressed in various cell types, including cells of the immune system. However, the specific roles of PrP{sup C} in the immune system have not been clearly elucidated. In the present study, we investigated the effects of a soluble form of recombinant PrP{sup C} protein on human natural killer (NK) cells. Recombinant soluble PrP{sup C} protein was generated by fusion of human PrP{sup C} with the Fc portion of human IgG{sub 1} (PrP{sup C}-Fc). PrP{sup C}-Fc binds to the surface of human NK cells, particularly to CD56{sup dim} NK cells. PrP{sup C}-Fc induced the production of cytokines and chemokines and the degranulation of granzyme B from NK cells. In addition, PrP{sup C}-Fc facilitated the IL-15-induced proliferation of NK cells. PrP{sup C}-Fc induced phosphorylation of ERK-1/2 and JNK in NK cells, and inhibitors of the ERK or the JNK pathways abrogated PrP{sup C}-Fc-induced cytokine production in NK cells. In conclusion, the soluble form of recombinant PrP{sup C}-Fc protein activates human NK cells via the ERK and JNK signaling pathways. - Highlights: • Recombinant soluble PrP{sup C} (PrP{sup C}-Fc) was generated by fusion of human PrP{sup C} with IgG1 Fc portion. • PrP{sup C}-Fc protein induces the production of cytokines and degranulation from human NK cells. • PrP{sup C}-Fc protein enhances the IL-15-induced proliferation of human NK cells. • PrP{sup C}-Fc protein activates human NK cells via the ERK and JNK signaling pathways.

  1. Activation of human natural killer cells by the soluble form of cellular prion protein

    International Nuclear Information System (INIS)

    Seong, Yeon-Jae; Sung, Pil Soo; Jang, Young-Soon; Choi, Young Joon; Park, Bum-Chan; Park, Su-Hyung; Park, Young Woo; Shin, Eui-Cheol

    2015-01-01

    Cellular prion protein (PrP C ) is widely expressed in various cell types, including cells of the immune system. However, the specific roles of PrP C in the immune system have not been clearly elucidated. In the present study, we investigated the effects of a soluble form of recombinant PrP C protein on human natural killer (NK) cells. Recombinant soluble PrP C protein was generated by fusion of human PrP C with the Fc portion of human IgG 1 (PrP C -Fc). PrP C -Fc binds to the surface of human NK cells, particularly to CD56 dim NK cells. PrP C -Fc induced the production of cytokines and chemokines and the degranulation of granzyme B from NK cells. In addition, PrP C -Fc facilitated the IL-15-induced proliferation of NK cells. PrP C -Fc induced phosphorylation of ERK-1/2 and JNK in NK cells, and inhibitors of the ERK or the JNK pathways abrogated PrP C -Fc-induced cytokine production in NK cells. In conclusion, the soluble form of recombinant PrP C -Fc protein activates human NK cells via the ERK and JNK signaling pathways. - Highlights: • Recombinant soluble PrP C (PrP C -Fc) was generated by fusion of human PrP C with IgG1 Fc portion. • PrP C -Fc protein induces the production of cytokines and degranulation from human NK cells. • PrP C -Fc protein enhances the IL-15-induced proliferation of human NK cells. • PrP C -Fc protein activates human NK cells via the ERK and JNK signaling pathways

  2. Structures of non-cellular tissues of the body and their importance in otorhinolaryngology

    Directory of Open Access Journals (Sweden)

    S. N. Shatokhina

    2016-01-01

    Full Text Available We present  the  results of our studies  in various pathological   conditions   in  otorhinolaryngology performed  with  a diagnostic  technology  of functional morphology  of non-cellular tissue structures  (mouth  fluid, surgical wound  exudation, blood  serum, and  others. With the  use of methods of cuniform and  marginal dehydration of biological fluids, the possibility of developing essentially novel criteria was shown, such as:• prediction  of complicated  course of post-operative wound  healing in subjects with a lamellar morphotype in the  wound  exudation  resulting from cholesterol residues due to massive cell death;• prediction  of a polypous  rhinosinusitis  relapse in subjects with an increase in the proliferation marker, anisotropic  parallels lines in the  dehydrated  serum  obtained from the  blood  taken from the inferior nasal turbinate;• diagnostics   of  the  middle  ear  cholesteatoma in children by combination of cuniform and marginal  dehydration of the  mouth  fluid. The singularity of the  technique  is based  on triple sampling  of the  fluid:  first sample  was  taken immediately  after awakening, the  second  one, after a few minutes  of active swallowing movements  and  the  third one,  after trans-tympanicair pumping.  Detection  of the  structural  signs of congestive effusion and the lamellar morphotype as a destruction marker in the third sample suggested the presence  of cholesteatoma;• assessment  of  treatment  efficacy  in  patients with chronic tonsillitis and of the indications to tonsillectomy in patients  with persisting pathological  characteristics   of  the  exudation   from the palatal tonsil lacunes throughout the whole course of conservative treatment;• determination of the grade of activity / absence of activity of laryngeal cancer by identification of a basic spherolith with various degrees  of anisotropy

  3. Click chemistry for the conservation of cellular structures and fluorescent proteins: ClickOx.

    Science.gov (United States)

    Löschberger, Anna; Niehörster, Thomas; Sauer, Markus

    2014-05-01

    Reactive oxygen species (ROS), including hydrogen peroxide, are known to cause structural damage not only in living, but also in fixed, cells. Copper-catalyzed azide-alkyne cycloaddition (click chemistry) is known to produce ROS. Therefore, fluorescence imaging of cellular structures, such as the actin cytoskeleton, remains challenging when combined with click chemistry protocols. In addition, the production of ROS substantially weakens the fluorescence signal of fluorescent proteins. This led us to develop ClickOx, which is a new click chemistry protocol for improved conservation of the actin structure and better conservation of the fluorescence signal of green fluorescent protein (GFP)-fusion proteins. Herein we demonstrate that efficient oxygen removal by addition of an enzymatic oxygen scavenger system (ClickOx) considerably reduces ROS-associated damage during labeling of nascent DNA with ATTO 488 azide by Cu(I)-catalyzed click chemistry. Standard confocal and super-resolution fluorescence images of phalloidin-labeled actin filaments and GFP/yellow fluorescent protein-labeled cells verify the conservation of the cytoskeleton microstructure and fluorescence intensity, respectively. Thus, ClickOx can be used advantageously for structure preservation in conventional and most notably in super-resolution microscopy methods. Copyright © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  4. Plant storage proteins – the main nourisching products – from biosynthesis to cellular storage depots

    Directory of Open Access Journals (Sweden)

    Agnieszka Chmielnicka

    2017-06-01

    Full Text Available Storage proteins of legumes are one of the main components of the human and animal diet. The substances collected in their seeds have the pro-health values, supporting the prevention of many civilization diseases. However, there are still many uncertainties about the mechanisms leading to the production of nutritious seeds. It is also difficult to identify which of their constituents and in what final form are responsible for the observed protective effects in vivo. In this work, on the background of different types of storage proteins, these deposited mainly in legumes were in the focus of interest. They were characterized on the example of pea (Pisum sativum proteins. Mechanisms associated with their biosynthesis and transport to specific cellular compartments was presented. Ways of their post-translational processing, segregation and storage in the specific vacuoles were also discussed. Therefore, the paper presents the state-of-the-art knowledge concerning the processes making the accumulated protein deposits ready to use by plants, animals and humans.

  5. A Graphical User Interface for Software-assisted Tracking of Protein Concentration in Dynamic Cellular Protrusions.

    Science.gov (United States)

    Saha, Tanumoy; Rathmann, Isabel; Galic, Milos

    2017-07-11

    Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex signaling mechanisms underlying filopodial initiation, elongation and subsequent stabilization or retraction, it is crucial to determine the spatio-temporal protein activity in these dynamic structures. To analyze protein function in filopodia, we recently developed a semi-automated tracking algorithm that adapts to filopodial shape-changes, thus allowing parallel analysis of protrusion dynamics and relative protein concentration along the whole filopodial length. Here, we present a detailed step-by-step protocol for optimized cell handling, image acquisition and software analysis. We further provide instructions for the use of optional features during image analysis and data representation, as well as troubleshooting guidelines for all critical steps along the way. Finally, we also include a comparison of the described image analysis software with other programs available for filopodia quantification. Together, the presented protocol provides a framework for accurate analysis of protein dynamics in filopodial protrusions using image analysis software.

  6. Dual personality of Mad1: regulation of nuclear import by a spindle assembly checkpoint protein.

    Science.gov (United States)

    Cairo, Lucas V; Ptak, Christopher; Wozniak, Richard W

    2013-01-01

    Nuclear transport is a dynamic process that can be modulated in response to changes in cellular physiology. We recently reported that the transport activity of yeast nuclear pore complexes (NPCs) is altered in response to kinetochore-microtubule (KT-MT) interaction defects. Specifically, KT detachment from MTs activates a signaling pathway that prevents the nuclear import of cargos by the nuclear transport factor Kap121p. This loss of Kap121p-mediated import is thought to influence the nuclear environment, including the phosphorylation state of nuclear proteins. A key regulator of this process is the spindle assembly checkpoint protein Mad1p. In response to unattached KTs, Mad1p dynamically cycles between NPCs and KTs. This cycling appears to induce NPC molecular rearrangements that prevent the nuclear import of Kap121p-cargo complexes. Here, we discuss the underlying mechanisms and the physiological relevance of Mad1p cycling and the inhibition of Kap121p-mediated nuclear import, focusing on outstanding questions within the pathway.

  7. Expression of the Major Vault Protein (MVP) and Cellular Vault Particles in Fish.

    Science.gov (United States)

    Margiotta, Alyssa L; Bain, Lisa J; Rice, Charles D

    2017-11-01

    Cellular vaults are ubiquitous 13 mega Da multi-subunit ribonuceloprotein particles that may have a role in nucleocytoplasmic transport. Seventy percent of the vault's mass consists of a ≈100 kDa protein, the major vault protein (MVP). In humans, a drug resistance-associated protein, originally identified as lung resistance protein in metastatic lung cancer, was ultimately shown to be the previously described MVP. In this study, a partial MVP sequence was cloned from channel catfish. Recombinant MVP (rMVP) was used to generate a monoclonal antibody that recognizes full length protein in distantly related fish species, as well as mice. MVP is expressed in fish spleen, liver, anterior kidney, renal kidney, and gills, with a consistent expression in epithelial cells, macrophages, or endothelium at the interface of the tissue and environment or vasculature. We show that vaults are distributed throughout cells of fish lymphoid cells, with nuclear and plasma membrane aggregations in some cells. Protein expression studies were extended to liver neoplastic lesions in Atlantic killifish collected in situ at the Atlantic Wood USA-EPA superfund site on the southern branch of the Elizabeth River, VA. MVP is highly expressed in these lesions, with intense staining at the nuclear membrane, similar to what is known about MVP expression in human liver neoplasia. Additionally, MVP mRNA expression was quantified in channel catfish ovarian cell line following treatment with different classes of pharmacological agents. Notably, mRNA expression is induced by ethidium bromide, which damages DNA. Anat Rec, 2017. © 2017 Wiley Periodicals, Inc. Anat Rec, 300:1981-1992, 2017. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

  8. Influence of HFE variants and cellular iron on monocyte chemoattractant protein-1

    Directory of Open Access Journals (Sweden)

    Simmons Zachary

    2009-02-01

    Full Text Available Abstract Background Polymorphisms in the MHC class 1-like gene known as HFE have been proposed as genetic modifiers of neurodegenerative diseases that include neuroinflammation as part of the disease process. Variants of HFE are relatively common in the general population and are most commonly associated with iron overload, but can promote subclinical cellular iron loading even in the absence of clinically identified disease. The effects of the variants as well as the resulting cellular iron dyshomeostasis potentially impact a number of disease-associated pathways. We tested the hypothesis that the two most common HFE variants, H63D and C282Y, would affect cellular secretion of cytokines and trophic factors. Methods We screened a panel of cytokines and trophic factors using a multiplexed immunoassay in human neuroblastoma SH-SY5Y cells expressing different variants of HFE. The influence of cellular iron secretion on the potent chemokine monocyte chemoattractant protein-1 (MCP-1 was assessed using ferric ammonium citrate and the iron chelator, desferroxamine. Additionally, an antioxidant, Trolox, and an anti-inflammatory, minocycline, were tested for their effects on MCP-1 secretion in the presence of HFE variants. Results Expression of the HFE variants altered the labile iron pool in SH-SY5Y cells. Of the panel of cytokines and trophic factors analyzed, only the release of MCP-1 was affected by the HFE variants. We further examined the relationship between iron and MCP-1 and found MCP-1 secretion tightly associated with intracellular iron status. A potential direct effect of HFE is considered because, despite having similar levels of intracellular iron, the association between HFE genotype and MCP-1 expression was different for the H63D and C282Y HFE variants. Moreover, HFE genotype was a factor in the effect of minocycline, a multifaceted antibiotic used in treating a number of neurologic conditions associated with inflammation, on MCP-1

  9. Localization of cellular retinol-binding protein and retinol-binding protein in cells comprising the blood-brain barrier of rat and human

    International Nuclear Information System (INIS)

    MacDonald, P.N.; Ong, D.E.; Bok, D.

    1990-01-01

    Brain is not generally recognized as an organ that requires vitamin A, perhaps because no obvious histologic lesions have been observed in severely vitamin A-deficient animals. However, brain tissue does contain cellular vitamin A-binding proteins and a nuclear receptor protein for retinoic acid. In the present study, immunohistochemical techniques were used to determine the cell-specific location of cellular retinol-binding protein in human and rat brain tissue. Cellular retinol-binding protein was localized specifically within the cuboidal epithelial cells of the choroid plexus, two primary sites of the mammalian blood-brain barrier. In addition, autoradiographic procedures demonstrated binding sites for serum retinol-binding protein in the choroidal epithelium. These observations suggest that a significant movement of retinol across the blood-brain barrier may occur

  10. LiZIP3 is a cellular zinc transporter that mediates the tightly regulated import of zinc in Leishmania infantum parasites

    Science.gov (United States)

    Carvalho, Sandra; da Silva, Rosa Barreira; Shawki, Ali; Castro, Helena; Lamy, Márcia; Eide, David; Costa, Vítor; Mackenzie, Bryan; Tomás, Ana M.

    2016-01-01

    Summary Cellular zinc homeostasis ensures that the intracellular concentration of this element is kept within limits that enable its participation in critical physiological processes without exerting toxic effects. We report here the identification and characterization of the first mediator of zinc homeostasis in Leishmania infantum, LiZIP3, a member of the ZIP family of divalent metal-ion transporters. The zinc transporter activity of LiZIP3 was first disclosed by its capacity to rescue the growth of Saccharomyces cerevisiae strains deficient in zinc acquisition. Subsequent expression of LiZIP3 in Xenopus laevis oocytes was shown to stimulate the uptake of a broad range of metal ions, among which Zn2+ was the preferred LiZIP3 substrate (K0.5 ≈ 0.1 μM). Evidence that LiZIP3 functions as a zinc importer in L. infantum came from the observations that the protein locates to the cell membrane and that its overexpression leads to augmented zinc internalization. Importantly, expression and cell-surface location of LiZIP3 are lost when parasites face high zinc bioavailability. LiZIP3 decline in response to zinc is regulated at the mRNA level in a process involving (a) short-lived protein(s). Collectively, our data reveal that LiZIP3 enables L. infantum to acquire zinc in a highly regulated manner, hence contributing to zinc homeostasis. PMID:25644708

  11. Cellular inhibitor of apoptosis protein 2 (cIAP2) controls human colonic epithelial restitution, migration and Rac1 activation

    DEFF Research Database (Denmark)

    Seidelin, JB; Larsen, Sylvester; Linnemann, D

    2015-01-01

    epithelial cells (IECs) was increased at the wound edge after 24 h (P 2 was induced in vitro in regenerating Caco2 IECs after wound infliction (P ...Identification of pathways involved in wound healing is important for understanding the pathogenesis of various intestinal diseases. Cellular inhibitor of apoptosis protein 2 (cIAP2) regulates proliferation and migration in nonepithelial cells and is expressed in human colonocytes. The aim...... of the study was to investigate the role of cIAP2 for wound healing in the normal human colon. Wound tissue was generated by taking rectosigmoidal biopsies across an experimental ulcer in healthy subjects after 5, 24, and 48 h. In experimental ulcers, the expression of cIAP2 in regenerating intestinal...

  12. Magnolol Affects Cellular Proliferation, Polyamine Biosynthesis and Catabolism-Linked Protein Expression and Associated Cellular Signaling Pathways in Human Prostate Cancer Cells in vitro

    Directory of Open Access Journals (Sweden)

    Brendan T. McKeown

    2015-01-01

    Full Text Available Background: Prostate cancer is the most commonly diagnosed form of cancer in men in Canada and the United States. Both genetic and environmental factors contribute to the development and progression of many cancers, including prostate cancer. Context and purpose of this study: This study investigated the effects of magnolol, a compound found in the roots and bark of the magnolia tree Magnolia officinalis, on cellular proliferation and proliferation-linked activities of PC3 human prostate cancer cells in vitro. Results: PC3 cells exposed to magnolol at a concentration of 80 μM for 6 hours exhibited decreased protein expression of ornithine decarboxylase, a key regulator in polyamine biosynthesis, as well as affecting the expression of other proteins involved in polyamine biosynthesis and catabolism. Furthermore, protein expression of the R2 subunit of ribonucleotide reductase, a key regulatory protein associated with DNA synthesis, was significantly decreased. Finally, the MAPK (mitogen-activated protein kinase, PI3K (phosphatidylinositol 3-kinase, NFκB (nuclear factor of kappa-light-chain-enhancer of activated B cells and AP-1 (activator protein 1 cellular signaling pathways were assayed to determine which, if any, of these pathways magnolol exposure would alter. Protein expressions of p-JNK-1 and c-jun were significantly increased while p-p38, JNK-1/2, PI3Kp85, p-PI3Kp85, p-Akt, NFκBp65, p-IκBα and IκBα protein expressions were significantly decreased. Conclusions: These alterations further support the anti-proliferative effects of magnolol on PC3 human prostate cancer cells in vitro and suggest that magnolol may have potential as a novel anti-prostate cancer agent.

  13. GCK-MODY diabetes associated with protein misfolding, cellular self-association and degradation.

    Science.gov (United States)

    Negahdar, Maria; Aukrust, Ingvild; Johansson, Bente B; Molnes, Janne; Molven, Anders; Matschinsky, Franz M; Søvik, Oddmund; Kulkarni, Rohit N; Flatmark, Torgeir; Njølstad, Pål Rasmus; Bjørkhaug, Lise

    2012-11-01

    GCK-MODY, dominantly inherited mild fasting hyperglycemia, has been associated with >600 different mutations in the glucokinase (GK)-encoding gene (GCK). When expressed as recombinant pancreatic proteins, some mutations result in enzymes with normal/near-normal catalytic properties. The molecular mechanism(s) of GCK-MODY due to these mutations has remained elusive. Here, we aimed to explore the molecular mechanisms for two such catalytically 'normal' GCK mutations (S263P and G264S) in the F260-L270 loop of GK. When stably overexpressed in HEK293 cells and MIN6 β-cells, the S263P- and G264S-encoded mutations generated misfolded proteins with an increased rate of degradation (S263P>G264S) by the protein quality control machinery, and a propensity to self-associate (G264S>S263P) and form dimers (SDS resistant) and aggregates (partly Triton X-100 insoluble), as determined by pulse-chase experiments and subcellular fractionation. Thus, the GCK-MODY mutations S263P and G264S lead to protein misfolding causing destabilization, cellular dimerization/aggregation and enhanced rate of degradation. In silico predicted conformational changes of the F260-L270 loop structure are considered to mediate the dimerization of both mutant proteins by a domain swapping mechanism. Thus, similar properties may represent the molecular mechanisms for additional unexplained GCK-MODY mutations, and may also contribute to the disease mechanism in other previously characterized GCK-MODY inactivating mutations. Copyright © 2012 Elsevier B.V. All rights reserved.

  14. Upon Infection the Cellular WD Repeat-containing Protein 5 (WDR5) Localizes to Cytoplasmic Inclusion Bodies and Enhances Measles Virus Replication.

    Science.gov (United States)

    Ma, Dzwokai; George, Cyril X; Nomburg, Jason; Pfaller, Christian K; Cattaneo, Roberto; Samuel, Charles E

    2017-12-13

    Replication of negative-strand RNA viruses occurs in association with discrete cytoplasmic foci called inclusion bodies. Whereas inclusion bodies represent a prominent subcellular structure induced by viral infection, our knowledge of the cellular protein components involved in inclusion body formation and function is limited. Using measles virus-infected HeLa cells, we found that the WD repeat-containing protein 5 (WDR5), a subunit of histone H3 lysine 4 methyltransferases, was selectively recruited to virus-induced inclusion bodies. Furthermore, WDR5 was found in complexes containing viral proteins associated with RNA replication. WDR5 was not detected with mitochondria, stress granules, or other known secretory or endocytic compartments of infected cells. WDR5 deficiency decreased both viral protein production and infectious virus yields. Interferon production was modestly increased in WDR5 deficient cells. Thus, our study identifies WDR5 as a novel viral inclusion body-associated cellular protein and suggests a role for WDR5 in promoting viral replication. IMPORTANCE Measles virus is a human pathogen that remains a global concern with more than 100,000 measles-related deaths annually despite the availability of an effective vaccine. As measles continues to cause significant morbidity and mortality, understanding the virus-host interactions at the molecular level that affect virus replication efficiency is important for development and optimization of treatment procedures. Measles virus is an RNA virus that encodes six genes and replicates in the cytoplasm of infected cells in discrete cytoplasmic replication bodies, though little is known of the biochemical nature of these structures. Here we show that the cellular protein WDR5 is enriched in the cytoplasmic viral replication factories and enhances virus growth. WDR5-containing protein complex includes viral proteins responsible for viral RNA replication. Thus, we have identified WDR5 as a host factor that

  15. Protein misfolding and cellular stress in disease and ageing - Concepts and protocols

    Directory of Open Access Journals (Sweden)

    Carlo Alberto Redi

    2011-09-01

    Full Text Available To those readers that already got the Protein misfolding and disease volume, this new title can sound as an update or a second edition of the previous volume: well, this is not the case. To those colleagues that would like to enter the fascinating field of protein’s misfolding this new volume constitutes an excellent opportunity to be driven on the causes and mechanisms that are actually know to produce the misfolding. For both types of scientists this volume is a must: the subtitle already sounds as a warning since it reads concepts and protocols rather than methods and protocols. In other words, there is an entire section (part I, chapters 1-8 devoted to explain the concepts and the approaches we have gathered in these last years on the misfolding. These chapters are presented in the review style so that the relevant bibliographies are all there; in addition, this section is presenting the conceptual paradigma linking protein misfolding to ageing by conceiving the disease itself as premature ageing processes. Thus, the reader can take profit from this first part and become acquiented on the molecular effects brought about by protein misfolding at a cellular level and, generally speaking, on the pathogenetic mechanisms thereafter triggered by soluble prefibrillar aggregates...

  16. Invited review: Whey proteins as antioxidants and promoters of cellular antioxidant pathways.

    Science.gov (United States)

    Corrochano, Alberto R; Buckin, Vitaly; Kelly, Phil M; Giblin, Linda

    2018-03-28

    Oxidative stress contributes to cell injury and aggravates several chronic diseases. Dietary antioxidants help the body to fight against free radicals and, therefore, avoid or reduce oxidative stress. Recently, proteins from milk whey liquid have been described as antioxidants. This review summarizes the evidence that whey products exhibit radical scavenging activity and reducing power. It examines the processing and treatment attempts to increase the antioxidant bioactivity and identifies 1 enzyme, subtilisin, which consistently produces the most potent whey fractions. The review compares whey from different milk sources and puts whey proteins in the context of other known food antioxidants. However, for efficacy, the antioxidant activity of whey proteins must not only survive processing, but also upper gut transit and arrival in the bloodstream, if whey products are to promote antioxidant levels in target organs. Studies reveal that direct cell exposure to whey samples increases intracellular antioxidants such as glutathione. However, the physiological relevance of these in vitro assays is questionable, and evidence is conflicting from dietary intervention trials, with both rats and humans, that whey products can boost cellular antioxidant biomarkers. Copyright © 2018 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

  17. Highly efficient enzyme encapsulation in a protein nanocage: towards enzyme catalysis in a cellular nanocompartment mimic

    Science.gov (United States)

    Schoonen, Lise; Nolte, Roeland J. M.; van Hest, Jan C. M.

    2016-07-01

    The study of enzyme behavior in small nanocompartments is crucial for the understanding of biocatalytic processes in the cellular environment. We have developed an enzymatic conjugation strategy to attach a model enzyme to the interior of a cowpea chlorotic mottle virus capsid. It is shown that with this methodology high encapsulation efficiencies can be achieved. Additionally, we demonstrate that the encapsulation does not affect the enzyme performance in terms of a decreased activity or a hampered substrate diffusion. Finally, it is shown that the encapsulated enzymes are protected against proteases. We believe that our strategy can be used to study enzyme kinetics in an environment that approaches physiological conditions.The study of enzyme behavior in small nanocompartments is crucial for the understanding of biocatalytic processes in the cellular environment. We have developed an enzymatic conjugation strategy to attach a model enzyme to the interior of a cowpea chlorotic mottle virus capsid. It is shown that with this methodology high encapsulation efficiencies can be achieved. Additionally, we demonstrate that the encapsulation does not affect the enzyme performance in terms of a decreased activity or a hampered substrate diffusion. Finally, it is shown that the encapsulated enzymes are protected against proteases. We believe that our strategy can be used to study enzyme kinetics in an environment that approaches physiological conditions. Electronic supplementary information (ESI) available: Experimental procedures for the cloning, expression, and purification of all proteins, as well as supplementary figures and calculations. See DOI: 10.1039/c6nr04181g

  18. A DEAD box protein facilitates HIV-1 replication as a cellular co-factor of Rev

    International Nuclear Information System (INIS)

    Fang Jianhua; Kubota, Satoshi; Yang Bin; Zhou Naiming; Zhang Hui; Godbout, Roseline; Pomerantz, Roger J.

    2004-01-01

    HIV-1 Rev escorts unspliced viral mRNAs out of the nucleus of infected cells, which allows formation of infectious HIV-1 virions. We have identified a putative DEAD box (Asp-Glu-Ala-Asp) RNA helicase, DDX1, as a cellular co-factor of Rev, through yeast and mammalian two-hybrid systems using the N-terminal motif of Rev as 'bait'. DDX1 is not a functional homolog of HIV-1 Rev, but down-regulation of DDX1 resulted in an alternative splicing pattern of Rev-responsive element (RRE)-containing mRNA, and attenuation of Gag p24 antigen production from HLfb rev(-) cells rescued by exogenous Rev. Co-transfection of a DDX1 expression vector with HIV-1 significantly increased viral production. DDX1 binding to Rev, as well as to the RRE, strongly suggest that DDX1 affects Rev function through the Rev-RRE axis. Moreover, down-regulation of DDX1 altered the steady state subcellular distribution of Rev, from nuclear/nucleolar to cytoplasmic dominance. These findings indicate that DDX1 is a critical cellular co-factor for Rev function, which maintains the proper subcellular distribution of this lentiviral regulatory protein. Therefore, alterations in DDX1-Rev interactions could induce HIV-1 persistence and targeting DDX1 may lead to rationally designed and novel anti-HIV-1 strategies and therapeutics

  19. The importance of protein in leaf selection of folivorous primates.

    Science.gov (United States)

    Ganzhorn, Joerg U; Arrigo-Nelson, Summer J; Carrai, Valentina; Chalise, Mukesh K; Donati, Giuseppe; Droescher, Iris; Eppley, Timothy M; Irwin, Mitchell T; Koch, Flávia; Koenig, Andreas; Kowalewski, Martin M; Mowry, Christopher B; Patel, Erik R; Pichon, Claire; Ralison, Jose; Reisdorff, Christoph; Simmen, Bruno; Stalenberg, Eleanor; Starrs, Danswell; Terboven, Juana; Wright, Patricia C; Foley, William J

    2017-04-01

    Protein limitation has been considered a key factor in hypotheses on the evolution of life history and animal communities, suggesting that animals should prioritize protein in their food choice. This contrasts with the limited support that food selection studies have provided for such a priority in nonhuman primates, particularly for folivores. Here, we suggest that this discrepancy can be resolved if folivores only need to select for high protein leaves when average protein concentration in the habitat is low. To test the prediction, we applied meta-analyses to analyze published and unpublished results of food selection for protein and fiber concentrations from 24 studies (some with multiple species) of folivorous primates. To counter potential methodological flaws, we differentiated between methods analyzing total nitrogen and soluble protein concentrations. We used a meta-analysis to test for the effect of protein on food selection by primates and found a significant effect of soluble protein concentrations, but a non-significant effect for total nitrogen. Furthermore, selection for soluble protein was reinforced in forests where protein was less available. Selection for low fiber content was significant but unrelated to the fiber concentrations in representative leaf samples of a given forest. There was no relationship (either negative or positive) between the concentration of protein and fiber in the food or in representative samples of leaves. Overall our study suggests that protein selection is influenced by the protein availability in the environment, explaining the sometimes contradictory results in previous studies on protein selection. Am. J. Primatol. 79:e22550, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  20. Interaction between cellular retinoic acid-binding protein II and histone hypoacetylation in renal cell carcinoma

    Directory of Open Access Journals (Sweden)

    Viroj Wiwanitkit

    2008-04-01

    Full Text Available Renal cell carcinoma is a rare but serious malignancy. Since a reduction in the level of retinoic acid receptor beta 2 (RARbeta2 expression in cancer cells due in part to histone hypoacetylation which is controlled by histone deacetylase (HD, the study on the interaction between cellular retinoic acid-binding proteins II (CRABP II, which is proposed to have its potential influence on retinoic acid (RA response, and HD can be useful. Comparing to CARBP II and HD, the CARBP II-HD poses the same function and biological process as HD. This can confirm that HD has a significant suppressive effect on the expression of CARBP II. Therefore, reduction in the level of RARbeta2 expression in cancer cells can be expected and this can lead to failure in treatment of renal cell carcinoma with RA. The author hereby purpose that additional HD inhibitor should be added into the regiment of RA to increase the effectiveness of treatment.

  1. The induced expression of heat shock proteins as a part of the early cellular response to gamma radiation

    International Nuclear Information System (INIS)

    Stankova, K.; Ivanova, K.; Georgieva, R.; Rupova, I.; Boteva, R.

    2008-01-01

    A variety of stressful stimuli including gamma radiation can induce increase in the synthesis of heat shock proteins (Hsp). This family of molecular chaperones includes members with molecular masses ranging from 10 to 150 kDa and has been identified in all organisms from bacteria to humans. Hsp70 chaperones are very important. The present study aimed to characterize the radiation-induced changes in Hsp70 synthesis in human lymphocytes as a part of the early cellular response to gamma irradiation. The expression of Hsp70 was determined with Western blot and the radiation-induced apoptotic changes were registered by staining with fluorescent dyes. Part of the experiments were performed in the presence of the organic solvent DMSO. At low concentrations this reagent shows antioxidant activity and can reduce the level of the radiation-induced oxidant stress which determines the predominant biological effects of the ionizing radiation. Irradiation with 0.5 to 8 Gy caused statistically significant increase in the synthesis of Hsp70 which was strongest after irradiation with 4 Gy. In the range 0.5-2 Gy the enhancement of the radiation-induced synthesis of Hsp70 reached 60%. Our experimental results characterize changes in the Hsp70 synthesis after gamma irradiation as a part of the early cellular stress response in lymphocytes. (authors)

  2. Drosophila melanogaster cellular repressor of E1A-stimulated genes is a lysosomal protein essential for fly development

    OpenAIRE

    Kowalewski-Nimmerfall, Elisabeth; Sch?hs, Philipp; Maresch, Daniel; Rendic, Dubravko; Kr?mer, Helmut; Mach, Lukas

    2014-01-01

    Mammalian cellular repressor of E1A-stimulated genes is a lysosomal glycoprotein implicated in cellular growth and differentiation. The genome of the fruit fly Drosophila melanogaster encodes a putative orthologue (dCREG), suggesting evolutionarily conserved physiological functions of this protein. In D. melanogaster S2 cells, dCREG was found to localize in lysosomes. Further studies revealed that intracellular dCREG is subject of proteolytic maturation. Processing and turnover could be subst...

  3. Involvement of Sib Proteins in the Regulation of Cellular Adhesion in Dictyostelium discoideum▿ †

    OpenAIRE

    Cornillon, Sophie; Froquet, Romain; Cosson, Pierre

    2008-01-01

    Molecular mechanisms ensuring cellular adhesion have been studied in detail in Dictyostelium amoebae, but little is known about the regulation of cellular adhesion in these cells. Here, we show that cellular adhesion is regulated in Dictyostelium, notably by the concentration of a cellular secreted factor accumulating in the medium. This constitutes a quorum-sensing mechanism allowing coordinated regulation of cellular adhesion in a Dictyostelium population. In order to understand the mechani...

  4. Engineered mutations in fibrillin-1 leading to Marfan syndrome act at the protein, cellular and organismal levels.

    Science.gov (United States)

    Zeyer, Karina A; Reinhardt, Dieter P

    2015-01-01

    Fibrillins are the major components of microfibrils in the extracellular matrix of elastic and non-elastic tissues. They are multi-domain proteins, containing primarily calcium binding epidermal growth factor-like (cbEGF) domains and 8-cysteine/transforming growth factor-beta binding protein-like (TB) domains. Mutations in the fibrillin-1 gene give rise to Marfan syndrome, a connective tissue disorder with clinical complications in the cardiovascular, skeletal, ocular and other organ systems. Here, we review the consequences of engineered Marfan syndrome mutations in fibrillin-1 at the protein, cellular and organismal levels. Representative point mutations associated with Marfan syndrome in affected individuals have been introduced and analyzed in recombinant fibrillin-1 fragments. Those mutations affect fibrillin-1 on a structural and functional level. Mutations which impair folding of cbEGF domains can affect protein trafficking. Protein folding disrupted by some mutations can lead to defective secretion in mutant fibrillin-1 fragments, whereas fragments with other Marfan mutations are secreted normally. Many Marfan mutations render fibrillin-1 more susceptible to proteolysis. There is also evidence that some mutations affect heparin binding. Few mutations have been further analyzed in mouse models. An extensively studied mouse model of Marfan syndrome expresses mouse fibrillin-1 with a missense mutation (p.C1039G). The mice display similar characteristics to human patients with Marfan syndrome. Overall, the analyses of engineered mutations leading to Marfan syndrome provide important insights into the pathogenic molecular mechanisms exerted by mutated fibrillin-1. Copyright © 2015 Elsevier B.V. All rights reserved.

  5. The yeast mitogen-activated protein kinase Slt2 is involved in the cellular response to genotoxic stress

    Directory of Open Access Journals (Sweden)

    Soriano-Carot María

    2012-02-01

    Full Text Available Abstract Background The maintenance of genomic integrity is essential for cell viability. Complex signalling pathways (DNA integrity checkpoints mediate the response to genotoxic stresses. Identifying new functions involved in the cellular response to DNA-damage is crucial. The Saccharomyces cerevisiae SLT2 gene encodes a member of the mitogen-activated protein kinase (MAPK cascade whose main function is the maintenance of the cell wall integrity. However, different observations suggest that SLT2 may also have a role related to DNA metabolism. Results This work consisted in a comprehensive study to connect the Slt2 protein to genome integrity maintenance in response to genotoxic stresses. The slt2 mutant strain was hypersensitive to a variety of genotoxic treatments, including incubation with hydroxyurea (HU, methylmetanosulfonate (MMS, phleomycin or UV irradiation. Furthermore, Slt2 was activated by all these treatments, which suggests that Slt2 plays a central role in the cellular response to genotoxic stresses. Activation of Slt2 was not dependent on the DNA integrity checkpoint. For MMS and UV, Slt2 activation required progression through the cell cycle. In contrast, HU also activated Slt2 in nocodazol-arrested cells, which suggests that Slt2 may respond to dNTP pools alterations. However, neither the protein level of the distinct ribonucleotide reductase subunits nor the dNTP pools were affected in a slt2 mutant strain. An analysis of the checkpoint function revealed that Slt2 was not required for either cell cycle arrest or the activation of the Rad53 checkpoint kinase in response to DNA damage. However, slt2 mutant cells showed an elongated bud and partially impaired Swe1 degradation after replicative stress, indicating that Slt2 could contribute, in parallel with Rad53, to bud morphogenesis control after genotoxic stresses. Conclusions Slt2 is activated by several genotoxic treatments and is required to properly cope with DNA damage. Slt

  6. Physiological Importance and Mechanisms of Protein Hydrolysate Absorption

    Science.gov (United States)

    Zhanghi, Brian M.; Matthews, James C.

    Understanding opportunities to maximize the efficient digestion and assimilation by production animals of plant- and animal-derived protein products is critical for farmers, nutritionists, and feed manufacturers to sustain and expand the affordable production of high quality animal products for human consumption. The challenge to nutritionists is to match gastrointestinal tract load to existing or ­inducible digestive and absorptive capacities. The challenge to feed manufacturers is to develop products that are efficient substrates for digestion, absorption, and/or both events. Ultimately, the efficient absorption of digesta proteins depends on the mediated passage (transport) of protein hydrosylate products as dipeptides and unbound amino acids across the lumen- and blood-facing membranes of intestinal absorptive cells. Data testing the relative efficiency of supplying protein as hydrolysates or specific dipeptides versus as free amino acids, and the response of animals in several physiological states to feeding of protein hydrolysates, are presented and reviewed in this chapter. Next, data describing the transport mechanisms responsible for absorbing protein hydrolysate digestion products, and the known and putative regulation of these mechanisms by their substrates (small peptides) and hormones are presented and reviewed. Several conclusions are drawn regarding the efficient use of protein hydrolysate-based diets for particular physiological states, the economically-practical application of which likely will depend on technological advances in the manufacture of protein hydrolysate products.

  7. Nuclear imprisonment of host cellular mRNA by nsp1β protein of porcine reproductive and respiratory syndrome virus

    International Nuclear Information System (INIS)

    Han, Mingyuan; Ke, Hanzhong; Zhang, Qingzhan; Yoo, Dongwan

    2017-01-01

    Positive-strand RNA genomes function as mRNA for viral protein synthesis which is fully reliant on host cell translation machinery. Competing with cellular protein translation apparatus needs to ensure the production of viral proteins, but this also stifles host innate defense. In the present study, we showed that porcine reproductive and respiratory syndrome virus (PRRSV), whose replication takes place in the cytoplasm, imprisoned host cell mRNA in the nucleus, which suggests a novel mechanism to enhance translation of PRRSV genome. PRRSV nonstructural protein (nsp) 1β was identified as the nuclear protein playing the role for host mRNA nuclear retention and subversion of host protein synthesis. A SAP (SAF-A/B, Acinus, and PIAS) motif was identified in nsp1β with the consensus sequence of 126 -LQxxLxxxGL- 135 . In situ hybridization unveiled that SAP mutants were unable to cause nuclear retention of host cell mRNAs and did not suppress host protein synthesis. In addition, these SAP mutants reverted PRRSV-nsp1β-mediated suppression of interferon (IFN) production, IFN signaling, and TNF-α production pathway. Using reverse genetics, a series of SAP mutant PRRS viruses, vK124A, vL126A, vG134A, and vL135A were generated. No mRNA nuclear retention was observed during vL126A and vL135A infections. Importantly, vL126A and vL135A did not suppress IFN production. For other arteriviruses, mRNA nuclear accumulation was also observed for LDV-nsp1β and SHFV-nsp1β. EAV-nsp1 was exceptional and did not block the host mRNA nuclear export. - Highlights: •PRRS virus blocks host mRNA nuclear export to the cytoplasm. •PRRSV nsp1β is the viral protein responsible for host mRNA nuclear retention. •SAP domain in nsp1β is essential for host mRNA nuclear retention and type I interferon suppression. •Mutation in the SAP domain of nsp1β causes the loss of function. •Host mRNA nuclear retention by nsp1β is common in the family Arteriviridae, except equine arteritis virus.

  8. Nuclear imprisonment of host cellular mRNA by nsp1β protein of porcine reproductive and respiratory syndrome virus

    Energy Technology Data Exchange (ETDEWEB)

    Han, Mingyuan, E-mail: hanming@umich.edu; Ke, Hanzhong; Zhang, Qingzhan; Yoo, Dongwan, E-mail: dyoo@illinois.edu

    2017-05-15

    Positive-strand RNA genomes function as mRNA for viral protein synthesis which is fully reliant on host cell translation machinery. Competing with cellular protein translation apparatus needs to ensure the production of viral proteins, but this also stifles host innate defense. In the present study, we showed that porcine reproductive and respiratory syndrome virus (PRRSV), whose replication takes place in the cytoplasm, imprisoned host cell mRNA in the nucleus, which suggests a novel mechanism to enhance translation of PRRSV genome. PRRSV nonstructural protein (nsp) 1β was identified as the nuclear protein playing the role for host mRNA nuclear retention and subversion of host protein synthesis. A SAP (SAF-A/B, Acinus, and PIAS) motif was identified in nsp1β with the consensus sequence of {sub 126}-LQxxLxxxGL-{sub 135}. In situ hybridization unveiled that SAP mutants were unable to cause nuclear retention of host cell mRNAs and did not suppress host protein synthesis. In addition, these SAP mutants reverted PRRSV-nsp1β-mediated suppression of interferon (IFN) production, IFN signaling, and TNF-α production pathway. Using reverse genetics, a series of SAP mutant PRRS viruses, vK124A, vL126A, vG134A, and vL135A were generated. No mRNA nuclear retention was observed during vL126A and vL135A infections. Importantly, vL126A and vL135A did not suppress IFN production. For other arteriviruses, mRNA nuclear accumulation was also observed for LDV-nsp1β and SHFV-nsp1β. EAV-nsp1 was exceptional and did not block the host mRNA nuclear export. - Highlights: •PRRS virus blocks host mRNA nuclear export to the cytoplasm. •PRRSV nsp1β is the viral protein responsible for host mRNA nuclear retention. •SAP domain in nsp1β is essential for host mRNA nuclear retention and type I interferon suppression. •Mutation in the SAP domain of nsp1β causes the loss of function. •Host mRNA nuclear retention by nsp1β is common in the family Arteriviridae, except equine

  9. EDTA treatment alters protein glycosylation in the cellular slime mold Dictyostelium discoideum

    International Nuclear Information System (INIS)

    West, C.M.; Brownstein, S.A.

    1988-01-01

    The authors have found that treatment of cells with EDTA resulted in the accumulation of lower molecular weight forms of two cell-type-specific glycoproteins. These new glycoproteins lacked a developmentally regulated glycoantigen defined by monoclonal antibody 54.2. Since EDTA dissociated the cells, the possible involvement of cell separation was tested by immobilizing cells in soft agarose. Glycoantigen expression on these proteins was found to be dependent on cAMP and high oxygen tension but not on cell contact, and was reversibly sensitive to EDTA regardless of the state of cell association. The EDTA effect was mimicked by other soluble, but not particulate, membrane impermeable chelators, could be completed by Zn 2+ better than Mg 2+ , and appeared to involve an intracellular mechanism. Studies with [ 14 C]EDTA showed that EDTA equilibrated with a cellular compartment in a temperature-dependent, Zn 2+ -insensitive fashion with half-time kinetics of loading and unloading of 30-40 min. The data suggest that this step in glycosylation, which was found to be delayed 1 or more hours subsequent to protein synthesis, involves an intracellular, transition metal-ion-dependent process which can be modulated by chelators entering the cell through the endocytic pathway

  10. Characterizing Protein Interactions Employing a Genome-Wide siRNA Cellular Phenotyping Screen

    Science.gov (United States)

    Suratanee, Apichat; Schaefer, Martin H.; Betts, Matthew J.; Soons, Zita; Mannsperger, Heiko; Harder, Nathalie; Oswald, Marcus; Gipp, Markus; Ramminger, Ellen; Marcus, Guillermo; Männer, Reinhard; Rohr, Karl; Wanker, Erich; Russell, Robert B.; Andrade-Navarro, Miguel A.; Eils, Roland; König, Rainer

    2014-01-01

    Characterizing the activating and inhibiting effect of protein-protein interactions (PPI) is fundamental to gain insight into the complex signaling system of a human cell. A plethora of methods has been suggested to infer PPI from data on a large scale, but none of them is able to characterize the effect of this interaction. Here, we present a novel computational development that employs mitotic phenotypes of a genome-wide RNAi knockdown screen and enables identifying the activating and inhibiting effects of PPIs. Exemplarily, we applied our technique to a knockdown screen of HeLa cells cultivated at standard conditions. Using a machine learning approach, we obtained high accuracy (82% AUC of the receiver operating characteristics) by cross-validation using 6,870 known activating and inhibiting PPIs as gold standard. We predicted de novo unknown activating and inhibiting effects for 1,954 PPIs in HeLa cells covering the ten major signaling pathways of the Kyoto Encyclopedia of Genes and Genomes, and made these predictions publicly available in a database. We finally demonstrate that the predicted effects can be used to cluster knockdown genes of similar biological processes in coherent subgroups. The characterization of the activating or inhibiting effect of individual PPIs opens up new perspectives for the interpretation of large datasets of PPIs and thus considerably increases the value of PPIs as an integrated resource for studying the detailed function of signaling pathways of the cellular system of interest. PMID:25255318

  11. Nitric oxide-mediated modulation of iron regulatory proteins: implication for cellular iron homeostasis.

    Science.gov (United States)

    Kim, Sangwon; Ponka, Prem

    2002-01-01

    Iron regulatory proteins (IRP1 and IRP2) control the synthesis of transferrin receptors (TfR) and ferritin by binding to iron-responsive elements (IREs) that are located in the 3' untranslated region (UTR) and the 5' UTR of their respective mRNAs. Cellular iron levels affect binding of IRPs to IREs and consequently expression of TfR and ferritin. Moreover, NO(.), a redox species of nitric oxide that interacts primarily with iron, can activate IRP1 RNA-binding activity resulting in an increase in TfR mRNA levels and a decrease in ferritin synthesis. We have shown that treatment of RAW 264.7 cells (a murine macrophage cell line) with NO(+) (nitrosonium ion, which causes S-nitrosylation of thiol groups) resulted in a rapid decrease in RNA-binding of IRP2, followed by IRP2 degradation, and these changes were associated with a decrease in TfR mRNA levels and a dramatic increase in ferritin synthesis. Moreover, we demonstrated that stimulation of RAW 264.7 cells with lipopolysaccharide (LPS) and interferon-gamma (IFN-gamma) increased IRP1 binding activity, whereas RNA-binding of IRP2 decreased and was followed by a degradation of this protein. Furthermore, the decrease of IRP2 binding/protein levels was associated with a decrease in TfR mRNA levels and an increase in ferritin synthesis in LPS/IFN-gamma-treated cells, and these changes were prevented by inhibitors of inducible nitric oxide synthase. These results suggest that NO(+)-mediated degradation of IRP2 plays a major role in iron metabolism during inflammation.

  12. Reduced Sleep During Social Isolation Leads to Cellular Stress and Induction of the Unfolded Protein Response.

    Science.gov (United States)

    Brown, Marishka K; Strus, Ewa; Naidoo, Nirinjini

    2017-07-01

    Social isolation has a multitude of negative consequences on human health including the ability to endure challenges to the immune system, sleep amount and efficiency, and general morbidity and mortality. These adverse health outcomes are conserved in other social species. In the fruit fly Drosophila melanogaster, social isolation leads to increased aggression, impaired memory, and reduced amounts of daytime sleep. There is a correlation between molecules affected by social isolation and those implicated in sleep in Drosophila. We previously demonstrated that acute sleep loss in flies and mice induced the unfolded protein response (UPR), an adaptive signaling pathway. One mechanism indicating UPR upregulation is elevated levels of the endoplasmic reticular chaperone BiP/GRP78. We previously showed that BiP overexpression in Drosophila led to increased sleep rebound. Increased rebound sleep has also been demonstrated in socially isolated (SI) flies. D. melanogaster were used to study the effect of social isolation on cellular stress. SI flies displayed an increase in UPR markers; there were higher BiP levels, increased phosphorylation of the translation initiation factor eIF2α, and increased splicing of xbp1. These are all indicators of UPR activation. In addition, the effects of isolation on the UPR were reversible; pharmacologically and genetically altering sleep in the flies modulated the UPR. The reduction in sleep observed in SI flies is a cellular stressor that results in UPR induction. © Sleep Research Society 2017. Published by Oxford University Press [on behalf of the Sleep Research Society]. All rights reserved. For permissions, please email: journals.permissions@oup.com

  13. Enhancement of antibody-dependent cellular cytotoxicity of cetuximab by a chimeric protein encompassing interleukin-15.

    Science.gov (United States)

    Ochoa, Maria Carmen; Minute, Luna; López, Ascensión; Pérez-Ruiz, Elisabeth; Gomar, Celia; Vasquez, Marcos; Inoges, Susana; Etxeberria, Iñaki; Rodriguez, Inmaculada; Garasa, Saray; Mayer, Jan-Peter Andreas; Wirtz, Peter; Melero, Ignacio; Berraondo, Pedro

    2018-01-01

    Enhancement of antibody-dependent cellular cytotoxicity (ADCC) may potentiate the antitumor efficacy of tumor-targeted monoclonal antibodies. Increasing the numbers and antitumor activity of NK cells is a promising strategy to maximize the ADCC of standard-of-care tumor-targeted antibodies. For this purpose, we have preclinically tested a recombinant chimeric protein encompassing the sushi domain of the IL15Rα, IL-15, and apolipoprotein A-I (Sushi-IL15-Apo) as produced in CHO cells. The size-exclusion purified monomeric fraction of this chimeric protein was stable and retained the IL-15 and the sushi domain bioactivity as measured by CTLL-2 and Mo-7e cell proliferation and STAT5 phosphorylation in freshly isolated human NK and CD8 + T cells. On cell cultures, Sushi-IL15-Apo increases NK cell proliferation and survival as well as spontaneous and antibody-mediated cytotoxicity. Scavenger receptor class B type I (SR-B1) is the receptor for ApoA-I and is expressed on the surface of tumor cells. SR-B1 can adsorb the chimeric protein on tumor cells and can transpresent IL-15 to NK and CD8 + T cells. A transient NK-humanized murine model was developed to test the increase of ADCC attained by the chimeric protein in vivo . The EGFR + human colon cancer cell line HT-29 was intraperitoneally inoculated in immune-deficient Rag2 -/- γc -/- mice that were reconstituted with freshly isolated PBMCs and treated with the anti-EGFR mAb cetuximab. The combination of the Sushi-IL15-Apo protein and cetuximab reduced the number of remaining tumor cells in the peritoneal cavity and delayed tumor engraftment in the peritoneum. Furthermore, Sushi-IL15-Apo increased the anti-tumor effect of a murine anti-EGFR mAb in Rag1 -/- mice bearing subcutaneous MC38 colon cancer transfected to express EGFR. Thus, Sushi-IL15-Apo is a potent tool to increase the number and the activation of NK cells to promote the ADCC activity of antibodies targeting tumor antigens.

  14. Delivery of kinesin spindle protein targeting siRNA in solid lipid nanoparticles to cellular models of tumor vasculature

    International Nuclear Information System (INIS)

    Ying, Bo; Campbell, Robert B.

    2014-01-01

    Highlights: • siRNA-lipid nanoparticles are solid particles not lipid bilayers with aqueous core. • High, but not low, PEG content can prevent nanoparticle encapsulation of siRNA. • PEG reduces cellular toxicity of cationic nanoparticles in vitro. • PEG reduces zeta potential while improving gene silencing of siRNA nanoparticles. • Kinesin spindle protein can be an effective target for tumor vascular targeting. - Abstract: The ideal siRNA delivery system should selectively deliver the construct to the target cell, avoid enzymatic degradation, and evade uptake by phagocytes. In the present study, we evaluated the importance of polyethylene glycol (PEG) on lipid-based carrier systems for encapsulating, and delivering, siRNA to tumor vessels using cellular models. Lipid nanoparticles containing different percentage of PEG were evaluated based on their physical chemical properties, density compared to water, siRNA encapsulation, toxicity, targeting efficiency and gene silencing in vitro. siRNA can be efficiently loaded into lipid nanoparticles (LNPs) when DOTAP is included in the formulation mixture. However, the total amount encapsulated decreased with increase in PEG content. In the presence of siRNA, the final formulations contained a mixed population of particles based on density. The major population which contains the majority of siRNA exhibited a density of 4% glucose, and the minor fraction associated with a decreased amount of siRNA had a density less than PBS. The inclusion of 10 mol% PEG resulted in a greater amount of siRNA associated with the minor fraction. Finally, when kinesin spindle protein (KSP) siRNA was encapsulated in lipid nanoparticles containing a modest amount of PEG, the proliferation of endothelial cells was inhibited due to the efficient knock down of KSP mRNA. The presence of siRNA resulted in the formation of solid lipid nanoparticles when prepared using the thin film and hydration method. LNPs with a relatively modest amount of

  15. Acrolein-exposed normal human lung fibroblasts in vitro: cellular senescence, enhanced telomere erosion, and degradation of Werner's syndrome protein.

    Science.gov (United States)

    Jang, Jun-Ho; Bruse, Shannon; Huneidi, Salam; Schrader, Ronald M; Monick, Martha M; Lin, Yong; Carter, A Brent; Klingelhutz, Aloysius J; Nyunoya, Toru

    2014-09-01

    Acrolein is a ubiquitous environmental hazard to human health. Acrolein has been reported to activate the DNA damage response and induce apoptosis. However, little is known about the effects of acrolein on cellular senescence. We examined whether acrolein induces cellular senescence in cultured normal human lung fibroblasts (NHLF). We cultured NHLF in the presence or absence of acrolein and determined the effects of acrolein on cell proliferative capacity, senescence-associated β-galactosidase activity, the known senescence-inducing pathways (e.g., p53, p21), and telomere length. We found that acrolein induced cellular senescence by increasing both p53 and p21. The knockdown of p53 mediated by small interfering RNA (siRNA) attenuated acrolein-induced cellular senescence. Acrolein decreased Werner's syndrome protein (WRN), a member of the RecQ helicase family involved in DNA repair and telomere maintenance. Acrolein-induced down-regulation of WRN protein was rescued by p53 knockdown or proteasome inhibition. Finally, we found that acrolein accelerated p53-mediated telomere shortening. These results suggest that acrolein induces p53-mediated cellular senescence accompanied by enhanced telomere attrition and WRN protein down-regulation.

  16. The cellular prion protein negatively regulates phagocytosis and cytokine expression in murine bone marrow-derived macrophages.

    Directory of Open Access Journals (Sweden)

    Min Wang

    Full Text Available The cellular prion protein (PrP(C is a glycosylphosphatidylinositol (GPI-anchored glycoprotein on the cell surface. Previous studies have demonstrated contradictory roles for PrP(C in connection with the phagocytic ability of macrophages. In the present work, we investigated the function of PrP(C in phagocytosis and cytokine expression in bone marrow-derived macrophages infected with Escherichia coli. E. coli infection induced an increase in the PRNP mRNA level. Knockout of PrP(C promoted bacterial uptake; upregulated Rab5, Rab7, and Eea1 mRNA expression; and increased the recruitment of lysosomal-associated membrane protein-2 to phagosomes, suggesting enhanced microbicidal activity. Remarkably, knockout of PrP(C suppressed the proliferation of internalized bacteria and increased the expression of cytokines such as interleukin-1β. Collectively, our data reveal an important role of PrP(C as a negative regulator for phagocytosis, phagosome maturation, cytokine expression, and macrophage microbicidal activity.

  17. Outer membrane protein functions as integrator of protein import and DNA inheritance in mitochondria

    Science.gov (United States)

    Käser, Sandro; Oeljeklaus, Silke; Týč, Jiří; Vaughan, Sue; Warscheid, Bettina; Schneider, André

    2016-01-01

    Trypanosomatids are one of the earliest diverging eukaryotes that have fully functional mitochondria. pATOM36 is a trypanosomatid-specific essential mitochondrial outer membrane protein that has been implicated in protein import. Changes in the mitochondrial proteome induced by ablation of pATOM36 and in vitro assays show that pATOM36 is required for the assembly of the archaic translocase of the outer membrane (ATOM), the functional analog of the TOM complex in other organisms. Reciprocal pull-down experiments and immunofluorescence analyses demonstrate that a fraction of pATOM36 interacts and colocalizes with TAC65, a previously uncharacterized essential component of the tripartite attachment complex (TAC). The TAC links the single-unit mitochondrial genome to the basal body of the flagellum and mediates the segregation of the replicated mitochondrial genomes. RNAi experiments show that pATOM36, in line with its dual localization, is not only essential for ATOM complex assembly but also for segregation of the replicated mitochondrial genomes. However, the two functions are distinct, as a truncated version of pATOM36 lacking the 75 C-terminal amino acids can rescue kinetoplast DNA missegregation but not the lack of ATOM complex assembly. Thus, pATOM36 has a dual function and integrates mitochondrial protein import with mitochondrial DNA inheritance. PMID:27436903

  18. Activation of the cellular unfolded protein response by recombinant adeno-associated virus vectors.

    Directory of Open Access Journals (Sweden)

    Balaji Balakrishnan

    Full Text Available The unfolded protein response (UPR is a stress-induced cyto-protective mechanism elicited towards an influx of large amount of proteins in the endoplasmic reticulum (ER. In the present study, we evaluated if AAV manipulates the UPR pathways during its infection. We first examined the role of the three major UPR axes, namely, endoribonuclease inositol-requiring enzyme-1 (IRE1α, activating transcription factor 6 (ATF6 and PKR-like ER kinase (PERK in AAV infected cells. Total RNA from mock or AAV infected HeLa cells were used to determine the levels of 8 different ER-stress responsive transcripts from these pathways. We observed a significant up-regulation of IRE1α (up to 11 fold and PERK (up to 8 fold genes 12-48 hours after infection with self-complementary (scAAV2 but less prominent with single-stranded (ssAAV2 vectors. Further studies demonstrated that scAAV1 and scAAV6 also induce cellular UPR in vitro, with AAV1 vectors activating the PERK pathway (3 fold while AAV6 vectors induced a significant increase on all the three major UPR pathways [6-16 fold]. These data suggest that the type and strength of UPR activation is dependent on the viral capsid. We then examined if transient inhibition of UPR pathways by RNA interference has an effect on AAV transduction. siRNA mediated silencing of PERK and IRE1α had a modest effect on AAV2 and AAV6 mediated gene expression (∼1.5-2 fold in vitro. Furthermore, hepatic gene transfer of scAAV2 vectors in vivo, strongly elevated IRE1α and PERK pathways (2 and 3.5 fold, respectively. However, when animals were pre-treated with a pharmacological UPR inhibitor (metformin during scAAV2 gene transfer, the UPR signalling and its subsequent inflammatory response was attenuated concomitant to a modest 2.8 fold increase in transgene expression. Collectively, these data suggest that AAV vectors activate the cellular UPR pathways and their selective inhibition may be beneficial during AAV mediated gene transfer.

  19. Nuclear protein accumulation in cellular senescence and organismal aging revealed with a novel single-cell resolution fluorescence microscopy assay.

    Science.gov (United States)

    De Cecco, Marco; Jeyapalan, Jessie; Zhao, Xiaoai; Tamamori-Adachi, Mimi; Sedivy, John M

    2011-10-01

    Replicative cellular senescence was discovered some 50 years ago. The phenotypes of senescent cells have been investigated extensively in cell culture, and found to affect essentially all aspects of cellular physiology. The relevance of cellular senescence in the context of age-associated pathologies as well as normal aging is a topic of active and ongoing interest. Considerable effort has been devoted to biomarker discovery to enable the microscopic detection of single senescent cells in tissues. One characteristic of senescent cells documented very early in cell culture studies was an increase in cell size and total protein content, but whether this occurs in vivo is not known. A limiting factor for studies of protein content and localization has been the lack of suitable fluorescence microscopy tools. We have developed an easy and flexible method, based on the merocyanine dye known as NanoOrange, to visualize and quantitatively measure total protein levels by high resolution fluorescence microscopy. NanoOrange staining can be combined with antibody-based immunofluorescence, thus providing both specific target and total protein information in the same specimen. These methods are optimally combined with automated image analysis platforms for high throughput analysis. We document here increasing protein content and density in nuclei of senescent human and mouse fibroblasts in vitro, and in liver nuclei of aged mice in vivo. Additionally, in aged liver nuclei NanoOrange revealed protein-dense foci that colocalize with centromeric heterochromatin.

  20. Photochemistry and stereoselectivity of cellular retinaldehyde-binding protein from bovine retina

    International Nuclear Information System (INIS)

    Saari, J.C.; Bredberg, D.L.

    1987-01-01

    11-cis-Retinaldehyde bound to cellular retinaldehyde-binding protein (CRALBP) is unaffected in bovine eyecup preparations by illumination that bleaches approximately 70% of the rhodopsin. Illumination of retinal homogenates to which CRALBP X [ 3 H]11-cis-retinaldehyde had been added did not result in a reduction of the specific activity of recovered 11-cis-retinaldehyde, ruling out a bleaching regeneration cycle. The quantum efficiency of photoisomerization for CRALBP X 11-cis-retinaldehyde was determined by comparing the rate of photoisomerization of 11-cis-retinaldehyde bound to purified CRALBP and opsin. The low value obtained (0.07), coupled with a low molar extinction coefficient (15,400 M-1 cm-1), results in a photosensitivity only about 4% that of rhodopsin. CRALBP binds 9-cis- and 11-cis-retinaldehyde, producing complexes with absorption maxima at 405 and 425 nm, respectively. No complexes were detected with 13-cis- and all-trans-retinaldehyde. Following incubation of CRALBP X 11-cis-retinol with an equimolar mixture of 9-, 11-, 13-cis-, and all-trans-retinaldehydes, only 11-cis-retinaldehyde and residual 11-cis-retinol are present on the protein following separation from excess retinoids. A similar result is obtained following incubation of CRALBP X 11-cis-retinol with mixtures of 9- and 11-cis-retinaldehyde ranging in composition from 9:1 to 1:9 (9-cis-:11-cis-,mol/mol). The results indicate that CRALBP X 11-cis-retinol is sufficiently stereoselective in its binding properties to warrant consideration as a component of the mechanism for the generation of 11-cis-retinaldehyde in the dark

  1. 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.

  2. Mapping of nuclear import signal and importin α3 binding regions of 52K protein of bovine adenovirus-3

    International Nuclear Information System (INIS)

    Paterson, Carolyn P.; Ayalew, Lisanework E.; Tikoo, Suresh K.

    2012-01-01

    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 ( 105 RKR 107 ) of the identified domain (amino acids 102 GMPRKRVLT 110 ) are essential for nuclear localization of 52K. The nuclear import of a GST-52K fusion protein utilizes the classical importin α/β-dependent nuclear transport pathway. The 52K protein is preferentially bound to the cellular nuclear import receptor importin α3. 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-α3 and localizing a cytoplasmic protein to the nucleus. These results suggest that 52K contains a bipartite NLS, which preferentially utilize an importin α3 nuclear import receptor-mediated pathway to transport 52K to the nucleus.

  3. The Cellular Prion Protein Prevents Copper-Induced Inhibition of P2X4 Receptors

    Directory of Open Access Journals (Sweden)

    Ramón A. Lorca

    2011-01-01

    Full Text Available Although the physiological function of the cellular prion protein (PrPC remains unknown, several evidences support the notion of its role in copper homeostasis. PrPC binds Cu2+ through a domain composed by four to five repeats of eight amino acids. Previously, we have shown that the perfusion of this domain prevents and reverses the inhibition by Cu2+ of the adenosine triphosphate (ATP-evoked currents in the P2X4 receptor subtype, highlighting a modulatory role for PrPC in synaptic transmission through regulation of Cu2+ levels. Here, we study the effect of full-length PrPC in Cu2+ inhibition of P2X4 receptor when both are coexpressed. PrPC expression does not significantly change the ATP concentration-response curve in oocytes expressing P2X4 receptors. However, the presence of PrPC reduces the inhibition by Cu2+ of the ATP-elicited currents in these oocytes, confirming our previous observations with the Cu2+ binding domain. Thus, our observations suggest a role for PrPC in modulating synaptic activity through binding of extracellular Cu2+.

  4. Melatonin Promotes Apoptosis of Oxaliplatin-resistant Colorectal Cancer Cells Through Inhibition of Cellular Prion Protein.

    Science.gov (United States)

    Lee, Jun Hee; Yoon, Yeo Min; Han, Yong-Seok; Yun, Chul Won; Lee, Sang Hun

    2018-04-01

    Drug resistance restricts the efficacy of chemotherapy in colorectal cancer. However, the detailed molecular mechanism of drug resistance in colorectal cancer cells remains unclear. The level of cellular prion protein (PrP C ) in oxaliplatin-resistant colorectal cancer (SNU-C5/Oxal-R) cells was assessed. PrP C level in SNU-C5/Oxal-R cells was significantly increased compared to that in wild-type (SNU-C5) cells. Superoxide dismutase and catalase activities were higher in SNU-C5/Oxal-R cells than in SNU-C5 cells. Treatment of SNU-C5/Oxal-R cells with oxaliplatin and melatonin reduced PrP C expression, while suppressing antioxidant enzyme activity and increasing superoxide anion generation. In SNU-C5/Oxal-R cells, endoplasmic reticulum stress and apoptosis were significantly increased following co-treatment with oxaliplatin and melatonin compared to treatment with oxaliplatin alone. Co-treatment with oxaliplatin and melatonin increased endoplasmic reticulum stress in and apoptosis of SNU-C5/Oxal-R cells through inhibition of PrP C , suggesting that PrP C could be a key molecule in oxaliplatin resistance of colorectal cancer cells. Copyright© 2018, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

  5. The Endoplasmic Reticulum Coat Protein II Transport Machinery Coordinates Cellular Lipid Secretion and Cholesterol Biosynthesis*

    Science.gov (United States)

    Fryer, Lee G. D.; Jones, Bethan; Duncan, Emma J.; Hutchison, Claire E.; Ozkan, Tozen; Williams, Paul A.; Alder, Olivia; Nieuwdorp, Max; Townley, Anna K.; Mensenkamp, Arjen R.; Stephens, David J.; Dallinga-Thie, Geesje M.; Shoulders, Carol C.

    2014-01-01

    Triglycerides and cholesterol are essential for life in most organisms. Triglycerides serve as the principal energy storage depot and, where vascular systems exist, as a means of energy transport. Cholesterol is essential for the functional integrity of all cellular membrane systems. The endoplasmic reticulum is the site of secretory lipoprotein production and de novo cholesterol synthesis, yet little is known about how these activities are coordinated with each other or with the activity of the COPII machinery, which transports endoplasmic reticulum cargo to the Golgi. The Sar1B component of this machinery is mutated in chylomicron retention disorder, indicating that this Sar1 isoform secures delivery of dietary lipids into the circulation. However, it is not known why some patients with chylomicron retention disorder develop hepatic steatosis, despite impaired intestinal fat malabsorption, and why very severe hypocholesterolemia develops in this condition. Here, we show that Sar1B also promotes hepatic apolipoprotein (apo) B lipoprotein secretion and that this promoting activity is coordinated with the processes regulating apoB expression and the transfer of triglycerides/cholesterol moieties onto this large lipid transport protein. We also show that although Sar1A antagonizes the lipoprotein secretion-promoting activity of Sar1B, both isoforms modulate the expression of genes encoding cholesterol biosynthetic enzymes and the synthesis of cholesterol de novo. These results not only establish that Sar1B promotes the secretion of hepatic lipids but also adds regulation of cholesterol synthesis to Sar1B's repertoire of transport functions. PMID:24338480

  6. A structural basis for cellular uptake of GST-fold proteins.

    Directory of Open Access Journals (Sweden)

    Melanie J Morris

    Full Text Available It has recently emerged that glutathione transferase enzymes (GSTs and other structurally related molecules can be translocated from the external medium into many different cell types. In this study we aim to explore in detail, the structural features that govern cell translocation and by dissecting the human GST enzyme GSTM2-2 we quantatively demonstrate that the α-helical C-terminal domain (GST-C is responsible for this property. Attempts to further examine the constituent helices within GST-C resulted in a reduction in cell translocation efficiency, indicating that the intrinsic GST-C domain structure is necessary for maximal cell translocation capacity. In particular, it was noted that the α-6 helix of GST-C plays a stabilising role in the fold of this domain. By destabilising the conformation of GST-C, an increase in cell translocation efficiency of up to ∼2-fold was observed. The structural stability profiles of these protein constructs have been investigated by circular dichroism and differential scanning fluorimetry measurements and found to impact upon their cell translocation efficiency. These experiments suggest that the globular, helical domain in the 'GST-fold' structural motif plays a role in influencing cellular uptake, and that changes that affect the conformational stability of GST-C can significantly influence cell translocation efficiency.

  7. Local sequence information in cellular retinoic acid-binding protein I: specific residue roles in beta-turns.

    Science.gov (United States)

    Rotondi, Kenneth S; Gierasch, Lila M

    2003-01-01

    We have recently shown that two of the beta-turns (III and IV) in the ten-stranded, beta-clam protein, cellular retinoic acid-binding protein I (CRABP I), are favored in short peptide fragments, arguing that they are encoded by local interactions (K. S. Rotondi and L. M. Gierasch, Biochemistry, 2003, Vol. 42, pp. 7976-7985). In this paper we examine these turns in greater detail to dissect the specific local interactions responsible for their observed native conformational biases. Conformations of peptides corresponding to the turn III and IV fragments were examined under conditions designed to selectively disrupt stabilizing interactions, using pH variation, chaotrope addition, or mutagenesis to probe specific side-chain influences. We find that steric constraints imposed by excluded volume effects between near neighbor residues (i,i+2), favorable polar (i,i+2) interactions, and steric permissiveness of glycines are the principal factors accounting for the observed native bias in these turns. Longer-range stabilizing interactions across the beta-turns do not appear to play a significant role in turn stability in these short peptides, in contrast to their importance in hairpins. Additionally, our data add to a growing number of examples of the 3:5 type I turn with a beta-bulge as a class of turns with high propensity to form locally defined structure. Current work is directed at the interplay between the local sequence information in the turns and more long-range influences in the mechanism of folding of this predominantly beta-sheet protein. Copyright 2004 Wiley Periodicals, Inc.

  8. SUN1 Regulates HIV-1 Nuclear Import in a Manner Dependent on the Interaction between the Viral Capsid and Cellular Cyclophilin A.

    Science.gov (United States)

    Luo, Xinlong; Yang, Wei; Gao, Guangxia

    2018-07-01

    Human immunodeficiency virus type 1 (HIV-1) can infect nondividing cells via passing through the nuclear pore complex. The nuclear membrane-imbedded protein SUN2 was recently reported to be involved in the nuclear import of HIV-1. Whether SUN1, which shares many functional similarities with SUN2, is involved in this process remained to be explored. Here we report that overexpression of SUN1 specifically inhibited infection by HIV-1 but not that by simian immunodeficiency virus (SIV) or murine leukemia virus (MLV). Overexpression of SUN1 did not affect reverse transcription but led to reduced accumulation of the 2-long-terminal-repeat (2-LTR) circular DNA and integrated viral DNA, suggesting a block in the process of nuclear import. HIV-1 CA was mapped as a determinant for viral sensitivity to SUN1. Treatment of SUN1-expressing cells with cyclosporine (CsA) significantly reduced the sensitivity of the virus to SUN1, and an HIV-1 mutant containing CA-G89A, which does not interact with cyclophilin A (CypA), was resistant to SUN1 overexpression. Downregulation of endogenous SUN1 inhibited the nuclear entry of the wild-type virus but not that of the G89A mutant. These results indicate that SUN1 participates in the HIV-1 nuclear entry process in a manner dependent on the interaction of CA with CypA. IMPORTANCE HIV-1 infects both dividing and nondividing cells. The viral preintegration complex (PIC) can enter the nucleus through the nuclear pore complex. It has been well known that the viral protein CA plays an important role in determining the pathways by which the PIC enters the nucleus. In addition, the interaction between CA and the cellular protein CypA has been reported to be important in the selection of nuclear entry pathways, though the underlying mechanisms are not very clear. Here we show that both SUN1 overexpression and downregulation inhibited HIV-1 nuclear entry. CA played an important role in determining the sensitivity of the virus to SUN1: the regulatory

  9. VP7: an attachment protein of bluetongue virus for cellular receptors in Culicoides variipennis.

    Science.gov (United States)

    Xu, G; Wilson, W; Mecham, J; Murphy, K; Zhou, E M; Tabachnick, W

    1997-07-01

    The importance of VP7 of bluetongue virus (BTV) in the binding of BTV to membrane proteins of the BTV vector Culicoides variipennis was investigated. Core BTV particles, prepared from whole viruses, lacked outer proteins VP2 and VP5 and had VP7 exposed. More core particles and whole viruses bound to membrane preparations of adults of C. variipennis and KC cells, which were cultured from this vector insect, than to membrane preparations of Manduca sexta larvae. More core particles than whole viruses bound to membrane preparations of adults of C. variipennis and KC cells. Polyclonal anti-idiotypic antibodies (anti-Id), which were made against an antigen-combining region of an anti-BTV-10 VP7 antibody and functionally mimicked VP7, bound more to the membrane preparations of adults of C. variipennis and KC cells, and less to cytosol preparations. In Western overalay analysis, the Culicoides plasma membrane preparation reduced binding of an anti-VP7 monoclonal antibody to VP7. Whole and core BTV particles and the anti-Id bound to a membrane protein with a molecular mass of 23 kDa that was present predominantly in membrane preparations of adults of C. variipennis and KC cells. This protein was present in much lower concentrations in membrane preparations of C6/36 and DM-2 insect cells.

  10. Exploring the diversity of protein modifications: special bacterial phosphorylation systems

    DEFF Research Database (Denmark)

    Mijakovic, Ivan; Grangeasse, Christophe; Turgay, Kürşad

    2016-01-01

    Protein modifications not only affect protein homeostasis but can also establish new cellular protein functions and are important components of complex cellular signal sensing and transduction networks. Among these post-translational modifications, protein phosphorylation represents the one that ...

  11. Identification of multidrug resistance protein 1 (MRP1/ABCC1) as a molecular gate for cellular export of cobalamin

    DEFF Research Database (Denmark)

    Beedholm-Ebsen, Rasmus; van de Wetering, Koen; Hardlei, Tore

    2010-01-01

    transporters by cellular gene silencing showed a role in cellular Cbl efflux of the ATP-binding cassette (ABC)-drug transporter, ABCC1, alias multidrug resistance protein 1 (MRP1), which is present in the basolateral membrane of intestinal epithelium and in other cells. The ability of MRP1 to mediate ATP...... and kidney. In contrast, Cbl accumulates in the terminal part of the intestine of these mice, suggesting a functional malabsorption because of a lower epithelial basolateral Cbl efflux. The identification of this Cbl export mechanism now allows the delineation of a coherent pathway for Cbl trafficking from...

  12. Potassium-transporting proteins in skeletal muscle: cellular location and fiber-type differences

    DEFF Research Database (Denmark)

    Kristensen, Michael; Juel, Carsten

    2010-01-01

    Potassium (K+) displacement in skeletal muscle may be an important factor in the development of muscle fatigue during intense exercise. It has been shown in vitro that an increase in the extracellular K+ concentration ([K+]e) to values higher than approx. 10 mm significantly reduce force developm......Potassium (K+) displacement in skeletal muscle may be an important factor in the development of muscle fatigue during intense exercise. It has been shown in vitro that an increase in the extracellular K+ concentration ([K+]e) to values higher than approx. 10 mm significantly reduce force......, but is suggested primarily to participate in K+ release to the interstitium. Because there is restricted diffusion of K+ to the interstitium, K+ released to the T-tubules during AP propagation will be removed primarily by reuptake mediated by transport proteins located in the T-tubule membrane. The most important...

  13. The surfactant protein C mutation A116D alters cellular processing, stress tolerance, surfactant lipid composition, and immune cell activation

    Directory of Open Access Journals (Sweden)

    Zarbock Ralf

    2012-03-01

    Full Text Available Abstract Background Surfactant protein C (SP-C is important for the function of pulmonary surfactant. Heterozygous mutations in SFTPC, the gene encoding SP-C, cause sporadic and familial interstitial lung disease (ILD in children and adults. Mutations mapping to the BRICHOS domain located within the SP-C proprotein result in perinuclear aggregation of the proprotein. In this study, we investigated the effects of the mutation A116D in the BRICHOS domain of SP-C on cellular homeostasis. We also evaluated the ability of drugs currently used in ILD therapy to counteract these effects. Methods SP-CA116D was expressed in MLE-12 alveolar epithelial cells. We assessed in vitro the consequences for cellular homeostasis, immune response and effects of azathioprine, hydroxychloroquine, methylprednisolone and cyclophosphamide. Results Stable expression of SP-CA116D in MLE-12 alveolar epithelial cells resulted in increased intracellular accumulation of proSP-C processing intermediates. SP-CA116D expression further led to reduced cell viability and increased levels of the chaperones Hsp90, Hsp70, calreticulin and calnexin. Lipid analysis revealed decreased intracellular levels of phosphatidylcholine (PC and increased lyso-PC levels. Treatment with methylprednisolone or hydroxychloroquine partially restored these lipid alterations. Furthermore, SP-CA116D cells secreted soluble factors into the medium that modulated surface expression of CCR2 or CXCR1 receptors on CD4+ lymphocytes and neutrophils, suggesting a direct paracrine effect of SP-CA116D on neighboring cells in the alveolar space. Conclusions We show that the A116D mutation leads to impaired processing of proSP-C in alveolar epithelial cells, alters cell viability and lipid composition, and also activates cells of the immune system. In addition, we show that some of the effects of the mutation on cellular homeostasis can be antagonized by application of pharmaceuticals commonly applied in ILD therapy

  14. Calicivirus 3C-like proteinase inhibits cellular translation by cleavage of poly(A)-binding protein.

    Science.gov (United States)

    Kuyumcu-Martinez, Muge; Belliot, Gaël; Sosnovtsev, Stanislav V; Chang, Kyeong-Ok; Green, Kim Y; Lloyd, Richard E

    2004-08-01

    Caliciviruses are single-stranded RNA viruses that cause a wide range of diseases in both humans and animals, but little is known about the regulation of cellular translation during infection. We used two distinct calicivirus strains, MD145-12 (genus Norovirus) and feline calicivirus (FCV) (genus Vesivirus), to investigate potential strategies used by the caliciviruses to inhibit cellular translation. Recombinant 3C-like proteinases (r3CL(pro)) from norovirus and FCV were found to cleave poly(A)-binding protein (PABP) in the absence of other viral proteins. The norovirus r3CL(pro) PABP cleavage products were indistinguishable from those generated by poliovirus (PV) 3C(pro) cleavage, while the FCV r3CL(pro) products differed due to cleavage at an alternate cleavage site 24 amino acids downstream of one of the PV 3C(pro) cleavage sites. All cleavages by calicivirus or PV proteases separated the C-terminal domain of PABP that binds translation factors eIF4B and eRF3 from the N-terminal RNA-binding domain of PABP. The effect of PABP cleavage by the norovirus r3CL(pro) was analyzed in HeLa cell translation extracts, and the presence of r3CL(pro) inhibited translation of both endogenous and exogenous mRNAs. Translation inhibition was poly(A) dependent, and replenishment of the extracts with PABP restored translation. Analysis of FCV-infected feline kidney cells showed that the levels of de novo cellular protein synthesis decreased over time as virus-specific proteins accumulated, and cleavage of PABP occurred in virus-infected cells. Our data indicate that the calicivirus 3CL(pro), like PV 3C(pro), mediates the cleavage of PABP as part of its strategy to inhibit cellular translation. PABP cleavage may be a common mechanism among certain virus families to manipulate cellular translation.

  15. Re-localization of Cellular Protein SRp20 during Poliovirus Infection: Bridging a Viral IRES to the Host Cell Translation Apparatus

    Science.gov (United States)

    Fitzgerald, Kerry D.; Semler, Bert L.

    2011-01-01

    Poliovirus IRES-mediated translation requires the functions of certain canonical as well as non-canonical factors for the recruitment of ribosomes to the viral RNA. The interaction of cellular proteins PCBP2 and SRp20 in extracts from poliovirus-infected cells has been previously described, and these two proteins were shown to function synergistically in viral translation. To further define the mechanism of ribosome recruitment for the initiation of poliovirus IRES-dependent translation, we focused on the role of the interaction between cellular proteins PCBP2 and SRp20. Work described here demonstrates that SRp20 dramatically re-localizes from the nucleus to the cytoplasm of poliovirus-infected neuroblastoma cells during the course of infection. Importantly, SRp20 partially co-localizes with PCBP2 in the cytoplasm of infected cells, corroborating our previous in vitro interaction data. In addition, the data presented implicate the presence of these two proteins in viral translation initiation complexes. We show that in extracts from poliovirus-infected cells, SRp20 is associated with PCBP2 bound to poliovirus RNA, indicating that this interaction occurs on the viral RNA. Finally, we generated a mutated version of SRp20 lacking the RNA recognition motif (SRp20ΔRRM) and found that this protein is localized similar to the full length SRp20, and also partially co-localizes with PCBP2 during poliovirus infection. Expression of this mutated version of SRp20 results in a ∼100 fold decrease in virus yield for poliovirus when compared to expression of wild type SRp20, possibly via a dominant negative effect. Taken together, these results are consistent with a model in which SRp20 interacts with PCBP2 bound to the viral RNA, and this interaction functions to recruit ribosomes to the viral RNA in a direct or indirect manner, with the participation of additional protein-protein or protein-RNA interactions. PMID:21779168

  16. Activated α2 -Macroglobulin Induces Mesenchymal Cellular Migration Of Raw264.7 Cells Through Low-Density Lipoprotein Receptor-Related Protein 1.

    Science.gov (United States)

    Ferrer, Darío G; Dato, Virginia Actis; Fincati, Javier R Jaldín; Lorenc, Valeria E; Sánchez, María C; Chiabrando, Gustavo A

    2017-07-01

    Distinct modes of cell migration contribute to diverse types of cell movements. The mesenchymal mode is characterized by a multistep cycle of membrane protrusion, the formation of focal adhesion, and the stabilization at the leading edge associated with the degradation of extracellular matrix (ECM) components and with regulated extracellular proteolysis. Both α 2 -Macroglobulin (α 2 M) and its receptor, low density lipoprotein receptor-related protein 1 (LRP1), play important roles in inflammatory processes, by controlling the extracellular activity of several proteases. The binding of the active form of α 2 M (α 2 M*) to LRP1 can also activate different signaling pathways in macrophages, thus inducing extracellular matrix metalloproteinase-9 (MMP-9) activation and cellular proliferation. In the present study, we investigated whether the α 2 M*/LRP1 interaction induces cellular migration of the macrophage-derived cell line, Raw264.7. By using the wound-scratch migration assay and confocal microscopy, we demonstrate that α 2 M* induces LRP1-mediated mesenchymal cellular migration. This migration exhibits the production of enlarged cellular protrusions, MT1-MMP distribution to these leading edge protrusions, actin polymerization, focal adhesion formation, and increased intracellular LRP1/β1-integrin colocalization. Moreover, the presence of calphostin-C blocked the α 2 M*-stimulated cellular protrusions, suggesting that the PKC activation is involved in the cellular motility of Raw264.7 cells. These findings could constitute a therapeutic target for inflammatory processes with deleterious consequences for human health, such as rheumatoid arthritis, atherosclerosis and cancer. J. Cell. Biochem. 118: 1810-1818, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  17. Threonine 89 Is an Important Residue of Profilin-1 That Is Phosphorylatable by Protein Kinase A.

    Directory of Open Access Journals (Sweden)

    David Gau

    Full Text Available Dynamic regulation of actin cytoskeleton is at the heart of all actin-based cellular events. In this study, we sought to identify novel post-translational modifications of Profilin-1 (Pfn1, an important regulator of actin polymerization in cells.We performed in vitro protein kinase assay followed by mass-spectrometry to identify Protein Kinase A (PKA phosphorylation sites of Pfn1. By two-dimensional gel electrophoresis (2D-GE analysis, we further examined the changes in the isoelectric profile of ectopically expressed Pfn1 in HEK-293 cells in response to forskolin (FSK, an activator of cAMP/PKA pathway. Finally, we combined molecular dynamics simulations (MDS, GST pull-down assay and F-actin analyses of mammalian cells expressing site-specific phosphomimetic variants of Pfn1 to predict the potential consequences of phosphorylation of Pfn1.We identified several PKA phosphorylation sites of Pfn1 including Threonine 89 (T89, a novel site. Consistent with PKA's ability to phosphorylate Pfn1 in vitro, FSK stimulation increased the pool of the most negatively charged form of Pfn1 in HEK-293 cells which can be attenuated by PKA inhibitor H89. MDS predicted that T89 phosphorylation destabilizes an intramolecular interaction of Pfn1, potentially increasing its affinity for actin. The T89D phosphomimetic mutation of Pfn1 elicits several changes that are hallmarks of proteins folded into alternative three-dimensional conformations including detergent insolubility, protein aggregation and accelerated proteolysis, suggesting that T89 is a structurally important residue of Pfn1. Expression of T89D-Pfn1 induces actin:T89D-Pfn1 co-clusters and dramatically reduces overall actin polymerization in cells, indicating an actin-sequestering action of T89D-Pfn1. Finally, rendering T89 non-phosphorylatable causes a positive charge shift in the isoelectric profile of Pfn1 in a 2D gel electrophoresis analysis of cell extracts, a finding that is consistent with

  18. The cellular Mre11 protein interferes with adenovirus E4 mutant DNA replication

    International Nuclear Information System (INIS)

    Mathew, Shomita S.; Bridge, Eileen

    2007-01-01

    Adenovirus type 5 (Ad5) relocalizes and degrades the host DNA repair protein Mre11, and efficiently initiates viral DNA replication. Mre11 associates with Ad E4 mutant DNA replication centers and is important for concatenating viral genomes. We have investigated the role of Mre11 in the E4 mutant DNA replication defect. RNAi-mediated knockdown of Mre11 dramatically rescues E4 mutant DNA replication in cells that do or do not concatenate viral genomes, suggesting that Mre11 inhibits DNA replication independent of genome concatenation. The mediator of DNA damage checkpoint 1 (Mdc1) protein is involved in recruiting and sustaining Mre11 at sites of DNA damage following ionizing radiation. We observe foci formation by Mdc1 in response to viral infection, indicating that this damage response protein is activated. However, knockdown of Mdc1 does not prevent Mre11 from localizing at viral DNA replication foci or rescue E4 mutant DNA replication. Our results are consistent with a model in which Mre11 interferes with DNA replication when it is localized at viral DNA replication foci

  19. Majority of cellular fatty acid acylated proteins are localized to the cytoplasmic surface of the plasma membrane

    International Nuclear Information System (INIS)

    Wilcox, C.A.; Olson, E.N.

    1987-01-01

    The BC 2 Hl muscle cell line was previously reported to contain a broad array of fatty acid acylated proteins. Palmitate was shown to be attached to membrane proteins posttranslationally through thiol ester linkages, whereas myristate was attached cotranslationally, or within seconds thereafter, to soluble and membrane-bound proteins through amide linkages. The temporal and subcellular differences between palmitate and myristate acylation suggested that these two classes of acyl proteins might follow different intracellular pathways to distinct subcellular membrane systems or organelles. In this study, the authors examined the subcellular localization of the major fatty acylated proteins in BC 4 Hl cells. Palmitate-containing proteins were localized to the plasma membrane, but only a subset of myristate-containing proteins was localized to this membrane fraction. The majority of acyl proteins were nonglycosylated and resistant to digestion with extracellular proteases, suggesting that they were not exposed to the external surface of the plasma membrane. Many proteins were, however, digested during incubation of isolated membranes with proteases, which indicates that these proteins were, however, digested during incubation of isolated membranes with proteases, which indicates that these proteins face the cytoplasm. Two-dimensional gel electrophoresis of proteins labeled with [ 3 H]palmitate and [ 3 H]myristate revealed that individual proteins were modified by only one of the two fatty acids and did not undergo both N-linked myristylation and ester-linked palmitylation. Together, these results suggest that the majority of cellular acyl proteins are routed to the cytoplasmic surface of the plasma membrane, and they raise the possibility that fatty acid acylation may play a role in intracellular sorting of nontransmembranous, nonglycosylated membrane proteins

  20. HSPB8 and BAG3 cooperate to promote spatial sequestration of ubiquitinated proteins and coordinate the cellular adaptive response to proteasome insufficiency.

    Science.gov (United States)

    Guilbert, Solenn M; Lambert, Herman; Rodrigue, Marc-Antoine; Fuchs, Margit; Landry, Jacques; Lavoie, Josée N

    2018-02-05

    BCL2-associated athanogene (BAG)-3 is viewed as a platform that would physically and functionally link distinct classes of molecular chaperones of the heat shock protein (HSP) family for the stabilization and clearance of damaged proteins. In this study, we show that HSPB8, a member of the small heat shock protein subfamily, cooperates with BAG3 to coordinate the sequestration of harmful proteins and the cellular adaptive response upon proteasome inhibition. Silencing of HSPB8, like depletion of BAG3, inhibited targeting of ubiquitinated proteins to the juxtanuclear aggresome, a mammalian system of spatial quality control. However, aggresome targeting was restored in BAG3-depleted cells by a mutant BAG3 defective in HSPB8 binding, uncoupling HSPB8 function from its binding to BAG3. Depletion of HSPB8 impaired formation of ubiquitinated microaggregates in an early phase and interfered with accurate modifications of the stress sensor p62/sequestosome (SQSTM)-1. This impairment correlated with decreased coupling of BAG3 to p62/SQSTM1 in response to stress, hindering Kelch-like ECH-associated protein (KEAP)-1 sequestration and stabilization of nuclear factor E2-related factor (Nrf)-2, an important arm of the antioxidant defense. Notably, the myopathy-associated mutation of BAG3 (P209L), which lies within the HSPB8-binding motif, deregulated the association between BAG3 and p62/SQSTM1 and the KEAP1-Nrf2 signaling axis. Together, our findings support a so-far-unrecognized role for the HSPB8-BAG3 connection in mounting of an efficient stress response, which may be involved in BAG3-related human diseases.-Guilbert, S. M., Lambert, H., Rodrigue, M.-A., Fuchs, M., Landry, J., Lavoie, J. N. HSPB8 and BAG3 cooperate to promote spatial sequestration of ubiquitinated proteins and coordinate the cellular adaptive response to proteasome insufficiency.

  1. The G-protein-coupled receptor, GPR84, is important for eye development in Xenopus laevis.

    Science.gov (United States)

    Perry, Kimberly J; Johnson, Verity R; Malloch, Erica L; Fukui, Lisa; Wever, Jason; Thomas, Alvin G; Hamilton, Paul W; Henry, Jonathan J

    2010-11-01

    G-protein-coupled receptors (GPCRs) represent diverse, multifamily groups of cell signaling receptors involved in many cellular processes. We identified Xenopus laevis GPR84 as a member of the A18 subfamily of GPCRs. During development, GPR84 is detected in the embryonic lens placode, differentiating lens fiber cells, retina, and cornea. Anti-sense morpholino oligonucleotide-mediated knockdown and RNA rescue experiments demonstrate GPR84's importance in lens, cornea, and retinal development. Examination of cell proliferation using an antibody against histone H3 S10P reveals significant increases in the lens and retina following GPR84 knockdown. Additionally, there was also an increase in apoptosis in the retina and lens, as revealed by TUNEL assay. Reciprocal transplantation of the presumptive lens ectoderm between uninjected controls and morpholino-injected embryos demonstrates that GPR84 is necessary in the retina for proper development of the retina, as well as other eye tissues including the lens and cornea. © 2010 Wiley-Liss, Inc.

  2. A mutation in human VAP-B--MSP domain, present in ALS patients, affects the interaction with other cellular proteins.

    Science.gov (United States)

    Mitne-Neto, M; Ramos, C R R; Pimenta, D C; Luz, J S; Nishimura, A L; Gonzales, F A; Oliveira, C C; Zatz, M

    2007-09-01

    Amyotrophic Lateral Sclerosis (ALS) is the most common adult-onset Motor Neuron Disease (MND), characterized by motor neurons death in the cortex, brainstem and spinal cord. Ten loci linked to Familial ALS have been mapped. ALS8 is caused by a substitution of a proline by a serine in the Vesicle-Associated Membrane Protein-Associated protein-B/C (VAP-B/C). VAP-B belongs to a highly conserved family of proteins implicated in Endoplasmic Reticulum-Golgi and intra-Golgi transport and microtubules stabilization. Previous studies demonstrated that the P56S mutation disrupts the subcellular localization of VAP-B and that this position would be essential for Unfolded Protein Response (UPR) induced by VAP-B. In the present work we expressed and purified recombinant wild-type and P56S mutant VAP-B-MSP domain for the analysis of its interactions with other cellular proteins. Our findings suggest that the P56S mutation may lead to a less stable interaction of this endoplasmic reticulum protein with at least two other proteins: tubulin and GAPDH. These two proteins have been previously related to other forms of neurodegenerative diseases and are potential key points to understand ALS8 pathogenesis and other forms of MND. Understanding the role of these protein interactions may help the treatment of this devastating disease in the future.

  3. Passive acquisition of leukocyte proteins is associated with changes in phosphorylation of cellular proteins and cell-cell adhesion properties.

    OpenAIRE

    Tabibzadeh, S. S.; Kong, Q. F.; Kapur, S.

    1994-01-01

    In this report, we show that interaction of neoplastic epithelial cells with vesicles derived from leukocytes results in passive acquisition by tumor cells of a diverse group of leukocyte proteins. Vesicles shed from leukocytes were heterogeneous and exhibited the specific proteins expressed on leukocyte subsets. Accordingly, epithelial cells differentially acquired leukocyte proteins associated with vesicles. Ultrastructural localization demonstrated that acquired proteins were associated wi...

  4. 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

  5. Domain-Specific Activation of Death-Associated Intracellular Signalling Cascades by the Cellular Prion Protein in Neuroblastoma Cells.

    Science.gov (United States)

    Vilches, Silvia; Vergara, Cristina; Nicolás, Oriol; Mata, Ágata; Del Río, José A; Gavín, Rosalina

    2016-09-01

    The biological functions of the cellular prion protein remain poorly understood. In fact, numerous studies have aimed to determine specific functions for the different protein domains. Studies of cellular prion protein (PrP(C)) domains through in vivo expression of molecules carrying internal deletions in a mouse Prnp null background have provided helpful data on the implication of the protein in signalling cascades in affected neurons. Nevertheless, understanding of the mechanisms underlying the neurotoxicity induced by these PrP(C) deleted forms is far from complete. To better define the neurotoxic or neuroprotective potential of PrP(C) N-terminal domains, and to overcome the heterogeneity of results due to the lack of a standardized model, we used neuroblastoma cells to analyse the effects of overexpressing PrP(C) deleted forms. Results indicate that PrP(C) N-terminal deleted forms were properly processed through the secretory pathway. However, PrPΔF35 and PrPΔCD mutants led to death by different mechanisms sharing loss of alpha-cleavage and activation of caspase-3. Our data suggest that both gain-of-function and loss-of-function pathogenic mechanisms may be associated with N-terminal domains and may therefore contribute to neurotoxicity in prion disease. Dissecting the molecular response induced by PrPΔF35 may be the key to unravelling the physiological and pathological functions of the prion protein.

  6. The Cellular Chaperone Heat Shock Protein 90 Is Required for Foot-and-Mouth Disease Virus Capsid Precursor Processing and Assembly of Capsid Pentamers.

    Science.gov (United States)

    Newman, Joseph; Asfor, Amin S; Berryman, Stephen; Jackson, Terry; Curry, Stephen; Tuthill, Tobias J

    2018-03-01

    Productive picornavirus infection requires the hijacking of host cell pathways to aid with the different stages of virus entry, synthesis of the viral polyprotein, and viral genome replication. Many picornaviruses, including foot-and-mouth disease virus (FMDV), assemble capsids via the multimerization of several copies of a single capsid precursor protein into a pentameric subunit which further encapsidates the RNA. Pentamer formation is preceded by co- and posttranslational modification of the capsid precursor (P1-2A) by viral and cellular enzymes and the subsequent rearrangement of P1-2A into a structure amenable to pentamer formation. We have developed a cell-free system to study FMDV pentamer assembly using recombinantly expressed FMDV capsid precursor and 3C protease. Using this assay, we have shown that two structurally different inhibitors of the cellular chaperone heat shock protein 90 (hsp90) impeded FMDV capsid precursor processing and subsequent pentamer formation. Treatment of FMDV permissive cells with the hsp90 inhibitor prior to infection reduced the endpoint titer by more than 10-fold while not affecting the activity of a subgenomic replicon, indicating that translation and replication of viral RNA were unaffected by the drug. IMPORTANCE FMDV of the Picornaviridae family is a pathogen of huge economic importance to the livestock industry due to its effect on the restriction of livestock movement and necessary control measures required following an outbreak. The study of FMDV capsid assembly, and picornavirus capsid assembly more generally, has tended to be focused upon the formation of capsids from pentameric intermediates or the immediate cotranslational modification of the capsid precursor protein. Here, we describe a system to analyze the early stages of FMDV pentameric capsid intermediate assembly and demonstrate a novel requirement for the cellular chaperone hsp90 in the formation of these pentameric intermediates. We show the added complexity

  7. The emergence of extracellular matrix mechanics and cell traction forces as important regulators of cellular self-organization.

    Science.gov (United States)

    Checa, Sara; Rausch, Manuel K; Petersen, Ansgar; Kuhl, Ellen; Duda, Georg N

    2015-01-01

    Physical cues play a fundamental role in a wide range of biological processes, such as embryogenesis, wound healing, tumour invasion and connective tissue morphogenesis. Although it is well known that during these processes, cells continuously interact with the local extracellular matrix (ECM) through cell traction forces, the role of these mechanical interactions on large scale cellular and matrix organization remains largely unknown. In this study, we use a simple theoretical model to investigate cellular and matrix organization as a result of mechanical feedback signals between cells and the surrounding ECM. The model includes bi-directional coupling through cellular traction forces to deform the ECM and through matrix deformation to trigger cellular migration. In addition, we incorporate the mechanical contribution of matrix fibres and their reorganization by the cells. We show that a group of contractile cells will self-polarize at a large scale, even in homogeneous environments. In addition, our simulations mimic the experimentally observed alignment of cells in the direction of maximum stiffness and the building up of tension as a consequence of cell and fibre reorganization. Moreover, we demonstrate that cellular organization is tightly linked to the mechanical feedback loop between cells and matrix. Cells with a preference for stiff environments have a tendency to form chains, while cells with a tendency for soft environments tend to form clusters. The model presented here illustrates the potential of simple physical cues and their impact on cellular self-organization. It can be used in applications where cell-matrix interactions play a key role, such as in the design of tissue engineering scaffolds and to gain a basic understanding of pattern formation in organogenesis or tissue regeneration.

  8. Vitamin A, Cancer Treatment and Prevention: The New Role of Cellular Retinol Binding Proteins

    Directory of Open Access Journals (Sweden)

    Elena Doldo

    2015-01-01

    Full Text Available Retinol and vitamin A derivatives influence cell differentiation, proliferation, and apoptosis and play an important physiologic role in a wide range of biological processes. Retinol is obtained from foods of animal origin. Retinol derivatives are fundamental for vision, while retinoic acid is essential for skin and bone growth. Intracellular retinoid bioavailability is regulated by the presence of specific cytoplasmic retinol and retinoic acid binding proteins (CRBPs and CRABPs. CRBP-1, the most diffuse CRBP isoform, is a small 15 KDa cytosolic protein widely expressed and evolutionarily conserved in many tissues. CRBP-1 acts as chaperone and regulates the uptake, subsequent esterification, and bioavailability of retinol. CRBP-1 plays a major role in wound healing and arterial tissue remodelling processes. In the last years, the role of CRBP-1-related retinoid signalling during cancer progression became object of several studies. CRBP-1 downregulation associates with a more malignant phenotype in breast, ovarian, and nasopharyngeal cancers. Reexpression of CRBP-1 increased retinol sensitivity and reduced viability of ovarian cancer cells in vitro. Further studies are needed to explore new therapeutic strategies aimed at restoring CRBP-1-mediated intracellular retinol trafficking and the meaning of CRBP-1 expression in cancer patients’ screening for a more personalized and efficacy retinoid therapy.

  9. Overproduction of a Model Sec- and Tat-Dependent Secretory Protein Elicits Different Cellular Responses in Streptomyces lividans.

    Directory of Open Access Journals (Sweden)

    Sonia Gullón

    Full Text Available Streptomyces lividans is considered an efficient host for the secretory production of homologous and heterologous proteins. To identify possible bottlenecks in the protein production process, a comparative transcriptomic approach was adopted to study cellular responses during the overproduction of a Sec-dependent model protein (alpha-amylase and a Tat-dependent model protein (agarase in Streptomyces lividans. The overproduction of the model secretory proteins via the Sec or the Tat route in S. lividans does elicit a different major cell response in the bacterium. The stringent response is a bacterial response to nutrients' depletion, which naturally occurs at late times of the bacterial cell growth. While the induction of the stringent response at the exponential phase of growth may limit overall productivity in the case of the Tat route, the induction of that response does not take place in the case of the Sec route, which comparatively is an advantage in secretory protein production processes. Hence, this study identifies a potential major drawback in the secretory protein production process depending on the secretory route, and provides clues to improving S. lividans as a protein production host.

  10. Identification of functionally important amino acid residues in the mitochondria targeting sequence of Hepatitis B virus X protein

    International Nuclear Information System (INIS)

    Li, Sai Kam; Ho, Sai Fan; Tsui, Kwok Wing; Fung, Kwok Pui; Waye, M.Y. Mary

    2008-01-01

    Chronic hepatitis B virus (HBV) infection has been strongly associated with hepatocellular carcinoma (HCC) and the X protein (HBx) is thought to mediate the cellular changes associated with carcinogenesis. Recently, isolation of the hepatitis B virus integrants from HCC tissue by others have established the fact that the X gene is often truncated at its C-terminus. Expression of the GFP fusion proteins of HBx and its truncation mutants with a GFP tag in human liver cell-lines in this study revealed that the C-terminus of HBx is indispensable for its specific localization in the mitochondria. A crucial region of seven amino acids at the C-terminus has been mapped out in which the cysteine residue at position 115 serves as the most important residue for the subcellular localization. When cysteine 115 of HBx is mutated to alanine the mitochondria targeting property of HBx is abrogated

  11. Mapping of immunogenic and protein-interacting regions at the surface of the seven-bladed β-propeller domain of the HIV-1 cellular interactor EED

    Directory of Open Access Journals (Sweden)

    Gouet Patrice

    2008-02-01

    Full Text Available Abstract Background The human EED protein, a member of the superfamily of Polycomb group proteins, is involved in multiple cellular protein complexes. Its C-terminal domain, which is common to the four EED isoforms, contains seven repeats of a canonical WD-40 motif. EED is an interactor of three HIV-1 proteins, matrix (MA, integrase (IN and Nef. An antiviral activity has been found to be associated with isoforms EED3 and EED4 at the late stage of HIV-1 replication, due to a negative effect on virus assembly and genomic RNA packaging. The aim of the present study was to determine the regions of the EED C-terminal core domain which were accessible and available to protein interactions, using three-dimensional (3D protein homology modelling with a WD-40 protein of known structure, and epitope mapping of anti-EED antibodies. Results Our data suggested that the C-terminal domain of EED was folded as a seven-bladed β-propeller protein. During the completion of our work, crystallographic data of EED became available from co-crystals of the EED C-terminal core with the N-terminal domain of its cellular partner EZH2. Our 3D-model was in good congruence with the refined structural model determined from crystallographic data, except for a unique α-helix in the fourth β-blade. More importantly, the position of flexible loops and accessible β-strands on the β-propeller was consistent with our mapping of immunogenic epitopes and sites of interaction with HIV-1 MA and IN. Certain immunoreactive regions were found to overlap with the EZH2, MA and IN binding sites, confirming their accessibility and reactivity at the surface of EED. Crystal structure of EED showed that the two discrete regions of interaction with MA and IN did not overlap with each other, nor with the EZH2 binding pocket, but were contiguous, and formed a continuous binding groove running along the lateral face of the β-propeller. Conclusion Identification of antibody-, MA-, IN- and EZH2

  12. Timing of postexercise protein intake is important for muscle hypertrophy with resistance training in elderly humans

    DEFF Research Database (Denmark)

    Esmarck, B.; Andersen, J.L.; Olsen, S.

    2001-01-01

    1. Age-associated loss of skeletal muscle mass and strength can partly be counteracted by resistance training, causing a net synthesis of muscular proteins. Protein synthesis is influenced synergistically by postexercise amino acid supplementation, but the importance of the timing of protein intake...

  13. Effects of cell culture media on the dynamic formation of protein-nanoparticle complexes and influence on the cellular response.

    Science.gov (United States)

    Maiorano, Gabriele; Sabella, Stefania; Sorce, Barbara; Brunetti, Virgilio; Malvindi, Maria Ada; Cingolani, Roberto; Pompa, Pier Paolo

    2010-12-28

    The development of appropriate in vitro protocols to assess the potential toxicity of the ever expanding range of nanoparticles represents a challenging issue, because of the rapid changes of their intrinsic physicochemical properties (size, shape, reactivity, surface area, etc.) upon dispersion in biological fluids. Dynamic formation of protein coating around nanoparticles is a key molecular event, which may strongly impact the biological response in nanotoxicological tests. In this work, by using citrate-capped gold nanoparticles (AuNPs) of different sizes as a model, we show, by several spectroscopic techniques (dynamic light scattering, UV-visible, plasmon resonance light scattering), that proteins-NP interactions are differently mediated by two widely used cellular media (i.e., Dulbecco Modified Eagle's medium (DMEM) and Roswell Park Memorial Institute medium (RPMI), supplemented with fetal bovine serum). We found that, while DMEM elicits the formation of a large time-dependent protein corona, RPMI shows different dynamics with reduced protein coating. Characterization of these nanobioentities was also performed by sodium dodecyl sulfate polyacrylamide gel electrophoresis and mass spectroscopy, revealing that the average composition of protein corona does not reflect the relative abundance of serum proteins. To evaluate the biological impact of such hybrid bionanostructures, several comparative viability assays onto two cell lines (HeLa and U937) were carried out in the two media, in the presence of 15 nm AuNPs. We observed that proteins/NP complexes formed in RPMI are more abundantly internalized in cells as compared to DMEM, overall exerting higher cytotoxic effects. These results show that, beyond an in-depth NPs characterization before cellular experiments, a detailed understanding of the effects elicited by cell culture media on NPs is crucial for standardized nanotoxicology tests.

  14. Silencing of ribosomal protein S9 elicits a multitude of cellular responses inhibiting the growth of cancer cells subsequent to p53 activation.

    Directory of Open Access Journals (Sweden)

    Mikael S Lindström

    Full Text Available BACKGROUND: Disruption of the nucleolus often leads to activation of the p53 tumor suppressor pathway through inhibition of MDM2 that is mediated by a limited set of ribosomal proteins including RPL11 and RPL5. The effects of ribosomal protein loss in cultured mammalian cells have not been thoroughly investigated. Here we characterize the cellular stress response caused by depletion of ribosomal protein S9 (RPS9. METHODOLOGY/PRINCIPAL FINDINGS: Depletion of RPS9 impaired production of 18S ribosomal RNA and induced p53 activity. It promoted p53-dependent morphological differentiation of U343MGa Cl2:6 glioma cells as evidenced by intensified expression of glial fibrillary acidic protein and profound changes in cell shape. U2OS osteosarcoma cells displayed a limited senescence response with increased expression of DNA damage response markers, whereas HeLa cervical carcinoma cells underwent cell death by apoptosis. Knockdown of RPL11 impaired p53-dependent phenotypes in the different RPS9 depleted cell cultures. Importantly, knockdown of RPS9 or RPL11 also markedly inhibited cell proliferation through p53-independent mechanisms. RPL11 binding to MDM2 was retained despite decreased levels of RPL11 protein following nucleolar stress. In these settings, RPL11 was critical for maintaining p53 protein stability but was not strictly required for p53 protein synthesis. CONCLUSIONS: p53 plays an important role in the initial restriction of cell proliferation that occurs in response to decreased level of RPS9. Our results do not exclude the possibility that other nucleolar stress sensing molecules act upstream or in parallel to RPL11 to activate p53. Inhibiting the expression of certain ribosomal proteins, such as RPS9, could be one efficient way to reinitiate differentiation processes or to induce senescence or apoptosis in rapidly proliferating tumor cells.

  15. Drosophila melanogaster cellular repressor of E1A-stimulated genes is a lysosomal protein essential for fly development.

    Science.gov (United States)

    Kowalewski-Nimmerfall, Elisabeth; Schähs, Philipp; Maresch, Daniel; Rendic, Dubravko; Krämer, Helmut; Mach, Lukas

    2014-12-01

    Mammalian cellular repressor of E1A-stimulated genes is a lysosomal glycoprotein implicated in cellular growth and differentiation. The genome of the fruit fly Drosophila melanogaster encodes a putative orthologue (dCREG), suggesting evolutionarily conserved physiological functions of this protein. In D. melanogaster S2 cells, dCREG was found to localize in lysosomes. Further studies revealed that intracellular dCREG is subject of proteolytic maturation. Processing and turnover could be substantially reduced by RNAi-mediated silencing of cathepsin L. In contrast to mammalian cells, lysosomal delivery of dCREG does not depend on its carbohydrate moiety. Furthermore, depletion of the putative D. melanogaster lysosomal sorting receptor lysosomal enzyme receptor protein did not compromise cellular retention of dCREG. We also investigated the developmental consequences of dCREG ablation in whole D. melanogaster flies. Ubiquitous depletion of dCREG proved lethal at the late pupal stage once a knock-down efficiency of >95% was achieved. These results demonstrate that dCREG is essential for proper completion of fly development. Copyright © 2014. Published by Elsevier B.V.

  16. Efficient in vitro import of a cytosolic heat shock protein into pea chloroplasts

    OpenAIRE

    Lubben, Thomas H.; Keegstra, Kenneth

    1986-01-01

    In order to further our understanding of the targeting of nuclear-encoded proteins into intracellular organelles, we have investigated the import of chimeric precursor proteins into pea chloroplasts. Two different chimeric precursor proteins were produced by in vitro expression of chimeric genes. One chimeric precursor contained the transit peptide of the small subunit of soybean ribulose 1,5-bisphosphate carboxylase and the mature peptide of the same protein from pea. The second contained th...

  17. Emerging roles for protein histidine phosphorylation in cellular signal transduction: lessons from the islet ?-cell

    OpenAIRE

    Kowluru, Anjaneyulu

    2008-01-01

    Protein phosphorylation represents one of the key regulatory events in physiological insulin secretion from the islet ?-cell. In this context, several classes of protein kinases (e.g. calcium-, cyclic nucleotide- and phospholipid-dependent protein kinases and tyrosine kinases) have been characterized in the ?-cell. The majority of phosphorylated amino acids identified include phosphoserine, phosphothreonine and phosphotyrosine. Protein histidine phosphorylation has been implicated in the prok...

  18. Topology and cellular localization of the small hydrophobic protein of avian metapneumovirus

    Science.gov (United States)

    The small hydrophobic protein (SH) is a type II integral membrane protein that is packaged into virions and is only present in certain paramyxoviruses including metapneumovirus. In addition to a highly divergent primary sequence, SH proteins vary significantly in size among the different viruses. Hu...

  19. What makes protein indigestible from tissue-related, cellular, and molecular aspects?

    NARCIS (Netherlands)

    Becker, P.M.; Yu, P.Q.

    2013-01-01

    This paper gives an insight into key factors, which impair enzymatic protein digestion. By nature, some proteins in raw products are already poorly digestible because of structural peculiarities, or due to their occurrence in plant cytoplasmic organelles or in cell membranes. In plant-based protein,

  20. Does constructive neutral evolution play an important role in the origin of cellular complexity? Making sense of the origins and uses of biological complexity

    NARCIS (Netherlands)

    Speijer, Dave

    2011-01-01

    Recently, constructive neutral evolution has been touted as an important concept for the understanding of the emergence of cellular complexity. It has been invoked to help explain the development and retention of, amongst others, RNA splicing, RNA editing and ribosomal and mitochondrial respiratory

  1. The endoplasmic reticulum coat protein II transport machinery coordinates cellular lipid secretion and cholesterol biosynthesis

    NARCIS (Netherlands)

    Fryer, Lee G. D.; Jones, Bethan; Duncan, Emma J.; Hutchison, Claire E.; Ozkan, Tozen; Williams, Paul A.; Alder, Olivia; Nieuwdorp, Max; Townley, Anna K.; Mensenkamp, Arjen R.; Stephens, David J.; Dallinga-Thie, Geesje M.; Shoulders, Carol C.

    2014-01-01

    Triglycerides and cholesterol are essential for life in most organisms. Triglycerides serve as the principal energy storage depot and, where vascular systems exist, as a means of energy transport. Cholesterol is essential for the functional integrity of all cellular membrane systems. The endoplasmic

  2. Super-resolution imaging and tracking of protein–protein interactions in sub-diffraction cellular space

    Science.gov (United States)

    Liu, Zhen; Xing, Dong; Su, Qian Peter; Zhu, Yun; Zhang, Jiamei; Kong, Xinyu; Xue, Boxin; Wang, Sheng; Sun, Hao; Tao, Yile; Sun, Yujie

    2014-01-01

    Imaging the location and dynamics of individual interacting protein pairs is essential but often difficult because of the fluorescent background from other paired and non-paired molecules, particularly in the sub-diffraction cellular space. Here we develop a new method combining bimolecular fluorescence complementation and photoactivated localization microscopy for super-resolution imaging and single-molecule tracking of specific protein–protein interactions. The method is used to study the interaction of two abundant proteins, MreB and EF-Tu, in Escherichia coli cells. The super-resolution imaging shows interesting distribution and domain sizes of interacting MreB–EF-Tu pairs as a subpopulation of total EF-Tu. The single-molecule tracking of MreB, EF-Tu and MreB–EF-Tu pairs reveals intriguing localization-dependent heterogonous dynamics and provides valuable insights to understanding the roles of MreB–EF-Tu interactions. PMID:25030837

  3. Involvement of the iron regulatory protein from Eisenia andrei earthworms in the regulation of cellular iron homeostasis.

    Directory of Open Access Journals (Sweden)

    Petra Procházková

    Full Text Available Iron homeostasis in cells is regulated by iron regulatory proteins (IRPs that exist in different organisms. IRPs are cytosolic proteins that bind to iron-responsive elements (IREs of the 5'- or 3'-untranslated regions (UTR of mRNAs that encode many proteins involved in iron metabolism. In this study, we have cloned and described a new regulatory protein belonging to the family of IRPs from the earthworm Eisenia andrei (EaIRP. The earthworm IRE site in 5'-UTR of ferritin mRNA most likely folds into a secondary structure that differs from the conventional IRE structures of ferritin due to the absence of a typically unpaired cytosine that participates in protein binding. Prepared recombinant EaIRP and proteins from mammalian liver extracts are able to bind both mammalian and Eisenia IRE structures of ferritin mRNA, although the affinity of the rEaIRP/Eisenia IRE structure is rather low. This result suggests the possible contribution of a conventional IRE structure. When IRP is supplemented with a Fe-S cluster, it can function as a cytosolic aconitase. Cellular cytosolic and mitochondrial fractions, as well as recombinant EaIRP, exhibit aconitase activity that can be abolished by the action of oxygen radicals. The highest expression of EaIRP was detected in parts of the digestive tract. We can assume that earthworms may possess an IRE/IRP regulatory network as a potential mechanism for maintaining cellular iron homeostasis, although the aconitase function of EaIRP is most likely more relevant.

  4. Involvement of the Iron Regulatory Protein from Eisenia andrei Earthworms in the Regulation of Cellular Iron Homeostasis

    Science.gov (United States)

    Procházková, Petra; Škanta, František; Roubalová, Radka; Šilerová, Marcela; Dvořák, Jiří; Bilej, Martin

    2014-01-01

    Iron homeostasis in cells is regulated by iron regulatory proteins (IRPs) that exist in different organisms. IRPs are cytosolic proteins that bind to iron-responsive elements (IREs) of the 5′- or 3′-untranslated regions (UTR) of mRNAs that encode many proteins involved in iron metabolism. In this study, we have cloned and described a new regulatory protein belonging to the family of IRPs from the earthworm Eisenia andrei (EaIRP). The earthworm IRE site in 5′-UTR of ferritin mRNA most likely folds into a secondary structure that differs from the conventional IRE structures of ferritin due to the absence of a typically unpaired cytosine that participates in protein binding. Prepared recombinant EaIRP and proteins from mammalian liver extracts are able to bind both mammalian and Eisenia IRE structures of ferritin mRNA, although the affinity of the rEaIRP/Eisenia IRE structure is rather low. This result suggests the possible contribution of a conventional IRE structure. When IRP is supplemented with a Fe-S cluster, it can function as a cytosolic aconitase. Cellular cytosolic and mitochondrial fractions, as well as recombinant EaIRP, exhibit aconitase activity that can be abolished by the action of oxygen radicals. The highest expression of EaIRP was detected in parts of the digestive tract. We can assume that earthworms may possess an IRE/IRP regulatory network as a potential mechanism for maintaining cellular iron homeostasis, although the aconitase function of EaIRP is most likely more relevant. PMID:25279857

  5. Multiple Functional Domains and Complexes of the Two Nonstructural Proteins of Human Respiratory Syncytial Virus Contribute to Interferon Suppression and Cellular Location▿

    Science.gov (United States)

    Swedan, Samer; Andrews, Joel; Majumdar, Tanmay; Musiyenko, Alla; Barik, Sailen

    2011-01-01

    Human respiratory syncytial virus (RSV), a major cause of severe respiratory diseases, efficiently suppresses cellular innate immunity, represented by type I interferon (IFN), using its two unique nonstructural proteins, NS1 and NS2. In a search for their mechanism, NS1 was previously shown to decrease levels of TRAF3 and IKKε, whereas NS2 interacted with RIG-I and decreased TRAF3 and STAT2. Here, we report on the interaction, cellular localization, and functional domains of these two proteins. We show that recombinant NS1 and NS2, expressed in lung epithelial A549 cells, can form homo- as well as heteromers. Interestingly, when expressed alone, substantial amounts of NS1 and NS2 localized to the nuclei and to the mitochondria, respectively. However, when coexpressed with NS2, as in RSV infection, NS1 could be detected in the mitochondria as well, suggesting that the NS1-NS2 heteromer localizes to the mitochondria. The C-terminal tetrapeptide sequence, DLNP, common to both NS1 and NS2, was required for some functions, but not all, whereas only the NS1 N-terminal region was important for IKKε reduction. Finally, NS1 and NS2 both interacted specifically with host microtubule-associated protein 1B (MAP1B). The contribution of MAP1B in NS1 function was not tested, but in NS2 it was essential for STAT2 destruction, suggesting a role of the novel DLNP motif in protein-protein interaction and IFN suppression. PMID:21795342

  6. Protein-protein association and cellular localization of four essential gene products encoded by tellurite resistance-conferring cluster "ter" from pathogenic Escherichia coli.

    Science.gov (United States)

    Valkovicova, Lenka; Vavrova, Silvia Minarikova; Mravec, Jozef; Grones, Jozef; Turna, Jan

    2013-12-01

    Gene cluster "ter" conferring high tellurite resistance has been identified in various pathogenic bacteria including Escherichia coli O157:H7. However, the precise mechanism as well as the molecular function of the respective gene products is unclear. Here we describe protein-protein association and localization analyses of four essential Ter proteins encoded by minimal resistance-conferring fragment (terBCDE) by means of recombinant expression. By using a two-plasmid complementation system we show that the overproduced single Ter proteins are not able to mediate tellurite resistance, but all Ter members play an irreplaceable role within the cluster. We identified several types of homotypic and heterotypic protein-protein associations among the Ter proteins by in vitro and in vivo pull-down assays and determined their cellular localization by cytosol/membrane fractionation. Our results strongly suggest that Ter proteins function involves their mutual association, which probably happens at the interface of the inner plasma membrane and the cytosol.

  7. Effect of electromagnetic fields at 2.45 GHz on the levels of cellular stress proteins HSP-90 and 70 in the rat thyroid

    International Nuclear Information System (INIS)

    Misa Agustino, M. J.; Alvarez-Folgueras, M.; Jorge-Mora, M. T.; Jorge Barreiro, F. J.; Ares Pena, F. J.; Lleiro, J.; Lopez Martin, M. E.

    2011-01-01

    In this study we analyzed the cellular stress levels achieved by heat shock proteins (HSP) 90 and 70 in rat thyroid tissue after exposure to radio waves in TWG experimental system. Parallel measurements of body stress in animals by rectal temperature probes allow us to determine whether there is any interaction between temperature increases and cellular stress.

  8. A Critical Appraisal of Quantitative Studies of Protein Degradation in the Framework of Cellular Proteostasis

    Directory of Open Access Journals (Sweden)

    Beatriz Alvarez-Castelao

    2012-01-01

    Full Text Available Protein homeostasis, proteostasis, is essential to understand cell function. Protein degradation is a crucial component of the proteostatic mechanisms of the cell. Experiments on protein degradation are nowadays present in many investigations in the field of molecular and cell biology. In the present paper, we focus on the different experimental approaches to study protein degradation and present a critical appraisal of the results derived from steady-state and kinetic experiments using detection of unlabelled and labelled protein methodologies with a proteostatic perspective. This perspective allows pinpointing the limitations in interpretation of results and the need of further experiments and/or controls to establish “definitive evidence” for the role of protein degradation in the proteostasis of a given protein or the entire proteome. We also provide a spreadsheet for simple calculations of mRNA and protein decays for mimicking different experimental conditions and a checklist for the analysis of experiments dealing with protein degradation studies that may be useful for researchers interested in the area of protein turnover.

  9. Telmisartan enhances mitochondrial activity and alters cellular functions in human coronary artery endothelial cells via AMP-activated protein kinase pathway.

    Science.gov (United States)

    Kurokawa, Hirofumi; Sugiyama, Seigo; Nozaki, Toshimitsu; Sugamura, Koichi; Toyama, Kensuke; Matsubara, Junichi; Fujisue, Koichiro; Ohba, Keisuke; Maeda, Hirofumi; Konishi, Masaaki; Akiyama, Eiichi; Sumida, Hitoshi; Izumiya, Yasuhiro; Yasuda, Osamu; Kim-Mitsuyama, Shokei; Ogawa, Hisao

    2015-04-01

    Mitochondrial dysfunction plays an important role in cellular senescence and impaired function of vascular endothelium, resulted in cardiovascular diseases. Telmisartan is a unique angiotensin II type I receptor blocker that has been shown to prevent cardiovascular events in high risk patients. AMP-activated protein kinase (AMPK) plays a critical role in mitochondrial biogenesis and endothelial function. This study assessed whether telmisartan enhances mitochondrial function and alters cellular functions via AMPK in human coronary artery endothelial cells (HCAECs). In cultured HCAECs, telmisartan significantly enhanced mitochondrial activity assessed by mitochondrial reductase activity and intracellular ATP production and increased the expression of mitochondria related genes. Telmisartan prevented cellular senescence and exhibited the anti-apoptotic and pro-angiogenic properties. The expression of genes related anti-oxidant and pro-angiogenic properties were increased by telmisartan. Telmisartan increased endothelial NO synthase and AMPK phosphorylation. Peroxisome proliferator-activated receptor gamma signaling was not involved in telmisartan-induced improvement of mitochondrial function. All of these effects were abolished by inhibition of AMPK. Telmisartan enhanced mitochondrial activity and exhibited anti-senescence effects and improving endothelial function through AMPK in HCAECs. Telmisartan could provide beneficial effects on vascular diseases via enhancement of mitochondrial activity and modulating endothelial function through AMPK activation. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

  10. A Plastid Protein That Evolved from Ubiquitin and Is Required for Apicoplast Protein Import in Toxoplasma gondii

    Directory of Open Access Journals (Sweden)

    Justin D. Fellows

    2017-06-01

    Full Text Available Apicomplexan parasites cause a variety of important infectious diseases, including malaria, toxoplasma encephalitis, and severe diarrhea due to Cryptosporidium. Most apicomplexans depend on an organelle called the apicoplast which is derived from a red algal endosymbiont. The apicoplast is essential for the parasite as the compartment of fatty acid, heme, and isoprenoid biosynthesis. The majority of the approximate 500 apicoplast proteins are nucleus encoded and have to be imported across the four membranes that surround the apicoplast. Import across the second outermost membrane of the apicoplast, the periplastid membrane, depends on an apicoplast-specific endoplasmic reticulum-associated protein degradation (ERAD complex and on enzymes of the associated ubiquitination cascade. However, identification of an apicoplast ubiquitin associated with this machinery has long been elusive. Here we identify a plastid ubiquitin-like protein (PUBL, an apicoplast protein that is derived from a ubiquitin ancestor but that has significantly changed in its primary sequence. PUBL is distinct from known ubiquitin-like proteins, and phylogenomic analyses suggest a clade specific to apicomplexans. We demonstrate that PUBL and the AAA ATPase CDC48AP both act to translocate apicoplast proteins across the periplastid membrane during protein import. Conditional null mutants and genetic complementation show that both proteins are critical for this process and for parasite survival. PUBL residues homologous to those that are required for ubiquitin conjugation onto target proteins are essential for this function, while those required for polyubiquitination and preprotein processing are dispensable. Our experiments provide a mechanistic understanding of the molecular machinery that drives protein import across the membranes of the apicoplast.

  11. A network biology approach to understanding the importance of chameleon proteins in human physiology and pathology.

    Science.gov (United States)

    Bahramali, Golnaz; Goliaei, Bahram; Minuchehr, Zarrin; Marashi, Sayed-Amir

    2017-02-01

    Chameleon proteins are proteins which include sequences that can adopt α-helix-β-strand (HE-chameleon) or α-helix-coil (HC-chameleon) or β-strand-coil (CE-chameleon) structures to operate their crucial biological functions. In this study, using a network-based approach, we examined the chameleon proteins to give a better knowledge on these proteins. We focused on proteins with identical chameleon sequences with more than or equal to seven residues long in different PDB entries, which adopt HE-chameleon, HC-chameleon, and CE-chameleon structures in the same protein. One hundred and ninety-one human chameleon proteins were identified via our in-house program. Then, protein-protein interaction (PPI) networks, Gene ontology (GO) enrichment, disease network, and pathway enrichment analyses were performed for our derived data set. We discovered that there are chameleon sequences which reside in protein-protein interaction regions between two proteins critical for their dual function. Analysis of the PPI networks for chameleon proteins introduced five hub proteins, namely TP53, EGFR, HSP90AA1, PPARA, and HIF1A, which were presented in four PPI clusters. The outcomes demonstrate that the chameleon regions are in critical domains of these proteins and are important in the development and treatment of human cancers. The present report is the first network-based functional study of chameleon proteins using computational approaches and might provide a new perspective for understanding the mechanisms of diseases helping us in developing new medical therapies along with discovering new proteins with chameleon properties which are highly important in cancer.

  12. Distinct cellular and subcellular distributions of G protein-coupled receptor kinase and arrestin isoforms in the striatum.

    Directory of Open Access Journals (Sweden)

    Evgeny Bychkov

    Full Text Available G protein-coupled receptor kinases (GRKs and arrestins mediate desensitization of G protein-coupled receptors (GPCR. Arrestins also mediate G protein-independent signaling via GPCRs. Since GRK and arrestins demonstrate no strict receptor specificity, their functions in the brain may depend on their cellular complement, expression level, and subcellular targeting. However, cellular expression and subcellular distribution of GRKs and arrestins in the brain is largely unknown. We show that GRK isoforms GRK2 and GRK5 are similarly expressed in direct and indirect pathway neurons in the rat striatum. Arrestin-2 and arrestin-3 are also expressed in neurons of both pathways. Cholinergic interneurons are enriched in GRK2, arrestin-3, and GRK5. Parvalbumin-positive interneurons express more of GRK2 and less of arrestin-2 than medium spiny neurons. The GRK5 subcellular distribution in the human striatal neurons is altered by its phosphorylation: unphosphorylated enzyme preferentially localizes to synaptic membranes, whereas phosphorylated GRK5 is found in plasma membrane and cytosolic fractions. Both GRK isoforms are abundant in the nucleus of human striatal neurons, whereas the proportion of both arrestins in the nucleus was equally low. However, overall higher expression of arrestin-2 yields high enough concentration in the nucleus to mediate nuclear functions. These data suggest cell type- and subcellular compartment-dependent differences in GRK/arrestin-mediated desensitization and signaling.

  13. MNF, an ankyrin repeat protein of myxoma virus, is part of a native cellular SCF complex during viral infection

    Directory of Open Access Journals (Sweden)

    Gelfi Jacqueline

    2010-03-01

    Full Text Available Abstract Myxoma virus (MYXV, a member of the Poxviridae family, is the agent responsible for myxomatosis, a fatal disease in the European rabbit (Oryctolagus cuniculus. Like all poxviruses, MYXV is known for encoding multiple proteins that regulate cellular signaling pathways. Among them, four proteins share the same ANK/PRANC structure: M148R, M149R, MNF (Myxoma Nuclear factor and M-T5, all of them described as virulence factors. This family of poxvirus proteins, recently identified, has drawn considerable attention for its potential role in modulating the host ubiquitin-proteasome system during viral infection. To date, many members of this novel protein family have been shown to interact with SCF components, in vitro. Here, we focus on MNF gene, which has been shown to express a nuclear protein presenting nine ANK repeats, one of which has been identified as a nuclear localization signal. In transfection, MNF has been shown to colocalise with the transcription factor NF-κB in the nucleus of TNFα-stimulated cells. Functionally, MNF is a critical virulence factor since its deletion generates an almost apathogenic virus. In this study, to pursue the investigation of proteins interacting with MNF and of its mechanism of action, we engineered a recombinant MYXV expressing a GFP-linked MNF under the control of MNF native promoter. Infection of rabbits with MYXV-GFPMNF recombinant virus provided the evidence that the GFP fusion does not disturb the main function of MNF. Hence, cells were infected with MYXV-GFPMNF and immunoprecipitation of the GFPMNF fusion protein was performed to identify MNF's partners. For the first time, endogenous components of SCF (Cullin-1 and Skp1 were co-precipitated with an ANK myxoma virus protein, expressed in an infectious context, and without over-expression of any protein.

  14. MNF, an ankyrin repeat protein of myxoma virus, is part of a native cellular SCF complex during viral infection

    Science.gov (United States)

    2010-01-01

    Myxoma virus (MYXV), a member of the Poxviridae family, is the agent responsible for myxomatosis, a fatal disease in the European rabbit (Oryctolagus cuniculus). Like all poxviruses, MYXV is known for encoding multiple proteins that regulate cellular signaling pathways. Among them, four proteins share the same ANK/PRANC structure: M148R, M149R, MNF (Myxoma Nuclear factor) and M-T5, all of them described as virulence factors. This family of poxvirus proteins, recently identified, has drawn considerable attention for its potential role in modulating the host ubiquitin-proteasome system during viral infection. To date, many members of this novel protein family have been shown to interact with SCF components, in vitro. Here, we focus on MNF gene, which has been shown to express a nuclear protein presenting nine ANK repeats, one of which has been identified as a nuclear localization signal. In transfection, MNF has been shown to colocalise with the transcription factor NF-κB in the nucleus of TNFα-stimulated cells. Functionally, MNF is a critical virulence factor since its deletion generates an almost apathogenic virus. In this study, to pursue the investigation of proteins interacting with MNF and of its mechanism of action, we engineered a recombinant MYXV expressing a GFP-linked MNF under the control of MNF native promoter. Infection of rabbits with MYXV-GFPMNF recombinant virus provided the evidence that the GFP fusion does not disturb the main function of MNF. Hence, cells were infected with MYXV-GFPMNF and immunoprecipitation of the GFPMNF fusion protein was performed to identify MNF's partners. For the first time, endogenous components of SCF (Cullin-1 and Skp1) were co-precipitated with an ANK myxoma virus protein, expressed in an infectious context, and without over-expression of any protein. PMID:20211013

  15. Degradation of Human PDZ-Proteins by Human Alphapapillomaviruses Represents an Evolutionary Adaptation to a Novel Cellular Niche.

    Science.gov (United States)

    Van Doorslaer, Koenraad; DeSalle, Rob; Einstein, Mark H; Burk, Robert D

    2015-06-01

    In order to complete their life cycle, papillomaviruses have evolved to manipulate a plethora of cellular pathways. The products of the human Alphapapillomavirus E6 proteins specifically interact with and target PDZ containing proteins for degradation. This viral phenotype has been suggested to play a role in viral oncogenesis. To analyze the association of HPV E6 mediated PDZ-protein degradation with cervical oncogenesis, a high-throughput cell culture assay was developed. Degradation of an epitope tagged human MAGI1 isoform was visualized by immunoblot. The correlation between HPV E6-induced degradation of hMAGI1 and epidemiologically determined HPV oncogenicity was evaluated using a Bayesian approach within a phylogenetic context. All tested oncogenic types degraded the PDZ-containing protein hMAGI1d; however, E6 proteins isolated from several related albeit non-oncogenic viral types were equally efficient at degrading hMAGI1. The relationship between both traits (oncogenicity and PDZ degradation potential) is best explained by a model in which the potential to degrade PDZ proteins was acquired prior to the oncogenic phenotype. This analysis provides evidence that the ancestor of both oncogenic and non-oncogenic HPVs acquired the potential to degrade human PDZ-containing proteins. This suggests that HPV E6 directed degradation of PDZ-proteins represents an ancient ecological niche adaptation. Phylogenetic modeling indicates that this phenotype is not specifically correlated with oncogenic risk, but may act as an enabling phenotype. The role of PDZ protein degradation in HPV fitness and oncogenesis needs to be interpreted in the context of Alphapapillomavirus evolution.

  16. Degradation of Human PDZ-Proteins by Human Alphapapillomaviruses Represents an Evolutionary Adaptation to a Novel Cellular Niche

    Science.gov (United States)

    Van Doorslaer, Koenraad; DeSalle, Rob; Einstein, Mark H.; Burk, Robert D.

    2015-01-01

    In order to complete their life cycle, papillomaviruses have evolved to manipulate a plethora of cellular pathways. The products of the human Alphapapillomavirus E6 proteins specifically interact with and target PDZ containing proteins for degradation. This viral phenotype has been suggested to play a role in viral oncogenesis. To analyze the association of HPV E6 mediated PDZ-protein degradation with cervical oncogenesis, a high-throughput cell culture assay was developed. Degradation of an epitope tagged human MAGI1 isoform was visualized by immunoblot. The correlation between HPV E6-induced degradation of hMAGI1 and epidemiologically determined HPV oncogenicity was evaluated using a Bayesian approach within a phylogenetic context. All tested oncogenic types degraded the PDZ-containing protein hMAGI1d; however, E6 proteins isolated from several related albeit non-oncogenic viral types were equally efficient at degrading hMAGI1. The relationship between both traits (oncogenicity and PDZ degradation potential) is best explained by a model in which the potential to degrade PDZ proteins was acquired prior to the oncogenic phenotype. This analysis provides evidence that the ancestor of both oncogenic and non-oncogenic HPVs acquired the potential to degrade human PDZ-containing proteins. This suggests that HPV E6 directed degradation of PDZ-proteins represents an ancient ecological niche adaptation. Phylogenetic modeling indicates that this phenotype is not specifically correlated with oncogenic risk, but may act as an enabling phenotype. The role of PDZ protein degradation in HPV fitness and oncogenesis needs to be interpreted in the context of Alphapapillomavirus evolution. PMID:26086730

  17. Degradation of Human PDZ-Proteins by Human Alphapapillomaviruses Represents an Evolutionary Adaptation to a Novel Cellular Niche.

    Directory of Open Access Journals (Sweden)

    Koenraad Van Doorslaer

    2015-06-01

    Full Text Available In order to complete their life cycle, papillomaviruses have evolved to manipulate a plethora of cellular pathways. The products of the human Alphapapillomavirus E6 proteins specifically interact with and target PDZ containing proteins for degradation. This viral phenotype has been suggested to play a role in viral oncogenesis. To analyze the association of HPV E6 mediated PDZ-protein degradation with cervical oncogenesis, a high-throughput cell culture assay was developed. Degradation of an epitope tagged human MAGI1 isoform was visualized by immunoblot. The correlation between HPV E6-induced degradation of hMAGI1 and epidemiologically determined HPV oncogenicity was evaluated using a Bayesian approach within a phylogenetic context. All tested oncogenic types degraded the PDZ-containing protein hMAGI1d; however, E6 proteins isolated from several related albeit non-oncogenic viral types were equally efficient at degrading hMAGI1. The relationship between both traits (oncogenicity and PDZ degradation potential is best explained by a model in which the potential to degrade PDZ proteins was acquired prior to the oncogenic phenotype. This analysis provides evidence that the ancestor of both oncogenic and non-oncogenic HPVs acquired the potential to degrade human PDZ-containing proteins. This suggests that HPV E6 directed degradation of PDZ-proteins represents an ancient ecological niche adaptation. Phylogenetic modeling indicates that this phenotype is not specifically correlated with oncogenic risk, but may act as an enabling phenotype. The role of PDZ protein degradation in HPV fitness and oncogenesis needs to be interpreted in the context of Alphapapillomavirus evolution.

  18. Cellular responses to the expression of unstable secretory proteins in the filamentous fungus Aspergillus oryzae.

    Science.gov (United States)

    Yokota, Jun-Ichi; Shiro, Daisuke; Tanaka, Mizuki; Onozaki, Yasumichi; Mizutani, Osamu; Kakizono, Dararat; Ichinose, Sakurako; Shintani, Tomoko; Gomi, Katsuya; Shintani, Takahiro

    2017-03-01

    Filamentous fungi are often used as cell factories for recombinant protein production because of their ability to secrete large quantities of hydrolytic enzymes. However, even using strong transcriptional promoters, yields of nonfungal proteins are generally much lower than those of fungal proteins. Recent analyses revealed that expression of certain nonfungal secretory proteins induced the unfolded protein response (UPR), suggesting that they are recognized as proteins with folding defects in filamentous fungi. More recently, however, even highly expressed endogenous secretory proteins were found to evoke the UPR. These findings raise the question of whether the unfolded or misfolded state of proteins is selectively recognized by quality control mechanisms in filamentous fungi. In this study, a fungal secretory protein (1,2-α-D-mannosidase; MsdS) with a mutation that decreases its thermostability was expressed at different levels in Aspergillus oryzae. We found that, at moderate expression levels, wild-type MsdS was secreted to the medium, while the mutant was not. In the strain with a deletion for the hrdA gene, which is involved in the endoplasmic reticulum-associated degradation pathway, mutant MsdS had specifically increased levels in the intracellular fraction but was not secreted. When overexpressed, the mutant protein was secreted to the medium to a similar extent as the wild-type protein; however, the mutant underwent hyperglycosylation and induced the UPR. Deletion of α-amylase (the most abundant secretory protein in A. oryzae) alleviated the UPR induction by mutant MsdS overexpression. These findings suggest that misfolded MsdS and unfolded species of α-amylase might act synergistically for UPR induction.

  19. Transcellular tunnel dynamics: Control of cellular dewetting by actomyosin contractility and I-BAR proteins.

    Science.gov (United States)

    Lemichez, Emmanuel; Gonzalez-Rodriguez, David; Bassereau, Patricia; Brochard-Wyart, Françoise

    2013-03-01

    Dewetting is the spontaneous withdrawal of a liquid film from a non-wettable surface by nucleation and growth of dry patches. Two recent reports now propose that the principles of dewetting explain the physical phenomena underpinning the opening of transendothelial cell macroaperture (TEM) tunnels, referred to as cellular dewetting. This was discovered by studying a group of bacterial toxins endowed with the property of corrupting actomyosin cytoskeleton contractility. For both liquid and cellular dewetting, the growth of holes is governed by a competition between surface forces and line tension. We also discuss how the dynamics of TEM opening and closure represent remarkable systems to investigate actin cytoskeleton regulation by sensors of plasma membrane curvature and investigate the impact on membrane tension and the role of TEM in vascular dysfunctions. Copyright © 2013 Soçiété Française des Microscopies and Soçiété de Biologie Cellulaire de France.

  20. Role of cellular FKBP52 protein in intracellular trafficking of recombinant adeno-associated virus 2 vectors

    International Nuclear Information System (INIS)

    Zhao Weihong; Zhong Li; Wu Jianqing; Chen Linyuan; Qing Keyun; Weigel-Kelley, Kirsten A.; Larsen, Steven H.; Shou Weinian; Warrington, Kenneth H.; Srivastava, Arun

    2006-01-01

    We have reported that tyrosine-phosphorylated forms of a cellular protein, FKBP52, inhibit the second-strand DNA synthesis of adeno-associated virus 2 (AAV), leading to inefficient transgene expression from recombinant AAV vectors. To further explore the role of FKBP52 in AAV-mediated transduction, we established murine embryo fibroblasts (MEFs) cultures from FKBP52 wild-type (WT), heterozygous (HE), and knockout (KO) mice. Conventional AAV vectors failed to transduce WT MEFs efficiently, and the transduction efficiency was not significantly increased in HE or KO MEFs. AAV vectors failed to traffic efficiently to the nucleus in these cells. Treatment with hydroxyurea (HU) increased the transduction efficiency of conventional AAV vectors by ∼25-fold in WT MEFs, but only by ∼4-fold in KO MEFs. The use of self-complementary AAV (scAAV) vectors, which bypass the requirement of viral second-strand DNA synthesis, revealed that HU treatment increased the transduction efficiency ∼23-fold in WT MEFs, but only ∼4-fold in KO MEFs, indicating that the lack of HU treatment-mediated increase in KO MEFs was not due to failure of AAV to undergo viral second-strand DNA synthesis. Following HU treatment, ∼59% of AAV genomes were present in the nuclear fraction from WT MEFs, but only ∼28% in KO MEFs, indicating that the pathway by which HU treatment mediates nuclear transport of AAV was impaired in KO MEFs. When KO MEFs were stably transfected with an FKBP52 expression plasmid, HU treatment-mediated increase in the transduction efficiency was restored in these cells, which correlated directly with improved intracellular trafficking. Intact AAV particles were also shown to interact with FKBP52 as well as with dynein, a known cellular protein involved in AAV trafficking. These studies suggest that FKBP52, being a cellular chaperone protein, facilitates intracellular trafficking of AAV, which has implications in the optimal use of recombinant AAV vectors in human gene

  1. The role of ORMDL proteins, guardians of cellular sphingolipids, in asthma

    Czech Academy of Sciences Publication Activity Database

    Paulenda, Tomáš; Dráber, Petr

    2016-01-01

    Roč. 71, č. 7 (2016), s. 918-930 ISSN 0105-4538 R&D Projects: GA ČR(CZ) GA14-00703S; GA ČR(CZ) GA14-09807S; GA ČR(CZ) GBP302/12/G101 Institutional support: RVO:68378050 Keywords : asthma * cellular membranes * endoplasmic * reticulum * ORMDL3 * sphingolipids Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 7.361, year: 2016

  2. Effect of perfluorohexane on the expression of cellular adhesion molecules and surfactant protein A in human mesothelial cells in vitro.

    Science.gov (United States)

    Haufe, Dirk; Dahmen, Klaus G; Tiebel, Oliver; Hübler, Matthias; Koch, Thea

    2011-08-01

    The intraperitoneal instillation of perfluorocarbons augmented systemic oxygenation and was protective in mesenteric ischemia-reperfusion and experimental lung injury. To study biocompatibility and potential anti-inflammatory effects of intraperitoneal perfluorocarbons, we evaluated the influence of perfluorohexane and/or inflammatory stimuli on human mesothelial cells in vitro. Perfluorohexane exposure neither impaired cell viability nor induced cellular activation. TNFα enhanced ICAM-1 expression, which was not attenuated by simultaneous perfluorohexane treatment. Concentration of intracellular surfactant protein A tended to be higher in perfluorohexane treated cells compared to controls. Our in vitro data add further evidence that intraperitoneal perfluorocarbon application is feasible without adverse local effects.

  3. Cellular recovery from exposure to sub-optimal concentrations of AB toxins that inhibit protein synthesis

    Science.gov (United States)

    Shiga toxin 1, exotoxin A, diphtheria toxin and ricin are all AB-type protein toxins that act within the host cytosol to kill the host cell through a pathway involving the inhibition of protein synthesis. It is thought that a single molecule of cytosolic toxin is sufficient to kill the host cell. In...

  4. Interactions between vertebrate hemoglobins and red cell proteins: Possible roles in regulating cellular metabolism and rheology

    DEFF Research Database (Denmark)

    Weber, Roy E.

    2007-01-01

    , chicken and human cdB3 peptides on O2 binding properties of fish, bird and mammalian Hbs are consistent with such a role in endothermic, but not in ectothermic, vertebrates3. Measurements of the interaction between Hbs and anionic domains of Band 3, other membrane proteins and intracellular proteins (band...

  5. Identification of dynamic changes in proteins associated with the cellular cytoskeleton after exposure to okadaic acid

    DEFF Research Database (Denmark)

    Opsahl, Jill A; Ljostveit, Sonja; Solstad, Therese

    2013-01-01

    be combined with control cells before the isolation of lipid rafts. Protein phosphorylation events and translocations induced by okadaic acid were identified by mass spectrometry. Okadaic acid was shown to regulate the phosphorylation status and location of proteins associated with the actin cytoskeleton...... of the cortical actin cytoskeleton and cell detachment....

  6. Bio-solubilization of Chinese lignite II: extra-cellular protein analysis

    Energy Technology Data Exchange (ETDEWEB)

    Tao, Xiu-xiang; Pan, Lan-ying; Shi, Kai-yi; Chen-hui; Yin, Su-dong; Luo, Zhen-fu [China University of Mining & Technology, Xuzhou (China). School of Chemical Engineering and Technology

    2009-05-15

    A white rot fungus strain, Trichoderma sp. AH, was isolated from rotten wood in Fushun and used to study the mechanism of lignite bio-solubilization. The results showed that nitric acid pretreated Fushun lignite was solubilized by T. sp. AH and that extracellular proteins from T. sp. AH were correlated with the lignite bio-solubilization results. In the presence of Fushun lignite the extracellular protein concentration from T. sp. AH was 4.5 g/L while the concentration was 3 g/L in the absence of Fushun lignite. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of the extracelular proteins detected at least four new protein bands after the T. sp. AH had solubilized the lignite. Enzyme color reactions showed that extracelular proteins from T. sp. AH mainly consisted of phenol-oxidases, but that lignin decomposition enzymes such as laccase, peroxidase and manganese peroxidases were not present. 9 refs., 8 figs.

  7. FINE SPECIFICITY OF CELLULAR IMMUNE-RESPONSES IN HUMANS TO HUMAN CYTOMEGALOVIRUS IMMEDIATE-EARLY 1-PROTEIN

    NARCIS (Netherlands)

    ALP, NJ; ALLPORT, TD; VANZANTEN, J; RODGERS, B; SISSONS, JGP; BORYSIEWICZ, LK

    Cell-mediated immunity is important in maintaining the virus-host equilibrium in persistent human cytomegalovirus (HCMV) infection. The HCMV 72-kDa major immediate early 1 protein (IE1) is a target for CD8+ cytotoxic T cells in humans, as is the equivalent 89-kDa protein in mouse. Less is known

  8. The human ubiquitin-conjugating enzyme Cdc34 controls cellular proliferation through regulation of p27Kip1 protein levels

    International Nuclear Information System (INIS)

    Butz, Nicole; Ruetz, Stephan; Natt, Francois; Hall, Jonathan; Weiler, Jan; Mestan, Juergen; Ducarre, Monique; Grossenbacher, Rita; Hauser, Patrick; Kempf, Dominique; Hofmann, Francesco

    2005-01-01

    Ubiquitin-mediated degradation of the cyclin-dependent kinase inhibitor p27 Kip1 was shown to be required for the activation of key cyclin-dependent kinases, thereby triggering the onset of DNA replication and cell cycle progression. Although the SCF Skp2 ubiquitin ligase has been reported to mediate p27 Kip1 degradation, the nature of the human ubiquitin-conjugating enzyme involved in this process has not yet been determined at the cellular level. Here, we show that antisense oligonucleotides targeting the human ubiquitin-conjugating enzyme Cdc34 downregulate its expression, inhibit the degradation of p27 Kip1 , and prevent cellular proliferation. Elevation of p27 Kip1 protein level is found to be the sole requirement for the inhibition of cellular proliferation induced upon downregulation of Cdc34. Indeed, reducing the expression of p27 Kip1 with a specific antisense oligonucleotide is sufficient to reverse the anti-proliferative phenotype elicited by the Cdc34 antisense. Furthermore, downregulation of Cdc34 is found to specifically increase the abundance of the SCF Skp2 ubiquitin ligase substrate p27 Kip1 , but has no concomitant effect on the level of IkBα and β-catenin, which are known substrates of a closely related SCF ligase

  9. Protein Modification with Amphiphilic Block Copoly(2-oxazoline)s as a New Platform for Enhanced Cellular Delivery

    KAUST Repository

    Tong, Jing

    2010-08-02

    Several homopolymers, random copolymers and block copolymers based on poly(2-oxazoline)s (POx) were synthesized and conjugated to horseradish peroxidase (HRP) using biodegradable and nonbiodegradable linkers. These conjugates were characterized by amino group titration, polyacrylamide gel electrophoresis (PAGE), isoelectric focusing, enzymatic activity assay and conformation analysis. The conjugates contained on average from about one to two polymer chains per enzyme. From 70% to 90% of enzymatic activity was retained in most cases. Circular dichroism (CD) analysis revealed that HRP modification affected the secondary structure of the apoprotein but did not affect the tertiary structure and heme environment. Enhanced cellular uptake was found in the conjugates of two block copolymers using both MDCK cells and Caco-2 cells, but not in the conjugates of random copolymer and homopolymer. Conjugation with a block copolymer of 2-methyl-2-oxazoline and 2-butyl-2-oxazoline led to the highest cellular uptake as compared to other conjugates. Our data indicates that modification with amphiphilic POx has the potential to modulate and enhance cellular delivery of proteins.

  10. Protein Modification with Amphiphilic Block Copoly(2-oxazoline)s as a New Platform for Enhanced Cellular Delivery

    KAUST Repository

    Tong, Jing; Luxenhofer, Robert; Yi, Xiang; Jordan, Rainer; Kabanov, Alexander V.

    2010-01-01

    Several homopolymers, random copolymers and block copolymers based on poly(2-oxazoline)s (POx) were synthesized and conjugated to horseradish peroxidase (HRP) using biodegradable and nonbiodegradable linkers. These conjugates were characterized by amino group titration, polyacrylamide gel electrophoresis (PAGE), isoelectric focusing, enzymatic activity assay and conformation analysis. The conjugates contained on average from about one to two polymer chains per enzyme. From 70% to 90% of enzymatic activity was retained in most cases. Circular dichroism (CD) analysis revealed that HRP modification affected the secondary structure of the apoprotein but did not affect the tertiary structure and heme environment. Enhanced cellular uptake was found in the conjugates of two block copolymers using both MDCK cells and Caco-2 cells, but not in the conjugates of random copolymer and homopolymer. Conjugation with a block copolymer of 2-methyl-2-oxazoline and 2-butyl-2-oxazoline led to the highest cellular uptake as compared to other conjugates. Our data indicates that modification with amphiphilic POx has the potential to modulate and enhance cellular delivery of proteins.

  11. Sulfolobus turreted icosahedral virus c92 protein responsible for the formation of pyramid-like cellular lysis structures.

    Science.gov (United States)

    Snyder, Jamie C; Brumfield, Susan K; Peng, Nan; She, Qunxin; Young, Mark J

    2011-07-01

    Host cells infected by Sulfolobus turreted icosahedral virus (STIV) have been shown to produce unusual pyramid-like structures on the cell surface. These structures represent a virus-induced lysis mechanism that is present in Archaea and appears to be distinct from the holin/endolysin system described for DNA bacteriophages. This study investigated the STIV gene products required for pyramid formation in its host Sulfolobus solfataricus. Overexpression of STIV open reading frame (ORF) c92 in S. solfataricus alone is sufficient to produce the pyramid-like lysis structures in cells. Gene disruption of c92 within STIV demonstrates that c92 is an essential protein for virus replication. Immunolocalization of c92 shows that the protein is localized to the cellular membranes forming the pyramid-like structures.

  12. Protein translocation channel of mitochondrial inner membrane and matrix-exposed import motor communicate via two-domain coupling protein.

    Science.gov (United States)

    Banerjee, Rupa; Gladkova, Christina; Mapa, Koyeli; Witte, Gregor; Mokranjac, Dejana

    2015-12-29

    The majority of mitochondrial proteins are targeted to mitochondria by N-terminal presequences and use the TIM23 complex for their translocation across the mitochondrial inner membrane. During import, translocation through the channel in the inner membrane is coupled to the ATP-dependent action of an Hsp70-based import motor at the matrix face. How these two processes are coordinated remained unclear. We show here that the two domain structure of Tim44 plays a central role in this process. The N-terminal domain of Tim44 interacts with the components of the import motor, whereas its C-terminal domain interacts with the translocation channel and is in contact with translocating proteins. Our data suggest that the translocation channel and the import motor of the TIM23 complex communicate through rearrangements of the two domains of Tim44 that are stimulated by translocating proteins.

  13. Proteome analyses of cellular proteins in methicillin-resistant Staphylococcus aureus treated with rhodomyrtone, a novel antibiotic candidate.

    Directory of Open Access Journals (Sweden)

    Wipawadee Sianglum

    Full Text Available The ethanolic extract from Rhodomyrtus tomentosa leaf exhibited good antibacterial activities against both methicillin-resistant Staphylococcus aureus (MRSA and S. aureus ATCC 29213. Its minimal inhibitory concentration (MIC values ranged from 31.25-62.5 µg/ml, and the minimal bactericidal concentration (MBC was 250 µg/ml. Rhodomyrtone, an acylphloroglucinol derivative, was 62.5-125 times more potent at inhibiting the bacteria than the ethanolic extract, the MIC and MBC values were 0.5 µg/ml and 2 µg/ml, respectively. To provide insights into antibacterial mechanisms involved, the effects of rhodomyrtone on cellular protein expression of MRSA have been investigated using proteomic approaches. Proteome analyses revealed that rhodomyrtone at subinhibitory concentration (0.174 µg/ml affected the expression of several major functional classes of whole cell proteins in MRSA. The identified proteins involve in cell wall biosynthesis and cell division, protein degradation, stress response and oxidative stress, cell surface antigen and virulence factor, and various metabolic pathways such as amino acid, carbohydrate, energy, lipid, and nucleotide metabolism. Transmission electron micrographs confirmed the effects of rhodomyrtone on morphological and ultrastructural alterations in the treated bacterial cells. Biological processes in cell wall biosynthesis and cell division were interrupted. Prominent changes including alterations in cell wall, abnormal septum formation, cellular disintegration, and cell lysis were observed. Unusual size and shape of staphylococcal cells were obviously noted in the treated MRSA. These pioneer findings on proteomic profiling and phenotypic features of rhodomyrtone-treated MRSA may resolve its antimicrobial mechanisms which could lead to the development of a new effective regimen for the treatment of MRSA infections.

  14. Complement factor H family proteins in their non-canonical role as modulators of cellular functions.

    Science.gov (United States)

    Józsi, Mihály; Schneider, Andrea E; Kárpáti, Éva; Sándor, Noémi

    2018-01-04

    Complement factor H is a major regulator of the alternative pathway of the complement system. The factor H-related proteins are less characterized, but recent data indicate that they rather promote complement activation. These proteins have some common ligands with factor H and have both overlapping and distinct functions depending on domain composition and the degree of conservation of amino acid sequence. Factor H and some of the factor H-related proteins also appear in a non-canonical function that is beyond their role in the modulation of complement activation. This review covers our current understanding on this emerging role of factor H family proteins in modulating the activation and function of various cells by binding to receptors or receptor ligands. Copyright © 2018 Elsevier Ltd. All rights reserved.

  15. Unraveling the cellular context of cyclic nucleotide signaling proteins by chemical proteomics

    NARCIS (Netherlands)

    Corradini, E.

    2015-01-01

    Understanding the molecular mechanisms which regulate signal transduction is fundamental to the development of therapeutic molecules for the treatment of several diseases. In particular, signaling proteins, such as cyclic nucleotide dependent enzymes are the orchestrators of many tissue functions.

  16. Replication protein A, the laxative that keeps DNA regular: The importance of RPA phosphorylation in maintaining genome stability.

    Science.gov (United States)

    Byrne, Brendan M; Oakley, Gregory G

    2018-04-20

    The eukaryotic ssDNA-binding protein, Replication protein A (RPA), was first discovered almost three decades ago. Since then, much progress has been made to elucidate the critical roles for RPA in DNA metabolic pathways that help promote genomic stability. The canonical RPA heterotrimer (RPA1-3) is an essential coordinator of DNA metabolism that interacts with ssDNA and numerous protein partners to coordinate its roles in DNA replication, repair, recombination and telomere maintenance. An alternative form of RPA, termed aRPA, is formed by a complex of RPA4 with RPA1 and RPA3. aRPA is expressed differentially in cells compared to canonical RPA and has been shown to inhibit canonical RPA function while allowing for regular maintenance of cell viability. Interestingly, while aRPA is defective in DNA replication and cell cycle progression, it was shown to play a supporting role in nucleotide excision repair and recombination. The binding domains of canonical RPA interact with a growing number of partners involved in numerous genome maintenance processes. The protein interactions of the RPA-ssDNA complex are not only governed by competition between the binding proteins but also by post-translation modifications such as phosphorylation. Phosphorylation of RPA2 is an important post-translational modification of the RPA complex, and is essential for directing context-specific functions of the RPA complex in the DNA damage response. Due to the importance of RPA in cellular metabolism, it was identified as an appealing target for chemotherapeutic drug development that could be used in future cancer treatment regimens. Copyright © 2018 Elsevier Ltd. All rights reserved.

  17. Cellular Cholesterol Regulates Ubiquitination and Degradation of the Cholesterol Export Proteins ABCA1 and ABCG1*

    Science.gov (United States)

    Hsieh, Victar; Kim, Mi-Jurng; Gelissen, Ingrid C.; Brown, Andrew J.; Sandoval, Cecilia; Hallab, Jeannette C.; Kockx, Maaike; Traini, Mathew; Jessup, Wendy; Kritharides, Leonard

    2014-01-01

    The objective of this study was to examine the influence of cholesterol in post-translational control of ABCA1 and ABCG1 protein expression. Using CHO cell lines stably expressing human ABCA1 or ABCG1, we observed that the abundance of these proteins is increased by cell cholesterol loading. The response to increased cholesterol is rapid, is independent of transcription, and appears to be specific for these membrane proteins. The effect is mediated through cholesterol-dependent inhibition of transporter protein degradation. Cell cholesterol loading similarly regulates degradation of endogenously expressed ABCA1 and ABCG1 in human THP-1 macrophages. Turnover of ABCA1 and ABCG1 is strongly inhibited by proteasomal inhibitors and is unresponsive to inhibitors of lysosomal proteolysis. Furthermore, cell cholesterol loading inhibits ubiquitination of ABCA1 and ABCG1. Our findings provide evidence for a rapid, cholesterol-dependent, post-translational control of ABCA1 and ABCG1 protein levels, mediated through a specific and sterol-sensitive mechanism for suppression of transporter protein ubiquitination, which in turn decreases proteasomal degradation. This provides a mechanism for acute fine-tuning of cholesterol transporter activity in response to fluctuations in cell cholesterol levels, in addition to the longer term cholesterol-dependent transcriptional regulation of these genes. PMID:24500716

  18. Discovery of Cellular Proteins Required for the Early Steps of HCV Infection Using Integrative Genomics

    Science.gov (United States)

    Yang, Jae-Seong; Kwon, Oh Sung; Kim, Sanguk; Jang, Sung Key

    2013-01-01

    Successful viral infection requires intimate communication between virus and host cell, a process that absolutely requires various host proteins. However, current efforts to discover novel host proteins as therapeutic targets for viral infection are difficult. Here, we developed an integrative-genomics approach to predict human genes involved in the early steps of hepatitis C virus (HCV) infection. By integrating HCV and human protein associations, co-expression data, and tight junction-tetraspanin web specific networks, we identified host proteins required for the early steps in HCV infection. Moreover, we validated the roles of newly identified proteins in HCV infection by knocking down their expression using small interfering RNAs. Specifically, a novel host factor CD63 was shown to directly interact with HCV E2 protein. We further demonstrated that an antibody against CD63 blocked HCV infection, indicating that CD63 may serve as a new therapeutic target for HCV-related diseases. The candidate gene list provides a source for identification of new therapeutic targets. PMID:23593195

  19. Membrane-bound heat shock proteins facilitate the uptake of dying cells and cross-presentation of cellular antigen.

    Science.gov (United States)

    Zhu, Haiyan; Fang, Xiaoyun; Zhang, Dongmei; Wu, Weicheng; Shao, Miaomiao; Wang, Lan; Gu, Jianxin

    2016-01-01

    Heat shock proteins (HSPs) were originally identified as stress-responsive proteins and serve as molecular chaperones in different intracellular compartments. Translocation of HSPs to the cell surface and release of HSPs into the extracellular space have been observed during the apoptotic process and in response to a variety of cellular stress. Here, we report that UV irradiation and cisplatin treatment rapidly induce the expression of membrane-bound Hsp60, Hsp70, and Hsp90 upstream the phosphatidylserine exposure. Membrane-bound Hsp60, Hsp70 and Hsp90 could promote the release of IL-6 and IL-1β as well as DC maturation by the evaluation of CD80 and CD86 expression. On the other hand, Hsp60, Hsp70 and Hsp90 on cells could facilitate the uptake of dying cells by bone marrow-derived dendritic cells. Lectin-like oxidized LDL receptor-1 (LOX-1), as a common receptor for Hsp60, Hsp70, and Hsp90, is response for their recognition and mediates the uptake of dying cells. Furthermore, membrane-bound Hsp60, Hsp70 and Hsp90 could promote the cross-presentation of OVA antigen from E.G7 cells and inhibition of the uptake of dying cells by LOX-1 decreases the cross-presentation of cellular antigen. Therefore, the rapid exposure of HSPs on dying cells at the early stage allows for the recognition by and confers an activation signal to the immune system.

  20. Cellular and Tumor Radiosensitivity is Correlated to Epidermal Growth Factor Receptor Protein Expression Level in Tumors Without EGFR Amplification

    International Nuclear Information System (INIS)

    Kasten-Pisula, Ulla; Saker, Jarob; Eicheler, Wolfgang; Krause, Mechthild; Yaromina, Ala; Meyer-Staeckling, Soenke; Scherkl, Benjamin; Kriegs, Malte; Brandt, Burkhard; Grenman, Reidar; Petersen, Cordula; Baumann, Michael; Dikomey, Ekkehard

    2011-01-01

    Purpose: There is conflicting evidence for whether the expression of epidermal growth factor receptor in human tumors can be used as a marker of radioresponse. Therefore, this association was studied in a systematic manner using squamous cell carcinoma (SCC) cell lines grown as cell cultures and xenografts. Methods and Materials: The study was performed with 24 tumor cell lines of different tumor types, including 10 SCC lines, which were also investigated as xenografts on nude mice. Egfr gene dose and the length of CA-repeats in intron 1 were determined by polymerase chain reaction, protein expression in vitro by Western blot and in vivo by enzyme-linked immunosorbent assay, and radiosensitivity in vitro by colony formation. Data were correlated with previously published tumor control dose 50% data after fractionated irradiation of xenografts of the 10 SCC. Results: EGFR protein expression varies considerably, with most tumor cell lines showing moderate and only few showing pronounced upregulation. EGFR upregulation could only be attributed to massive gene amplification in the latter. In the case of little or no amplification, in vitro EGFR expression correlated with both cellular and tumor radioresponse. In vivo EGFR expression did not show this correlation. Conclusions: Local tumor control after the fractionated irradiation of tumors with little or no gene amplification seems to be dependent on in vitro EGFR via its effect on cellular radiosensitivity.

  1. Recombinant proteins of Zaire ebolavirus induce potent humoral and cellular immune responses and protect against live virus infection in mice.

    Science.gov (United States)

    Lehrer, Axel T; Wong, Teri-Ann S; Lieberman, Michael M; Humphreys, Tom; Clements, David E; Bakken, Russell R; Hart, Mary Kate; Pratt, William D; Dye, John M

    2018-05-24

    Infections with filoviruses in humans are highly virulent, causing hemorrhagic fevers which result in up to 90% mortality. In addition to natural infections, the ability to use these viruses as bioterrorist weapons is of significant concern. Currently, there are no licensed vaccines or therapeutics available to combat these infections. The pathogenesis of disease involves the dysregulation of the host's immune system, which results in impairment of the innate and adaptive immune responses, with subsequent development of lymphopenia, thrombocytopenia, hemorrhage, and death. Questions remain with regard to the few survivors of infection, who manage to mount an effective adaptive immune response. These questions concern the humoral and cellular components of this response, and whether such a response can be elicited by an appropriate prophylactic vaccine. The data reported herein describe the production and evaluation of a recombinant subunit Ebola virus vaccine candidate consisting of insect cell expressed Zaire ebolavirus (EBOV) surface glycoprotein (GP) and the matrix proteins VP24 and VP40. The recombinant subunit proteins are shown to be highly immunogenic in mice, yielding both humoral and cellular responses, as well as highly efficacious, providing up to 100% protection against a lethal challenge with live virus. These results demonstrate proof of concept for such a recombinant non-replicating vaccine candidate in the mouse model of EBOV which helps to elucidate immune correlates of protection and warrants further development. Copyright © 2017 Elsevier Ltd. All rights reserved.

  2. The 'sweet' spot of cellular pluripotency: protein glycosylation in human pluripotent stem cells and its applications in regenerative medicine.

    Science.gov (United States)

    Wang, Yu-Chieh; Lin, Victor; Loring, Jeanne F; Peterson, Suzanne E

    2015-05-01

    Human pluripotent stem cells (hPSCs) promise for the future of regenerative medicine. The structural and biochemical diversity associated with glycans makes them a unique type of macromolecule modification that is involved in the regulation of a vast array of biochemical events and cellular activities including pluripotency in hPSCs. The primary focus of this review article is to highlight recent advances in stem cell research from a glycobiological perspective. We also discuss how our understanding of glycans and glycosylation may help overcome barriers hindering the clinical application of hPSC-derived cells. A literature survey using NCBI-PubMed and Google Scholar was performed in 2014. Regenerative medicine hopes to provide novel strategies to combat human disease and tissue injury that currently lack effective therapies. Although progress in this field is accelerating, many critical issues remain to be addressed in order for cell-based therapy to become a practical and safe treatment option. Emerging evidence suggests that protein glycosylation may significantly influence the regulation of cellular pluripotency, and that the exploitation of protein glycosylation in hPSCs and their differentiated derivatives may lead to transformative and translational discoveries for regenerative medicine. In addition, hPSCs represent a novel research platform for investigating glycosylation-related disease.

  3. TOM9.2 Is a Calmodulin-Binding Protein Critical for TOM Complex Assembly but Not for Mitochondrial Protein Import in Arabidopsis thaliana.

    Science.gov (United States)

    Parvin, Nargis; Carrie, Chris; Pabst, Isabelle; Läßer, Antonia; Laha, Debabrata; Paul, Melanie V; Geigenberger, Peter; Heermann, Ralf; Jung, Kirsten; Vothknecht, Ute C; Chigri, Fatima

    2017-04-03

    The translocon on the outer membrane of mitochondria (TOM) facilitates the import of nuclear-encoded proteins. The principal machinery of mitochondrial protein transport seems conserved in eukaryotes; however, divergence in the composition and structure of TOM components has been observed between mammals, yeast, and plants. TOM9, the plant homolog of yeast Tom22, is significantly smaller due to a truncation in the cytosolic receptor domain, and its precise function is not understood. Here we provide evidence showing that TOM9.2 from Arabidopsis thaliana is involved in the formation of mature TOM complex, most likely by influencing the assembly of the pore-forming subunit TOM40. Dexamethasone-induced RNAi gene silencing of TOM9.2 results in a severe reduction in the mature TOM complex, and the assembly of newly imported TOM40 into the complex is impaired. Nevertheless, mutant plants are fully viable and no obvious downstream effects of the loss of TOM complex, i.e., on mitochondrial import capacity, were observed. Furthermore, we found that TOM9.2 can bind calmodulin (CaM) in vitro and that CaM impairs the assembly of TOM complex in the isolated wild-type mitochondria, suggesting a regulatory role of TOM9.2 and a possible integration of TOM assembly into the cellular calcium signaling network. Copyright © 2017 The Author. Published by Elsevier Inc. All rights reserved.

  4. Stimulation of the human immunodeficiency virus type 1 enhancer by the human T-cell leukemia virus type I tax gene product involves the action of inducible cellular proteins.

    Science.gov (United States)

    Böhnlein, E; Siekevitz, M; Ballard, D W; Lowenthal, J W; Rimsky, L; Bogérd, H; Hoffman, J; Wano, Y; Franza, B R; Greene, W C

    1989-04-01

    The human immunodeficiency virus type 1 (HIV-1) preferentially infects CD4+ T lymphocytes and may exist as a latent provirus within these cells for extended periods. The transition to a productive retroviral infection results in T-cell death and clinically may lead to the acquired immune deficiency syndrome. Accelerated production of infectious HIV-1 virions appears to be closely linked to a heightened state of T-cell activation. The transactivator (Tax) protein of the type I human T-cell leukemia virus (HTLV-I) can produce such an activated T-cell phenotype and augments activity of the HIV-1 long terminal repeat. One Tax-responsive region within the HIV-1 long terminal repeat has been mapped to a locus composed of two 10-base-pair direct repeats sharing homology with the binding site for the eucaryotic transcription factor NF-kappaB (GGGACTTTCC). Tax-expressing Jurkat T cells contain one or more inducible cellular proteins that specifically associate with the HIV-1 enhancer at these binding sites. Microscale DNA affinity precipitation assays identified a Tax-inducible 86-kilodalton protein, HIVEN86A, as one of these HIV-1 enhancer-binding factors. The interaction of HIVEN86A, and presumably other cellular proteins, with the HIV-1 enhancer appears functionally important as oligonucleotides corresponding to this enhancer were sufficient to impart Tax inducibility to an unresponsive heterologous promoter. These findings suggest that the Tax-inducible cellular protein HIVEN86A plays an important role in the transcriptional activation of the HIV-1 enhancer. These specific protein-DNA interactions may also be important for the transition of HIV-1 from a latent to a productive mode of infection. Furthermore, these findings highlight an intriguing biological interplay between HTLV-1 and HIV-1 through a cellular transcriptional pathway that is normally involved in T-cell activation and growth.

  5. Participation of 14-3-3ε and 14-3-3ζ proteins in the phagocytosis, component of cellular immune response, in Aedes mosquito cell lines.

    Science.gov (United States)

    Trujillo-Ocampo, Abel; Cázares-Raga, Febe Elena; Del Angel, Rosa María; Medina-Ramírez, Fernando; Santos-Argumedo, Leopoldo; Rodríguez, Mario H; Hernández-Hernández, Fidel de la Cruz

    2017-08-01

    Better knowledge of the innate immune system of insects will improve our understanding of mosquitoes as potential vectors of diverse pathogens. The ubiquitously expressed 14-3-3 protein family is evolutionarily conserved from yeast to mammals, and at least two isoforms of 14-3-3, the ε and ζ, have been identified in insects. These proteins have been shown to participate in both humoral and cellular immune responses in Drosophila. As mosquitoes of the genus Aedes are the primary vectors for arboviruses, causing several diseases such as dengue fever, yellow fever, Zika and chikungunya fevers, cell lines derived from these mosquitoes, Aag-2 from Aedes aegypti and C6/36 HT from Aedes albopictus, are currently used to study the insect immune system. Here, we investigated the role of 14-3-3 proteins (ε and ζ isoform) in phagocytosis, the main cellular immune responses executed by the insects, using Aedes spp. cell lines. We evaluated the mRNA and protein expression of 14-3-3ε and 14-3-3ζ in C6/36 HT and Aag-2 cells, and demonstrated that both proteins were localised in the cytoplasm. Further, in C6/36 HT cells treated with a 14-3-3 specific inhibitor we observed a notable modification of cell morphology with filopodia-like structure caused through cytoskeleton reorganisation (co-localization of 14-3-3 proteins with F-actin), more importantly the decrease in Salmonella typhimurium, Staphylococcus aureus and E. coli phagocytosis and reduction in phagolysosome formation. Additionally, silencing of 14-3-3ε and 14-3-3ζ expression by mean of specific DsiRNA confirmed the decreased phagocytosis and phagolysosome formation of pHrodo labelled E. coli and S. aureus bacteria by Aag-2 cells. The 14-3-3ε and 14-3-3ζ proteins modulate cytoskeletal remodelling, and are essential for phagocytosis of Gram-positive and Gram-negative bacteria in Aedes spp. cell lines.

  6. Cellular protein receptors of maculosin, a host specific phytotoxin of spotted knapweed (Centaurea maculosa L.).

    Science.gov (United States)

    Park, S H; Strobel, G A

    1994-01-05

    Maculosin (the diketopiperazine, cyclo (L-Pro-L-Tyr)) is a host specific phytotoxin produced by Alternaria alternata on spotted knapweed (Centaurea maculosa L.). Receptors for this phytotoxin have been isolated from spotted knapweed. Knapweed leaves possess most of the maculosin-binding activity in the cytosolic fraction. However, activity was also observed in the whole membrane fraction of the leaf. The binding component of the cytosolic fraction was identified as a protein(s) because of its heat-lability and sensitivity to proteases. A 16-fold purification of a toxin-binding protein was carried out by ammonium sulfate fractionation, and Sephadex G-200, and maculosin-affinity column chromatography. The affinity column was prepared with epoxy activated Sepharose 6B to which the phenolic group of maculosin was attached. The receptor was estimated to contain more than one binding protein by native and SDS-PAGE. At least one of the maculosin-binding proteins was identified as ribulose-1,5-biphosphate carboxylase (RuBPcase).

  7. In vitro phosphorylation of the movement protein of tomato mosaic tobamovirus by a cellular kinase.

    Science.gov (United States)

    Matsushita, Y; Hanazawa, K; Yoshioka, K; Oguchi, T; Kawakami, S; Watanabe, Y; Nishiguchi, M; Nyunoya, H

    2000-08-01

    The movement protein (MP) of tomato mosaic virus (ToMV) was produced in E. coli as a soluble fusion protein with glutathione S-transferase. When immobilized on glutathione affinity beads, the recombinant protein was phosphorylated in vitro by incubating with cell extracts of Nicotiana tabacum and tobacco suspension culture cells (BY-2) in the presence of [gamma-(32)P]ATP. Phosphorylation occurred even after washing the beads with a detergent-containing buffer, indicating that the recombinant MP formed a stable complex with some protein kinase(s) during incubation with the cell extract. Phosphoamino acid analysis revealed that the MP was phosphorylated on serine and threonine residues. Phosphorylation of the MP was decreased by addition of kinase inhibitors such as heparin, suramin and quercetin, which are known to be effective for casein kinase II (CK II). The phosphorylation level was not changed by other types of inhibitor. In addition, as shown for animal and plant CK II, [gamma-(32)P]GTP was efficiently used as a phosphoryl donor. Phosphorylation was not affected by amino acid replacements at serine-37 and serine-238, but was completely inhibited by deletion of the carboxy-terminal 9 amino acids, including threonine-256, serine-257, serine-261 and serine-263. These results suggest that the MP of ToMV could be phosphorylated in plant cells by a host protein kinase that is closely related to CK II.

  8. An Amphipathic Helix Directs Cellular Membrane Curvature Sensing and Function of the BAR Domain Protein PICK1.

    Science.gov (United States)

    Herlo, Rasmus; Lund, Viktor K; Lycas, Matthew D; Jansen, Anna M; Khelashvili, George; Andersen, Rita C; Bhatia, Vikram; Pedersen, Thomas S; Albornoz, Pedro B C; Johner, Niklaus; Ammendrup-Johnsen, Ina; Christensen, Nikolaj R; Erlendsson, Simon; Stoklund, Mikkel; Larsen, Jannik B; Weinstein, Harel; Kjærulff, Ole; Stamou, Dimitrios; Gether, Ulrik; Madsen, Kenneth L

    2018-05-15

    BAR domains are dimeric protein modules that sense, induce, and stabilize lipid membrane curvature. Here, we show that membrane curvature sensing (MCS) directs cellular localization and function of the BAR domain protein PICK1. In PICK1, and the homologous proteins ICA69 and arfaptin2, we identify an amphipathic helix N-terminal to the BAR domain that mediates MCS. Mutational disruption of the helix in PICK1 impaired MCS without affecting membrane binding per se. In insulin-producing INS-1E cells, super-resolution microscopy revealed that disruption of the helix selectively compromised PICK1 density on insulin granules of high curvature during their maturation. This was accompanied by reduced hormone storage in the INS-1E cells. In Drosophila, disruption of the helix compromised growth regulation. By demonstrating size-dependent binding on insulin granules, our finding highlights the function of MCS for BAR domain proteins in a biological context distinct from their function, e.g., at the plasma membrane during endocytosis. Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.

  9. Conformational landscape of an amyloid intra-cellular domain and Landau-Ginzburg-Wilson paradigm in protein dynamics

    International Nuclear Information System (INIS)

    Dai, Jin; He, Jianfeng; Niemi, Antti J.

    2016-01-01

    The Landau-Ginzburg-Wilson paradigm is proposed as a framework, to investigate the conformational landscape of intrinsically unstructured proteins. A universal Cα-trace Landau free energy is deduced from general symmetry considerations, with the ensuing all-atom structure modeled using publicly available reconstruction programs Pulchra and Scwrl. As an example, the conformational stability of an amyloid precursor protein intra-cellular domain (AICD) is inspected; the reference conformation is the crystallographic structure with code 3DXC in Protein Data Bank (PDB) that describes a heterodimer of AICD and a nuclear multi-domain adaptor protein Fe65. Those conformations of AICD that correspond to local or near-local minima of the Landau free energy are identified. For this, the response of the original 3DXC conformation to variations in the ambient temperature is investigated, using the Glauber algorithm. The conclusion is that in isolation the AICD conformation in 3DXC must be unstable. A family of degenerate conformations that minimise the Landau free energy is identified, and it is proposed that the native state of an isolated AICD is a superposition of these conformations. The results are fully in line with the presumed intrinsically unstructured character of isolated AICD and should provide a basis for a systematic analysis of AICD structure in future NMR experiments.

  10. Conformational landscape of an amyloid intra-cellular domain and Landau-Ginzburg-Wilson paradigm in protein dynamics

    Energy Technology Data Exchange (ETDEWEB)

    Dai, Jin; He, Jianfeng, E-mail: Antti.Niemi@physics.uu.se, E-mail: hjf@bit.edu.cn [School of Physics, Beijing Institute of Technology, Beijing 100081 (China); Niemi, Antti J., E-mail: Antti.Niemi@physics.uu.se, E-mail: hjf@bit.edu.cn [School of Physics, Beijing Institute of Technology, Beijing 100081 (China); Department of Physics and Astronomy, Uppsala University, P.O. Box 803, S-75108 Uppsala (Sweden); Laboratoire de Mathematiques et Physique Theorique CNRS UMR 6083, Fédération Denis Poisson, Université de Tours, Parc de Grandmont, F37200 Tours (France)

    2016-07-28

    The Landau-Ginzburg-Wilson paradigm is proposed as a framework, to investigate the conformational landscape of intrinsically unstructured proteins. A universal Cα-trace Landau free energy is deduced from general symmetry considerations, with the ensuing all-atom structure modeled using publicly available reconstruction programs Pulchra and Scwrl. As an example, the conformational stability of an amyloid precursor protein intra-cellular domain (AICD) is inspected; the reference conformation is the crystallographic structure with code 3DXC in Protein Data Bank (PDB) that describes a heterodimer of AICD and a nuclear multi-domain adaptor protein Fe65. Those conformations of AICD that correspond to local or near-local minima of the Landau free energy are identified. For this, the response of the original 3DXC conformation to variations in the ambient temperature is investigated, using the Glauber algorithm. The conclusion is that in isolation the AICD conformation in 3DXC must be unstable. A family of degenerate conformations that minimise the Landau free energy is identified, and it is proposed that the native state of an isolated AICD is a superposition of these conformations. The results are fully in line with the presumed intrinsically unstructured character of isolated AICD and should provide a basis for a systematic analysis of AICD structure in future NMR experiments.

  11. Mycoplasma hyopneumoniae and Mycoplasma flocculare differential domains from orthologous surface proteins induce distinct cellular immune responses in mice.

    Science.gov (United States)

    Leal, Fernanda Munhoz Dos Anjos; Virginio, Veridiana Gomes; Martello, Carolina Lumertz; Paes, Jéssica Andrade; Borges, Thiago J; Jaeger, Natália; Bonorino, Cristina; Ferreira, Henrique Bunselmeyer

    2016-07-15

    Mycoplasma hyopneumoniae and Mycoplasma flocculare are two genetically close species found in the swine respiratory tract. Despite their similarities, while M. hyopneumoniae is the causative agent of porcine enzootic pneumonia, M. flocculare is a commensal bacterium. Genomic and transcriptional comparative analyses so far failed to explain the difference in pathogenicity between these two species. We then hypothesized that such difference might be, at least in part, explained by amino acid sequence and immunological or functional differences between ortholog surface proteins. In line with that, it was verified that approximately 85% of the ortholog surface proteins from M. hyopneumoniae 7448 and M. flocculare present one or more differential domains. To experimentally assess possible immunological implications of this kind of difference, the extracellular differential domains from one pair of orthologous surface proteins (MHP7448_0612, from M. hyopneumoniae, and MF_00357, from M. flocculare) were expressed in E. coli and used to immunize mice. The recombinant polypeptides (rMHP61267-169 and rMF35767-196, respectively) induced distinct cellular immune responses. While, rMHP61267-169 induced both Th1 and Th2 responses, rMF35767-196 induced just an early pro-inflammatory response. These results indicate that immunological properties determined by differential domains in orthologous surface protein might play a role in pathogenicity, contributing to elicit specific and differential immune responses against each species. Copyright © 2016 Elsevier B.V. All rights reserved.

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

    International Nuclear Information System (INIS)

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

    2009-01-01

    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.

  13. Quantifying the global cellular thiol-disulfide status

    DEFF Research Database (Denmark)

    Hansen, Rosa E; Roth, Doris; Winther, Jakob R

    2009-01-01

    It is widely accepted that the redox status of protein thiols is of central importance to protein structure and folding and that glutathione is an important low-molecular-mass redox regulator. However, the total cellular pools of thiols and disulfides and their relative abundance have never been...... determined. In this study, we have assembled a global picture of the cellular thiol-disulfide status in cultured mammalian cells. We have quantified the absolute levels of protein thiols, protein disulfides, and glutathionylated protein (PSSG) in all cellular protein, including membrane proteins. These data...... cell types. However, when cells are exposed to a sublethal dose of the thiol-specific oxidant diamide, PSSG levels increase to >15% of all protein cysteine. Glutathione is typically characterized as the "cellular redox buffer"; nevertheless, our data show that protein thiols represent a larger active...

  14. Cellular and molecular biology of orphan G protein-coupled receptors.

    Science.gov (United States)

    Oh, Da Young; Kim, Kyungjin; Kwon, Hyuk Bang; Seong, Jae Young

    2006-01-01

    The superfamily of G protein-coupled receptors (GPCRs) is the largest and most diverse group of membrane-spanning proteins. It plays a variety of roles in pathophysiological processes by transmitting extracellular signals to cells via heterotrimeric G proteins. Completion of the human genome project revealed the presence of approximately 168 genes encoding established nonsensory GPCRs, as well as 207 genes predicted to encode novel GPCRs for which the natural ligands remained to be identified, the so-called orphan GPCRs. Eighty-six of these orphans have now been paired to novel or previously known molecules, and 121 remain to be deorphaned. A better understanding of the GPCR structures and classification; knowledge of the receptor activation mechanism, either dependent on or independent of an agonist; increased understanding of the control of GPCR-mediated signal transduction; and development of appropriate ligand screening systems may improve the probability of discovering novel ligands for the remaining orphan GPCRs.

  15. CK2 phosphorylation of Schistosoma mansoni HMGB1 protein regulates its cellular traffic and secretion but not its DNA transactions.

    Science.gov (United States)

    de Abreu da Silva, Isabel Caetano; Carneiro, Vitor Coutinho; Maciel, Renata de Moraes; da Costa, Rodrigo Furtado Madeiro; Furtado, Daniel Rodrigues; de Oliveira, Francisco Meirelles Bastos; da Silva-Neto, Mário Alberto Cardoso; Rumjanek, Franklin David; Fantappié, Marcelo Rosado

    2011-01-01

    The helminth Schistosoma mansoni parasite resides in mesenteric veins where fecundated female worms lay hundred of eggs daily. Some of the egg antigens are trapped in the liver and induce a vigorous granulomatous response. High Mobility Group Box 1 (HMGB1), a nuclear factor, can also be secreted and act as a cytokine. Schistosome HMGB1 (SmHMGB1) is secreted by the eggs and stimulate the production of key cytokines involved in the pathology of schistosomiasis. Thus, understanding the mechanism of SmHMGB1 release becomes mandatory. Here, we addressed the question of how the nuclear SmHMGB1 can reach the extracellular space. We showed in vitro and in vivo that CK2 phosphorylation was involved in the nucleocytoplasmic shuttling of SmHMGB1. By site-directed mutagenesis we mapped the two serine residues of SmHMGB1 that were phosphorylated by CK2. By DNA bending and supercoiling assays we showed that CK2 phosphorylation of SmHMGB1 had no effect in the DNA binding activities of the protein. We showed by electron microscopy, as well as by cell transfection and fluorescence microscopy that SmHMGB1 was present in the nucleus and cytoplasm of adult schistosomes and mammalian cells. In addition, we showed that treatments of the cells with either a phosphatase or a CK2 inhibitor were able to enhance or block, respectively, the cellular traffic of SmHMGB1. Importantly, we showed by confocal microscopy and biochemically that SmHMGB1 is significantly secreted by S. mansoni eggs of infected animals and that SmHMGB1 that were localized in the periovular schistosomotic granuloma were phosphorylated. We showed that secretion of SmHMGB1 is regulated by phosphorylation. Moreover, our results suggest that egg-secreted SmHMGB1 may represent a new egg antigen. Therefore, the identification of drugs that specifically target phosphorylation of SmHMGB1 might block its secretion and interfere with the pathogenesis of schistosomiasis.

  16. CK2 phosphorylation of Schistosoma mansoni HMGB1 protein regulates its cellular traffic and secretion but not its DNA transactions.

    Directory of Open Access Journals (Sweden)

    Isabel Caetano de Abreu da Silva

    Full Text Available BACKGROUND: The helminth Schistosoma mansoni parasite resides in mesenteric veins where fecundated female worms lay hundred of eggs daily. Some of the egg antigens are trapped in the liver and induce a vigorous granulomatous response. High Mobility Group Box 1 (HMGB1, a nuclear factor, can also be secreted and act as a cytokine. Schistosome HMGB1 (SmHMGB1 is secreted by the eggs and stimulate the production of key cytokines involved in the pathology of schistosomiasis. Thus, understanding the mechanism of SmHMGB1 release becomes mandatory. Here, we addressed the question of how the nuclear SmHMGB1 can reach the extracellular space. PRINCIPAL FINDINGS: We showed in vitro and in vivo that CK2 phosphorylation was involved in the nucleocytoplasmic shuttling of SmHMGB1. By site-directed mutagenesis we mapped the two serine residues of SmHMGB1 that were phosphorylated by CK2. By DNA bending and supercoiling assays we showed that CK2 phosphorylation of SmHMGB1 had no effect in the DNA binding activities of the protein. We showed by electron microscopy, as well as by cell transfection and fluorescence microscopy that SmHMGB1 was present in the nucleus and cytoplasm of adult schistosomes and mammalian cells. In addition, we showed that treatments of the cells with either a phosphatase or a CK2 inhibitor were able to enhance or block, respectively, the cellular traffic of SmHMGB1. Importantly, we showed by confocal microscopy and biochemically that SmHMGB1 is significantly secreted by S. mansoni eggs of infected animals and that SmHMGB1 that were localized in the periovular schistosomotic granuloma were phosphorylated. CONCLUSIONS: We showed that secretion of SmHMGB1 is regulated by phosphorylation. Moreover, our results suggest that egg-secreted SmHMGB1 may represent a new egg antigen. Therefore, the identification of drugs that specifically target phosphorylation of SmHMGB1 might block its secretion and interfere with the pathogenesis of schistosomiasis.

  17. Altered protein networks and cellular pathways in severe west nile disease in mice.

    Directory of Open Access Journals (Sweden)

    Christophe Fraisier

    Full Text Available BACKGROUND: The recent West Nile virus (WNV outbreaks in developed countries, including Europe and the United States, have been associated with significantly higher neuropathology incidence and mortality rate than previously documented. The changing epidemiology, the constant risk of (re-emergence of more virulent WNV strains, and the lack of effective human antiviral therapy or vaccines makes understanding the pathogenesis of severe disease a priority. Thus, to gain insight into the pathophysiological processes in severe WNV infection, a kinetic analysis of protein expression profiles in the brain of WNV-infected mice was conducted using samples prior to and after the onset of clinical symptoms. METHODOLOGY/PRINCIPAL FINDINGS: To this end, 2D-DIGE and gel-free iTRAQ labeling approaches were combined, followed by protein identification by mass spectrometry. Using these quantitative proteomic approaches, a set of 148 proteins with modified abundance was identified. The bioinformatics analysis (Ingenuity Pathway Analysis of each protein dataset originating from the different time-point comparisons revealed that four major functions were altered during the course of WNV-infection in mouse brain tissue: i modification of cytoskeleton maintenance associated with virus circulation; ii deregulation of the protein ubiquitination pathway; iii modulation of the inflammatory response; and iv alteration of neurological development and neuronal cell death. The differential regulation of selected host protein candidates as being representative of these biological processes were validated by western blotting using an original fluorescence-based method. CONCLUSION/SIGNIFICANCE: This study provides novel insights into the in vivo kinetic host reactions against WNV infection and the pathophysiologic processes involved, according to clinical symptoms. This work offers useful clues for anti-viral research and further evaluation of early biomarkers for the diagnosis

  18. Optimizing the protein switch: altering nuclear import and export signals, and ligand binding domain

    Science.gov (United States)

    Kakar, Mudit; Davis, James R.; Kern, Steve E.; Lim, Carol S.

    2007-01-01

    Ligand regulated localization controllable protein constructs were optimized in this study. Several constructs were made from a classical nuclear export signal (HIV-rev, MAPKK, or progesterone receptor) in combination with a SV40 T-antigen type nuclear import signal. Different ligand binding domains (LBDs from glucocorticoid receptor or progesterone receptor) were also tested for their ability to impart control over localization of proteins. This study was designed to create constructs which are cytoplasmic in the absence of ligand and nuclear in the presence of ligand, and also to regulate the amount of protein translocating to the nucleus on ligand induction. The balance between the strengths of import and export signals was critical for overall localization of proteins. The amount of protein entering the nucleus was also affected by the dose of ligand (10-100nM). However, the overall import characteristics were determined by the strengths of localization signals and the inherent localization properties of the LBD used. This study established that the amount of protein present in a particular compartment can be regulated by the use of localization signals of various strengths. These optimized localization controllable protein constructs can be used to correct for diseases due to aberrant localization of proteins. PMID:17574289

  19. Using Cellular Proteins to Reveal Mechanisms of HIV Infection | Center for Cancer Research

    Science.gov (United States)

    A vital step in HIV infection is the insertion of viral DNA into the genome of the host cell. In order for the insertion to occur, viral nucleic acid must be transported through the membrane that separates the main cellular compartment (the cytoplasm) from the nucleus, where the host DNA is located. Scientists are actively studying the mechanism used to transport viral DNA into the nucleus in the hopes of targeting this step with future anti-HIV treatments. Up to this point, researchers have identified some of the viral components that play a role in nuclear transport, but they have not determined how viral interactions with other molecules in the cell contribute to the process.

  20. Biogenesis of mitochondrial carrier proteins: molecular mechanisms of import into mitochondria.

    Science.gov (United States)

    Ferramosca, Alessandra; Zara, Vincenzo

    2013-03-01

    Mitochondrial metabolite carriers are hydrophobic proteins which catalyze the flux of several charged or hydrophilic substrates across the inner membrane of mitochondria. These proteins, like most mitochondrial proteins, are nuclear encoded and after their synthesis in the cytosol are transported into the inner mitochondrial membrane. Most metabolite carriers, differently from other nuclear encoded mitochondrial proteins, are synthesized without a cleavable presequence and contain several, poorly characterized, internal targeting signals. However, an interesting aspect is the presence of a positively charged N-terminal presequence in a limited number of mitochondrial metabolite carriers. Over the last few years the molecular mechanisms of import of metabolite carrier proteins into mitochondria have been thoroughly investigated. This review summarizes the present knowledge and discusses recent advances on the import and sorting of mitochondrial metabolite carriers. Copyright © 2012 Elsevier B.V. All rights reserved.

  1. Sheep scrapie susceptibility-linked polymorphisms do not modulate the initial binding of cellular to disease-associated prion protein prior to conversion

    NARCIS (Netherlands)

    Rigter, A.; Bossers, A.

    2005-01-01

    Conversion of the host-encoded protease-sensitive cellular prion protein (PrPC) into the scrapie-associated protease-resistant isoform (PrPSc) of prion protein (PrP) is the central event in transmissible spongiform encephalopathies or prion diseases. Differences in transmissibility and

  2. PIN proteins perform a rate-limiting function in cellular auxin efflux

    Czech Academy of Sciences Publication Activity Database

    Petrášek, Jan; Mravec, J.; Bouchard, R.; Blakeslee, J.J.; Abas, M.; Seifertová, Daniela; Wisniewska, J.; Tadele, Z.; Kubeš, Martin; Čovanová, Milada; Dhonukshe, P.; Skůpa, Petr; Benková, E.; Perry, Lucie; Křeček, Pavel; Lee, O.R.; Fink, G.R.; Geisler, M.; Murphy, A.S.; Luschnig, C.; Zažímalová, Eva; Friml, J.

    2006-01-01

    Roč. 312, č. 5775 (2006), s. 914-918 ISSN 0036-8075 R&D Projects: GA AV ČR IAA6038303; GA MŠk(CZ) LC06034 Institutional research plan: CEZ:AV0Z50380511 Keywords : auxin transport * PIN proteins * PGP transporters * MDR Subject RIV: ED - Physiology Impact factor: 30.028, year: 2006

  3. The Intracellular Destiny of the Protein Corona : A Study on its Cellular Internalization and Evolution

    NARCIS (Netherlands)

    Bertoli, Filippo; Garry, David; Monopoli, Marco P.; Salvati, Anna; Dawson, Kenneth A.

    2016-01-01

    It has been well established that the early stages of nanoparticle cell interactions are governed, at least in part, by the layer of proteins and other biomolecules adsorbed and slowly exchanged with the surrounding biological media (biomolecular corona). Subsequent to membrane interactions,

  4. Protein production from whey using Penicillium cyclopium; growth parameters and cellular composition

    Energy Technology Data Exchange (ETDEWEB)

    Kim, J H; Lebeault, J M

    1981-01-01

    The growth parameters of Penicillium cyclopium were evaluated in a continuous culture system for the production of fungal protein from whey. Dilution rates were 0.05-0.20/h under constant conditions of temperature (28 degrees) and pH (3.5). The saturation coefficients in the Monod equation were 0.74 g/l for lactose and 0.14 mg/l for O/sub 2/. For a wide range of dilution rates, the yield was 0.68 g biomass/g lactose and the maintenance coefficient 0.005 g lactose/g biomass-h. The maximum biomass productivity achieved was 2 g biomass/l-h at dilution rates of 0.16-0.17/h with a lactose concentration of 20 g/l in the feed. The crude protein and total nucleic acid contents increased with a dilution rate, crude protein content was 43-54%, and total nucleic acids were 6-9% at dilution rates of 0.05-0.2/h, while the Lowry protein content was almost constant at 37.5% of dry matter.

  5. Ligand Binding Induces Conformational Changes in Human Cellular Retinol-binding Protein 1 (CRBP1) Revealed by Atomic Resolution Crystal Structures.

    Science.gov (United States)

    Silvaroli, Josie A; Arne, Jason M; Chelstowska, Sylwia; Kiser, Philip D; Banerjee, Surajit; Golczak, Marcin

    2016-04-15

    Important in regulating the uptake, storage, and metabolism of retinoids, cellular retinol-binding protein 1 (CRBP1) is essential for trafficking vitamin A through the cytoplasm. However, the molecular details of ligand uptake and targeted release by CRBP1 remain unclear. Here we report the first structure of CRBP1 in a ligand-free form as well as ultra-high resolution structures of this protein bound to either all-trans-retinol or retinylamine, the latter a therapeutic retinoid that prevents light-induced retinal degeneration. Superpositioning of human apo- and holo-CRBP1 revealed major differences within segments surrounding the entrance to the retinoid-binding site. These included α-helix II and hairpin turns between β-strands βC-βD and βE-βF as well as several side chains, such as Phe-57, Tyr-60, and Ile-77, that change their orientations to accommodate the ligand. Additionally, we mapped hydrogen bond networks inside the retinoid-binding cavity and demonstrated their significance for the ligand affinity. Analyses of the crystallographic B-factors indicated several regions with higher backbone mobility in the apoprotein that became more rigid upon retinoid binding. This conformational flexibility of human apo-CRBP1 facilitates interaction with the ligands, whereas the more rigid holoprotein structure protects the labile retinoid moiety during vitamin A transport. These findings suggest a mechanism of induced fit upon ligand binding by mammalian cellular retinol-binding proteins. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  6. Cellular protein quality control and the evolution of aggregates in spinocerebellar ataxia type 3 (SCA3).

    Science.gov (United States)

    Seidel, K; Meister, M; Dugbartey, G J; Zijlstra, M P; Vinet, J; Brunt, E R P; van Leeuwen, F W; Rüb, U; Kampinga, H H; den Dunnen, W F A

    2012-10-01

    A characteristic of polyglutamine diseases is the increased propensity of disease proteins to aggregate, which is thought to be a major contributing factor to the underlying neurodegeneration. Healthy cells contain mechanisms for handling protein damage, the protein quality control, which must be impaired or inefficient to permit proteotoxicity under pathological conditions. We used a quantitative analysis of immunohistochemistry of the pons of eight patients with the polyglutamine disorder spinocerebellar ataxia type 3. We employed the anti-polyglutamine antibody 1C2, antibodies against p62 that is involved in delivering ubiquitinated protein aggregates to autophagosomes, antibodies against the chaperones HSPA1A and DNAJB1 and the proteasomal stress marker UBB⁺¹. The 1C2 antibody stained neuronal nuclear inclusions (NNIs), diffuse nuclear staining (DNS), granular cytoplasmic staining (GCS) and combinations, with reproducible distribution. P62 always co-localized with 1C2 in NNI. DNS and GCS co-stained with a lower frequency. UBB⁺¹ was present in a subset of neurones with NNI. A subset of UBB⁺¹-containing neurones displayed increased levels of HSPA1A, while DNAJB1 was sequestered into the NNI. Based on our results, we propose a model for the aggregation-associated pathology of spinocerebellar ataxia type 3: GCS and DNS aggregation likely represents early stages of pathology, which progresses towards formation of p62-positive NNI. A fraction of NNI exhibits UBB⁺¹ staining, implying proteasomal overload at a later stage. Subsequently, the stress-inducible HSPA1A is elevated while DNAJB1 is recruited into NNIs. This indicates that the stress response is only induced late when all endogenous protein quality control systems have failed. © 2011 The Authors. Neuropathology and Applied Neurobiology © 2011 British Neuropathological Society.

  7. Importin α5 negatively regulates importin β1-mediated nuclear import of Newcastle disease virus matrix protein and viral replication and pathogenicity in chicken fibroblasts.

    Science.gov (United States)

    Duan, Zhiqiang; Xu, Haixu; Ji, Xinqin; Zhao, Jiafu; Xu, Houqiang; Hu, Yan; Deng, Shanshan; Hu, Shunlin; Liu, Xiufan

    2018-12-31

    The matrix (M) protein of Newcastle disease virus (NDV) is demonstrated to localize in the nucleus via intrinsic nuclear localization signal (NLS), but cellular proteins involved in the nuclear import of NDV M protein and the role of M's nuclear localization in the replication and pathogenicity of NDV remain unclear. In this study, importin β1 was screened to interact with NDV M protein by yeast two-hybrid screening. This interaction was subsequently confirmed by co-immunoprecipitation and pull-down assays. In vitro binding studies indicated that the NLS region of M protein and the amino acids 336-433 of importin β1 that belonged to the RanGTP binding region were important for binding. Importantly, a recombinant virus with M/NLS mutation resulted in a pathotype change of NDV and attenuated viral replication and pathogenicity in chicken fibroblasts and SPF chickens. In agreement with the binding data, nuclear import of NDV M protein in digitonin-permeabilized HeLa cells required both importin β1 and RanGTP. Interestingly, importin α5 was verified to interact with M protein through binding importin β1. However, importin β1 or importin α5 depletion by siRNA resulted in different results, which showed the obviously cytoplasmic or nuclear accumulation of M protein and the remarkably decreased or increased replication ability and pathogenicity of NDV in chicken fibroblasts, respectively. Our findings therefore demonstrate for the first time the nuclear import mechanism of NDV M protein and the negative regulation role of importin α5 in importin β1-mediated nuclear import of M protein and the replication and pathogenicity of a paramyxovirus.

  8. Cloning, characterization and sub-cellular localization of gamma subunit of T-complex protein-1 (chaperonin) from Leishmania donovani

    Energy Technology Data Exchange (ETDEWEB)

    Bhaskar,; Kumari, Neeti [Division of Biochemistry, CSIR-Central Drug Research Institute, Chattar Manzil Palace, PO Box 173, Lucknow (India); Goyal, Neena, E-mail: neenacdri@yahoo.com [Division of Biochemistry, CSIR-Central Drug Research Institute, Chattar Manzil Palace, PO Box 173, Lucknow (India)

    2012-12-07

    Highlights: Black-Right-Pointing-Pointer The study presents cloning and characterization of TCP1{gamma} gene from L. donovani. Black-Right-Pointing-Pointer TCP1{gamma} is a subunit of T-complex protein-1 (TCP1), a chaperonin class of protein. Black-Right-Pointing-Pointer LdTCP{gamma} exhibited differential expression in different stages of promastigotes. Black-Right-Pointing-Pointer LdTCP{gamma} co-localized with actin, a cytoskeleton protein. Black-Right-Pointing-Pointer The data suggests that this gene may have a role in differentiation/biogenesis. Black-Right-Pointing-Pointer First report on this chapronin in Leishmania. -- Abstract: T-complex protein-1 (TCP1) complex, a chaperonin class of protein, ubiquitous in all genera of life, is involved in intracellular assembly and folding of various proteins. The gamma subunit of TCP1 complex (TCP1{gamma}), plays a pivotal role in the folding and assembly of cytoskeleton protein(s) as an individual or complexed with other subunits. Here, we report for the first time cloning, characterization and expression of the TCP1{gamma} of Leishmania donovani (LdTCP1{gamma}), the causative agent of Indian Kala-azar. Primary sequence analysis of LdTCP1{gamma} revealed the presence of all the characteristic features of TCP1{gamma}. However, leishmanial TCP1{gamma} represents a distinct kinetoplastid group, clustered in a separate branch of the phylogenic tree. LdTCP1{gamma} exhibited differential expression in different stages of promastigotes. The non-dividing stationary phase promastigotes exhibited 2.5-fold less expression of LdTCP1{gamma} as compared to rapidly dividing log phase parasites. The sub-cellular distribution of LdTCP1{gamma} was studied in log phase promastigotes by employing indirect immunofluorescence microscopy. The protein was present not only in cytoplasm but it was also localized in nucleus, peri-nuclear region, flagella, flagellar pocket and apical region. Co-localization of LdTCP1{gamma} with actin suggests

  9. Cloning, characterization and sub-cellular localization of gamma subunit of T-complex protein-1 (chaperonin) from Leishmania donovani

    International Nuclear Information System (INIS)

    Bhaskar,; Kumari, Neeti; Goyal, Neena

    2012-01-01

    Highlights: ► The study presents cloning and characterization of TCP1γ gene from L. donovani. ► TCP1γ is a subunit of T-complex protein-1 (TCP1), a chaperonin class of protein. ► LdTCPγ exhibited differential expression in different stages of promastigotes. ► LdTCPγ co-localized with actin, a cytoskeleton protein. ► The data suggests that this gene may have a role in differentiation/biogenesis. ► First report on this chapronin in Leishmania. -- Abstract: T-complex protein-1 (TCP1) complex, a chaperonin class of protein, ubiquitous in all genera of life, is involved in intracellular assembly and folding of various proteins. The gamma subunit of TCP1 complex (TCP1γ), plays a pivotal role in the folding and assembly of cytoskeleton protein(s) as an individual or complexed with other subunits. Here, we report for the first time cloning, characterization and expression of the TCP1γ of Leishmania donovani (LdTCP1γ), the causative agent of Indian Kala-azar. Primary sequence analysis of LdTCP1γ revealed the presence of all the characteristic features of TCP1γ. However, leishmanial TCP1γ represents a distinct kinetoplastid group, clustered in a separate branch of the phylogenic tree. LdTCP1γ exhibited differential expression in different stages of promastigotes. The non-dividing stationary phase promastigotes exhibited 2.5-fold less expression of LdTCP1γ as compared to rapidly dividing log phase parasites. The sub-cellular distribution of LdTCP1γ was studied in log phase promastigotes by employing indirect immunofluorescence microscopy. The protein was present not only in cytoplasm but it was also localized in nucleus, peri-nuclear region, flagella, flagellar pocket and apical region. Co-localization of LdTCP1γ with actin suggests that, this gene may have a role in maintaining the structural dynamics of cytoskeleton of parasite.

  10. Does constructive neutral evolution play an important role in the origin of cellular complexity? Making sense of the origins and uses of biological complexity.

    Science.gov (United States)

    Speijer, Dave

    2011-05-01

    Recently, constructive neutral evolution has been touted as an important concept for the understanding of the emergence of cellular complexity. It has been invoked to help explain the development and retention of, amongst others, RNA splicing, RNA editing and ribosomal and mitochondrial respiratory chain complexity. The theory originated as a welcome explanation of isolated small scale cellular idiosyncrasies and as a reaction to 'overselectionism'. Here I contend, that in its extended form, it has major conceptual problems, can not explain observed patterns of complex processes, is too easily dismissive of alternative selectionist models, underestimates the creative force of complexity as such, and--if seen as a major evolutionary mechanism for all organisms--could stifle further thought regarding the evolution of highly complex biological processes. Copyright © 2011 WILEY Periodicals, Inc.

  11. Cellular localization of Sun4p and its interaction with proteins in the yeast birth scar

    Czech Academy of Sciences Publication Activity Database

    Kuznetsov, E.; Váchová, Libuše; Palková, Z.

    2016-01-01

    Roč. 15, č. 14 (2016), s. 1898-1907 ISSN 1538-4101 R&D Projects: GA ČR GA13-08605S; GA MŠk(CZ) ED1.1.00/02.0109 Institutional support: RVO:61388971 Keywords : cell wall * glucanases * SUN family of proteins * yeast birth scar Subject RIV: EE - Microbiology, Virology Impact factor: 3.530, year: 2016

  12. Human myosin VIIa is a very slow processive motor protein on various cellular actin structures.

    Science.gov (United States)

    Sato, Osamu; Komatsu, Satoshi; Sakai, Tsuyoshi; Tsukasaki, Yoshikazu; Tanaka, Ryosuke; Mizutani, Takeomi; Watanabe, Tomonobu M; Ikebe, Reiko; Ikebe, Mitsuo

    2017-06-30

    Human myosin VIIa (MYO7A) is an actin-linked motor protein associated with human Usher syndrome (USH) type 1B, which causes human congenital hearing and visual loss. Although it has been thought that the role of human myosin VIIa is critical for USH1 protein tethering with actin and transportation along actin bundles in inner-ear hair cells, myosin VIIa's motor function remains unclear. Here, we studied the motor function of the tail-truncated human myosin VIIa dimer (HM7AΔTail/LZ) at the single-molecule level. We found that the HM7AΔTail/LZ moves processively on single actin filaments with a step size of 35 nm. Dwell-time distribution analysis indicated an average waiting time of 3.4 s, yielding ∼0.3 s -1 for the mechanical turnover rate; hence, the velocity of HM7AΔTail/LZ was extremely slow, at 11 nm·s -1 We also examined HM7AΔTail/LZ movement on various actin structures in demembranated cells. HM7AΔTail/LZ showed unidirectional movement on actin structures at cell edges, such as lamellipodia and filopodia. However, HM7AΔTail/LZ frequently missed steps on actin tracks and exhibited bidirectional movement at stress fibers, which was not observed with tail-truncated myosin Va. These results suggest that the movement of the human myosin VIIa motor protein is more efficient on lamellipodial and filopodial actin tracks than on stress fibers, which are composed of actin filaments with different polarity, and that the actin structures influence the characteristics of cargo transportation by human myosin VIIa. In conclusion, myosin VIIa movement appears to be suitable for translocating USH1 proteins on stereocilia actin bundles in inner-ear hair cells. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  13. DNA replication restart and cellular dynamics of Hef helicase/nuclease protein in Haloferax volcanii.

    Science.gov (United States)

    Lestini, Roxane; Delpech, Floriane; Myllykallio, Hannu

    2015-11-01

    Understanding how frequently spontaneous replication arrests occur and how archaea deal with these arrests are very interesting and challenging research topics. Here we will described how genetic and imaging studies have revealed the central role of the archaeal helicase/nuclease Hef belonging to the XPF/MUS81/FANCM family of endonucleases in repair of arrested replication forks. Special focus will be on description of a recently developed combination of genetic and imaging tools to study the dynamic localization of a functional Hef::GFP (Green Fluorescent Protein) fusion protein in the living cells of halophilic archaea Haloferax volcanii. As Archaea provide an excellent and unique model for understanding how DNA replication is regulated to allow replication of a circular DNA molecule either from single or multiple replication origins, we will also summarize recent studies that have revealed peculiar features regarding DNA replication, particularly in halophilic archaea. We strongly believe that fundamental knowledge of our on-going studies will shed light on the evolutionary history of the DNA replication machinery and will help to establish general rules concerning replication restart and the key role of recombination proteins not only in bacteria, yeast and higher eukaryotes but also in archaea. Copyright © 2015 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.

  14. Bacillus anthracis TIR Domain-Containing Protein Localises to Cellular Microtubule Structures and Induces Autophagy.

    Science.gov (United States)

    Carlsson, Emil; Thwaite, Joanne E; Jenner, Dominic C; Spear, Abigail M; Flick-Smith, Helen; Atkins, Helen S; Byrne, Bernadette; Ding, Jeak Ling

    2016-01-01

    Toll-like receptors (TLRs) recognise invading pathogens and mediate downstream immune signalling via Toll/IL-1 receptor (TIR) domains. TIR domain proteins (Tdps) have been identified in multiple pathogenic bacteria and have recently been implicated as negative regulators of host innate immune activation. A Tdp has been identified in Bacillus anthracis, the causative agent of anthrax. Here we present the first study of this protein, designated BaTdp. Recombinantly expressed and purified BaTdp TIR domain interacted with several human TIR domains, including that of the key TLR adaptor MyD88, although BaTdp expression in cultured HEK293 cells had no effect on TLR4- or TLR2- mediated immune activation. During expression in mammalian cells, BaTdp localised to microtubular networks and caused an increase in lipidated cytosolic microtubule-associated protein 1A/1B-light chain 3 (LC3), indicative of autophagosome formation. In vivo intra-nasal infection experiments in mice showed that a BaTdp knockout strain colonised host tissue faster with higher bacterial load within 4 days post-infection compared to the wild type B. anthracis. Taken together, these findings indicate that BaTdp does not play an immune suppressive role, but rather, its absence increases virulence. BaTdp present in wild type B. anthracis plausibly interact with the infected host cell, which undergoes autophagy in self-defence.

  15. Abalone Protein Hydrolysates: Preparation, Angiotensin I Converting Enzyme Inhibition and Cellular Antioxidant Activity.

    Science.gov (United States)

    Park, Soo Yeon; Je, Jae-Young; Hwang, Joung-Youl; Ahn, Chang-Bum

    2015-09-01

    Abalone protein was hydrolyzed by enzymatic hydrolysis and the optimal enzyme/substrate (E/S) ratios were determined. Abalone protein hydrolysates (APH) produced by Protamex at E/S ratio of 1:100 showed angiotensin I converting enzyme inhibitory activity with IC50 of 0.46 mg/mL, and APH obtained by Flavourzyme at E/S ratio of 1:100 possessed the oxygen radical absorbance capacity value of 457.6 μM trolox equivalent/mg sample. Flavourzyme abalone protein hydrolysates (FAPH) also exhibited H2O2 scavenging activity with IC50 of 0.48 mg/mL and Fe(2+) chelating activity with IC50 of 2.26 mg/mL as well as high reducing power. FAPH significantly (P<0.05) protected H2O2-induced hepatic cell damage in cultured hepatocytes, and the cell viability was restored to 90.27% in the presence of FAPH. FAPH exhibited 46.20% intracellular ROS scavenging activity and 57.89% lipid peroxidation inhibition activity in cultured hepatocytes. Overall, APH may be useful as an ingredient for functional foods.

  16. Viral and cellular SOS-regulated motor proteins: dsDNA translocation mechanisms with divergent functions.

    Science.gov (United States)

    Wolfe, Annie; Phipps, Kara; Weitao, Tao

    2014-01-01

    DNA damage attacks on bacterial cells have been known to activate the SOS response, a transcriptional response affecting chromosome replication, DNA recombination and repair, cell division and prophage induction. All these functions require double-stranded (ds) DNA translocation by ASCE hexameric motors. This review seeks to delineate the structural and functional characteristics of the SOS response and the SOS-regulated DNA translocases FtsK and RuvB with the phi29 bacteriophage packaging motor gp16 ATPase as a prototype to study bacterial motors. While gp16 ATPase, cellular FtsK and RuvB are similarly comprised of hexameric rings encircling dsDNA and functioning as ATP-driven DNA translocases, they utilize different mechanisms to accomplish separate functions, suggesting a convergent evolution of these motors. The gp16 ATPase and FtsK use a novel revolution mechanism, generating a power stroke between subunits through an entropy-DNA affinity switch and pushing dsDNA inward without rotation of DNA and the motor, whereas RuvB seems to employ a rotation mechanism that remains to be further characterized. While FtsK and RuvB perform essential tasks during the SOS response, their roles may be far more significant as SOS response is involved in antibiotic-inducible bacterial vesiculation and biofilm formation as well as the perspective of the bacteria-cancer evolutionary interaction.

  17. Microstructure and in vitro cellular response to novel soy protein-based porous structures for tissue regeneration applications.

    Science.gov (United States)

    Olami, Hilla; Zilberman, Meital

    2016-02-01

    Interest in the development of new bioresorbable structures for various tissue engineering applications is on the rise. In the current study, we developed and studied novel soy protein-based porous blends as potential new scaffolds for such applications. Soy protein has several advantages over the various types of natural proteins employed for biomedical applications due to its low price, non-animal origin and relatively long storage time and stability. In the present study, blends of soy protein with other polymers (gelatin, pectin and alginate) were added and chemically cross-linked using the cross-linking agents carbodiimide or glyoxal, and the porous structure was obtained through lyophilization. The resulting blend porous structures were characterized using environmental scanning microscopy, and the cytotoxicity of these scaffolds was examined in vitro. The biocompatibility of the scaffolds was also evaluated in vitro by seeding and culturing human fibroblasts on these scaffolds. Cell growth morphology and adhesion were examined histologically. The results show that these blends can be assembled into porous three-dimensional structures by combining chemical cross-linking with freeze-drying. The achieved blend structures combine suitable porosity with a large pore size (100-300 µm). The pore structure in the soy-alginate scaffolds possesses adequate interconnectivity compared to that of the soy-gelatin scaffolds. However, porous structure was not observed for the soy-pectin blend, which presented a different structure with significantly lower porosities than all other groups. The in vitro evaluation of these porous soy blends demonstrated that soy-alginate blends are advantageous over soy-gelatin blends and exhibited adequate cytocompatibility along with better cell infiltration and stability. These soy protein scaffolds may be potentially useful as a cellular/acellular platform for skin regeneration applications. © The Author(s) 2015.

  18. Analyzing import intermediates of mitochondrial proteins by blue native gel electrophoresis.

    Science.gov (United States)

    Waizenegger, Thomas; Rapaport, Doron

    2007-01-01

    Blue native gel electrophoresis (BNGE) is a powerful tool for analyzing native protein complexes from biological membranes as well as water-soluble proteins. It can be used for determining relative molecular masses of protein complexes and their subunit composition and for the detection of subcomplexes. We describe the analysis by BNGE of in vitro import reactions composed of radiolabeled precursor proteins and isolated mitochondria. Such an analysis is a powerful tool to follow import intermediates and to study assembly of protein complexes. Analysis of import reactions by BNGE provides information on the molecular mass of the complex with which the imported precursor is associated. In addition, components of such a complex can be identified by incubating the mitochondrial lysate with either soluble antibodies or antibodies coupled to protein A matrix. The binding of soluble antibodies to specific complexes results in an observed shift in their apparent molecular mass (antibody shift). Alternatively, addition of matrix-bound antibodies followed by removal of the matrix from the mixture will result in depletion of the specific complex from the mitochondrial lysate (antibody depletion). The experimental details of these techniques are described.

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

    Science.gov (United States)

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

    2014-01-01

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

  20. Monoacylated Cellular Prion Proteins Reduce Amyloid-β-Induced Activation of Cytoplasmic Phospholipase A2 and Synapse Damage

    Directory of Open Access Journals (Sweden)

    Ewan West

    2015-06-01

    Full Text Available Alzheimer’s disease (AD is a progressive neurodegenerative disease characterized by the accumulation of amyloid-β (Aβ and the loss of synapses. Aggregation of the cellular prion protein (PrPC by Aβ oligomers induced synapse damage in cultured neurons. PrPC is attached to membranes via a glycosylphosphatidylinositol (GPI anchor, the composition of which affects protein targeting and cell signaling. Monoacylated PrPC incorporated into neurons bound “natural Aβ”, sequestering Aβ outside lipid rafts and preventing its accumulation at synapses. The presence of monoacylated PrPC reduced the Aβ-induced activation of cytoplasmic phospholipase A2 (cPLA2 and Aβ-induced synapse damage. This protective effect was stimulus specific, as treated neurons remained sensitive to α-synuclein, a protein associated with synapse damage in Parkinson’s disease. In synaptosomes, the aggregation of PrPC by Aβ oligomers triggered the formation of a signaling complex containing the cPLA2.a process, disrupted by monoacylated PrPC. We propose that monoacylated PrPC acts as a molecular sponge, binding Aβ oligomers at the neuronal perikarya without activating cPLA2 or triggering synapse damage.

  1. Proteomic analysis of the dysferlin protein complex unveils its importance for sarcolemmal maintenance and integrity.

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    Antoine de Morrée

    Full Text Available Dysferlin is critical for repair of muscle membranes after damage. Mutations in dysferlin lead to a progressive muscular dystrophy. Recent studies suggest additional roles for dysferlin. We set out to study dysferlin's protein-protein interactions to obtain comprehensive knowledge of dysferlin functionalities in a myogenic context. We developed a robust and reproducible method to isolate dysferlin protein complexes from cells and tissue. We analyzed the composition of these complexes in cultured myoblasts, myotubes and skeletal muscle tissue by mass spectrometry and subsequently inferred potential protein functions through bioinformatics analyses. Our data confirm previously reported interactions and support a function for dysferlin as a vesicle trafficking protein. In addition novel potential functionalities were uncovered, including phagocytosis and focal adhesion. Our data reveal that the dysferlin protein complex has a dynamic composition as a function of myogenic differentiation. We provide additional experimental evidence and show dysferlin localization to, and interaction with the focal adhesion protein vinculin at the sarcolemma. Finally, our studies reveal evidence for cross-talk between dysferlin and its protein family member myoferlin. Together our analyses show that dysferlin is not only a membrane repair protein but also important for muscle membrane maintenance and integrity.

  2. An integrated approach to elucidate the intra-viral and viral-cellular protein interaction networks of a gamma-herpesvirus.

    Directory of Open Access Journals (Sweden)

    Shaoying Lee

    2011-10-01

    Full Text Available Genome-wide yeast two-hybrid (Y2H screens were conducted to elucidate the molecular functions of open reading frames (ORFs encoded by murine γ-herpesvirus 68 (MHV-68. A library of 84 MHV-68 genes and gene fragments was generated in a Gateway entry plasmid and transferred to Y2H vectors. All possible pair-wise interactions between viral proteins were tested in the Y2H assay, resulting in the identification of 23 intra-viral protein-protein interactions (PPIs. Seventy percent of the interactions between viral proteins were confirmed by co-immunoprecipitation experiments. To systematically investigate virus-cellular protein interactions, the MHV-68 Y2H constructs were screened against a cellular cDNA library, yielding 243 viral-cellular PPIs involving 197 distinct cellar proteins. Network analyses indicated that cellular proteins targeted by MHV-68 had more partners in the cellular PPI network and were located closer to each other than expected by chance. Taking advantage of this observation, we scored the cellular proteins based on their network distances from other MHV-68-interacting proteins and segregated them into high (Y2H-HP and low priority/not-scored (Y2H-LP/NS groups. Significantly more genes from Y2H-HP altered MHV-68 replication when their expression was inhibited with siRNAs (53% of genes from Y2H-HP, 21% of genes from Y2H-LP/NS, and 16% of genes randomly chosen from the human PPI network; p<0.05. Enriched Gene Ontology (GO terms in the Y2H-HP group included regulation of apoptosis, protein kinase cascade, post-translational protein modification, transcription from RNA polymerase II promoter, and IκB kinase/NFκB cascade. Functional validation assays indicated that PCBP1, which interacted with MHV-68 ORF34, may be involved in regulating late virus gene expression in a manner consistent with the effects of its viral interacting partner. Our study integrated Y2H screening with multiple functional validation approaches to create

  3. Cell segmentation in time-lapse fluorescence microscopy with temporally varying sub-cellular fusion protein patterns.

    Science.gov (United States)

    Bunyak, Filiz; Palaniappan, Kannappan; Chagin, Vadim; Cardoso, M

    2009-01-01

    Fluorescently tagged proteins such as GFP-PCNA produce rich dynamically varying textural patterns of foci distributed in the nucleus. This enables the behavioral study of sub-cellular structures during different phases of the cell cycle. The varying punctuate patterns of fluorescence, drastic changes in SNR, shape and position during mitosis and abundance of touching cells, however, require more sophisticated algorithms for reliable automatic cell segmentation and lineage analysis. Since the cell nuclei are non-uniform in appearance, a distribution-based modeling of foreground classes is essential. The recently proposed graph partitioning active contours (GPAC) algorithm supports region descriptors and flexible distance metrics. We extend GPAC for fluorescence-based cell segmentation using regional density functions and dramatically improve its efficiency for segmentation from O(N(4)) to O(N(2)), for an image with N(2) pixels, making it practical and scalable for high throughput microscopy imaging studies.

  4. Cellular prion protein (PrPc and hypoxia: true to each other in good times and in bad, in sickness and in health

    Directory of Open Access Journals (Sweden)

    Sanja Ramljak

    2016-12-01

    Full Text Available The cellular prion protein (PrPc and hypoxia appear to be tightly intertwined. Beneficial effects of PrPc on neuronal survival under hypoxic conditions such as focal cerebral ischemia are strongly supported. Conversely, increasing evidence indicates detrimental effects of increased PrPc expression on cancer progression, another condition accompanied by low oxygen tensions. A switch between anaerobic and aerobic metabolism characterizes both conditions. A cellular process that might unite both is glycolysis. Putative role of PrPc in stimulation of glycolysis in times of need is indeed thought provoking. A significance of astrocytic PrPc expression for neuronal survival under hypoxic conditions and possible association of PrPc with the astrocyte-neuron lactate shuttle (ANLS is considered. We posit PrPc-induced lactate production via transactivation of lactate dehydrogenase A by hypoxia inducible factor 1α as an important factor for survival of both neurons and tumor cells in hypoxic microenvironment. Concomitantly, we discuss a cross-talk between Wnt/ß-catenin and PI-3K/Akt signaling pathways in executing PrPc-induced activation of glycolysis. Finally, we would like to emphasize that we see a great potential in joining expertise from both fields, neuroscience and cancer research in revealing the mechanisms underlying hypoxia-related pathologies. PrPc may prove focal point for future research.

  5. HTLV-1 Tax Oncoprotein Subverts the Cellular DNA Damage Response via Binding to DNA-dependent Protein Kinase*S⃞

    Science.gov (United States)

    Durkin, Sarah S.; Guo, Xin; Fryrear, Kimberly A.; Mihaylova, Valia T.; Gupta, Saurabh K.; Belgnaoui, S. Mehdi; Haoudi, Abdelali; Kupfer, Gary M.; Semmes, O. John

    2008-01-01

    Human T-cell leukemia virus type-1 is the causative agent for adult T-cell leukemia. Previous research has established that the viral oncoprotein Tax mediates the transformation process by impairing cell cycle control and cellular response to DNA damage. We showed previously that Tax sequesters huChk2 within chromatin and impairs the response to ionizing radiation. Here we demonstrate that DNA-dependent protein kinase (DNA-PK) is a member of the Tax·Chk2 nuclear complex. The catalytic subunit, DNA-PKcs, and the regulatory subunit, Ku70, were present. Tax-containing nuclear extracts showed increased DNA-PK activity, and specific inhibition of DNA-PK prevented Tax-induced activation of Chk2 kinase activity. Expression of Tax induced foci formation and phosphorylation of H2AX. However, Tax-induced constitutive signaling of the DNA-PK pathway impaired cellular response to new damage, as reflected in suppression of ionizing radiation-induced DNA-PK phosphorylation and γH2AX stabilization. Tax co-localized with phospho-DNA-PK into nuclear speckles and a nuclear excluded Tax mutant sequestered endogenous phospho-DNA-PK into the cytoplasm, suggesting that Tax interaction with DNA-PK is an initiating event. We also describe a novel interaction between DNA-PK and Chk2 that requires Tax. We propose that Tax binds to and stabilizes a protein complex with DNA-PK and Chk2, resulting in a saturation of DNA-PK-mediated damage repair response. PMID:18957425

  6. The nuclear import of the human T lymphotropic virus type I (HTLV-1) tax protein is carrier- and energy-independent.

    Science.gov (United States)

    Tsuji, Takahiro; Sheehy, Noreen; Gautier, Virginie W; Hayakawa, Hitoshi; Sawa, Hirofumi; Hall, William W

    2007-05-04

    HTLV-1 is the etiologic agent of the adult T cell leukemialymphoma (ATLL). The viral regulatory protein Tax plays a central role in leukemogenesis as a transcriptional transactivator of both viral and cellular gene expression, and this requires Tax activity in both the cytoplasm and the nucleus. In the present study, we have investigated the mechanisms involved in the nuclear localization of Tax. Employing a GFP fusion expression system and a range of Tax mutants, we could confirm that the N-terminal 60 amino acids, and specifically residues within the zinc finger motif in this region, are important for nuclear localization. Using an in vitro nuclear import assay, it could be demonstrated that the transportation of Tax to the nucleus required neither energy nor carrier proteins. Specific and direct binding between Tax and p62, a nucleoporin with which the importin beta family of proteins have been known to interact was also observed. The nuclear import activity of wild type Tax and its mutants and their binding affinity for p62 were also clearly correlated, suggesting that the entry of Tax into the nucleus involves a direct interaction with nucleoporins within the nuclear pore complex (NPC). The nuclear export of Tax was also shown to be carrier independent. It could be also demonstrated that Tax it self may have a carrier function and that the NF-kappaB subunit p65 could be imported into the nucleus by Tax. These studies suggest that Tax could alter the nucleocytoplasmic distribution of cellular proteins, and this could contribute to the deregulation of cellular processes observed in HTLV-1 infection.

  7. Identification of Litopenaeus vannamei BiP as a novel cellular attachment protein for white spot syndrome virus by using a biotinylation based affinity chromatography method.

    Science.gov (United States)

    Yuan, Zengzhi; Chen, Meng; Wang, Jingting; Li, Zhuoyu; Geng, Xuyun; Sun, Jinsheng

    2018-05-05

    White spot syndrome virus (WSSV) is a dangerous threat to shrimp farming that also attacks a wide range of crustaceans. Knowledge of the surface protein-protein interactions between the pathogen and host is very crucial to unraveling the molecular pathogenesis mechanisms of WSSV. In this study, LvBiP (Litopenaeus vannamei immunoglobulin heavy-chain-binding protein) was identified as a novel WSSV binding protein of L. vannamei by a biotinylation based affinity chromatography method. By using pull-down and ELISA assays, the binding of recombinant LvBiP to WSSV was proved to be specific and ATP- dependent. The interaction was also confirmed by the result of co-immunoprecipitation assay. Immunofluorescence studies revealed the co-localization of LvBiP with WSSV on the cell surface of shrimp haemocytes. Additionally, LvBiP is likely to play an important role in WSSV infection. Treatment of gill cellular membrane proteins (CMPs) with purified rLvBiP and antibody that specifically recognizes LvBiP, led to a significant reduction in the binding of WSSV to gill CMPs. In the in vivo neutralization assay, rLvBiP and anti-LvBiP polyclonal antibody partially blocked the infection of WSSV. Taken together, the results indicate that LvBiP, a molecular chaperon of the HSP70 family, is a novel host factor involved at the step of attachment of the WSSV to the host cells and a potential candidate of therapeutic target. Copyright © 2018. Published by Elsevier Ltd.

  8. The F box protein Fbx6 regulates Chk1 stability and cellular sensitivity to replication stress.

    Science.gov (United States)

    Zhang, You-Wei; Brognard, John; Coughlin, Chris; You, Zhongsheng; Dolled-Filhart, Marisa; Aslanian, Aaron; Manning, Gerard; Abraham, Robert T; Hunter, Tony

    2009-08-28

    ATR and Chk1 are two key protein kinases in the replication checkpoint. Activation of ATR-Chk1 has been extensively investigated, but checkpoint termination and replication fork restart are less well understood. Here, we report that DNA damage not only activates Chk1, but also exposes a degron-like region at the carboxyl terminus of Chk1 to an Fbx6-containing SCF (Skp1-Cul1-F box) E3 ligase, which mediates the ubiquitination and degradation of Chk1 and, in turn, terminates the checkpoint. The protein levels of Chk1 and Fbx6 showed an inverse correlation in both cultured cancer cells and in human breast tumor tissues. Further, we show that low levels of Fbx6 and consequent impairment of replication stress-induced Chk1 degradation are associated with cancer cell resistance to the chemotherapeutic agent, camptothecin. We propose that Fbx6-dependent Chk1 degradation contributes to S phase checkpoint termination and that a defect in this mechanism might increase tumor cell resistance to certain anticancer drugs.

  9. The importance of adding EDTA for the nanopore analysis of proteins.

    Science.gov (United States)

    Krasniqi, Besnik; Lee, Jeremy S

    2012-06-01

    Nanopore analysis is a promising technique for studying the conformation of proteins and protein/protein interactions. Two proteins (bacterial thioredoxin and maltose binding protein) were subjected to nanopore analysis with α-hemolysin. Two types of events were observed; bumping events with a blockade current less than -40 pA and intercalation events with blockade currents between -40 pA and -100 pA. In potassium phosphate buffer, pH 7.8, both proteins gave intercalation events but the frequency of these events was significantly reduced in TRIS or HEPES buffers especially in the presence of 0.01 mM divalent metal ions. The frequency of events was restored by the addition of EDTA. For maltose binding protein, the frequency of intercalation events was also decreased in the presence of maltose but not lactose to which it does not bind. It is proposed that the events with large blockade currents represent transient intercalation of a loop or end of the protein into the pore and that divalent metal ions inhibit this process. The results demonstrate that the choice of buffer and the effects of metal ion contamination are important considerations in nanopore analysis.

  10. Signal transduction in neurons: effects of cellular prion protein on fyn kinase and ERK1/2 kinase

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    Tomasi Vittorio

    2010-12-01

    Full Text Available Abstract Background It has been reported that cellular prion protein (PrPc co-localizes with caveolin-1 and participates to signal transduction events by recruiting Fyn kinase. As PrPc is a secreted protein anchored to the outer surface membrane through a glycosylphosphatidylinositol (GPI anchor (secPrP and caveolin-1 is located in the inner leaflet of plasma membrane, there is a problem of how the two proteins can physically interact each other and transduce signals. Results By using the GST-fusion proteins system we observed that PrPc strongly interacts with caveolin-1 scaffolding domain and with a caveolin-1 hydrophilic C-terminal region, but not with the caveolin-1 N-terminal region. In vitro binding experiments were also performed to define the site(s of PrPc interacting with cav-1. The results are consistent with a participation of PrPc octapeptide repeats motif in the binding to caveolin-1 scaffolding domain. The caveolar localization of PrPc was ascertained by co-immunoprecipitation, by co-localization after flotation in density gradients and by confocal microscopy analysis of PrPc and caveolin-1 distributions in a neuronal cell line (GN11 expressing caveolin-1 at high levels. Conclusions We observed that, after antibody-mediated cross-linking or copper treatment, PrPc was internalized probably into caveolae. We propose that following translocation from rafts to caveolae or caveolae-like domains, secPrP could interact with caveolin-1 and induce signal transduction events.

  11. Signal transduction in neurons: effects of cellular prion protein on fyn kinase and ERK1/2 kinase.

    Science.gov (United States)

    Tomasi, Vittorio

    2010-12-16

    It has been reported that cellular prion protein (PrPc) co-localizes with caveolin-1 and participates to signal transduction events by recruiting Fyn kinase. As PrPc is a secreted protein anchored to the outer surface membrane through a glycosylphosphatidylinositol (GPI) anchor (secPrP) and caveolin-1 is located in the inner leaflet of plasma membrane, there is a problem of how the two proteins can physically interact each other and transduce signals. By using the GST-fusion proteins system we observed that PrPc strongly interacts with caveolin-1 scaffolding domain and with a caveolin-1 hydrophilic C-terminal region, but not with the caveolin-1 N-terminal region. In vitro binding experiments were also performed to define the site(s) of PrPc interacting with cav-1. The results are consistent with a participation of PrPc octapeptide repeats motif in the binding to caveolin-1 scaffolding domain. The caveolar localization of PrPc was ascertained by co-immunoprecipitation, by co-localization after flotation in density gradients and by confocal microscopy analysis of PrPc and caveolin-1 distributions in a neuronal cell line (GN11) expressing caveolin-1 at high levels. We observed that, after antibody-mediated cross-linking or copper treatment, PrPc was internalized probably into caveolae. We propose that following translocation from rafts to caveolae or caveolae-like domains, secPrP could interact with caveolin-1 and induce signal transduction events.

  12. Humoral and cellular immune responses to glucose regulated protein 78 - a novel Leishmania donovani antigen

    DEFF Research Database (Denmark)

    Jensen, Anja T R; Ismail, Ahmed; Gaafar, Ameera

    2002-01-01

    The recently cloned glucose regulated protein 78 (GRP78) of Leishmania donovani has been suggested as a new and promising Leishmania vaccine candidate. We assessed antibody and T-cell reactivity to GRP78 in an enzyme-linked immunosorbent assay (ELISA) and in lymphoproliferative assays. Serological...... with a positive leishmanin skin test showed antibody reactivity to recombinant GRP78 (rGRP78). In lymphoproliferative assays, 9 of 13 isolates of peripheral blood mononuclear cells (PBMC) from individuals previously infected with L. donovani and one of three individuals previously infected with L. major showed...... in an area endemic for malaria but free of leishmaniasis and plasma from healthy Danes was negative in the assay. GRP78 antibody was detected in 10% and 5% of plasma samples from Sudanese and Ghanaian malaria patients, respectively, whereas 35% of plasma samples from otherwise healthy Sudanese individuals...

  13. Maintenance of asymmetric cellular localization of an auxin transport protein through interaction with the actin cytoskeleton

    Science.gov (United States)

    Muday, G. K.

    2000-01-01

    In shoots, polar auxin transport is basipetal (that is, from the shoot apex toward the base) and is driven by the basal localization of the auxin efflux carrier complex. The focus of this article is to summarize the experiments that have examined how the asymmetric distribution of this protein complex is controlled and the significance of this polar distribution. Experimental evidence suggests that asymmetries in the auxin efflux carrier may be established through localized secretion of Golgi vesicles, whereas an attachment of a subunit of the efflux carrier to the actin cytoskeleton may maintain this localization. In addition, the idea that this localization of the efflux carrier may control both the polarity of auxin movement and more globally regulate developmental polarity is explored. Finally, evidence indicating that the gravity vector controls auxin transport polarity is summarized and possible mechanisms for the environmentally induced changes in auxin transport polarity are discussed.

  14. A single peroxisomal targeting signal mediates matrix protein import in diatoms.

    Directory of Open Access Journals (Sweden)

    Nicola H Gonzalez

    Full Text Available Peroxisomes are single membrane bound compartments. They are thought to be present in almost all eukaryotic cells, although the bulk of our knowledge about peroxisomes has been generated from only a handful of model organisms. Peroxisomal matrix proteins are synthesized cytosolically and posttranslationally imported into the peroxisomal matrix. The import is generally thought to be mediated by two different targeting signals. These are respectively recognized by the two import receptor proteins Pex5 and Pex7, which facilitate transport across the peroxisomal membrane. Here, we show the first in vivo localization studies of peroxisomes in a representative organism of the ecologically relevant group of diatoms using fluorescence and transmission electron microscopy. By expression of various homologous and heterologous fusion proteins we demonstrate that targeting of Phaeodactylum tricornutum peroxisomal matrix proteins is mediated only by PTS1 targeting signals, also for proteins that are in other systems imported via a PTS2 mode of action. Additional in silico analyses suggest this surprising finding may also apply to further diatoms. Our data suggest that loss of the PTS2 peroxisomal import signal is not reserved to Caenorhabditis elegans as a single exception, but has also occurred in evolutionary divergent organisms. Obviously, targeting switching from PTS2 to PTS1 across different major eukaryotic groups might have occurred for different reasons. Thus, our findings question the widespread assumption that import of peroxisomal matrix proteins is generally mediated by two different targeting signals. Our results implicate that there apparently must have been an event causing the loss of one targeting signal even in the group of diatoms. Different possibilities are discussed that indicate multiple reasons for the detected targeting switching from PTS2 to PTS1.

  15. Structural analysis of site-directed mutants of cellular retinoic acid-binding protein II addresses the relationship between structural integrity and ligand binding

    International Nuclear Information System (INIS)

    Vaezeslami, Soheila; Jia, Xiaofei; Vasileiou, Chrysoula; Borhan, Babak; Geiger, James H.

    2008-01-01

    A water network stabilizes the structure of cellular retionic acid binding protein II. The structural integrity of cellular retinoic acid-binding protein II (CRABPII) has been investigated using the crystal structures of CRABPII mutants. The overall fold was well maintained by these CRABPII mutants, each of which carried multiple different mutations. A water-mediated network is found to be present across the large binding cavity, extending from Arg111 deep inside the cavity to the α2 helix at its entrance. This chain of interactions acts as a ‘pillar’ that maintains the integrity of the protein. The disruption of the water network upon loss of Arg111 leads to decreased structural integrity of the protein. A water-mediated network can be re-established by introducing the hydrophilic Glu121 inside the cavity, which results in a rigid protein with the α2 helix adopting an altered conformation compared with wild-type CRABPII

  16. Cyclosporin A associated helicase-like protein facilitates the association of hepatitis C virus RNA polymerase with its cellular cyclophilin B.

    Directory of Open Access Journals (Sweden)

    Kengo Morohashi

    Full Text Available BACKGROUND: Cyclosporin A (CsA is well known as an immunosuppressive drug useful for allogeneic transplantation. It has been reported that CsA inhibits hepatitis C virus (HCV genome replication, which indicates that cellular targets of CsA regulate the viral replication. However, the regulation mechanisms of HCV replication governed by CsA target proteins have not been fully understood. PRINCIPAL FINDINGS: Here we show a chemical biology approach that elucidates a novel mechanism of HCV replication. We developed a phage display screening to investigate compound-peptide interaction and identified a novel cellular target molecule of CsA. This protein, named CsA associated helicase-like protein (CAHL, possessed RNA-dependent ATPase activity that was negated by treatment with CsA. The downregulation of CAHL in the cells resulted in a decrease of HCV genome replication. CAHL formed a complex with HCV-derived RNA polymerase NS5B and host-derived cyclophilin B (CyPB, known as a cellular cofactor for HCV replication, to regulate NS5B-CyPB interaction. CONCLUSIONS: We found a cellular factor, CAHL, as CsA associated helicase-like protein, which would form trimer complex with CyPB and NS5B of HCV. The strategy using a chemical compound and identifying its target molecule by our phage display analysis is useful to reveal a novel mechanism underlying cellular and viral physiology.

  17. Cyclosporin A associated helicase-like protein facilitates the association of hepatitis C virus RNA polymerase with its cellular cyclophilin B.

    Science.gov (United States)

    Morohashi, Kengo; Sahara, Hiroeki; Watashi, Koichi; Iwabata, Kazuki; Sunoki, Takashi; Kuramochi, Kouji; Takakusagi, Kaori; Miyashita, Hiroki; Sato, Noriyuki; Tanabe, Atsushi; Shimotohno, Kunitada; Kobayashi, Susumu; Sakaguchi, Kengo; Sugawara, Fumio

    2011-04-29

    Cyclosporin A (CsA) is well known as an immunosuppressive drug useful for allogeneic transplantation. It has been reported that CsA inhibits hepatitis C virus (HCV) genome replication, which indicates that cellular targets of CsA regulate the viral replication. However, the regulation mechanisms of HCV replication governed by CsA target proteins have not been fully understood. Here we show a chemical biology approach that elucidates a novel mechanism of HCV replication. We developed a phage display screening to investigate compound-peptide interaction and identified a novel cellular target molecule of CsA. This protein, named CsA associated helicase-like protein (CAHL), possessed RNA-dependent ATPase activity that was negated by treatment with CsA. The downregulation of CAHL in the cells resulted in a decrease of HCV genome replication. CAHL formed a complex with HCV-derived RNA polymerase NS5B and host-derived cyclophilin B (CyPB), known as a cellular cofactor for HCV replication, to regulate NS5B-CyPB interaction. We found a cellular factor, CAHL, as CsA associated helicase-like protein, which would form trimer complex with CyPB and NS5B of HCV. The strategy using a chemical compound and identifying its target molecule by our phage display analysis is useful to reveal a novel mechanism underlying cellular and viral physiology.

  18. Many Saccharomyces cerevisiae Cell Wall Protein Encoding Genes Are Coregulated by Mss11, but Cellular Adhesion Phenotypes Appear Only Flo Protein Dependent.

    Science.gov (United States)

    Bester, Michael C; Jacobson, Dan; Bauer, Florian F

    2012-01-01

    The outer cell wall of the yeast Saccharomyces cerevisiae serves as the interface with the surrounding environment and directly affects cell-cell and cell-surface interactions. Many of these interactions are facilitated by specific adhesins that belong to the Flo protein family. Flo mannoproteins have been implicated in phenotypes such as flocculation, substrate adhesion, biofilm formation, and pseudohyphal growth. Genetic data strongly suggest that individual Flo proteins are responsible for many specific cellular adhesion phenotypes. However, it remains unclear whether such phenotypes are determined solely by the nature of the expressed FLO genes or rather as the result of a combination of FLO gene expression and other cell wall properties and cell wall proteins. Mss11 has been shown to be a central element of FLO1 and FLO11 gene regulation and acts together with the cAMP-PKA-dependent transcription factor Flo8. Here we use genome-wide transcription analysis to identify genes that are directly or indirectly regulated by Mss11. Interestingly, many of these genes encode cell wall mannoproteins, in particular, members of the TIR and DAN families. To examine whether these genes play a role in the adhesion properties associated with Mss11 expression, we assessed deletion mutants of these genes in wild-type and flo11Δ genetic backgrounds. This analysis shows that only FLO genes, in particular FLO1/10/11, appear to significantly impact on such phenotypes. Thus adhesion-related phenotypes are primarily dependent on the balance of FLO gene expression.

  19. Sulfolobus Turreted Icosahedral Virus c92 Protein Responsible for the Formation of Pyramid-Like Cellular Lysis Structures

    DEFF Research Database (Denmark)

    Snyder, Jamie C; Brumfield, Susan K; Peng, Nan

    2011-01-01

    Host cells infected by Sulfolobus turreted icosahedral virus (STIV) have been shown to produce unusual pyramid-like structures on the cell surface. These structures represent a virus-induced lysis mechanism that is present in Archaea and appears to be distinct from the holin/endolysin system desc...... disruption of c92 within STIV demonstrates that c92 is an essential protein for virus replication. Immunolocalization of c92 shows that the protein is localized to the cellular membranes forming the pyramid-like structures.......Host cells infected by Sulfolobus turreted icosahedral virus (STIV) have been shown to produce unusual pyramid-like structures on the cell surface. These structures represent a virus-induced lysis mechanism that is present in Archaea and appears to be distinct from the holin/endolysin system...... described for DNA bacteriophages. This study investigated the STIV gene products required for pyramid formation in its host Sulfolobus solfataricus. Overexpression of STIV open reading frame (ORF) c92 in S. solfataricus alone is sufficient to produce the pyramid-like lysis structures in cells. Gene...

  20. Identification of a tripartite import signal in the Ewing Sarcoma protein (EWS)

    International Nuclear Information System (INIS)

    Shaw, Debra J.; Morse, Robert; Todd, Adrian G.; Eggleton, Paul; Lorson, Christian L.; Young, Philip J.

    2009-01-01

    The Ewing Sarcoma (EWS) protein is a ubiquitously expressed RNA processing factor that localises predominantly to the nucleus. However, the mechanism through which EWS enters the nucleus remains unclear, with differing reports identifying three separate import signals within the EWS protein. Here we have utilized a panel of truncated EWS proteins to clarify the reported nuclear localisation signals. We describe three C-terminal domains that are important for efficient EWS nuclear localization: (1) the third RGG-motif; (2) the last 10 amino acids (known as the PY-import motif); and (3) the zinc-finger motif. Although these three domains are involved in nuclear import, they are not independently capable of driving the efficient import of a GFP-moiety. However, collectively they form a complex tripartite signal that efficiently drives GFP-import into the nucleus. This study helps clarify the EWS import signal, and the identification of the involvement of both the RGG- and zinc-finger motifs has wide reaching implications.

  1. Identification of a tripartite import signal in the Ewing Sarcoma protein (EWS)

    Energy Technology Data Exchange (ETDEWEB)

    Shaw, Debra J.; Morse, Robert; Todd, Adrian G. [Clinical Neurobiology, IBCS, Peninsula College of Medicine and Dentistry, Exeter EX1 2LU (United Kingdom); Eggleton, Paul [Inflammation and Musculoskeletal Disease, IBCS, Peninsula College of Medicine and Dentistry, Exeter EX1 2LU (United Kingdom); MRC Immunochemistry Unit, University of Oxford, Oxford OX1 3QU (United Kingdom); Lorson, Christian L. [Department of Veterinary Pathobiology, Bond Life Sciences Center, 1201 Rollins Road, University of Missouri, Columbia, MO 65211 (United States); Young, Philip J., E-mail: philip.young@pms.ac.uk [Clinical Neurobiology, IBCS, Peninsula College of Medicine and Dentistry, Exeter EX1 2LU (United Kingdom)

    2009-12-25

    The Ewing Sarcoma (EWS) protein is a ubiquitously expressed RNA processing factor that localises predominantly to the nucleus. However, the mechanism through which EWS enters the nucleus remains unclear, with differing reports identifying three separate import signals within the EWS protein. Here we have utilized a panel of truncated EWS proteins to clarify the reported nuclear localisation signals. We describe three C-terminal domains that are important for efficient EWS nuclear localization: (1) the third RGG-motif; (2) the last 10 amino acids (known as the PY-import motif); and (3) the zinc-finger motif. Although these three domains are involved in nuclear import, they are not independently capable of driving the efficient import of a GFP-moiety. However, collectively they form a complex tripartite signal that efficiently drives GFP-import into the nucleus. This study helps clarify the EWS import signal, and the identification of the involvement of both the RGG- and zinc-finger motifs has wide reaching implications.

  2. Futile import of tRNAs and proteins into the mitochondrion of Trypanosoma brucei evansi

    Czech Academy of Sciences Publication Activity Database

    Paris, Zdeněk; Hashimi, Hassan; Lun, Sijia; Alfonzo, J. D.; Lukeš, Julius

    2011-01-01

    Roč. 176, č. 2 (2011), 116-120 ISSN 0166-6851 R&D Projects: GA ČR GA204/09/1667; GA MŠk LC07032 Institutional research plan: CEZ:AV0Z60220518 Keywords : Trypanosoma * tRNA * Protein import * Mitochondrion * Kinetoplast Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 2.551, year: 2011

  3. Yeast two-hybrid screens imply involvement of Fanconi anemia proteins in transcription regulation, cell signaling, oxidative metabolism, and cellular transport.

    Science.gov (United States)

    Reuter, Tanja Y; Medhurst, Annette L; Waisfisz, Quinten; Zhi, Yu; Herterich, Sabine; Hoehn, Holger; Gross, Hans J; Joenje, Hans; Hoatlin, Maureen E; Mathew, Christopher G; Huber, Pia A J

    2003-10-01

    Mutations in one of at least eight different genes cause bone marrow failure, chromosome instability, and predisposition to cancer associated with the rare genetic syndrome Fanconi anemia (FA). The cloning of seven genes has provided the tools to study the molecular pathway disrupted in Fanconi anemia patients. The structure of the genes and their gene products provided few clues to their functional role. We report here the use of 3 FA proteins, FANCA, FANCC, and FANCG, as "baits" in the hunt for interactors to obtain clues for FA protein functions. Using five different human cDNA libraries we screened 36.5x10(6) clones with the technique of the yeast two-hybrid system. We identified 69 proteins which have not previously been linked to the FA pathway as direct interactors of FANCA, FANCC, or FANCG. Most of these proteins are associated with four functional classes including transcription regulation (21 proteins), signaling (13 proteins), oxidative metabolism (10 proteins), and intracellular transport (11 proteins). Interaction with 6 proteins, DAXX, Ran, IkappaBgamma, USP14, and the previously reported SNX5 and FAZF, was additionally confirmed by coimmunoprecipitation and/or colocalization studies. Taken together, our data strongly support the hypothesis that FA proteins are functionally involved in several complex cellular pathways including transcription regulation, cell signaling, oxidative metabolism, and cellular transport.

  4. Alzheimer's Disease Brain-Derived Amyloid-{beta}-Mediated Inhibition of LTP In Vivo Is Prevented by Immunotargeting Cellular Prion Protein.

    LENUS (Irish Health Repository)

    Barry, Andrew E

    2011-05-18

    Synthetic amyloid-β protein (Aβ) oligomers bind with high affinity to cellular prion protein (PrP(C)), but the role of this interaction in mediating the disruption of synaptic plasticity by such soluble Aβ in vitro is controversial. Here we report that intracerebroventricular injection of Aβ-containing aqueous extracts of Alzheimer\\'s disease (AD) brain robustly inhibits long-term potentiation (LTP) without significantly affecting baseline excitatory synaptic transmission in the rat hippocampus in vivo. Moreover, the disruption of LTP was abrogated by immunodepletion of Aβ. Importantly, intracerebroventricular administration of antigen-binding antibody fragment D13, directed to a putative Aβ-binding site on PrP(C), prevented the inhibition of LTP by AD brain-derived Aβ. In contrast, R1, a Fab directed to the C terminus of PrP(C), a region not implicated in binding of Aβ, did not significantly affect the Aβ-mediated inhibition of LTP. These data support the pathophysiological significance of SDS-stable Aβ dimer and the role of PrP(C) in mediating synaptic plasticity disruption by soluble Aβ.

  5. Identification of a Protein with Antioxidant Activity that is Important for the Protection against Beer Ageing

    Science.gov (United States)

    Wu, Ming J.; Clarke, Frank M.; Rogers, Peter J.; Young, Paul; Sales, Narelle; O’Doherty, Patrick J.; Higgins, Vincent J.

    2011-01-01

    This study was carried out with fresh Australian lager beer which was sampled directly off the production line, the same samples aged for 12 weeks at 30 °C, and the vintage beer which was kept at 20 °C for 5 years. Characteristic Australian lager flavour was maintained in the fresh and vintage beers but was lost in the aged beer. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and free thiol group labelling analyses of beer proteins found that this flavour stability correlated with the presence of an unknown 10 kilodaltons (kDa) protein with a higher level of free thiols. The protein was purified by size-exclusion chromatography, then peptide sequencing and database matching identified it as the barley lipid transfer protein (LTP1). Further characterisation using diphenylpicrylhydrazyl (DPPH) free radical scavenging and a Saccharomyces cerevisiae-based antioxidant screening assay demonstrated that the LTP1 protein was active in DPPH reduction and antioxidant activity. The absence of free thiol in the aged beer indicates that the thiol functional groups within the LTP1 protein were saturated and suggests that it is important in the flavour stability of beer by maintaining reduction capacity during the ageing process. PMID:22016646

  6. Is chloroplast import of photosynthesis proteins facilitated by an actin-TOC-TIC-VIPP1 complex?

    Science.gov (United States)

    Jouhet, Juliette; Gray, John C

    2009-10-01

    Actin filaments are major components of the cytoskeleton that interact with chloroplast envelope membranes to allow chloroplast positioning and movement, stromule mobility and gravitropism perception. We recently reported that Toc159, a component of the TOC complex of the chloroplast protein import apparatus, interacts directly with actin. The interaction of Toc159 and actin was identified by co-immunoprecipitation and co-sedimentation experiments with detergent-solubilised pea chloroplast envelope membranes. In addition, many of the components of the TOC-TIC protein import apparatus and VIPP1 (vesicle-inducing protein in plastids 1) were identified by mass spectroscopy in the material co-immunoprecipitated with antibodies to actin. Toc159 is the receptor for the import of photosynthesis proteins and VIPP1 is involved in thylakoid membrane formation by inducing vesicle formation from the chloroplast inner envelope membrane, suggesting we may have identified an actin-TOC-TIC-VIPP1 complex that may provide a means of channeling cytosolic preproteins to the thylakoid membrane. The interaction of Toc159 with actin may facilitate exchange between the putative soluble and membrane forms of Toc159 and promote the interaction of cytosolic preproteins with the TOC complex.

  7. On the importance of polar interactions for complexes containing intrinsically disordered proteins.

    Directory of Open Access Journals (Sweden)

    Eric T C Wong

    Full Text Available There is a growing recognition for the importance of proteins with large intrinsically disordered (ID segments in cell signaling and regulation. ID segments in these proteins often harbor regions that mediate molecular recognition. Coupled folding and binding of the recognition regions has been proposed to confer high specificity to interactions involving ID segments. However, researchers recently questioned the origin of the interaction specificity of ID proteins because of the overrepresentation of hydrophobic residues in their interaction interfaces. Here, we focused on the role of polar and charged residues in interactions mediated by ID segments. Making use of the extended nature of most ID segments when in complex with globular proteins, we first identified large numbers of complexes between globular proteins and ID segments by using radius-of-gyration-based selection criteria. Consistent with previous studies, we found the interfaces of these complexes to be enriched in hydrophobic residues, and that these residues contribute significantly to the stability of the interaction interface. However, our analyses also show that polar interactions play a larger role in these complexes than in structured protein complexes. Computational alanine scanning and salt-bridge analysis indicate that interfaces in ID complexes are highly complementary with respect to electrostatics, more so than interfaces of globular proteins. Follow-up calculations of the electrostatic contributions to the free energy of binding uncovered significantly stronger Coulombic interactions in complexes harbouring ID segments than in structured protein complexes. However, they are counter-balanced by even higher polar-desolvation penalties. We propose that polar interactions are a key contributing factor to the observed high specificity of ID segment-mediated interactions.

  8. GCK-MODY diabetes as a protein misfolding disease: the mutation R275C promotes protein misfolding, self-association and cellular degradation.

    Science.gov (United States)

    Negahdar, Maria; Aukrust, Ingvild; Molnes, Janne; Solheim, Marie H; Johansson, Bente B; Sagen, Jørn V; Dahl-Jørgensen, Knut; Kulkarni, Rohit N; Søvik, Oddmund; Flatmark, Torgeir; Njølstad, Pål R; Bjørkhaug, Lise

    2014-01-25

    GCK-MODY, dominantly inherited mild hyperglycemia, is associated with more than 600 mutations in the glucokinase gene. Different molecular mechanisms have been shown to explain GCK-MODY. Here, we report a Pakistani family harboring the glucokinase mutation c.823C>T (p.R275C). The recombinant and in cellulo expressed mutant pancreatic enzyme revealed slightly increased enzyme activity (kcat) and normal affinity for α-D-glucose, and resistance to limited proteolysis by trypsin comparable with wild-type. When stably expressed in HEK293 cells and MIN6 β-cells (at different levels), the mutant protein appeared misfolded and unstable with a propensity to form dimers and aggregates. Its degradation rate was increased, involving the lysosomal and proteasomal quality control systems. On mutation, a hydrogen bond between the R275 side-chain and the carbonyl oxygen of D267 is broken, destabilizing the F260-L271 loop structure and the protein. This promotes the formation of dimers/aggregates and suggests that an increased cellular degradation is the molecular mechanism by which R275C causes GCK-MODY. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

  9. Prolonged exposure to particulate chromate inhibits RAD51 nuclear import mediator proteins.

    Science.gov (United States)

    Browning, Cynthia L; Wise, John Pierce

    2017-09-15

    Particulate hexavalent chromium (Cr(VI)) is a human lung carcinogen and a human health concern. The induction of structural chromosome instability is considered to be a driving mechanism of Cr(VI)-induced carcinogenesis. Homologous recombination repair protects against Cr(VI)-induced chromosome damage, due to its highly accurate repair of Cr(VI)-induced DNA double strand breaks. However, recent studies demonstrate Cr(VI) inhibits homologous recombination repair through the misregulation of RAD51. RAD51 is an essential protein in HR repair that facilitates the search for a homologous sequence. Recent studies show prolonged Cr(VI) exposure prevents proper RAD51 subcellular localization, causing it to accumulate in the cytoplasm. Since nuclear import of RAD51 is crucial to its function, this study investigated the effect of Cr(VI) on the RAD51 nuclear import mediators, RAD51C and BRCA2. We show acute (24h) Cr(VI) exposure induces the proper localization of RAD51C and BRCA2. In contrast, prolonged (120h) exposure increased the cytoplasmic localization of both proteins, although RAD51C localization was more severely impaired. These results correlate temporally with the previously reported Cr(VI)-induced RAD51 cytoplasmic accumulation. In addition, we found Cr(VI) does not inhibit interaction between RAD51 and its nuclear import mediators. Altogether, our results suggest prolonged Cr(VI) exposure inhibits the nuclear import of RAD51C, and to a lesser extent, BRCA2, which results in the cytoplasmic accumulation of RAD51. Cr(VI)-induced inhibition of nuclear import may play a key role in its carcinogenic mechanism since the nuclear import of many tumor suppressor proteins and DNA repair proteins is crucial to their function. Copyright © 2017 Elsevier Inc. All rights reserved.

  10. Inhibition of Poliovirus-Induced Cleavage of Cellular Protein PCBP2 Reduces the Levels of Viral RNA Replication

    Science.gov (United States)

    Chase, Amanda J.; Daijogo, Sarah

    2014-01-01

    ABSTRACT Due to their small genome size, picornaviruses must utilize host proteins to mediate cap-independent translation and viral RNA replication. The host RNA-binding protein poly(rC) binding protein 2 (PCBP2) is involved in both processes in poliovirus infected cells. It has been shown that the viral proteinase 3CD cleaves PCBP2 and contributes to viral translation inhibition. However, cleaved PCBP2 remains active in viral RNA replication. This would suggest that both cleaved and intact forms of PCBP2 have a role in the viral RNA replication cycle. The picornavirus genome must act as a template for both translation and RNA replication. However, a template that is actively being translated cannot function as a template for RNA replication, suggesting that there is a switch in template usage from translation to RNA replication. We demonstrate that the cleavage of PCBP2 by the poliovirus 3CD proteinase is a necessary step for efficient viral RNA replication and, as such, may be important for mediating a switch in template usage from translation to RNA replication. IMPORTANCE Poliovirus, like all positive-strand RNA viruses that replicate in the cytoplasm of eukaryotic cells, uses its genomic RNA as a template for both viral protein synthesis and RNA replication. Given that these processes cannot occur simultaneously on the same template, poliovirus has evolved a mechanism(s) to facilitate the switch from using templates for translation to using them for RNA synthesis. This study explores one possible scenario for how the virus alters the functions of a host cell RNA binding protein to mediate, in part, this important transition. PMID:24371074

  11. Localization of the cellular retinoic acid binding protein (CRABP) gene relative to the acute promyelocytic leukemia-associated breakpoint on human chromosome 15

    NARCIS (Netherlands)

    A.H.M. Geurts van Kessel (Ad); H. de Leeuw (H.); E.J. Dekker (Erik Jan); J.M. Rijks (Jolianne); N. Spurr (N.); A.M. Ledbetter (Andrew M.); E. Kootwijk (E.); M.J. Vaessen (Marie-Josée)

    1991-01-01

    textabstractA human genomic fragment comprising the cellular retinoic acid binding protein (CRABP) gene was isolated. By using a panel of somatic cell hybrids, this gene could be assigned to human chromosome 15. Subsequently, a possible involvement of the CRABP gene in translocation (15;17)

  12. Acrolein-Exposed Normal Human Lung Fibroblasts in Vitro: Cellular Senescence, Enhanced Telomere Erosion, and Degradation of Werner’s Syndrome Protein

    Science.gov (United States)

    Jang, Jun-Ho; Bruse, Shannon; Huneidi, Salam; Schrader, Ronald M.; Monick, Martha M.; Lin, Yong; Carter, A. Brent; Klingelhutz, Aloysius J.

    2014-01-01

    Background: Acrolein is a ubiquitous environmental hazard to human health. Acrolein has been reported to activate the DNA damage response and induce apoptosis. However, little is known about the effects of acrolein on cellular senescence. Objectives: We examined whether acrolein induces cellular senescence in cultured normal human lung fibroblasts (NHLF). Methods: We cultured NHLF in the presence or absence of acrolein and determined the effects of acrolein on cell proliferative capacity, senescence-associated β-galactosidase activity, the known senescence-inducing pathways (e.g., p53, p21), and telomere length. Results: We found that acrolein induced cellular senescence by increasing both p53 and p21. The knockdown of p53 mediated by small interfering RNA (siRNA) attenuated acrolein-induced cellular senescence. Acrolein decreased Werner’s syndrome protein (WRN), a member of the RecQ helicase family involved in DNA repair and telomere maintenance. Acrolein-induced down-regulation of WRN protein was rescued by p53 knockdown or proteasome inhibition. Finally, we found that acrolein accelerated p53-mediated telomere shortening. Conclusions: These results suggest that acrolein induces p53-mediated cellular senescence accompanied by enhanced telomere attrition and WRN protein down-regulation. Citation: Jang JH, Bruse S, Huneidi S, Schrader RM, Monick MM, Lin Y, Carter AB, Klingelhutz AJ, Nyunoya T. 2014. Acrolein-exposed normal human lung fibroblasts in vitro: cellular senescence, enhanced telomere erosion, and degradation of Werner’s syndrome protein. Environ Health Perspect 122:955–962; http://dx.doi.org/10.1289/ehp.1306911 PMID:24747221

  13. Protease resistance of infectious prions is suppressed by removal of a single atom in the cellular prion protein.

    Science.gov (United States)

    Leske, Henning; Hornemann, Simone; Herrmann, Uli Simon; Zhu, Caihong; Dametto, Paolo; Li, Bei; Laferriere, Florent; Polymenidou, Magdalini; Pelczar, Pawel; Reimann, Regina Rose; Schwarz, Petra; Rushing, Elisabeth Jane; Wüthrich, Kurt; Aguzzi, Adriano

    2017-01-01

    Resistance to proteolytic digestion has long been considered a defining trait of prions in tissues of organisms suffering from transmissible spongiform encephalopathies. Detection of proteinase K-resistant prion protein (PrPSc) still represents the diagnostic gold standard for prion diseases in humans, sheep and cattle. However, it has become increasingly apparent that the accumulation of PrPSc does not always accompany prion infections: high titers of prion infectivity can be reached also in the absence of protease resistant PrPSc. Here, we describe a structural basis for the phenomenon of protease-sensitive prion infectivity. We studied the effect on proteinase K (PK) resistance of the amino acid substitution Y169F, which removes a single oxygen atom from the β2-α2 loop of the cellular prion protein (PrPC). When infected with RML or the 263K strain of prions, transgenic mice lacking wild-type (wt) PrPC but expressing MoPrP169F generated prion infectivity at levels comparable to wt mice. The newly generated MoPrP169F prions were biologically indistinguishable from those recovered from prion-infected wt mice, and elicited similar pathologies in vivo. Surprisingly, MoPrP169F prions showed greatly reduced PK resistance and density gradient analyses showed a significant reduction in high-density aggregates. Passage of MoPrP169F prions into mice expressing wt MoPrP led to full recovery of protease resistance, indicating that no strain shift had taken place. We conclude that a subtle structural variation in the β2-α2 loop of PrPC affects the sensitivity of PrPSc to protease but does not impact prion replication and infectivity. With these findings a specific structural feature of PrPC can be linked to a physicochemical property of the corresponding PrPSc.

  14. Mutations in the Schmallenberg Virus Gc Glycoprotein Facilitate Cellular Protein Synthesis Shutoff and Restore Pathogenicity of NSs Deletion Mutants in Mice.

    Science.gov (United States)

    Varela, Mariana; Pinto, Rute Maria; Caporale, Marco; Piras, Ilaria M; Taggart, Aislynn; Seehusen, Frauke; Hahn, Kerstin; Janowicz, Anna; de Souza, William Marciel; Baumgärtner, Wolfgang; Shi, Xiaohong; Palmarini, Massimo

    2016-06-01

    Serial passage of viruses in cell culture has been traditionally used to attenuate virulence and identify determinants of viral pathogenesis. In a previous study, we found that a strain of Schmallenberg virus (SBV) serially passaged in tissue culture (termed SBVp32) unexpectedly displayed increased pathogenicity in suckling mice compared to wild-type SBV. In this study, we mapped the determinants of SBVp32 virulence to the viral genome M segment. SBVp32 virulence is associated with the capacity of this virus to reach high titers in the brains of experimentally infected suckling mice. We also found that the Gc glycoprotein, encoded by the M segment of SBVp32, facilitates host cell protein shutoff in vitro Interestingly, while the M segment of SBVp32 is a virulence factor, we found that the S segment of the same virus confers by itself an attenuated phenotype to wild-type SBV, as it has lost the ability to block the innate immune system of the host. Single mutations present in the Gc glycoprotein of SBVp32 are sufficient to compensate for both the attenuated phenotype of the SBVp32 S segment and the attenuated phenotype of NSs deletion mutants. Our data also indicate that the SBVp32 M segment does not act as an interferon (IFN) antagonist. Therefore, SBV mutants can retain pathogenicity even when they are unable to fully control the production of IFN by infected cells. Overall, this study suggests that the viral glycoprotein of orthobunyaviruses can compensate, at least in part, for the function of NSs. In addition, we also provide evidence that the induction of total cellular protein shutoff by SBV is determined by multiple viral proteins, while the ability to control the production of IFN maps to the NSs protein. The identification of viral determinants of pathogenesis is key to the development of prophylactic and intervention measures. In this study, we found that the bunyavirus Gc glycoprotein is a virulence factor. Importantly, we show that mutations in the Gc

  15. Toxic influence of organophosphate, carbamate, and organochlorine pesticides on cellular metabolism of lipids, proteins, and carbohydrates: a systematic review.

    Science.gov (United States)

    Karami-Mohajeri, Somayyeh; Abdollahi, Mohammad

    2011-09-01

    Pesticides, including organophosphate (OP), organochlorine (OC), and carbamate (CB) compounds, are widely used in agricultural and indoor purposes. OP and CB act as acetyl cholinesterase (AChE) inhibitors that affect lots of organs such as peripheral and central nervous systems, muscles, liver, pancreas, and brain, whereas OC are neurotoxic involved in alteration of ion channels. There are several reports about metabolic disorders, hyperglycemia, and also oxidative stress in acute and chronic exposures to pesticides that are linked with diabetes and other metabolic disorders. In this respect, there are several in vitro and in vivo but few clinical studies about mechanism underlying these effects. Bibliographic databases were searched for the years 1963-2010 and resulted in 1652 articles. After elimination of duplicates or irrelevant papers, 204 papers were included and reviewed. Results indicated that OP and CB impair the enzymatic pathways involved in metabolism of carbohydrates, fats and protein within cytoplasm, mitochondria, and proxisomes. It is believed that OP and CB show this effect through inhibition of AChE or affecting target organs directly. OC mostly affect lipid metabolism in the adipose tissues and change glucose pathway in other cells. As a shared mechanism, all OP, CB and OC induce cellular oxidative stress via affecting mitochondrial function and therefore disrupt neuronal and hormonal status of the body. Establishing proper epidemiological studies to explore exact relationships between exposure levels to these pesticides and rate of resulted metabolic disorders in human will be helpful.

  16. Whole Body Hyperthermia in Mice Confers Delayed Radioprotection at Cellular and Tissue Levels: Inducible Heat Shock Proteins as Endogenous Radioprotectors

    International Nuclear Information System (INIS)

    Malytina, Y. V.; Sements, T. N.; Semina, O. V.; Mosin, A. F.; Kabakov, A.

    2004-01-01

    It was previously shown on heat shock protein (Hsp)-over expressing cell lines that the increased intracellular content of Hsp 70 or Hsp27 is associated with the elevated radioresistance. However, it was so far unknown whether the in vivo Fsp induction by stressful preconditioning can confer radioprotection at the tissue and cellular levels. In the present study, we examined how the in vivo up-regulation of the Hsp expression in response to mild whole body hyperthermia (42 degrees C, 10 min) in mice changes susceptibility of their bone marrow stem cells and thymocytes to subsequent gamma-irradiation. to assess the expectable contribution of stress-inducible Hsp we used injections with Quercetin, a flavonoid inhibiting the stress-responsive Hsp induction. The results demonstrate that the bone marrow stem cells and thymocytes from heat-preconditioned mice were more radioresistant than those from the non-preconditioned animals. the radioprotection was well manifested if mice or their isolated thymocytes were irradiated 18-25 h after the in vivo hyperthermia. This delayed radioprotection resulting from the heat preconditioning was suppressed in Quercetin-injected mice. The revealed correlation between the intracellular Hsp accumulation and the acquired Quercetin-sensitive radioprotection suggests a beneficial role of Hsps as of endogenous radioprotectors. Our finding discovers new ways for artificial modulation of effects of irradiation on target cells via manipulating the Hsp expression. (Author) 17 refs

  17. No Effect of the Transforming Growth Factor {beta}1 Promoter Polymorphism C-509T on TGFB1 Gene Expression, Protein Secretion, or Cellular Radiosensitivity

    Energy Technology Data Exchange (ETDEWEB)

    Reuther, Sebastian; Metzke, Elisabeth [Laboratory of Radiobiology and Experimental Radiooncology, University Hospital Hamburg-Eppendorf, Hamburg (Germany); Bonin, Michael [Department of Medical Genetics, University of Tuebingen (Germany); Petersen, Cordula [Clinic of Radiotherapy and Radiooncology, University Hospital Hamburg-Eppendorf, Hamburg (Germany); Dikomey, Ekkehard, E-mail: dikomey@uke.de [Laboratory of Radiobiology and Experimental Radiooncology, University Hospital Hamburg-Eppendorf, Hamburg (Germany); Raabe, Annette [Laboratory of Radiobiology and Experimental Radiooncology, University Hospital Hamburg-Eppendorf, Hamburg (Germany)

    2013-02-01

    Purpose: To study whether the promoter polymorphism (C-509T) affects transforming growth factor {beta}1 gene (TGFB1) expression, protein secretion, and/or cellular radiosensitivity for both human lymphocytes and fibroblasts. Methods and Materials: Experiments were performed with lymphocytes taken either from 124 breast cancer patients or 59 pairs of normal monozygotic twins. We used 15 normal human primary fibroblast strains as controls. The C-509T genotype was determined by polymerase chain reaction-restriction fragment length polymorphism or TaqMan single nucleotide polymorphism (SNP) genotyping assay. The cellular radiosensitivity of lymphocytes was measured by G0/1 assay and that of fibroblasts by colony assay. The amount of extracellular TGFB1 protein was determined by enzyme-linked immunosorbent assay, and TGFB1 expression was assessed via microarray analysis or reverse transcription-polymerase chain reaction. Results: The C-509T genotype was found not to be associated with cellular radiosensitivity, neither for lymphocytes (breast cancer patients, P=.811; healthy donors, P=.181) nor for fibroblasts (P=.589). Both TGFB1 expression and TGFB1 protein secretion showed considerable variation, which, however, did not depend on the C-509T genotype (protein secretion: P=.879; gene expression: lymphocytes, P=.134, fibroblasts, P=.605). There was also no general correlation between TGFB1 expression and cellular radiosensitivity (lymphocytes, P=.632; fibroblasts, P=.573). Conclusion: Our data indicate that any association between the SNP C-509T of TGFB1 and risk of normal tissue toxicity cannot be ascribed to a functional consequence of this SNP, either on the level of gene expression, protein secretion, or cellular radiosensitivity.

  18. The requirement of matrix ATP for the import of precursor proteins into the mitochondrial matrix and intermembrane space

    NARCIS (Netherlands)

    Stuart, Rosemary A.; Gruhler, Albrecht; Klei, Ida van der; Guiard, Bernard; Koll, Hans; Neupert, Walter

    1994-01-01

    The role of ATP in the matrix for the import of precursor proteins into the various mitochondrial subcompartments was investigated by studying protein translocation at experimentally defined ATP levels. Proteins targeted to the matrix were neither imported or processed when matrix ATP was depleted.

  19. Np9, a cellular protein of retroviral ancestry restricted to human, chimpanzee and gorilla, binds and regulates ubiquitin ligase MDM2

    Science.gov (United States)

    Heyne, Kristina; Kölsch, Kathrin; Bruand, Marine; Kremmer, Elisabeth; Grässer, Friedrich A; Mayer, Jens; Roemer, Klaus

    2015-01-01

    Humans and primates are long-lived animals with long reproductive phases. One factor that appears to contribute to longevity and fertility in humans, as well as to cancer-free survival, is the transcription factor and tumor suppressor p53, controlled by its main negative regulator MDM2. However, p53 and MDM2 homologs are found throughout the metazoan kingdom from Trichoplacidae to Hominidae. Therefore the question arises, if p53/MDM2 contributes to the shaping of primate features, then through which mechanisms. Previous findings have indicated that the appearances of novel p53-regulated genes and wild-type p53 variants during primate evolution are important in this context. Here, we report on another mechanism of potential relevance. Human endogenous retrovirus K subgroup HML-2 (HERV-K(HML-2)) type 1 proviral sequences were formed in the genomes of the predecessors of contemporary Hominoidea and can be identified in the genomes of Nomascus leucogenys (gibbon) up to Homo sapiens. We previously reported on an alternative splicing event in HERV-K(HML-2) type 1 proviruses that can give rise to nuclear protein of 9 kDa (Np9). We document here the evolution of Np9-coding capacity in human, chimpanzee and gorilla, and show that the C-terminal half of Np9 binds directly to MDM2, through a domain of MDM2 that is known to be contacted by various cellular proteins in response to stress. Np9 can inhibit the MDM2 ubiquitin ligase activity toward p53 in the cell nucleus, and can support the transactivation of genes by p53. Our findings point to the possibility that endogenous retrovirus protein Np9 contributes to the regulation of the p53-MDM2 pathway specifically in humans, chimpanzees and gorillas. PMID:26103464

  20. A peptide export-import control circuit modulating bacterial development regulates protein phosphatases of the phosphorelay.

    Science.gov (United States)

    Perego, M

    1997-08-05

    The phosphorelay signal transduction system activates developmental transcription in sporulation of Bacillus subtilis by phosphorylation of aspartyl residues of the Spo0F and Spo0A response regulators. The phosphorylation level of these response regulators is determined by the opposing activities of protein kinases and protein aspartate phosphatases that interpret positive and negative signals for development in a signal integration circuit. The RapA protein aspartate phosphatase of the phosphorelay is regulated by a peptide that directly inhibits its activity. This peptide is proteolytically processed from an inactive pre-inhibitor protein encoded in the phrA gene. The pre-inhibitor is cleaved by the protein export apparatus to a putative pro-inhibitor that is further processed to the active inhibitor peptide and internalized by the oligopeptide permease. This export-import circuit is postulated to be a mechanism for timing phosphatase activity where the processing enzymes regulate the rate of formation of the active inhibitor. The processing events may, in turn, be controlled by a regulatory hierarchy. Chromosome sequencing has revealed several other phosphatase-prepeptide gene pairs in B. subtilis, suggesting that the use of this mechanism may be widespread in signal transduction.

  1. Proteins Play Important Role in Intercellular Adhesion Affecting on Fruit Textural Quality

    DEFF Research Database (Denmark)

    Bahadur Adhikari, Khem; Shomer, Ilan

    2012-01-01

    Fruit textural quality is becoming a major quality parameter for export, postharvest preservation, handling and processing. The main determinant of textural quality is intercellular adhesion (ICA) as attributed by the cell wall (CW) and its components. The importance of CW protein in ICA strength......Fruit textural quality is becoming a major quality parameter for export, postharvest preservation, handling and processing. The main determinant of textural quality is intercellular adhesion (ICA) as attributed by the cell wall (CW) and its components. The importance of CW protein in ICA...... strengthening was exempli ed in Medjoul date (Phoenix dactylifera L.) fruit, as a model. Fruit mesocarp sensitively responded to culture environment which was assayed in vitro at pH 3.5( pKa) in presence of organic acid molecules. The max penetration force, as a measure of ICA strength, of p...

  2. Molecular characterization and expression analysis of chloroplast protein import components in tomato (Solanum lycopersicum.

    Directory of Open Access Journals (Sweden)

    Jianmin Yan

    Full Text Available The translocon at the outer envelope membrane of chloroplasts (Toc mediates the recognition and initial import into the organelle of thousands of nucleus-encoded proteins. These proteins are translated in the cytosol as precursor proteins with cleavable amino-terminal targeting sequences called transit peptides. The majority of the known Toc components that mediate chloroplast protein import were originally identified in pea, and more recently have been studied most extensively in Arabidopsis. With the completion of the tomato genome sequencing project, it is now possible to identify putative homologues of the chloroplast import components in tomato. In the work reported here, the Toc GTPase cDNAs from tomato were identified, cloned and analyzed. The analysis revealed that there are four Toc159 homologues (slToc159-1, -2, -3 and -4 and two Toc34 homologues (slToc34-1 and -2 in tomato, and it was shown that tomato Toc159 and Toc34 homologues share high sequence similarity with the comparable import apparatus components from Arabidopsis and pea. Thus, tomato is a valid model for further study of this system. The expression level of Toc complex components was also investigated in different tissues during tomato development. The two tomato Toc34 homologues are expressed at higher levels in non-photosynthetic tissues, whereas, the expression of two tomato Toc159 homologues, slToc159-1 and slToc159-4, were higher in photosynthetic tissues, and the expression patterns of slToc159-2 was not significantly different in photosynthetic and non-photosynthetic tissues, and slToc159-3 expression was limited to a few select tissues.

  3. Cellular repressor of E1A-stimulated genes is a bona fide lysosomal protein which undergoes proteolytic maturation during its biosynthesis

    International Nuclear Information System (INIS)

    Schaehs, Philipp; Weidinger, Petra; Probst, Olivia C.; Svoboda, Barbara; Stadlmann, Johannes; Beug, Hartmut; Waerner, Thomas; Mach, Lukas

    2008-01-01

    Cellular repressor of E1A-stimulated genes (CREG) has been reported to be a secretory glycoprotein implicated in cellular growth and differentiation. We now show that CREG is predominantly localized within intracellular compartments. Intracellular CREG was found to lack an N-terminal peptide present in the secreted form of the protein. In contrast to normal cells, CREG is largely secreted by fibroblasts missing both mannose 6-phosphate receptors. This is not observed in cells lacking only one of them. Mass spectrometric analysis of recombinant CREG revealed that the protein contains phosphorylated oligosaccharides at either of its two N-glycosylation sites. Cellular CREG was found to cosediment with lysosomal markers upon subcellular fractionation by density-gradient centrifugation. In fibroblasts expressing a CREG-GFP fusion construct, the heterologous protein was detected in compartments containing lysosomal proteins. Immunolocalization of endogenous CREG confirmed that intracellular CREG is localized in lysosomes. Proteolytic processing of intracellular CREG involves the action of lysosomal cysteine proteinases. These results establish that CREG is a lysosomal protein that undergoes proteolytic maturation in the course of its biosynthesis, carries the mannose 6-phosphate recognition marker and depends on the interaction with mannose 6-phosphate receptors for efficient delivery to lysosomes

  4. Iron-Restricted Diet Affects Brain Ferritin Levels, Dopamine Metabolism and Cellular Prion Protein in a Region-Specific Manner

    Directory of Open Access Journals (Sweden)

    Jessica M. V. Pino

    2017-05-01

    Full Text Available Iron is an essential micronutrient for several physiological functions, including the regulation of dopaminergic neurotransmission. On the other hand, both iron, and dopamine can affect the folding and aggregation of proteins related with neurodegenerative diseases, such as cellular prion protein (PrPC and α-synuclein, suggesting that deregulation of iron homeostasis and the consequential disturbance of dopamine metabolism can be a risk factor for conformational diseases. These proteins, in turn, are known to participate in the regulation of iron and dopamine metabolism. In this study, we evaluated the effects of dietary iron restriction on brain ferritin levels, dopamine metabolism, and the expression levels of PrPC and α-synuclein. To achieve this goal, C57BL/6 mice were fed with iron restricted diet (IR or with normal diet (CTL for 1 month. IR reduced iron and ferritin levels in liver. Ferritin reduction was also observed in the hippocampus. However, in the striatum of IR group, ferritin level was increased, suggesting that under iron-deficient condition, each brain area might acquire distinct capacity to store iron. Increased lipid peroxidation was observed only in hippocampus of IR group, where ferritin level was reduced. IR also generated discrete results regarding dopamine metabolism of distinct brain regions: in striatum, the level of dopamine metabolites (DOPAC and HVA was reduced; in prefrontal cortex, only HVA was increased along with the enhanced MAO-A activity; in hippocampus, no alterations were observed. PrPC levels were increased only in the striatum of IR group, where ferritin level was also increased. PrPC is known to play roles in iron uptake. Thus, the increase of PrPC in striatum of IR group might be related to the increased ferritin level. α-synuclein was not altered in any regions. Abnormal accumulation of ferritin, increased MAO-A activity or lipid peroxidation are molecular features observed in several neurological

  5. The cellular prion protein interacts with the tissue non-specific alkaline phosphatase in membrane microdomains of bioaminergic neuronal cells.

    Directory of Open Access Journals (Sweden)

    Myriam Ermonval

    Full Text Available BACKGROUND: The cellular prion protein, PrP(C, is GPI anchored and abundant in lipid rafts. The absolute requirement of PrP(C in neurodegeneration associated to prion diseases is well established. However, the function of this ubiquitous protein is still puzzling. Our previous work using the 1C11 neuronal model, provided evidence that PrP(C acts as a cell surface receptor. Besides a ubiquitous signaling function of PrP(C, we have described a neuronal specificity pointing to a role of PrP(C in neuronal homeostasis. 1C11 cells, upon appropriate induction, engage into neuronal differentiation programs, giving rise either to serotonergic (1C11(5-HT or noradrenergic (1C11(NE derivatives. METHODOLOGY/PRINCIPAL FINDINGS: The neuronal specificity of PrP(C signaling prompted us to search for PrP(C partners in 1C11-derived bioaminergic neuronal cells. We show here by immunoprecipitation an association of PrP(C with an 80 kDa protein identified by mass spectrometry as the tissue non-specific alkaline phosphatase (TNAP. This interaction occurs in lipid rafts and is restricted to 1C11-derived neuronal progenies. Our data indicate that TNAP is implemented during the differentiation programs of 1C11(5-HT and 1C11(NE cells and is active at their cell surface. Noteworthy, TNAP may contribute to the regulation of serotonin or catecholamine synthesis in 1C11(5-HT and 1C11(NE bioaminergic cells by controlling pyridoxal phosphate levels. Finally, TNAP activity is shown to modulate the phosphorylation status of laminin and thereby its interaction with PrP. CONCLUSION/SIGNIFICANCE: The identification of a novel PrP(C partner in lipid rafts of neuronal cells favors the idea of a role of PrP in multiple functions. Because PrP(C and laminin functionally interact to support neuronal differentiation and memory consolidation, our findings introduce TNAP as a functional protagonist in the PrP(C-laminin interplay. The partnership between TNAP and PrP(C in neuronal cells may

  6. Changes in Cellular mRNA Stability, Splicing, and Polyadenylation through HuR Protein Sequestration by a Cytoplasmic RNA Virus

    Directory of Open Access Journals (Sweden)

    Michael D. Barnhart

    2013-11-01

    Full Text Available The impact of RNA viruses on the posttranscriptional regulation of cellular gene expression is unclear. Sindbis virus causes a dramatic relocalization of the cellular HuR protein from the nucleus to the cytoplasm in infected cells. This is to the result of the expression of large amounts of viral RNAs that contain high-affinity HuR binding sites in their 3′ UTRs effectively serving as a sponge for the HuR protein. Sequestration of HuR by Sindbis virus is associated with destabilization of cellular mRNAs that normally bind HuR and rely on it to regulate their expression. Furthermore, significant changes can be observed in nuclear alternative polyadenylation and splicing events on cellular pre-mRNAs as a result of sequestration of HuR protein by the 3′ UTR of transcripts of this cytoplasmic RNA virus. These studies suggest a molecular mechanism of virus-host interaction that probably has a significant impact on virus replication, cytopathology, and pathogenesis.

  7. Luminescent cyclometalated iridium(III) polypyridine indole complexes--synthesis, photophysics, electrochemistry, protein-binding properties, cytotoxicity, and cellular uptake.

    Science.gov (United States)

    Lau, Jason Shing-Yip; Lee, Pui-Kei; Tsang, Keith Hing-Kit; Ng, Cyrus Ho-Cheong; Lam, Yun-Wah; Cheng, Shuk-Han; Lo, Kenneth Kam-Wing

    2009-01-19

    A series of luminescent cyclometalated iridium(III) polypyridine indole complexes, [Ir(N--C)(2)(N--N)](PF(6)) (HN--C = 2-phenylpyridine (Hppy), N--N = 4-((2-(indol-3-yl)ethyl)aminocarbonyl)-4'-methyl-2,2'-bipyridine (bpy-ind) (1a), N--N = 4-((5-((2-(indol-3-yl)ethyl)aminocarbonyl)pentyl)aminocarbonyl)-4'-methyl-2,2'-bipyridine (bpy-C6-ind) (1b); HN--C = 7,8-benzoquinoline (Hbzq), N--N = bpy-ind (2a), N--N = bpy-C6-ind (2b); and HN--C = 2-phenylquinoline (Hpq), N--N = bpy-ind (3a), N--N = bpy-C6-ind (3b)), have been synthesized, characterized, and their photophysical and electrochemical properties and lipophilicity investigated. Photoexcitation of the complexes in fluid solutions at 298 K and in alcohol glass at 77 K resulted in intense and long-lived luminescence (lambda(em) = 540-616 nm, tau(o) = 0.13-5.15 mus). The emission of the complexes has been assigned to a triplet metal-to-ligand charge-transfer ((3)MLCT) (dpi(Ir) --> pi*(N--N)) excited state, probably with some mixing of triplet intraligand ((3)IL) (pi --> pi*) (pq) character for complexes 3a,b. Electrochemical measurements revealed that all the complexes showed an irreversible indole oxidation wave at ca. +1.1 V versus SCE, a quasi-reversible iridium(IV/III) couple at ca. +1.3 V, and a reversible diimine reduction couple at ca. -1.3 V. The interactions of these complexes with an indole-binding protein, bovine serum albumin (BSA), have been studied by emission titrations, and the K(a) values are on the order of 10(4) M(-1). Additionally, the cytotoxicity of the complexes toward human cervix epithelioid carcinoma (HeLa) cells has been examined by the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide (MTT) assay. The IC(50) values of the complexes ranged from 1.1 to 6.3 microM, which are significantly smaller than that of cisplatin (30.7 microM) under the same experimental conditions. Furthermore, the cellular uptake of the complexes has been investigated by flow cytometry and laser

  8. Spindle assembly checkpoint protein expression correlates with cellular proliferation and shorter time to recurrence in ovarian cancer.

    LENUS (Irish Health Repository)

    McGrogan, Barbara

    2014-07-01

    Ovarian carcinoma (OC) is the most lethal of the gynecological malignancies, often presenting at an advanced stage. Treatment is hampered by high levels of drug resistance. The taxanes are microtubule stabilizing agents, used as first-line agents in the treatment of OC that exert their apoptotic effects through the spindle assembly checkpoint. BUB1-related protein kinase (BUBR1) and mitotic arrest deficient 2 (MAD2), essential spindle assembly checkpoint components, play a key role in response to taxanes. BUBR1, MAD2, and Ki-67 were assessed on an OC tissue microarray platform representing 72 OC tumors of varying histologic subtypes. Sixty-one of these patients received paclitaxel and platinum agents combined; 11 received platinum alone. Overall survival was available for all 72 patients, whereas recurrence-free survival (RFS) was available for 66 patients. Increased BUBR1 expression was seen in serous carcinomas, compared with other histologies (P = .03). Increased BUBR1 was significantly associated with tumors of advanced stage (P = .05). Increased MAD2 and BUBR1 expression also correlated with increased cellular proliferation (P < .0002 and P = .02, respectively). Reduced MAD2 nuclear intensity was associated with a shorter RFS (P = .03), in ovarian tumors of differing histologic subtype (n = 66). In this subgroup, for those women who received paclitaxel and platinum agents combined (n = 57), reduced MAD2 intensity also identified women with a shorter RFS (P < .007). For the entire cohort of patients, irrespective of histologic subtype or treatment, MAD2 nuclear intensity retained independent significance in a multivariate model, with tumors showing reduced nuclear MAD2 intensity identifying patients with a poorer RFS (P = .05).

  9. Loss of cellular FLICE-inhibitory protein promotes acute cholestatic liver injury and inflammation from bile duct ligation.

    Science.gov (United States)

    Gehrke, Nadine; Nagel, Michael; Straub, Beate K; Wörns, Marcus A; Schuchmann, Marcus; Galle, Peter R; Schattenberg, Jörn M

    2018-03-01

    Cholestatic liver injury results from impaired bile flow or metabolism and promotes hepatic inflammation and fibrogenesis. Toxic bile acids that accumulate in cholestasis induce apoptosis and contribute to early cholestatic liver injury, which is amplified by accompanying inflammation. The aim of the current study was to evaluate the role of the antiapoptotic caspase 8-homolog cellular FLICE-inhibitory (cFLIP) protein during acute cholestatic liver injury. Transgenic mice exhibiting hepatocyte-specific deletion of cFLIP (cFLIP -/- ) were used for in vivo and in vitro analysis of cholestatic liver injury using bile duct ligation (BDL) and the addition of bile acids ex vivo. Loss of cFLIP in hepatocytes promoted acute cholestatic liver injury early after BDL, which was characterized by a rapid release of proinflammatory and chemotactic cytokines (TNF, IL-6, IL-1β, CCL2, CXCL1, and CXCL2), an increased presence of CD68 + macrophages and an influx of neutrophils in the liver, and resulting apoptotic and necrotic hepatocyte cell death. Mechanistically, liver injury in cFLIP -/- mice was aggravated by reactive oxygen species, and sustained activation of the JNK signaling pathway. In parallel, cytoprotective NF-κB p65, A20, and the MAPK p38 were inhibited. Increased injury in cFLIP -/- mice was accompanied by activation of hepatic stellate cells and profibrogenic regulators. The antagonistic caspase 8-homolog cFLIP is a critical regulator of acute, cholestatic liver injury. NEW & NOTEWORTHY The current paper explores the role of a classical modulator of hepatocellular apoptosis in early, cholestatic liver injury. These include activation of NF-κB and MAPK signaling, production of inflammatory cytokines, and recruitment of neutrophils in response to cholestasis. Because these signaling pathways are currently exploited in clinical trials for the treatment of nonalcoholic steatohepatitis and cirrhosis, the current data will help in the development of novel pharmacological

  10. Presence of viral RNA and proteins in exosomes from the cellular clones resistant to Rift Valley Fever Virus infection.

    Directory of Open Access Journals (Sweden)

    Noor eAhsan

    2016-02-01

    Full Text Available Rift Valley Fever Virus (RVFV is a RNA virus that belongs to the genus Phlebovirus, family Bunyaviridae. It infects humans and livestock and causes Rift Valley fever. RVFV is considered an agricultural pathogen by the USDA, as it can cause up to 100% abortion in cattle and extensive death of newborns. In addition, it is designated as Category A pathogen by the CDC and the NIAID. In some human cases of RVFV infection, the virus causes fever, ocular damage, liver damage, hemorrhagic fever, and death. There are currently limited options for vaccine candidates, which include the MP-12 and clone 13 versions of RVFV. Viral infections often deregulate multiple cellular pathways that contribute to replication and host pathology. We have previously shown that latent HIV-1 and HTLV-1 infected cells secrete exosomes that contain short viral RNAs, limited number of genomic RNAs, and viral proteins. These exosomes largely target neighboring cells and activate the NF-кB pathway, leading to cell proliferation and overall better viral replication. In this manuscript, we studied the effects of exosome formation from RVFV infected cells and their function on recipient cells. We initially infected cells, isolated resistant clones, and further purified using dilution cloning. We then characterized these cells as resistant to new RVFV infection, but sensitive to other viral infections, including Venezuelan Equine Encephalitis Virus (VEEV. These clones contained normal markers (i.e. CD63 for exosomes and were able to activate the TLR pathway in recipient reporter cells. Interestingly, the exosome rich preparations, much like their host cell, contained viral RNA (L, M, and S genome. The RNAs were detected using qRT-PCR in both parental and exosomal preparations as well as in CD63 immunoprecipitates. Viral proteins such as N and a modified form of NSs were present in some of these exosomes. Finally, treatment of recipient cells (T- cells and monocytic cells showed

  11. Critical Importance of Protein 4.1 in Centrosome and Mitiotic Spindle Aberrations in Breast Cancer Pathogenesis

    National Research Council Canada - National Science Library

    Krauss, Sharon W

    2005-01-01

    Important pathological hallmarks of many breast cancers include centrosome amplification, spindle pole defects leading to aberrant chromosome segregation, altered nucleoskeletal proteins and perturbed cytokinesis...

  12. Disease-associated mutations disrupt functionally important regions of intrinsic protein disorder.

    Directory of Open Access Journals (Sweden)

    Vladimir Vacic

    Full Text Available The effects of disease mutations on protein structure and function have been extensively investigated, and many predictors of the functional impact of single amino acid substitutions are publicly available. The majority of these predictors are based on protein structure and evolutionary conservation, following the assumption that disease mutations predominantly affect folded and conserved protein regions. However, the prevalence of the intrinsically disordered proteins (IDPs and regions (IDRs in the human proteome together with their lack of fixed structure and low sequence conservation raise a question about the impact of disease mutations in IDRs. Here, we investigate annotated missense disease mutations and show that 21.7% of them are located within such intrinsically disordered regions. We further demonstrate that 20% of disease mutations in IDRs cause local disorder-to-order transitions, which represents a 1.7-2.7 fold increase compared to annotated polymorphisms and neutral evolutionary substitutions, respectively. Secondary structure predictions show elevated rates of transition from helices and strands into loops and vice versa in the disease mutations dataset. Disease disorder-to-order mutations also influence predicted molecular recognition features (MoRFs more often than the control mutations. The repertoire of disorder-to-order transition mutations is limited, with five most frequent mutations (R→W, R→C, E→K, R→H, R→Q collectively accounting for 44% of all deleterious disorder-to-order transitions. As a proof of concept, we performed accelerated molecular dynamics simulations on a deleterious disorder-to-order transition mutation of tumor protein p63 and, in agreement with our predictions, observed an increased α-helical propensity of the region harboring the mutation. Our findings highlight the importance of mutations in IDRs and refine the traditional structure-centric view of disease mutations. The results of this study

  13. Dual inhibition of γ-oryzanol on cellular melanogenesis: inhibition of tyrosinase activity and reduction of melanogenic gene expression by a protein kinase A-dependent mechanism.

    Science.gov (United States)

    Jun, Hee-jin; Lee, Ji Hae; Cho, Bo-Ram; Seo, Woo-Duck; Kang, Hang-Won; Kim, Dong-Woo; Cho, Kang-Jin; Lee, Sung-Joon

    2012-10-26

    The in vitro effects on melanogenesis of γ-oryzanol (1), a rice bran-derived phytosterol, were investigated. The melanin content in B16F1 cells was significantly and dose-dependently reduced (-13% and -28% at 3 and 30 μM, respectively). Tyrosinase enzyme activity was inhibited by 1 both in a cell-free assay and when analyzed based on the measurement of cellular tyrosinase activity. Transcriptome analysis was performed to investigate the biological pathways altered by 1, and it was found that gene expression involving protein kinase A (PKA) signaling was markedly altered. Subsequent analyses revealed that 1 stimulation in B16 cells reduced cytosolic cAMP concentrations, PKA activity (-13% for cAMP levels and -40% for PKA activity), and phosphorylation of the cAMP-response element binding protein (-57%), which, in turn, downregulated the expression of microphthalmia-associated transcription factor (MITF; -59% for mRNA and -64% for protein), a key melanogenic gene transcription factor. Accordingly, tyrosinase-related protein 1 (TRP-1; -69% for mRNA and -82% for protein) and dopachrome tautomerase (-51% for mRNA and -92% for protein) in 1-stimulated B16F1 cells were also downregulated. These results suggest that 1 has dual inhibitory activities for cellular melanogenesis by inhibiting tyrosinase enzyme activity and reducing MITF and target genes in the PKA-dependent pathway.

  14. Expression of cellular prion protein in the frontal and occipital lobe in Alzheimer's disease, diffuse Lewy body disease, and in normal brain: an immunohistochemical study.

    Science.gov (United States)

    Rezaie, Payam; Pontikis, Charlie C; Hudson, Lance; Cairns, Nigel J; Lantos, Peter L

    2005-08-01

    Cellular prion protein (PrP(c)) is a glycoprotein expressed at low to moderate levels within the nervous system. Recent studies suggest that PrP(c) may possess neuroprotective functions and that its expression is upregulated in certain neurodegenerative disorders. We investigated whether PrP(c) expression is altered in the frontal and occipital cortex in two well-characterized neurodegenerative disorders--Alzheimer's disease (AD) and diffuse Lewy body disease (DLBD)--compared with that in normal human brain using immunohistochemistry and computerized image analysis. The distribution of PrP(c) was further tested for correlation with glial reactivity. We found that PrP(c) was localized mainly in the gray matter (predominantly in neurons) and expressed at higher levels within the occipital cortex in the normal human brain. Image analysis revealed no significant variability in PrP(c) expression between DLBD and control cases. However, blood vessels within the white matter of DLBD cases showed immunoreactivity to PrP(c). By contrast, this protein was differentially expressed in the frontal and occipital cortex of AD cases; it was markedly overexpressed in the former and significantly reduced in the latter. Epitope specificity of antibodies appeared important when detecting PrP(c). The distribution of PrP(c) did not correlate with glial immunoreactivity. In conclusion, this study supports the proposal that regional changes in expression of PrP(c) may occur in certain neurodegenerative disorders such as AD, but not in other disorders such as DLBD.

  15. Immunization with Clinical HIV-1 Env Proteins Induces Broad Antibody Dependent Cellular Cytotoxicity-Mediating Antibodies in a Rabbit Vaccination Model.

    Science.gov (United States)

    Karlsson, Ingrid; Borggren, Marie; Jensen, Sanne Skov; Heyndrickx, Leo; Stewart-Jones, Guillaume; Scarlatti, Gabriella; Fomsgaard, Anders

    2017-11-17

    The induction of both neutralizing antibodies and non-neutralizing antibodies with effector functions, for example, antibody-dependent cellular cytotoxicity (ADCC), is desired in the search for effective vaccines against HIV-1. In the pursuit of novel immunogens capable of inducing an efficient antibody response, rabbits were immunized with selected antigens using different prime-boost strategies. We immunized 35 different groups of rabbits with Env antigens from clinical HIV-1 subtypes A and B, including immunization with DNA alone, protein alone, and DNA prime with protein boost. The rabbit sera were screened for ADCC activity using a GranToxiLux-based assay with human peripheral blood mononuclear cells as effector cells and CEM.NKR CCR5 cells coated with HIV-1 envelope as target cells. The groups with the highest ADCC activity were further characterized for cross-reactivity between HIV-1 subtypes. The immunogen inducing the most potent and broadest ADCC response was a trimeric gp140. The ADCC activity was highest against the HIV-1 subtype corresponding to the immunogen. The ADCC activity did not necessarily reflect neutralizing activity in the pseudovirus-TZMbl assay, but there was an overall correlation between the two antiviral activities. We present a rabbit vaccination model and an assay suitable for screening HIV-1 vaccine candidates for the induction of ADCC-mediating antibodies in addition to neutralizing antibodies. The antigens and/or immunization strategies capable of inducing antibodies with ADCC activity did not necessarily induce neutralizing activity and vice versa. Nevertheless, we identified vaccine candidates that were able to concurrently induce both types of responses and that had ADCC activity that was cross-reactive between different subtypes. When searching for an effective vaccine candidate, it is important to evaluate the antibody response using a model and an assay measuring the desired function.

  16. Colostrum and milk protein rankings and ratios of importance to neonatal calf health using a proteomics approach

    DEFF Research Database (Denmark)

    Nissen, Asger; Andersen, Pia Haubro; Bendixen, Emøke

    2017-01-01

    Administration of colostrum to the newborn calf before gut closure is pivotal to its health, because of the transfer of passive immunity. Traditionally, passive immunity has been attributed to the transfer of immunoglobulins although it is increasingly clear that multiple other factors contribute......, including innate immune proteins, developmental factors, immunomodulatory factors, and the presence of cellular immunity. The objective of this study was to produce a comprehensive comparison of the bovine colostrum proteome and the milk proteome by applying 2-dimensional liquid chromatography-tandem mass...... spectrometry. Further, the objectives were to rank proteins mutually and generate protein ratios from the spectral counts of the 2 proteomes and ELISA to gain insight into which proteins could be of most relevance to neonatal calf health. To obtain an in-depth picture of the bovine colostrum and milk proteome...

  17. Importance of a Conserved Lys/Arg Residue for Ligand/PDZ Domain Interactions as Examined by Protein Semisynthesis

    DEFF Research Database (Denmark)

    Pedersen, Søren W; Moran, Griffin E; Sereikaité, Vita

    2016-01-01

    PDZ domains are ubiquitous small protein domains that are mediators of numerous protein-protein interactions, and play a pivotal role in protein trafficking, synaptic transmission, and the assembly of signaling-transduction complexes. In recent years, PDZ domains have emerged as novel and exciting...... drug targets for diseases (in the brain in particular), so understanding the molecular details of PDZ domain interactions is of fundamental importance. PDZ domains bind to a protein partner at either a C-terminal peptide or internal peptide motifs. Here, we examined the importance of a conserved Lys...

  18. Nuclear magnetic resonance provides a quantitative description of protein conformational flexibility on physiologically important time scales.

    Science.gov (United States)

    Salmon, Loïc; Bouvignies, Guillaume; Markwick, Phineus; Blackledge, Martin

    2011-04-12

    A complete description of biomolecular activity requires an understanding of the nature and the role of protein conformational dynamics. In recent years, novel nuclear magnetic resonance-based techniques that provide hitherto inaccessible detail concerning biomolecular motions occurring on physiologically important time scales have emerged. Residual dipolar couplings (RDCs) provide precise information about time- and ensemble-averaged structural and dynamic processes with correlation times up to the millisecond and thereby encode key information for understanding biological activity. In this review, we present the application of two very different approaches to the quantitative description of protein motion using RDCs. The first is purely analytical, describing backbone dynamics in terms of diffusive motions of each peptide plane, using extensive statistical analysis to validate the proposed dynamic modes. The second is based on restraint-free accelerated molecular dynamics simulation, providing statistically sampled free energy-weighted ensembles that describe conformational fluctuations occurring on time scales from pico- to milliseconds, at atomic resolution. Remarkably, the results from these two approaches converge closely in terms of distribution and absolute amplitude of motions, suggesting that this kind of combination of analytical and numerical models is now capable of providing a unified description of protein conformational dynamics in solution.

  19. Proteomic amino-termini profiling reveals targeting information for protein import into complex plastids.

    Directory of Open Access Journals (Sweden)

    Pitter F Huesgen

    Full Text Available In organisms with complex plastids acquired by secondary endosymbiosis from a photosynthetic eukaryote, the majority of plastid proteins are nuclear-encoded, translated on cytoplasmic ribosomes, and guided across four membranes by a bipartite targeting sequence. In-depth understanding of this vital import process has been impeded by a lack of information about the transit peptide part of this sequence, which mediates transport across the inner three membranes. We determined the mature N-termini of hundreds of proteins from the model diatom Thalassiosira pseudonana, revealing extensive N-terminal modification by acetylation and proteolytic processing in both cytosol and plastid. We identified 63 mature N-termini of nucleus-encoded plastid proteins, deduced their complete transit peptide sequences, determined a consensus motif for their cleavage by the stromal processing peptidase, and found evidence for subsequent processing by a plastid methionine aminopeptidase. The cleavage motif differs from that of higher plants, but is shared with other eukaryotes with complex plastids.

  20. Roles of viral and cellular proteins in the expression of alternatively spliced HTLV-1 pX mRNAs

    International Nuclear Information System (INIS)

    Princler, Gerald L.; Julias, John G.; Hughes, Stephen H.; Derse, David

    2003-01-01

    The human T cell leukemia virus type 1 (HTLV-1) genome contains a cluster of at least five open reading frames (ORFs) near the 3' terminus within the pX region. The pX ORFs are encoded by mono- or bicistronic mRNAs that are generated by alternative splicing. The various pX mRNAs result from skipping of the internal exon (2-exon versus 3-exon isofoms) or from the utilization of alternative splice acceptor sites in the terminal exon. The Rex and Tax proteins, encoded by ORFs X-III and X-IV, have been studied intensively and are encoded by the most abundant of the alternative 3-exon mRNAs. The protein products of the other pX ORFs have not been detected in HTLV-1-infected cell lines and the levels of the corresponding mRNAs have not been accurately established. We have used real-time RT-PCR with splice-site specific primers to accurately measure the levels of individual pX mRNA species in chronically infected T cell lines. We have asked whether virus regulatory proteins or ectopic expression of cellular factors influence pX mRNA splicing in cells that were transfected with HTLV-1 provirus clones. In chronically infected cell lines, the pX-tax/rex mRNA was present at 500- to 2500-fold higher levels than the pX-tax-orfII mRNA and at approximately 1000-fold higher levels than pX-rex-orfI mRNA. Chronically infected cell lines that contain numerous defective proviruses expressed 2-exon forms of pX mRNAs at significantly higher levels compared to cell lines that contain a single full-length provirus. Cells transfected with provirus expression plasmids expressed similar relative amounts of 3-exon pX mRNAs but lower levels of 2-exon mRNA forms compared to cells containing a single, full-length provirus. The pX mRNA expression patterns were nearly identical in cells transfected with wild-type, Tax-minus, or Rex-minus proviruses. Cotransfection of cells with HTLV-1 provirus in combination with SF2/ASF expression plasmid resulted in a relative increase in pX-tax/rex m

  1. Western blot data using two distinct anti-O-GlcNAc monoclonal antibodies showing unique glycosylation status on cellular proteins under 2-deoxy-d-glucose treatment

    Directory of Open Access Journals (Sweden)

    Tetsuya Okuda

    2017-02-01

    Full Text Available Protein modification by O-linked N-acetylglucosamine (O-GlcNAcylation is one of the post transcriptional modifications occurring on cellular proteins. This paper provides a data set relating to the O-GlcNAcylation of cellular proteins detected by RL2 and CTD110.6 antibodies, which are commonly used for detection of protein O-GlcNAcylation, in 2-deoxy-d-glucose (2DG-treated human teratocarcinoma NCCIT cells in support of the research article entitled “A novel, promoter-based, target-specific assay identifies 2-deoxy-d-glucose as an inhibitor of globotriaosylceramide biosynthesis” (Okuda et al., 2009 [1]. The main article described a suppressive effect of 2DG on an Sp1 target gene in NCCIT cells and discussed the relationship between the effect of 2DG and O-GlcNAcylation status of Sp1. The data in this paper complements this relationship by Western blotting and clearly showed that the 2DG treatment increased O-GlcNAcylation of cellular proteins in NCCIT cells, whereas the RL2 and CTD110.6 epitopes were detected in a different manner. The RL2 epitope was detected on Sp1 during 2DG treatment, and the level was transiently increased at 24 h. In contrast, the CTD110.6 epitope became detectable on Sp1 over 72 h after 2DG treatment, and then the other proteins containing CTD110.6 epitopes also appeared in the cell lysates and the anti-Sp1 antibody precipitates.

  2. Border control: selectivity of chloroplast protein import and regulation at the TOC-complex.

    Science.gov (United States)

    Demarsy, Emilie; Lakshmanan, Ashok M; Kessler, Felix

    2014-01-01

    Plants have evolved complex and sophisticated molecular mechanisms to regulate their development and adapt to their surrounding environment. Particularly the development of their specific organelles, chloroplasts and other plastid-types, is finely tuned in accordance with the metabolic needs of the cell. The normal development and functioning of plastids require import of particular subsets of nuclear encoded proteins. Most preproteins contain a cleavable sequence at their N terminal (transit peptide) serving as a signal for targeting to the organelle and recognition by the translocation machinery TOC-TIC (translocon of outer membrane complex-translocon of inner membrane complex) spanning the dual membrane envelope. The plastid proteome needs constant remodeling in response to developmental and environmental factors. Therefore selective regulation of preprotein import plays a crucial role in plant development. In this review we describe the diversity of transit peptides and TOC receptor complexes, and summarize the current knowledge and potential directions for future research concerning regulation of the different Toc isoforms.

  3. Analysis of A549 cell proteome alteration in response to recombinant influenza A virus nucleoprotein and its interaction with cellular proteins, a preliminary study.

    Science.gov (United States)

    Kumar, D; Tiwari, K; Rajala, M S

    Influenza A virus undergoes frequent changes of antigenicity and contributes to seasonal epidemics or unpredictable pandemics. Nucleoprotein, encoded by gene segment 5, is an internal protein of the virus and is conserved among strains of different host origins. In the current study, we analyzed the differentially expressed proteins in A549 cells transiently transfected with the recombinant nucleoprotein of influenza A virus by 2D gel electrophoresis. The resolved protein spots on gel were identified by MALDI-TOF/Mass spectrometry analysis. The majority of the host proteins detected to be differentially abundant in recombinant nucleoprotein-expressing cells as compared to vector-transfected cells are the proteins of metabolic pathways, glycolytic enzymes, molecular chaperones and cytoskeletal proteins. We further demonstrated the interaction of virus nucleoprotein with some of the identified host cellular proteins. In vitro binding assay carried out using the purified recombinant nucleoprotein (pET29a+NP-His) and A549 cell lysate confirmed the interaction between nucleoprotein and host proteins, such as alpha enolase 1, pyruvate kinase and β-actin. The preliminary data of our study provides the information on virus nucleoprotein interaction with proteins involved in glycolysis. However, studies are ongoing to understand the significance of these interactions in modulating the host factors during virus replication.

  4. Processing of the glycosomal matrix-protein import receptor PEX5 of Trypanosoma brucei

    International Nuclear Information System (INIS)

    Gualdrón-López, Melisa; Michels, Paul A.M.

    2013-01-01

    Highlights: ► Most eukaryotic cells have a single gene for the peroxin PEX5. ► PEX5 is sensitive to in vitro proteolysis in distantly related organisms. ► TbPEX5 undergoes N-terminal truncation in vitro and possibly in vivo. ► Truncated TbPEX5 is still capable of binding PTS1-containing proteins. ► PEX5 truncation is physiologically relevant or an evolutionary conserved artifact. -- Abstract: Glycolysis in kinetoplastid protists such as Trypanosoma brucei is compartmentalized in peroxisome-like organelles called glycosomes. Glycosomal matrix-protein import involves a cytosolic receptor, PEX5, which recognizes the peroxisomal-targeting signal type 1 (PTS1) present at the C-terminus of the majority of matrix proteins. PEX5 appears generally susceptible to in vitro proteolytic processing. On western blots of T. brucei, two PEX5 forms are detected with apparent M r of 100 kDa and 72 kDa. 5′-RACE-PCR showed that TbPEX5 is encoded by a unique transcript that can be translated into a protein of maximally 72 kDa. However, recombinant PEX5 migrates aberrantly in SDS–PAGE with an apparent M r of 100 kDa, similarly as observed for the native peroxin. In vitro protease susceptibility analysis of native and 35 S-labelled PEX5 showed truncation of the 100 kDa form at the N-terminal side by unknown parasite proteases, giving rise to the 72 kDa form which remains functional for PTS1 binding. The relevance of these observations is discussed

  5. Processing of the glycosomal matrix-protein import receptor PEX5 of Trypanosoma brucei

    Energy Technology Data Exchange (ETDEWEB)

    Gualdrón-López, Melisa [Research Unit for Tropical Diseases, de Duve Institute, Université catholique de Louvain, Brussels (Belgium); Michels, Paul A.M., E-mail: paul.michels@uclouvain.be [Research Unit for Tropical Diseases, de Duve Institute, Université catholique de Louvain, Brussels (Belgium)

    2013-02-01

    Highlights: ► Most eukaryotic cells have a single gene for the peroxin PEX5. ► PEX5 is sensitive to in vitro proteolysis in distantly related organisms. ► TbPEX5 undergoes N-terminal truncation in vitro and possibly in vivo. ► Truncated TbPEX5 is still capable of binding PTS1-containing proteins. ► PEX5 truncation is physiologically relevant or an evolutionary conserved artifact. -- Abstract: Glycolysis in kinetoplastid protists such as Trypanosoma brucei is compartmentalized in peroxisome-like organelles called glycosomes. Glycosomal matrix-protein import involves a cytosolic receptor, PEX5, which recognizes the peroxisomal-targeting signal type 1 (PTS1) present at the C-terminus of the majority of matrix proteins. PEX5 appears generally susceptible to in vitro proteolytic processing. On western blots of T. brucei, two PEX5 forms are detected with apparent M{sub r} of 100 kDa and 72 kDa. 5′-RACE-PCR showed that TbPEX5 is encoded by a unique transcript that can be translated into a protein of maximally 72 kDa. However, recombinant PEX5 migrates aberrantly in SDS–PAGE with an apparent M{sub r} of 100 kDa, similarly as observed for the native peroxin. In vitro protease susceptibility analysis of native and {sup 35}S-labelled PEX5 showed truncation of the 100 kDa form at the N-terminal side by unknown parasite proteases, giving rise to the 72 kDa form which remains functional for PTS1 binding. The relevance of these observations is discussed.

  6. Importance of dispersion and electron correlation in ab initio protein folding.

    Science.gov (United States)

    He, Xiao; Fusti-Molnar, Laszlo; Cui, Guanglei; Merz, Kenneth M

    2009-04-16

    Dispersion is well-known to be important in biological systems, but the effect of electron correlation in such systems remains unclear. In order to assess the relationship between the structure of a protein and its electron correlation energy, we employed both full system Hartree-Fock (HF) and second-order Møller-Plesset perturbation (MP2) calculations in conjunction with the Polarizable Continuum Model (PCM) on the native structures of two proteins and their corresponding computer-generated decoy sets. Because of the expense of the MP2 calculation, we have utilized the fragment molecular orbital method (FMO) in this study. We show that the sum of the Hartree-Fock (HF) energy and force field (LJ6)-derived dispersion energy (HF + LJ6) is well correlated with the energies obtained using second-order Møller-Plesset perturbation (MP2) theory. In one of the two examples studied, the correlation energy as well as the empirical dispersive energy term was able to discriminate between native and decoy structures. On the other hand, for the second protein we studied, neither the correlation energy nor dispersion energy showed discrimination capabilities; however, the ab initio MP2 energy and the HF+LJ6 both ranked the native structure correctly. Furthermore, when we randomly scrambled the Lennard-Jones parameters, the correlation between the MP2 energy and the sum of the HF energy and dispersive energy (HF+LJ6) significantly drops, which indicates that the choice of Lennard-Jones parameters is important.

  7. The cellular RNA-binding protein EAP recognizes a conserved stem-loop in the Epstein-Barr virus small RNA EBER 1.

    Science.gov (United States)

    Toczyski, D P; Steitz, J A

    1993-01-01

    EAP (EBER-associated protein) is an abundant, 15-kDa cellular RNA-binding protein which associates with certain herpesvirus small RNAs. We have raised polyclonal anti-EAP antibodies against a glutathione S-transferase-EAP fusion protein. Analysis of the RNA precipitated by these antibodies from Epstein-Barr virus (EBV)- or herpesvirus papio (HVP)-infected cells shows that > 95% of EBER 1 (EBV-encoded RNA 1) and the majority of HVP 1 (an HVP small RNA homologous to EBER 1) are associated with EAP. RNase protection experiments performed on native EBER 1 particles with affinity-purified anti-EAP antibodies demonstrate that EAP binds a stem-loop structure (stem-loop 3) of EBER 1. Since bacterially expressed glutathione S-transferase-EAP fusion protein binds EBER 1, we conclude that EAP binding is independent of any other cellular or viral protein. Detailed mutational analyses of stem-loop 3 suggest that EAP recognizes the majority of the nucleotides in this hairpin, interacting with both single-stranded and double-stranded regions in a sequence-specific manner. Binding studies utilizing EBER 1 deletion mutants suggest that there may also be a second, weaker EAP-binding site on stem-loop 4 of EBER 1. These data and the fact that stem-loop 3 represents the most highly conserved region between EBER 1 and HVP 1 suggest that EAP binding is a critical aspect of EBER 1 and HVP 1 function. Images PMID:8380232

  8. Adamantane-based amphiphiles (ADAs) for membrane protein study: importance of a detergent hydrophobic group in membrane protein solubilisation.

    Science.gov (United States)

    Chae, Pil Seok; Bae, Hyoung Eun; Das, Manabendra

    2014-10-21

    We prepared adamantane-containing amphiphiles and evaluated them using a large membrane protein complex in terms of protein solubilisation and stabilization efficacy. These agents were superior to conventional detergents, especially in terms of the membrane protein solubilisation efficiency, implying a new detergent structure-property relationship.

  9. Porcine Reproductive and Respiratory Syndrome Virus Nucleocapsid Protein Interacts with Nsp9 and Cellular DHX9 To Regulate Viral RNA Synthesis.

    Science.gov (United States)

    Liu, Long; Tian, Jiao; Nan, Hao; Tian, Mengmeng; Li, Yuan; Xu, Xiaodong; Huang, Baicheng; Zhou, Enmin; Hiscox, Julian A; Chen, Hongying

    2016-06-01

    Porcine reproductive and respiratory syndrome virus (PRRSV) nucleocapsid (N) protein is the main component of the viral capsid to encapsulate viral RNA, and it is also a multifunctional protein involved in the regulation of host cell processes. Nonstructural protein 9 (Nsp9) is the RNA-dependent RNA polymerase that plays a critical role in viral RNA transcription and replication. In this study, we demonstrate that PRRSV N protein is bound to Nsp9 by protein-protein interaction and that the contacting surface on Nsp9 is located in the two predicted α-helixes formed by 48 residues at the C-terminal end of the protein. Mutagenesis analyses identified E646, E608, and E611 on Nsp9 and Q85 on the N protein as the pivotal residues participating in the N-Nsp9 interaction. By overexpressing the N protein binding fragment of Nsp9 in infected Marc-145 cells, the synthesis of viral RNAs, as well as the production of infectious progeny viruses, was dramatically inhibited, suggesting that Nsp9-N protein association is involved in the process of viral RNA production. In addition, we show that PRRSV N interacts with cellular RNA helicase DHX9 and redistributes the protein into the cytoplasm. Knockdown of DHX9 increased the ratio of short subgenomic mRNAs (sgmRNAs); in contrast, DHX9 overexpression benefited the synthesis of longer sgmRNAs and the viral genomic RNA (gRNA). These results imply that DHX9 is recruited by the N protein in PRRSV infection to regulate viral RNA synthesis. We postulate that N and DHX9 may act as antiattenuation factors for the continuous elongation of nascent transcript during negative-strand RNA synthesis. It is unclear whether the N protein of PRRSV is involved in regulation of the viral RNA production process. In this report, we demonstrate that the N protein of the arterivirus PRRSV participates in viral RNA replication and transcription through interacting with Nsp9 and its RdRp and recruiting cellular RNA helicase to promote the production of

  10. Gravity Plays an Important Role in Muscle Development and the Differentiation of Contractile Protein Phenotype

    Science.gov (United States)

    Adams, Gregory A.; Haddad, Fadia; Baldwin, Kenneth M.

    2003-01-01

    Several muscles in the body exist mainly to work against gravity. Whether gravity is important in the development of these muscles is not known. By examining the basic proteins that compose muscle, questions about the role of gravity in muscle development can be answered. Myosin heavy chains (MHCs) are a family of proteins critically important for muscle contraction. Several types of MHCs exist (e.g., neonatal, slow, fast), and each type is produced by a particular gene. Neonatal MHCs are produced early in life. Slow MHCs are important in antigravity muscles, and fast MHCs are found in fast-twitch power muscles. The gene that is turned on or expressed will determine which MHC is produced. Early in development, antigravity skeletal muscles (muscles that work against gravity) normally produce a combination of the neonatal/embryonic MHCs. The expression of these primitive MHCs is repressed early in development; and the adult slow and fast MHC genes become fully expressed. We tested the hypothesis that weightbearing activity is critical for inducing the normal expression of the slow MHC gene typically expressed in adult antigravity muscles. Also, we hypothesized that thyroid hormone, but not opposition to gravity, is necessary for expressing the adult fast IIb MHC gene essential for high-intensity muscle performance. Groups of normal thyroid and thyroid-deficient neonatal rats were studied after their return from the 16-day Neurolab mission and compared to matched controls. The results suggest: (1) Weightlessness impaired body and limb skeletal muscle growth in both normal and thyroid-deficient animals. Antigravity muscles were impaired more than those used primarily for locomotion andor nonweightbearing activity. (2) Systemic and muscle expression of insulin-like growth factor-I (IGF-I), an important body and tissue growth factor, was depressed in flight animals. (3) Normal slow, type I MHC gene expression was markedly repressed in the normal thyroid flight group. (4

  11. Peroxisomal matrix protein import - Suppression of protein import defects in Hansenula polymorpha pex mutants by overproduction of the PTS1 receptor pex5p

    NARCIS (Netherlands)

    Kiel, JAKW; Veenhuis, M

    2000-01-01

    In the past decade, much progress has been made in understanding the mechanisms that govern sorting of proteins to the peroxisomal lumen. This article summarizes the principal features of how peroxisomal matrix enzymes are thought to reach the peroxisome. In addition, it describes recent data that

  12. Phg1/TM9 proteins control intracellular killing of bacteria by determining cellular levels of the Kil1 sulfotransferase in Dictyostelium.

    Directory of Open Access Journals (Sweden)

    Marion Le Coadic

    Full Text Available Dictyostelium discoideum has largely been used to study phagocytosis and intracellular killing of bacteria. Previous studies have shown that Phg1A, Kil1 and Kil2 proteins are necessary for efficient intracellular killing of Klebsiella bacteria. Here we show that in phg1a KO cells, cellular levels of lysosomal glycosidases and lysozyme are decreased, and lysosomal pH is increased. Surprisingly, overexpression of Kil1 restores efficient killing in phg1a KO cells without correcting these lysosomal anomalies. Conversely, kil1 KO cells are defective for killing, but their enzymatic content and lysosomal pH are indistinguishable from WT cells. The killing defect of phg1a KO cells can be accounted for by the observation that in these cells the stability and the cellular amount of Kil1 are markedly reduced. Since Kil1 is the only sulfotransferase characterized in Dictyostelium, an (unidentified sulfated factor, defective in both phg1a and kil1 KO cells, may play a key role in intracellular killing of Klebsiella bacteria. In addition, Phg1B plays a redundant role with Phg1A in controlling cellular amounts of Kil1 and intracellular killing. Finally, cellular levels of Kil1 are unaffected in kil2 KO cells, and Kil1 overexpression does not correct the killing defect of kil2 KO cells, suggesting that Kil2 plays a distinct role in intracellular killing.

  13. Proteomic investigation of Vibrio alginolyticus challenged Caenorhabditis elegans revealed regulation of cellular homeostasis proteins and their role in supporting innate immune system.

    Science.gov (United States)

    Durai, Sellegounder; Singh, Nirpendra; Kundu, Suman; Balamurugan, Krishnaswamy

    2014-08-01

    Caenorhabditis elegans has been the preferred model system for many investigators to study pathogenesis. In the present investigation, regulation of C. elegans proteome was explored against V. alginolyticus infection using quantitative proteomics approach. Proteins were separated using 2D-DIGE and the differentially regulated proteins were identified using PMF and MALDI TOF/TOF analysis. The results thus obtained were validated using Western blotting for candidate proteins. The corresponding transcriptional regulation was quantified subsequently using real-time PCR. Interaction network for candidate proteins was predicted using search tool for the retrieval of interacting genes/proteins (STRING) and functional validation was performed using respective mutant strains. Out of the 25 proteins identified, 21 proteins appeared to be upregulated while four were downregulated. Upregulated proteins included those involved in stress-response (PDI-2, HSP-6), immune-response (protein kinase -18, GST-8) and energy-production (ATP-2) while proteins involved in structural maintenance (IFB-2) and lipid metabolism (SODH-1) were downregulated. The roles of these players in the host system during Vibrio infection was analyzed in vivo using wild type and mutant C. elegans. Survival assays using mutants lacking pdi-2, ire-1, and xbp-1 displayed enhanced susceptibility to V. alginolyticus. Cellular stress generated by V. alginolyticus was determined using ROS assay. This is the first report of proteome changes in C. elegans against V. alginolyticus challenge and highlights the significance of unfolded protein response (UPR) pathway during bacterial infection. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  14. The novel chloroplast outer membrane kinase KOC1 is a required component of the plastid protein import machinery.

    Science.gov (United States)

    Zufferey, Mónica; Montandon, Cyrille; Douet, Véronique; Demarsy, Emilie; Agne, Birgit; Baginsky, Sacha; Kessler, Felix

    2017-04-28

    The biogenesis and maintenance of cell organelles such as mitochondria and chloroplasts require the import of many proteins from the cytosol, a process that is controlled by phosphorylation. In the case of chloroplasts, the import of hundreds of different proteins depends on translocons at the outer and inner chloroplast membrane (TOC and TIC, respectively) complexes. The essential protein TOC159 functions thereby as an import receptor. It has an N-terminal acidic (A-) domain that extends into the cytosol, controls receptor specificity, and is highly phosphorylated in vivo However, kinases that phosphorylate the TOC159 A-domain to enable protein import have remained elusive. Here, using co-purification with TOC159 from Arabidopsis , we discovered a novel component of the chloroplast import machinery, the regulatory kinase at the outer chloroplast membrane 1 (KOC1). We found that KOC1 is an integral membrane protein facing the cytosol and stably associates with TOC. Moreover, KOC1 phosphorylated the A-domain of TOC159 in vitro , and in mutant koc1 chloroplasts, preprotein import efficiency was diminished. koc1 Arabidopsis seedlings had reduced survival rates after transfer from the dark to the light in which protein import into plastids is required to rapidly complete chloroplast biogenesis. In summary, our data indicate that KOC1 is a functional component of the TOC machinery that phosphorylates import receptors, supports preprotein import, and contributes to efficient chloroplast biogenesis. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  15. The Force Exerted by the Membrane Potential During Protein Import into the Mitochondrial Matrix

    Science.gov (United States)

    Shariff, Karim; Ghosal, Sandip; Matouschek, Andreas

    2002-01-01

    The electrostatic force exerted on a targeting sequence by the electrical potential across the inner mitochondrial membrane is calculated and found to vary from 1.4 pN to 2.2 pN (per unit elementary charge) as the radius of the inner membrane pore (assumed aqueous) is varied from 12 to 6.5 Angstroms, its measured range. Since the pore is not very much wider than the distance between water molecules, the full shielding effect of water may not be present; the extreme case of a nonaqueous pore gives a force of 3.1 pN per unit charge, which represents an upper limit. When applied to mitochondrial import experiments on the protein harness, these results imply that a force of 11 plus or minus 4 pN is sufficient to catalyze the unfolding of harness during import. Comparison of these results with unfolding forces measured using atomic force microscopy suggests that the two are not inconsistent.

  16. The Rieske Iron-Sulfur Protein: Import and Assembly into the Cytochrome bc 1 Complex of Yeast Mitochondria

    Science.gov (United States)

    Conte, Laura; Zara, Vincenzo

    2011-01-01

    The Rieske iron-sulfur protein, one of the catalytic subunits of the cytochrome bc 1 complex, is involved in electron transfer at the level of the inner membrane of yeast mitochondria. The Rieske iron-sulfur protein is encoded by nuclear DNA and, after being synthesized in the cytosol, is imported into mitochondria with the help of a cleavable N-terminal presequence. The imported protein, besides incorporating the 2Fe-2S cluster, also interacts with other catalytic and non-catalytic subunits of the cytochrome bc 1 complex, thereby assembling into the mature and functional respiratory complex. In this paper, we summarize the most recent findings on the import and assembly of the Rieske iron-sulfur protein into Saccharomyces cerevisiae mitochondria, also discussing a possible role of this protein both in the dimerization of the cytochrome bc 1 complex and in the interaction of this homodimer with other complexes of the mitochondrial respiratory chain. PMID:21716720

  17. The Rieske Iron-Sulfur Protein: Import and Assembly into the Cytochrome bc(1) Complex of Yeast Mitochondria.

    Science.gov (United States)

    Conte, Laura; Zara, Vincenzo

    2011-01-01

    The Rieske iron-sulfur protein, one of the catalytic subunits of the cytochrome bc(1) complex, is involved in electron transfer at the level of the inner membrane of yeast mitochondria. The Rieske iron-sulfur protein is encoded by nuclear DNA and, after being synthesized in the cytosol, is imported into mitochondria with the help of a cleavable N-terminal presequence. The imported protein, besides incorporating the 2Fe-2S cluster, also interacts with other catalytic and non-catalytic subunits of the cytochrome bc(1) complex, thereby assembling into the mature and functional respiratory complex. In this paper, we summarize the most recent findings on the import and assembly of the Rieske iron-sulfur protein into Saccharomyces cerevisiae mitochondria, also discussing a possible role of this protein both in the dimerization of the cytochrome bc(1) complex and in the interaction of this homodimer with other complexes of the mitochondrial respiratory chain.

  18. Identification of a Golgi apparatus protein complex important for the asexual erythrocytic cycle of the malaria parasite Plasmodium falciparum.

    Science.gov (United States)

    Hallée, Stéphanie; Thériault, Catherine; Gagnon, Dominic; Kehrer, Jessica; Frischknecht, Friedrich; Mair, Gunnar R; Richard, Dave

    2018-03-26

    Compared with other eukaryotic cell types, malaria parasites appear to possess a more rudimentary Golgi apparatus being composed of dispersed, unstacked cis and trans-cisternae. Despite playing a central role in the secretory pathway of the parasite, few Plasmodium Golgi resident proteins have been characterised. We had previously identified a new Golgi resident protein of unknown function, which we had named Golgi Protein 1, and now show that it forms a complex with a previously uncharacterised transmembrane protein (Golgi Protein 2, GP2). The Golgi Protein complex localises to the cis-Golgi throughout the erythrocytic cycle and potentially also during the mosquito stages. Analysis of parasite strains where GP1 expression is conditionally repressed and/or the GP2 gene is inactivated reveals that though the Golgi protein complex is not essential at any stage of the parasite life cycle, it is important for optimal asexual development in the blood stages. © 2018 John Wiley & Sons Ltd.

  19. In vivo import of plastocyanin and a fusion protein into developmentally different plastids of transgenic plants

    NARCIS (Netherlands)

    Boer, Douwe de; Cremers, Fons; Teertstra, Renske; Smits, Lianne; Hille, Jacques; Smeekens, Sjef; Weisbeek, Peter

    1988-01-01

    Transgenic tomato plants that constitutively express a foreign plastocyanin gene were used to study protein transport in different tissues. Normally expression of endogenous plastocyanin genes in plants is restricted to photosynthetic tissues only, whereas this foreign plastocyanin protein is found

  20. Sub-cellular localisation studies may spuriously detect the Yes-associated protein, YAP, in nucleoli leading to potentially invalid conclusions of its function.

    Science.gov (United States)

    Finch, Megan L; Passman, Adam M; Strauss, Robyn P; Yeoh, George C; Callus, Bernard A

    2015-01-01

    The Yes-associated protein (YAP) is a potent transcriptional co-activator that functions as a nuclear effector of the Hippo signaling pathway. YAP is oncogenic and its activity is linked to its cellular abundance and nuclear localisation. Activation of the Hippo pathway restricts YAP nuclear entry via its phosphorylation by Lats kinases and consequent cytoplasmic retention bound to 14-3-3 proteins. We examined YAP expression in liver progenitor cells (LPCs) and surprisingly found that transformed LPCs did not show an increase in YAP abundance compared to the non-transformed LPCs from which they were derived. We then sought to ascertain whether nuclear YAP was more abundant in transformed LPCs. We used an antibody that we confirmed was specific for YAP by immunoblotting to determine YAP's sub-cellular localisation by immunofluorescence. This antibody showed diffuse staining for YAP within the cytosol and nuclei, but, noticeably, it showed intense staining of the nucleoli of LPCs. This staining was non-specific, as shRNA treatment of cells abolished YAP expression to undetectable levels by Western blot yet the nucleolar staining remained. Similar spurious YAP nucleolar staining was also seen in mouse embryonic fibroblasts and mouse liver tissue, indicating that this antibody is unsuitable for immunological applications to determine YAP sub-cellular localisation in mouse cells or tissues. Interestingly nucleolar staining was not evident in D645 cells suggesting the antibody may be suitable for use in human cells. Given the large body of published work on YAP in recent years, many of which utilise this antibody, this study raises concerns regarding its use for determining sub-cellular localisation. From a broader perspective, it serves as a timely reminder of the need to perform appropriate controls to ensure the validity of published data.

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

    International Nuclear Information System (INIS)

    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.

  2. Sleep, Plasticity and the Pathophysiology of Neurodevelopmental Disorders: The Potential Roles of Protein Synthesis and Other Cellular Processes

    Directory of Open Access Journals (Sweden)

    Dante Picchioni

    2014-03-01

    Full Text Available Sleep is important for neural plasticity, and plasticity underlies sleep-dependent memory consolidation. It is widely appreciated that protein synthesis plays an essential role in neural plasticity. Studies of sleep-dependent memory and sleep-dependent plasticity have begun to examine alterations in these functions in populations with neurological and psychiatric disorders. Such an approach acknowledges that disordered sleep may have functional consequences during wakefulness. Although neurodevelopmental disorders are not considered to be sleep disorders per se, recent data has revealed that sleep abnormalities are among the most prevalent and common symptoms and may contribute to the progression of these disorders. The main goal of this review is to highlight the role of disordered sleep in the pathology of neurodevelopmental disorders and to examine some potential mechanisms by which sleep-dependent plasticity may be altered. We will also briefly attempt to extend the same logic to the other end of the developmental spectrum and describe a potential role of disordered sleep in the pathology of neurodegenerative diseases. We conclude by discussing ongoing studies that might provide a more integrative approach to the study of sleep, plasticity, and neurodevelopmental disorders.

  3. Immobilization of bioactive fibroblast growth factor-2 into cubic proteinous microcrystals (Bombyx mori cypovirus polyhedra) that are insoluble in a physiological cellular environment.

    Science.gov (United States)

    Mori, Hajime; Shukunami, Chisa; Furuyama, Akiko; Notsu, Hiroyuki; Nishizaki, Yuriko; Hiraki, Yuji

    2007-06-08

    The supramolecular architecture of the extracellular matrix and the disposition of its specific accessory molecules give rise to variable heterotopic signaling cues for single cells. Here we have described the successful occlusion of human fibroblast growth factor-2 (FGF-2) into the cubic inclusion bodies (FGF-2 polyhedra) of the Bombyx mori cytoplasmic polyhedrosis virus (BmCPV). The polyhedra are proteinous cubic crystals of several microns in size that are insoluble in the extracellular milieu. Purified FGF-2 polyhedra were found to stimulate proliferation and phosphorylation of p44/p42 mitogen-activated protein kinase in cultured fibroblasts. Moreover, cellular responses were blocked by a synthetic inhibitor of the FGF signaling pathway, SU5402, suggesting that FGF-2 polyhedra indeed act through FGF receptors. Furthermore, FGF-2 polyhedra retained potent growth stimulatory properties even after desiccation. We have demonstrated that BmCPV polyhedra microcrystals that occlude extracellular signaling proteins are a novel and versatile tool that can be employed to analyze cellular behavior at the single cell level.

  4. The MAP kinase-activated protein kinase Rck2p regulates cellular responses to cell wall stresses, filamentation and virulence in the human fungal pathogen Candida albicans.

    Science.gov (United States)

    Li, Xichuan; Du, Wei; Zhao, Jingwen; Zhang, Lilin; Zhu, Zhiyan; Jiang, Linghuo

    2010-06-01

    Rck2p is the Hog1p-MAP kinase-activated protein kinase required for the attenuation of protein synthesis in response to an osmotic challenge in Saccharomyces cerevisiae. Rck2p also regulates rapamycin sensitivity in both S. cerevisiae and Candida albicans. In this study, we demonstrate that the deletion of CaRCK2 renders C. albicans cells sensitive to, and CaRck2p translocates from the cytosol to the nucleus in response to, cell wall stresses caused by Congo red, Calcoflor White, elevated heat and zymolyase. However, the kinase activity of CaRck2p is not required for the cellular response to these cell wall stresses. Furthermore, transcripts of cell wall protein-encoding genes CaBGL2, CaHWP1 and CaXOG1 are reduced in C. albicans cells lacking CaRCK2. The deletion of CaRCK2 also reduces the in vitro filamentation of C. albicans and its virulence in a mouse model of systemic candidasis. The kinase activity of CaRck2p is required for the virulence, but not for the in vitro filamentation, in C. albicans. Therefore, Rck2p regulates cellular responses to cell wall stresses, filamentation and virulence in the human fungal pathogen C. albicans.

  5. The Epstein-Barr virus BFRF1 and BFLF2 proteins interact and coexpression alters their cellular localization

    International Nuclear Information System (INIS)

    Lake, Cathleen M.; Hutt-Fletcher, Lindsey M.

    2004-01-01

    The BFRF1 protein of Epstein-Barr virus (EBV) is a recently identified membrane protein that is the homolog of the alphaherpesvirus UL34 gene product. We report here that a yeast two-hybrid screen identified the BFLF2 gene product, a homolog of alphaherpesvirus UL31, as a protein that interacts with BFRF1. Expression of BFLF2 in mammalian cells revealed a protein of approximately 28 kDa that associated with BFRF1 in a noncovalently linked complex. When expressed alone, the BFRF1 protein was found in the cytoplasm and perinuclear region. BFLF2 was found diffusely in the nucleus in the absence of BFRF1, but coexpression of BFRF1 and BFLF2 resulted in colocalization of the two proteins at the nuclear rim. These data recapitulate the behavior of the alphaherpesvirus homologs of BFRF1 and BFLF2 and suggest that functional as well as structural and positional homology may be conserved

  6. Important mitochondrial proteins in human omental adipose tissue show reduced expression in obesity

    Directory of Open Access Journals (Sweden)

    Peter W. Lindinger

    2015-09-01

    Full Text Available Obesity is associated with impaired mitochondrial function. This study compares mitochondrial protein expression in omental fat in obese and non-obese humans. Omental adipose tissue was obtained by surgical biopsy, adipocytes were purified and mitochondria isolated. Using anion-exchange chromatography, SDS-PAGE and mass-spectrometry, 128 proteins with potentially different abundances in patient groups were identified, 62 of the 128 proteins are mainly localized in the mitochondria. Further quantification of 12 of these 62 proteins by immune dot blot analysis revealed four proteins citrate synthase, HADHA, LETM1 and mitofilin being inversely associated with BMI, and mitofilin being inversely correlated with gender.

  7. Important mitochondrial proteins in human omental adipose tissue show reduced expression in obesity.

    Science.gov (United States)

    Lindinger, Peter W; Christe, Martine; Eberle, Alex N; Kern, Beatrice; Peterli, Ralph; Peters, Thomas; Jayawardene, Kamburapola J I; Fearnley, Ian M; Walker, John E

    2015-09-01

    Obesity is associated with impaired mitochondrial function. This study compares mitochondrial protein expression in omental fat in obese and non-obese humans. Omental adipose tissue was obtained by surgical biopsy, adipocytes were purified and mitochondria isolated. Using anion-exchange chromatography, SDS-PAGE and mass-spectrometry, 128 proteins with potentially different abundances in patient groups were identified, 62 of the 128 proteins are mainly localized in the mitochondria. Further quantification of 12 of these 62 proteins by immune dot blot analysis revealed four proteins citrate synthase, HADHA, LETM1 and mitofilin being inversely associated with BMI, and mitofilin being inversely correlated with gender.

  8. Critical Importance of Protein 4.1 in Centrosome and Mitotic Spindle Aberrations in Breast Cancer Pathogenesis

    National Research Council Canada - National Science Library

    Krauss, Sharon W

    2006-01-01

    We proposed to test the novel hypothesis that protein 4.1 is of critical importance to centrosome and mitotic spindle aberrations that directly impact aspects of breast cancer pathogenesis. We characterized...

  9. Mutation of a Conserved Nuclear Export Sequence in Chikungunya Virus Capsid Protein Disrupts Host Cell Nuclear Import.

    Science.gov (United States)

    Jacobs, Susan C; Taylor, Adam; Herrero, Lara J; Mahalingam, Suresh; Fazakerley, John K

    2017-10-20

    Transmitted by mosquitoes; chikungunya virus (CHIKV) is responsible for frequent outbreaks of arthritic disease in humans. CHIKV is an arthritogenic alphavirus of the Togaviridae family. Capsid protein, a structural protein encoded by the CHIKV RNA genome, is able to translocate to the host cell nucleus. In encephalitic alphaviruses nuclear translocation induces host cell shut off; however, the role of capsid protein nuclear localisation in arthritogenic alphaviruses remains unclear. Using replicon systems, we investigated a nuclear export sequence (NES) in the N-terminal region of capsid protein; analogous to that found in encephalitic alphavirus capsid but uncharacterised in CHIKV. The chromosomal maintenance 1 (CRM1) export adaptor protein mediated CHIKV capsid protein export from the nucleus and a region within the N-terminal part of CHIKV capsid protein was required for active nuclear targeting. In contrast to encephalitic alphaviruses, CHIKV capsid protein did not inhibit host nuclear import; however, mutating the NES of capsid protein (∆NES) blocked host protein access to the nucleus. Interactions between capsid protein and the nucleus warrant further investigation.

  10. Proteomic analysis of HIV-1 Nef cellular binding partners reveals a role for exocyst complex proteins in mediating enhancement of intercellular nanotube formation

    Directory of Open Access Journals (Sweden)

    Mukerji Joya

    2012-06-01

    Full Text Available Abstract Background HIV-1 Nef protein contributes to pathogenesis via multiple functions that include enhancement of viral replication and infectivity, alteration of intracellular trafficking, and modulation of cellular signaling pathways. Nef stimulates formation of tunneling nanotubes and virological synapses, and is transferred to bystander cells via these intercellular contacts and secreted microvesicles. Nef associates with and activates Pak2, a kinase that regulates T-cell signaling and actin cytoskeleton dynamics, but how Nef promotes nanotube formation is unknown. Results To identify Nef binding partners involved in Pak2-association dependent Nef functions, we employed tandem mass spectrometry analysis of Nef immunocomplexes from Jurkat cells expressing wild-type Nef or Nef mutants defective for the ability to associate with Pak2 (F85L, F89H, H191F and A72P, A75P in NL4-3. We report that wild-type, but not mutant Nef, was associated with 5 components of the exocyst complex (EXOC1, EXOC2, EXOC3, EXOC4, and EXOC6, an octameric complex that tethers vesicles at the plasma membrane, regulates polarized exocytosis, and recruits membranes and proteins required for nanotube formation. Additionally, Pak2 kinase was associated exclusively with wild-type Nef. Association of EXOC1, EXOC2, EXOC3, and EXOC4 with wild-type, but not mutant Nef, was verified by co-immunoprecipitation assays in Jurkat cells. Furthermore, shRNA-mediated depletion of EXOC2 in Jurkat cells abrogated Nef-mediated enhancement of nanotube formation. Using bioinformatic tools, we visualized protein interaction networks that reveal functional linkages between Nef, the exocyst complex, and the cellular endocytic and exocytic trafficking machinery. Conclusions Exocyst complex proteins are likely a key effector of Nef-mediated enhancement of nanotube formation, and possibly microvesicle secretion. Linkages revealed between Nef and the exocyst complex suggest a new paradigm of

  11. Comparative proteomic analysis of Xanthomonas citri ssp. citri periplasmic proteins reveals changes in cellular envelope metabolism during in vitro pathogenicity induction.

    Science.gov (United States)

    Artier, Juliana; da Silva Zandonadi, Flávia; de Souza Carvalho, Flávia Maria; Pauletti, Bianca Alves; Leme, Adriana Franco Paes; Carnielli, Carolina Moretto; Selistre-de-Araujo, Heloisa Sobreiro; Bertolini, Maria Célia; Ferro, Jesus Aparecido; Belasque Júnior, José; de Oliveira, Julio Cezar Franco; Novo-Mansur, Maria Teresa Marques

    2018-01-01

    Citrus canker is a plant disease caused by Gram-negative bacteria from the genus Xanthomonas. The most virulent species is Xanthomonas citri ssp. citri (XAC), which attacks a wide range of citrus hosts. Differential proteomic analysis of the periplasm-enriched fraction was performed for XAC cells grown in pathogenicity-inducing (XAM-M) and pathogenicity-non-inducing (nutrient broth) media using two-dimensional electrophoresis combined with liquid chromatography-tandem mass spectrometry. Amongst the 40 proteins identified, transglycosylase was detected in a highly abundant spot in XAC cells grown under inducing condition. Additional up-regulated proteins related to cellular envelope metabolism included glucose-1-phosphate thymidylyltransferase, dTDP-4-dehydrorhamnose-3,5-epimerase and peptidyl-prolyl cis-trans-isomerase. Phosphoglucomutase and superoxide dismutase proteins, known to be involved in pathogenicity in other Xanthomonas species or organisms, were also detected. Western blot and quantitative real-time polymerase chain reaction analyses for transglycosylase and superoxide dismutase confirmed that these proteins were up-regulated under inducing condition, consistent with the proteomic results. Multiple spots for the 60-kDa chaperonin and glyceraldehyde-3-phosphate dehydrogenase were identified, suggesting the presence of post-translational modifications. We propose that substantial alterations in cellular envelope metabolism occur during the XAC infectious process, which are related to several aspects, from defence against reactive oxygen species to exopolysaccharide synthesis. Our results provide new candidates for virulence-related proteins, whose abundance correlates with the induction of pathogenicity and virulence genes, such as hrpD6, hrpG, hrpB7, hpa1 and hrpX. The results present new potential targets against XAC to be investigated in further functional studies. © 2016 BSPP AND JOHN WILEY & SONS LTD.

  12. NK cells of the oldest seniors represent constant and resistant to stimulation high expression of cellular protective proteins SIRT1 and HSP70.

    Science.gov (United States)

    Kaszubowska, Lucyna; Foerster, Jerzy; Kaczor, Jan Jacek; Schetz, Daria; Ślebioda, Tomasz Jerzy; Kmieć, Zbigniew

    2018-01-01

    Natural killer cells (NK cells) are cytotoxic lymphocytes of innate immunity that reveal some immunoregulatory properties, however, their role in the process of ageing is not completely understood. The study aimed to analyze the expression of proteins involved in cellular stress response: sirtuin 1 (SIRT1), heat shock protein 70 (HSP70) and manganese superoxide dismutase (SOD2) in human NK cells with reference to the process of ageing. Non-stimulated and stimulated with IL-2, LPS or PMA with ionomycin cells originated from peripheral blood samples of: seniors aged over 85 ('the oldest'; n  = 25; 88.5 ± 0.5 years, mean ± SEM), seniors aged under 85 ('the old'; n  = 30; 75.6 ± 0.9 years) and the young ( n  = 31; 20.9 ± 0.3 years). The relationships between the levels of expression of cellular protective proteins in the studied population were also analyzed. The concentrations of carbonyl groups and 8-isoprostanes, markers of oxidative stress, in both stimulated and non-stimulated cultured NK cells were measured to assess the level of the oxidative stress in the cells. The oldest seniors varied from the other age groups by significantly higher expression of SIRT1 and HSP70 both in non-stimulated and stimulated NK cells. These cells also appeared to be resistant to further stimulations with IL-2, LPS or PMA with ionomycin. Highly positive correlations between SIRT1 and intracellular HSP70 in both stimulated and non-stimulated NK cells were observed. SOD2 presented low expression in non-stimulated cells, whereas its sensitivity to stimulation increased with age of donors. High positive correlations between SOD2 and surface HSP70 were observed. We found that the markers of oxidative stress in NK cells did not change with ageing. The oldest seniors revealed well developed adaptive stress response in NK cells with increased, constant levels of SIRT1 and intracellular HSP70. They presented also very high positive correlations between

  13. The rational design of a novel potent analogue of the 5’-AMP-activated protein kinase inhibitor compound C with improved selectivity and cellular activity

    Science.gov (United States)

    Machrouhi, Fouzia; Ouhamou, Nouara; Laderoute, Keith; Calaoagan, Joy; Bukhtiyarova, Marina; Ehrlich, Paula J.; Klon, Anthony E.

    2010-01-01

    We have designed and synthesized analogues of compound C, a non-specific inhibitor of 5’-AMP-activated protein kinase (AMPK), using a computational fragment-based drug design (FBDD) approach. Synthesizing only twenty-seven analogues yielded a compound that was equipotent to compound C in the inhibition of the human AMPK (hAMPK) α2 subunit in the heterotrimeric complex in vitro, exhibited significantly improved selectivity against a subset of relevant kinases, and demonstrated enhanced cellular inhibition of AMPK. PMID:20932747

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

    Directory of Open Access Journals (Sweden)

    St-Laurent Georges

    2006-11-01

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

  15. The plant i-AAA protease controls the turnover of an essential mitochondrial protein import component.

    Science.gov (United States)

    Opalińska, Magdalena; Parys, Katarzyna; Murcha, Monika W; Jańska, Hanna

    2018-01-29

    Mitochondria are multifunctional organelles that play a central role in energy metabolism. Owing to the life-essential functions of these organelles, mitochondrial content, quality and dynamics are tightly controlled. Across the species, highly conserved ATP-dependent proteases prevent malfunction of mitochondria through versatile activities. This study focuses on a molecular function of the plant mitochondrial inner membrane-embedded AAA protease (denoted i -AAA) FTSH4, providing its first bona fide substrate. Here, we report that the abundance of the Tim17-2 protein, an essential component of the TIM17:23 translocase (Tim17-2 together with Tim50 and Tim23), is directly controlled by the proteolytic activity of FTSH4. Plants that are lacking functional FTSH4 protease are characterized by significantly enhanced capacity of preprotein import through the TIM17:23-dependent pathway. Taken together, with the observation that FTSH4 prevents accumulation of Tim17-2, our data point towards the role of this i -AAA protease in the regulation of mitochondrial biogenesis in plants. © 2018. Published by The Company of Biologists Ltd.

  16. Cellular uptake of beta-carotene from protein stabilized solid lipid nano-particles prepared by homogenization-evaporation method

    Science.gov (United States)

    Using a homogenization-evaporation method, beta-carotene (BC) loaded nano-particles were prepared with different ratios of food-grade sodium caseinate (SC), whey protein isolate (WPI), or soy protein isolate (SPI) to BC and evaluated for their physiochemical stability, in vitro cytotoxicity, and cel...

  17. The L1TD1 Protein Interactome Reveals the Importance of Post-transcriptional Regulation in Human Pluripotency

    Directory of Open Access Journals (Sweden)

    Maheswara Reddy Emani

    2015-03-01

    Full Text Available The RNA-binding protein L1TD1 is one of the most specific and abundant proteins in pluripotent stem cells and is essential for the maintenance of pluripotency in human cells. Here, we identify the protein interaction network of L1TD1 in human embryonic stem cells (hESCs and provide insights into the interactome network constructed in human pluripotent cells. Our data reveal that L1TD1 has an important role in RNA splicing, translation, protein traffic, and degradation. L1TD1 interacts with multiple stem-cell-specific proteins, many of which are still uncharacterized in the context of development. Further, we show that L1TD1 is a part of the pluripotency interactome network of OCT4, SOX2, and NANOG, bridging nuclear and cytoplasmic regulation and highlighting the importance of RNA biology in pluripotency.

  18. The Arabidopsis ppi1 Mutant Is Specifically Defective in the Expression, Chloroplast Import, and Accumulation of Photosynthetic ProteinsW⃞

    Science.gov (United States)

    Kubis, Sybille; Baldwin, Amy; Patel, Ramesh; Razzaq, Azam; Dupree, Paul; Lilley, Kathryn; Kurth, Joachim; Leister, Dario; Jarvis, Paul

    2003-01-01

    The import of nucleus-encoded proteins into chloroplasts is mediated by translocon complexes in the envelope membranes. A component of the translocon in the outer envelope membrane, Toc34, is encoded in Arabidopsis by two homologous genes, atTOC33 and atTOC34. Whereas atTOC34 displays relatively uniform expression throughout development, atTOC33 is strongly upregulated in rapidly growing, photosynthetic tissues. To understand the reason for the existence of these two related genes, we characterized the atTOC33 knockout mutant ppi1. Immunoblotting and proteomics revealed that components of the photosynthetic apparatus are deficient in ppi1 chloroplasts and that nonphotosynthetic chloroplast proteins are unchanged or enriched slightly. Furthermore, DNA array analysis of 3292 transcripts revealed that photosynthetic genes are moderately, but specifically, downregulated in ppi1. Proteome differences in ppi1 could be correlated with protein import rates: ppi1 chloroplasts imported the ribulose-1,5-bisphosphate carboxylase/oxygenase small subunit and 33-kD oxygen-evolving complex precursors at significantly reduced rates, but the import of a 50S ribosomal subunit precursor was largely unaffected. The ppi1 import defect occurred at the level of preprotein binding, which is consistent with a role for atToc33 during preprotein recognition. The data suggest that atToc33 is involved preferentially in the import of photosynthetic proteins and, by extension, that atToc34 is involved in the import of nonphotosynthetic chloroplast proteins. PMID:12897258

  19. Viral capsid assembly as a model for protein aggregation diseases: Active processes catalyzed by cellular assembly machines comprising novel drug targets.

    Science.gov (United States)

    Marreiros, Rita; Müller-Schiffmann, Andreas; Bader, Verian; Selvarajah, Suganya; Dey, Debendranath; Lingappa, Vishwanath R; Korth, Carsten

    2015-09-02

    Viruses can be conceptualized as self-replicating multiprotein assemblies, containing coding nucleic acids. Viruses have evolved to exploit host cellular components including enzymes to ensure their replicative life cycle. New findings indicate that also viral capsid proteins recruit host factors to accelerate their assembly. These assembly machines are RNA-containing multiprotein complexes whose composition is governed by allosteric sites. In the event of viral infection, the assembly machines are recruited to support the virus over the host and are modified to achieve that goal. Stress granules and processing bodies may represent collections of such assembly machines, readily visible by microscopy but biochemically labile and difficult to isolate by fractionation. We hypothesize that the assembly of protein multimers such as encountered in neurodegenerative or other protein conformational diseases, is also catalyzed by assembly machines. In the case of viral infection, the assembly machines have been modified by the virus to meet the virus' need for rapid capsid assembly rather than host homeostasis. In the case of the neurodegenerative diseases, it is the monomers and/or low n oligomers of the so-called aggregated proteins that are substrates of assembly machines. Examples for substrates are amyloid β peptide (Aβ) and tau in Alzheimer's disease, α-synuclein in Parkinson's disease, prions in the prion diseases, Disrupted-in-schizophrenia 1 (DISC1) in subsets of chronic mental illnesses, and others. A likely continuum between virus capsid assembly and cell-to-cell transmissibility of aggregated proteins is remarkable. Protein aggregation diseases may represent dysfunction and dysregulation of these assembly machines analogous to the aberrations induced by viral infection in which cellular homeostasis is pathologically reprogrammed. In this view, as for viral infection, reset of assembly machines to normal homeostasis should be the goal of protein aggregation

  20. Metabolic labeling of cellular glycoproteins with glucosamine: potential for erroneous interpretations due to nonenzymatic radiolabeling of proteins

    International Nuclear Information System (INIS)

    Briles, E.I.B.; Updyke, T.V.

    1986-01-01

    Proteins, including serum proteins of culture media, become nonenzymatically radiolabeled under conditions used for metabolic labeling of cultured cells with glucosamine. This occurs even under sterile conditions in the absence of cells. Various commercial lots of 3 H or 14 C glcN gave similar results: ∼ 0.7% of total label was incorporated into 20% serum (14 mg/ml protein) in 48 h at 37 0 C. By SDS-PAGE fluorography, labeled serum bands correspond to Coomassie stained bands. Incorporation is linear with protein concentration and label input, shows biphasic kinetics (initial rapid rate within first 3 hr, followed by slower linear rate with no sign of saturation through 120 hr), and is temperature-dependent (no reaction at 0 0 C; incorporation at 20 0 C is ∼ 45% of that at 37 0 C). Poly-D-lysine is a better acceptor than protein: 0.5 mg/ml PL accepts as much label as 7 mg/ml protein. Incorporation is inhibited by excess unlabeled glcN and ethanolamine, but not by man, gal or glucose. However, when proteins were incubated with 160 mM glcN, SDS-PAGE bands were yellow-brown, suggesting the occurrence of Maillard-type reactions. Although the chemical mechanism(s) responsible for nonmetabolic radiolabeling by glcN are not clear at this point, the fact that it occurs represents a serious artifact which may lead to erroneous interpretation of data

  1. Intestinal cellular localization of PCNA protein and CYP1A mRNA in Atlantic salmon Salmo salar L. exposed to a model toxicant

    Directory of Open Access Journals (Sweden)

    Olsvik Pål A

    2009-03-01

    Full Text Available Abstract Background The aim of the study was to examine the intestinal cellular localization of proliferating cell nuclear antigen (PCNA and cytochrome P450 A1 (CYP1A expression in Atlantic salmon Salmo salar L. exposed to a model toxicant. The stress response was induced by intraperitoneal injection of four salmon with a single dose (50 mg/kg of the CYP1A inducer β-naphthoflavone (BNF and intestinal tissue (mid and distal intestine; MI and DI was sampled seven days later. Samples for histology and gene transcription analysis were collected from four exposed fish and four control fish. PCNA was assessed by immunohistochemistry, CYP1A mRNA was studied by in situ hybridization (ISH and finally the transcription of five genes was quantified by real-time quantitative RT-PCR (real-time RT-PCR; two detoxifying genes (CYP1A and glutathione S-transferase; GST, a stress marker gene (heat shock protein 70; HSP70, PCNA and a gene marker of apoptosis (caspase 6A. Results PCNA protein and CYP1A mRNA were successfully localized in the intestinal cells (MI of both experimental groups. At the cellular level, BNF significantly lowered intestinal cell proliferation and increased the CYP1A mRNA levels compared to the control group. The real-time RT-PCR data, which showed an increased mRNA expression both in the MI and DI of 139- and 62-fold, respectively, confirmed the increased cellular CYP1A mRNA levels detected using ISH. HSP70 expression was also up-regulated in the exposed fish. The other examined genes did not show any differential regulation in the experimental fish group. Conclusion This study showed that CYP1A mRNA had a specific intestinal cellular transcription pattern in Atlantic salmon exposed to BNF. At the cellular level CYP1A mRNA expression was always observed at or around the cell nucleus close to the basolateral cell membrane and at the tissue level CYP1A mRNA expression was most frequently observed in the basal and apex area of the intestinal

  2. Contrasting evolutionary patterns of spore coat proteins in two Bacillus species groups are linked to a difference in cellular structure

    Science.gov (United States)

    2013-01-01

    Background The Bacillus subtilis-group and the Bacillus cereus-group are two well-studied groups of species in the genus Bacillus. Bacteria in this genus can produce a highly resistant cell type, the spore, which is encased in a complex protective protein shell called the coat. Spores in the B. cereus-group contain an additional outer layer, the exosporium, which encircles the coat. The coat in B. subtilis spores possesses inner and outer layers. The aim of this study is to investigate whether differences in the spore structures influenced the divergence of the coat protein genes during the evolution of these two Bacillus species groups. Results We designed and implemented a computational framework to compare the evolutionary histories of coat proteins. We curated a list of B. subtilis coat proteins and identified their orthologs in 11 Bacillus species based on phylogenetic congruence. Phylogenetic profiles of these coat proteins show that they can be divided into conserved and labile ones. Coat proteins comprising the B. subtilis inner coat are significantly more conserved than those comprising the outer coat. We then performed genome-wide comparisons of the nonsynonymous/synonymous substitution rate ratio, dN/dS, and found contrasting patterns: Coat proteins have significantly higher dN/dS in the B. subtilis-group genomes, but not in the B. cereus-group genomes. We further corroborated this contrast by examining changes of dN/dS within gene trees, and found that some coat protein gene trees have significantly different dN/dS between the B subtilis-clade and the B. cereus-clade. Conclusions Coat proteins in the B. subtilis- and B. cereus-group species are under contrasting selective pressures. We speculate that the absence of the exosporium in the B. subtilis spore coat effectively lifted a structural constraint that has led to relaxed negative selection pressure on the outer coat. PMID:24283940

  3. DNA mismatch repair proteins MLH1 and PMS2 can be imported to the nucleus by a classical nuclear import pathway.

    Science.gov (United States)

    de Barros, Andrea C; Takeda, Agnes A S; Dreyer, Thiago R; Velazquez-Campoy, Adrian; Kobe, Boštjan; Fontes, Marcos R M

    2018-03-01

    MLH1 and PMS2 proteins form the MutLα heterodimer, which plays a major role in DNA mismatch repair (MMR) in humans. Mutations in MMR-related proteins are associated with cancer, especially with colon cancer. The N-terminal region of MutLα comprises the N-termini of PMS2 and MLH1 and, similarly, the C-terminal region of MutLα is composed by the C-termini of PMS2 and MLH1, and the two are connected by linker region. The nuclear localization sequences (NLSs) necessary for the nuclear transport of the two proteins are found in this linker region. However, the exact NLS sequences have been controversial, with different sequences reported, particularly for MLH1. The individual components are not imported efficiently, presumably due to their C-termini masking their NLSs. In order to gain insights into the nuclear transport of these proteins, we solved the crystal structures of importin-α bound to peptides corresponding to the supposed NLSs of MLH1 and PMS2 and performed isothermal titration calorimetry to study their binding affinities. Both putative MLH1 and PMS2 NLSs can bind to importin-α as monopartite NLSs, which is in agreement with some previous studies. However, MLH1-NLS has the highest affinity measured by a natural NLS peptide, suggesting a major role of MLH1 protein in nuclear import compared to PMS2. Finally, the role of MLH1 and PMS2 in the nuclear transport of the MutLα heterodimer is discussed. Copyright © 2017 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.

  4. Down-regulation of cellular FLICE-inhibitory protein (Long Form contributes to apoptosis induced by Hsp90 inhibition in human lung cancer cells

    Directory of Open Access Journals (Sweden)

    Wang Qilin

    2012-12-01

    Full Text Available Abstract Background Cellular FLICE-Inhibitory Protein (long form, c-FLIPL is a critical negative regulator of death receptor-mediated apoptosis. Overexpression of c-FLIPL has been reported in many cancer cell lines and is associated with chemoresistance. In contrast, down-regulation of c-FLIP may drive cancer cells into cellular apoptosis. This study aims to demonstrate that inhibition of the heat shock protein 90 (Hsp90 either by inhibitors geldanamycin/17-N-Allylamino-17-demethoxygeldanamycin (GA/17-AAG or siRNA technique in human lung cancer cells induces c-FLIPL degradation and cellular apoptosis through C-terminus of Hsp70-interacting protein (CHIP-mediated mechanisms. Methods Calu-1 and H157 cell lines (including H157-c-FLIPL overexpressing c-FLIPL and control cell H157-lacZ were treated with 17-AAG and the cell lysates were prepared to detect the given proteins by Western Blot and the cell survival was assayed by SRB assay. CHIP and Hsp90 α/β proteins were knocked down by siRNA technique. CHIP and c-FLIPL plasmids were transfected into cells and immunoprecipitation experiments were performed to testify the interactions between c-FLIPL, CHIP and Hsp90. Results c-FLIPL down-regulation induced by 17-AAG can be reversed with the proteasome inhibitor MG132, which suggested that c-FLIPL degradation is mediated by a ubiquitin-proteasome system. Inhibition of Hsp90α/β reduced c-FLIPL level, whereas knocking down CHIP expression with siRNA technique inhibited c-FLIPL degradation. Furthermore, c-FLIPL and CHIP were co-precipitated in the IP complexes. In addition, overexpression of c-FLIPL can rescue cancer cells from apoptosis. When 17-AAG was combined with an anti-cancer agent celecoxib(CCB, c-FLIPL level declined further and there was a higher degree of caspase activation. Conclusion We have elucidated c-FLIPL degradation contributes to apoptosis induced by Hsp90 inhibition, suggesting c-FLIP and Hsp90 may be the promising combined targets

  5. Direct protein-protein interaction between PLCγ1 and the bradykinin B2 receptor-Importance of growth conditions

    International Nuclear Information System (INIS)

    Duchene, Johan; Chauhan, Sharmila D.; Lopez, Frederic; Pecher, Christiane; Esteve, Jean-Pierre; Girolami, Jean-Pierre; Bascands, Jean-Loup; Schanstra, Joost P.

    2005-01-01

    Recently, we have described a novel protein-protein interaction between the G-protein coupled bradykinin B2 receptor and tyrosine phosphatase SHP-2 via an immunoreceptor tyrosine-based inhibition motif (ITIM) sequence located in the C-terminal part of the B2 receptor and the Src homology (SH2) domains of SHP-2. Here we show that phospholipase C (PLC)γ1, another SH2 domain containing protein, can also interact with this ITIM sequence. Using surface plasmon resonance analysis, we observed that PLCγ1 interacted with a peptide containing the phosphorylated form of the bradykinin B2 receptor ITIM sequence. In CHO cells expressing the wild-type B2 receptor, bradykinin-induced transient recruitment and activation of PLCγ1. Interestingly, this interaction was only observed in quiescent and not in proliferating cells. Mutation of the key ITIM residue abolished this interaction with and activation of PLCγ1. Finally we also identified bradykinin-induced PLCγ1 recruitment and activation in primary culture renal mesangial cells

  6. Identification of Proteins Bound to Dengue Viral RNA In Vivo Reveals New Host Proteins Important for Virus Replication

    Directory of Open Access Journals (Sweden)

    Stacia L. Phillips

    2016-01-01

    Full Text Available Dengue virus is the most prevalent cause of arthropod-borne infection worldwide. Due to the limited coding capacity of the viral genome and the complexity of the viral life cycle, host cell proteins play essential roles throughout the course of viral infection. Host RNA-binding proteins mediate various aspects of virus replication through their physical interactions with viral RNA. Here we describe a technique designed to identify such interactions in the context of infected cells using UV cross-linking followed by antisense-mediated affinity purification and mass spectrometry. Using this approach, we identified interactions, several of them novel, between host proteins and dengue viral RNA in infected Huh7 cells. Most of these interactions were subsequently validated using RNA immunoprecipitation. Using small interfering RNA (siRNA-mediated gene silencing, we showed that more than half of these host proteins are likely involved in regulating virus replication, demonstrating the utility of this method in identifying biologically relevant interactions that may not be identified using traditional in vitro approaches.

  7. Ebselen impairs cellular oxidative state and induces endoplasmic reticulum stress and activation of crucial mitogen-activated protein kinases in pancreatic tumour AR42J cells.

    Science.gov (United States)

    Santofimia-Castaño, Patricia; Izquierdo-Alvarez, Alicia; Plaza-Davila, María; Martinez-Ruiz, Antonio; Fernandez-Bermejo, Miguel; Mateos-Rodriguez, Jose M; Salido, Gines M; Gonzalez, Antonio

    2018-01-01

    Ebselen (2-phenyl-1,2-benzisoselenazol-3(2H)-one) is an organoselenium radical scavenger compound, which has strong antioxidant and anti-inflammatory effects. However, evidence suggests that this compound could exert deleterious actions on cell physiology. In this study, we have analyzed the effect of ebselen on rat pancreatic AR42J cells. Cytosolic free-Ca 2+ concentration ([Ca 2+ ] c ), cellular oxidative status, setting of endoplasmic reticulum stress, and phosphorylation of major mitogen-activated protein kinases were analyzed. Our results show that ebselen evoked a concentration-dependent increase in [Ca 2+ ] c . The compound induced an increase in the generation of reactive oxygen species in the mitochondria. We also observed an increase in global cysteine oxidation in the presence of ebselen. In the presence of ebselen an impairment of cholecystokinin-evoked amylase release was noted. Moreover, involvement of the unfolded protein response markers, ER chaperone and signaling regulator GRP78/BiP, eukaryotic translation initiation factor 2α and X-box binding protein 1 was detected. Finally, increases in the phosphorylation of SAPK/JNK, p38 MAPK, and p44/42 MAPK in the presence of ebselen were also observed. Our results provide evidences for an impairment of cellular oxidative state and enzyme secretion, the induction of endoplasmic reticulum stress and the activation of crucial mitogen-activated protein kinases in the presence of ebselen. As a consequence ebselen exerts a potential toxic effect on AR42J cells. © 2017 Wiley Periodicals, Inc.

  8. The Cellular DNA Helicase ChlR1 Regulates Chromatin and Nuclear Matrix Attachment of the Human Papillomavirus 16 E2 Protein and High-Copy-Number Viral Genome Establishment.

    Science.gov (United States)

    Harris, Leanne; McFarlane-Majeed, Laura; Campos-León, Karen; Roberts, Sally; Parish, Joanna L

    2017-01-01

    In papillomavirus infections, the viral genome is established as a double-stranded DNA episome. To segregate the episomes into daughter cells during mitosis, they are tethered to cellular chromatin by the viral E2 protein. We previously demonstrated that the E2 proteins of diverse papillomavirus types, including bovine papillomavirus (BPV) and human papillomavirus 16 (HPV16), associate with the cellular DNA helicase ChlR1. This virus-host interaction is important for the tethering of BPV E2 to mitotic chromatin and the stable maintenance of BPV episomes. The role of the association between E2 and ChlR1 in the HPV16 life cycle is unresolved. Here we show that an HPV16 E2 Y131A mutant (E2 Y131A ) had significantly reduced binding to ChlR1 but retained transcriptional activation and viral origin-dependent replication functions. Subcellular fractionation of keratinocytes expressing E2 Y131A showed a marked change in the localization of the protein. Compared to that of wild-type E2 (E2 WT ), the chromatin-bound pool of E2 Y131A was decreased, concomitant with an increase in nuclear matrix-associated protein. Cell cycle synchronization indicated that the shift in subcellular localization of E2 Y131A occurred in mid-S phase. A similar alteration between the subcellular pools of the E2 WT protein occurred upon ChlR1 silencing. Notably, in an HPV16 life cycle model in primary human keratinocytes, mutant E2 Y131A genomes were established as episomes, but at a markedly lower copy number than that of wild-type HPV16 genomes, and they were not maintained upon cell passage. Our studies indicate that ChlR1 is an important regulator of the chromatin association of E2 and of the establishment and maintenance of HPV16 episomes. Infections with high-risk human papillomaviruses (HPVs) are a major cause of anogenital and oropharyngeal cancers. During infection, the circular DNA genome of HPV persists within the nucleus, independently of the host cell chromatin. Persistence of infection

  9. Cellular La protein shields nonsegmented negative-strand RNA viral leader RNA from RIG-I and enhances virus growth by diverse mechanisms.

    Science.gov (United States)

    Bitko, Vira; Musiyenko, Alla; Bayfield, Mark A; Maraia, Richard J; Barik, Sailen

    2008-08-01

    The La antigen (SS-B) associates with a wide variety of cellular and viral RNAs to affect gene expression in multiple systems. We show that La is the major cellular protein found to be associated with the abundant 44-nucleotide viral leader RNA (leRNA) early after infection with respiratory syncytial virus (RSV), a nonsegmented negative-strand RNA virus. Consistent with this, La redistributes from the nucleus to the cytoplasm in RSV-infected cells. Upon RNA interference knockdown of La, leRNA is redirected to associate with the RNA-binding protein RIG-I, a known activator of interferon (IFN) gene expression, and this is accompanied by the early induction of IFN mRNA. These results suggest that La shields leRNA from RIG-I, abrogating the early viral activation of type I IFN. We mapped the leRNA binding function to RNA recognition motif 1 of La and showed that while wild-type La greatly enhanced RSV growth, a La mutant defective in RSV leRNA binding also did not support RSV growth. Comparative studies of RSV and Sendai virus and the use of IFN-negative Vero cells indicated that La supports the growth of nonsegmented negative-strand RNA viruses by both IFN suppression and a potentially novel IFN-independent mechanism.

  10. The Effect of a p38 Mitogen-Activated Protein Kinase Inhibitor on Cellular Senescence of Cultivated Human Corneal Endothelial Cells.

    Science.gov (United States)

    Hongo, Akane; Okumura, Naoki; Nakahara, Makiko; Kay, EunDuck P; Koizumi, Noriko

    2017-07-01

    We have begun a clinical trial of a cell-based therapy for corneal endothelial dysfunction in Japan. The purpose of this study was to investigate the usefulness of a p38 MAPK inhibitor for prevention cellular senescence in cultivated human corneal endothelial cells (HCECs). HCECs of 10 donor corneas were divided and cultured with or without SB203580 (a p38 MAPK inhibitor). Cell density and morphology were evaluated by phase-contrast microscopy. Expression of function-related proteins was examined by immunofluorescent staining. Cellular senescence was evaluated by SA-β-gal staining and Western blotting for p16 and p21. Senescence-associated factors were evaluated by membrane blotting array, quantitative PCR, and ELISA. Phase-contrast microscopy showed a significantly higher cell density for HCECs cultured with SB203580 than without SB203580 (2623 ± 657 cells/mm2 and 1752 ± 628 cells/mm2, respectively). The HCECs cultured with SB203580 maintained a hexagonal morphology and expressed ZO-1, N-cadherin, and Na+/K+-ATPase in the plasma membrane, whereas the control HCECs showed an altered staining pattern for these marker proteins. HCECs cultured without SB203580 showed high positive SA-β-gal staining, a low nuclear/cytoplasm ratio, and expression of p16 and p21. IL-6, IL-8, CCL2, and CXCL1 were observed at high levels in low cell density HCECs cultured without SB203580. Activation of p38 MAPK signaling due to culture stress might be a causative factor that induces cellular senescence; therefore, the use of p38 MAPK inhibitor to counteract senescence may achieve sufficient numbers of HCECs for tissue engineering therapy for corneal endothelial dysfunction.

  11. Mechanisms and Effects on HBV Replication of the Interaction between HBV Core Protein and Cellular Filamin B.

    Science.gov (United States)

    Li, Yilin; Sun, Yishuang; Sun, Fuyun; Hua, Rong; Li, Chenlin; Chen, Lang; Guo, Deyin; Mu, Jingfang

    2018-03-28

    Hepatitis B virus (HBV) infection is one of the major problems that threatens global health. There have been many studies on HBV, but the relationship between HBV and host factors is largely unexplored and more studies are needed to clarify these interactions. Filamin B is an actin-binding protein that acts as a cytoskeleton protein, and it is involved in cell development and several signaling pathways. In this study, we showed that filamin B interacted with HBV core protein, and the interaction promoted HBV replication. The interaction between filamin B and core protein was observed in HEK 293T, Huh7 and HepG2 cell lines by co-immunoprecipitation and co-localization immnofluoresence. Overexpression of filamin B increased the levels of HBV total RNAs and pre-genome RNA (pgRNA), and improved the secretion level of hepatitis B surface antigen (HBsAg) and hepatitis B e antigen (HBeAg). In contrast, filamin B knockdown inhibited HBV replication, decreased the level of HBV total RNAs and pgRNA, and reduced the secretion level of HBsAg and HBeAg. In addition, we found that filamin B and core protein may interact with each other via four blocks of argentine residues at the C-terminus of core protein. In conclusion, we identify filamin B as a novel host factor that can interact with core protein to promote HBV replication in hepatocytes. Our study provides new insights into the relationship between HBV and host factors and may provide new strategies for the treatment of HBV infection.

  12. Quantum dot bio-conjugate: as a western blot probe for highly sensitive detection of cellular proteins

    Energy Technology Data Exchange (ETDEWEB)

    Kale, Sonia [Agharkar Research Institute (India); Kale, Anup [University of Alabama, Center for Materials for Information Technology (United States); Gholap, Haribhau; Rana, Abhimanyu [National Chemical Laboratory, Physical and Materials Chemistry Division (India); Desai, Rama [National Centre for Cell Science (India); Banpurkar, Arun [University of Pune, Department of Physics (India); Ogale, Satishchandra, E-mail: sb.ogale@ncl.res.in [National Chemical Laboratory, Physical and Materials Chemistry Division (India); Shastry, Padma, E-mail: padma@nccs.res.in [National Centre for Cell Science (India)

    2012-03-15

    In the present study, we report a quantum dot (QD)-tailored western blot analysis for a sensitive, rapid and flexible detection of the nuclear and cytoplasmic proteins. Highly luminescent CdTe and (CdTe)ZnS QDs are synthesized by aqueous method. High resolution transmission electron microscopy, Raman spectroscopy, fourier transform infrared spectroscopy, fluorescence spectroscopy and X-ray diffraction are used to characterize the properties of the quantum dots. The QDs are functionalized with antibodies of prostate apoptosis response-4 (Par-4), poly(ADP-ribose) polymerases and {beta} actin to specifically bind with the proteins localized in the nucleus and cytoplasm of the cells, respectively. The QD-conjugated antibodies are used to overcome the limitations of conventional western blot technique. The sensitivity and rapidity of protein detection in QD-based approach is very high, with detection limits up to 10 pg of protein. In addition, these labels provide the capability of enhanced identification and localization of marker proteins in intact cells by confocal laser scanning microscopy.

  13. Programming Post-Translational Control over the Metabolic Labeling of Cellular Proteins with a Noncanonical Amino Acid.

    Science.gov (United States)

    Thomas, Emily E; Pandey, Naresh; Knudsen, Sarah; Ball, Zachary T; Silberg, Jonathan J

    2017-08-18

    Transcriptional control can be used to program cells to label proteins with noncanonical amino acids by regulating the expression of orthogonal aminoacyl tRNA synthetases (aaRSs). However, we cannot yet program cells to control labeling in response to aaRS and ligand binding. To identify aaRSs whose activities can be regulated by interactions with ligands, we used a combinatorial approach to discover fragmented variants of Escherichia coli methionyl tRNA synthetase (MetRS) that require fusion to associating proteins for maximal activity. We found that these split proteins could be leveraged to create ligand-dependent MetRS using two approaches. When a pair of MetRS fragments was fused to FKBP12 and the FKBP-rapamycin binding domain (FRB) of mTOR and mutations were introduced that direct substrate specificity toward azidonorleucine (Anl), Anl metabolic labeling was significantly enhanced in growth medium containing rapamycin, which stabilizes the FKBP12-FRB complex. In addition, fusion of MetRS fragments to the termini of the ligand-binding domain of the estrogen receptor yielded proteins whose Anl metabolic labeling was significantly enhanced when 4-hydroxytamoxifen (4-HT) was added to the growth medium. These findings suggest that split MetRS can be fused to a range of ligand-binding proteins to create aaRSs whose metabolic labeling activities depend upon post-translational interactions with ligands.

  14. The small delta antigen of hepatitis delta virus is an acetylated protein and acetylation of lysine 72 may influence its cellular localization and viral RNA synthesis

    International Nuclear Information System (INIS)

    Mu, J.-J.; Tsay, Y.-G.; Juan, L.-J.; Fu, T.-F.; Huang, W.-H.; Chen, D.-S.; Chen, P.-J.

    2004-01-01

    Hepatitis delta virus (HDV) is a single-stranded RNA virus that encodes two viral nucleocapsid proteins named small and large form hepatitis delta antigen (S-HDAg and L-HDAg). The S-HDAg is essential for viral RNA replication while the L-HDAg is required for viral assembly. In this study, we demonstrated that HDAg are acetylated proteins. Metabolic labeling with [ 3 H]acetate revealed that both forms of HDAg could be acetylated in vivo. The histone acetyltransferase (HAT) domain of cellular acetyltransferase p300 could acetylate the full-length and the N-terminal 88 amino acids of S-HDAg in vitro. By mass spectrometric analysis of the modified protein, Lys-72 of S-HDAg was identified as one of the acetylation sites. Substitution of Lys-72 to Arg caused the mutant S-HDAg to redistribute from the nucleus to the cytoplasm. The mutant reduced viral RNA accumulation and resulted in the earlier appearance of L-HDAg. These results demonstrated that HDAg is an acetylated protein and mutation of HDAg at Lys-72 modulates HDAg subcellular localization and may participate in viral RNA nucleocytoplasmic shuttling and replication

  15. The challenges in and importance of analysing protein structure and physical stability in complex formulations

    DEFF Research Database (Denmark)

    Jorgensen, L.; Jensen, Minna Grønning; Roest, N.

    2013-01-01

    In this review several analytical challenges that may be encountered during protein formulation development of complex formulations are discussed through recent examples. These examples show how selected advanced biophysical methods can greatly increase our understanding of the system under...

  16. Myristic acid, a rare fatty acid, is the lipid attached to the transforming protein of Rous sarcoma virus and its cellular homolog

    International Nuclear Information System (INIS)

    Buss, J.E.; Sefton, B.M.

    1985-01-01

    The lipid bound to p60/sub src/, the transforming protein of Rous sarcoma virus, has been identified by gas and thin-layer chromatography as the 14-carbon saturated fatty acid, myristic acid. The protein can be labeled biosynthetically with either [ 3 H]myristic acid or [ 3 H]palmitic acid. Incorporation of [ 3 H]myristic acid was noticeably greater than incorporation of [ 3 H]palmitic acid. All of the [ 3 H]myristic acid-derived label in p60/sub src/ was present as myristic acid. In contrast, none of the radioactivity derived from [ 3 H]palmitic acid was recovered as palmitic acid. Instead, all 3 H incorporated into p60/sub src/ from [ 3 H]palmitic acid arose by metabolism to myristic acid. The cellular tyrosine kinase, p60c-/sub src/ also contains myristic acid. By comparison of the extent of myristylation of p60v-/sub src/ with that of the Moloney murine leukemia virus structural protein precursor, Pr65gag, the authors estimate that greater than 80% of the molecules of p60v-/sub src/ contain one molecule of this fatty acid. Myristylation is a rare form of protein modification. p60v-/sub src/ contains 10 to 40% of the myristic acid bound to protein in cells transformed by Rous sarcoma virus and is easily identified in total cell lysates when [ 3 H]myristic acid-labeled proteins are separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Comparison of the amount of [ 3 H]myristic acid-labeled p60/sub src/ in total cell lysates and in immunoprecipitates suggests that immunoprecipitation with rabbit anti-Rous sarcoma virus tumor sera detects ca. 25% of the p60/sub src/ present in cells

  17. Radiation-induced structural changes in membrane proteins of human erythrocytes and ghosts and the relation to cellular morphology

    Energy Technology Data Exchange (ETDEWEB)

    Schuurhuis, G.J.; Hommes, J.; Vos, J.; Molenaar, I.; Konings, A.W.T. (Rijksuniversiteit Groningen (Netherlands))

    1984-02-01

    Isolated human erythrocytes and ghosts were irradiated with X-rays under different experimental conditions and the effect examined with regard to the structure of membrane proteins and morphology of whole cells and ghosts. From sodium dodecyl sulphate/polyacrylamide gel electrophoresis it is concluded that spectrin (band 1 and 2) is the most radiosensitive of the membrane proteins examined. X-irradiation of cells and ghosts induced covalent cross-linking of a small fraction of membrane proteins. In the protein aggregates thus formed spectrin was found to be the major component. Molecular disulphide (-SS-) bridges seemed to account for part of the cross-links observed. Some nondisulphide cross-links were found, especially when ghosts were irradiated. Significant amounts of spectrin aggregates were formed during post-irradiation incubation at 37/sup 0/C but not at 4/sup 0/C. In the intact cell a transformation in shape from discocyte to echinocyte accompanied the process of post-irradiation spectrin aggregation. The characteristics of both processes, such as their reversibility with adenosine, point to a metabolic involvement. It is shown that there is no causal relationship between the two phenomena observed. Possible causes of the post-irradiation effects and the parallelism with similar processes in non-irradiated metabolically depleted cells are discussed.

  18. Radiation-induced structural changes in membrane proteins of human erythrocytes and ghosts and the relation to cellular morphology

    International Nuclear Information System (INIS)

    Schuurhuis, G.J.; Hommes, J.; Vos, J.; Molenaar, I.; Konings, A.W.T.

    1984-01-01

    Isolated human erythrocytes and ghosts were irradiated with X-rays under different experimental conditions and the effect examined with regard to the structure of membrane proteins and morphology of whole cells and ghosts. From sodium dodecyl sulphate/polyacrylamide gel electrophoresis it is concluded that spectrin (band 1 and 2) is the most radiosensitive of the membrane proteins examined. X-irradiation of cells and ghosts induced covalent cross-linking of a small fraction of membrane proteins. In the protein aggregates thus formed spectrin was found to be the major component. Molecular disulphide (-SS-) bridges seemed to account for part of the cross-links observed. Some nondisulphide cross-links were found, especially when ghosts were irradiated. Significant amounts of spectrin aggregates were formed during post-irradiation incubation at 37 0 C but not at 4 0 C. In the intact cell a transformation in shape from discocyte to echinocyte accompanied the process of post-irradiation spectrin aggregation. The characteristics of both processes, such as their reversibility with adenosine, point to a metabolic involvement. It is shown that there is no causal relationship between the two phenomena observed. Possible causes of the post-irradiation effects and the parallelism with similar processes in non-irradiated metabolically depleted cells are discussed. (author)

  19. Optimized labeling of membrane proteins for applications to super-resolution imaging in confined cellular environments using monomeric streptavidin.

    Science.gov (United States)

    Chamma, Ingrid; Rossier, Olivier; Giannone, Grégory; Thoumine, Olivier; Sainlos, Matthieu

    2017-04-01

    Recent progress in super-resolution imaging (SRI) has created a strong need to improve protein labeling with probes of small size that minimize the target-to-label distance, increase labeling density, and efficiently penetrate thick biological tissues. This protocol describes a method for labeling genetically modified proteins incorporating a small biotin acceptor peptide with a 3-nm fluorescent probe, monomeric streptavidin. We show how to express, purify, and conjugate the probe to organic dyes with different fluorescent properties, and how to label selectively biotinylated membrane proteins for SRI techniques (point accumulation in nanoscale topography (PAINT), stimulated emission depletion (STED), stochastic optical reconstruction microscopy (STORM)). This method is complementary to the previously described anti-GFP-nanobody/SNAP-tag strategies, with the main advantage being that it requires only a short 15-amino-acid tag, and can thus be used with proteins resistant to fusion with large tags and for multicolor imaging. The protocol requires standard molecular biology/biochemistry equipment, making it easily accessible for laboratories with only basic skills in cell biology and biochemistry. The production/purification/conjugation steps take ∼5 d, and labeling takes a few minutes to an hour.

  20. Decreased PARP and procaspase-2 protein levels are associated with cellular drug resistance in childhood acute lymphoblastic leukemia

    NARCIS (Netherlands)

    A. Holleman (Amy); M.L. den Boer (Monique); K.M. Kazemier (Karin); H.B. Beverloo (Berna); A.R.M. von Bergh (Anne); G.E. Janka-Schaub (Gritta); R. Pieters (Rob)

    2005-01-01

    textabstractDrug resistance in childhood acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML) is associated with impaired ability to induce apoptosis. To elucidate causes of apoptotic defects, we studied the protein expression of Apaf-1, procaspases-2, -3, -6, -7,

  1. Accessible surface area of proteins from purely sequence information and the importance of global features

    Science.gov (United States)

    Faraggi, Eshel; Zhou, Yaoqi; Kloczkowski, Andrzej

    2014-03-01

    We present a new approach for predicting the accessible surface area of proteins. The novelty of this approach lies in not using residue mutation profiles generated by multiple sequence alignments as descriptive inputs. Rather, sequential window information and the global monomer and dimer compositions of the chain are used. We find that much of the lost accuracy due to the elimination of evolutionary information is recouped by the use of global features. Furthermore, this new predictor produces similar results for proteins with or without sequence homologs deposited in the Protein Data Bank, and hence shows generalizability. Finally, these predictions are obtained in a small fraction (1/1000) of the time required to run mutation profile based prediction. All these factors indicate the possible usability of this work in de-novo protein structure prediction and in de-novo protein design using iterative searches. Funded in part by the financial support of the National Institutes of Health through Grants R01GM072014 and R01GM073095, and the National Science Foundation through Grant NSF MCB 1071785.

  2. A 7-day high protein hypocaloric diet promotes cellular metabolic adaptations and attenuates lean mass loss in healthy males

    Directory of Open Access Journals (Sweden)

    Matthew Furber

    2017-08-01

    Full Text Available Mitochondrial quantity and density are associated with increased oxidative metabolism. It has been demonstrated that a hypocaloric high fat/low carbohydrate (HF/LC diet can up-regulate transcriptional markers of mitochondrial biogenesis; this was yet to be explored in vivo subsequent to a high protein/low carbohydrate (HP/LC diet. Thus the aims of the study were to explore such diets on transcriptional markers or mitochondrial biogenesis, body composition and resting metabolic rate (RMR. Forty-five healthy male participants were randomly assigned one of four intervention diets: eucaloric high protein low carbohydrate (PRO-EM, hypocaloric high protein low carbohydrate (PRO-ER, eucaloric high carbohydrate (CHO-EM or hypocaloric high carbohydrate (CHO-ER. The macronutrient ratio of the high protein diet and high carbohydrate diets was 40:30:30% and 10:60:30% (PRO:CHO:FAT respectively. Energy intake for the hypocaloric diets were calculated to match resting metabolic rate. Participants visited the laboratory on 3 occasions each separated by 7 days. On each visit body composition, resting metabolic rate and a muscle biopsy from the vastus lateralis was collected. Prior to visit 1 and 2 habitual diet was consumed which was used as a control, between visit 2 and 3 the intervention diet was consumed continuously for 7-days. No group × time effect was observed, however in the PRO-ER group a significant increase in AMPK, PGC-1α, SIRT1 and SIRT3 mRNA expression was observed post diet intervention groups (p < 0.05. No change was observed in any of the transcriptional markers in the other 3 groups. Despite ∼30% reduction in calorie intake no difference in lean mass (LM loss was observed between the PRO-ER and CHO-EM groups. The results from this study suggest that a 7-day a high protein low carbohydrate hypocaloric diet increased AMPK, SIRT1 and PGC-1 α mRNA expression at rest, and also suggest that increased dietary protein may attenuate LM mass

  3. Phosphorylation near nuclear localization signal regulates nuclear import of adenomatous polyposis coli protein

    OpenAIRE

    Zhang, Fang; White, Raymond L.; Neufeld, Kristi L.

    2000-01-01

    Mutation of the adenomatous polyposis coli (APC) gene is an early step in the development of colorectal carcinomas. APC protein is located in both the cytoplasm and the nucleus. The objective of this study was to define the nuclear localization signals (NLSs) in APC protein. APC contains two potential NLSs comprising amino acids 1767–1772 (NLS1APC) and 2048–2053 (NLS2APC). Both APC NLSs are well conserved among human, mouse, rat, and fly. NLS1APC and NLS2APC each w...

  4. Cellular gravity

    NARCIS (Netherlands)

    F.C. Gruau; J.T. Tromp (John)

    1999-01-01

    textabstractWe consider the problem of establishing gravity in cellular automata. In particular, when cellular automata states can be partitioned into empty, particle, and wall types, with the latter enclosing rectangular areas, we desire rules that will make the particles fall down and pile up on

  5. An early function of the adenoviral E1B 55 kDa protein is required for the nuclear relocalization of the cellular p53 protein in adenovirus-infected normal human cells

    International Nuclear Information System (INIS)

    Cardoso, F.M.; Kato, Sayuri E.M.; Huang Wenying; Flint, S. Jane; Gonzalez, Ramon A.

    2008-01-01

    It is well established that the human subgroup C adenovirus type 5 (Ad5) E1B 55 kDa protein can regulate the activity and concentration of the cellular tumor suppressor, p53. However, the contribution(s) of these functions of the E1B protein to viral reproduction remains unclear. To investigate this issue, we examined properties of p53 in normal human cells infected by E1B mutant viruses that display defective entry into the late phase or viral late mRNA export. The steady-state concentrations of p53 were significantly higher in cells infected by the E1B 55 kDa null mutant Hr6 or three mutants carrying small insertions in the E1B 55 kDa protein coding sequence than in Ad5-infected cells. Nevertheless, none of the mutants induced apoptosis in infected cells. Rather, the localization of p53 to E1B containing nuclear sites observed during infection by Ad5 was prevented by mutations that impair interaction of the E1B protein with p53 and/or with the E4 Orf6 protein. These results indicate that the E1B protein fulfills an early function that correlates efficient entry into the late phase with the localization of E1B and p53 in the nucleus of Ad5-infected normal human cells

  6. A 7-day high protein hypocaloric diet promotes cellular metabolic adaptations and attenuates lean mass loss in healthy males

    OpenAIRE

    Matthew Furber; Ana Anton-Solanas; Emma Koppe; Charlotte Ashby; Michael Roberts; Justin Roberts

    2017-01-01

    Mitochondrial quantity and density are associated with increased oxidative metabolism. It has been demonstrated that a hypocaloric high fat/low carbohydrate (HF/LC) diet can up-regulate transcriptional markers of mitochondrial biogenesis; this was yet to be explored in vivo subsequent to a high protein/low carbohydrate (HP/LC) diet. Thus the aims of the study were to explore such diets on transcriptional markers or mitochondrial biogenesis, body composition and resting metabolic rate (RMR). F...

  7. Conformational Profiling of the AT1 Angiotensin II Receptor Reflects Biased Agonism, G Protein Coupling, and Cellular Context.

    Science.gov (United States)

    Devost, Dominic; Sleno, Rory; Pétrin, Darlaine; Zhang, Alice; Shinjo, Yuji; Okde, Rakan; Aoki, Junken; Inoue, Asuka; Hébert, Terence E

    2017-03-31

    Here, we report the design and use of G protein-coupled receptor-based biosensors to monitor ligand-mediated conformational changes in receptors in intact cells. These biosensors use bioluminescence resonance energy transfer with Renilla luciferase (RlucII) as an energy donor, placed at the distal end of the receptor C-tail, and the small fluorescent molecule FlAsH as an energy acceptor, its binding site inserted at different positions throughout the intracellular loops and C-terminal tail of the angiotensin II type I receptor. We verified that the modifications did not compromise receptor localization or function before proceeding further. Our biosensors were able to capture effects of both canonical and biased ligands, even to the extent of discriminating between different biased ligands. Using a combination of G protein inhibitors and HEK 293 cell lines that were CRISPR/Cas9-engineered to delete Gα q , Gα 11 , Gα 12 , and Gα 13 or β-arrestins, we showed that Gα q and Gα 11 are required for functional responses in conformational sensors in ICL3 but not ICL2. Loss of β-arrestin did not alter biased ligand effects on ICL2P2. We also demonstrate that such biosensors are portable between different cell types and yield context-dependent readouts of G protein-coupled receptor conformation. Our study provides mechanistic insights into signaling events that depend on either G proteins or β-arrestin. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  8. The pleiotropic effect of WD-40 domain containing proteins on cellular differentiation and production of secondary metabolites in Streptomyces coelicolor

    Czech Academy of Sciences Publication Activity Database

    Ulrych, Aleš; Goldová, Jana; Petříček, Miroslav; Benada, Oldřich; Kofroňová, Olga; Rampírová, Petra; Petříčková, Kateřina; Branny, Pavel

    2013-01-01

    Roč. 9, č. 6 (2013), s. 1453-1469 ISSN 1742-206X R&D Projects: GA MŠk LH12191; GA MŠk LH12055; GA MŠk 2B08064 Institutional support: RVO:61388971 Keywords : AERIAL MYCELIUM FORMATION * WD-REPEAT PROTEINS * BIOSYNTHESIS GENE-CLUSTER Subject RIV: EE - Microbiology, Virology Impact factor: 3.183, year: 2013

  9. Use of Nucleic Acid-Protein Cytophotometry and Cytointerferometry in Characterizing Cellular Mode of Trichothecene Toxicant Action

    Science.gov (United States)

    1986-10-01

    dose of 0.5 mg/k, the predominant bistopathic alterations included serosal petechial hemorrhages, moderate edema of the submucosa with Larea~e in...by a disruption of endothelial cell membranes, pycnotic nuclei, presence of edema in the interstitium and petechial hemorrhages within 2 h after T-2...Townsend. 1982. Inhibition, by trichothecene antibiotics, of brain protein synthesis and fever in rabbits. J. Physiol., Lond. 322:447. 21. Yarom, R

  10. Involvement of the Iron Regulatory Protein from Eisenia andrei Earthworms in the Regulation of Cellular Iron Homeostasis

    Czech Academy of Sciences Publication Activity Database

    Procházková, Petra; Škanta, František; Roubalová, Radka; Šilerová, Marcela; Dvořák, Jiří; Bilej, Martin

    2014-01-01

    Roč. 9, č. 10 (2014) E-ISSN 1932-6203 R&D Projects: GA MŠk(CZ) ED1.1.00/02.0109; GA MŠk(CZ) EE2.3.20.0055 Institutional support: RVO:61388971 Keywords : MULTIPLE SEQUENCE ALIGNMENT * ELEMENT-BINDING PROTEIN * FERRITIN MESSENGER-RNA Subject RIV: EE - Microbiology, Virology Impact factor: 3.234, year: 2014

  11. Impact of high hydrostatic pressure processing on individual cellular resuscitation times and protein aggregates in Escherichia coli.

    Science.gov (United States)

    Govers, Sander K; Aertsen, Abram

    2015-11-20

    Live cell biology approaches can contribute to a more comprehensive understanding of heterogeneous injury and resuscitation phenomena in stressed populations of foodborne pathogens and spoilage microorganisms, and in turn lead to better insights in the mechanisms and dynamics of inactivation that can improve food safety and preservation measures. Especially in the context of designing minimal processing strategies, which depend on a synergistic combination of different mild stresses to ensure sufficient microbial reduction, a more profound understanding of the impact of each such stress or hurdle is mandatory. High hydrostatic pressure (HHP) stress is an interesting hurdle in this concept since cells that manage to survive this stress nevertheless tend to be injured and sensitized to subsequent stresses. In this study, populations of Escherichia coli were subjected to different HHP intensities and studied at the single-cell level with time-lapse fluorescence microscopy while monitoring resuscitation times and protein aggregate integrity at the single-cell level. This approach revealed that higher pressure intensities lead to longer and more variable resuscitation times of surviving cells as well as an increased dispersal of intracellular protein aggregates. Interestingly, at mild HHP exposure, cells within the population incurring less dispersion of protein aggregates appeared to have a higher probability of survival. Copyright © 2015 Elsevier B.V. All rights reserved.

  12. The Fanconi anemia proteins FAA and FAC function in different cellular compartments to protect against cross-linking agent cytotoxicity.

    Science.gov (United States)

    Kruyt, F A; Youssoufian, H

    1998-10-01

    Fanconi anemia (FA) is an autosomal recessive disease characterized by chromosomal instability, bone marrow failure, and a high risk of developing malignancies. Although the disorder is genetically heterogeneous, all FA cells are defined by their sensitivity to the apoptosis-inducing effect of cross-linking agents, such as mitomycin C (MMC). The cloned FA disease genes, FAC and FAA, encode proteins with no homology to each other or to any known protein. We generated a highly specific antibody against FAA and found the protein in both the cytoplasm and nucleus of mammalian cells. By subcellular fractionation, FAA is also associated with intracellular membranes. To identify the subcellular compartment that is relevant for FAA activity, we appended nuclear export and nuclear localization signals to the carboxy terminus of FAA and enriched its localization in either the cytoplasm or the nucleus. Nuclear localization of FAA was both necessary and sufficient to correct MMC sensitivity in FA-A cells. In addition, we found no evidence for an interaction between FAA and FAC either in vivo or in vitro. Together with a previous finding that FAC is active in the cytoplasm but not in the nucleus, our results indicate that FAA and FAC function in separate subcellular compartments. Thus, FAA and FAC, if functionally linked, are more likely to be in a linear pathway rather than form a macromolecular complex to protect against cross-linker cytotoxicity.

  13. Clinical importance of non-specific lipid transfer proteins as food allergens

    NARCIS (Netherlands)

    van Ree, R.

    2002-01-01

    Non-specific lipid transfer proteins (nsLTPs) have recently been identified as plant food allergens. They are good examples of true food allergens, in the sense that they are capable of sensitizing, i.e. inducing specific IgE, as well as of eliciting severe symptoms. This is in contrast with most

  14. Structure of Human Tyrosinase Related Protein 1 Reveals a Binuclear Zinc Active Site Important for Melanogenesis

    NARCIS (Netherlands)

    Lai, Xuelei; Wichers, Harry J.; Soler-Lopez, Montserrat; Dijkstra, Bauke W.

    2017-01-01

    Tyrosinase-related protein 1 (TYRP1) is one of three tyrosinase-like glycoenzymes in human melanocytes that are key to the production of melanin, the compound responsible for the pigmentation of skin, eye, and hair. Difficulties with producing these enzymes in pure form have hampered the

  15. ZNF143 protein is an important regulator of the myeloid transcription factor C/EBP

    Czech Academy of Sciences Publication Activity Database

    Gonzalez, D.; Luyten, A.; Bartholdy, B.; Zhou, Q.; Kardošová, Miroslava; Ebralidze, A.; Swanson, K.D.; Radomska, H.S.; Zhang, P.; Kobayashi, S.S.; Welner, R.S.; Levantini, E.; Steidl, U.; Chong, G.; Collombet, S.; Choi, M.H.; Friedman, A.D.; Scott, L.M.; Alberich-Jorda, Meritxell; Tenen, D.G.

    2017-01-01

    Roč. 292, č. 46 (2017), s. 18924-18936 ISSN 0021-9258 Institutional support: RVO:68378050 Keywords : CCAAT-enhancer-binding protein * gene regulation * hematopoiesis * promoter * transcription factor * EBPalpha * ZNF143 Subject RIV: EB - Genetics ; Molecular Biology OBOR OECD: Cell biology Impact factor: 4.125, year: 2016

  16. Outer membrane protein A (OmpA: a new player in shigella flexneri protrusion formation and inter-cellular spreading.

    Directory of Open Access Journals (Sweden)

    Cecilia Ambrosi

    Full Text Available Outer membrane protein A (OmpA is a multifaceted predominant outer membrane protein of Escherichia coli and other Enterobacteriaceae whose role in the pathogenesis of various bacterial infections has recently been recognized. Here, the role of OmpA on the virulence of Shigella flexneri has been investigated. An ompA mutant of wild-type S. flexneri 5a strain M90T was constructed (strain HND92 and it was shown to be severely impaired in cell-to-cell spreading since it failed to plaque on HeLa cell monolayers. The lack of OmpA significantly reduced the levels of IcsA while the levels of cell associated and released IcsP-cleaved 95 kDa amino-terminal portion of the mature protein were similar. Nevertheless, the ompA mutant displayed IcsA exposed across the entire bacterial surface. Surprisingly, the ompA mutant produced proper F-actin comet tails, indicating that the aberrant IcsA exposition at bacterial lateral surface did not affect proper activation of actin-nucleating proteins, suggesting that the absence of OmpA likely unmasks mature or cell associated IcsA at bacterial lateral surface. Moreover, the ompA mutant was able to invade and to multiply within HeLa cell monolayers, although internalized bacteria were found to be entrapped within the host cell cytoplasm. We found that the ompA mutant produced significantly less protrusions than the wild-type strain, indicating that this defect could be responsible of its inability to plaque. Although we could not definitely rule out that the ompA mutation might exert pleiotropic effects on other S. flexneri genes, complementation of the ompA mutation with a recombinant plasmid carrying the S. flexneri ompA gene clearly indicated that a functional OmpA protein is required and sufficient for proper IcsA exposition, plaque and protrusion formation. Moreover, an independent ompA mutant was generated. Since we found that both mutants displayed identical virulence profile, these results further supported the

  17. Control of eIF4E cellular localization by eIF4E-binding proteins, 4E-BPs.

    Science.gov (United States)

    Rong, Liwei; Livingstone, Mark; Sukarieh, Rami; Petroulakis, Emmanuel; Gingras, Anne-Claude; Crosby, Katherine; Smith, Bradley; Polakiewicz, Roberto D; Pelletier, Jerry; Ferraiuolo, Maria A; Sonenberg, Nahum

    2008-07-01

    Eukaryotic initiation factor (eIF) 4E, the mRNA 5'-cap-binding protein, mediates the association of eIF4F with the mRNA 5'-cap structure to stimulate cap-dependent translation initiation in the cytoplasm. The assembly of eIF4E into the eIF4F complex is negatively regulated through a family of repressor proteins, called the eIF4E-binding proteins (4E-BPs). eIF4E is also present in the nucleus, where it is thought to stimulate nuclear-cytoplasmic transport of certain mRNAs. eIF4E is transported to the nucleus via its interaction with 4E-T (4E-transporter), but it is unclear how it is retained in the nucleus. Here we show that a sizable fraction (approximately 30%) of 4E-BP1 is localized to the nucleus, where it binds eIF4E. In mouse embryo fibroblasts (MEFs) subjected to serum starvation and/or rapamycin treatment, nuclear 4E-BPs sequester eIF4E in the nucleus. A dramatic loss of nuclear 4E-BP1 occurs in c-Ha-Ras-expressing MEFs, which fail to show starvation-induced nuclear accumulation of eIF4E. Therefore, 4E-BP1 is a regulator of eIF4E cellular l