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Sample records for 12s-lipoxygenase protein associates

  1. 12S-lipoxygenase protein associates with {alpha}-actin fibers in human umbilical artery vascular smooth muscle cells

    SciTech Connect

    Weisinger, Gary . E-mail: gary_w@tasmc.health.gov.il; Limor, Rona; Marcus-Perlman, Yonit; Knoll, Esther; Kohen, Fortune; Schinder, Vera; Firer, Michael; Stern, Naftali

    2007-05-11

    The current study sets out to characterize the intracellular localization of the platelet-type 12S-lipoxygenase (12-LO), an enzyme involved in angiotensin-II induced signaling in vascular smooth muscle cells (VSMC). Immunohistochemical analysis of VSMC in vitro or human umbilical arteries in vivo showed a clear cytoplasmic localization. On immunogold electron microscopy, 12-LO was found primarily associated with cytoplasmic VSMC muscle fibrils. Upon angiotensin-II treatment of cultured VSMC, immunoprecipitated 12-LO was found bound to {alpha}-actin, a component of the cytoplasmic myofilaments. 12-LO/{alpha}-actin binding was blocked by VSMC pretreatment with the 12-LO inhibitors, baicalien or esculetine and the protein synthesis inhibitor, cycloheximide. Moreover, the binding of 12-LO to {alpha}-actin was not associated with 12-LO serine or tyrosine phosphorylation. These observations suggest a previously unrecognized angiotensin-II dependent protein interaction in VSMC through which 12-LO protein may be trafficked, for yet undiscovered purposes towards the much more abundantly expressed cytoskeletal protein {alpha}-actin.

  2. Interfacial Protein-Protein Associations

    PubMed Central

    Langdon, Blake B.; Kastantin, Mark; Walder, Robert; Schwartz, Daniel K.

    2014-01-01

    While traditional models of protein adsorption focus primarily on direct protein-surface interactions, recent findings suggest that protein-protein interactions may play a central role. Using high-throughput intermolecular resonance energy transfer (RET) tracking, we directly observed dynamic, protein-protein associations of bovine serum albumin on poly(ethylene glycol) modified surfaces. The associations were heterogeneous and reversible, and associating molecules resided on the surface for longer times. The appearance of three distinct RET states suggested a spatially heterogeneous surface – with areas of high protein density (i.e. strongly-interacting clusters) coexisting with mobile monomers. Distinct association states exhibited characteristic behavior, i.e. partial-RET (monomer-monomer) associations were shorter-lived than complete-RET (protein-cluster) associations. While the fractional surface area covered by regions with high protein density (i.e. clusters) increased with increasing concentration, the distribution of contact times between monomers and clusters was independent of solution concentration, suggesting that associations were a local phenomenon, and independent of the global surface coverage. PMID:24274729

  3. Designed protein-protein association.

    PubMed

    Grueninger, Dirk; Treiber, Nora; Ziegler, Mathias O P; Koetter, Jochen W A; Schulze, Monika-Sarah; Schulz, Georg E

    2008-01-11

    The analysis of natural contact interfaces between protein subunits and between proteins has disclosed some general rules governing their association. We have applied these rules to produce a number of novel assemblies, demonstrating that a given protein can be engineered to form contacts at various points of its surface. Symmetry plays an important role because it defines the multiplicity of a designed contact and therefore the number of required mutations. Some of the proteins needed only a single side-chain alteration in order to associate to a higher-order complex. The mobility of the buried side chains has to be taken into account. Four assemblies have been structurally elucidated. Comparisons between the designed contacts and the results will provide useful guidelines for the development of future architectures. PMID:18187656

  4. Microtubules, Tubulins and Associated Proteins.

    ERIC Educational Resources Information Center

    Raxworthy, Michael J.

    1988-01-01

    Reviews much of what is known about microtubules, which are biopolymers consisting predominantly of subunits of the globular protein, tubulin. Describes the functions of microtubules, their structure and assembly, microtube associated proteins, and microtubule-disrupting agents. (TW)

  5. A Bayesian Estimator of Protein-Protein Association Probabilities

    SciTech Connect

    Gilmore, Jason M.; Auberry, Deanna L.; Sharp, Julia L.; White, Amanda M.; Anderson, Kevin K.; Daly, Don S.

    2008-07-01

    The Bayesian Estimator of Protein-Protein Association Probabilities (BEPro3) is a software tool for estimating probabilities of protein-protein association between bait and prey protein pairs using data from multiple-bait, multiple-replicate, protein pull-down LC-MS assay experiments. BEPro3 is open source software that runs on both Windows XP and Mac OS 10.4 or newer versions, and is freely available from http://www.pnl.gov/statistics/BEPro3.

  6. Bayesian Estimator of Protein-Protein Association Probabilities

    Energy Science and Technology Software Center (ESTSC)

    2008-05-28

    The Bayesian Estimator of Protein-Protein Association Probabilities (BEPro3) is a software tool for estimating probabilities of protein-protein association between bait and prey protein pairs using data from multiple-bait, multiple-replicate, protein LC-MS/MS affinity isolation experiments. BEPro3 is public domain software, has been tested on Windows XP and version 10.4 or newer of the Mac OS 10.4, and is freely available. A user guide, example dataset with analysis and additional documentation are included with the BEPro3 download.

  7. DAPD: A Knowledgebase for Diabetes Associated Proteins.

    PubMed

    Gopinath, Krishnasamy; Jayakumararaj, Ramaraj; Karthikeyan, Muthusamy

    2015-01-01

    Recent advancements in genomics and proteomics provide a solid foundation for understanding the pathogenesis of diabetes. Proteomics of diabetes associated pathways help to identify the most potent target for the management of diabetes. The relevant datasets are scattered in various prominent sources which takes much time to select the therapeutic target for the clinical management of diabetes. However, additional information about target proteins is needed for validation. This lacuna may be resolved by linking diabetes associated genes, pathways and proteins and it will provide a strong base for the treatment and planning management strategies of diabetes. Thus, a web source "Diabetes Associated Proteins Database (DAPD)" has been developed to link the diabetes associated genes, pathways and proteins using PHP, MySQL. The current version of DAPD has been built with proteins associated with different types of diabetes. In addition, DAPD has been linked to external sources to gain the access to more participatory proteins and their pathway network. DAPD will reduce the time and it is expected to pave the way for the discovery of novel anti-diabetic leads using computational drug designing for diabetes management. DAPD is open accessed via following url www.mkarthikeyan.bioinfoau.org/dapd. PMID:26357271

  8. Targeted knock-down of a structurally atypical zebrafish 12S-lipoxygenase leads to severe impairment of embryonic development

    PubMed Central

    Haas, Ulrike; Raschperger, Elisabeth; Hamberg, Mats; Samuelsson, Bengt; Tryggvason, Karl; Haeggström, Jesper Z.

    2011-01-01

    Lipoxygenases (LO) are a class of dioxygenases, which form hydroperoxy, hydroxy, and epoxy derivatives of arachidonic acid with distinct positional and stereochemical configurations. In man, there are two known types of 12-LO that are distinguished by their expression patterns and catalytic properties. The platelet 12S-LO plays a role in platelet aggregation and 12R-LO seems to be important for normal skin function. Using BLAST searches of the zebrafish (zf) genome we identified one candidate zf12-LO gene with 43% identity with human 12R-LO at the mRNA level and the deduced primary sequence carried the so called “Coffa” structural determinant (Gly residue) for R stereoselectivity of LOs. However, incubations of recombinant, purified, zf12-LO with arachidonic acid revealed exclusive formation of 12(S)-hydroperoxy-eicosatetraenoic acid. Further studies with immunohistochemistry showed prominent expression of zf12-LO in the cell nuclei of skin epithelium, the epithelial lining of the stomodeum, and the pharyngeal pouches in zf embryos. To probe its function, zf12-LO was subjected to targeted knock-down in zf embryos, resulting in the development of a severe phenotype, characterized by abnormal development of the brain, the eyes, and the tail as well as pericardial and yolk sac edema. Hence, we have identified a unique vertebrate 12S-LO that breaks the current structure-function paradigms for S and R stereo-specificity and with critical roles in normal embryonic development. PMID:22143766

  9. Tracking Membrane Protein Association in Model Membranes

    PubMed Central

    Reffay, Myriam; Gambin, Yann; Benabdelhak, Houssain; Phan, Gilles; Taulier, Nicolas; Ducruix, Arnaud; Hodges, Robert S.; Urbach, Wladimir

    2009-01-01

    Membrane proteins are essential in the exchange processes of cells. In spite of great breakthrough in soluble proteins studies, membrane proteins structures, functions and interactions are still a challenge because of the difficulties related to their hydrophobic properties. Most of the experiments are performed with detergent-solubilized membrane proteins. However widely used micellar systems are far from the biological two-dimensions membrane. The development of new biomimetic membrane systems is fundamental to tackle this issue. We present an original approach that combines the Fluorescence Recovery After fringe Pattern Photobleaching technique and the use of a versatile sponge phase that makes it possible to extract crucial informations about interactions between membrane proteins embedded in the bilayers of a sponge phase. The clear advantage lies in the ability to adjust at will the spacing between two adjacent bilayers. When the membranes are far apart, the only possible interactions occur laterally between proteins embedded within the same bilayer, whereas when membranes get closer to each other, interactions between proteins embedded in facing membranes may occur as well. After validating our approach on the streptavidin-biotinylated peptide complex, we study the interactions between two membrane proteins, MexA and OprM, from a Pseudomonas aeruginosa efflux pump. The mode of interaction, the size of the protein complex and its potential stoichiometry are determined. In particular, we demonstrate that: MexA is effectively embedded in the bilayer; MexA and OprM do not interact laterally but can form a complex if they are embedded in opposite bilayers; the population of bound proteins is at its maximum for bilayers separated by a distance of about 200 Å, which is the periplasmic thickness of Pseudomonas aeruginosa. We also show that the MexA-OprM association is enhanced when the position and orientation of the protein is restricted by the bilayers. We

  10. Multifunctional Microtubule-Associated Proteins in Plants

    PubMed Central

    Krtková, Jana; Benáková, Martina; Schwarzerová, Kateřina

    2016-01-01

    Microtubules (MTs) are involved in key processes in plant cells, including cell division, growth and development. MT-interacting proteins modulate MT dynamics and organization, mediating functional and structural interaction of MTs with other cell structures. In addition to conventional microtubule-associated proteins (MAPs) in plants, there are many other MT-binding proteins whose primary function is not related to the regulation of MTs. This review focuses on enzymes, chaperones, or proteins primarily involved in other processes that also bind to MTs. The MT-binding activity of these multifunctional MAPs is often performed only under specific environmental or physiological conditions, or they bind to MTs only as components of a larger MT-binding protein complex. The involvement of multifunctional MAPs in these interactions may underlie physiological and morphogenetic events, e.g., under specific environmental or developmental conditions. Uncovering MT-binding activity of these proteins, although challenging, may contribute to understanding of the novel functions of the MT cytoskeleton in plant biological processes. PMID:27148302

  11. Adaptable Lipid Matrix Promotes Protein-Protein Association in Membranes.

    PubMed

    Kuznetsov, Andrey S; Polyansky, Anton A; Fleck, Markus; Volynsky, Pavel E; Efremov, Roman G

    2015-09-01

    The cell membrane is "stuffed" with proteins, whose transmembrane (TM) helical domains spontaneously associate to form functionally active complexes. For a number of membrane receptors, a modulation of TM domains' oligomerization has been shown to contribute to the development of severe pathological states, thus calling for detailed studies of the atomistic aspects of the process. Despite considerable progress achieved so far, several crucial questions still remain: How do the helices recognize each other in the membrane? What is the driving force of their association? Here, we assess the dimerization free energy of TM helices along with a careful consideration of the interplay between the structure and dynamics of protein and lipids using atomistic molecular dynamics simulations in the hydrated lipid bilayer for three different model systems - TM fragments of glycophorin A, polyalanine and polyleucine peptides. We observe that the membrane driven association of TM helices exhibits a prominent entropic character, which depends on the peptide sequence. Thus, a single TM peptide of a given composition induces strong and characteristic perturbations in the hydrophobic core of the bilayer, which may facilitate the initial "communication" between TM helices even at the distances of 20-30 Å. Upon tight helix-helix association, the immobilized lipids accommodate near the peripheral surfaces of the dimer, thus disturbing the packing of the surrounding. The dimerization free energy of the modeled peptides corresponds to the strength of their interactions with lipids inside the membrane being the lowest for glycophorin A and similarly higher for both homopolymers. We propose that the ability to accommodate lipid tails determines the dimerization strength of TM peptides and that the lipid matrix directly governs their association. PMID:26575933

  12. Protein-protein interactions of mitochondrial-associated protein via bioluminescence resonance energy transfer

    PubMed Central

    Koshiba, Takumi

    2015-01-01

    Protein-protein interactions are essential biological reactions occurring at inter- and intra-cellular levels. The analysis of their mechanism is generally required in order link to understand their various cellular functions. Bioluminescence resonance energy transfer (BRET), which is based on an enzymatic activity of luciferase, is a useful tool for investigating protein-protein interactions in live cells. The combination of the BRET system and biomolecular fluorescence complementation (BiFC) would provide us a better understanding of the hetero-oligomeric structural states of protein complexes. In this review, we discuss the application of BRET to the protein-protein interactions of mitochondrial-associated proteins and discuss its physiological relevance. PMID:27493852

  13. Lipid droplets and associated proteins in sebocytes.

    PubMed

    Schneider, Marlon R

    2016-01-15

    Mammalian skin is characterized by the presence of sebaceous glands (SGs), which develop with the hair follicle and whose predominant cell type is the sebocyte. Sebocytes are epithelial cells that progressively accumulate lipids and eventually release their content (sebum) by holocrine secretion as cells disrupt. In addition to thermoregulatory and pheromonal actions, numerous additional functions have been demonstrated or postulated for sebum, including antimicrobial and antioxidant activities. The SG has also been involved in the pathogenesis of skin diseases as acne vulgaris and some forms of alopecia. Although lipid accumulation culminating in cell disruption and content release is the hallmark of sebocyte differentiation, only a surprisingly low number of studies have so far focused on sebocyte lipid droplets and their associated proteins. PMID:26571075

  14. Structure prediction of magnetosome-associated proteins.

    PubMed

    Nudelman, Hila; Zarivach, Raz

    2014-01-01

    Magnetotactic bacteria (MTB) are Gram-negative bacteria that can navigate along geomagnetic fields. This ability is a result of a unique intracellular organelle, the magnetosome. These organelles are composed of membrane-enclosed magnetite (Fe3O4) or greigite (Fe3S4) crystals ordered into chains along the cell. Magnetosome formation, assembly, and magnetic nano-crystal biomineralization are controlled by magnetosome-associated proteins (MAPs). Most MAP-encoding genes are located in a conserved genomic region - the magnetosome island (MAI). The MAI appears to be conserved in all MTB that were analyzed so far, although the MAI size and organization differs between species. It was shown that MAI deletion leads to a non-magnetic phenotype, further highlighting its important role in magnetosome formation. Today, about 28 proteins are known to be involved in magnetosome formation, but the structures and functions of most MAPs are unknown. To reveal the structure-function relationship of MAPs we used bioinformatics tools in order to build homology models as a way to understand their possible role in magnetosome formation. Here we present a predicted 3D structural models' overview for all known Magnetospirillum gryphiswaldense strain MSR-1 MAPs. PMID:24523717

  15. Structure prediction of magnetosome-associated proteins

    PubMed Central

    Nudelman, Hila; Zarivach, Raz

    2014-01-01

    Magnetotactic bacteria (MTB) are Gram-negative bacteria that can navigate along geomagnetic fields. This ability is a result of a unique intracellular organelle, the magnetosome. These organelles are composed of membrane-enclosed magnetite (Fe3O4) or greigite (Fe3S4) crystals ordered into chains along the cell. Magnetosome formation, assembly, and magnetic nano-crystal biomineralization are controlled by magnetosome-associated proteins (MAPs). Most MAP-encoding genes are located in a conserved genomic region – the magnetosome island (MAI). The MAI appears to be conserved in all MTB that were analyzed so far, although the MAI size and organization differs between species. It was shown that MAI deletion leads to a non-magnetic phenotype, further highlighting its important role in magnetosome formation. Today, about 28 proteins are known to be involved in magnetosome formation, but the structures and functions of most MAPs are unknown. To reveal the structure–function relationship of MAPs we used bioinformatics tools in order to build homology models as a way to understand their possible role in magnetosome formation. Here we present a predicted 3D structural models’ overview for all known Magnetospirillum gryphiswaldense strain MSR-1 MAPs. PMID:24523717

  16. A marginal band-associated protein has properties of both microtubule- and microfilament-associated proteins

    PubMed Central

    1989-01-01

    The marginal band of nucleated erythrocytes is a microtubule organelle under rigorous quantitative and spatial control, with properties quite different from those of the microtubule organelles of cultured cells. Previous results suggest that proteins other than tubulin may participate in organizing the marginal band, and may interact with elements of the erythrocyte cytoskeleton in addition to microtubules. To identify such species, we raised mAbs against the proteins that assemble from chicken brain homogenates with tubulin. One such antibody binds to a single protein in chicken erythrocytes, and produces an immunofluorescence pattern colocalizing with marginal band microtubules. Several properties of this protein are identical to those of ezrin, a protein isolated from brush border and localized to motile elements of cultured cells. A significant proportion of the antigen is not soluble in erythrocytes, as determined by extraction with nonionic detergent. This cytoskeleton-associated fraction is unaffected by treatments that solubilize the marginal band microtubules. The protein has properties of both microtubule- and microfilament-associated proteins. In the accompanying manuscript (Goslin, K., E. Birgbauer, G. Banker, and F. Solomon. 1989. J. Cell Biol. 109:1621-1631), we show that the same antibody recognizes a component of growth cones with a similar dual nature. In early embryonic red blood cells, the antigen is dispersed throughout the cell and does not colocalize with assembled tubulin. Its confinement to the marginal band during development follows rather than precedes that of microtubules. These results, along with previous work, suggest models for the formation of the marginal band. PMID:2677023

  17. Shedding Light on Selenium Biomineralization: Proteins Associated with Bionanominerals ▿

    PubMed Central

    Lenz, Markus; Kolvenbach, Boris; Gygax, Benjamin; Moes, Suzette; Corvini, Philippe F. X.

    2011-01-01

    Selenium-reducing microorganisms produce elemental selenium nanoparticles with particular physicochemical properties due to an associated organic fraction. This study identified high-affinity proteins associated with such bionanominerals and with nonbiogenic elemental selenium. Proteins with an anticipated functional role in selenium reduction, such as a metalloid reductase, were found to be associated with nanoparticles formed by one selenium respirer, Sulfurospirillum barnesii. PMID:21602371

  18. Expression of two membrane fusion proteins, synaptosome-associated protein of 25 kDa and vesicle-associated membrane protein, in choroid plexus epithelium.

    PubMed

    Chung, I; Burkart, A; Szmydynger-Chodobska, J; Dodd, K A; Trimble, W S; Miller, K V; Shim, M; Chodobski, A

    2003-01-01

    In addition to being the major site of cerebrospinal fluid formation, the choroid plexus epithelium emerges as an important source of polypeptides in the brain. Physiologically regulated release of some polypeptides synthesized by the choroid plexus has been shown. The molecular mechanisms underlying this polypeptide secretion have not been characterized, however. In the present study, synaptosome-associated protein of 25 kDa and vesicle-associated membrane protein, two membrane fusion proteins playing a critical role in exocytosis in neurons and endocrine cells, were found to be expressed in the choroid plexus epithelium. It was also shown that in choroidal epithelium, synaptosome-associated protein of 25 kDa and vesicle-associated membrane protein stably interact. Two members of the vesicle-associated membrane protein family, vesicle-associated membrane protein-1 and vesicle-associated membrane protein-2, were expressed in the rat choroid plexus at the messenger RNA and protein level. However, their newly discovered isoforms, vesicle-associated membrane protein-1b and vesicle-associated membrane protein-2b, produced by alternative RNA splicing, were not detected in choroidal tissue. Immunohistochemistry demonstrated that vesicle-associated membrane protein is confined to the cytoplasm of choroidal epithelium, whereas synaptosome-associated protein of 25 kDa is associated with plasma membranes, albeit with a varied cellular distribution among species studied. Specifically, in the rat choroid plexus, synaptosome-associated protein of 25 kDa was localized to the basolateral membrane domain of choroidal epithelium and was expressed in small groups of cells. In comparison, in ovine and human choroidal tissues, apical staining for synaptosome-associated protein of 25 kDa was found in the majority of epithelial cells. These species-related differences in cellular synaptosome-associated protein of 25 kDa distribution suggested that the synaptosome-associated protein of

  19. Neurodegenerative diseases and widespread aggregation are associated with supersaturated proteins

    PubMed Central

    Ciryam, Prajwal; Tartaglia, Gian Gaetano; Morimoto, Richard I.; Dobson, Christopher M.; Vendruscolo, Michele

    2013-01-01

    Summary The maintenance of protein solubility is a fundamental aspect of protein homeostasis, as aggregation is associated with cytotoxicity and a variety of human diseases. Numerous proteins unrelated in sequence and structure, however, can misfold and aggregate, and widespread aggregation can occur in living systems under stress or ageing. A crucial question in this context is why only certain proteins aggregate in vivo while others do not. We identify here the proteins most vulnerable to aggregation as those whose cellular concentrations are high relative to their solubilities. These supersaturated proteins represent a metastable sub-proteome involved in pathological aggregation during stress and ageing, and are overrepresented in biochemical processes associated with neurodegenerative disorders. Consequently, such cellular processes become dysfunctional when the ability to keep intrinsically supersaturated proteins soluble is compromised. Thus, the simultaneous analysis of abundance and solubility can rationalize the diverse cellular pathologies linked to neurodegenerative diseases and aging. PMID:24183671

  20. 48-Kilodalton intermediate-filament-associated protein in astrocytes.

    PubMed

    Abd-el-Basset, E M; Kalnins, V I; Subrahmanyan, L; Ahmed, I; Fedoroff, S

    1988-01-01

    We provide evidence that a protein of 48 kilodaltons (KD), recognized by a normal rabbit serum (F2N), is associated with intermediate filaments (IF) of astrocytes both in cell cultures and in situ. Immunofluorescence staining shows that the F2N serum gives a fibrous staining pattern similar to that seen with anti-serum to glial filament protein (GFP), a protein specific for IF of astrocytes, and that both proteins are present in the perinuclear fibrous aggregates of IF produced by treating the cells with colchicine. At the ultrastructural level the gold particles decorating the 48-KD protein are localized in clusters along the IF, whereas the gold particles decorating the GFP are localized on the IF in a linear pattern. This difference in distribution and the fact that the two proteins have different electrophoretic mobilities on SDS gels indicates that the 48-KD protein although associated with IF is different from GFP. The 48-KD protein appears to be a distinct, developmentally regulated intermediate-filament-associated protein (IFAP), different from other IFAPs reported to date and the first IFAP described in astrocytes. Its appearance in late developmental stages when motile astroblasts are changing into nonmotile stellate cells suggests that the 48-KD protein may be involved in cross-linking the GFP-containing IF to provide more tensile strength to the cytoplasm at the expense of flexibility. PMID:2449542

  1. Proteins associated with human parainfluenza virus type 3.

    PubMed Central

    Jambou, R C; Elango, N; Venkatesan, S

    1985-01-01

    The polypeptides associated with human parainfluenza virus type 3 were identified. Five proteins were present in detergent- and salt-resistant viral cores. Of these, three proteins designated NP0, NP1, and NP2 of 68,000, 58,000, and 52,000 daltons, respectively, were stably associated with 50S RNA in CsCl gradient-purified nucleocapsids. The amounts of NP1 and NP2 were variable, and these proteins were shown to be structurally related to the major nucleocapsid protein (NP0) by partial Staphylococcus aureus V8 protease mapping. The other core proteins included a 240K protein designated L (candidate for the viral polymerase) and an 84K protein designated as the phosphoprotein (P) on the basis of a predominant incorporation of Pi. The viral envelope had four prominent proteins (72, 53, 40, and 12K) under reducing conditions of electrophoresis. The 72 and 53K proteins were specifically labeled with [3H]glucosamine and [3H]mannose. When sulfhydryl reagents were removed, a new 62K protein was visualized in place of the 72, 53, and 12K proteins. The 53 and 12K proteins were interpreted to be the two subunits (F1 and F2) of the fusion protein, and the 72K protein was designated as the HN (hemagglutinin-neuraminidase) glycoprotein. The unglycosylated 40K protein represented the viral matrix protein (M). Immunoprecipitation of infected cell lysates with rabbit hyperimmune antiserum against purified virus confirmed the viral origin of these polypeptides. Images PMID:2993658

  2. Programming Molecular Association and Viscoelastic Behavior in Protein Networks.

    PubMed

    Dooling, Lawrence J; Buck, Maren E; Zhang, Wen-Bin; Tirrell, David A

    2016-06-01

    A set of recombinant artificial proteins that can be cross-linked, by either covalent bonds or association of helical domains or both, is described. The designed proteins can be used to construct molecular networks in which the mechanism of crosslinking determines the time-dependent responses to mechanical deformation. PMID:27061171

  3. Expression of microtubule-associated protein 2 by reactive astrocytes.

    PubMed Central

    Geisert, E E; Johnson, H G; Binder, L I

    1990-01-01

    After an injury to the central nervous system, a dramatic change in the astrocytes bordering the wound occurs. The most characteristic feature of this process, termed reactive gliosis, is the upregulation of the intermediate filament protein, glial fibrillary acidic protein. In the present study, we show that reactive astrocytes express high levels of microtubule-associated protein 2 (MAP-2), a protein normally found in the somatodendritic compartment of neurons. When sections of injured brain are double-stained with antibodies directed against MAP-2 and glial fibrillary protein, all of the reactive astrocytes are found to contain MAP-2. The high levels of this protein appear to represent a permanent change in reactive astrocytes. In parallel quantitative studies, an elevated level of MAP-2 in the injured brain is confirmed by an immunoblot analysis of injured and normal white matter. This report demonstrates the direct involvement of a microtubule protein in the process of reactive gliosis. Images PMID:1692628

  4. BioID Identification of Lamin-Associated Proteins.

    PubMed

    Mehus, Aaron A; Anderson, Ruthellen H; Roux, Kyle J

    2016-01-01

    A- and B-type lamins support the nuclear envelope, contribute to heterochromatin organization, and regulate a myriad of nuclear processes. The mechanisms by which lamins function in different cell types and the mechanisms by which lamin mutations cause over a dozen human diseases (laminopathies) remain unclear. The identification of proteins associated with lamins is likely to provide fundamental insight into these mechanisms. BioID (proximity-dependent biotin identification) is a unique and powerful method for identifying protein-protein and proximity-based interactions in living cells. BioID utilizes a mutant biotin ligase from bacteria that is fused to a protein of interest (bait). When expressed in living cells and stimulated with excess biotin, this BioID-fusion protein promiscuously biotinylates directly interacting and vicinal endogenous proteins. Following biotin-affinity capture, the biotinylated proteins can be identified using mass spectrometry. BioID thus enables screening for physiologically relevant protein associations that occur over time in living cells. BioID is applicable to insoluble proteins such as lamins that are often refractory to study by other methods and can identify weak and/or transient interactions. We discuss the use of BioID to elucidate novel lamin-interacting proteins and its applications in a broad range of biological systems, and provide detailed protocols to guide new applications. PMID:26778550

  5. Identification of a Novel Inhibitory Actin-capping Protein Binding Motif in CD2-associated Protein*

    PubMed Central

    Bruck, Serawit; Huber, Tobias B.; Ingham, Robert J.; Kim, Kyoungtae; Niederstrasser, Hanspeter; Allen, Paul M.; Pawson, Tony; Cooper, John A.; Shaw, Andrey S.

    2008-01-01

    CD2-associated protein (CD2AP) is a scaffold molecule that plays a critical role in the maintenance of the kidney filtration barrier. Little, however, is understood about its mechanism of function. We used mass spectrometry to identify CD2AP-interacting proteins. Many of the proteins that we identified suggest a role for CD2AP in endocytosis and actin regulation. To address the role of CD2AP in regulation of the actin cytoskeleton, we focused on characterizing the interaction of CD2AP with actin-capping protein CP. We identified a novel binding motif LXHXTXXRPK(X)6P present in CD2AP that is also found in its homolog Cin85 and other capping protein-associated proteins such as CARMIL and CKIP-1. CD2AP inhibits the function of capping protein in vitro. Therefore, our results support a role of CD2AP in the regulation of the actin cytoskeleton. PMID:16707503

  6. Low protein silage associated with rumen impaction in suckler cows.

    PubMed

    2016-04-23

    Rumen impaction associated with low protein diets in a suckler cowCampylobacteriosis in suckler cowsPlant toxicity in ewesListerial encephalitis in ewes ITALIC! Chorioptes bovis-associated infertility in ramsThese are among matters discussed in the disease surveillance report for January 2016 from SAC Consulting: Veterinary Services (SAC C VS). PMID:27103691

  7. Cullin Family Proteins and Tumorigenesis: Genetic Association and Molecular Mechanisms

    PubMed Central

    Chen, Zhi; Sui, Jie; Zhang, Fan; Zhang, Caiguo

    2015-01-01

    Cullin family proteins function as scaffolds to form numerous E3 ubiquitin ligases with RING proteins, adaptor proteins and substrate recognition receptors. These E3 ligases further recognize numerous substrates to participate in a variety of cellular processes, such as DNA damage and repair, cell death and cell cycle progression. Clinically, cullin-associated E3 ligases have been identified to involve numerous human diseases, especially with regard to multiple cancer types. Over the past few years, our understanding of cullin proteins and their functions in genome stability and tumorigenesis has expanded enormously. Herein, this review briefly provides current perspectives on cullin protein functions, and mainly summarizes and discusses molecular mechanisms of cullin proteins in tumorigenesis. PMID:25663940

  8. Nanoparticles-cell association predicted by protein corona fingerprints

    NASA Astrophysics Data System (ADS)

    Palchetti, S.; Digiacomo, L.; Pozzi, D.; Peruzzi, G.; Micarelli, E.; Mahmoudi, M.; Caracciolo, G.

    2016-06-01

    In a physiological environment (e.g., blood and interstitial fluids) nanoparticles (NPs) will bind proteins shaping a ``protein corona'' layer. The long-lived protein layer tightly bound to the NP surface is referred to as the hard corona (HC) and encodes information that controls NP bioactivity (e.g. cellular association, cellular signaling pathways, biodistribution, and toxicity). Decrypting this complex code has become a priority to predict the NP biological outcomes. Here, we use a library of 16 lipid NPs of varying size (Ø ~ 100-250 nm) and surface chemistry (unmodified and PEGylated) to investigate the relationships between NP physicochemical properties (nanoparticle size, aggregation state and surface charge), protein corona fingerprints (PCFs), and NP-cell association. We found out that none of the NPs' physicochemical properties alone was exclusively able to account for association with human cervical cancer cell line (HeLa). For the entire library of NPs, a total of 436 distinct serum proteins were detected. We developed a predictive-validation modeling that provides a means of assessing the relative significance of the identified corona proteins. Interestingly, a minor fraction of the HC, which consists of only 8 PCFs were identified as main promoters of NP association with HeLa cells. Remarkably, identified PCFs have several receptors with high level of expression on the plasma membrane of HeLa cells.In a physiological environment (e.g., blood and interstitial fluids) nanoparticles (NPs) will bind proteins shaping a ``protein corona'' layer. The long-lived protein layer tightly bound to the NP surface is referred to as the hard corona (HC) and encodes information that controls NP bioactivity (e.g. cellular association, cellular signaling pathways, biodistribution, and toxicity). Decrypting this complex code has become a priority to predict the NP biological outcomes. Here, we use a library of 16 lipid NPs of varying size (Ø ~ 100-250 nm) and surface

  9. The Cdc48 machine in endoplasmic reticulum associated protein degradation.

    PubMed

    Wolf, Dieter H; Stolz, Alexandra

    2012-01-01

    The AAA-type ATPase Cdc48 (named p97/VCP in mammals) is a molecular machine in all eukaryotic cells that transforms ATP hydrolysis into mechanic power to unfold and pull proteins against physical forces, which make up a protein's structure and hold it in place. From the many cellular processes, Cdc48 is involved in, its function in endoplasmic reticulum associated protein degradation (ERAD) is understood best. This quality control process for proteins of the secretory pathway scans protein folding and discovers misfolded proteins in the endoplasmic reticulum (ER), the organelle, destined for folding of these proteins and their further delivery to their site of action. Misfolded lumenal and membrane proteins of the ER are detected by chaperones and lectins and retro-translocated out of the ER for degradation. Here the Cdc48 machinery, recruited to the ER membrane, takes over. After polyubiquitylation of the protein substrate, Cdc48 together with its dimeric co-factor complex Ufd1-Npl4 pulls the misfolded protein out and away from the ER membrane and delivers it to down-stream components for degradation by a cytosolic proteinase machine, the proteasome. The known details of the Cdc48-Ufd1-Npl4 motor complex triggered process are subject of this review article. PMID:21945179

  10. Analysis of alpha-synuclein-associated proteins by quantitative proteomics.

    PubMed

    Zhou, Yong; Gu, Guangyu; Goodlett, David R; Zhang, Terry; Pan, Catherine; Montine, Thomas J; Montine, Kathleen S; Aebersold, Ruedi H; Zhang, Jing

    2004-09-10

    To identify the proteins associated with soluble alpha-synuclein (AS) that might promote AS aggregation, a key event leading to neurodegeneration, we quantitatively compared protein profiles of AS-associated protein complexes in MES cells exposed to rotenone, a pesticide that produces parkinsonism in animals and induces Lewy body (LB)-like inclusions in the remaining dopaminergic neurons, and to vehicle. We identified more than 250 proteins associated with Nonidet P-40 soluble AS, and demonstrated that at least 51 of these proteins displayed significant differences in their relative abundance in AS complexes under conditions where rotenone was cytotoxic and induced formation of cytoplasmic inclusions immunoreactive to anti-AS. Overexpressing one of these proteins, heat shock protein (hsp) 70, not only protected cells from rotenone-mediated cytotoxicity but also decreased soluble AS aggregation. Furthermore, the protection afforded by hsp70 transfection appeared to be related to suppression of rotenone-induced oxidative stress as well as mitochondrial and proteasomal dysfunction. PMID:15234983

  11. Urease-associated heat shock protein of Helicobacter pylori.

    PubMed Central

    Evans, D J; Evans, D G; Engstrand, L; Graham, D Y

    1992-01-01

    Helicobacter pylori urease is an extracellular, cell-bound enzyme with a molecular weight of approximately 600,000 (600K enzyme) comprising six 66K and six 31K subunits. A 62K protein is closely associated with the H. pylori urease, both in crude preparations and after gel filtration; this protein can be removed from the urease by ion-exchange chromatography without inactivating the enzyme. We purified this urease-associated protein and determined its N-terminal amino acid sequence. The sequence is 80% homologous (identical plus conserved amino acid residues) to the Escherichia coli GroEL heat shock protein (HSP), 75% homologous to the human homolog, and 84% homologous to the HSP homolog found in species of Chlamydia. Thus, the 62K urease-associated protein of H. pylori belongs to the HSP60 family of stress proteins known as chaperonins. Evidently this protein, HSP62, participates in the extracellular assembly and/or protection of the urease against inactivation in the hostile environment of the stomach. Images PMID:1348725

  12. The association between glycosylphosphatidylinositol-anchored proteins and heterotrimeric G protein alpha subunits in lymphocytes.

    PubMed Central

    Solomon, K R; Rudd, C E; Finberg, R W

    1996-01-01

    Glycosylphosphatidylinositol (GPI)-anchored proteins are nonmembrane spanning cell surface proteins that have been demonstrated to be signal transduction molecules. Because these proteins do not extend into the cytoplasm, the mechanism by which cross-linking of these molecules leads to intracellular signal transduction events is obscure. Previous analysis has indicated that these proteins are associated with src family member tyrosine kinases; however, the role this interaction plays in the generation of intracellular signals is not clear. Here we show that GPI-anchored proteins are associated with alpha subunits of heterotrimeric GTP binding proteins (G proteins) in both human and murine lymphocytes. When the GPI-anchored proteins CD59, CD48, and Thy-1 were immunoprecipitated from various cell lines or freshly isolated lymphocytes, all were found to be associated with a 41-kDa phosphoprotein that we have identified, by using specific antisera, as a mixture of tyrosine phosphorylated G protein alpha subunits: a small amount of Gialpha1, and substantial amounts of Gialpha2 and Gialpha3. GTP binding assays performed with immunoprecipitations of CD59 indicated that there was GTP-binding activity associated with this molecule. Thus, we have shown by both immunochemical and functional criteria that GPI-anchored proteins are physically associated with G proteins. These experiments suggest a potential role of G proteins in the transduction of signals generated by GPI-anchored molecules expressed on lymphocytes of both mouse and human. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 Fig. 6 PMID:8650218

  13. Engineering nanoparticle-protein associations for protein crystal nucleation and nanoparticle arrangement

    NASA Astrophysics Data System (ADS)

    Benoit, Denise N.

    Engineering the nanoparticle - protein association offers a new way to form protein crystals as well as new approaches for arrangement of nanoparticles. Central to this control is the nanoparticle surface. By conjugating polymers on the surface with controlled molecular weights many properties of the nanoparticle can be changed including its size, stability in buffers and the association of proteins with its surface. Large molecular weight poly(ethylene glycol) (PEG) coatings allow for weak associations between proteins and nanoparticles. These interactions can lead to changes in how proteins crystallize. In particular, they decrease the time to nucleation and expand the range of conditions over which protein crystals form. Interestingly, when PEG chain lengths are too short then protein association is minimized and these effects are not observed. One important feature of protein crystals nucleated with nanoparticles is that the nanoparticles are incorporated into the crystals. What results are nanoparticles placed at well-defined distances in composite protein-nanoparticle crystals. Crystals on the size scale of 10 - 100 micrometers exhibit optical absorbance, fluorescence and super paramagnetic behavior derivative from the incorporated nanomaterials. The arrangement of nanoparticles into three dimensional arrays also gives rise to new and interesting physical and chemical properties, such as fluorescence enhancement and varied magnetic response. In addition, anisotropic nanomaterials aligned throughout the composite crystal have polarization dependent optical properties.

  14. Nanoparticles-cell association predicted by protein corona fingerprints.

    PubMed

    Palchetti, S; Digiacomo, L; Pozzi, D; Peruzzi, G; Micarelli, E; Mahmoudi, M; Caracciolo, G

    2016-07-01

    In a physiological environment (e.g., blood and interstitial fluids) nanoparticles (NPs) will bind proteins shaping a "protein corona" layer. The long-lived protein layer tightly bound to the NP surface is referred to as the hard corona (HC) and encodes information that controls NP bioactivity (e.g. cellular association, cellular signaling pathways, biodistribution, and toxicity). Decrypting this complex code has become a priority to predict the NP biological outcomes. Here, we use a library of 16 lipid NPs of varying size (Ø≈ 100-250 nm) and surface chemistry (unmodified and PEGylated) to investigate the relationships between NP physicochemical properties (nanoparticle size, aggregation state and surface charge), protein corona fingerprints (PCFs), and NP-cell association. We found out that none of the NPs' physicochemical properties alone was exclusively able to account for association with human cervical cancer cell line (HeLa). For the entire library of NPs, a total of 436 distinct serum proteins were detected. We developed a predictive-validation modeling that provides a means of assessing the relative significance of the identified corona proteins. Interestingly, a minor fraction of the HC, which consists of only 8 PCFs were identified as main promoters of NP association with HeLa cells. Remarkably, identified PCFs have several receptors with high level of expression on the plasma membrane of HeLa cells. PMID:27279572

  15. A novel single-molecule study to determine protein--protein association constants.

    PubMed

    Ratcliff, G C; Erie, D A

    2001-06-20

    Atomic force microscopy (AFM) is traditionally used as an imaging technique to gain qualitative information for a biological system. We have successfully used the imaging capabilities of the AFM to determine protein-protein association constants. We have developed a method to measure the molecular weight of a protein based on its volume determined from AFM images. Our volume determination method allows for rapid, accurate analysis of large protein populations. On the basis of the measured volume, the fraction of monomers as dimers was determined for the DNA helicase UvrD, and the dissociation constant (K(d)) for the helicase was calculated. We determined a K(d) for UvrD of 1.4 microM, which is in good agreement with published K(d) data obtained from analytical ultracentrifugation (AUC) studies. Our method provides a rapid method for determining protein-protein association constants. PMID:11403593

  16. VAMP-1: a synaptic vesicle-associated integral membrane protein.

    PubMed

    Trimble, W S; Cowan, D M; Scheller, R H

    1988-06-01

    Several proteins are associated with, or are integral components of, the lipid bilayer that forms the delineating membrane of neuronal synaptic vesicles. To characterize these molecules, we used a polyclonal antiserum raised against purified cholinergic synaptic vesicles from Torpedo to screen a cDNA expression library constructed from mRNA of the electromotor nucleus. One clone encodes VAMP-1 (vesicle-associated membrane protein 1), a nervous-system-specific protein of 120 amino acids whose primary sequence can be divided into three domains: a proline-rich amino terminus, a highly charged internal region, and a hydrophobic carboxyl-terminal domain that is predicted to comprise a membrane anchor. Tryptic digestion of intact and lysed vesicles suggests that the protein faces the cytoplasm, where it may play a role in packaging, transport, or release of neurotransmitters. PMID:3380805

  17. Radioimmunoassay for pregnancy-associated plasma protein A

    SciTech Connect

    Sinosich, M.J.; Teisner, B.; Folkerson, J.; Saunders, D.M.; Grudzinskas, J.G.

    1982-01-01

    A specific and highly sensitive radioimmunoassay for determination of pregnancy-associated plasma protein A in human serum is described. The minimum detection limit for this protein was 2.9 ..mu..g/L. The within- and between-assay coefficients of variation were 4.0 and 4.5%, respectively. The circulating protein was detected within 32 days of conception in eight normal pregnancies and within 21 days in a twin pregnancy. Circulating concentrations in the mother at term were consistently higher (10-fold) than in matched amniotic fluid; none was detected in the umbilical circulation. This protein was also detected in the circulation of patients with hydatidiform mole. This assay will permit investigations into the clinical evaluation of measurements of the protein during early pregnancy and trophoblastic disease.

  18. Proteins associated with RNase E in a multicomponent ribonucleolytic complex.

    PubMed Central

    Miczak, A; Kaberdin, V R; Wei, C L; Lin-Chao, S

    1996-01-01

    The Escherichia coli endoribonuclease RNase E is essential for RNA processing and degradation. Earlier work provided evidence that RNase E exists intracellularly as part of a multicomponent complex and that one of the components of this complex is a 3'-to-5' exoribonuclease, polynucleotide phosphorylase (EC 2.7.7.8). To isolate and identify other components of the RNase E complex, FLAG-epitope-tagged RNase E (FLAG-Rne) fusion protein was purified on a monoclonal antibody-conjugated agarose column. The FLAG-Rne fusion protein, eluted by competition with the synthetic FLAG peptide, was found to be associated with other proteins. N-terminal sequencing of these proteins revealed the presence in the RNase E complex not only of polynucleotide phosphorylase but also of DnaK, RNA helicase, and enolase (EC 4.2.1.11). Another protein associated only with epitope-tagged temperature-sensitive (Rne-3071) mutant RNase E but not with the wild-type enzyme is GroEL. The FLAG-Rne complex has RNase E activity in vivo and in vitro. The relative amount of proteins associated with wild-type and Rne-3071 expressed at an elevated temperature differed. Images Fig. 1 Fig. 2 PMID:8632981

  19. Protein-Based Three-Dimensional Memories and Associative Processors

    NASA Astrophysics Data System (ADS)

    Birge, Robert

    2008-03-01

    The field of bioelectronics has benefited from the fact that nature has often solved problems of a similar nature to those which must be solved to create molecular electronic or photonic devices that operate with efficiency and reliability. Retinal proteins show great promise in bioelectronic devices because they operate with high efficiency (˜0.65%), high cyclicity (>10^7), operate over an extended wavelength range (360 -- 630 nm) and can convert light into changes in voltage, pH, absorption or refractive index. This talk will focus on a retinal protein called bacteriorhodopsin, the proton pump of the organism Halobacterium salinarum. Two memories based on this protein will be described. The first is an optical three-dimensional memory. This memory stores information using volume elements (voxels), and provides as much as a thousand-fold improvement in effective capacity over current technology. A unique branching reaction of a variant of bacteriorhodopsin is used to turn each protein into an optically addressed latched AND gate. Although three working prototypes have been developed, a number of cost/performance and architectural issues must be resolved prior to commercialization. The major issue is that the native protein provides a very inefficient branching reaction. Genetic engineering has improved performance by nearly 500-fold, but a further order of magnitude improvement is needed. Protein-based holographic associative memories will also be discussed. The human brain stores and retrieves information via association, and human intelligence is intimately connected to the nature and enormous capacity of this associative search and retrieval process. To a first order approximation, creativity can be viewed as the association of two seemingly disparate concepts to form a totally new construct. Thus, artificial intelligence requires large scale associative memories. Current computer hardware does not provide an optimal environment for creating artificial

  20. Dickkopf-related protein 3 is a potential Aβ-associated protein in Alzheimer's Disease.

    PubMed

    Bruggink, Kim A; Kuiperij, H Bea; Gloerich, Jolein; Otte-Höller, Irene; Rozemuller, Annemieke J M; Claassen, Jurgen A H R; Küsters, Benno; Verbeek, Marcel M

    2015-09-01

    Amyloid-β (Aβ) is the most prominent protein in Alzheimer's disease (AD) senile plaques. In addition, Aβ interacts with a variety of Aβ-associated proteins (AAPs), some of which can form complexes with Aβ and influence its clearance, aggregation or toxicity. Identification of novel AAPs may shed new light on the pathophysiology of AD and the metabolic fate of Aβ. In this study, we aimed to identify new AAPs by searching for proteins that may form soluble complexes with Aβ in CSF, using a proteomics approach. We identified the secreted Wnt pathway protein Dickkopf-related protein 3 (Dkk-3) as a potential Aβ-associated protein. Using immunohistochemistry on human AD brain tissue, we observed that (i) Dkk-3 co-localizes with Aβ in the brain, both in diffuse and classic plaques. (ii) Dkk-3 is expressed in neurons and in blood vessel walls in the brain and (iii) is secreted by leptomeningeal smooth muscle cells in vitro. Finally, measurements using ELISA revealed that (iv) Dkk-3 protein is abundantly present in both cerebrospinal fluid and serum, but its levels are similar in non-demented controls and patients with AD, Lewy body dementia, and frontotemporal dementia. Our study demonstrates that Dkk-3 is a hitherto unidentified Aβ-associated protein which, given its relatively high cerebral concentrations and co-localization with Aβ, is potentially involved in AD pathology. In this study, we propose that Dickkopf-related protein-3 (Dkk-3) might be a novel Amyloid-β (Aβ) associated protein. We demonstrate that Dkk-3 is expressed in the brain, especially in vessel walls, and co-localizes with Aβ in senile plaques. Furthermore, Dkk-3 levels in cerebrospinal fluid strongly correlate with Aβ40 levels, but were not suitable to discriminate non-demented controls and patients with dementia. PMID:26119087

  1. Encounter complexes and dimensionality reduction in protein–protein association

    PubMed Central

    Kozakov, Dima; Li, Keyong; Hall, David R; Beglov, Dmitri; Zheng, Jiefu; Vakili, Pirooz; Schueler-Furman, Ora; Paschalidis, Ioannis Ch; Clore, G Marius; Vajda, Sandor

    2014-01-01

    An outstanding challenge has been to understand the mechanism whereby proteins associate. We report here the results of exhaustively sampling the conformational space in protein–protein association using a physics-based energy function. The agreement between experimental intermolecular paramagnetic relaxation enhancement (PRE) data and the PRE profiles calculated from the docked structures shows that the method captures both specific and non-specific encounter complexes. To explore the energy landscape in the vicinity of the native structure, the nonlinear manifold describing the relative orientation of two solid bodies is projected onto a Euclidean space in which the shape of low energy regions is studied by principal component analysis. Results show that the energy surface is canyon-like, with a smooth funnel within a two dimensional subspace capturing over 75% of the total motion. Thus, proteins tend to associate along preferred pathways, similar to sliding of a protein along DNA in the process of protein-DNA recognition. DOI: http://dx.doi.org/10.7554/eLife.01370.001 PMID:24714491

  2. Thermodynamics of folding and association of lattice-model proteins

    NASA Astrophysics Data System (ADS)

    Cellmer, Troy; Bratko, Dusan; Prausnitz, John M.; Blanch, Harvey

    2005-05-01

    Closely related to the "protein folding problem" is the issue of protein misfolding and aggregation. Protein aggregation has been associated with the pathologies of nearly 20 human diseases and presents serious difficulties during the manufacture of pharmaceutical proteins. Computational studies of multiprotein systems have recently emerged as a powerful complement to experimental efforts aimed at understanding the mechanisms of protein aggregation. We describe the thermodynamics of systems containing two lattice-model 64-mers. A parallel tempering algorithm abates problems associated with glassy systems and the weighted histogram analysis method improves statistical quality. The presence of a second chain has a substantial effect on single-chain conformational preferences. The melting temperature is substantially reduced, and the increase in the population of unfolded states is correlated with an increase in interactions between chains. The transition from two native chains to a non-native aggregate is entropically favorable. Non-native aggregates receive ˜25% of their stabilizing energy from intraprotein contacts not found in the lowest-energy structure. Contact maps show that for non-native dimers, nearly 50% of the most probable interprotein contacts involve pairs of residues that form native contacts, suggesting that a domain-swapping mechanism is involved in self-association.

  3. Protein landscape at Drosophila melanogaster telomere-associated sequence repeats.

    PubMed

    Antão, José M; Mason, James M; Déjardin, Jérôme; Kingston, Robert E

    2012-06-01

    The specific set of proteins bound at each genomic locus contributes decisively to regulatory processes and to the identity of a cell. Understanding of the function of a particular locus requires the knowledge of what factors interact with that locus and how the protein composition changes in different cell types or during the response to internal and external signals. Proteomic analysis of isolated chromatin segments (PICh) was developed as a tool to target, purify, and identify proteins associated with a defined locus and was shown to allow the purification of human telomeric chromatin. Here we have developed this method to identify proteins that interact with the Drosophila telomere-associated sequence (TAS) repeats. Several of the purified factors were validated as novel TAS-bound proteins by chromatin immunoprecipitation, and the Brahma complex was confirmed as a dominant modifier of telomeric position effect through the use of a genetic test. These results offer information on the efficacy of applying the PICh protocol to loci with sequence more complex than that found at human telomeres and identify proteins that bind to the TAS repeats, which might contribute to TAS biology and chromatin silencing. PMID:22493064

  4. Virulent strain associated outer membrane proteins of Borrelia burgdorferi.

    PubMed Central

    Skare, J T; Shang, E S; Foley, D M; Blanco, D R; Champion, C I; Mirzabekov, T; Sokolov, Y; Kagan, B L; Miller, J N; Lovett, M A

    1995-01-01

    We have isolated and purified outer membrane vesicles (OMV) from Borrelia burgdorferi strain B31 based on methods developed for isolation of Treponema pallidum OMV. Purified OMV exhibited distinct porin activities with conductances of 0.6 and 12.6 nano-Siemen and had no detectable beta-NADH oxidase activity indicating their outer membrane origin and their lack of inner membrane contamination, respectively. Hydrophobic proteins were identified by phase partitioning with Triton X-114. Most of these hydrophobic membrane proteins were not acylated, suggesting that they are outer membrane-spanning proteins. Identification of palmitate-labeled lipoproteins revealed that several were enriched in the OMV, several were enriched in the protoplasmic cylinder inner membrane fraction, and others were found exclusively associated with the inner membrane. The protein composition of OMV changed significantly with successive in vitro cultivation of strain B31. Using antiserum with specificity for virulent strain B31, we identified OMV antigens on the surface of the spirochete and identified proteins whose presence in OMV could be correlated with virulence and protective immunity in the rabbit Lyme disease model. These virulent strain associated outer membrane-spanning proteins may provide new insight into the pathogenesis of Lyme disease. Images PMID:7593626

  5. Nanoforms: a new type of protein-associated mineralization

    NASA Astrophysics Data System (ADS)

    Vali, Hojatollah; McKee, Marc D.; Çiftçioglu, Neva; Sears, S. Kelly; Plows, Fiona L.; Chevet, Eric; Ghiabi, Pegah; Plavsic, Marc; Kajander, E. Olavi; Zare, Richard N.

    2001-01-01

    Controversy surrounds the interpretation of various nano-phenomena as being living organisms. Incubation of fetal bovine serum under standard cell culture conditions results in the formation of free entities in solution, here referred to as nanoforms. These nanoforms, when examined by transmission electron microscopy, have a distinct ovoid morphology ranging in size from tens to hundreds of nanometers. They are composed of hydroxyapatite and proteins and constitute a novel form of protein-associated mineralization. No detectable cell structure resembling bacteria is apparent. However, immunodetection of the proteins associated with the nanoforms, by two specific monoclonal antibodies, suggests a possible biogenic origin. The significance of nanoforms for the recognition of biological activity in ancient geological systems is discussed. The mode of mineralization in nanoforms is also compared to matrix-mediated calcification in vertebrates.

  6. Proteins that associate with lamins: Many faces, many functions

    SciTech Connect

    Schirmer, Eric C. . E-mail: e.schirmer@ed.ac.uk; Foisner, Roland . E-mail: roland.foisner@meduniwien.ac.at

    2007-06-10

    Lamin-associated polypeptides (LAPs) comprise inner nuclear membrane proteins tightly associated with the peripheral lamin scaffold as well as proteins forming stable complexes with lamins in the nucleoplasm. The involvement of LAPs in a wide range of human diseases may be linked to an equally bewildering range of their functions, including sterol reduction, histone modification, transcriptional repression, and Smad- and {beta}-catenin signaling. Many LAPs are likely to be at the center of large multi-protein complexes, components of which may dictate their functions, and a few LAPs have defined enzymatic activities. Here we discuss the definition of LAPs, review their many binding partners, elaborate their functions in nuclear architecture, chromatin organization, gene expression and signaling, and describe what is currently known about their links to human disease.

  7. Matrix Gla Protein polymorphisms are associated with coronary artery calcification

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Matrix Gla Protein (MGP) is a key regulator of vascular calcification. Genetic variation at the MGP locus could modulate the development of coronary artery calcification (CAC). We examined the cross-sectional association between MGP SNPs [rs1800802 (T-138C), rs1800801 (G-7A),and rs4236 (Ala102Thr)...

  8. Characterization of Disease-Associated Mutations in Human Transmembrane Proteins

    PubMed Central

    Molnár, János; Szakács, Gergely; Tusnády, Gábor E.

    2016-01-01

    Transmembrane protein coding genes are commonly associated with human diseases. We characterized disease causing mutations and natural polymorphisms in transmembrane proteins by mapping missense genetic variations from the UniProt database on the transmembrane protein topology listed in the Human Transmembrane Proteome database. We found characteristic differences in the spectrum of amino acid changes within transmembrane regions: in the case of disease associated mutations the non-polar to non-polar and non-polar to charged amino acid changes are equally frequent. In contrast, in the case of natural polymorphisms non-polar to charged amino acid changes are rare while non-polar to non-polar changes are common. The majority of disease associated mutations result in glycine to arginine and leucine to proline substitutions. Mutations to positively charged amino acids are more common in the center of the lipid bilayer, where they cause more severe structural and functional anomalies. Our analysis contributes to the better understanding of the effect of disease associated mutations in transmembrane proteins, which can help prioritize genetic variations in personal genomic investigations. PMID:26986070

  9. Intrinsic membrane association of Drosophila cysteine string proteins.

    PubMed

    Mastrogiacomo, A; Kohan, S A; Whitelegge, J P; Gundersen, C B

    1998-09-25

    Cysteine string proteins (csps) are highly conserved constituents of vertebrate and invertebrate secretory organelles. Biochemical and immunoprecipitation experiments implied that vertebrate csps were integral membrane proteins that were tethered to the outer leaflet of secretory vesicles via the fatty acyl residues of their extensively acylated cysteine string. Independently, work of others suggested that Drosophila csps were peripheral membrane proteins that were anchored to membranes by a mechanism that was independent of the cysteine string and its fatty acyl residues. We extended these investigation and found first that sodium carbonate treatment partially stripped both csps and the integral membrane protein, synaptotagmin, from Drosophila membranes. Concomitantly, carbonate released fatty acids into the medium, arguing that it has a mild, solubilizing effect on these membranes. Second, we observed that Drosophila csps behaved like integral membrane proteins in Triton X-114 partitioning experiments. Third, we found that when membrane-bound csps were deacylated, they remained membrane bound. Moreover, it appeared that hydrophobic interactions were necessary for this persistent membrane association of csps. Thus, neither reducing conditions, urea, nor chaotropic agents displaced deacylated csps from membranes. Only detergents were effective in solubilizing deacylated csps. Finally, by virtue of the inaccessibility of deacylated csps to thiol alkylation by the membrane-impermeant alkylating reagent, iodoacetic acid, we inferred that it was the cysteine string domain that mediated the membrane association of deacylated csps. Thus, we conclude that under physiological conditions csps are integral membrane proteins of secretory organelles, and that the cysteine string domain plays a vital role in the membrane association of these proteins. PMID:9771899

  10. Building a Hierarchical Organization of Protein Complexes Out of Protein Association Data

    PubMed Central

    Stojmirović, Aleksandar; Yu, Yi-Kuo

    2014-01-01

    Organizing experimentally determined protein associations as a hierarchy can be a good approach to elucidating the content of protein complexes and the modularity of subcomplexes. Several challenges exist. First, intrinsically sticky proteins, such as chaperones, are often falsely assigned to many functionally unrelated complexes. Second, the reported collections of proteins may not be true “complexes” in the sense that they bind together and perform a joint cellular function. Third, due to imperfect sensitivity of protein detection methods, both false positive and false negative assignments of a protein to complexes may occur. We mitigate the first issue by down-weighting sticky proteins by their occurrence frequencies. We approach the other two problems by merging nearly identical complexes and by constructing a directed acyclic graph (DAG) based on the relationship of partial inclusion. The constructed DAG, within which smaller complexes form parts of the larger, can reveal how different complexes are joined. By merging almost identical complexes one can deemphasize the influence of false positives, while allowing false negatives to be rescued by other nearly identical association data. We investigate several protein weighting schemes and compare their corresponding DAGs using yeast and human complexes. We find that the scheme incorporating weights based on information flow in the network of direct protein–protein interactions produces biologically most meaningful DAGs. In either yeast or human, isolated nodes form a large proportion of the final hierarchy. While most connected components encompass very few nodes, the largest one for each species contains a sizable portion of all nodes. By considering examples of subgraphs composed of nodes containing a specified protein, we illustrate that the graphs' topological features can correctly suggest the biological roles of protein complexes. The input data, final results and the source code are available at ftp

  11. Association of ebola virus matrix protein VP40 with microtubules.

    PubMed

    Ruthel, Gordon; Demmin, Gretchen L; Kallstrom, George; Javid, Melodi P; Badie, Shirin S; Will, Amy B; Nelle, Timothy; Schokman, Rowena; Nguyen, Tam L; Carra, John H; Bavari, Sina; Aman, M Javad

    2005-04-01

    Viruses exploit a variety of cellular components to complete their life cycles, and it has become increasingly clear that use of host cell microtubules is a vital part of the infection process for many viruses. A variety of viral proteins have been identified that interact with microtubules, either directly or via a microtubule-associated motor protein. Here, we report that Ebola virus associates with microtubules via the matrix protein VP40. When transfected into mammalian cells, a fraction of VP40 colocalized with microtubule bundles and VP40 coimmunoprecipitated with tubulin. The degree of colocalization and microtubule bundling in cells was markedly intensified by truncation of the C terminus to a length of 317 amino acids. Further truncation to 308 or fewer amino acids abolished the association with microtubules. Both the full-length and the 317-amino-acid truncation mutant stabilized microtubules against depolymerization with nocodazole. Direct physical interaction between purified VP40 and tubulin proteins was demonstrated in vitro. A region of moderate homology to the tubulin binding motif of the microtubule-associated protein MAP2 was identified in VP40. Deleting this region resulted in loss of microtubule stabilization against drug-induced depolymerization. The presence of VP40-associated microtubules in cells continuously treated with nocodazole suggested that VP40 promotes tubulin polymerization. Using an in vitro polymerization assay, we demonstrated that VP40 directly enhances tubulin polymerization without any cellular mediators. These results suggest that microtubules may play an important role in the Ebola virus life cycle and potentially provide a novel target for therapeutic intervention against this highly pathogenic virus. PMID:15795257

  12. Chicken Egg Shell Membrane Associated Proteins and Peptides.

    PubMed

    Makkar, Sarbjeet; Liyanage, Rohana; Kannan, Lakshmi; Packialakshmi, Balamurugan; Lay, Jack O; Rath, Narayan C

    2015-11-11

    Egg shells are poultry industry byproducts with potential for use in various biological and agricultural applications. We have been interested in the membranes underlying the calcareous shell as a feed supplement, which showed potential to improve immunity and performance of post hatch poultry. Therefore, to determine their protein and peptide profiles, we extracted the egg shell membranes (ESM) from fresh unfertilized eggs with methanol and guanidine hydrochloride (GdHCl) to obtain soluble proteins for analysis by mass spectrometry. The methanol extract was subjected to matrix-assisted laser desorption ionization (MALDI), electrospray ionization (ESI), high-performance reverse phase liquid chromatographic separation (HPLC), and tandem mass spectrometry (MS/MS) to determine its peptide and protein profiles. The GdHCl extract was subjected to ESI-HPLC-MS/MS following trypsin digestion of reduced/alkylated proteins. Nine proteins from the methanol extract and >275 proteins from the GdHCl extract were tentatively identified. The results suggested the presence of several abundant proteins from egg whites, such as ovoalbumin, ovotransferrin, and lysozyme as well as many others associated with antimicrobial, biomechanical, cytoskeletal organizational, cell signaling, and enzyme activities. Collagens, keratin, agrin, and laminin were some of the structural proteins present in the ESM. The methanol-soluble fraction contained several clusterin peptides and defensins, particularly, two isoforms of gallin. The ratios of the two isoforms of gallin differed between the membranes obtained from brown and white eggs. The high abundance of several antimicrobial, immunomodulatory, and other bioactive proteins in the ESM along with its potential to entrap various microbes and antigens may make it a suitable vehicle for oral immunization of post hatch poultry and improve their disease resistance. PMID:26485361

  13. Association of the P6 protein of Cauliflower mosaic virus with plasmodesmata and plasmodesmal proteins.

    PubMed

    Rodriguez, Andres; Angel, Carlos A; Lutz, Lindy; Leisner, Scott M; Nelson, Richard S; Schoelz, James E

    2014-11-01

    The P6 protein of Cauliflower mosaic virus (CaMV) is responsible for the formation of inclusion bodies (IBs), which are the sites for viral gene expression, replication, and virion assembly. Moreover, recent evidence indicates that ectopically expressed P6 inclusion-like bodies (I-LBs) move in association with actin microfilaments. Because CaMV virions accumulate preferentially in P6 IBs, we hypothesized that P6 IBs have a role in delivering CaMV virions to the plasmodesmata. We have determined that the P6 protein interacts with a C2 calcium-dependent membrane-targeting protein (designated Arabidopsis [Arabidopsis thaliana] Soybean Response to Cold [AtSRC2.2]) in a yeast (Saccharomyces cerevisiae) two-hybrid screen and have confirmed this interaction through coimmunoprecipitation and colocalization assays in the CaMV host Nicotiana benthamiana. An AtSRC2.2 protein fused to red fluorescent protein (RFP) was localized to the plasma membrane and specifically associated with plasmodesmata. The AtSRC2.2-RFP fusion also colocalized with two proteins previously shown to associate with plasmodesmata: the host protein Plasmodesmata-Localized Protein1 (PDLP1) and the CaMV movement protein (MP). Because P6 I-LBs colocalized with AtSRC2.2 and the P6 protein had previously been shown to interact with CaMV MP, we investigated whether P6 I-LBs might also be associated with plasmodesmata. We examined the colocalization of P6-RFP I-LBs with PDLP1-green fluorescent protein (GFP) and aniline blue (a stain for callose normally observed at plasmodesmata) and found that P6-RFP I-LBs were associated with each of these markers. Furthermore, P6-RFP coimmunoprecipitated with PDLP1-GFP. Our evidence that a portion of P6-GFP I-LBs associate with AtSRC2.2 and PDLP1 at plasmodesmata supports a model in which P6 IBs function to transfer CaMV virions directly to MP at the plasmodesmata. PMID:25239023

  14. Root carbon and protein metabolism associated with heat tolerance.

    PubMed

    Huang, Bingru; Rachmilevitch, Shimon; Xu, Jichen

    2012-05-01

    Extensive past efforts have been taken toward understanding heat tolerance mechanisms of the aboveground organs. Root systems play critical roles in whole-plant adaptation to heat stress, but are less studied. This review discusses recent research results revealing some critical physiological and metabolic factors underlying root thermotolerance, with a focus on temperate perennial grass species. Comparative analysis of differential root responses to supraoptimal temperatures by a heat-adapted temperate C3 species, Agrostis scabra, which can survive high soil temperatures up to 45 °C in geothermal areas in Yellow Stone National Park, and a heat-sensitive cogeneric species, Agrostis stolonifera, suggested that efficient carbon and protein metabolism is critical for root thermotolerance. Superior root thermotolerance in a perennial grass was associated with a greater capacity to control respiratory costs through respiratory acclimation, lowering carbon investment in maintenance for protein turnover, and efficiently partitioning carbon into different metabolic pools and alternative respiration pathways. Proteomic analysis demonstrated that root thermotolerance was associated with an increased maintenance of stability and less degradation of proteins, particularly those important for metabolism and energy production. In addition, thermotolerant roots are better able to maintain growth and activity during heat stress by activating stress defence proteins such as those participating in antioxidant defence (i.e. superoxide dismutase, peroxidase, glutathione S-transferase) and chaperoning protection (i.e. heat shock protein). PMID:22328905

  15. Heat Shock Proteins in Association with Heat Tolerance in Grasses

    PubMed Central

    Xu, Yan; Zhan, Chenyang; Huang, Bingru

    2011-01-01

    The grass family Poaceae includes annual species cultivated as major grain crops and perennial species cultivated as forage or turf grasses. Heat stress is a primary factor limiting growth and productivity of cool-season grass species and is becoming a more significant problem in the context of global warming. Plants have developed various mechanisms in heat-stress adaptation, including changes in protein metabolism such as the induction of heat shock proteins (HSPs). This paper summarizes the structure and function of major HSPs, recent research progress on the association of HSPs with grass tolerance to heat stress, and incorporation of HSPs in heat-tolerant grass breeding. PMID:22084689

  16. Balanced Protein–Water Interactions Improve Properties of Disordered Proteins and Non-Specific Protein Association

    PubMed Central

    2015-01-01

    Some frequently encountered deficiencies in all-atom molecular simulations, such as nonspecific protein–protein interactions being too strong, and unfolded or disordered states being too collapsed, suggest that proteins are insufficiently well solvated in simulations using current state-of-the-art force fields. To address these issues, we make the simplest possible change, by modifying the short-range protein–water pair interactions, and leaving all the water–water and protein–protein parameters unchanged. We find that a modest strengthening of protein–water interactions is sufficient to recover the correct dimensions of intrinsically disordered or unfolded proteins, as determined by direct comparison with small-angle X-ray scattering (SAXS) and Förster resonance energy transfer (FRET) data. The modification also results in more realistic protein-protein affinities, and average solvation free energies of model compounds which are more consistent with experiment. Most importantly, we show that this scaling is small enough not to affect adversely the stability of the folded state, with only a modest effect on the stability of model peptides forming α-helix and β-sheet structures. The proposed adjustment opens the way to more accurate atomistic simulations of proteins, particularly for intrinsically disordered proteins, protein–protein association, and crowded cellular environments. PMID:25400522

  17. Repeat-containing protein effectors of plant-associated organisms

    PubMed Central

    Mesarich, Carl H.; Bowen, Joanna K.; Hamiaux, Cyril; Templeton, Matthew D.

    2015-01-01

    Many plant-associated organisms, including microbes, nematodes, and insects, deliver effector proteins into the apoplast, vascular tissue, or cell cytoplasm of their prospective hosts. These effectors function to promote colonization, typically by altering host physiology or by modulating host immune responses. The same effectors however, can also trigger host immunity in the presence of cognate host immune receptor proteins, and thus prevent colonization. To circumvent effector-triggered immunity, or to further enhance host colonization, plant-associated organisms often rely on adaptive effector evolution. In recent years, it has become increasingly apparent that several effectors of plant-associated organisms are repeat-containing proteins (RCPs) that carry tandem or non-tandem arrays of an amino acid sequence or structural motif. In this review, we highlight the diverse roles that these repeat domains play in RCP effector function. We also draw attention to the potential role of these repeat domains in adaptive evolution with regards to RCP effector function and the evasion of effector-triggered immunity. The aim of this review is to increase the profile of RCP effectors from plant-associated organisms. PMID:26557126

  18. Adeno-associated virus rep protein synthesis during productive infection

    SciTech Connect

    Redemann, B.E.; Mendelson, E.; Carter, B.J.

    1989-02-01

    Adeno-associated virus (AAV) Rep proteins mediate viral DNA replication and can regulate expression from AAV genes. The authors studied the kinetics of synthesis of the four Rep proteins, Rep78, Rep68, Rep52, and Rep40, during infection of human 293 or KB cells with AAV and helper adenovirus by in vivo labeling with (/sup 35/S)methionine, immunoprecipitation, and immunoblotting analyses. Rep78 and Rep52 were readily detected concomitantly with detection of viral monomer duplex DNA replicating about 10 to 12 h after infection, and Rep68 and Rep40 were detected 2 h later. Rep78 and Rep52 were more abundant than Rep68 and Rep40 owing to a higher synthesis rate throughout the infectious cycle. In some experiments, very low levels of Rep78 could be detected as early as 4 h after infection. The synthesis rates of Rep proteins were maximal between 14 and 24 h and then decreased later after infection. Isotopic pulse-chase experiments showed that each of the Rep proteins was synthesized independently and was stable for at least 15 h. A slower-migrating, modified form of Rep78 was identified late after infection. AAV capsid protein synthesis was detected at 10 to 12 h after infection and also exhibited synthesis kinetics similar to those of the Rep proteins. AAV DNA replication showed at least two clearly defined stages. Bulk duplex replicating DNA accumulation began around 10 to 12 h and reached a maximum level at about 20 h when Rep and capsid protein synthesis was maximal. Progeny single-stranded DNA accumulation began about 12 to 13 h, but most of this DNA accumulated after 24 h when Rep and capsid protein synthesis had decreased.

  19. Protein-associated water and secondary structure effect removal of blood proteins from metallic substrates.

    PubMed

    Anand, Gaurav; Zhang, Fuming; Linhardt, Robert J; Belfort, Georges

    2011-03-01

    Removing adsorbed protein from metals has significant health and industrial consequences. There are numerous protein-adsorption studies using model self-assembled monolayers or polymeric substrates but hardly any high-resolution measurements of adsorption and removal of proteins on industrially relevant transition metals. Surgeons and ship owners desire clean metal surfaces to reduce transmission of disease via surgical instruments and minimize surface fouling (to reduce friction and corrosion), respectively. A major finding of this work is that, besides hydrophobic interaction adhesion energy, water content in an adsorbed protein layer and secondary structure of proteins determined the access and hence ability to remove adsorbed proteins from metal surfaces with a strong alkaline-surfactant solution (NaOH and 5 mg/mL SDS in PBS at pH 11). This is demonstrated with three blood proteins (bovine serum albumin, immunoglobulin, and fibrinogen) and four transition metal substrates and stainless steel (platinum (Pt), gold (Au), tungsten (W), titanium (Ti), and 316 grade stainless steel (SS)). All the metallic substrates were checked for chemical contaminations like carbon and sulfur and were characterized using X-ray photoelectron spectroscopy (XPS). While Pt and Au surfaces were oxide-free (fairly inert elements), W, Ti, and SS substrates were associated with native oxide. Difference measurements between a quartz crystal microbalance with dissipation (QCM-D) and surface plasmon resonance spectroscopy (SPR) provided a measure of the water content in the protein-adsorbed layers. Hydrophobic adhesion forces, obtained with atomic force microscopy, between the proteins and the metals correlated with the amount of the adsorbed protein-water complex. Thus, the amount of protein adsorbed decreased with Pt, Au, W, Ti and SS, in this order. Neither sessile contact angle nor surface roughness of the metal substrates was useful as predictors here. All three globular proteins

  20. Large-scale de novo prediction of physical protein-protein association.

    PubMed

    Elefsinioti, Antigoni; Saraç, Ömer Sinan; Hegele, Anna; Plake, Conrad; Hubner, Nina C; Poser, Ina; Sarov, Mihail; Hyman, Anthony; Mann, Matthias; Schroeder, Michael; Stelzl, Ulrich; Beyer, Andreas

    2011-11-01

    Information about the physical association of proteins is extensively used for studying cellular processes and disease mechanisms. However, complete experimental mapping of the human interactome will remain prohibitively difficult in the near future. Here we present a map of predicted human protein interactions that distinguishes functional association from physical binding. Our network classifies more than 5 million protein pairs predicting 94,009 new interactions with high confidence. We experimentally tested a subset of these predictions using yeast two-hybrid analysis and affinity purification followed by quantitative mass spectrometry. Thus we identified 462 new protein-protein interactions and confirmed the predictive power of the network. These independent experiments address potential issues of circular reasoning and are a distinctive feature of this work. Analysis of the physical interactome unravels subnetworks mediating between different functional and physical subunits of the cell. Finally, we demonstrate the utility of the network for the analysis of molecular mechanisms of complex diseases by applying it to genome-wide association studies of neurodegenerative diseases. This analysis provides new evidence implying TOMM40 as a factor involved in Alzheimer's disease. The network provides a high-quality resource for the analysis of genomic data sets and genetic association studies in particular. Our interactome is available via the hPRINT web server at: www.print-db.org. PMID:21836163

  1. Large-scale De Novo Prediction of Physical Protein-Protein Association*

    PubMed Central

    Elefsinioti, Antigoni; Saraç, Ömer Sinan; Hegele, Anna; Plake, Conrad; Hubner, Nina C.; Poser, Ina; Sarov, Mihail; Hyman, Anthony; Mann, Matthias; Schroeder, Michael; Stelzl, Ulrich; Beyer, Andreas

    2011-01-01

    Information about the physical association of proteins is extensively used for studying cellular processes and disease mechanisms. However, complete experimental mapping of the human interactome will remain prohibitively difficult in the near future. Here we present a map of predicted human protein interactions that distinguishes functional association from physical binding. Our network classifies more than 5 million protein pairs predicting 94,009 new interactions with high confidence. We experimentally tested a subset of these predictions using yeast two-hybrid analysis and affinity purification followed by quantitative mass spectrometry. Thus we identified 462 new protein-protein interactions and confirmed the predictive power of the network. These independent experiments address potential issues of circular reasoning and are a distinctive feature of this work. Analysis of the physical interactome unravels subnetworks mediating between different functional and physical subunits of the cell. Finally, we demonstrate the utility of the network for the analysis of molecular mechanisms of complex diseases by applying it to genome-wide association studies of neurodegenerative diseases. This analysis provides new evidence implying TOMM40 as a factor involved in Alzheimer's disease. The network provides a high-quality resource for the analysis of genomic data sets and genetic association studies in particular. Our interactome is available via the hPRINT web server at: www.print-db.org. PMID:21836163

  2. Phytoferritin association induced by EGCG inhibits protein degradation by proteases.

    PubMed

    Wang, Aidong; Zhou, Kai; Qi, Xin; Zhao, Guanghua

    2014-12-01

    Phytoferritin is a promising resource of non-heme iron supplementation, but it is not stable against degradation by proteases in the gastrointestinal tract. Therefore, how to improve the stability of ferritin in the presence of proteases is a challenge. Since (-)-epigallocatechin-3-gallate (EGCG) is rich in phenolic-hydroxyl groups, it could interact with ferritin through hydrogen bonds, thereby preventing protein from degradation. To confirm this idea, we focus on the interaction between EGCG and phytoferritin, and the consequence of such interaction. Results demonstrated that EGCG did interact with ferritin, and such interaction induced the change in the tertiary/quaternary structure of protein but not in its secondary structure. Furthermore, stopped-flow and dynamic light scattering (DLS) results showed that EGCG could trigger ferritin association. Consequently, such protein association markedly inhibited protein digestion by pepsin at pH 4.0 and by trypsin at pH 7.5. These findings raise the possibility to improve the stability of phytoferritin in the presence of proteases. PMID:25384342

  3. Assessing association between protein truncating variants and quantitative traits

    PubMed Central

    Rivas, Manuel A.; Pirinen, Matti; Neville, Matthew J.; Gaulton, Kyle J.; Moutsianas, Loukas; Lindgren, Cecilia M.; Karpe, Fredrik; McCarthy, Mark I.; Donnelly, Peter

    2013-01-01

    Motivation: In sequencing studies of common diseases and quantitative traits, power to test rare and low frequency variants individually is weak. To improve power, a common approach is to combine statistical evidence from several genetic variants in a region. Major challenges are how to do the combining and which statistical framework to use. General approaches for testing association between rare variants and quantitative traits include aggregating genotypes and trait values, referred to as ‘collapsing’, or using a score-based variance component test. However, little attention has been paid to alternative models tailored for protein truncating variants. Recent studies have highlighted the important role that protein truncating variants, commonly referred to as ‘loss of function’ variants, may have on disease susceptibility and quantitative levels of biomarkers. We propose a Bayesian modelling framework for the analysis of protein truncating variants and quantitative traits. Results: Our simulation results show that our models have an advantage over the commonly used methods. We apply our models to sequence and exome-array data and discover strong evidence of association between low plasma triglyceride levels and protein truncating variants at APOC3 (Apolipoprotein C3). Availability: Software is available from http://www.well.ox.ac.uk/~rivas/mamba Contact: donnelly@well.ox.ac.uk PMID:23860716

  4. Altered sodium channel-protein associations in critical illness myopathy

    PubMed Central

    2012-01-01

    Background During the acute phase of critical illness myopathy (CIM) there is inexcitability of skeletal muscle. In a rat model of CIM, muscle inexcitability is due to inactivation of sodium channels. A major contributor to this sodium channel inactivation is a hyperpolarized shift in the voltage dependence of sodium channel inactivation. The goal of the current study was to find a biochemical correlate of the hyperpolarized shift in sodium channel inactivation. Methods The rat model of CIM was generated by cutting the sciatic nerve and subsequent injections of dexamethasone for 7 days. Skeletal muscle membranes were prepared from gastrocnemius muscles, and purification and biochemical analyses carried out. Immunoprecipitations were performed with a pan-sodium channel antibody, and the resulting complexes probed in Western blots with various antibodies. Results We carried out analyses of sodium channel glycosylation, phosphorylation, and association with other proteins. Although there was some loss of channel glycosylation in the disease, as assessed by size analysis of glycosylated and de-glycosylated protein in control and CIM samples, previous work by other investigators suggest that such loss would most likely shift channel inactivation gating in a depolarizing direction; thus such loss was viewed as compensatory rather than causative of the disease. A phosphorylation site at serine 487 was identified on the NaV 1.4 sodium channel α subunit, but there was no clear evidence of altered phosphorylation in the disease. Co-immunoprecipitation experiments carried out with a pan-sodium channel antibody confirmed that the sodium channel was associated with proteins of the dystrophin associated protein complex (DAPC). This complex differed between control and CIM samples. Syntrophin, dystrophin, and plectin associated strongly with sodium channels in both control and disease conditions, while β-dystroglycan and neuronal nitric oxide synthase (nNOS) associated

  5. 14-3-3 protein targets misfolded chaperone-associated proteins to aggresomes

    PubMed Central

    Xu, Zhe; Graham, Kourtney; Foote, Molly; Liang, Fengshan; Rizkallah, Raed; Hurt, Myra; Wang, Yanchang; Wu, Yuying; Zhou, Yi

    2013-01-01

    Summary The aggresome is a key cytoplasmic organelle for sequestration and clearance of toxic protein aggregates. Although loading misfolded proteins cargos to dynein motors has been recognized as an important step in the aggresome formation process, the molecular machinery that mediates the association of cargos with the dynein motor is poorly understood. Here, we report a new aggresome-targeting pathway that involves isoforms of 14-3-3, a family of conserved regulatory proteins. 14-3-3 interacts with both the dynein-intermediate chain (DIC) and an Hsp70 co-chaperone Bcl-2-associated athanogene 3 (BAG3), thereby recruiting chaperone-associated protein cargos to dynein motors for their transport to aggresomes. This molecular cascade entails functional dimerization of 14-3-3, which we show to be crucial for the formation of aggresomes in both yeast and mammalian cells. These results suggest that 14-3-3 functions as a molecular adaptor to promote aggresomal targeting of misfolded protein aggregates and may link such complexes to inclusion bodies observed in various neurodegenerative diseases. PMID:23843611

  6. Protein 600 is a microtubule/endoplasmic reticulum-associated protein in CNS neurons.

    PubMed

    Shim, Su Yeon; Wang, Jian; Asada, Naoyuki; Neumayer, Gernot; Tran, Hong Chi; Ishiguro, Kei-ichiro; Sanada, Kamon; Nakatani, Yoshihiro; Nguyen, Minh Dang

    2008-04-01

    There is an increasing body of literature pointing to cytoskeletal proteins as spatial organizers and interactors of organelles. In this study, we identified protein 600 (p600) as a novel microtubule-associated protein (MAP) developmentally regulated in neurons. p600 exhibits the unique feature to interact with the endoplasmic reticulum (ER). Silencing of p600 by RNA interference (RNAi) destabilizes neuronal processes in young primary neurons undergoing neurite extension and containing scarce staining of the ER marker Bip. Furthermore, in utero electroporation of p600 RNAi alters neuronal migration, a process that depends on synergistic actions of microtubule dynamics and ER functions. p600-depleted migrating neurons display thin, crooked, and "zigzag" leading process with very few ER membranes. Thus, p600 constitutes the only known MAP to associate with the ER in neurons, and this interaction may impact on multiple cellular processes ranging from neuronal development to neuronal maturation and plasticity. PMID:18385319

  7. Altered surfactant protein A gene expression and protein metabolism associated with repeat exposure to inhaled endotoxin.

    PubMed

    George, Caroline L S; White, Misty L; O'Neill, Marsha E; Thorne, Peter S; Schwartz, David A; Snyder, Jeanne M

    2003-12-01

    Chronically inhaled endotoxin, which is ubiquitous in many occupational and domestic environments, can adversely affect the respiratory system resulting in an inflammatory response and decreased lung function. Surfactant-associated protein A (SP-A) is part of the lung innate immune system and may attenuate the inflammatory response in various types of lung injury. Using a murine model to mimic occupational exposures to endotoxin, we hypothesized that SP-A gene expression and protein would be elevated in response to repeat exposure to inhaled grain dust and to purified lipopolysaccharide (LPS). Our results demonstrate that repeat exposure to inhaled endotoxin, either in the form of grain dust or purified LPS, results in increased whole lung SP-A gene expression and type II alveolar epithelial cell hyperplasia, whereas SP-A protein levels in lung lavage fluid are decreased. Furthermore, these alterations in SP-A gene activity and protein metabolism are dependent on an intact endotoxin signaling system. PMID:12922979

  8. Why are proteins with glutamine- and asparagine-rich regions associated with protein misfolding diseases?

    NASA Astrophysics Data System (ADS)

    Cruzeiro, Leonor

    2005-12-01

    The possibility that vibrational excited states (VESs) are the drivers of protein folding and function (the VES hypothesis) is explored to explain the reason why Gln- and Asn-rich proteins are associated with degenerative diseases. The Davydov/Scott model is extended to describe energy transfer from the water solution to the protein and vice versa. Computer simulations show that, on average, Gln and Asn residues lead to an initial larger absorption of energy from the environment to the protein, something that can explain the greater structural instability of prions. The sporadic, inherited and infectious character of prion diseases is discussed in the light of the VES hypothesis. An alternative treatment for prion diseases is suggested.

  9. Electrostatic Rate Enhancement and Transient Complex of Protein-Protein Association

    PubMed Central

    Alsallaq, Ramzi; Zhou, Huan-Xiang

    2012-01-01

    The association of two proteins is bounded by the rate at which they, via diffusion, find each other while in appropriate relative orientations. Orientational constraints restrict this rate to ~105 – 106 M−1s−1. Proteins with higher association rates generally have complementary electrostatic surfaces; proteins with lower association rates generally are slowed down by conformational changes upon complex formation. Previous studies (Zhou, Biophys. J. 1997;73:2441–2445) have shown that electrostatic enhancement of the diffusion-limited association rate can be accurately modeled by kD = kD0 exp(−*/ kBT), where kD and kD0 are the rates in the presence and absence of electrostatic interactions, respectively, * is the average electrostatic interaction energy in a “transient-complex” ensemble, and kBT is thermal energy. The transient-complex ensemble separates the bound state from the unbound state. Predictions of the transient-complex theory on four protein complexes were found to agree well with experiment when the electrostatic interaction energy was calculated with the linearized Poisson-Boltzmann (PB) equation (Alsallaq and Zhou, Structure 2007, 15:215–224). Here we show that the agreement is further improved when the nonlinear PB equation is used. These predictions are obtained with the dielectric boundary defined as the protein van der Waals surface. When the dielectric boundary is instead specified as the molecular surface, electrostatic interactions in the transient complex become repulsive and are thus predicted to retard association. Together these results demonstrate that the transient-complex theory is predictive of electrostatic rate enhancement and can help parameterize PB calculations. PMID:17932929

  10. Carboxy terminus heat shock protein 70 interacting protein reduces tau-associated degenerative changes.

    PubMed

    Saidi, Laiq-Jan; Polydoro, Manuela; Kay, Kevin R; Sanchez, Laura; Mandelkow, Eva-Maria; Hyman, Bradley T; Spires-Jones, Tara L

    2015-01-01

    One of the hallmarks of Alzheimer's disease is the formation of neurofibrillary tangles, intracellular aggregates of hyperphosphorylated, mislocalized tau protein, which are associated with neuronal loss. Changes in tau are known to impair cellular transport (including that of mitochondria) and are associated with cell death in cell culture and mouse models of tauopathy. Thus clearing pathological forms of tau from cells is a key therapeutic strategy. One critical modulator in the degradation and clearance of misfolded proteins is the co-chaperone CHIP (Carboxy terminus Hsp70 interacting Protein), which is known to play a role in refolding and clearance of hyperphosphorylated tau. Here, we tested the hypothesis that CHIP could ameliorate pathological changes associated with tau. We find that co-expressing CHIP with full-length tau, tau truncated at D421 mimicking caspase cleavage, or the short tauRDΔK280 tau construct containing only the tau repeat domain with a tauopathy mutation, decreases tau protein levels in human H4 neuroglioma cells in a manner dependent on the Hsp70-binding TPR domain of CHIP. The observed reduction in tau levels by CHIP is associated with a decrease of tau phosphorylation and reduced levels of cleaved Caspase 3 indicating that CHIP plays an important role in preventing tau-induced pathological changes. Furthermore, tau-associated mitochondrial transport deficits are rescued by CHIP co-expression in H4 cells. Together, these data suggest that the co-chaperone CHIP can rescue the pathological effects of tau, and indicate that other diseases of protein misfolding and accumulation may also benefit from CHIP upregulation. PMID:25374103

  11. An aquaporin protein is associated with drought stress tolerance.

    PubMed

    Li, Jun; Ban, Liping; Wen, Hongyu; Wang, Zan; Dzyubenko, Nikolay; Chapurin, Vladimir; Gao, Hongwen; Wang, Xuemin

    2015-04-01

    Water channel proteins known as aquaporins (AQPs) regulate the movement of water and other small molecules across plant vacuolar and plasma membranes; they are associated with plant tolerance of biotic and abiotic stresses. In this study, a PIP type AQPs gene, designated as GoPIP1, was cloned from Galega orientalis, a high value leguminous forage crop. The GoPIP1 gene consists of an 870 bp open reading frame encoding a protein of 289 amino acids, and belongs to the PIP1 subgroup of the PIP subfamily. The transcript level of GoPIP1 was higher in the root of G. orientalis than in the leaf and stem. The level of GoPIP1 transcript increased significantly when treated with 200 mM NaCl or 20% polyethylene glycol (PEG) 6000. Transient expression of GoPIP1 in onion epidermal cells revealed that the GoPIP1 protein was localized to the plasma membrane. Over-expression of GoPIP1 increased the rosette/root ratio and increased sensitivity to drought in transgenic Arabidopsis plants. However, GoPIP1 over-expression in Arabidopsis had no significant effect under saline condition. The present data provides a gene resource that contributes to furthering our understanding of water channel protein and their application in plant stress tolerance. PMID:25701792

  12. Association between milk protein gene variants and protein composition traits in dairy cattle.

    PubMed

    Huang, W; Peñagaricano, F; Ahmad, K R; Lucey, J A; Weigel, K A; Khatib, H

    2012-01-01

    The objective of this study was to identify DNA markers in the 4 casein genes (CSN1S1, CSN1S2, CSN2, and CSN3) and the 2 major whey protein genes (LALBA and LGB) that show associations with milk protein profile measured by reverse-phase HPLC. Fifty-three single nucleotide polymorphisms (SNP) were genotyped for cows in a unique resource population consisting of purebred Holstein and (Holstein × Jersey) × Holstein crossbred animals. Seven traits were analyzed, including concentrations of α(S)-casein (CN), β-CN, κ-CN, α-lactalbumin, β-lactoglobulin, and 2 additional secondary traits, the total concentration of the above 5 milk proteins and the α(S)-CN to β-CN ratio. A substantial fraction of phenotypic variation could be explained by the additive genetic component for the 7 milk protein composition traits studied. Moreover, several SNP were significantly associated with all examined traits at an experiment-wise error rate of 0.05, except for α-lactalbumin. Importantly, the significant SNP explained a large proportion of the phenotypic variation of milk protein composition. Our findings could be used for selecting animals that produce milk with desired composition or desired processing and manufacturing properties. PMID:22192223

  13. Detecting protein association at the T cell plasma membrane.

    PubMed

    Baumgart, Florian; Schütz, Gerhard J

    2015-04-01

    At the moment, many models on T cell signaling rely on results obtained via rather indirect methodologies, which makes direct comparison and conclusions to the in vivo situation difficult. Recently, a variety of new imaging methods were developed, which have the potential to directly shed light onto the mysteries of protein association at the T cell membrane. While the new modalities are extremely promising, for a broad readership it may be difficult to judge the results, since technological shortcomings are not always obvious. In this review article, we put key questions on the mechanism of protein interactions in the T cell plasma membrane into relation with techniques that allow to address such questions. We discuss applicability of the techniques, their strengths and weaknesses. This article is part of a Special Issue entitled: Nanoscale membrane organisation and signalling. PMID:25300585

  14. Inferring drug-disease associations based on known protein complexes

    PubMed Central

    2015-01-01

    Inferring drug-disease associations is critical in unveiling disease mechanisms, as well as discovering novel functions of available drugs, or drug repositioning. Previous work is primarily based on drug-gene-disease relationship, which throws away many important information since genes execute their functions through interacting others. To overcome this issue, we propose a novel methodology that discover the drug-disease association based on protein complexes. Firstly, the integrated heterogeneous network consisting of drugs, protein complexes, and disease are constructed, where we assign weights to the drug-disease association by using probability. Then, from the tripartite network, we get the indirect weighted relationships between drugs and diseases. The larger the weight, the higher the reliability of the correlation. We apply our method to mental disorders and hypertension, and validate the result by using comparative toxicogenomics database. Our ranked results can be directly reinforced by existing biomedical literature, suggesting that our proposed method obtains higher specificity and sensitivity. The proposed method offers new insight into drug-disease discovery. Our method is publicly available at http://1.complexdrug.sinaapp.com/Drug_Complex_Disease/Data_Download.html. PMID:26044949

  15. Hematopoietic lineage cell specific protein 1 associates with and down-regulates protein kinase CK2.

    PubMed

    Ruzzene, M; Brunati, A M; Sarno, S; Donella-Deana, A; Pinna, L A

    1999-11-12

    The catalytic (alpha) subunit of protein kinase CK2 and the hematopoietic specific protein 1 (HS1) display opposite effects on Ha-ras induced fibroblast transformation, by enhancing and counteracting it, respectively. Here we show the occurrence of physical association between HS1 and CK2alpha as judged from both far Western blot and plasmon resonance (BIAcore) analysis. Association of HS1 with CK2alpha is drastically reduced by the deletion of the HS1 C-terminal region (403-486) containing an SH3 domain. HS1, but not its deletion mutant HS1 Delta324-393, lacking a sequence similar to an acidic stretch of the regulatory beta-subunit of CK2, inhibits calmodulin phosphorylation by CK2alpha. These data indicate that HS1 physically interacts with CK2alpha and down-regulates its activity by a mechanism similar to the beta-subunit. PMID:10561491

  16. Protein aggregates are associated with replicative aging without compromising protein quality control

    PubMed Central

    Saarikangas, Juha; Barral, Yves

    2015-01-01

    Differentiation of cellular lineages is facilitated by asymmetric segregation of fate determinants between dividing cells. In budding yeast, various aging factors segregate to the aging (mother)-lineage, with poorly understood consequences. In this study, we show that yeast mother cells form a protein aggregate during early replicative aging that is maintained as a single, asymmetrically inherited deposit over the remaining lifespan. Surprisingly, deposit formation was not associated with stress or general decline in proteostasis. Rather, the deposit-containing cells displayed enhanced degradation of cytosolic proteasome substrates and unimpaired clearance of stress-induced protein aggregates. Deposit formation was dependent on Hsp42, which collected non-random client proteins of the Hsp104/Hsp70-refolding machinery, including the prion Sup35. Importantly, loss of Hsp42 resulted in symmetric inheritance of its constituents and prolonged the lifespan of the mother cell. Together, these data suggest that protein aggregation is an early aging-associated differentiation event in yeast, having a two-faceted role in organismal fitness. DOI: http://dx.doi.org/10.7554/eLife.06197.001 PMID:26544680

  17. Protein kinase activity associated with simian virus 40 T antigen.

    PubMed Central

    Griffin, J D; Spangler, G; Livingston, D M

    1979-01-01

    Incubation of simian virus 40 (SV40) tumor (T) antigen-containing immunoprecipitates with [gamma-32P]ATP results in the incorporation of radioactive phosphate into large T antigen. Highly purified preparations of large T antigen from a SV40-transformed cell line, SV80, are able to catalyze the phosphorylation of a known phosphate acceptor, casein. The kinase activity migrates with large T antigen through multiple purification steps. Sedimentation analysis under non-T-antigen-aggregating conditions reveals that kinase activity and the immunoreactive protein comigrate as a 6S structure. The kinase activity of purified preparations of large T antigen can be specifically adsorbed to solid-phase anti-T IgG, and partially purified T antigen from a SV40 tsA transformation is thermolabile in its ability to phosphorylate casein when compared to comparably purified wild-type T antigen. These observations indicate that the SV40 large T antigen is closely associated with protein kinase (ATP:protein phosphotransferase, EC 2.7.1.37) activity. Images PMID:223152

  18. The evolution and diversification of plant microtubule-associated proteins.

    PubMed

    Gardiner, John

    2013-07-01

    Plant evolution is marked by major advances in structural characteristics that facilitated the highly successful colonization of dry land. Underlying these advances is the evolution of genes encoding specialized proteins that form novel microtubular arrays of the cytoskeleton. This review investigates the evolution of plant families of microtubule-associated proteins (MAPs) through the recently sequenced genomes of Arabidopsis thaliana, Oryza sativa, Selaginella moellendorffii, Physcomitrella patens, Volvox carteri and Chlamydomonas reinhardtii. The families of MAPs examined are AIR9, CLASP, CRIPT, MAP18, MOR1, TON, EB1, AtMAP70, SPR2, SPR1, WVD2 and MAP65 families (abbreviations are defined in the footnote to Table 1). Conjectures are made regarding the evolution of MAPs in plants in relation to the evolution of multicellularity, oriented cell division and vasculature. Angiosperms in particular have high numbers of proteins that are involved in promotion of helical growth or its suppression, and novel plant microtubular structures may have acted as a catalyst for the development of novel plant MAPs. Comparisons of plant MAP gene families with those of animals show that animals may have more flexibility in the structure of their microtubule cytoskeletons than plants, but with both plants and animals possessing many MAP splice variants. PMID:23551562

  19. Protein kinase CK2 and protein kinase D are associated with the COP9 signalosome

    PubMed Central

    Uhle, Stefan; Medalia, Ohad; Waldron, Richard; Dumdey, Renate; Henklein, Peter; Bech-Otschir, Dawadschargal; Huang, Xiaohua; Berse, Matthias; Sperling, Joseph; Schade, Rüdiger; Dubiel, Wolfgang

    2003-01-01

    The COP9 signalosome (CSN) purified from human erythrocytes possesses kinase activity that phosphoryl ates proteins such as c-Jun and p53 with consequence for their ubiquitin (Ub)-dependent degradation. Here we show that protein kinase CK2 (CK2) and protein kinase D (PKD) co-purify with CSN. Immunoprecipi tation and far-western blots reveal that CK2 and PKD are in fact associated with CSN. As indicated by electron microscopy with gold-labeled ATP, at least 10% of CSN particles are associated with kinases. Kinase activity, most likely due to CK2 and PKD, co-immuno precipitates with CSN from HeLa cells. CK2 binds to ΔCSN3(111–403) and CSN7, whereas PKD interacts with full-length CSN3. CK2 phosphorylates CSN2 and CSN7, and PKD modifies CSN7. Both CK2 and PKD phosphorylate c-Jun as well as p53. CK2 phosphoryl ates Thr155, which targets p53 to degradation by the Ub system. Curcumin, emodin, DRB and resveratrol block CSN-associated kinases and induce degradation of c-Jun in HeLa cells. Curcumin treatment results in elevated amounts of c-Jun–Ub conjugates. We conclude that CK2 and PKD are recruited by CSN in order to regulate Ub conjugate formation. PMID:12628923

  20. Hepatitis B virus X protein mediates yes-associated protein 1 upregulation in hepatocellular carcinoma

    PubMed Central

    Wu, Yuzhuo; Zhang, Junhe; Zhang, Huaihong; Zhai, Yufeng

    2016-01-01

    Hepatitis B virus (HBV) X protein (HBx) is implicated in the development of hepatocellular carcinoma (HCC). Yes-associated protein 1 (YAP) is an important proto-oncogene, which is a downstream effector molecule in the Hippo signaling pathway. The aim of the present study was to investigate the association between HBx expression in HCC samples and YAP expression in the Hippo pathway. A total of 20 pathologically confirmed HCC samples, 20 corresponding adjacent non-tumor liver tissues and 5 normal liver tissue samples were collected. The expression of HBx and YAP in the tissues was analyzed by quantitative reverse transcription-polymerase chain reaction and western blot analysis. The intensity and location of YAP expression were analyzed by immunohistochemistry. YAP mRNA and protein expression levels in HCC samples infected with HBV were significantly higher than those of normal liver tissues. Furthermore, YAP expression was positively correlated with HBx expression in HBV-positive HCC samples. Immunohistochemical staining revealed that YAP was predominantly expressed in the nuclei in HBV-positive HCC tissues. YAP expression was significantly decreased in the normal liver tissue and corresponding adjacent liver tissue when compared with the HCC tissues and by contrast to HCC tissues, YAP was predominantly located in the cytoplasm. In conclusion, these results indicate that the YAP gene is a key driver of HBx-induced liver cancer. Therefore, YAP may present a novel target in the treatment of HBV-associated HCC.

  1. Conservation of Oxidative Protein Stabilization in an Insect Homologue of Parkinsonism-Associated Protein DJ-1

    SciTech Connect

    Lin, Jiusheng; Prahlad, Janani; Wilson, Mark A.

    2012-08-21

    DJ-1 is a conserved, disease-associated protein that protects against oxidative stress and mitochondrial damage in multiple organisms. Human DJ-1 contains a functionally essential cysteine residue (Cys106) whose oxidation is important for regulating protein function by an unknown mechanism. This residue is well-conserved in other DJ-1 homologues, including two (DJ-1{alpha} and DJ-1{beta}) in Drosophila melanogaster. Because D. melanogaster is a powerful model system for studying DJ-1 function, we have determined the crystal structure and impact of cysteine oxidation on Drosophila DJ-1{beta}. The structure of D. melanogaster DJ-1{beta} is similar to that of human DJ-1, although two important residues in the human protein, Met26 and His126, are not conserved in DJ-1{beta}. His126 in human DJ-1 is substituted with a tyrosine in DJ-1{beta}, and this residue is not able to compose a putative catalytic dyad with Cys106 that was proposed to be important in the human protein. The reactive cysteine in DJ-1 is oxidized readily to the cysteine-sulfinic acid in both flies and humans, and this may regulate the cytoprotective function of the protein. We show that the oxidation of this conserved cysteine residue to its sulfinate form (Cys-SO{sub 2{sup -}}) results in considerable thermal stabilization of both Drosophila DJ-1{beta} and human DJ-1. Therefore, protein stabilization is one potential mechanism by which cysteine oxidation may regulate DJ-1 function in vivo. More generally, most close DJ-1 homologues are likely stabilized by cysteine-sulfinic acid formation but destabilized by further oxidation, suggesting that they are biphasically regulated by oxidative modification.

  2. Atomistic Simulation of Lignocellulosic Biomass and Associated Cellulosomal Protein Complexes

    SciTech Connect

    Petridis, Loukas; Crowley, Michael F; Smith, Jeremy C

    2010-01-01

    Computer simulations have been performed to obtain an atomic-level understanding of lignocellulose structure and the assembly of its associated cellulosomal protein complexes. First, a CHARMM molecular mechanics force field for lignin is derived and validated by performing a molecular dynamics simulation of a crystal of a lignin fragment molecule and comparing simulation-derived structural features with experimental results. Together with the existing force field for polysaccharides, this work provides the basis for full simulations of lignocellulose. Second, the underlying molecular mechanism governing the assembly of various cellulosomal modules is investigated by performing a novel free-energy calculation of the cohesin-dockerin dissociation. Our calculation indicates a free-energy barrier of ~17 kcal/mol and further reveals a stepwise dissociation pathway involving both the central -sheet interface and its adjacent solvent-exposed loop/turn regions clustered at both ends of the -barrel structure.

  3. Golgi protein ACBD3 mediates neurotoxicity associated with Huntington's Disease

    PubMed Central

    Sbodio, Juan I.; Paul, Bindu D.; Machamer, Carolyn E.; Snyder, Solomon H.

    2013-01-01

    Summary Huntington's disease (HD) is an autosomal dominant neurodegenerative disease caused by expansion of polyglutamine repeats in the gene for huntingtin (Htt). In HD the corpus striatum selectively degenerates despite uniform expression of mutant huntingtin (mHtt) throughout the brain and body. Striatal selectivity reflects the binding of the striatal-selective protein Rhes to mHtt to augment cytotoxicity, but molecular mechanisms underlying the toxicity have been elusive. Here we report that the Golgi protein ACBD3 (Acyl-CoA binding Domain Containing 3) mediates mHtt cytotoxicity via a Rhes/mHtt/ACBD3 complex. ACBD3 levels are markedly elevated in the striatum of HD patients, in a striatal cell line harboring polyglutamine repeats, and in the brains of HD mice. Moreover, ACBD3 deletion abolishes HD neurotoxicity, which is increased by ACBD3 overexpression. Enhanced levels of ACBD3 elicited by ER, mitochondrial and Golgi stresses may account for HD associated augmentation of ACBD3 and neurodegeneration. PMID:24012756

  4. Staphylococcus saprophyticus surface-associated protein (Ssp) is associated with lifespan reduction in Caenorhabditis elegans

    PubMed Central

    Szabados, Florian; Mohner, Amelie; Kleine, Britta; Gatermann, Sören G

    2013-01-01

    Staphylococcal lipases have been proposed as pathogenicity factors. In Staphylococcus saprophyticus the surface-associated protein (Ssp) has been previously characterized as a cell wall-associated true lipase. A S. saprophyticus Δssp::ermB mutant has been described as less virulent in an in vivo model of urinary tract infection compared with its wild-type. This is the first report showing that S. saprophyticus induced a lifespan reduction in Caenorhabditis elegans similar to that of S. aureus RN4220. In two S. saprophyticus Δssp::ermB mutants lifespan reduction in C. elegans was partly abolished. In order to attribute virulence to the lipase activity itself and distinguish this phenomenon from the presence of the Ssp-protein, the conserved active site of the lipase was modified by site-directed ligase-independent mutagenesis and lipase activity-deficient mutants were constructed. These results indicate that the Ssp is associated with pathogenicity in C. elegans and one could speculate that the lipase activity itself is responsible for this virulence. PMID:23959029

  5. Heavy path mining of protein-protein associations in the malaria parasite.

    PubMed

    Yu, Xinran; Korkmaz, Turgay; Lilburn, Timothy G; Cai, Hong; Gu, Jianying; Wang, Yufeng

    2015-07-15

    Annotating and understanding the function of proteins and other elements in a genome can be difficult in the absence of a well-studied and evolutionarily close relative. The causative agent of malaria, one of the oldest and most deadly global infectious diseases, is a good example of this problem. The burden of malaria is huge and there is a pressing need for new, more effective antimalarial strategies. However, techniques such as homology-dependent annotation transfer are severely impaired in this parasite because there are no well-understood close relatives. To circumvent this approach we developed a network-based method that uses a heavy path network-mining algorithm. We uncovered the protein-protein associations that are implicated in important cellular processes including genome integrity, DNA repair, transcriptional regulation, invasion, and pathogenesis, thus demonstrating the utility of this method. The URL of the source code for super-sequence mining method is http://www.cs.utsa.edu/~korkmaz/research/heavy-path-mining/. PMID:25861922

  6. Integrated protein function prediction by mining function associations, sequences, and protein–protein and gene–gene interaction networks

    PubMed Central

    Cao, Renzhi; Cheng, Jianlin

    2016-01-01

    Motivations Protein function prediction is an important and challenging problem in bioinformatics and computational biology. Functionally relevant biological information such as protein sequences, gene expression, and protein–protein interactions has been used mostly separately for protein function prediction. One of the major challenges is how to effectively integrate multiple sources of both traditional and new information such as spatial gene–gene interaction networks generated from chromosomal conformation data together to improve protein function prediction. Results In this work, we developed three different probabilistic scores (MIS, SEQ, and NET score) to combine protein sequence, function associations, and protein–protein interaction and spatial gene–gene interaction networks for protein function prediction. The MIS score is mainly generated from homologous proteins found by PSI-BLAST search, and also association rules between Gene Ontology terms, which are learned by mining the Swiss-Prot database. The SEQ score is generated from protein sequences. The NET score is generated from protein–protein interaction and spatial gene–gene interaction networks. These three scores were combined in a new Statistical Multiple Integrative Scoring System (SMISS) to predict protein function. We tested SMISS on the data set of 2011 Critical Assessment of Function Annotation (CAFA). The method performed substantially better than three base-line methods and an advanced method based on protein profile–sequence comparison, profile–profile comparison, and domain co-occurrence networks according to the maximum F-measure. PMID:26370280

  7. Immunoprecipitation of Plasma Membrane Receptor-Like Kinases for Identification of Phosphorylation Sites and Associated Proteins.

    PubMed

    Kadota, Yasuhiro; Macho, Alberto P; Zipfel, Cyril

    2016-01-01

    Membrane proteins are difficult to study for numerous reasons. The surface of membrane proteins is relatively hydrophobic and sometimes very unstable, additionally requiring detergents for their extraction from the membrane. This leads to challenges at all levels, including expression, solubilization, purification, identification of associated proteins, and the identification of post-translational modifications. However, recent advances in immunoprecipitation technology allow to isolate membrane proteins efficiently, facilitating the study of protein-protein interactions, the identification of novel associated proteins, and to identify post-translational modifications, such as phosphorylation. Here, we describe an optimized immunoprecipitation protocol for plant plasma membrane receptor-like kinases. PMID:26577786

  8. Nesprin-3, a novel outer nuclear membrane protein, associates with the cytoskeletal linker protein plectin

    PubMed Central

    Wilhelmsen, Kevin; Litjens, Sandy H.M.; Kuikman, Ingrid; Tshimbalanga, Ntambua; Janssen, Hans; van den Bout, Iman; Raymond, Karine; Sonnenberg, Arnoud

    2005-01-01

    Despite their importance in cell biology, the mechanisms that maintain the nucleus in its proper position in the cell are not well understood. This is primarily the result of an incomplete knowledge of the proteins in the outer nuclear membrane (ONM) that are able to associate with the different cytoskeletal systems. Two related ONM proteins, nuclear envelope spectrin repeat (nesprin)–1 and –2, are known to make direct connections with the actin cytoskeleton through their NH2-terminal actin-binding domain (ABD). We have now isolated a third member of the nesprin family that lacks an ABD and instead binds to the plakin family member plectin, which can associate with the intermediate filament (IF) system. Overexpression of nesprin-3 results in a dramatic recruitment of plectin to the nuclear perimeter, which is where these two molecules are colocalized with both keratin-6 and -14. Importantly, plectin binds to the integrin α6β4 at the cell surface and to nesprin-3 at the ONM in keratinocytes, suggesting that there is a continuous connection between the nucleus and the extracellular matrix through the IF cytoskeleton. PMID:16330710

  9. Establishing an osteosarcoma associated protein-protein interaction network to explore the pathogenesis of osteosarcoma

    PubMed Central

    2013-01-01

    Background The aim of this study was to establish an osteosarcoma (OS) associated protein-protein interaction network and explore the pathogenesis of osteosarcoma. Methods The gene expression profile GSE9508 was downloaded from the Gene Expression Omnibus database, including five samples of non-malignant bone (the control), seven samples for non-metastatic patients (six of which were analyzed in duplicate), and 11 samples for metastatic patients (10 of which were analyzed in duplicate). Differentially expressed genes (DEGs) between osteosarcoma and control samples were identified by packages in R with the threshold of |logFC (fold change)| > 1 and false discovery rate < 0.05. Osprey software was used to construct the interaction network of DEGs, and genes at protein-protein interaction (PPI) nodes with high degrees were identified. The Database for Annotation, Visualization and Integrated Discovery and WebGestalt software were then used to perform functional annotation and pathway enrichment analyses for PPI networks, in which P < 0.05 was considered statistically significant. Results Compared to the control samples, the expressions of 42 and 341 genes were altered in non-metastatic OS and metastatic OS samples, respectively. A total of 15 significantly enriched functions were obtained with Gene Ontology analysis (P < 0.05). The DEGs were classified and significantly enriched in three pathways, including the tricarboxylic acid cycle, lysosome and axon guidance. Genes such as HRAS, IDH3A, ATP6ap1, ATP6V0D2, SEMA3F and SEMA3A were involved in the enriched pathways. Conclusions The hub genes from metastatic OS samples are not only bio-markers of OS, but also help to improve therapies for OS. PMID:24330838

  10. Uncoupling protein 2 gene polymorphisms are associated with obesity

    PubMed Central

    2012-01-01

    Background Uncoupling protein 2 (UCP2) gene polymorphisms have been reported as genetic risk factors for obesity and type 2 diabetes mellitus (T2DM). We examined the association of commonly observed UCP2 G(−866)A (rs659366) and Ala55Val (C > T) (rs660339) single nucleotide polymorphisms (SNPs) with obesity, high fasting plasma glucose, and serum lipids in a Balinese population. Methods A total of 603 participants (278 urban and 325 rural subjects) were recruited from Bali Island, Indonesia. Fasting plasma glucose (FPG), triglyceride (TG), high density lipoprotein cholesterol (HDL-C), low density lipoprotein cholesterol (LDL-C) and total cholesterol (TC) were measured. Obesity was determined based on WHO classifications for adult Asians. Participants were genotyped for G(−866)A and Ala55Val polymorphisms of the UCP2 gene. Results Obesity prevalence was higher in urban subjects (51%) as compared to rural subjects (23%). The genotype, minor allele (MAF), and heterozygosity frequencies were similar between urban and rural subjects for both SNPs. All genotype frequencies were in Hardy-Weinberg equilibrium. A combined analysis of genotypes and environment revealed that the urban subjects carrying the A/A genotype of the G(−866)A SNP have higher BMI than the rural subjects with the same genotype. Since the two SNPs showed strong linkage disequilibrium (D’ = 0.946, r2 = 0.657), a haplotype analysis was performed. We found that the AT haplotype was associated with high BMI only when the urban environment was taken into account. Conclusions We have demonstrated the importance of environmental settings in studying the influence of the common UCP2 gene polymorphisms in the development of obesity in a Balinese population. PMID:22533685

  11. Extracellular matrix-associated proteins form an integral and dynamic system during Pseudomonas aeruginosa biofilm development

    PubMed Central

    Zhang, Weipeng; Sun, Jin; Ding, Wei; Lin, Jinshui; Tian, Renmao; Lu, Liang; Liu, Xiaofen; Shen, Xihui; Qian, Pei-Yuan

    2015-01-01

    Though the essential role of extracellular matrix in biofilm development has been extensively documented, the function of matrix-associated proteins is elusive. Determining the dynamics of matrix-associated proteins would be a useful way to reveal their functions in biofilm development. Therefore, we applied iTRAQ-based quantitative proteomics to evaluate matrix-associated proteins isolated from different phases of Pseudomonas aeruginosa ATCC27853 biofilms. Among the identified 389 proteins, 54 changed their abundance significantly. The increased abundance of stress resistance and nutrient metabolism-related proteins over the period of biofilm development was consistent with the hypothesis that biofilm matrix forms micro-environments in which cells are optimally organized to resist stress and use available nutrients. Secreted proteins, including novel putative effectors of the type III secretion system were identified, suggesting that the dynamics of pathogenesis-related proteins in the matrix are associated with biofilm development. Interestingly, there was a good correlation between the abundance changes of matrix-associated proteins and their expression. Further analysis revealed complex interactions among these modulated proteins, and the mutation of selected proteins attenuated biofilm development. Collectively, this work presents the first dynamic picture of matrix-associated proteins during biofilm development, and provides evidences that the matrix-associated proteins may form an integral and well regulated system that contributes to stress resistance, nutrient acquisition, pathogenesis and the stability of the biofilm. PMID:26029669

  12. Analysis of p107-associated proteins: p107 associates with a form of E2F that differs from pRB-associated E2F-1.

    PubMed Central

    Dyson, N; Dembski, M; Fattaey, A; Ngwu, C; Ewen, M; Helin, K

    1993-01-01

    The binding of viral oncogenes to cellular proteins is thought to modulate the activities of these cellular targets. The p107 protein is targeted by many viral proteins, including adenovirus E1A, simian virus 40 large T antigen, and human papillomavirus type 16 E7 protein. A panel of monoclonal antibodies against p107 was raised and used to identify cellular proteins that interact with the p107 protein in vivo. p107-associated proteins included cyclin A, cyclin E, and cdk2. In addition, p107 was found to associate with 62- to 65- and 50-kDa phosphoproteins in ML-1 cells, a human myeloid leukemia cell line. The 62- to 65-kDa proteins have many of the properties of the transcription factor E2F but were distinguished from pRB-associated E2F-1 by both immunologic and biochemical properties. Images PMID:8230483

  13. Lipid droplet-associated proteins in atherosclerosis (Review).

    PubMed

    Plakkal Ayyappan, Janeesh; Paul, Antoni; Goo, Young-Hwa

    2016-06-01

    Accumulation of atherosclerotic plaques in arterial walls leads to major cardiovascular diseases and stroke. Macrophages/foam cells are central components of atherosclerotic plaques, which populate the arterial wall in order to remove harmful modified low‑density lipoprotein (LDL) particles, resulting in the accumulation of lipids, mostly LDL‑derived cholesterol ester, in cytosolic lipid droplets (LDs). At present, LDs are recognized as dynamic organelles that govern cellular metabolic processes. LDs consist of an inner core of neutral lipids surrounded by a monolayer of phospholipids and free cholesterol, and contain LD‑associated proteins (LDAPs) that regulate LD functions. Foam cells are characterized by an aberrant accumulation of cytosolic LDs, and are considered a hallmark of atherosclerotic lesions through all stages of development. Previous studies have investigated the mechanisms underlying foam cell formation, aiming to discover therapeutic strategies that target foam cells and intervene against atherosclerosis. It is well established that LDAPs have a major role in the pathogenesis of metabolic diseases caused by dysfunction of lipid metabolism, and several studies have linked LDAPs to the development of atherosclerosis. In this review, several foam cell‑targeting pathways have been described, with an emphasis on the role of LDAPs in cholesterol mobilization from macrophages. In addition, the potential of LDAPs as therapeutic targets to prevent the progression and/or facilitate the regression of the disease has been discussed. PMID:27082419

  14. Lipid droplet-associated proteins in atherosclerosis (Review)

    PubMed Central

    AYYAPPAN, JANEESH PLAKKAL; PAUL, ANTONI; GOO, YOUNG-HWA

    2016-01-01

    Accumulation of atherosclerotic plaques in arterial walls leads to major cardiovascular diseases and stroke. Macrophages/foam cells are central components of atherosclerotic plaques, which populate the arterial wall in order to remove harmful modified low-density lipoprotein (LDL) particles, resulting in the accumulation of lipids, mostly LDL-derived cholesterol ester, in cytosolic lipid droplets (LDs). At present, LDs are recognized as dynamic organelles that govern cellular metabolic processes. LDs consist of an inner core of neutral lipids surrounded by a monolayer of phospholipids and free cholesterol, and contain LD-associated proteins (LDAPs) that regulate LD functions. Foam cells are characterized by an aberrant accumulation of cytosolic LDs, and are considered a hallmark of atherosclerotic lesions through all stages of development. Previous studies have investigated the mechanisms underlying foam cell formation, aiming to discover therapeutic strategies that target foam cells and intervene against atherosclerosis. It is well established that LDAPs have a major role in the pathogenesis of metabolic diseases caused by dysfunction of lipid metabolism, and several studies have linked LDAPs to the development of atherosclerosis. In this review, several foam cell-targeting pathways have been described, with an emphasis on the role of LDAPs in cholesterol mobilization from macrophages. In addition, the potential of LDAPs as therapeutic targets to prevent the progression and/or facilitate the regression of the disease has been discussed. PMID:27082419

  15. Lacritin and other autophagy associated proteins in ocular surface health.

    PubMed

    Karnati, Roy; Talla, Venu; Peterson, Katherine; Laurie, Gordon W

    2016-03-01

    Advantage may be taken of macroautophagy ('autophagy') to promote ocular health. Autophagy continually captures aged or damaged cellular material for lysosomal degradation and recyling. When autophagic flux is chronically elevated, or alternatively deficient, health suffers. Chronic elevation of flux and stress are the consequence of inflammatory cytokines or of dry eye tears but not normal tears invitro. Exogenous tear protein lacritin transiently accelerates flux to restore homeostasis invitro and corneal health invivo, and yet the monomeric active form of lacritin appears to be selectively deficient in dry eye. Tissue transglutaminase-dependent cross-linking of monomer decreases monomer quantity and monomer affinity for coreceptor syndecan-1 thereby abrogating activity. Tissue transglutaminase is elevated in dry eye. Mutation of arylsulfatase A, arylsulfatase B, ceroid-lipofuscinosis neuronal 3, mucolipin, or Niemann-Pick disease type C1 respectively underlie several diseases of apparently insufficient autophagic flux that affect the eye, including: metachromatic leukodystrophy, mucopolysaccharidosis type VI, juvenile-onset Batten disease, mucolipidosis IV, and Niemann-Pick type C associated with myelin sheath destruction of corneal sensory and ciliary nerves and of the optic nerve; corneal clouding, ocular hypertension, glaucoma and optic nerve atrophy; accumulation of 'ceroid-lipofuscin' in surface conjunctival cells, and in ganglion and neuronal cells; decreased visual acuity and retinal dystrophy; and neurodegeneration. For some, enzyme or gene replacement, or substrate reduction, therapy is proving to be successful. Here we discuss examples of restoring ocular surface homeostasis through alteration of autophagy, with particular attention to lacritin. PMID:26318608

  16. HIV Genome-Wide Protein Associations: a Review of 30 Years of Research.

    PubMed

    Li, Guangdi; De Clercq, Erik

    2016-09-01

    The HIV genome encodes a small number of viral proteins (i.e., 16), invariably establishing cooperative associations among HIV proteins and between HIV and host proteins, to invade host cells and hijack their internal machineries. As a known example, the HIV envelope glycoprotein GP120 is closely associated with GP41 for viral entry. From a genome-wide perspective, a hypothesis can be worked out to determine whether 16 HIV proteins could develop 120 possible pairwise associations either by physical interactions or by functional associations mediated via HIV or host molecules. Here, we present the first systematic review of experimental evidence on HIV genome-wide protein associations using a large body of publications accumulated over the past 3 decades. Of 120 possible pairwise associations between 16 HIV proteins, at least 34 physical interactions and 17 functional associations have been identified. To achieve efficient viral replication and infection, HIV protein associations play essential roles (e.g., cleavage, inhibition, and activation) during the HIV life cycle. In either a dispensable or an indispensable manner, each HIV protein collaborates with another viral protein to accomplish specific activities that precisely take place at the proper stages of the HIV life cycle. In addition, HIV genome-wide protein associations have an impact on anti-HIV inhibitors due to the extensive cross talk between drug-inhibited proteins and other HIV proteins. Overall, this study presents for the first time a comprehensive overview of HIV genome-wide protein associations, highlighting meticulous collaborations between all viral proteins during the HIV life cycle. PMID:27357278

  17. Calculating the Bimolecular Rate of Protein-Protein Association with Interacting Crowders.

    PubMed

    Yap, Eng-Hui; Head-Gordon, Teresa

    2013-05-14

    We have recently introduced a method termed Poisson-Boltzmann semianalytical method (PB-SAM) for solving the linearized Poisson-Boltzmann equation for large numbers of arbitrarily shaped dielectric cavities with controlled precision. In this work we extend the applicability of the PB-SAM approach by deriving force and torque expressions that fully account for mutual polarization in both the zero- and first-order derivatives of the surface charges, that can now be embedded into a Brownian dynamics scheme to look at electrostatic-driven mesoscale assembly and kinetics. We demonstrate the capabilities of the PB-SAM approach by simulating the protein concentration effects on the bimolecular rate of association of barnase and barstar, under periodic boundary conditions and evaluated through mean first passage times. We apply PB-SAM to the pseudo-first-order reaction rate conditions in which either barnase or barstar are in great excess relative to the other protein (124:1). This can be considered a specific case in which the PB-SAM approach can be applied to crowding conditions in which crowders are not inert but can form interactions with other molecules. PMID:26583736

  18. Role of Carbonyl Modifications on Aging-Associated Protein Aggregation

    NASA Astrophysics Data System (ADS)

    Tanase, Maya; Urbanska, Aleksandra M.; Zolla, Valerio; Clement, Cristina C.; Huang, Liling; Morozova, Kateryna; Follo, Carlo; Goldberg, Michael; Roda, Barbara; Reschiglian, Pierluigi; Santambrogio, Laura

    2016-01-01

    Protein aggregation is a common biological phenomenon, observed in different physiological and pathological conditions. Decreased protein solubility and a tendency to aggregate is also observed during physiological aging but the causes are currently unknown. Herein we performed a biophysical separation of aging-related high molecular weight aggregates, isolated from the bone marrow and splenic cells of aging mice and followed by biochemical and mass spectrometric analysis. The analysis indicated that compared to younger mice an increase in protein post-translational carbonylation was observed. The causative role of these modifications in inducing protein misfolding and aggregation was determined by inducing carbonyl stress in young mice, which recapitulated the increased protein aggregation observed in old mice. Altogether our analysis indicates that oxidative stress-related post-translational modifications accumulate in the aging proteome and are responsible for increased protein aggregation and altered cell proteostasis.

  19. Role of Carbonyl Modifications on Aging-Associated Protein Aggregation

    PubMed Central

    Tanase, Maya; Urbanska, Aleksandra M.; Zolla, Valerio; Clement, Cristina C.; Huang, Liling; Morozova, Kateryna; Follo, Carlo; Goldberg, Michael; Roda, Barbara; Reschiglian, Pierluigi; Santambrogio, Laura

    2016-01-01

    Protein aggregation is a common biological phenomenon, observed in different physiological and pathological conditions. Decreased protein solubility and a tendency to aggregate is also observed during physiological aging but the causes are currently unknown. Herein we performed a biophysical separation of aging-related high molecular weight aggregates, isolated from the bone marrow and splenic cells of aging mice and followed by biochemical and mass spectrometric analysis. The analysis indicated that compared to younger mice an increase in protein post-translational carbonylation was observed. The causative role of these modifications in inducing protein misfolding and aggregation was determined by inducing carbonyl stress in young mice, which recapitulated the increased protein aggregation observed in old mice. Altogether our analysis indicates that oxidative stress-related post-translational modifications accumulate in the aging proteome and are responsible for increased protein aggregation and altered cell proteostasis. PMID:26776680

  20. Surface Association of Pht Proteins of Streptococcus pneumoniae

    PubMed Central

    Plumptre, Charles D.; Ogunniyi, Abiodun D.

    2013-01-01

    Streptococcus pneumoniae is a major human pathogen responsible for massive global morbidity and mortality. The pneumococcus attaches a variety of proteins to its cell surface, many of which contribute to virulence; one such family are the polyhistidine triad (Pht) proteins PhtA, PhtB, PhtD, and PhtE. In this study, we have examined the mechanism of Pht surface attachment using PhtD as a model. Analysis of deletion and point mutants identified a three-amino-acid region of PhtD (Q27-H28-R29) that is critical for the process. The analogous region in PhtE was also necessary for its attachment to the cell surface. Furthermore, we show that a large proportion of the total amount of each Pht protein is released into bacterial culture supernatants. Other surface proteins were also released, albeit to lesser extents, and this was not due to pneumococcal autolysis. The extent of release of surface proteins was strain dependent and was not affected by the capsule. Lastly, we compared the fitness of wild-type and ΔphtABDE pneumococci in vivo in a mouse coinfection model. Release of Pht proteins by the wild type did not complement the mutant strain, consistent with surface-attached rather than soluble forms of the Pht proteins playing the major role in virulence. The significant degree of release of Pht proteins from intact bacteria may have implications for the use of these proteins in novel vaccines. PMID:23876799

  1. Simulations of HIV Capsid Protein Dimerization Reveal the Effect of Chemistry and Topography on the Mechanism of Hydrophobic Protein Association

    NASA Astrophysics Data System (ADS)

    Yu, Naiyin; Hagan, Michael F.

    2012-09-01

    Recent work has shown that the hydrophobic protein surfaces in aqueous solution sit near a drying transition. The tendency for these surfaces to expel water from their vicinity leads to self assembly of macromolecular complexes. In this article we show with a realistic model for a biologically pertinent system how this phenomenon appears at the molecular level. We focus on the association of the C-terminal domain (CA-C) of the human immunodeficiency virus (HIV) capsid protein. By combining all-atom simulations with specialized sampling techniques we measure the water density distribution during the approach of two CA-C proteins as a function of separation and amino acid sequence in the interfacial region. The simulations demonstrate that CA-C protein-protein interactions sit at the edge of a dewetting transition and that this mesoscopic manifestation of the underlying liquid-vapor phase transition can be readily manipulated by biology or protein engineering to significantly affect association behavior. While the wild type protein remains wet until contact, we identify a set of in silico mutations, in which three hydrophilic amino acids are replaced with nonpolar residues, that leads to dewetting prior to association. The existence of dewetting depends on the size and relative locations of substituted residues separated by nm length scales, indicating long range cooperativity and a sensitivity to surface topography. These observations identify important details which are missing from descriptions of protein association based on buried hydrophobic surface area.

  2. Proteomic analysis and identification of cell surface-associated proteins of Clostridium chauvoei.

    PubMed

    Jayaramaiah, Usharani; Singh, Neetu; Thankappan, Sabarinath; Mohanty, Ashok Kumar; Chaudhuri, Pallab; Singh, Vijendra Pal; Nagaleekar, Viswas Konasagara

    2016-06-01

    Blackleg is a highly fatal disease of cattle and sheep, caused by Clostridium chauvoei, a Gram positive, anaerobic, spore forming bacteria. Cell surface-associated proteins play a major role in inducing the protective immunity. However, the identity of a majority of cell surface-associated proteins of C. chauvoei is not known. In the present investigation, we have used SDS-PAGE, 2D-gel electrophoresis and Western blotting followed by mass spectrometry to identify cell surface-associated proteins of C. chauvoei. Among the identified proteins, which have shown to offer protective antigencity in other bacteria, Enolase, Chaperonin, Ribosomal protein L10, Glycosyl Hydrolase and Flavoprotein were characterized by sequencing and their overexpression in Escherichia coli. In conclusion, cell surface-associated proteins were identified using proteomic approach and the genes for the immunoreactive proteins were expressed, which may prove to be potential diagnostic or vaccine candidates. PMID:26971466

  3. Polysomes of Trypanosoma brucei: Association with Initiation Factors and RNA-Binding Proteins

    PubMed Central

    Klein, Cornelia; Terrao, Monica; Inchaustegui Gil, Diana; Clayton, Christine

    2015-01-01

    We report here the results of experiments designed to identify RNA-binding proteins that might be associated with Trypanosoma brucei polysomes. After some preliminary mass spectrometry of polysomal fractions, we investigated the distributions of selected tagged proteins using sucrose gradients and immunofluorescence. As expected, the polysomal fractions contained nearly all annotated ribosomal proteins, the translation-associated protein folding complex, and many translation factors, but also many other abundant proteins. Results suggested that cap-binding proteins EIF4E3 and EIF4E4 were associated with both free and membrane-bound polysomes. The EIF4E binding partners EIF4G4 and EIF4G3 were present but the other EIF4E and EIF4G paralogues were not detected. The dominant EIF4E in the polysomal fraction is EIF4E4 and very few polysomal mRNAs are associated with EIF4G. Thirteen potential mRNA-binding proteins were detected in the polysomes, including the known polysome-associated protein RBP42. The locations of two of the other proteins were tested after epitope tagging: RBP29 was in the nucleus and ZC3H29 was in the cytoplasm. Quantitative analyses showed that specific association of an RNA-binding protein with the polysome fraction in sucrose gradients will not be detected if the protein is in more than 25-fold molar excess over its target binding sites. PMID:26287607

  4. Proteome analysis of microtubule-associated proteins and their interacting partners from mammalian brain.

    PubMed

    Kozielski, Frank; Riaz, Tahira; DeBonis, Salvatore; Koehler, Christian J; Kroening, Mario; Panse, Isabel; Strozynski, Margarita; Donaldson, Ian M; Thiede, Bernd

    2011-07-01

    The microtubule (MT) cytoskeleton is essential for a variety of cellular processes. MTs are finely regulated by distinct classes of MT-associated proteins (MAPs), which themselves bind to and are regulated by a large number of additional proteins. We have carried out proteome analyses of tubulin-rich and tubulin-depleted MAPs and their interacting partners isolated from bovine brain. In total, 573 proteins were identified giving us unprecedented access to brain-specific MT-associated proteins from mammalian brain. Most of the standard MAPs were identified and at least 500 proteins have been reported as being associated with MTs. We identified protein complexes with a large number of subunits such as brain-specific motor/adaptor/cargo complexes for kinesins, dynein, and dynactin, and proteins of an RNA-transporting granule. About 25% of the identified proteins were also found in the synaptic vesicle proteome. Analysis of the MS/MS data revealed many posttranslational modifications, amino acid changes, and alternative splice variants, particularly in tau, a key protein implicated in Alzheimer's disease. Bioinformatic analysis of known protein-protein interactions of the identified proteins indicated that the number of MAPs and their associated proteins is larger than previously anticipated and that our database will be a useful resource to identify novel binding partners. PMID:20567863

  5. Matrix Gla Protein polymorphism, but not concentrations, is associated with radiographic hand osteoarthritis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Objective. Factors associated with mineralization and osteophyte formation in osteoarthritis (OA) are incompletely understood. Genetic polymorphisms of matrix Gla protein (MGP), a mineralization inhibitor, have been associated clinically with conditions of abnormal calcification. We therefore evalua...

  6. Protein kinase activity associated with pancreatic zymogen granules.

    PubMed

    Burnham, D B; Munowitz, P; Thorn, N; Williams, J A

    1985-05-01

    Purified zymogen granules were prepared from rat pancreas by using an iso-osmotic Percoll gradient. In the presence of [gamma-32P]ATP, phosphorylation of several granule proteins was induced by Ca2+, most notably a Mr-13 000 protein, whereas addition of cyclic AMP was without effect. When phosphatidylserine was also added, Ca2+ increased the phosphorylation of additional proteins, with the largest effect on a protein of Mr 62 000. Purified granules were also able to phosphorylate exogenous substrates. Ca2+-induced phosphorylation of lysine-rich histone was enhanced over 3-fold in the presence of phosphatidylserine, and cyclic AMP-activated protein kinase activity was revealed with mixed histone as substrate. The concentrations of free Ca2+ and cyclic AMP required for half-maximal phosphorylation of both endogenous and exogenous proteins were 1-3 microM and 57 nM respectively. Treatment of granules with 0.25 M-KCl resulted in the release of phosphatidylserine-dependent kinase activity into a high-speed granule supernatant. In contrast, granule-protein substrates of Ca2+-activated kinase activity were resistant to KCl extraction, and in fact were present in purified granule membranes. Kinase activity activated by cyclic AMP was not extracted by KCl treatment. It is concluded that phosphorylation of integral membrane proteins in the zymogen granule can be induced by one or more Ca2+-activated protein kinases. Such a reaction is a potential mechanism by which exocytosis may be regulated in the exocrine pancreas by Ca2+-mediated secretagogues. PMID:4004796

  7. Protein kinase activity associated with pancreatic zymogen granules.

    PubMed Central

    Burnham, D B; Munowitz, P; Thorn, N; Williams, J A

    1985-01-01

    Purified zymogen granules were prepared from rat pancreas by using an iso-osmotic Percoll gradient. In the presence of [gamma-32P]ATP, phosphorylation of several granule proteins was induced by Ca2+, most notably a Mr-13 000 protein, whereas addition of cyclic AMP was without effect. When phosphatidylserine was also added, Ca2+ increased the phosphorylation of additional proteins, with the largest effect on a protein of Mr 62 000. Purified granules were also able to phosphorylate exogenous substrates. Ca2+-induced phosphorylation of lysine-rich histone was enhanced over 3-fold in the presence of phosphatidylserine, and cyclic AMP-activated protein kinase activity was revealed with mixed histone as substrate. The concentrations of free Ca2+ and cyclic AMP required for half-maximal phosphorylation of both endogenous and exogenous proteins were 1-3 microM and 57 nM respectively. Treatment of granules with 0.25 M-KCl resulted in the release of phosphatidylserine-dependent kinase activity into a high-speed granule supernatant. In contrast, granule-protein substrates of Ca2+-activated kinase activity were resistant to KCl extraction, and in fact were present in purified granule membranes. Kinase activity activated by cyclic AMP was not extracted by KCl treatment. It is concluded that phosphorylation of integral membrane proteins in the zymogen granule can be induced by one or more Ca2+-activated protein kinases. Such a reaction is a potential mechanism by which exocytosis may be regulated in the exocrine pancreas by Ca2+-mediated secretagogues. Images Fig. 1. Fig. 2. Fig. 7. Fig. 8. PMID:4004796

  8. G-protein coupled receptor-associated sorting protein 1 (GASP-1), a ubiquitous tumor marker.

    PubMed

    Zheng, Xiaoyi; Chang, Frank; Zhang, Xinmin; Rothman, Vicki L; Tuszynski, George P

    2012-08-01

    Using an innovative "2-D high performance liquid electrophoresis" (2-D HPLE) technology we identified that a specific fragment of G-protein coupled receptor-associated sorting protein 1 (GASP-1) was present in the sera of breast cancer patients and was over-expressed in early and late stage breast tumors (Tuszynski, G.P. et al., 2011). In this study we further investigated the significance of GASP-1 as a tumor marker by investigating the expression GASP-1 in different kinds of tumors as well as in the sera of patients with various cancers. Over expression of GASP-1 was detected in brain, pancreatic, and breast cancers as compared to their respective normal tissues as assessed by immunohistochemical staining of tissue arrays using a "peptide specific" GASP-1 antibody. We found that across these cancers, GASP-1 was expressed approximately 10 fold more in the cancer as compared to normal tissue. The increase in GASP-1 expression was also seen in hyperplastic and inflammatory lesions of breast and pancreatic cancers as compared to normal tissue. GASP-1 was primarily expressed in the tumor epithelium of the epithelial-derived cancers and in the transformed glial cells of the brain tumors. Using a sensitive "competitive ELISA" for GASP-1, we found that sera from patients with brain, liver, breast and lung cancers expressed 4-7 fold more GASP-1 peptide than sera from normal healthy individuals. These studies identify GASP-1 as a potential new serum and tumor biomarker for several cancers and suggest that GASP-1 may be a novel target for development of cancer therapeutics. PMID:22483848

  9. Identification of novel proteins associated with both alpha-synuclein and DJ-1.

    PubMed

    Jin, Jinghua; Li, G Jane; Davis, Jeanne; Zhu, David; Wang, Yan; Pan, Catherine; Zhang, Jing

    2007-05-01

    The molecular mechanisms leading to neurodegeneration in Parkinson disease (PD) remain elusive, although many lines of evidence have indicated that alpha-synuclein and DJ-1, two critical proteins in PD pathogenesis, interact with each other functionally. The investigation on whether alpha-synuclein directly interacts with DJ-1 has been controversial. In the current study, we analyzed proteins associated with alpha-synuclein and/or DJ-1 with a robust proteomics technique called stable isotope labeling by amino acids in cell culture (SILAC) in dopaminergic MES cells exposed to rotenone versus controls. We identified 324 and 306 proteins in the alpha-synuclein- and DJ-1-associated protein complexes, respectively. Among alpha-synuclein-associated proteins, 141 proteins displayed significant changes in the relative abundance (increase or decrease) after rotenone treatment; among DJ-1-associated proteins, 119 proteins displayed significant changes in the relative abundance after rotenone treatment. Although no direct interaction was observed between alpha-synuclein and DJ-1, whether analyzed by affinity purification followed by mass spectrometry or subsequent direct co-immunoprecipitation, 144 proteins were seen in association with both alpha-synuclein and DJ-1. Of those, 114 proteins displayed significant changes in the relative abundance in the complexes associated with alpha-synuclein, DJ-1, or both after rotenone treatment. A subset of these proteins (mortalin, nucleolin, grp94, calnexin, and clathrin) was further validated for their association with both alpha-synuclein and DJ-1 using confocal microscopy, Western blot, and/or immunoprecipitation. Thus, we not only confirmed that there was no direct interaction between alpha-synuclein and DJ-1 but also, for the first time, report these five novel proteins to be associating with both alpha-synuclein and DJ-1. Further characterization of these docking proteins will likely shed more light on the mechanisms by which DJ-1

  10. [Cytoskeletal actin and its associated proteins. Some examples in Protista].

    PubMed

    Guillén, N; Carlier, M F; Brugerolle, G; Tardieux, I; Ausseil, J

    1998-06-01

    Many processes, cell motility being an example, require cells to remodel the actin cytoskeleton in response to both intracellular and extracellular signals. Reorganization of the actin cytoskeleton involves the rapid disassembly and reassembly of actin filaments, a phenomenon regulated by the action of particular actin-binding proteins. In recent years, an interest in studying actin regulation in unicellular organisms has arisen. Parasitic protozoan are among these organisms and studies of the cytoskeleton functions of these protozoan are relevant related to either cell biology or pathogenicity. To discuss recent data in this field, a symposium concerning "Actin and actin-binding proteins in protists" was held on May 8-11 in Paris, France, during the XXXV meeting of the French Society of Protistology. As a brief summary of the symposium we report here findings concerning the in vitro actin dynamic assembly, as well as the characterization of several actin-binding proteins from the parasitic protozoan Entamoeba histolytica, Trichomonas vaginalis and Plasmodium knowlesi. In addition, localization of actin in non-pathogen protists such as Prorocentrum micans and Crypthecodinium cohnii is also presented. The data show that some actin-binding proteins facilitate organization of filaments into higher order structures as pseudopods, while others have regulatory functions, indicating very particular roles for actin-binding proteins. One of the proteins discussed during the symposium, the actin depolymerizing factor ADF, was shown to enhance the treadmilling rate of actin filaments. In vitro, ADF binds to the ADP-bound forms of G-actin and F-actin, thereby participating in and changing the rate of actin assembly. Biochemical approaches allowed the identification of a protein complex formed by HSP/C70-cap32-34 which might also be involved in depolymerization of F-actin in P. knowlesi. Molecular and cellular approaches were used to identify proteins such as ABP-120 and myosin

  11. Methyl-accepting protein associated with bacterial sensory phodopsin I

    SciTech Connect

    Spudich, E.N.; Hasselbacher, C.A. ); Spudich, J.L. )

    1988-09-01

    In vivo radiolabeling of Halaobacterium halobium phototaxis mutants and revertants with L-(methyl-{sup 3}H) methionine implicated seven methyl-accepting protein bands with apparent molecular masses from 65 to 150 kilodaltons (kDa) in adaptation of the organism to chemo and photo stimuli, and one of these (94 kDa) was specifically implicated in photoaxis. The lability of the radiolabeled bands to mild base treatment indicated the the methyl linkages are carboxylmethylesters, as is the case in the eubacterial chemotaxis receptor-transducers. The 94-kDa protein was present in increased amounts in an overproducer of the apoprotein of sensory rhodopsin I, one of two retinal-containing photoaxis receptors in H. halobium. It was absent in a strain the contained sensory rhodopsin II and that lacked sensory rhodopsin I and was also absent in a mutant that lacked both photoreceptors. Based in the role of methyl-accepting proteins in chemotaxis in other bacteria, we suggest that the 94-kDa protein is the signal transducer for sensory rhodopsin I. By ({sup 3}H)retinal labeling studies, we previously identified a 25-kDa retinal-binding polypeptide that was derived from photochemically reactive sensory rhodopsin I. When H. halobium membranes containing sensory rhodopsin I were treated by a procedure that stably reduced ({sup 3}H) retinal onto the 25-kDa apoprotein, a 94-kDa protein was also found to be radiolabeled. Protease digestion confirmed that the 94-kDa retinal-labeled protein was the same as the methyl-accepting protein that was suggested above to be the siginal transducer for sensory rhodopsin I. Possible models are that the 25- and 94-kDa proteins are tightly interacting components of the photosensory signaling machinery or that both are forms of sensory rhodopsin I.

  12. Associations between heat shock protein 70 genetic polymorphisms and calving traits in crossbred Brahman cows

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Stressors such as heat, cold, toxins, and oxygen deprivation are known to induce heat shock proteins. Genetic polymorphisms associated with heat shock protein genes have been associated with decreased male and female fertility. Our objectives were to 1) confirm single nucleotide polymorphisms (SNP) ...

  13. SIMILARITIES BETWEEN PROTEIN IIIA AND PROTEIN IIIB, TWO PROMINENT SYNAPTIC VESICLE-ASSOCIATED PHOSPHOPROTEINS (JOURNAL VERSION)

    EPA Science Inventory

    Protein IIIa (Mr 74,000) and protein IIIb (Mr 55,000) are two major phosphoproteins found in mammalian brain. It was previously shown in intact nerve cells that the phosphorylation state of these two proteins could be increased by electrical stimulation, by depolarizing agents in...

  14. A protein kinase associated with paired helical filaments in Alzheimer disease.

    PubMed Central

    Vincent, I J; Davies, P

    1992-01-01

    We have identified a protein kinase in immunoaffinity-purified preparations of paired helical filaments from brain tissue of individuals with Alzheimer disease. The kinase phosphorylates the filament proteins in vitro in a manner independent of second messenger regulation or of modulation by heparin and polyamines. Physiological concentrations of hemin, an oxidized heme porphyrin, inhibit the kinase and abolish Alz-50 immunoreactivity of the proteins. Since paired helical filaments are composed of hyperphosphorylated proteins, association of a protein kinase with the filaments provides a mechanism for abnormal processing of the proteins in disease. Images PMID:1557394

  15. LUD, a new protein domain associated with lactate utilization

    PubMed Central

    2013-01-01

    Background A novel highly conserved protein domain, DUF162 [Pfam: PF02589], can be mapped to two proteins: LutB and LutC. Both proteins are encoded by a highly conserved LutABC operon, which has been implicated in lactate utilization in bacteria. Based on our analysis of its sequence, structure, and recent experimental evidence reported by other groups, we hereby redefine DUF162 as the LUD domain family. Results JCSG solved the first crystal structure [PDB:2G40] from the LUD domain family: LutC protein, encoded by ORF DR_1909, of Deinococcus radiodurans. LutC shares features with domains in the functionally diverse ISOCOT superfamily. We have observed that the LUD domain has an increased abundance in the human gut microbiome. Conclusions We propose a model for the substrate and cofactor binding and regulation in LUD domain. The significance of LUD-containing proteins in the human gut microbiome, and the implication of lactate metabolism in the radiation-resistance of Deinococcus radiodurans are discussed. PMID:24274019

  16. Programmable protein arrays for immunoprofiling HPV-associated cancers.

    PubMed

    Ewaisha, Radwa; Meshay, Ian; Resnik, Jack; Katchman, Benjamin A; Anderson, Karen S

    2016-04-01

    Over 600,000 cancers each year are attributed to the human papillomavirus (HPV), including cervical, anogenital and oropharyngeal cancers (OPC). A key challenge in understanding HPV immunobiology is the diversity of oncogenic HPV types and the need for multiplexed display of HPV antigens to measure antibody responses. We have generated custom HPV protein microarrays displaying 98 proteins as C-terminal GST fusion proteins, representing eight antigens of two low-risk HPV types (HPV6 and 11) and ten oncogenic high-risk HPV types (HPV16, 18, 31, 33, 35, 39, 45, 51, 52 and 58). We demonstrate robust and reproducible protein expression of 96/98 of the antigens using a human cell lysate expression system. The target epitopes and specificities of four monoclonal antibodies were identified. Using sera from ten patients with newly diagnosed OPC and ten controls, we demonstrate specific IgG seroreactivity to HPV16 E1, E2, and E7 (a fold increase of 1.52, 2.19 and 1.35 in cases vs. controls, respectively, all p < 0.005), confirming our prior data on an ELISA platform. We also detect HPV52 E7 Abs in serum from a patient with cervical cancer. The HPV protein array has potential for rapid identification of serologic responses to 12 HPV types. PMID:27089055

  17. Expressions of Senescence-Associated β-Galactosidase and Senescence Marker Protein-30 are Associated with Lens Epithelial Cell Apoptosis

    PubMed Central

    Zhou, Dan; Yin, Dan; Xiao, Fang; Hao, Jie

    2015-01-01

    Background To investigate associations of senescence marker protein-30 and senescence-associated β-galactosidase expression with lens epithelial cells apoptosis among Chinese age-related cataract patients. Material/Methods A total of 145 age-related cataract patients (69 cases with nuclear cataract in 91 eyes and 76 cases of cortical cataract with 102 eyes) were enrolled in our study. An annular tear of the central part of anterior lens capsules was performed for each patient. Immunohistochemical staining and real-time PCR were used to detect the protein and mRNA expression levels, and TUNEL was used to assess lens epithelial cells apoptosis. Comparisons of protein expression levels and lens epithelial cells apoptosis were made between the 2 groups. Results The results showed a higher protein expression level of senescence marker protein-30 in surrounding parts of the anterior lens capsule compared with the central part of the anterior lens capsule; however, the positive rate of senescence-associated β-galactosidase was remarkably higher in the central part than in the surrounding part. Compared with cortical cataract patients, nuclear cataract patients had elevated senescence marker protein-30 protein and mRNA expression levels, but had a decreased positive rate of senescence-associated β-galactosidase. TUNEL results showed that the lens epithelial cell apoptosis rate was higher in the central part of the anterior lens capsule than in the surrounding part in both groups. Within either central or surrounding area of anterior lens capsule, cortical cataract patients exhibited a significantly higher lens epithelial cell apoptosis rate in contrast with nuclear cataract patients. Conclusions Our study results suggest that senescence marker protein-30 and senescence-associated β-galactosidase expressions in both nuclear cataract and cortical cataract patients were associated with lens epithelial cells apoptosis. PMID:26619319

  18. Association of Influenza Virus Proteins with Membrane Rafts

    PubMed Central

    Veit, Michael; Thaa, Bastian

    2011-01-01

    Assembly and budding of influenza virus proceeds in the viral budozone, a domain in the plasma membrane with characteristics of cholesterol/sphingolipid-rich membrane rafts. The viral transmembrane glycoproteins hemagglutinin (HA) and neuraminidase (NA) are intrinsically targeted to these domains, while M2 is seemingly targeted to the edge of the budozone. Virus assembly is orchestrated by the matrix protein M1, binding to all viral components and the membrane. Budding progresses by protein- and lipid-mediated membrane bending and particle scission probably mediated by M2. Here, we summarize the experimental evidence for this model with emphasis on the raft-targeting features of HA, NA, and M2 and review the functional importance of raft domains for viral protein transport, assembly and budding, environmental stability, and membrane fusion. PMID:22312341

  19. Possible association between phages, Hoc protein, and the immune system.

    PubMed

    Dabrowska, K; Switała-Jeleń, K; Opolski, A; Górski, A

    2006-02-01

    Mammals have become "an environment" for enterobacterial phage life cycles. Therefore it could be expected that bacteriophages adapt to them. This adaptation must comprise bacteriophage proteins. Gp Hoc seems to have significance neither for phage particle structure nor for phage antibacterial activity. It is evidently not necessary for the "typical" antibacterial actions of bacteriophages. But the rules of evolution make it improbable that gp Hoc really has no function, and non-essential genes of T4-type phages are probably important for phages' adaptation to their particular lifestyle. More interesting is the eukaryotic origin of gp Hoc: a resemblance to immunoglobulin-like proteins that reflects their evolutionary relation. Substantial differences in biological activity between T4 and a mutant that lacks gp Hoc were observed in a mammalian system. Hoc protein seems to be one of the molecules predicted to interact with mammalian organisms and/or modulate these interactions. PMID:16195787

  20. Localized mRNA translation and protein association

    NASA Astrophysics Data System (ADS)

    Zhdanov, Vladimir

    2014-08-01

    Recent direct observations of localization of mRNAs and proteins both in prokaryotic and eukaryotic cells can be related to slowdown of diffusion of these species due to macromolecular crowding and their ability to aggregate and form immobile or slowly mobile complexes. Here, a generic kinetic model describing both these factors is presented and comprehensively analyzed. Although the model is non-linear, an accurate self-consistent analytical solution of the corresponding reaction-diffusion equation has been constructed, the types of localized protein distributions have been explicitly shown, and the predicted kinetic regimes of gene expression have been classified.

  1. Association of the P6 Protein of Cauliflower mosaic virus with Plasmodesmata and Plasmodesmal Proteins1[W][OPEN

    PubMed Central

    Rodriguez, Andres; Angel, Carlos A.; Lutz, Lindy; Leisner, Scott M.; Nelson, Richard S.; Schoelz, James E.

    2014-01-01

    The P6 protein of Cauliflower mosaic virus (CaMV) is responsible for the formation of inclusion bodies (IBs), which are the sites for viral gene expression, replication, and virion assembly. Moreover, recent evidence indicates that ectopically expressed P6 inclusion-like bodies (I-LBs) move in association with actin microfilaments. Because CaMV virions accumulate preferentially in P6 IBs, we hypothesized that P6 IBs have a role in delivering CaMV virions to the plasmodesmata. We have determined that the P6 protein interacts with a C2 calcium-dependent membrane-targeting protein (designated Arabidopsis [Arabidopsis thaliana] Soybean Response to Cold [AtSRC2.2]) in a yeast (Saccharomyces cerevisiae) two-hybrid screen and have confirmed this interaction through coimmunoprecipitation and colocalization assays in the CaMV host Nicotiana benthamiana. An AtSRC2.2 protein fused to red fluorescent protein (RFP) was localized to the plasma membrane and specifically associated with plasmodesmata. The AtSRC2.2-RFP fusion also colocalized with two proteins previously shown to associate with plasmodesmata: the host protein Plasmodesmata-Localized Protein1 (PDLP1) and the CaMV movement protein (MP). Because P6 I-LBs colocalized with AtSRC2.2 and the P6 protein had previously been shown to interact with CaMV MP, we investigated whether P6 I-LBs might also be associated with plasmodesmata. We examined the colocalization of P6-RFP I-LBs with PDLP1-green fluorescent protein (GFP) and aniline blue (a stain for callose normally observed at plasmodesmata) and found that P6-RFP I-LBs were associated with each of these markers. Furthermore, P6-RFP coimmunoprecipitated with PDLP1-GFP. Our evidence that a portion of P6-GFP I-LBs associate with AtSRC2.2 and PDLP1 at plasmodesmata supports a model in which P6 IBs function to transfer CaMV virions directly to MP at the plasmodesmata. PMID:25239023

  2. Decreased activity of Blastocladiella emersonii zoospore ribosomes: correlation with developmental changes in ribosome-associated proteins.

    PubMed

    Jaworski, A J; Wilson, J B

    1989-10-01

    Ribosomal proteins isolated from dormant zoospores were compared to the ribosomal proteins found in the active growth phase by two-dimensional polyacrylamide gel electrophoresis. Zoospore ribosomes were found to contain a set of five proteins, designated Z1 to Z5, which were not present in growth phase ribosomes. The Z1-Z5 proteins were not removed by high-salt washes using either 1 M KCl or 1 M NH4 Cl. The Z1 protein is found associated with zoospore 60 S subunits while Z2-Z5 are bound to 40 S subunits. Zoospore monoribosomes and polyribosomes contain comparable levels of each of the five proteins. Approximately 60 min. after sporulation is induced, the Z1-Z5 proteins begin to accumulate on the ribosomes with the highest levels of these proteins found associated with ribosomes at the zoospore stage. During germination, the proteins gradually disappear and are not detectable on the ribosomes after 4 hr of germination. The presence of the Z1-Z5 proteins correlates with a decrease in in vitro protein synthetic activity of the fungal ribosomes. The data are consistent with the hypothesis that the proteins regulate translation by completely blocking protein synthesis on a subset of ribosomes while the remainder of the ribosomes function at normal rates. PMID:2776972

  3. Studies on gonococcus infection. XV. Identification of surface proteins of Neisseria gonorrhoeae correlated with leukocyte association.

    PubMed Central

    King, G J; Swanson, J

    1978-01-01

    Neisseria gonorrhoeae which exhibit high levels of leukocyte association have a surface protein which is considerably diminished in isogenic gonococci which exhibit low levels of leukocyte association (LA). The LA protein exhibits strain variation in molecular weight and immunogenicity. Membranes derived from LA+ and LA- organisms show quantitative differences in their adsorption to leukocytes; these differences are analogous to those found for the intact organisms regarding their association with leukocytes. Images PMID:211086

  4. Computational Framework for Prediction of Peptide Sequences That May Mediate Multiple Protein Interactions in Cancer-Associated Hub Proteins

    PubMed Central

    Sarkar, Debasree; Patra, Piya; Ghosh, Abhirupa; Saha, Sudipto

    2016-01-01

    A considerable proportion of protein-protein interactions (PPIs) in the cell are estimated to be mediated by very short peptide segments that approximately conform to specific sequence patterns known as linear motifs (LMs), often present in the disordered regions in the eukaryotic proteins. These peptides have been found to interact with low affinity and are able bind to multiple interactors, thus playing an important role in the PPI networks involving date hubs. In this work, PPI data and de novo motif identification based method (MEME) were used to identify such peptides in three cancer-associated hub proteins—MYC, APC and MDM2. The peptides corresponding to the significant LMs identified for each hub protein were aligned, the overlapping regions across these peptides being termed as overlapping linear peptides (OLPs). These OLPs were thus predicted to be responsible for multiple PPIs of the corresponding hub proteins and a scoring system was developed to rank them. We predicted six OLPs in MYC and five OLPs in MDM2 that scored higher than OLP predictions from randomly generated protein sets. Two OLP sequences from the C-terminal of MYC were predicted to bind with FBXW7, component of an E3 ubiquitin-protein ligase complex involved in proteasomal degradation of MYC. Similarly, we identified peptides in the C-terminal of MDM2 interacting with FKBP3, which has a specific role in auto-ubiquitinylation of MDM2. The peptide sequences predicted in MYC and MDM2 look promising for designing orthosteric inhibitors against possible disease-associated PPIs. Since these OLPs can interact with other proteins as well, these inhibitors should be specific to the targeted interactor to prevent undesired side-effects. This computational framework has been designed to predict and rank the peptide regions that may mediate multiple PPIs and can be applied to other disease-associated date hub proteins for prediction of novel therapeutic targets of small molecule PPI modulators. PMID

  5. Identification of proteins associated with RNA polymerase III using a modified tandem chromatin affinity purification.

    PubMed

    Nguyen, Ngoc-Thuy-Trinh; Saguez, Cyril; Conesa, Christine; Lefebvre, Olivier; Acker, Joël

    2015-02-01

    To identify the proteins associated with the RNA polymerase III (Pol III) machinery in exponentially growing yeast cells, we developed our own tandem chromatin affinity purification procedure (TChAP) after in vivo cross-link, allowing a reproducible and good recovery of the protein bait and its associated partners. In contrast to TFIIIA that could only be purified as a free protein, this protocol allows us to capture free Pol III together with Pol III bound on its target genes. Transcription factors, elongation factors, RNA-associated proteins and proteins involved in Pol III biogenesis were identified by mass spectrometry. Interestingly, the presence of all the TFIIIB subunits found associated with Pol III together with the absence of TFIIIC and chromatin factors including histones suggest that DNA-bound Pol III purified using TChAP is mainly engaged in transcription reinitiation. PMID:25086199

  6. Effect of the microtubule-associated protein tau on dynamics of single-headed motor proteins KIF1A

    NASA Astrophysics Data System (ADS)

    Sparacino, J.; Farías, M. G.; Lamberti, P. W.

    2014-02-01

    Intracellular transport based on molecular motors and its regulation are crucial to the functioning of cells. Filamentary tracks of the cells are abundantly decorated with nonmotile microtubule-associated proteins, such as tau. Motivated by experiments on kinesin-tau interactions [Dixit et al., Science 319, 1086 (2008), 10.1126/science.1152993] we developed a stochastic model of interacting single-headed motor proteins KIF1A that also takes into account the interactions between motor proteins and tau molecules. Our model reproduces experimental observations and predicts significant effects of tau on bound time and run length which suggest an important role of tau in regulation of kinesin-based transport.

  7. Transmembrane Protein (Perfringolysin O) Association with Ordered Membrane Domains (Rafts) Depends Upon the Raft-Associating Properties of Protein-Bound Sterol

    PubMed Central

    Lin, Qingqing; London, Erwin

    2013-01-01

    Because transmembrane (TM) protein localization, or nonlocalization, in ordered membrane domains (rafts) is a key to understanding membrane domain function, it is important to define the origin of protein-raft interaction. One hypothesis is that a tight noncovalent attachment of TM proteins to lipids that have a strong affinity for ordered domains can be sufficient to induce raft-protein interaction. The sterol-binding protein perfringolysin O (PFO) was used to test this hypothesis. PFO binds both to sterols that tend to localize in ordered domains (e.g., cholesterol), and to those that do not (e.g., coprostanol), but it does not bind to epicholesterol, a raft-promoting 3α-OH sterol. Using a fluorescence resonance energy transfer assay in model membrane vesicles containing coexisting ordered and disordered lipid domains, both TM and non-TM forms of PFO were found to concentrate in ordered domains in vesicles containing high and low-Tm lipids plus cholesterol or 1:1 (mol/mol) cholesterol/epicholesterol, whereas they concentrate in disordered domains in vesicles containing high-Tm and low-Tm lipids plus 1:1 (mol/mol) coprostanol/epicholesterol. Combined with previous studies this behavior indicates that TM protein association with ordered domains is dependent upon both the association of the protein-bound sterol with ordered domains and hydrophobic match between TM segments and rafts. PMID:24359745

  8. Identification of a novel protein-protein interaction motif mediating interaction of GPCR-associated sorting proteins with G protein-coupled receptors.

    PubMed

    Bornert, Olivier; Møller, Thor C; Boeuf, Julien; Candusso, Marie-Pierre; Wagner, Renaud; Martinez, Karen L; Simonin, Frederic

    2013-01-01

    GPCR desensitization and down-regulation are considered key molecular events underlying the development of tolerance in vivo. Among the many regulatory proteins that are involved in these complex processes, GASP-1 have been shown to participate to the sorting of several receptors toward the degradation pathway. This protein belongs to the recently identified GPCR-associated sorting proteins (GASPs) family that comprises ten members for which structural and functional details are poorly documented. We present here a detailed structure-function relationship analysis of the molecular interaction between GASPs and a panel of GPCRs. In a first step, GST-pull down experiments revealed that all the tested GASPs display significant interactions with a wide range of GPCRs. Importantly, the different GASP members exhibiting the strongest interaction properties were also characterized by the presence of a small, highly conserved and repeated "GASP motif" of 15 amino acids. We further showed using GST-pull down, surface plasmon resonance and co-immunoprecipitation experiments that the central domain of GASP-1, which contains 22 GASP motifs, is essential for the interaction with GPCRs. We then used site directed mutagenesis and competition experiments with synthetic peptides to demonstrate that the GASP motif, and particularly its highly conserved core sequence SWFW, is critically involved in the interaction with GPCRs. Overall, our data show that several members of the GASP family interact with GPCRs and highlight the presence within GASPs of a novel protein-protein interaction motif that might represent a new target to investigate the involvement of GASPs in the modulation of the activity of GPCRs. PMID:23441177

  9. Axoplasmic transport of microtubule-associated proteins in the rat sciatic nerve

    SciTech Connect

    Takenaka, T.; Inomata, K.

    1981-09-01

    /sup 32/P-ATP was injected into the L5 dorsal root ganglion and axoplasmic transport of the phosphorylate MA proteins 2, microtubule-associated proteins 2, was observed. After the injection of /sup 32/P-ATP, the nerve was dissected out at prescribed time intervals and sliced into 5-mm pieces. Each segment was electrophoresed on an SDS-polyacrylamide slab gel and subjected to autoradiography. A protein of 310,000 dalton was transported at a velocity of 6.6-10.6 mm/day in the axon with the electrophoretic mobility identical to that of MA proteins 2, one of the key components associated with the microtubules.

  10. Protein 4.1: its association with the human erythrocyte membrane.

    PubMed Central

    Shiffer, K A; Goodman, S R

    1984-01-01

    125I-labeled protein 4.1a and 4.1b have equal ability to reassociate with inside-out erythrocyte vesicles that were depleted of protein 4.1 in addition to other peripheral membrane proteins. The reassociation of 125I-labeled protein 4.1 to protein 4.1-depleted vesicles at 4 degrees C is salt dependent, pH dependent, and saturable with a Kd of 42-50 nM and an extrapolated maximal binding capacity of 120-140 micrograms of protein 4.1 bound per mg of vesicle protein or 60-70 micrograms of protein 4.1 bound per mg of ghost protein, correlating with the protein 4.1 content in the erythrocyte membrane (6-7% of the total membrane protein). Selective proteolytic cleavage of these vesicles with papain (5 micrograms/ml at 4 degrees C) eliminates greater than 60% of the high-affinity binding sites; therefore, we conclude that the interaction of protein 4.1 with the cytoplasmic membrane surface is through a specific high-affinity protein-protein association. Images PMID:6589603

  11. Brownian dynamics study of the influences of electrostatic interaction and diffusion on protein-protein association kinetics.

    PubMed Central

    Zhou, H X

    1993-01-01

    A unified model is presented for protein-protein association processes that are under the influences of electrostatic interaction and diffusion (e.g., protein oligomerization, enzyme catalysis, electron and energy transfer). The proteins are modeled as spheres that bear point charges and undergo translational and rotational Brownian motion. Before association can occur the two spheres have to be aligned properly to form a reaction complex via diffusion. The reaction complex can either go on to form the product or it can dissociate into the separate reactants through diffusion. The electrostatic interaction, like diffusion, influences every step except the one that brings the reaction complex into the product. The interaction potential is obtained by extending the Kirkwood-Tanford protein model (Tanford, C., and J. G. Kirkwood. 1957. J. Am. Chem. Soc. 79:5333-5339) to two charge-embedded spheres and solving the consequent equations under a particular basis set. The time-dependent association rate coefficient is then obtained through Brownian dynamics simulations according an algorithm developed earlier (Zhou, H.-X. 1990. J. Phys. Chem. 94:8794-8800). This method is applied to a model system of the cytochrome c and cytochrome c peroxidase association process and the results confirm the experimental dependence of the association rate constant on the solution ionic strength. An important conclusion drawn from this study is that when the product is formed by very specific alignment of the reactants, as is often the case, the effect of the interaction potential is simply to scale the association rate constant by a Boltzmann factor. This explains why mutations in the interface of the reaction complex have strong influences on the association rate constant whereas those away from the interface have minimal effects. It comes about because the former mutations change the interaction potential of the reaction complex significantly and the latter ones do not. PMID:8396447

  12. Type VI secretion apparatus and phage tail-associated protein complexes share a common evolutionary origin

    SciTech Connect

    Leiman, Petr G.; Basler, Marek; Ramagopal, Udupi A.; Bonanno, Jeffrey B.; Sauder, J. Michael; Pukatzki, Stefan; Burley, Stephen K.; Almo, Steven C.; Mekalanos, John J.

    2009-04-22

    Protein secretion is a common property of pathogenic microbes. Gram-negative bacterial pathogens use at least 6 distinct extracellular protein secretion systems to export proteins through their multilayered cell envelope and in some cases into host cells. Among the most widespread is the newly recognized Type VI secretion system (T6SS) which is composed of 15--20 proteins whose biochemical functions are not well understood. Using crystallographic, biochemical, and bioinformatic analyses, we identified 3 T6SS components, which are homologous to bacteriophage tail proteins. These include the tail tube protein; the membrane-penetrating needle, situated at the distal end of the tube; and another protein associated with the needle and tube. We propose that T6SS is a multicomponent structure whose extracellular part resembles both structurally and functionally a bacteriophage tail, an efficient machine that translocates proteins and DNA across lipid membranes into cells.

  13. Detergent-associated Solution Conformations of Helical and Beta-barrel Membrane Proteins

    SciTech Connect

    Mo, Yiming; Lee, Byung-Kwon; Ankner, John Francis; Becker, Jeffrey Marvin; Heller, William T

    2008-01-01

    Membrane proteins present major challenges for structural biology. In particular, the production of suitable crystals for high-resolution structural determination continues to be a significant roadblock for developing an atomic-level understanding of these vital cellular systems. The use of detergents for extracting membrane proteins from the native membrane for either crystallization or reconstitution into model lipid membranes for further study is assumed to leave the protein with the proper fold with a belt of detergent encompassing the membrane-spanning segments of the structure. Small-angle X-ray scattering was used to probe the detergent-associated solution conformations of three membrane proteins, namely bacteriorhodopsin (BR), the Ste2p G-protein coupled receptor from Saccharomyces cerevisiae, and the Escherichia coli porin OmpF. The results demonstrate that, contrary to the traditional model of a detergent-associated membrane protein, the helical proteins BR and Ste2p are not in the expected, compact conformation and associated with detergent micelles, while the ?-barrel OmpF is indeed embedded in a disk-like micelle in a properly folded state. The comparison provided by the BR and Ste2p, both members of the 7TM family of helical membrane proteins, further suggests that the interhelical interactions between the transmembrane helices of the two proteins differ, such that BR, like other rhodopsins, can properly refold to crystallize, while Ste2p continues to prove resistant to crystallization from an initially detergent-associated state.

  14. Deficiency of cyclase-associated protein 2 promotes arrhythmias associated with connexin43 maldistribution and fibrosis

    PubMed Central

    Peche, Vivek Shahaji; Linhart, Markus; Nickenig, Georg; Noegel, Angelika Anna; Schrickel, Jan Wilko

    2016-01-01

    Introduction Cyclase-associated protein 2 (CAP2) plays a major role in regulating the actin cytoskeleton. Since inactivation of CAP2 in a mouse model by a gene trap approach (Cap2gt/gt) results in cardiomyopathy and increased mortality, we hypothesized that CAP2 has a major impact on arrhythmias and electrophysiological parameters. Material and methods We performed long-term-ECG recordings in transgenic CAP2 deficient mice (C57BL/6) to detect spontaneous arrhythmias. In vivo electrophysiological studies by right heart catheterization and ex vivo epicardial mapping were used to analyze electrophysiological parameters, the inducibility of arrhythmias, and conduction velocities. Expression and distribution of cardiac connexins and the amount of cardiac fibrosis were evaluated. Results Spontaneous ventricular arrhythmias could be detected in Cap2gt/gt during the long-term-ECG recording. Cap2gt/gt showed marked conduction delays at atrial and ventricular levels, including a reduced heart rate (421.0 ±40.6 bpm vs. 450.8 ±27.9 bpm; p < 0.01), and prolongations of PQ (46.3 ±4.1 ms vs. 38.6 ±6.5 ms; p < 0.01), QRS (16.2 ±2.6 ms vs. 12.6 ±1.4 ms; p < 0.01), and QTc interval (55.8 ±6.0 ms vs. 45.2 ±3.3 ms; p = 0.02) in comparison to wild type mice. The PQ prolongation was due to an infra-Hisian conduction delay (HV: 9.7 ±2.1 ms vs. 6.5 ±3.1 ms; p = 0.02). The inducibility of ventricular tachycardias during the electrophysiological studies was significantly elevated in the mutant mice (inducible animals: 88% vs. 33%; p = 0.04). Cap2gt/gt showed more abnormal distribution of connexin43 compared to WT (23.0 ±4.7% vs. 2.9 ±0.8%; p < 0.01). Myocardial fibrosis was elevated in Cap2gt/gt hearts (9.1 ±6.7% vs. 5.5 ±3.3%; p < 0.01). Conclusions Loss of CAP2 results in marked electrophysiological disturbances including impaired sinus node function, conduction delays, and susceptibility to malignant arrhythmias. Structural changes in Cap2gt/gt are associated with

  15. Analysis of low-density lipoprotein-associated proteins using the method of digitized native protein mapping.

    PubMed

    Jin, Ya; Chen, Jin; Wang, Ahui; Zhang, Jun; Chen, Shumin; Manabe, Takashi; Tan, Wen

    2016-07-01

    The method of digitized native protein mapping, combining nondenaturing micro 2DE, grid gel-cutting, and quantitative LC-MS/MS (in data-independent acquisition mode, or MS(E) ), was improved by using a new MS/MS mode, ion mobility separation enhanced-MS(E) (HDMS(E) ), and applied to analyze the area of human plasma low-density lipoprotein (LDL). An 18 mm × 4.8 mm rectangular area which included LDL on a nondenaturing micro 2D gel of human plasma was grid-cut into 72 square gel pieces and subjected to quantitative LC-MS/MS. Compared with MS(E) , HDMS(E) showed significantly higher performance, by assigning 50% more proteins and detecting each protein in more squares. A total of 253 proteins were assigned with LC-HDMS(E) and the quantity distribution of each was reconstructed as a native protein map. The maps showed that Apo B-100 was the most abundant protein in the grid-cut area, concentrated at pI ca. 5.4-6.1 and apparent mass ca. 1000 kDa, which corresponded to four gel pieces, squares 39-42. An Excel macro was prepared to search protein maps which showed protein quantity peaks localized within this concentrated region of Apo B-100. Twenty-two proteins out of the 252 matched this criterion, in which 19 proteins have been reported to be associated with LDL. This method only requires several microliters of a plasma sample and the principle of the protein separation is totally different from the commonly used ultracentrifugation. The results obtained by this method would provide new insights on the structure and function of LDL. PMID:27174546

  16. RAID: a comprehensive resource for human RNA-associated (RNA-RNA/RNA-protein) interaction.

    PubMed

    Zhang, Xiaomeng; Wu, Deng; Chen, Liqun; Li, Xiang; Yang, Jinxurong; Fan, Dandan; Dong, Tingting; Liu, Mingyue; Tan, Puwen; Xu, Jintian; Yi, Ying; Wang, Yuting; Zou, Hua; Hu, Yongfei; Fan, Kaili; Kang, Juanjuan; Huang, Yan; Miao, Zhengqiang; Bi, Miaoman; Jin, Nana; Li, Kongning; Li, Xia; Xu, Jianzhen; Wang, Dong

    2014-07-01

    Transcriptomic analyses have revealed an unexpected complexity in the eukaryote transcriptome, which includes not only protein-coding transcripts but also an expanding catalog of noncoding RNAs (ncRNAs). Diverse coding and noncoding RNAs (ncRNAs) perform functions through interaction with each other in various cellular processes. In this project, we have developed RAID (http://www.rna-society.org/raid), an RNA-associated (RNA-RNA/RNA-protein) interaction database. RAID intends to provide the scientific community with all-in-one resources for efficient browsing and extraction of the RNA-associated interactions in human. This version of RAID contains more than 6100 RNA-associated interactions obtained by manually reviewing more than 2100 published papers, including 4493 RNA-RNA interactions and 1619 RNA-protein interactions. Each entry contains detailed information on an RNA-associated interaction, including RAID ID, RNA/protein symbol, RNA/protein categories, validated method, expressing tissue, literature references (Pubmed IDs), and detailed functional description. Users can query, browse, analyze, and manipulate RNA-associated (RNA-RNA/RNA-protein) interaction. RAID provides a comprehensive resource of human RNA-associated (RNA-RNA/RNA-protein) interaction network. Furthermore, this resource will help in uncovering the generic organizing principles of cellular function network. PMID:24803509

  17. Cilia and cilia-associated proteins in cancer

    PubMed Central

    Seeger-Nukpezah, Tamina; Little, Joy L.; Serzhanova, Victoria; Golemis, Erica A.

    2013-01-01

    The primary cilium is a well-established target in the pathogenesis of numerous developmental and chronic disorders, and more recently is attracting interest as a structure relevant to cancer. Here we discuss mechanisms by which changes in cilia can contribute to the formation and growth of tumors. We emphasize the cancer-relevance of cilia-dependent signaling pathways and proteins including mTOR, VHL, TSC, WNT, Aurora-A, NEDD9, and Hedgehog, and highlight the emerging role of ciliary dysfunction in renal cell carcinoma, medulloblastoma, and breast cancer. PMID:24982684

  18. Genome-Wide Association Study of CSF Levels of 59 Alzheimer's Disease Candidate Proteins: Significant Associations with Proteins Involved in Amyloid Processing and Inflammation

    PubMed Central

    Kauwe, John S. K.; Bailey, Matthew H.; Ridge, Perry G.; Perry, Rachel; Wadsworth, Mark E.; Hoyt, Kaitlyn L.; Staley, Lyndsay A.; Karch, Celeste M.; Harari, Oscar; Cruchaga, Carlos; Ainscough, Benjamin J.; Bales, Kelly; Pickering, Eve H.; Bertelsen, Sarah; Fagan, Anne M.; Holtzman, David M.; Morris, John C.; Goate, Alison M.

    2014-01-01

    Cerebrospinal fluid (CSF) 42 amino acid species of amyloid beta (Aβ42) and tau levels are strongly correlated with the presence of Alzheimer's disease (AD) neuropathology including amyloid plaques and neurodegeneration and have been successfully used as endophenotypes for genetic studies of AD. Additional CSF analytes may also serve as useful endophenotypes that capture other aspects of AD pathophysiology. Here we have conducted a genome-wide association study of CSF levels of 59 AD-related analytes. All analytes were measured using the Rules Based Medicine Human DiscoveryMAP Panel, which includes analytes relevant to several disease-related processes. Data from two independently collected and measured datasets, the Knight Alzheimer's Disease Research Center (ADRC) and Alzheimer's Disease Neuroimaging Initiative (ADNI), were analyzed separately, and combined results were obtained using meta-analysis. We identified genetic associations with CSF levels of 5 proteins (Angiotensin-converting enzyme (ACE), Chemokine (C-C motif) ligand 2 (CCL2), Chemokine (C-C motif) ligand 4 (CCL4), Interleukin 6 receptor (IL6R) and Matrix metalloproteinase-3 (MMP3)) with study-wide significant p-values (p<1.46×10−10) and significant, consistent evidence for association in both the Knight ADRC and the ADNI samples. These proteins are involved in amyloid processing and pro-inflammatory signaling. SNPs associated with ACE, IL6R and MMP3 protein levels are located within the coding regions of the corresponding structural gene. The SNPs associated with CSF levels of CCL4 and CCL2 are located in known chemokine binding proteins. The genetic associations reported here are novel and suggest mechanisms for genetic control of CSF and plasma levels of these disease-related proteins. Significant SNPs in ACE and MMP3 also showed association with AD risk. Our findings suggest that these proteins/pathways may be valuable therapeutic targets for AD. Robust associations in cognitively normal

  19. Proteome-wide identification of poly(ADP-ribose) binding proteins and poly(ADP-ribose)-associated protein complexes.

    PubMed

    Gagné, Jean-Philippe; Isabelle, Maxim; Lo, Ken Sin; Bourassa, Sylvie; Hendzel, Michael J; Dawson, Valina L; Dawson, Ted M; Poirier, Guy G

    2008-12-01

    Poly(ADP-ribose) (pADPr) is a polymer assembled from the enzymatic polymerization of the ADP-ribosyl moiety of NAD by poly(ADP-ribose) polymerases (PARPs). The dynamic turnover of pADPr within the cell is essential for a number of cellular processes including progression through the cell cycle, DNA repair and the maintenance of genomic integrity, and apoptosis. In spite of the considerable advances in the knowledge of the physiological conditions modulated by poly(ADP-ribosyl)ation reactions, and notwithstanding the fact that pADPr can play a role of mediator in a wide spectrum of biological processes, few pADPr binding proteins have been identified so far. In this study, refined in silico prediction of pADPr binding proteins and large-scale mass spectrometry-based proteome analysis of pADPr binding proteins were used to establish a comprehensive repertoire of pADPr-associated proteins. Visualization and modeling of these pADPr-associated proteins in networks not only reflect the widespread involvement of poly(ADP-ribosyl)ation in several pathways but also identify protein targets that could shed new light on the regulatory functions of pADPr in normal physiological conditions as well as after exposure to genotoxic stimuli. PMID:18981049

  20. Proteome-wide identification of poly(ADP-ribose) binding proteins and poly(ADP-ribose)-associated protein complexes

    PubMed Central

    Gagné, Jean-Philippe; Isabelle, Maxim; Lo, Ken Sin; Bourassa, Sylvie; Hendzel, Michael J.; Dawson, Valina L.; Dawson, Ted M.; Poirier, Guy G.

    2008-01-01

    Poly(ADP-ribose) (pADPr) is a polymer assembled from the enzymatic polymerization of the ADP-ribosyl moiety of NAD by poly(ADP-ribose) polymerases (PARPs). The dynamic turnover of pADPr within the cell is essential for a number of cellular processes including progression through the cell cycle, DNA repair and the maintenance of genomic integrity, and apoptosis. In spite of the considerable advances in the knowledge of the physiological conditions modulated by poly(ADP-ribosyl)ation reactions, and notwithstanding the fact that pADPr can play a role of mediator in a wide spectrum of biological processes, few pADPr binding proteins have been identified so far. In this study, refined in silico prediction of pADPr binding proteins and large-scale mass spectrometry-based proteome analysis of pADPr binding proteins were used to establish a comprehensive repertoire of pADPr-associated proteins. Visualization and modeling of these pADPr-associated proteins in networks not only reflect the widespread involvement of poly(ADP-ribosyl)ation in several pathways but also identify protein targets that could shed new light on the regulatory functions of pADPr in normal physiological conditions as well as after exposure to genotoxic stimuli. PMID:18981049

  1. Interaction of a 14-3-3 protein with the plant microtubule-associated protein EDE1

    PubMed Central

    Pignocchi, Cristina; Doonan, John H.

    2011-01-01

    Background and Aims The cell cycle-regulated protein ENDOSPERM DEFECTIVE 1 (EDE1) is a novel plant microtubule-associated protein essential for plant cell division and for microtubule organization in endosperm. EDE1 is only present on microtubules at mitosis and its expression is highly cell cycle regulated both at the protein and the transcript levels. Methods To search for EDE1-interacting proteins, a yeast two-hybrid screen was used in which EDE1 was fused with GAL4 DNA binding domain and expressed in a yeast strain that was then mated with a strain carrying a cDNA library fused to the GAL4 transactivation domain. Candidate interacting proteins were identified and confirmed in vitro. Key Results 14-3-3 upsilon was isolated several times from the library screen. In in vitro tests, it also interacted with EDE1: 14-3-3 upsilon most strongly associates with EDE1 in its free form, but also weakly when EDE1 is bound to microtubules. This study shows that EDE1 is a cyclin-dependent kinase substrate but that phosphorylation is not required for interaction with 14-3-3 upsilon. Conclusions The results suggest that 14-3-3 proteins may play a role in cytoskeletal organization of plant cells. The potential role of this interaction in the dynamics of EDE1 during the cell cycle is discussed. PMID:21558460

  2. Peroxymonosulfate Rapidly Inactivates the Disease-Associated Prion Protein.

    PubMed

    Chesney, Alexandra R; Booth, Clarissa J; Lietz, Christopher B; Li, Lingjun; Pedersen, Joel A

    2016-07-01

    Prions, the etiological agents in transmissible spongiform encephalopathies, exhibit remarkable resistance to most methods of inactivation that are effective against conventional pathogens. Prions are composed of pathogenic conformers of the prion protein (PrP(TSE)). Some prion diseases are transmitted, in part, through environmental routes. The recalcitrance of prions to inactivation may lead to a persistent reservoir of infectivity that contributes to the environmental maintenance of epizootics. At present, few methods exist to remediate prion-contaminated land surfaces. Here we conducted a proof-of-principle study to examine the ability of peroxymonosulfate to degrade PrP(TSE). We find that peroxymonosulfate rapidly degrades PrP(TSE) from two species. Transition-metal-catalyzed decomposition of peroxymonosulfate to produce sulfate radicals appears to enhance degradation. We further demonstrate that exposure to peroxymonosulfate significantly reduced PrP(C) to PrP(TSE) converting ability as measured by protein misfolding cyclic amplification, used as a proxy for infectivity. Liquid chromatography-tandem mass spectrometry revealed that exposure to peroxymonosulfate results in oxidative modifications to methionine and tryptophan residues. This study indicates that peroxymonosulfate may hold promise for decontamination of prion-contaminated surfaces. PMID:27247993

  3. Proteins Associated with SF3a60 in T. brucei

    PubMed Central

    Nyambega, Benson; Helbig, Claudia; Masiga, Daniel K.; Clayton, Christine; Levin, Mariano J.

    2014-01-01

    Trypanosoma brucei relies on Spliced leader trans splicing to generate functional messenger RNAs. Trans splicing joins the specialized SL exon from the SL RNA to pre-mRNAs and is mediated by the trans-spliceosome, which is made up of small nuclear ribonucleoprotein particles and non-snRNP factors. Although the trans spliceosome is essential for trypanosomatid gene expression, not all spliceosomal protein factors are known and of these, only a few are completely characterized. In this study, we have characterized the trypanosome Splicing Factor, SF3a60, the only currently annotated SF3a component. As expected, epitope-tagged SF3a60 localizes in the trypanosome nucleus. SF3a60 is essential for cell viability but its depletion seem to have no detectable effect on trans-splicing. In addition, we used SF3a60 as bait in a Yeast-2-hybrid system screen and identified its interacting protein factors. The interactions with SF3a120, SF3a66 and SAP130 were confirmed by tandem affinity purification and mass spectrometry. PMID:24651488

  4. Kaposi's sarcoma-associated herpesvirus latency-associated nuclear antigen interacts with bromodomain protein Brd4 on host mitotic chromosomes.

    PubMed

    You, Jianxin; Srinivasan, Viswanathan; Denis, Gerald V; Harrington, William J; Ballestas, Mary E; Kaye, Kenneth M; Howley, Peter M

    2006-09-01

    The latency-associated nuclear antigen (LANA) of Kaposi's sarcoma-associated herpesvirus (KSHV) is required for viral episome maintenance in host cells during latent infection. Two regions of the protein have been implicated in tethering LANA/viral episomes to the host mitotic chromosomes, and LANA chromosome-binding sites are subjects of high interest. Because previous studies had identified bromodomain protein Brd4 as the mitotic chromosome anchor for the bovine papillomavirus E2 protein, which tethers the viral episomes to host mitotic chromosomes (J. You, J. L. Croyle, A. Nishimura, K. Ozato, and P. M. Howley, Cell 117:349-360, 2004, and J. You, M. R. Schweiger, and P. M. Howley, J. Virol. 79:14956-14961, 2005), we examined whether KSHV LANA interacts with Brd4. We found that LANA binds Brd4 in vivo and in vitro and that the binding is mediated by a direct protein-protein interaction between the ET (extraterminal) domain of Brd4 and a carboxyl-terminal region of LANA previously implicated in chromosome binding. Brd4 associates with mitotic chromosomes throughout mitosis and demonstrates a strong colocalization with LANA and the KSHV episomes on host mitotic chromosomes. Although another bromodomain protein, RING3/Brd2, binds to LANA in a similar fashion in vitro, it is largely excluded from the mitotic chromosomes in KSHV-uninfected cells and is partially recruited to the chromosomes in KSHV-infected cells. These data identify Brd4 as an interacting protein for the carboxyl terminus of LANA on mitotic chromosomes and suggest distinct functional roles for the two bromodomain proteins RING3/Brd2 and Brd4 in LANA binding. Additionally, because Brd4 has recently been shown to have a role in transcription, we examined whether Brd4 can regulate the CDK2 promoter, which can be transactivated by LANA. PMID:16940503

  5. Bisphenol A accelerates capacitation-associated protein tyrosine phosphorylation of rat sperm by activating protein kinase A.

    PubMed

    Wan, Xiaofeng; Ru, Yanfei; Chu, Chen; Ni, Zimei; Zhou, Yuchuan; Wang, Shoulin; Zhou, Zuomin; Zhang, Yonglian

    2016-06-01

    Bisphenol A (BPA) is a synthetic estrogen-mimic chemical. It has been shown to affect many reproductive endpoints. However, the effect of BPA on the mature sperm and the mechanism of its action are not clear yet. Here, our in vitro studies indicated that BPA could accelerate sperm capacitation-associated protein tyrosine phosphorylation in time- and dose-dependent manners. In vivo, the adult male rats exposed to a high dose of BPA could result in a significant increase in sperm activity. Further investigation demonstrated that BPA could accelerate capacitation-associated protein tyrosine phosphorylation even if sperm were incubated in medium devoid of BSA, HCO3 (-), and Ca(2+) However, this action of BPA stimulation could be blocked by H89, a highly selective blocker of protein kinase A (PKA), but not by KH7, a specific inhibitor of adenylyl cyclase. These data suggest that BPA may activate PKA to affect sperm functions and male fertility. PMID:27174873

  6. Targeting of Pseudorabies Virus Structural Proteins to Axons Requires Association of the Viral Us9 Protein with Lipid Rafts

    PubMed Central

    Lyman, Mathew G.; Curanovic, Dusica; Enquist, Lynn W.

    2008-01-01

    The pseudorabies virus (PRV) Us9 protein plays a central role in targeting viral capsids and glycoproteins to axons of dissociated sympathetic neurons. As a result, Us9 null mutants are defective in anterograde transmission of infection in vivo. However, it is unclear how Us9 promotes axonal sorting of so many viral proteins. It is known that the glycoproteins gB, gC, gD and gE are associated with lipid raft microdomains on the surface of infected swine kidney cells and monocytes, and are directed into the axon in a Us9-dependent manner. In this report, we determined that Us9 is associated with lipid rafts, and that this association is critical to Us9-mediated sorting of viral structural proteins. We used infected non-polarized and polarized PC12 cells, a rat pheochromocytoma cell line that acquires many of the characteristics of sympathetic neurons in the presence of nerve growth factor (NGF). In these cells, Us9 is highly enriched in detergent-resistant membranes (DRMs). Moreover, reducing the affinity of Us9 for lipid rafts inhibited anterograde transmission of infection from sympathetic neurons to epithelial cells in vitro. We conclude that association of Us9 with lipid rafts is key for efficient targeting of structural proteins to axons and, as a consequence, for directional spread of PRV from pre-synaptic to post-synaptic neurons and cells of the mammalian nervous system. PMID:18483549

  7. Quantification of protein group coherence and pathway assignment using functional association

    PubMed Central

    2011-01-01

    Background Genomics and proteomics experiments produce a large amount of data that are awaiting functional elucidation. An important step in analyzing such data is to identify functional units, which consist of proteins that play coherent roles to carry out the function. Importantly, functional coherence is not identical with functional similarity. For example, proteins in the same pathway may not share the same Gene Ontology (GO) terms, but they work in a coordinated fashion so that the aimed function can be performed. Thus, simply applying existing functional similarity measures might not be the best solution to identify functional units in omics data. Results We have designed two scores for quantifying the functional coherence by considering association of GO terms observed in two biological contexts, co-occurrences in protein annotations and co-mentions in literature in the PubMed database. The counted co-occurrences of GO terms were normalized in a similar fashion as the statistical amino acid contact potential is computed in the protein structure prediction field. We demonstrate that the developed scores can identify functionally coherent protein sets, i.e. proteins in the same pathways, co-localized proteins, and protein complexes, with statistically significant score values showing a better accuracy than existing functional similarity scores. The scores are also capable of detecting protein pairs that interact with each other. It is further shown that the functional coherence scores can accurately assign proteins to their respective pathways. Conclusion We have developed two scores which quantify the functional coherence of sets of proteins. The scores reflect the actual associations of GO terms observed either in protein annotations or in literature. It has been shown that they have the ability to accurately distinguish biologically relevant groups of proteins from random ones as well as a good discriminative power for detecting interacting pairs of

  8. Association of lipids with integral membrane surface proteins of Mycoplasma hyorhinis

    SciTech Connect

    Bricker, T.M.; Boyer, M.J.; Keith, J.; Watson-McKown, R.; Wise, K.S.

    1988-02-01

    Triton X-114 (TX-114)-phase fractionation was used to identify and characterize integral membrane surface proteins of the wall-less procaryote Mycoplasma hyorhinis GDL. Phase fractionation of mycoplasmas followed by analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed selective partitioning of approximately 30 (/sup 35/S)methionine-labeled intrinsic membrane proteins into the TX-114 phase. Similar analysis of (/sup 3/H)palmitate-labeled cells showed that approximately 20 proteins of this organism were associated with lipid, all of which also efficiently partitioned as integral membrane components into the detergent phase. Immunoblotting and immunoprecipitation of TX-114-phase proteins from /sup 125/I-surface-labeled cells with four monoclonal antibodies to distinct surface epitopes of M. hyorhinis identified surface proteins p120, p70, p42, and p23 as intrinsic membrane components. Immunoprecipitation of (/sup 3/H)palmitate-labeled TX-114-phase proteins further established that surface proteins p120, p70, and p23 (a molecule that mediates complement-dependent mycoplasmacidal monoclonal antibody activity) were among the lipid-associated proteins of this organism. Two of these proteins, p120 and p123, were acidic (pI less than or equal to 4.5), as shown by two-dimensional isoelectric focusing. This study established that M. hyorhinis contains an abundance of integral membrane proteins tightly associated with lipids and that many of these proteins are exposed at the external surface of the single limiting plasma membrane. Monoclonal antibodies are reported that will allow detailed analysis of the structure and processing of lipid-associated mycoplasma proteins.

  9. The Golgi-Associated Hook3 Protein Is a Member of a Novel Family of Microtubule-Binding Proteins

    PubMed Central

    Walenta, Jason H.; Didier, Aaron J.; Liu, Xinran; Krämer, Helmut

    2001-01-01

    Microtubules are central to the spatial organization of diverse membrane-trafficking systems. Here, we report that Hook proteins constitute a novel family of cytosolic coiled coil proteins that bind to organelles and to microtubules. The conserved NH2-terminal domains of Hook proteins mediate attachment to microtubules, whereas the more divergent COOH-terminal domains mediate the binding to organelles. Human Hook3 bound to Golgi membranes in vitro and was enriched in the cis-Golgi in vivo. Unlike other cis-Golgi–associated proteins, however, a large fraction of Hook3 maintained its juxtanuclear localization after Brefeldin A treatment, indicating a Golgi-independent mechanism for Hook3 localization. Because overexpression of Hook3 caused fragmentation of the Golgi complex, we propose that Hook3 participates in defining the architecture and localization of the mammalian Golgi complex. PMID:11238449

  10. Cardiac arrhythmias associated with a liquid protein diet for the treatment of obesity

    SciTech Connect

    Lantigua, R.A.; Amatruda, J.M.; Biddle, T.L.; Forbes, G.B.; Lockwood, D.H.

    1980-09-25

    Our data demonstrate that a liquid protein diet is frequently associated with potentially life-threatening arrhythmias that are not detected on routine electrocardiography. Several studies of metabolic balance failed to reveal a cause for these arrhythmias. We recommended that the use of liquid protein diets should be terminated pending further investigation of the causes and prevention of the cardiac toxicity.

  11. IDENTIFICATION OF MAIZE KERNEL ENDOSPERM PROTEINS ASSOCIATED WITH RESISTANCE TO AFLATOXIN CONTAMINATION BY ASPERGILLUS FLAVUS

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Aflatoxins are carcinogens produced mainly by Aspergillus flavus during infection of susceptible crops, such as maize (Zea mays L.). Previously, embryo proteins from maize genotypes resistant or susceptible to A. flavus infection were compared using proteomics and resistance-associated proteins wer...

  12. The surface-associated proteins of wheat starch granules: suitability of wheat starch for celiac patients

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Wheat starch is used to make baked products for celiac patients in several European countries, but is avoided in the US because of uncertainty about the amounts of associated grain storage (gluten) proteins. People with celiac disease (CD) must avoid wheat, rye and barley proteins and products that...

  13. Electrostatic effects in the brownian dynamics of association and orientation of heme proteins

    SciTech Connect

    Northrup, S.H.; Boles, J.O.; Reynolds, J.C.L.

    1987-11-05

    The Brownian dynamics (BD) method is applied to the diffusional association of electron-transfer proteins cytochrome c (CYTC) and cytochrome c peroxidase (CYP). They examine the role of protein electrostatic charge distribution and solvent mediation in the facilitation of protein-protein docking prior to the electron-transfer step. Accurate interaction potentials are computed by iterating the linearized Poisson-Boltzmann equation around the larger protein CYP. The lower dielectric constant inside proteins and their irregular surface topography are taken into account. Realistic criteria for determining the successful docking of the proteins are based on a combination of mutual orientation of heme planes and heme edge-to-edge distance. The proteins successfully meet the most stringent of these criteria in two distinct regions of relative separation space which coincide with two electrostatically attractive regions. The existence of a large ensemble of electrostatically stable encounter complexes seemingly with acceptable geometric requirements for electron transfer is observed rather than a single dominant complex. The reaction criteria matching the experimental association rate is the case in which the heme edge distance is inside 20 A and the heme planes are coparallel to within 60/sup 0/. Ionic strength dependence of the association rate for this case agrees with that observed in experiment in the physiological regime. Association at random heme plane orientation results in a rate constant which is almost twice as large as that from experiment.

  14. Proteomic analysis reveals the dynamic association of proteins with translated mRNAs in Trypanosoma cruzi.

    PubMed

    Alves, Lysangela R; Avila, Andréa R; Correa, Alejandro; Holetz, Fabíola B; Mansur, Fernanda C B; Manque, Patrício A; de Menezes, Juliana P B; Buck, Gregory A; Krieger, Marco A; Goldenberg, Samuel

    2010-03-01

    Gene regulation is mainly post-transcriptional in trypanosomatids. The stability of mRNA and access to polysomes are thought to be tightly regulated, allowing Trypanosoma cruzi to adapt to the different environmental conditions during its life cycle. Post-transcriptional regulation requires the association between mRNAs and certain proteins to form mRNP complexes. We investigated the dynamic association between proteins and mRNAs, using poly(T) beads to isolate and characterize proteins and protein complexes bound to poly-A+ mRNAs. The protein content of these fractions was analyzed by mass spectrometry (LC-MS/MS). We identified 542 protein component of the mRNP complexes associated with mRNAs. Twenty-four of the proteins obtained were present in all fractions, whereas some other proteins were exclusive to a particular fraction: epimastigote polysomal (0.37%) and post-polysomal (2.95%) fractions; stress polysomal (13.8%) and post-polysomal (40.78%) fractions. Several proteins known to be involved in mRNA metabolism were identified, and this was considered important as it made it possible to confirm the reliability of our mRNP isolation approach. This procedure allowed us to have a first insight into the composition and dynamics of mRNPs in T. cruzi. PMID:20060445

  15. Association of Bovine Papillomavirus E2 Protein with Nuclear Structures In Vivo

    PubMed Central

    Kurg, Reet; Sild, Kristiina; Ilves, Aigi; Sepp, Mari; Ustav, Mart

    2005-01-01

    Papillomaviruses are small DNA viruses which have the capacity to establish a persistent infection in mammalian epithelial cells. The papillomavirus E2 protein is a central coordinator of viral gene expression, genome replication, and maintenance. We have investigated the distribution of bovine papillomavirus E2 protein in nuclei of proliferating cells and found that E2 is associated with cellular chromatin. This distribution does not change during the entire cell cycle. The N-terminal transactivation domain, but not the C-terminal DNA-binding domain, of the E2 protein is responsible for this association. The majority of the full-length E2 protein can only be detected in chromatin-enriched fractions but not as a free protein in the nucleus. Limited micrococcal nuclease digestion revealed that the E2 protein partitioned to different chromatin regions. A fraction of the E2 protein was located at nuclear sites that are resistant against nuclease attack, whereas the remaining E2 resided on compact chromatin accessible to micrococcal nuclease. These data suggest that there are two pools of E2 in the cell nucleus: one that localizes on transcriptionally inactive compact chromatin and the other, which compartmentalizes to transcriptionally active nuclear structures of the cell. Our data also suggest that E2 associates with chromatin through cellular protein(s), which in turn is released from chromatin at 0.4 M salt. PMID:16051845

  16. Autoimmunoreactive IgGs Against Cardiac Lipid Raft-Associated Proteins in Patients with POTS

    PubMed Central

    Wang, Xiao-Li; Ling, Tian-You; Charlesworth, M. Cristine; Figueroa, Juan J.; Low, Phillip; Shen, Win-Kuang; Lee, Hon-Chi

    2013-01-01

    Lipid rafts are specialized plasma membrane microdomains that serve as platforms for integrating cellular signal transductions. We have recently reported that autoantibodies against cardiac membrane proteins are present in patients with postural orthostatic tachycardia syndrome (POTS). In this study, we examined the presence of autoimmunoreactive IgGs against lipid raft proteins in these patients. IgGs were purified from the sera of 10 patients and 7 normal controls. Cardiac lipid raft preparations were isolated from normal human heart tissue. The lipid raft-associated proteins were resolved by 2DE and immunoblotted against IgGs from each subject. Protein spots that reacted specifically with patient IgGs were identified by nanoLC-MS/MS. Thirty-four such protein spots, and 72 unique proteins were identified. The targets of autoimmunoreactive IgGs include proteins associated with caveolae structure, adrenergic signaling, calcium signaling, cytostructures, chaperone and energy metabolism. Multiple pathways were involved including those that regulate caveolae-mediated signaling, oxidative phosphorylation, fatty acid metabolism, protein ubiquitination, and cardiac β-adrenergic signaling. Our results suggest that cardiac lipid raft-associated proteins are targets of autoimmunoreactive IgGs from patients with POTS. Autoimmunity may play a role in the pathogenesis of POTS. PMID:23562385

  17. Association of bovine papillomavirus E2 protein with nuclear structures in vivo.

    PubMed

    Kurg, Reet; Sild, Kristiina; Ilves, Aigi; Sepp, Mari; Ustav, Mart

    2005-08-01

    Papillomaviruses are small DNA viruses which have the capacity to establish a persistent infection in mammalian epithelial cells. The papillomavirus E2 protein is a central coordinator of viral gene expression, genome replication, and maintenance. We have investigated the distribution of bovine papillomavirus E2 protein in nuclei of proliferating cells and found that E2 is associated with cellular chromatin. This distribution does not change during the entire cell cycle. The N-terminal transactivation domain, but not the C-terminal DNA-binding domain, of the E2 protein is responsible for this association. The majority of the full-length E2 protein can only be detected in chromatin-enriched fractions but not as a free protein in the nucleus. Limited micrococcal nuclease digestion revealed that the E2 protein partitioned to different chromatin regions. A fraction of the E2 protein was located at nuclear sites that are resistant against nuclease attack, whereas the remaining E2 resided on compact chromatin accessible to micrococcal nuclease. These data suggest that there are two pools of E2 in the cell nucleus: one that localizes on transcriptionally inactive compact chromatin and the other, which compartmentalizes to transcriptionally active nuclear structures of the cell. Our data also suggest that E2 associates with chromatin through cellular protein(s), which in turn is released from chromatin at 0.4 M salt. PMID:16051845

  18. A Model of Protein Association Based on Their Hydrophobic and Electric Interactions

    PubMed Central

    Mozo-Villarías, Angel; Cedano, Juan; Querol, Enrique

    2014-01-01

    The propensity of many proteins to oligomerize and associate to form complex structures from their constituent monomers, is analyzed in terms of their hydrophobic (H), and electric pseudo-dipole (D) moment vectors. In both cases these vectors are defined as the product of the distance between their positive and negative centroids, times the total hydrophobicity or total positive charge of the protein. Changes in the magnitudes and directions of H and D are studied as monomers associate to form larger complexes. We use these descriptors to study similarities and differences in two groups of associations: a) open associations such as polymers with an undefined number of monomers (i.e. actin polymerization, amyloid and HIV capsid assemblies); b) closed symmetrical associations of finite size, like spherical virus capsids and protein cages. The tendency of the hydrophobic moments of the monomers in an association is to align in parallel arrangements following a pattern similar to those of phospholipids in a membrane. Conversely, electric dipole moments of monomers tend to align in antiparallel associations. The final conformation of a given assembly is a fine-tuned combination of these forces, limited by steric constraints. This determines whether the association will be open (indetermined number of monomers) or closed (fixed number of monomers). Any kinetic, binding or molecular peculiarities that characterize a protein assembly, comply with the vector rules laid down in this paper. These findings are also independent of protein size and shape. PMID:25329830

  19. A model of protein association based on their hydrophobic and electric interactions.

    PubMed

    Mozo-Villarías, Angel; Cedano, Juan; Querol, Enrique

    2014-01-01

    The propensity of many proteins to oligomerize and associate to form complex structures from their constituent monomers, is analyzed in terms of their hydrophobic (H), and electric pseudo-dipole (D) moment vectors. In both cases these vectors are defined as the product of the distance between their positive and negative centroids, times the total hydrophobicity or total positive charge of the protein. Changes in the magnitudes and directions of H and D are studied as monomers associate to form larger complexes. We use these descriptors to study similarities and differences in two groups of associations: a) open associations such as polymers with an undefined number of monomers (i.e. actin polymerization, amyloid and HIV capsid assemblies); b) closed symmetrical associations of finite size, like spherical virus capsids and protein cages. The tendency of the hydrophobic moments of the monomers in an association is to align in parallel arrangements following a pattern similar to those of phospholipids in a membrane. Conversely, electric dipole moments of monomers tend to align in antiparallel associations. The final conformation of a given assembly is a fine-tuned combination of these forces, limited by steric constraints. This determines whether the association will be open (indetermined number of monomers) or closed (fixed number of monomers). Any kinetic, binding or molecular peculiarities that characterize a protein assembly, comply with the vector rules laid down in this paper. These findings are also independent of protein size and shape. PMID:25329830

  20. Thanatos-associated protein 7 associates with template activating factor-Ibeta and inhibits histone acetylation to repress transcription.

    PubMed

    Macfarlan, Todd; Parker, J Brandon; Nagata, Kyosuke; Chakravarti, Debabrata

    2006-02-01

    The posttranslational modifications of histones on chromatin or a lack thereof is critical in transcriptional regulation. Emerging studies indicate a role for histone-binding proteins in transcriptional activation and repression. We have previously identified template-activating factor-Ibeta (TAF-Ibeta, also called PHAPII, SET, and I(2)(pp2A)) as a component of a cellular complex called inhibitor of acetyltransferases (INHAT) that masks histone acetylation in vitro and blocks histone acetyltransferase (HAT)-dependent transcription in living cells. TAF-Ibeta has also been shown to associate with transcription factors, including nuclear receptors, to regulate their activities. To identify novel interactors of TAF-Ibeta, we employed a yeast two-hybrid screen and identified a previously uncharacterized human protein called thanatos-associated protein-7 (THAP7), a member of a large family of THAP domain-containing putative DNA-binding proteins. In this study we demonstrate that THAP7 associates with TAF-Ibeta in vitro and map their association domains to a C-terminal predicted coiled-coil motif on THAP7 and the central region of TAF-Ibeta. Similarly, stably transfected THAP7 associates with endogenous TAF-Ibeta in intact cells. Like TAF-Ibeta, THAP7 associates with histone H3 and histone H4 and inhibits histone acetylation. The histone-interacting domain of THAP7 is sufficient for this activity in vitro. Promoter-targeted THAP7 can also recruit TAF-Ibeta and silencing mediator of retinoid and thyroid receptors/nuclear hormone receptor corepressor (NCoR) proteins to promoters, and knockdown of TAF-Ibeta by small interfering RNA relieves THAP7-mediated repression, indicating that, like nuclear hormone receptors, THAP7 may represent a novel class of transcription factor that uses TAF-Ibeta as a corepressor to maintain histones in a hypoacetylated, repressed state. PMID:16195249

  1. NMR structure of the forkhead-associated domain from the Arabidopsis receptor kinase-associated protein phosphatase

    PubMed Central

    Lee, Gui-in; Ding, Zhaofeng; Walker, John C.; Van Doren, Steven R.

    2003-01-01

    Forkhead-associated (FHA) domains are phosphoprotein-binding modules found in diverse signaling proteins that bind partners phosphorylated on threonine or serine. Kinase-associated protein phosphatase from Arabidopsis employs its FHA domain for negative regulation of receptor-like kinase signaling pathways, which are important in plant development. The solution structure of the free state of kinase-interacting FHA domain (KI-FHA) of kinase-associated protein phosphatase has been determined with high precision and accuracy using residual dipolar couplings. KI-FHA is a sandwich of a five-stranded mixed β-sheet with a six-stranded antiparallel β-sheet. Despite homology only in the recognition loops, this fold is shared with FHA domains from checkpoint proteins from yeast and humans, as well as with nonhomologous MH2 domains of Smad tumor suppressors. A shared pattern of hydrophobicity throughout FHA domains and Smad MH2 domains may stabilize the core of the β-sandwich. Evolutionary trace analysis of FHA domains suggests class-specific residues in the recognition loops that could tune their phosphoprotein-binding specificity. This surface agrees with that of KI-FHA in contact with a phosphothreonine peptide ligand. Evolutionary trace analysis also predicts an unexpected swath of class-specific residues on another face of FHA domains. Protein interactions with these faces may affect assembly of transmembrane signaling complexes in plants, and in other FHA domain-containing assemblies. PMID:14500786

  2. Systematic Analysis of Endometrial Cancer-Associated Hub Proteins Based on Text Mining

    PubMed Central

    Gao, Huiqiao; Zhang, Zhenyu

    2015-01-01

    Objective. The aim of this study was to systematically characterize the expression of endometrial cancer- (EC-) associated genes and to analysis the functions, pathways, and networks of EC-associated hub proteins. Methods. Gene data for EC were extracted from the PubMed (MEDLINE) database using text mining based on NLP. PPI networks and pathways were integrated and obtained from the KEGG and other databases. Proteins that interacted with at least 10 other proteins were identified as the hub proteins of the EC-related genes network. Results. A total of 489 genes were identified as EC-related with P < 0.05, and 32 pathways were identified as significant (P < 0.05, FDR < 0.05). A network of EC-related proteins that included 271 interactions was constructed. The 17 proteins that interact with 10 or more other proteins (P < 0.05, FDR < 0.05) were identified as the hub proteins of this PPI network of EC-related genes. These 17 proteins are EGFR, MET, PDGFRB, CCND1, JUN, FGFR2, MYC, PIK3CA, PIK3R1, PIK3R2, KRAS, MAPK3, CTNNB1, RELA, JAK2, AKT1, and AKT2. Conclusion. Our data may help to reveal the molecular mechanisms of EC development and provide implications for targeted therapy for EC. However, corrections between certain proteins and EC continue to require additional exploration. PMID:26366417

  3. Comparative proteomics analysis of oral cancer cell lines: identification of cancer associated proteins

    PubMed Central

    2014-01-01

    Background A limiting factor in performing proteomics analysis on cancerous cells is the difficulty in obtaining sufficient amounts of starting material. Cell lines can be used as a simplified model system for studying changes that accompany tumorigenesis. This study used two-dimensional gel electrophoresis (2DE) to compare the whole cell proteome of oral cancer cell lines vs normal cells in an attempt to identify cancer associated proteins. Results Three primary cell cultures of normal cells with a limited lifespan without hTERT immortalization have been successfully established. 2DE was used to compare the whole cell proteome of these cells with that of three oral cancer cell lines. Twenty four protein spots were found to have changed in abundance. MALDI TOF/TOF was then used to determine the identity of these proteins. Identified proteins were classified into seven functional categories – structural proteins, enzymes, regulatory proteins, chaperones and others. IPA core analysis predicted that 18 proteins were related to cancer with involvements in hyperplasia, metastasis, invasion, growth and tumorigenesis. The mRNA expressions of two proteins – 14-3-3 protein sigma and Stress-induced-phosphoprotein 1 – were found to correlate with the corresponding proteins’ abundance. Conclusions The outcome of this analysis demonstrated that a comparative study of whole cell proteome of cancer versus normal cell lines can be used to identify cancer associated proteins. PMID:24422745

  4. The association between phosphatidylinositol phosphodiesterase activity and a specific subunit of microtubular protein in rat brain

    PubMed Central

    Quinn, P. J.

    1973-01-01

    1. Supernatant proteins from rat brain were separated into two fractions containing phosphatidylinositol phosphodiesterase activity by chromatography on DEAE-Sephadex A-50. 2. The first fraction sediments in linear sucrose density gradients in two bands corresponding to molecular weights of 66000 and 36000. There was presumptive evidence that the lighter protein constituted the monomeric form of the enzyme. The second fraction sediments predominantly as a single protein of molecular weight 86000. 3. Treatment of rat brain supernatant with [3H]colchicine abolished the second DEAE-Sephadex peak and removed the lighter protein from the first peak. These proteins emerged in the same position as the protein binding [3H]colchicine at high salt concentration; phospholipase activity was recovered from linear sucrose density gradients in positions corresponding to molecular weights 88000 and 43000, together with an aggregate of molecular weight 140000. Electrophoresis on sodium dodecyl sulphate–urea–polyacrylamide gels of this fraction revealed only three proteins: the α and β-subunits of microtubular protein, of molecular weights 56000 and 52000 respectively, and a protein of molecular weight 38000. 4. A sample of microtubular protein from mouse, labelled in vivo with [3H]proline and 32Pi, was added to rat brain supernatant together with an equal amount of the same microtubular protein treated with cyclic AMP and [γ-32P]ATP and the mixture subsequently characterized by ion-exchange chromatography. Some phospholipase activity characteristic of the second peak from DEAE-Sephadex was associated with one fraction of added microtubular protein. This fraction was identified on the basis of the 3H:32P ratio as the β subunit of the protein treated with ATP and cyclic AMP. The subunit of added microtubular protein untreated with nucleotides was not associated with phospholipase activity. PMID:4353236

  5. A Protein Domain and Family Based Approach to Rare Variant Association Analysis

    PubMed Central

    Richardson, Tom G.; Shihab, Hashem A.; Rivas, Manuel A.; McCarthy, Mark I.; Campbell, Colin; Timpson, Nicholas J.; Gaunt, Tom R.

    2016-01-01

    Background It has become common practice to analyse large scale sequencing data with statistical approaches based around the aggregation of rare variants within the same gene. We applied a novel approach to rare variant analysis by collapsing variants together using protein domain and family coordinates, regarded to be a more discrete definition of a biologically functional unit. Methods Using Pfam definitions, we collapsed rare variants (Minor Allele Frequency ≤ 1%) together in three different ways 1) variants within single genomic regions which map to individual protein domains 2) variants within two individual protein domain regions which are predicted to be responsible for a protein-protein interaction 3) all variants within combined regions from multiple genes responsible for coding the same protein domain (i.e. protein families). A conventional collapsing analysis using gene coordinates was also undertaken for comparison. We used UK10K sequence data and investigated associations between regions of variants and lipid traits using the sequence kernel association test (SKAT). Results We observed no strong evidence of association between regions of variants based on Pfam domain definitions and lipid traits. Quantile-Quantile plots illustrated that the overall distributions of p-values from the protein domain analyses were comparable to that of a conventional gene-based approach. Deviations from this distribution suggested that collapsing by either protein domain or gene definitions may be favourable depending on the trait analysed. Conclusion We have collapsed rare variants together using protein domain and family coordinates to present an alternative approach over collapsing across conventionally used gene-based regions. Although no strong evidence of association was detected in these analyses, future studies may still find value in adopting these approaches to detect previously unidentified association signals. PMID:27128313

  6. Identification of a novel matrix protein contained in a protein aggregate associated with collagen in fish otoliths.

    PubMed

    Tohse, Hidekazu; Takagi, Yasuaki; Nagasawa, Hiromichi

    2008-05-01

    In the biomineralization processes, proteins are thought to control the polymorphism and morphology of the crystals by forming complexes of structural and mineral-associated proteins. To identify such proteins, we have searched for proteins that may form high-molecular-weight (HMW) aggregates in the matrix of fish otoliths that have aragonite and vaterite as their crystal polymorphs. By screening a cDNA library of the trout inner ear using an antiserum raised against whole otolith matrix, a novel protein, named otolith matrix macromolecule-64 (OMM-64), was identified. The protein was found to have a molecular mass of 64 kDa, and to contain two tandem repeats and a Glu-rich region. The structure of the protein and that of its DNA are similar to those of starmaker, a protein involved in the polymorphism control in the zebrafish otoliths [Söllner C, Burghammer M, Busch-Nentwich E, Berger J, Schwarz H, Riekel C & Nicolson T (2003) Science302, 282-286]. (45)Ca overlay analysis revealed that the Glu-rich region has calcium-binding activity. Combined analysis by western blotting and deglycosylation suggested that OMM-64 is present in an HMW aggregate with heparan sulfate chains. Histological observations revealed that OMM-64 is expressed specifically in otolith matrix-producing cells and deposited onto the otolith. Moreover, the HMW aggregate binds to the inner ear-specific short-chain collagen otolin-1, and the resulting complex forms ring-like structures in the otolith matrix. Overall, OMM-64, by forming a calcium-binding aggregate that binds to otolin-1 and forming matrix protein architectures, may be involved in the control of crystal morphology during otolith biomineralization. PMID:18410381

  7. Association of protein structure, protein and carbohydrate subfractions with bioenergy profiles and biodegradation functions in modeled forage.

    PubMed

    Ji, Cuiying; Zhang, Xuewei; Yu, Peiqiang

    2016-03-15

    The objectives of this study were to detect unique aspects and association of forage protein inherent structure, biological compounds, protein and carbohydrate subfractions, bioenergy profiles, and biodegradation features. In this study, common available alfalfa hay from two different sourced-origins (FSO vs. CSO) was used as a modeled forage for inherent structure profile, bioenergy, biodegradation and their association between their structure and bio-functions. The molecular spectral profiles were determined using non-invasive molecular spectroscopy. The parameters included: protein structure amide I group, amide II group and their ratios; protein subfractions (PA1, PA2, PB1, PB2, PC); carbohydrate fractions (CA1, CA2, CA3, CA4, CB1, CB2, CC); biodegradable and undegradable fractions of protein (RDPA2, RDPB1, RDPB2, RDP; RUPA2 RUPB1, RUPB2, RUPC, RUP); biodegradable and undegradable fractions of carbohydrate (RDCA4, RDCB1, RDCB2, RDCB3, RDCHO; RUCA4, RUCB1; RUCB2; RUCB3 RUCC, RUCHO) and bioenergy profiles (tdNDF, tdFA, tdCP, tdNFC, TDN1×, DE3×, ME3×, NEL3×; NEm, NEg). The results show differences in protein and carbohydrate (CHO) subfractions in the moderately degradable true protein fraction (PB1: 502 vs. 420 g/kg CP, P=0.09), slowly degraded true protein fraction (PB2: 45 vs. 96 g/kg CP, P=0.02), moderately degradable CHO fraction (CB2: 283 vs. 223 g/kg CHO, P=0.06) and slowly degraded CHO fraction (CB3: 369 vs. 408 g/kg CHO) between the two sourced origins. As to biodegradable (RD) fractions of protein and CHO in rumen, there were differences in RD of PB1 (417 vs. 349 g/kg CP, P=0.09), RD of PB2 (29 vs. 62 g/kg CP, P=0.02), RD of CB2 (251 vs. 198 g/kg DM, P=0.06), RD of CB3 (236 vs. 261 g/kg CHO, P=0.08). As to bioenergy profile, there were differences in total digestible nutrient (TDN: 551 vs. 537 g/kg DM, P=0.06), and metabolic bioenergy (P=0.095). As to protein molecular structure, there were differences in protein structure 1st and 2nd amide groups (P

  8. Association of protein structure, protein and carbohydrate subfractions with bioenergy profiles and biodegradation functions in modeled forage

    NASA Astrophysics Data System (ADS)

    Ji, Cuiying; Zhang, Xuewei; Yu, Peiqiang

    2016-03-01

    The objectives of this study were to detect unique aspects and association of forage protein inherent structure, biological compounds, protein and carbohydrate subfractions, bioenergy profiles, and biodegradation features. In this study, common available alfalfa hay from two different sourced-origins (FSO vs. CSO) was used as a modeled forage for inherent structure profile, bioenergy, biodegradation and their association between their structure and bio-functions. The molecular spectral profiles were determined using non-invasive molecular spectroscopy. The parameters included: protein structure amide I group, amide II group and their ratios; protein subfractions (PA1, PA2, PB1, PB2, PC); carbohydrate fractions (CA1, CA2, CA3, CA4, CB1, CB2, CC); biodegradable and undegradable fractions of protein (RDPA2, RDPB1, RDPB2, RDP; RUPA2 RUPB1, RUPB2, RUPC, RUP); biodegradable and undegradable fractions of carbohydrate (RDCA4, RDCB1, RDCB2, RDCB3, RDCHO; RUCA4, RUCB1; RUCB2; RUCB3 RUCC, RUCHO) and bioenergy profiles (tdNDF, tdFA, tdCP, tdNFC, TDN1 ×, DE3 ×, ME3 ×, NEL3 ×; NEm, NEg). The results show differences in protein and carbohydrate (CHO) subfractions in the moderately degradable true protein fraction (PB1: 502 vs. 420 g/kg CP, P = 0.09), slowly degraded true protein fraction (PB2: 45 vs. 96 g/kg CP, P = 0.02), moderately degradable CHO fraction (CB2: 283 vs. 223 g/kg CHO, P = 0.06) and slowly degraded CHO fraction (CB3: 369 vs. 408 g/kg CHO) between the two sourced origins. As to biodegradable (RD) fractions of protein and CHO in rumen, there were differences in RD of PB1 (417 vs. 349 g/kg CP, P = 0.09), RD of PB2 (29 vs. 62 g/kg CP, P = 0.02), RD of CB2 (251 vs. 198 g/kg DM, P = 0.06), RD of CB3 (236 vs. 261 g/kg CHO, P = 0.08). As to bioenergy profile, there were differences in total digestible nutrient (TDN: 551 vs. 537 g/kg DM, P = 0.06), and metabolic bioenergy (P = 0.095). As to protein molecular structure, there were differences in protein structure 1st

  9. G-protein from Medicago sativa: functional association to photoreceptors.

    PubMed Central

    Muschietti, J P; Martinetto, H E; Coso, O A; Farber, M D; Torres, H N; Flawia, M M

    1993-01-01

    G-protein subunits were characterized from Medicago sativa (alfalfa) seedlings. Crude membranes and GTP-Sepharose-purified fractions were electrophoresed on SDS/polyacrylamide gels and analysed by Western blotting with 9193 (anti-alpha common) and AS/7 (anti-alpha t, anti-alpha i1 and anti-alpha i2) polyclonal antibodies. These procedures led to the identification of a specific polypeptide band of about 43 kDa. Another polypeptide reacting with the SW/1 (anti-beta) antibody, of about 37 kDa, was also detected. The 43 kDa polypeptide bound specifically [alpha-32P]GTP by a photoaffinity reaction and was ADP-ribosylated by activated cholera toxin, but not by pertussis toxin. Irradiation of etiolated Medicago sativa protoplast preparations at 660 nm for 1 min produced a maximal increase in the guanosine 5'-[gamma-thio]triphosphate (GTP[35S])-binding rate. After this period of irradiation, the binding rate tended to decrease. The effect of a red-light (660 nm) pulse on the binding rate was reversed when it was immediately followed by a period of far-red (> 730 nm) illumination. These results may suggest that activation of GTP[S]-binding rate was a consequence of conversion of phytochrome Pr into the Ptr form. Images Figure 1 Figure 2 Figure 3 PMID:8484719

  10. Effect of Acidic pH on Expression of Surface-Associated Proteins of Streptococcus oralis

    PubMed Central

    Wilkins, Joanna C.; Beighton, David; Homer, Karen A.

    2003-01-01

    Streptococcus oralis, a member of the mitis group of oral streptococci, is implicated in the pathogenesis of infective endocarditis and is the predominant aciduric non-mutans-group streptococcus in dental plaque. We undertook to identify the most abundant surface-associated proteins of S. oralis and to investigate changes in protein expression when the organism was grown under acidic culture conditions. Surface-associated proteins were extracted from cells grown in batch culture, separated by two-dimensional gel electrophoresis, excised, digested with trypsin, and analyzed by matrix-assisted laser desorption ionization-time of flight mass spectrometry and liquid chromatography-tandem mass spectrometry. Putative functions were assigned by homology to a translated genomic database of Streptococcus pneumoniae. A total of 27 proteins were identified; these included a lipoprotein, a ribosome recycling factor, and the glycolytic enzymes phosphoglycerate kinase, fructose bisphosphate aldolase, glyceraldehyde-3-phosphate dehydrogenase, and enolase. The most abundant protein, phosphocarrier protein HPr, was present as three isoforms. Neither lactate dehydrogenase nor pyruvate oxidase, dominant intracellular proteins, were present among the proteins on the gels, demonstrating that proteins in the surface-associated pool did not arise as a result of cell lysis. Eleven of the proteins identified were differentially expressed when cells were grown at pH 5.2 versus pH 7.0, and these included superoxide dismutase, a homologue of dipeptidase V from Lactococcus lactis, and the protein translation elongation factors G, Tu, and Ts. This study has extended the range of streptococcal proteins known to be expressed at the cell surface. Further investigations are required to ascertain their functions at this extracellular location and determine how their expression is influenced by other environmental conditions. PMID:12957916

  11. Huntington’s Disease Protein Huntingtin Associates with its own mRNA

    PubMed Central

    Culver, Brady P.; DeClercq, Josh; Dolgalev, Igor; Yu, Man Shan; Ma, Bin; Heguy, Adriana; Tanese, Naoko

    2016-01-01

    Background: The Huntington’s disease (HD) protein huntingtin (Htt) plays a role in multiple cellular pathways. Deregulation of one or more of these pathways by the mutant Htt protein has been suggested to contribute to the disease pathogenesis. Our recent discovery-based proteomics studies have uncovered RNA binding proteins and translation factors associated with the endogenous Htt protein purified from mouse brains, suggesting a potential new role for Htt in RNA transport and translation. Objective: To investigate how Htt might affect RNA metabolism we set out to purify and analyze RNA associated with Htt. Methods: RNA was extracted from immunopurified Htt-containing protein complexes and analyzed by microarrays and RNA-Seq. Results: Surprisingly, the most enriched mRNA that co-purified with Htt was Htt mRNA itself. The association of Htt protein and Htt mRNA was detected independent of intact ribosomes suggesting that it is not an RNA undergoing translation. Furthermore, we identified the recently reported mis-spliced Htt mRNA encoding a truncated protein comprised of exon 1 and a portion of the downstream intron in the immunoprecipitates containing mutant Htt protein. We show that Htt protein co-localizes with Htt mRNA and that wild-type Htt reduces expression of a reporter construct harboring the Htt 3’ UTR. Conclusions: HD protein is found in a complex with its own mRNA and RNA binding proteins and translation factors. Htt may be involved in modulating its expression through post-transcriptional pathways. It is possible that Htt shares mechanistic properties similar to RNA binding proteins such as TDP-43 and FUS implicated in other neurodegenerative diseases. PMID:26891106

  12. Regulation of Beclin 1 Protein Phosphorylation and Autophagy by Protein Phosphatase 2A (PP2A) and Death-associated Protein Kinase 3 (DAPK3).

    PubMed

    Fujiwara, Nobuyuki; Usui, Tatsuya; Ohama, Takashi; Sato, Koichi

    2016-05-13

    Autophagy is an evolutionarily conserved intracellular degradation system that is involved in cell survival and activated in various diseases, including cancer. Beclin 1 is a central scaffold protein that assembles components for promoting or inhibiting autophagy. Association of Beclin 1 with its interacting proteins is regulated by the phosphorylation of Beclin 1 by various Ser/Thr kinases, but the Ser/Thr phosphatases that regulate these phosphorylation events remain unknown. Here we identify Ser-90 in Beclin 1 as a regulatory site whose phosphorylation is markedly enhanced in cells treated with okadaic acid, an inhibitor of protein phosphatase 2A (PP2A). Beclin 1 Ser-90 phosphorylation is induced in skeletal muscle tissues isolated from starved mice. The Beclin 1 S90A mutant blocked starvation-induced autophagy. We found association of PP2A B55α with Beclin 1, which dissociate by starvation. We also found that death-associated protein kinase 3 directly phosphorylates Beclin 1 Ser-90. We propose that physiological regulation of Beclin 1 Ser-90 phosphorylation by PP2A and death-associated protein kinase 3 controls autophagy. PMID:26994142

  13. Heterodimeric Capping Protein from Arabidopsis Is a Membrane-Associated, Actin-Binding Protein1[W][OPEN

    PubMed Central

    Jimenez-Lopez, Jose C.; Wang, Xia; Kotchoni, Simeon O.; Huang, Shanjin; Szymanski, Daniel B.; Staiger, Christopher J.

    2014-01-01

    The actin cytoskeleton is a major regulator of cell morphogenesis and responses to biotic and abiotic stimuli. The organization and activities of the cytoskeleton are choreographed by hundreds of accessory proteins. Many actin-binding proteins are thought to be stimulus-response regulators that bind to signaling phospholipids and change their activity upon lipid binding. Whether these proteins associate with and/or are regulated by signaling lipids in plant cells remains poorly understood. Heterodimeric capping protein (CP) is a conserved and ubiquitous regulator of actin dynamics. It binds to the barbed end of filaments with high affinity and modulates filament assembly and disassembly reactions in vitro. Direct interaction of CP with phospholipids, including phosphatidic acid, results in uncapping of filament ends in vitro. Live-cell imaging and reverse-genetic analyses of cp mutants in Arabidopsis (Arabidopsis thaliana) recently provided compelling support for a model in which CP activity is negatively regulated by phosphatidic acid in vivo. Here, we used complementary biochemical, subcellular fractionation, and immunofluorescence microscopy approaches to elucidate CP-membrane association. We found that CP is moderately abundant in Arabidopsis tissues and present in a microsomal membrane fraction. Sucrose density gradient separation and immunoblotting with known compartment markers were used to demonstrate that CP is enriched on membrane-bound organelles such as the endoplasmic reticulum and Golgi. This association could facilitate cross talk between the actin cytoskeleton and a wide spectrum of essential cellular functions such as organelle motility and signal transduction. PMID:25201878

  14. EFhd2 is a novel amyloid protein associated with pathological tau in Alzheimer's disease.

    PubMed

    Ferrer-Acosta, Yancy; Rodríguez-Cruz, Eva N; Orange, François; De Jesús-Cortés, Hector; Madera, Bismark; Vaquer-Alicea, Jaime; Ballester, Juan; Guinel, Maxime J-F; Bloom, George S; Vega, Irving E

    2013-06-01

    EFhd2 is a conserved calcium-binding protein, abundant within the central nervous system. Previous studies identified EFhd2 associated with pathological forms of tau proteins in the tauopathy mouse model JNPL3, which expresses the human tau(P301L) mutant. This association was validated in human tauopathies, such as Alzheimer's disease (AD). However, the role that EFhd2 may play in tauopathies is still unknown. Here, we show that EFhd2 formed amyloid structures in vitro, a capability that is reduced by calcium ions. Electron microscopy (EM) analyses demonstrated that recombinant EFhd2 formed filamentous structures. EM analyses of sarkosyl-insoluble fractions derived from human AD brains also indicated that EFhd2 co-localizes with aggregated tau proteins and formed granular structures. Immunohistological analyses of brain slices demonstrated that EFhd2 co-localizes with pathological tau proteins in AD brains, confirming the co-aggregation of EFhd2 and pathological tau. Furthermore, EFhd2's coiled-coil domain mediated its self-oligomerization in vitro and its association with tau proteins in JNPL3 mouse brain extracts. The results demonstrate that EFhd2 is a novel amyloid protein associated with pathological tau proteins in AD brain and that calcium binding may regulate the formation of EFhd2's amyloid structures. Hence, EFhd2 may play an important role in the pathobiology of tau-mediated neurodegeneration. PMID:23331044

  15. DnaJ/Hsc70 chaperone complexes control the extracellular release of neurodegenerative-associated proteins.

    PubMed

    Fontaine, Sarah N; Zheng, Dali; Sabbagh, Jonathan J; Martin, Mackenzie D; Chaput, Dale; Darling, April; Trotter, Justin H; Stothert, Andrew R; Nordhues, Bryce A; Lussier, April; Baker, Jeremy; Shelton, Lindsey; Kahn, Mahnoor; Blair, Laura J; Stevens, Stanley M; Dickey, Chad A

    2016-07-15

    It is now known that proteins associated with neurodegenerative disease can spread throughout the brain in a prionlike manner. However, the mechanisms regulating the trans-synaptic spread propagation, including the neuronal release of these proteins, remain unknown. The interaction of neurodegenerative disease-associated proteins with the molecular chaperone Hsc70 is well known, and we hypothesized that much like disaggregation, refolding, degradation, and even normal function, Hsc70 may dictate the extracellular fate of these proteins. Here, we show that several proteins, including TDP-43, α-synuclein, and the microtubule-associated protein tau, can be driven out of the cell by an Hsc70 co-chaperone, DnaJC5. In fact, DnaJC5 overexpression induced tau release in cells, neurons, and brain tissue, but only when activity of the chaperone Hsc70 was intact and when tau was able to associate with this chaperone. Moreover, release of tau from neurons was reduced in mice lacking the DnaJC5 gene and when the complement of DnaJs in the cell was altered. These results demonstrate that the dynamics of DnaJ/Hsc70 complexes are critically involved in the release of neurodegenerative disease proteins. PMID:27261198

  16. Prediction of nanoparticles-cell association based on corona proteins and physicochemical properties.

    PubMed

    Liu, Rong; Jiang, Wen; Walkey, Carl D; Chan, Warren C W; Cohen, Yoram

    2015-06-01

    Cellular association of nanoparticles (NPs) in biological fluids is affected by proteins adsorbed onto the NP surface, forming a "protein corona", thereby impacting cellular bioactivity. Here we investigate, based on an extensive gold NPs protein corona dataset, the relationships between NP-cell association and protein corona fingerprints (PCFs) as well as NP physicochemical properties. Accordingly, quantitative structure-activity relationships (QSARs) were developed based on both linear and non-linear support vector regression (SVR) models making use of a sequential forward floating selection of descriptors. The SVR model with only 6 serum proteins and zeta potential had higher accuracy (R(2) = 0.895) relative to the linear model (R(2) = 0.850) with 11 PCFs. Considering the initial pool of 148 descriptors, the APOB, A1AT, ANT3, and PLMN serum proteins along with NP zeta potential were identified as most significant to correlating NP-cell association. The present study suggests that QSARs exploration of NP-cell association data, considering the role of both NP protein corona and physicochemical properties, can support the planning and interpretation of toxicity studies and guide the design of NPs for biomedical applications. PMID:25959034

  17. Identification of a new class of lipid droplet-associated proteins in plants.

    PubMed

    Horn, Patrick J; James, Christopher N; Gidda, Satinder K; Kilaru, Aruna; Dyer, John M; Mullen, Robert T; Ohlrogge, John B; Chapman, Kent D

    2013-08-01

    Lipid droplets in plants (also known as oil bodies, lipid bodies, or oleosomes) are well characterized in seeds, and oleosins, the major proteins associated with their surface, were shown to be important for stabilizing lipid droplets during seed desiccation and rehydration. However, lipid droplets occur in essentially all plant cell types, many of which may not require oleosin-mediated stabilization. The proteins associated with the surface of nonseed lipid droplets, which are likely to influence the formation, stability, and turnover of this compartment, remain to be elucidated. Here, we have combined lipidomic, proteomic, and transcriptomic studies of avocado (Persea americana) mesocarp to identify two new lipid droplet-associated proteins, which we named LDAP1 and LDAP2. These proteins are highly similar to each other and also to the small rubber particle proteins that accumulate in rubber-producing plants. An Arabidopsis (Arabidopsis thaliana) homolog to LDAP1 and LDAP2, At3g05500, was localized to the surface of lipid droplets after transient expression in tobacco (Nicotiana tabacum) cells that were induced to accumulate triacylglycerols. We propose that small rubber particle protein-like proteins are involved in the general process of binding and perhaps the stabilization of lipid-rich particles in the cytosol of plant cells and that the avocado and Arabidopsis protein members reveal a new aspect of the cellular machinery that is involved in the packaging of triacylglycerols in plant tissues. PMID:23821652

  18. IDENTIFICATION OF SUMO-2/3 MODIFIED PROTEINS ASSOCIATED WITH MITOTIC CHROMOSOMES

    PubMed Central

    Cubeñas-Potts, Caelin; Srikumar, Tharan; Lee, Christine; Osula, Omoruyi; Subramonian, Divya; Zhang, Xiang-Dong; Cotter, Robert J.; Raught, Brian; Matunis, Michael J.

    2015-01-01

    Sumoylation is essential for progression through mitosis, but the specific protein targets and functions remain poorly understood. In this study, we used chromosome spreads to more precisely define the localization of SUMO-2/3 to the inner-centromere and protein scaffold of mitotic chromosomes. We also developed methods to immunopurify proteins modified by endogenous, untagged SUMO-2/3 from mitotic chromosomes. Using these methods we identified 149 chromosome-associated SUMO-2/3 substrates by nLC-ESI-MS/MS. Approximately one-third of the identified proteins have reported functions in mitosis. Consistent with SUMO-2/3 immunolocalization, we identified known centromere and kinetochore associated proteins, as well as chromosome scaffold associated proteins. Notably, >30 proteins involved in chromatin modification or remodeling were identified. Our results provide insights into the roles of sumoylation as a regulator of chromatin structure and other diverse processes in mitosis. Furthermore, our purification and fractionation methodologies represent an important compliment to existing approaches to identify sumoylated proteins using exogenously expressed and tagged SUMOs. PMID:25367092

  19. Human fallopian tube proteome shows high coverage of mesenchymal stem cells associated proteins.

    PubMed

    Wang, Chenyuan; Liu, Yang; Chang, Cheng; Wu, Songfeng; Gao, Jie; Zhang, Yang; Chen, Yingjie; Zhong, Fan; Deng, Gaopi

    2016-01-01

    The object of this research was to report a draft proteome of human fallopian tube (hFT) comprises 5416 identified proteins, which could be considered as a physiological reference to complement Human Proteome Draft. The proteomic raw data and metadata were stored in an integrated proteome resources centre iProX (IPX00034300). This hFT proteome contains many hFT markers newly identified by mass spectrum. This hFT proteome comprises 660 high-, 3605 medium- and 1181 low-abundant proteins. Ribosome, cytoskeleton, vesicle and protein folding associated proteins showed obvious tendency to be higher abundance in hFT. The extraordinary high coverage of mesenchymal stem cells (MSCs)-associated proteins were identified in this hFT proteome, which highly supported that hFT should contain a plenty of MSCs. PMID:26759384

  20. Human fallopian tube proteome shows high coverage of mesenchymal stem cells associated proteins

    PubMed Central

    Wang, Chenyuan; Liu, Yang; Chang, Cheng; Wu, Songfeng; Gao, Jie; Zhang, Yang; Chen, Yingjie; Zhong, Fan; Deng, Gaopi

    2016-01-01

    The object of this research was to report a draft proteome of human fallopian tube (hFT) comprises 5416 identified proteins, which could be considered as a physiological reference to complement Human Proteome Draft. The proteomic raw data and metadata were stored in an integrated proteome resources centre iProX (IPX00034300). This hFT proteome contains many hFT markers newly identified by mass spectrum. This hFT proteome comprises 660 high-, 3605 medium- and 1181 low-abundant proteins. Ribosome, cytoskeleton, vesicle and protein folding associated proteins showed obvious tendency to be higher abundance in hFT. The extraordinary high coverage of mesenchymal stem cells (MSCs)-associated proteins were identified in this hFT proteome, which highly supported that hFT should contain a plenty of MSCs. PMID:26759384

  1. The La RNA-binding protein interacts with the vault RNA and is a vault-associated protein.

    PubMed

    Kickhoefer, Valerie A; Poderycki, Michael J; Chan, Edward K L; Rome, Leonard H

    2002-10-25

    Vaults are highly conserved ubiquitous ribonucleoprotein particles with an undefined function. Three protein species (p240/TEP1, p193/VPARP, and p100/MVP) and a small RNA comprise the 13-MDa vault particle. The expression of the unique 100-kDa major vault protein is sufficient to form the basic vault structure. Previously, we have shown that stable association of the vault RNA with the vault particle is dependent on its interaction with the p240/TEP1 protein. To identify other proteins that interact with the vault RNA, we used a UV-cross-linking assay. We find that a portion of the vault RNA is complexed with the La autoantigen in a separate smaller ribonucleoprotein particle. La interacts with the vault RNA (both in vivo and in vitro) presumably through binding to 3'-uridylates. Moreover, we also demonstrate that the La autoantigen is the 50-kDa protein that we have previously reported as a protein that co-purifies with vaults. PMID:12196535

  2. Immunogenic membrane-associated proteins of Mycobacterium tuberculosis revealed by proteomics.

    PubMed

    Sinha, Sudhir; Kosalai, K; Arora, Shalini; Namane, Abdelkader; Sharma, Pawan; Gaikwad, Anil N; Brodin, Priscille; Cole, Stewart T

    2005-07-01

    Membrane-associated proteins of Mycobacterium tuberculosis offer a challenge, as well as an opportunity, in the quest for better therapeutic and prophylactic interventions against tuberculosis. The authors have previously reported that extraction with the detergent Triton X-114 (TX-114) is a useful step in proteomic analysis of mycobacterial cell membranes, and detergent-soluble membrane proteins of mycobacteria are potent stimulators of human T cells. In this study 1-D and 2-D gel electrophoresis-based protocols were used for the analysis of proteins in the TX-114 extract of M. tuberculosis membranes. Peptide mass mapping (using MALDI-TOF-MS, matrix assisted laser desorption/ionization time of flight mass spectrometry) of 116 samples led to the identification of 105 proteins, 9 of which were new to the M. tuberculosis proteome. Functional orthologues of 73 of these proteins were also present in Mycobacterium leprae, suggesting their relative importance. Bioinformatics predicted that as many as 73% of the proteins had a hydrophobic disposition. 1-D gel electrophoresis revealed more hydrophobic/transmembrane and basic proteins than 2-D gel electrophoresis. Identified proteins fell into the following major categories: protein synthesis, cell wall biogenesis/architecture and conserved hypotheticals/unknowns. To identify immunodominant proteins of the detergent phase (DP), 14 low-molecular-mass fractions prepared by continuous-elution gel electrophoresis were subjected to T cell activation assays using blood samples from BCG-vaccinated healthy donors from a tuberculosis endemic area. Analysis of the responses (cell proliferation and IFN-gamma production) showed that the immunodominance of certain DP fractions was most probably due to ribosomal proteins, which is consistent with both their specificity for mycobacteria and their abundance. Other membrane-associated proteins, including transmembrane proteins/lipoproteins and ESAT-6, did not appear to contribute

  3. The Parkinsonism-associated protein DJ-1/Park7 prevents glycation damage in human keratinocyte.

    PubMed

    Advedissian, Tamara; Deshayes, Frédérique; Poirier, Françoise; Viguier, Mireille; Richarme, Gilbert

    2016-04-22

    Reducing sugars and dicarbonyls form covalent adducts with proteins through a nonenzymatic process known as glycation, which inactivates proteins, is increased in diabetic patients and is associated with diabetic complications, including retinopathy, cataracts, nephropathy, neuropathy, cardiomyopathy and skin defects. We recently characterized DJ-1/Park7 as a protein deglycase that repairs proteins from glycation by glyoxal and methylglyoxal, two major glycating agents which are responsible for up to 65% of glycation events. In this study, we investigated the ability of DJ-1 to prevent protein glycation in keratinocytes. Glycation of collagen and keratinocyte proteins was tested by measuring ultraviolet absorption and fluorescence emission. Protein glycation in HaCaT keratinocytes was investigated by immunodetection with anti-advanced glycation endproduct antibodies, after DJ-1 depletion or overexpression. In vitro, DJ-1 prevented glycation of collagen and keratinocyte protein extracts. In cell culture, DJ-1 depletion by small interfering RNAs resulted in a 3-fold increase in protein glycation levels. Moreover, protein glycation levels were decreased several-fold in cells overexpressing DJ-1 after addition of the Nrf2 inducer sulforaphane or after transfection with a DJ-1 plasmid. Thus, the DJ-1 deglycase plays a major role in preventing protein glycation in eukaryotic cells and might be important for preventing skin glycation. PMID:26995087

  4. Conformational modulation mediated by polyglutamine expansion in CAG repeat expansion disease-associated proteins.

    PubMed

    Verani, Margherita; Bustamante, Maria; Martufi, Paola; Daldin, Manuel; Cariulo, Cristina; Azzollini, Lucia; Fodale, Valentina; Puglisi, Francesca; Weiss, Andreas; Macdonald, Douglas; Petricca, Lara; Caricasole, Andrea

    2016-09-16

    We have previously reported TR-FRET based immunoassays to detect a conformational change imparted on huntingtin protein by the polyglutamine expansion, which we confirmed using biophysical methodologies. Using these immunoassays, we now report that polyglutamine expansion influences the conformational properties of other polyglutamine disease proteins, exemplified by the androgen receptor (associated with spinal bulbar muscular atrophy) and TATA binding protein (associated with spinocerebellar ataxia 17). Using artificial constructs bearing short or long polyglutamine expansions or a multimerized, unrelated epitope (mimicking the increase in anti-polyglutamine antibody epitopes present in polyglutamine repeats of increasing length) we confirmed that the conformational TR-FRET based immunoassay detects an intrinsic conformational property of polyglutamine repeats. The TR-FRET based conformational immunoassay may represent a rapid, scalable tool to identify modulators of polyglutamine-mediated conformational change in different proteins associated with CAG triplet repeat disorders. PMID:27520369

  5. Protein profiles associated with context fear conditioning and their modulation by memantine.

    PubMed

    Ahmed, Md Mahiuddin; Dhanasekaran, A Ranjitha; Block, Aaron; Tong, Suhong; Costa, Alberto C S; Gardiner, Katheleen J

    2014-04-01

    Analysis of the molecular basis of learning and memory has revealed details of the roles played by many genes and the proteins they encode. Because most individual studies focus on a small number of proteins, many complexities of the relationships among proteins and their dynamic responses to stimulation are not known. We have used the technique of reverse phase protein arrays (RPPA) to assess the levels of more than 80 proteins/protein modifications in subcellular fractions from hippocampus and cortex of mice trained in Context Fear Conditioning (CFC). Proteins include components of signaling pathways, several encoded by immediate early genes or involved in apoptosis and inflammation, and subunits of glutamate receptors. At one hour after training, levels of more than half the proteins had changed in one or more fractions, among them multiple components of the Mitogen-activated protein kinase, MAPK, and Mechanistic Target of Rapamycin, MTOR, pathways, subunits of glutamate receptors, and the NOTCH pathway modulator, NUMB homolog (Drosophila). Levels of 37 proteins changed in the nuclear fraction of hippocampus alone. Abnormalities in levels of thirteen proteins analyzed have been reported in brains of patients with Alzheimer's Disease. We therefore further investigated the protein profiles of mice treated with memantine, a drug approved for treatment of AD. In hippocampus, memantine alone induced many changes similar to those seen after CFC and altered the levels of seven proteins associated with Alzheimer's Disease abnormalities. Lastly, to further explore the relevance of these datasets, we superimposed responses to CFC and memantine onto components of the long term potentiation pathway, a process subserving learning and memory formation. Fourteen components of the long term potentiation pathway and 26 proteins interacting with components responded to CFC and/or memantine. Together, these datasets provide a novel view of the diversity and complexity in protein

  6. Protein Profiles Associated With Context Fear Conditioning and Their Modulation by Memantine*

    PubMed Central

    Ahmed, Md. Mahiuddin; Dhanasekaran, A. Ranjitha; Block, Aaron; Tong, Suhong; Costa, Alberto C. S.; Gardiner, Katheleen J.

    2014-01-01

    Analysis of the molecular basis of learning and memory has revealed details of the roles played by many genes and the proteins they encode. Because most individual studies focus on a small number of proteins, many complexities of the relationships among proteins and their dynamic responses to stimulation are not known. We have used the technique of reverse phase protein arrays (RPPA) to assess the levels of more than 80 proteins/protein modifications in subcellular fractions from hippocampus and cortex of mice trained in Context Fear Conditioning (CFC). Proteins include components of signaling pathways, several encoded by immediate early genes or involved in apoptosis and inflammation, and subunits of glutamate receptors. At one hour after training, levels of more than half the proteins had changed in one or more fractions, among them multiple components of the Mitogen-activated protein kinase, MAPK, and Mechanistic Target of Rapamycin, MTOR, pathways, subunits of glutamate receptors, and the NOTCH pathway modulator, NUMB homolog (Drosophila). Levels of 37 proteins changed in the nuclear fraction of hippocampus alone. Abnormalities in levels of thirteen proteins analyzed have been reported in brains of patients with Alzheimer's Disease. We therefore further investigated the protein profiles of mice treated with memantine, a drug approved for treatment of AD. In hippocampus, memantine alone induced many changes similar to those seen after CFC and altered the levels of seven proteins associated with Alzheimer's Disease abnormalities. Lastly, to further explore the relevance of these datasets, we superimposed responses to CFC and memantine onto components of the long term potentiation pathway, a process subserving learning and memory formation. Fourteen components of the long term potentiation pathway and 26 proteins interacting with components responded to CFC and/or memantine. Together, these datasets provide a novel view of the diversity and complexity in protein

  7. CARDIO-PRED: an in silico tool for predicting cardiovascular-disorder associated proteins.

    PubMed

    Jain, Prerna; Thukral, Nitin; Gahlot, Lokesh Kumar; Hasija, Yasha

    2015-06-01

    Interactions between proteins largely govern cellular processes and this has led to numerous efforts culminating in enormous information related to the proteins, their interactions and the function which is determined by their interactions. The main concern of the present study is to present interface analysis of cardiovascular-disorder (CVD) related proteins to shed lights on details of interactions and to emphasize the importance of using structures in network studies. This study combines the network-centred approach with three dimensional studies to comprehend the fundamentals of biology. Interface properties were used as descriptors to classify the CVD associated proteins and non-CVD associated proteins. Machine learning algorithm was used to generate a classifier based on the training set which was then used to predict potential CVD related proteins from a set of polymorphic proteins which are not known to be involved in any disease. Among several classifying algorithms applied to generate models, best performance was achieved using Random Forest with an accuracy of 69.5 %. The tool named CARDIO-PRED, based on the prediction model is present at http://www.genomeinformatics.dce.edu/CARDIO-PRED/. The predicted CVD related proteins may not be the causing factor of particular disease but can be involved in pathways and reactions yet unknown to us thus permitting a more rational analysis of disease mechanism. Study of their interactions with other proteins can significantly improve our understanding of the molecular mechanism of diseases. PMID:25972989

  8. Matrix Gla Protein is Associated with Risk Factors for Atherosclerosis but not with Coronary Artery Calcification

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Objectives: Atherosclerotic coronary artery calcification (CAC) is associated with increased coronary heart disease (CHD) risk. Matrix Gla Protein (MGP) is an inhibitor of calcification in vivo. However, little is known regarding the distribution of circulating MGP, and its associations with CHD...

  9. Translocation of botulinum neurotoxin serotype a and associated proteins across the intestinal epithelia

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Botulinum neurotoxins (BoNTs) are some of the most poisonous natural toxins and considered to be a major venue of bioterrorist threat. BoNTs associate with neurotoxin associated proteins (NAPs), forming large complexes. NAPs have been shown to shield the BoNT holotoxin from the harsh environment of ...

  10. An analysis of gene/protein associations at PubMed scale

    PubMed Central

    2011-01-01

    Background Event extraction following the GENIA Event corpus and BioNLP shared task models has been a considerable focus of recent work in biomedical information extraction. This work includes efforts applying event extraction methods to the entire PubMed literature database, far beyond the narrow subdomains of biomedicine for which annotated resources for extraction method development are available. Results In the present study, our aim is to estimate the coverage of all statements of gene/protein associations in PubMed that existing resources for event extraction can provide. We base our analysis on a recently released corpus automatically annotated for gene/protein entities and syntactic analyses covering the entire PubMed, and use named entity co-occurrence, shortest dependency paths and an unlexicalized classifier to identify likely statements of gene/protein associations. A set of high-frequency/high-likelihood association statements are then manually analyzed with reference to the GENIA ontology. Conclusions We present a first estimate of the overall coverage of gene/protein associations provided by existing resources for event extraction. Our results suggest that for event-type associations this coverage may be over 90%. We also identify several biologically significant associations of genes and proteins that are not addressed by these resources, suggesting directions for further extension of extraction coverage. PMID:22166173

  11. Live-cell imaging of microtubules and microtubule-associated proteins in Arabidopsis thaliana.

    PubMed

    Lucas, Jessica

    2013-01-01

    Microtubules and microtubule-associated proteins (MAPs) play fundamental roles in plant growth and morphogenesis. The ability to observe microtubules and MAPs in living cells using fluorescent protein fusions has propelled plant scientists forward and given them the opportunity to answer longstanding biological questions. In combination with the genetic resources available in the model plant Arabidopsis thaliana, our mechanistic understanding of how the microtubule cytoskeleton affects plant life has dramatically increased. It is a simple process to construct transgenic A. thaliana plants that express fluorescent protein fusions by using the disarmed plant pathogen Agrobacterium tumefaciens. Several screening steps are necessary to ensure that the fusion protein accurately mimics the native protein because transgenes are inserted randomly into the A. thaliana genome. To image the fluorescent proteins in planta, confocal microscopy is used to alleviate issues caused by specimen thickness and autofluorescence. PMID:23973076

  12. The 43-K protein, v1, associated with acetylcholine receptor containing membrane fragments is an actin-binding protein.

    PubMed Central

    Walker, J H; Boustead, C M; Witzemann, V

    1984-01-01

    Acetylcholine receptor enriched membrane fragments were obtained from the electric organs of Torpedo marmorata. The purified membrane fragments contained several proteins in addition to the acetylcholine receptor subunits. One of these was shown to be actin by means of immune blotting with a monoclonal antibody. Brief treatment of the membranes with pH 11.0 buffer removed actin and the other non-receptor proteins including the receptor-associated 43 000 mol. wt. polypeptide. This polypeptide was shown to bind actin after transferring the proteins from one- and two-dimensional polyacrylamide gels to nitrocellulose paper and incubating the nitrocellulose blots with actin. Specifically bound actin was demonstrated using the monoclonal antibodies to actin. No calcium or calmodulin dependency of binding was observed. The findings suggest that the 43 000 mol. wt. polypeptide is a link between the membrane-bound acetylcholine receptor and the cytoskeleton. Images Fig. 1. Fig. 2. Fig. 3. Fig. 4. PMID:6389118

  13. Structure of the GH1 domain of guanylate kinase-associated protein from Rattus norvegicus

    SciTech Connect

    Tong, Junsen; Yang, Huiseon; Eom, Soo Hyun; Chun, ChangJu; Im, Young Jun

    2014-09-12

    Graphical abstract: - Highlights: • The crystal structure of GKAP homology domain 1 (GH1) was determined. • GKAP GH1 is a three-helix bundle connected by short flexible loops. • The predicted helix α4 associates weakly with the helix α3, suggesting dynamic nature of the GH1 domain. - Abstract: Guanylate-kinase-associated protein (GKAP) is a scaffolding protein that links NMDA receptor-PSD-95 to Shank–Homer complexes by protein–protein interactions at the synaptic junction. GKAP family proteins are characterized by the presence of a C-terminal conserved GKAP homology domain 1 (GH1) of unknown structure and function. In this study, crystal structure of the GH1 domain of GKAP from Rattus norvegicus was determined in fusion with an N-terminal maltose-binding protein at 2.0 Å resolution. The structure of GKAP GH1 displays a three-helix bundle connected by short flexible loops. The predicted helix α4 which was not visible in the crystal structure associates weakly with the helix α3 suggesting dynamic nature of the GH1 domain. The strict conservation of GH1 domain across GKAP family members and the lack of a catalytic active site required for enzyme activity imply that the GH1 domain might serve as a protein–protein interaction module for the synaptic protein clustering.

  14. Functional genomics of intraflagellar transport-associated proteins in C. elegans.

    PubMed

    Inglis, Peter N; Blacque, Oliver E; Leroux, Michel R

    2009-01-01

    The nematode Caenorhabditis elegans presents numerous advantages for the identification and molecular analysis of intraflagellar transport (IFT)-associated proteins, which play a critical role in the formation of cilia. Many proteins were first described as participating in IFT in this organism, including IFTA-1 (IFT121), DYF-1 (fleer/IFT70), DYF-2 (IFT144), DYF-3 (Qilin), DYF-11 (MIP-T3/IFT54), DYF-13, XBX-1 (dynein light intermediate chain), XBX-2 (dynein light chain), CHE-13 (IFT57/HIPPI), orthologs of Bardet-Biedl syndrome proteins, and potential regulatory protein, IFTA-2 (RABL5/IFT22). Transgenic animals bearing green fluorescent protein (GFP)-tagged proteins can be generated with ease, and in vivo imaging of IFT in both wild-type and cilia mutant strains can be performed quickly. The analyses permit detailed information on the localization and dynamic properties (velocities along the ciliary axoneme) of the relevant proteins, providing insights into their potential functions in processes such as anterograde and retrograde transport and cilium formation, as well as association with distinct modules of the IFT machinery (e.g., IFT subcomplexes A or B). Behavioral studies of the corresponding IFT-associated gene mutants further enable an understanding of the ciliary role of the proteins-e.g., in chemosensation, lipid homeostasis, lifespan control, and signaling-in a multicellular animal. In this chapter, we discuss how C. elegans can be used for the identification and characterization of IFT-associated proteins, focusing on methods for the generation of GFP-tagged IFT reporter strains, time-lapse microscopy, and IFT rate measurements. PMID:20409822

  15. Characterization of proteins associating with 5' terminus of PGHS-1 mRNA.

    PubMed

    Bunimov, Natalia; Laneuville, Odette

    2010-06-01

    Induction of Prostaglandin Endoperoxide H Synthase-1 (PGHS-1) gene has been previously documented in a few studies during events such as development and cellular differentiation. However, molecular mechanisms governing the regulation of PGHS-1 gene expression and contributing to changes in protein levels are poorly understood. Using the MEG-01 cell model of PGHS-1 gene induction, our laboratory has previously demonstrated that the 5'UTR and the first two exons of PGHS-1 mRNA had a significant impact on decreasing the translational efficiency of a reporter gene and suggested that the presence of a secondary structure is required for conservation of this activity. This 5'end of PGHS-1 mRNA sequence has also been shown to associate with nucleolin protein. In the current study, we set to investigate the protein composition of the mRNP (messenger ribonucleoprotein) associating with the 5'end of PGHS-1 mRNA and to identify its protein members. RNA/protein binding assays coupled with LC-MS analysis identified serpin B1 and NF45 (nuclear factor 45) proteins as potential members of PGHS-1 mRNP complex. Immunoprecipitation experiments using MEG-01 protein extracts validated mass spectrometry data and confirmed binding of nucleolin, serpin B1, NF45 and NF90. The RNA fraction was extracted from immunoprecipitated mRNP complexes and association of RNA binding proteins, serpin B1, NF45 and NF90, to PGHS-1 mRNA target sequence was confirmed by RT-PCR. Together these data suggest that serpin B1, NF45 and NF90 associate with PGHS-1 mRNA and can potentially participate in the formation a single or a number of PGHS-1 ribonucleoprotein complexes, through nucleolin that possibly serves as a docking base for other protein complex members. PMID:20112001

  16. Insights into the quaternary association of proteins through structure graphs: a case study of lectins

    PubMed Central

    2005-01-01

    The unique three-dimensional structure of both monomeric and oligomeric proteins is encoded in their sequence. The biological functions of proteins are dependent on their tertiary and quaternary structures, and hence it is important to understand the determinants of quaternary association in proteins. Although a large number of investigations have been carried out in this direction, the underlying principles of protein oligomerization are yet to be completely understood. Recently, new insights into this problem have been gained from the analysis of structure graphs of proteins belonging to the legume lectin family. The legume lectins are an interesting family of proteins with very similar tertiary structures but varied quaternary structures. Hence they have become a very good model with which to analyse the role of primary structures in determining the modes of quaternary association. The present review summarizes the results of a legume lectin study as well as those obtained from a similar analysis carried out here on the animal lectins, namely galectins, pentraxins, calnexin, calreticulin and rhesus rotavirus Vp4 sialic-acid-binding domain. The lectin structure graphs have been used to obtain clusters of non-covalently interacting amino acid residues at the intersubunit interfaces. The present study, performed along with traditional sequence alignment methods, has provided the signature sequence motifs for different kinds of quaternary association seen in lectins. Furthermore, the network representation of the lectin oligomers has enabled us to detect the residues which make extensive interactions (‘hubs’) across the oligomeric interfaces that can be targetted for interface-destabilizing mutations. The present review also provides an overview of the methodology involved in representing oligomeric protein structures as connected networks of amino acid residues. Further, it illustrates the potential of such a representation in elucidating the structural

  17. Highly efficient extraction of cellular nucleic acid associated proteins in vitro with magnetic oxidized carbon nanotubes.

    PubMed

    Zhang, Yi; Hu, Zhengyan; Qin, Hongqiang; Wei, Xiaoluan; Cheng, Kai; Liu, Fangjie; Wu, Ren'an; Zou, Hanfa

    2012-12-01

    Nucleic acid associated proteins (NAaP) play the essential roles in gene regulation and protein expression. The global analysis of cellular NAaP would give a broad insight to understand the interaction between nucleic acids and the associated proteins, such as the important proteinous regulation factors on nucleic acids. Proteomic analysis presents a novel strategy to investigate a group of proteins. However, the large scale analysis of NAaP is yet impossible due to the lack of approaches to harvest target protein groups with a high efficiency. Herein, a simple and efficient method was developed to collect cellular NAaP using magnetic oxidized carbon nanotubes based on the strong interaction between carbon nanotubes and nucleic acids along with corresponding associated proteins. We found that the magnetic oxidized carbon nanotubes demonstrated a nearly 100% extraction efficiency for intracellular nucleic acids from cells in vitro. Importantly, the proteins associated on nucleic acids could be highly efficiently harvested using magnetic oxidized carbon nanotubes due to the binding of NAaP on nucleic acids. 1594 groups of nuclear NAaP and 2595 groups of cellular NAaP were extracted and identified from about 1,000,000 cells, and 803 groups of NAaP were analyzed with only about 10,000 cells, showing a promising performance for the proteomic analysis of NAaP from minute cellular samples. This highly efficient extraction strategy for NAaP is a simple approach to identify cellular nucleic acid associated proteome, and we believed this strategy could be further applied in systems biology to understand the gene expression and regulation. PMID:23121485

  18. Predicting Cell Association of Surface-Modified Nanoparticles Using Protein Corona Structure - Activity Relationships (PCSAR).

    PubMed

    Kamath, Padmaja; Fernandez, Alberto; Giralt, Francesc; Rallo, Robert

    2015-01-01

    Nanoparticles are likely to interact in real-case application scenarios with mixtures of proteins and biomolecules that will absorb onto their surface forming the so-called protein corona. Information related to the composition of the protein corona and net cell association was collected from literature for a library of surface-modified gold and silver nanoparticles. For each protein in the corona, sequence information was extracted and used to calculate physicochemical properties and statistical descriptors. Data cleaning and preprocessing techniques including statistical analysis and feature selection methods were applied to remove highly correlated, redundant and non-significant features. A weighting technique was applied to construct specific signatures that represent the corona composition for each nanoparticle. Using this basic set of protein descriptors, a new Protein Corona Structure-Activity Relationship (PCSAR) that relates net cell association with the physicochemical descriptors of the proteins that form the corona was developed and validated. The features that resulted from the feature selection were in line with already published literature, and the computational model constructed on these features had a good accuracy (R(2)LOO=0.76 and R(2)LMO(25%)=0.72) and stability, with the advantage that the fingerprints based on physicochemical descriptors were independent of the specific proteins that form the corona. PMID:25961528

  19. A yeast metal resistance protein similar to human cystic fibrosis transmembrane conductance regulator (CFTR) and multidrug resistance-associated protein.

    PubMed

    Szczypka, M S; Wemmie, J A; Moye-Rowley, W S; Thiele, D J

    1994-09-01

    Members of the ATP binding cassette (ABC) protein superfamily transport a variety of substances across biological membranes, including drugs, ions, and peptides. The yeast cadmium factor (YCF1) gene from Saccharomyces cerevisiae is required for cadmium resistance and encodes a 1,515 amino acid protein with extensive homology to both the human multidrug resistance-associated protein (MRP1) and the cystic fibrosis transmembrane conductance regulator (hCFTR). S. cerevisiae cells harboring a deletion of the YCF1 gene are hypersensitive to cadmium compared with wild type cells. Mutagenesis experiments demonstrate that conserved amino acid residues, functionally critical in hCFTR, play a vital role in YCF1-mediated cadmium resistance. Mutagenesis of phenylalanine 713 in the YCF1 nucleotide binding fold 1, which correlates with the delta F508 mutation found in the most common form of cystic fibrosis, completely abolished YCF1 function in cadmium detoxification. Furthermore, substitution of a serine to alanine residue in a potential protein kinase A phosphorylation site in a central region of YCF1, which displays sequence similarity to the central regulatory domain of hCFTR, also rendered YCF1 nonfunctional. These results suggest that YCF1 is composed of modular domains found in human proteins which function in drug and ion transport. PMID:7521334

  20. Specific Polymorphisms in Hepatitis C Virus Genotype 3 Core Protein Associated with Intracellular Lipid Accumulation

    PubMed Central

    Jhaveri, Ravi; McHutchison, John; Patel, Keyur; Qiang, Guan; Diehl, Anna Mae

    2008-01-01

    Background Steatosis is a common histological finding and a poor prognostic indicator in patients with hepatitis C virus (HCV) infection. In HCV genotype 3–infected patients, the etiology of steatosis appears to be closely correlated with unknown viral factors that increase intracellular lipid levels. We hypothesize that specific sequence polymorphisms in HCV genotype 3 core protein may be associated with hepatic intracellular lipid accumulation. Methods Using selected serum samples from 8 HCV genotype 3–infected patients with or without steatosis, we sequenced the HCV core gene to identify candidate polymorphisms associated with increased intracellular lipid levels. Results Two polymorphisms at positions 182 and 186 of the core protein correlated with the presence (P = .03) and absence (P = .005) of intrahepatic steatosis. Transfected liver cell lines expressing core protein with steatosis-associated polymorphisms had increased intracellular lipid levels compared with non–steatosis-associated core isolates, as measured by oil red O staining (P = .02). Site-specific mutagenesis performed at positions 182 and 186 in steatosis-associated core genes yielded proteins that had decreased intracellular lipid levels in transfected cells (P = .03). Conclusions We have identified polymorphisms in HCV core protein genotype 3 that produce increased intracellular lipid levels and thus may play a significant role in lipid metabolism or trafficking, contributing to steatosis. PMID:18177246

  1. Immunogold localization of tobravirus 2b nematode transmission helper protein associated with virus particles.

    PubMed

    Vellios, Evangelos; Duncan, George; Brown, Derek; MacFarlane, Stuart

    2002-08-15

    Transmission of the tobraviruses Tobacco rattle virus (TRV) and Pea early-browning virus (PEBV) by trichodorid vector nematodes requires the viral coat protein (CP) and the 2b protein, a nonstructural protein encoded by RNA2, the smaller of the two viral genomic RNAs. It is hypothesized that the 2b protein functions by interacting with a small, flexible domain located at the C-terminus of the CP, forming a bridge between the virus particle and the internal surface of the vector nematode feeding apparatus. Antibodies specific for the 2b protein of PEBV or TRV did not bind to virus particles that were adsorbed to electron microscope grids and were not able to trap virus particles from extracts of infected plants. However, electron microscopy of thin sections of plants infected with PEBV probed with 2b-specific antibodies which were further gold-labeled showed that the 2b protein localizes exclusively to virus particles. Similarly, immunogold localization studies showed that the 2b protein of TRV isolate PaY4 is associated only with TRV PaY4 virus particles. When a recombinant TRV encoding the PaY4 2b protein and the CP from TRV isolate PpK20 was examined, the 2b protein could not be detected by Western blotting and in IGL experiments was not associated with virus particles. These results suggest that in the absence of a specific interaction between compatible CP and 2b proteins, the 2b protein does not accumulate. PMID:12202212

  2. DPP6 Localization in Brain Supports Function as a Kv4 Channel Associated Protein

    PubMed Central

    Clark, Brian D.; Kwon, Elaine; Maffie, Jon; Jeong, Hyo-Young; Nadal, Marcela; Strop, Pavel; Rudy, Bernardo

    2008-01-01

    The gene encoding the dipeptidyl peptidase-like protein DPP6 (also known as DPPX) has been associated with human neural disease. However, until recently no function had been found for this protein. It has been proposed that DPP6 is an auxiliary subunit of neuronal Kv4 K+ channels, the ion channels responsible for the somato-dendritic A-type K+ current, an ionic current with crucial roles in the regulation of firing frequency, dendritic integration and synaptic plasticity. This view has been supported mainly by studies showing that DPP6 is necessary to generate channels with biophysical properties resembling the native channels in some neurons. However, independent evidence that DPP6 is a component of neuronal Kv4 channels in the brain, and whether this protein has other functions in the CNS is still lacking. We generated antibodies to DPP6 proteins to compare their distribution in brain with that of the Kv4 pore-forming subunits. DPP6 proteins were prominently expressed in neuronal populations expressing Kv4.2 proteins and both types of protein were enriched in the dendrites of these cells, strongly supporting the hypothesis that DPP6 is an associated protein of Kv4 channels in brain neurons. The observed similarity in the cellular and subcellular patterns of expression of both proteins suggests that this is the main function of DPP6 in brain. However, we also found that DPP6 antibodies intensely labeled the hippocampal mossy fiber axons, which lack Kv4 proteins, suggesting that DPP6 proteins may have additional, Kv4-unrelated functions. PMID:18978958

  3. Heterochromatin-Associated Proteins HP1a and Piwi Collaborate to Maintain the Association of Achiasmate Homologs in Drosophila Oocytes.

    PubMed

    Giauque, Christopher C; Bickel, Sharon E

    2016-05-01

    Accurate segregation of homologous chromosomes during meiosis depends on their ability to remain physically connected throughout prophase I. For homologs that achieve a crossover, sister chromatid cohesion distal to the chiasma keeps them attached until anaphase I. However, in Drosophila melanogaster wild-type oocytes, chromosome 4 never recombines, and the X chromosome fails to cross over in 6-10% of oocytes. Proper segregation of these achiasmate homologs relies on their pericentric heterochromatin-mediated association, but the mechanism(s) underlying this attachment remains poorly understood. Using an inducible RNA interference (RNAi) strategy combined with fluorescence in situ hybridization (FISH) to monitor centromere proximal association of the achiasmate FM7a/X homolog pair, we asked whether specific heterochromatin-associated proteins are required for the association and proper segregation of achiasmate homologs in Drosophila oocytes. When we knock down HP1a, H3K9 methytransferases, or the HP1a binding partner Piwi during mid-prophase, we observe significant disruption of pericentric heterochromatin-mediated association of FM7a/X homologs. Furthermore, for both HP1a and Piwi knockdown oocytes, transgenic coexpression of the corresponding wild-type protein is able to rescue RNAi-induced defects, but expression of a mutant protein with a single amino acid change that disrupts the HP1a-Piwi interaction is unable to do so. We show that Piwi is stably bound to numerous sites along the meiotic chromosomes, including centromere proximal regions. In addition, reduction of HP1a or Piwi during meiotic prophase induces a significant increase in FM7a/X segregation errors. We present a speculative model outlining how HP1a and Piwi could collaborate to keep achiasmate chromosomes associated in a homology-dependent manner. PMID:26984058

  4. Integrating gene expression and protein-protein interaction network to prioritize cancer-associated genes

    PubMed Central

    2012-01-01

    Background To understand the roles they play in complex diseases, genes need to be investigated in the networks they are involved in. Integration of gene expression and network data is a promising approach to prioritize disease-associated genes. Some methods have been developed in this field, but the problem is still far from being solved. Results In this paper, we developed a method, Networked Gene Prioritizer (NGP), to prioritize cancer-associated genes. Applications on several breast cancer and lung cancer datasets demonstrated that NGP performs better than the existing methods. It provides stable top ranking genes between independent datasets. The top-ranked genes by NGP are enriched in the cancer-associated pathways. The top-ranked genes by NGP-PLK1, MCM2, MCM3, MCM7, MCM10 and SKP2 might coordinate to promote cell cycle related processes in cancer but not normal cells. Conclusions In this paper, we have developed a method named NGP, to prioritize cancer-associated genes. Our results demonstrated that NGP performs better than the existing methods. PMID:22838965

  5. Wool Keratin-Associated Protein Genes in Sheep-A Review.

    PubMed

    Gong, Hua; Zhou, Huitong; Forrest, Rachel H J; Li, Shaobin; Wang, Jiqing; Dyer, Jolon M; Luo, Yuzhu; Hickford, Jon G H

    2016-01-01

    The importance of sheep's wool in making textiles has inspired extensive research into its structure and the underlying genetics since the 1960s. Wool keratin-associated proteins (KAPs) are a key structural component of the wool fibre. The characterisation of the genes encoding these proteins has progressed rapidly with advances in the nucleotide and protein sequencing. This review describes our knowledge of ovine KAPs, their categorisation into families, polymorphism in the proteins and genes, the clustering and chromosomal location of the genes, some characteristics of gene expression and some potential effects of the KAPs on wool traits. The extent and nature of genetic variation in wool KAP genes and its association with fibre characteristics, provides an opportunity for the development of gene-markers for selective breeding of sheep to produce better wool with properties highly matched to specific end-uses. PMID:27240405

  6. Degradation of lipid droplet-associated proteins by chaperone-mediated autophagy facilitates lipolysis

    PubMed Central

    Kaushik, Susmita; Cuervo, Ana Maria

    2015-01-01

    Chaperone-mediated autophagy (CMA) selectively degrades a subset of cytosolic proteins in lysosomes. A potent physiological activator of CMA is nutrient deprivation, a condition in which intracellular triglyceride stores or lipid droplets (LD) also undergo hydrolysis (lipolysis) to generate free fatty acids for energetic purposes. Here we report that LD-associated proteins perilipin 2 (PLIN2) and perilipin 3 (PLIN3) are CMA substrates and their degradation via CMA precedes lipolysis. In vivo studies revealed that CMA degradation of PLIN2 and PLIN3 was enhanced during starvation, concurrent with elevated levels of cytosolic adipose triglyceride lipase (ATGL) and macroautophagy proteins on LD. CMA blockage both in cultured cells and mouse liver or expression of CMA-resistant PLINs lead to reduced association of ATGL and macrolipophagy-related proteins with LD and the subsequent decrease in lipid oxidation and accumulation of LD. We propose a role of CMA in LD biology and in the maintenance of lipid homeostasis. PMID:25961502

  7. The Peptide Network between Tetanus Toxin and Human Proteins Associated with Epilepsy.

    PubMed

    Lucchese, Guglielmo; Spinosa, Jean Pierre; Kanduc, Darja

    2014-01-01

    Sequence matching analyses show that Clostridium tetani neurotoxin shares numerous pentapeptides (68, including multiple occurrences) with 42 human proteins that, when altered, have been associated with epilepsy. Such a peptide sharing is higher than expected, nonstochastic, and involves tetanus toxin-derived epitopes that have been validated as immunopositive in the human host. Of note, an unexpected high level of peptide matching is found in mitogen-activated protein kinase 10 (MK10), a protein selectively expressed in hippocampal areas. On the whole, the data indicate a potential for cross-reactivity between the neurotoxin and specific epilepsy-associated proteins and may help evaluate the potential risk for epilepsy following immune responses induced by tetanus infection. Moreover, this study may contribute to clarifying the etiopathogenesis of the different types of epilepsy. PMID:24982805

  8. Proteins that appear to be associated with pili in Neisseria gonorrhoeae.

    PubMed Central

    Muir, L L; Strugnell, R A; Davies, J K

    1988-01-01

    Pili of Neisseria gonorrhoeae are thought to be composed entirely of identical subunits, called pilin, that self-assemble in vitro. Previous pilus purification methods have relied on this latter point, and dissociation and reassociation of pilin subunits has yielded pilin preparations of high purity. Such a procedure could result in the loss of any pilus-associated proteins. We have developed a procedure for the isolation of intact native pili in a deoxycholate-urea buffer in which the pili are fractionated on the basis of size and hydrophobicity. Electron microscopy indicates that the pili are largely free from outer membrane vesicles and other cellular material. Electrophoretic analysis has shown that a number of proteins copurify with pilin. Antibodies to these proteins could be removed from an antiserum against whole piliated cells by absorption with piliated cells but not by absorption with nonpiliated cells. Hence, our results indicate that these proteins could be pilus associated. Images PMID:2898429

  9. Wool Keratin-Associated Protein Genes in Sheep—A Review

    PubMed Central

    Gong, Hua; Zhou, Huitong; Forrest, Rachel H. J.; Li, Shaobin; Wang, Jiqing; Dyer, Jolon M.; Luo, Yuzhu; Hickford, Jon G. H.

    2016-01-01

    The importance of sheep’s wool in making textiles has inspired extensive research into its structure and the underlying genetics since the 1960s. Wool keratin-associated proteins (KAPs) are a key structural component of the wool fibre. The characterisation of the genes encoding these proteins has progressed rapidly with advances in the nucleotide and protein sequencing. This review describes our knowledge of ovine KAPs, their categorisation into families, polymorphism in the proteins and genes, the clustering and chromosomal location of the genes, some characteristics of gene expression and some potential effects of the KAPs on wool traits. The extent and nature of genetic variation in wool KAP genes and its association with fibre characteristics, provides an opportunity for the development of gene-markers for selective breeding of sheep to produce better wool with properties highly matched to specific end-uses. PMID:27240405

  10. Herpes simplex virus protein UL11 but not UL51 is associated with lipid rafts.

    PubMed

    Koshizuka, Tetsuo; Kawaguchi, Yasushi; Nozawa, Naoki; Mori, Isamu; Nishiyama, Yukihiro

    2007-12-01

    The UL11 and UL51 gene products of herpes simplex virus (HSV) are membrane-associated tegument proteins that are incorporated into the HSV virion. UL11 and UL51 are conserved throughout the herpesvirus family. Both UL11 and UL51, either singly or in combination, are involved in virion envelopment and/or egress. Both proteins are fatty acylated: UL11 is both acylated by myristoic and palmitoic acids and UL51 is monoacylated by palmitoic acid. Using confocal microscopy and sucrose gradient fractionations in transfected or HSV-infected cells, we found that HSV-2 UL11 but not UL51 was associated with lipid rafts. The dual acylation of UL11 was necessary for lipid raft association, as mutations in the myristoylation or palmitoylation sites prevented lipid raft association. These differences in lipid raft association may contribute to the functional differences between UL11 and UL51. PMID:17694428

  11. The CCN Family Proteins: Modulators of Bone Development and Novel Targets in Bone-Associated Tumors

    PubMed Central

    Chen, Po-Chun; Cheng, Hsu-Chen; Yang, Shun-Fa; Tang, Chih-Hsin

    2014-01-01

    The CCN family of proteins is composed of six extracellular matrix-associated proteins that play crucial roles in skeletal development, wound healing, fibrosis, and cancer. Members of the CCN family share four conserved cysteine-rich modular domains that trigger signal transduction in cell adhesion, migration, proliferation, differentiation, and survival through direct binding to specific integrin receptors and heparan sulfate proteoglycans. In the present review, we discuss the roles of the CCN family proteins in regulating resident cells of the bone microenvironment. In vertebrate development, the CCN family plays a critical role in osteo/chondrogenesis and vasculo/angiogenesis. These effects are regulated through signaling via integrins, bone morphogenetic protein, vascular endothelial growth factor, Wnt, and Notch via direct binding to CCN family proteins. Due to the important roles of CCN family proteins in skeletal development, abnormal expression of CCN proteins is related to the tumorigenesis of primary bone tumors such as osteosarcoma, Ewing sarcoma, and chondrosarcoma. Additionally, emerging studies have suggested that CCN proteins may affect progression of secondary metastatic bone tumors by moderating the bone microenvironment. CCN proteins could therefore serve as potential therapeutic targets for drug development against primary and metastatic bone tumors. PMID:24551846

  12. Comprehensive proteome analysis of an Apc mouse model uncovers proteins associated with intestinal tumorigenesis.

    PubMed

    Hung, Kenneth E; Faca, Vitor; Song, Kenneth; Sarracino, David A; Richard, Larissa Georgeon; Krastins, Bryan; Forrester, Sara; Porter, Andrew; Kunin, Alexandra; Mahmood, Umar; Haab, Brian B; Hanash, Samir M; Kucherlapati, Raju

    2009-03-01

    Tumor-derived proteins may occur in the circulation as a result of secretion, shedding from the cell surface, or cell turnover. We have applied an in-depth comprehensive proteomic strategy to plasma from intestinal tumor-bearing Apc mutant mice to identify proteins associated with tumor development. We used quantitative tandem mass spectrometry of fractionated mouse plasma to identify differentially expressed proteins in plasma from intestinal tumor-bearing Apc mutant mice relative to matched controls. Up-regulated proteins were assessed for the expression of corresponding genes in tumor tissue. A subset of proteins implicated in colorectal cancer were selected for further analysis at the tissue level using antibody microarrays, Western blotting, tumor immunohistochemistry, and novel fluorescent imaging. We identified 51 proteins that were elevated in plasma with concordant up-regulation at the RNA level in tumor tissue. The list included multiple proteins involved in colon cancer pathogenesis: cathepsin B and cathepsin D, cullin 1, Parkinson disease 7, muscle pyruvate kinase, and Ran. Of these, Parkinson disease 7, muscle pyruvate kinase, and Ran were also found to be up-regulated in human colon adenoma samples. We have identified proteins with direct relevance to colorectal carcinogenesis that are present both in plasma and in tumor tissue in intestinal tumor-bearing mice. Our results show that integrated analysis of the plasma proteome and tumor transcriptome of genetically engineered mouse models is a powerful approach for the identification of tumor-related plasma proteins. PMID:19240248

  13. Postranslational modifications significantly alter the binding-folding pathways of proteins associating with DNA

    NASA Astrophysics Data System (ADS)

    Papoian, Garegin

    2012-02-01

    Many important regulators of gene activity are natively disordered, but fully or partially order when they bind to their targets on DNA. Interestingly, the ensembles of disordered states for such free proteins are not structurally featureless, but can qualitatively differ from protein to protein. In particular, in random coil like states the chains are swollen, making relatively few contacts, while in molten globule like states a significant collapse occurs, with ensuing high density of intra-protein interactions. Furthermore, since many DNA binding proteins are positively charged polyelectrolytes, the electrostatic self-repulsion also influences the degree of collapse of the chain and its conformational preferences in the free state and upon binding to DNA. In our work, we have found that the nature of the natively disordered ensemble significantly affects the way the protein folds upon binding to DNA. In particular, we showed that posttranslational modifications of amino acid residues, such as lysine acetylation, can alter the degree of collapse and conformational preferences for a free protein, and also profoundly impact the binding affinity and pathways for the protein DNA association. These trends will be discussed in the context of DNA interacting with various histone tails and the p53 protein.

  14. Evolutionary Diversification of the Sm Family of RNA-Associated Proteins

    PubMed Central

    Lynch, Michael

    2008-01-01

    The Sm family of proteins is closely associated with RNA metabolism throughout all life. These proteins form homomorphic and heteromorphic rings consisting of six or seven subunits with a characteristic central pore, the presence of which is critical for binding U-rich regions of single-stranded RNA. Eubacteria and Archaea typically carry one or two forms of Sm proteins and assemble one homomorphic ring per Sm protein. Eukaryotes typically carry 16 or more Sm proteins that assemble to form heteromorphic rings which lie at the center of a number of critical RNA-associated small nuclear ribonucleoproteins (snRNPs). High Sm protein diversity and heteromorphic Sm rings are features stretching back to the origin of eukaryotes; very deep phylogenetic divisions among existing Sm proteins indicate simultaneous evolution across essentially all existing eukaryotic life. Two basic forms of heteromorphic Sm rings are found in eukaryotes. Fixed Sm rings are highly stable and static and are assembled around an RNA cofactor. Flexible Sm rings also stabilize and chaperone RNA but assemble in the absence of an RNA substrate and, more significantly, associate with and dissociate from RNA substrates more freely than fixed rings. This suggests that the conformation of flexible Sm rings might be modified in some specific manner to facilitate association and dissociation with RNA. Diversification of eukaryotic Sm proteins may have been initiated by gene transfers and/or genome clashes that accompanied the origin of the eukaryotic cell itself, with further diversification driven by a greater need for steric specificity within increasingly complex snRNPs. PMID:18687770

  15. Global deletion of BCATm increases expression of skeletal muscle genes associated with protein turnover.

    PubMed

    Lynch, Christopher J; Kimball, Scot R; Xu, Yuping; Salzberg, Anna C; Kawasawa, Yuka Imamura

    2015-11-01

    Consumption of a protein-containing meal by a fasted animal promotes protein accretion in skeletal muscle, in part through leucine stimulation of protein synthesis and indirectly through repression of protein degradation mediated by its metabolite, α-ketoisocaproate. Mice lacking the mitochondrial branched-chain aminotransferase (BCATm/Bcat2), which interconverts leucine and α-ketoisocaproate, exhibit elevated protein turnover. Here, the transcriptomes of gastrocnemius muscle from BCATm knockout (KO) and wild-type mice were compared by next-generation RNA sequencing (RNA-Seq) to identify potential adaptations associated with their persistently altered nutrient signaling. Statistically significant changes in the abundance of 1,486/∼39,010 genes were identified. Bioinformatics analysis of the RNA-Seq data indicated that pathways involved in protein synthesis [eukaryotic initiation factor (eIF)-2, mammalian target of rapamycin, eIF4, and p70S6K pathways including 40S and 60S ribosomal proteins], protein breakdown (e.g., ubiquitin mediated), and muscle degeneration (apoptosis, atrophy, myopathy, and cell death) were upregulated. Also in agreement with our previous observations, the abundance of mRNAs associated with reduced body size, glycemia, plasma insulin, and lipid signaling pathways was altered in BCATm KO mice. Consistently, genes encoding anaerobic and/or oxidative metabolism of carbohydrate, fatty acids, and branched chain amino acids were modestly but systematically reduced. Although there was no indication that muscle fiber type was different between KO and wild-type mice, a difference in the abundance of mRNAs associated with a muscular dystrophy phenotype was observed, consistent with the published exercise intolerance of these mice. The results suggest transcriptional adaptations occur in BCATm KO mice that along with altered nutrient signaling may contribute to their previously reported protein turnover, metabolic and exercise phenotypes. PMID

  16. C-reactive protein gene variants: independent association with late-life depression and circulating protein levels.

    PubMed

    Ancelin, M-L; Farré, A; Carrière, I; Ritchie, K; Chaudieu, I; Ryan, J

    2015-01-01

    C-reactive protein (CRP) is a heritable biomarker of systemic inflammation that is commonly elevated in depressed patients. Variants in the CRP gene that influence protein levels could thus be associated with depression but this has seldom been examined, especially in the elderly. Depression was assessed in 990 people aged at least 65 years as part of the ESPRIT study. A clinical level of depression (DEP) was defined as having a score of ⩾16 on The Center for Epidemiologic Studies Depression scale or a diagnosis of current major depression based on the Mini-International Neuropsychiatric Interview and according to Diagnostic and Statistical Manual of Mental Disorders-IV criteria. Five single-nucleotide polymorphisms spanning the CRP gene were genotyped, and circulating levels of high-sensitivity CRP were determined. Multivariable analyses adjusted for socio-demographic characteristics, smoking, ischemic pathologies, cognitive impairment and inflammation-related chronic pathologies. The minor alleles of rs1130864 and rs1417938 were associated with a decreased risk of depression in women at Bonferroni-corrected significance levels (P=0.002). CRP gene variants were associated with serum levels in a gender-specific manner, but only rs1205 was found to be nominally associated with both an increased risk of DEP and lower circulating CRP levels in women. Variants of the CRP gene thus influence circulating CRP levels and appear as independent susceptibility factors for late-life depression. PMID:25603415

  17. Nectin-like molecule 1 is a protein 4.1N associated protein and recruits protein 4.1N from cytoplasm to the plasma membrane.

    PubMed

    Zhou, Yan; Du, Guangwei; Hu, Xiaoyan; Yu, Shun; Liu, Yaobo; Xu, Yaqin; Huang, Xiaowei; Liu, Jin; Yin, Bin; Fan, Ming; Peng, Xiaozhong; Qiang, Boqin; Yuan, Jiangang

    2005-05-20

    Nectins are immunoglobulin superfamily adhesion molecules that participate in the organization of epithelial and endothelial junctions. Sharing high homology with the poliovirus receptor (PVR/CD155), nectins were also named poliovirus receptor-related proteins (PRRs). Four nectins and five nectin-like molecules have been identified. Here we describe the cloning and characterization of human and mouse nectin-like molecular 1 (NECL1). Human and mouse NECL1 share 87.3% identity at the amino acid level. NECL1 contains an ectodomain made of three immunoglobulin-like domains, and a cytoplasmic region homologous to those of glycophorin C and contactin-associated protein. RNA blot and in situ hybridization analysis showed that NECL1 predominantly expressed in the central nervous system, mainly in neuronal cell bodies in a variety of brain regions including the cerebellum, cerebral cortex and hippocampus. In vitro binding assay proved the association of NECL1 with protein 4.1N. NECL1 localizes to the cell-cell junctions and recruits protein 4.1N to the plasma membranes through its C-terminus, thus may regulate the function of the cell-cell junction. We propose that the NECL1 and protein 4.1N complex is involved in the morphological development, stability, and dynamic plasticity of the nervous system. PMID:15893517

  18. Function of nucleoid-associated proteins in chromosome structuring and transcriptional regulation.

    PubMed

    Dorman, Charles J

    2014-01-01

    Nucleoid-associated proteins typically are abundant, low-molecular-mass polypeptides that bind DNA and alter its shape and its ability to participate in transactions such as transcription. Some can bind RNA and influence the gene expression profile of the cell at a posttranscriptional level. They also have the potential to model and remodel the structure of the nucleoid, contributing to chromosome packaging within the cell. Some nucleoid-associated proteins have been implicated in the facilitation of chromosome evolution through their ability to silence transcription, allowing new genes to be integrated into the nucleoid both physically and in a regulatory sense. The dynamic composition of the population of nucleoid-associated proteins in model bacteria such as Escherichia coli and Salmonella enterica links nucleoid structure and the global regulation of gene expression, enhancing microbial competitive fitness and survival in complex environments. PMID:25732335

  19. Protein-like fully reversible tetramerisation and super-association of an aminocellulose

    NASA Astrophysics Data System (ADS)

    Nikolajski, Melanie; Adams, Gary G.; Gillis, Richard B.; Besong, David Tabot; Rowe, Arthur J.; Heinze, Thomas; Harding, Stephen E.

    2014-01-01

    Unusual protein-like, partially reversible associative behaviour has recently been observed in solutions of the water soluble carbohydrates known as 6-deoxy-6-(ω-aminoalkyl)aminocelluloses, which produce controllable self-assembling films for enzyme immobilisation and other biotechnological applications. Now, for the first time, we have found a fully reversible self-association (tetramerisation) within this family of polysaccharides. Remarkably these carbohydrate tetramers are then seen to associate further in a regular way into supra-molecular complexes. Fully reversible oligomerisation has been hitherto completely unknown for carbohydrates and instead resembles in some respects the assembly of polypeptides and proteins like haemoglobin and its sickle cell mutation. Our traditional perceptions as to what might be considered ``protein-like'' and what might be considered as ``carbohydrate-like'' behaviour may need to be rendered more flexible, at least as far as interaction phenomena are concerned.

  20. Arabinogalactan protein and wall-associated kinase in a plasmalemmal reticulum with specialized vertices

    NASA Technical Reports Server (NTRS)

    Gens, J. S.; Fujiki, M.; Pickard, B. G.; Evans, M. L. (Principal Investigator)

    2000-01-01

    Arabinogalactan protein and wall-associated kinase (WAK) are suspected to be regulatory players at the interface between cytoplasm and cell wall. Both WAK(s) and arabinogalactan shown likely to represent arabinogalactan protein(s) have been visualized there with computational optical-sectioning microscopy. The arabinogalactan occurs in a polyhedral array at the external face of the cell membrane. WAK, and other proteins as yet unidentified, appear to fasten the membrane to the wall at vertices of the array. Evidence is presented that the array bears an important part of the mechanical stress experienced by the membrane, and it is speculated that the architectural organization of arabinogalactan protein, WAK, and other components of the array is critical for coordination of endomembrane activities, growth, and differentiation. The array has been named the plasmalemmal reticulum.

  1. In-depth mapping of human testicular and epididymal proteins and their functional association with spermatozoa

    PubMed Central

    LIU, XUEXIA; LIU, FUJUN

    2015-01-01

    The mammalian testis and epididymis are responsible for spermatozoa production and maturation, which contributes to male fertility. Predominantly expressed proteins in the testis and epididymis were suggested to be involved in the key functions or pathways in spermatogenesis and sperm maturation. To further investigate these proteins and their associations with sperm, large protein profiles of human testis and epididymis were mapped. Predominantly-expressed testicular (173) and epididymal (244) secreted proteins were further screened and functionally characterized. Differential expression levels of solute carrier family 2 (facilitated glucose transporter), member 3, solute carrier family 25 (carnitine/acylcarnitine translocase), member 20, WAP-type four-disulfide core domain protein 8 and prostate and testis expressed 1 were validated using western blot and immunohistochemical analyses. The results may provide novel insight into the understanding of testicular and epididymal physiology and function, and facilitate sperm maturation research. PMID:25760095

  2. Protein–Protein and Protein–Membrane Associations in the Lignin Pathway[W][OA

    PubMed Central

    Bassard, Jean-Etienne; Richert, Ludovic; Geerinck, Jan; Renault, Hugues; Duval, Frédéric; Ullmann, Pascaline; Schmitt, Martine; Meyer, Etienne; Mutterer, Jerôme; Boerjan, Wout; De Jaeger, Geert; Mely, Yves; Goossens, Alain; Werck-Reichhart, Danièle

    2012-01-01

    Supramolecular organization of enzymes is proposed to orchestrate metabolic complexity and help channel intermediates in different pathways. Phenylpropanoid metabolism has to direct up to 30% of the carbon fixed by plants to the biosynthesis of lignin precursors. Effective coupling of the enzymes in the pathway thus seems to be required. Subcellular localization, mobility, protein–protein, and protein–membrane interactions of four consecutive enzymes around the main branch point leading to lignin precursors was investigated in leaf tissues of Nicotiana benthamiana and cells of Arabidopsis thaliana. CYP73A5 and CYP98A3, the two Arabidopsis cytochrome P450s (P450s) catalyzing para- and meta-hydroxylations of the phenolic ring of monolignols were found to colocalize in the endoplasmic reticulum (ER) and to form homo- and heteromers. They moved along with the fast remodeling plant ER, but their lateral diffusion on the ER surface was restricted, likely due to association with other ER proteins. The connecting soluble enzyme hydroxycinnamoyltransferase (HCT), was found partially associated with the ER. Both HCT and the 4-coumaroyl-CoA ligase relocalized closer to the membrane upon P450 expression. Fluorescence lifetime imaging microscopy supports P450 colocalization and interaction with the soluble proteins, enhanced by the expression of the partner proteins. Protein relocalization was further enhanced in tissues undergoing wound repair. CYP98A3 was the most effective in driving protein association. PMID:23175744

  3. Isolation and Analysis of Keratins and Keratin-Associated Proteins from Hair and Wool.

    PubMed

    Deb-Choudhury, Santanu; Plowman, Jeffrey E; Harland, Duane P

    2016-01-01

    The presence of highly cross-linked protein networks in hair and wool makes them very difficult substrates for protein extraction, a prerequisite for further protein analysis and characterization. It is therefore imperative that these cross-links formed by disulfide bridges are first disrupted for the efficient extraction of proteins. Chaotropes such as urea are commonly used as efficient extractants. However, a combination of urea and thiourea not only improves recovery of proteins but also results in improved resolution of the keratins in 2DE gels. Reductants also play an important role in protein dissolution. Dithiothreitol effectively removes keratinous material from the cortex, whereas phosphines, like Tris(2-carboxyethyl)phosphine, remove material from the exocuticle. The relative extractability of the keratins and keratin-associated proteins is also dependent on the concentration of chaotropes, reductants, and pH, thus providing a means to preferentially extract these proteins. Ionic liquids such as 1-butyl-3-methylimidazolium chloride (BMIM(+)[Cl](-)) are known to solubilize wool by disrupting noncovalent interactions, specifically intermolecular hydrogen bonds. BMIM(+)[Cl](-) proved to be an effective extractant of wool proteins and complementary in nature to chaotropes such as urea and thiourea for identifying unique peptides of wool proteins using mass spectrometry (MS). Successful identification of proteins resolved by one- or two-dimensional electrophoresis and MS is highly dependent on the optimal recovery of its protease-digested peptides with an efficient removal of interfering substances. The detergent sodium deoxycholate used in conjunction with Empore™ disks improved identification of proteins by mass spectrometry leading to higher percentage sequence coverage, identification of unique peptides and higher score. PMID:26795475

  4. Interaction with the Yes-associated protein (YAP) allows TEAD1 to positively regulate NAIP expression.

    PubMed

    Landin Malt, André; Georges, Adrien; Silber, Joël; Zider, Alain; Flagiello, Domenico

    2013-10-01

    Although the expression of the neuronal apoptosis inhibitory protein (NAIP) gene is considered involved in apoptosis suppression as well as in inflammatory response, the molecular basis of the NAIP gene expression is poorly understood. Here we show that the TEA domain protein 1 (TEAD1) is able to positively activate the transcription of NAIP. We further demonstrate that this regulation is mediated by the presence of the endogenous Yes associated protein (YAP) cofactor, and requires the interaction with YAP. We finally identified an intronic region of the NAIP gene responding to TEAD1/YAP activity, suggesting that regulation of NAIP by TEAD1/YAP is at the transcriptional level. PMID:23994529

  5. Identification of a cellulose synthase-associated protein required for cellulose biosynthesis

    PubMed Central

    Gu, Ying; Kaplinsky, Nick; Bringmann, Martin; Cobb, Alex; Carroll, Andrew; Sampathkumar, Arun; Baskin, Tobias I.; Persson, Staffan; Somerville, Chris R.

    2010-01-01

    Cellulose synthase-interactive protein 1 (CSI1) was identified in a two-hybrid screen for proteins that interact with cellulose synthase (CESA) isoforms involved in primary plant cell wall synthesis. CSI1 encodes a 2,150-amino acid protein that contains 10 predicted Armadillo repeats and a C2 domain. Mutations in CSI1 cause defective cell elongation in hypocotyls and roots and reduce cellulose content. CSI1 is associated with CESA complexes, and csi1 mutants affect the distribution and movement of CESA complexes in the plasma membrane. PMID:20616083

  6. Laser capture microdissection to identify septum-associated proteins in Aspergillus nidulans.

    PubMed

    Zhang, Ying; Fischer, Reinhard; Teichert, Ines; Kück, Ulrich

    2016-01-01

    To spatially resolve genetic differences at the cellular level, the laser-capture microdissection technique was developed. With this method cells can be cut from tissues with a laser beam and analyzed for DNA, RNA or protein composition. Here we adapted the technique to isolate septal microtubule-organizing center (MTOC)-associated proteins in Aspergillus nidulans About 3000 septa were collected and subjected to peptide fingerprinting by mass-spectrometric analysis. We identified the microtubule polymerase AlpA and found it interacts with ApsB specifically at sMTOCs, suggesting that AlpA might be involved in the assembly or the functioning of this protein complex. PMID:26951366

  7. Association of polyalanine and polyglutamine coiled coils mediates expansion disease-related protein aggregation and dysfunction

    PubMed Central

    Pelassa, Ilaria; Corà, Davide; Cesano, Federico; Monje, Francisco J.; Montarolo, Pier Giorgio; Fiumara, Ferdinando

    2014-01-01

    The expansion of homopolymeric glutamine (polyQ) or alanine (polyA) repeats in certain proteins owing to genetic mutations induces protein aggregation and toxicity, causing at least 18 human diseases. PolyQ and polyA repeats can also associate in the same proteins, but the general extent of their association in proteomes is unknown. Furthermore, the structural mechanisms by which their expansion causes disease are not well understood, and these repeats are generally thought to misfold upon expansion into aggregation-prone β-sheet structures like amyloids. However, recent evidence indicates a critical role for coiled-coil (CC) structures in triggering aggregation and toxicity of polyQ-expanded proteins, raising the possibility that polyA repeats may as well form these structures, by themselves or in association with polyQ. We found through bioinformatics screenings that polyA, polyQ and polyQA repeats have a phylogenetically graded association in human and non-human proteomes and associate/overlap with CC domains. Circular dichroism and cross-linking experiments revealed that polyA repeats can form—alone or with polyQ and polyQA—CC structures that increase in stability with polyA length, forming higher-order multimers and polymers in vitro. Using structure-guided mutagenesis, we studied the relevance of polyA CCs to the in vivo aggregation and toxicity of RUNX2—a polyQ/polyA protein associated with cleidocranial dysplasia upon polyA expansion—and found that the stability of its polyQ/polyA CC controls its aggregation, localization and toxicity. These findings indicate that, like polyQ, polyA repeats form CC structures that can trigger protein aggregation and toxicity upon expansion in human genetic diseases. PMID:24497578

  8. Association of polyalanine and polyglutamine coiled coils mediates expansion disease-related protein aggregation and dysfunction.

    PubMed

    Pelassa, Ilaria; Corà, Davide; Cesano, Federico; Monje, Francisco J; Montarolo, Pier Giorgio; Fiumara, Ferdinando

    2014-07-01

    The expansion of homopolymeric glutamine (polyQ) or alanine (polyA) repeats in certain proteins owing to genetic mutations induces protein aggregation and toxicity, causing at least 18 human diseases. PolyQ and polyA repeats can also associate in the same proteins, but the general extent of their association in proteomes is unknown. Furthermore, the structural mechanisms by which their expansion causes disease are not well understood, and these repeats are generally thought to misfold upon expansion into aggregation-prone β-sheet structures like amyloids. However, recent evidence indicates a critical role for coiled-coil (CC) structures in triggering aggregation and toxicity of polyQ-expanded proteins, raising the possibility that polyA repeats may as well form these structures, by themselves or in association with polyQ. We found through bioinformatics screenings that polyA, polyQ and polyQA repeats have a phylogenetically graded association in human and non-human proteomes and associate/overlap with CC domains. Circular dichroism and cross-linking experiments revealed that polyA repeats can form--alone or with polyQ and polyQA--CC structures that increase in stability with polyA length, forming higher-order multimers and polymers in vitro. Using structure-guided mutagenesis, we studied the relevance of polyA CCs to the in vivo aggregation and toxicity of RUNX2--a polyQ/polyA protein associated with cleidocranial dysplasia upon polyA expansion--and found that the stability of its polyQ/polyA CC controls its aggregation, localization and toxicity. These findings indicate that, like polyQ, polyA repeats form CC structures that can trigger protein aggregation and toxicity upon expansion in human genetic diseases. PMID:24497578

  9. PTMcode: a database of known and predicted functional associations between post-translational modifications in proteins

    PubMed Central

    Minguez, Pablo; Letunic, Ivica; Parca, Luca; Bork, Peer

    2013-01-01

    Post-translational modifications (PTMs) are involved in the regulation and structural stabilization of eukaryotic proteins. The combination of individual PTM states is a key to modulate cellular functions as became evident in a few well-studied proteins. This combinatorial setting, dubbed the PTM code, has been proposed to be extended to whole proteomes in eukaryotes. Although we are still far from deciphering such a complex language, thousands of protein PTM sites are being mapped by high-throughput technologies, thus providing sufficient data for comparative analysis. PTMcode (http://ptmcode.embl.de) aims to compile known and predicted PTM associations to provide a framework that would enable hypothesis-driven experimental or computational analysis of various scales. In its first release, PTMcode provides PTM functional associations of 13 different PTM types within proteins in 8 eukaryotes. They are based on five evidence channels: a literature survey, residue co-evolution, structural proximity, PTMs at the same residue and location within PTM highly enriched protein regions (hotspots). PTMcode is presented as a protein-based searchable database with an interactive web interface providing the context of the co-regulation of nearly 75 000 residues in >10 000 proteins. PMID:23193284

  10. Topography of Lipid Droplet-Associated Proteins: Insights from Freeze-Fracture Replica Immunogold Labeling

    PubMed Central

    Robenek, Horst; Buers, Insa; Robenek, Mirko J.; Hofnagel, Oliver; Ruebel, Anneke; Troyer, David; Severs, Nicholas J.

    2011-01-01

    Lipid droplets are not merely storage depots for superfluous intracellular lipids in times of hyperlipidemic stress, but metabolically active organelles involved in cellular homeostasis. Our concepts on the metabolic functions of lipid droplets have come from studies on lipid droplet-associated proteins. This realization has made the study of proteins, such as PAT family proteins, caveolins, and several others that are targeted to lipid droplets, an intriguing and rapidly developing area of intensive inquiry. Our existing understanding of the structure, protein organization, and biogenesis of the lipid droplet has relied heavily on microscopical techniques that lack resolution and the ability to preserve native cellular and protein composition. Freeze-fracture replica immunogold labeling overcomes these disadvantages and can be used to define at high resolution the precise location of lipid droplet-associated proteins. In this paper illustrative examples of how freeze-fracture immunocytochemistry has contributed to our understanding of the spatial organization in the membrane plane and function of PAT family proteins and caveolin-1 are presented. By revisiting the lipid droplet with freeze-fracture immunocytochemistry, new perspectives have emerged which challenge prevailing concepts of lipid droplet biology and may hopefully provide a timely impulse for many ongoing studies. PMID:21490801

  11. Several novel nuclear envelope transmembrane proteins identified in skeletal muscle have cytoskeletal associations.

    PubMed

    Wilkie, Gavin S; Korfali, Nadia; Swanson, Selene K; Malik, Poonam; Srsen, Vlastimil; Batrakou, Dzmitry G; de las Heras, Jose; Zuleger, Nikolaj; Kerr, Alastair R W; Florens, Laurence; Schirmer, Eric C

    2011-01-01

    Nuclear envelopes from liver and a neuroblastoma cell line have previously been analyzed by proteomics; however, most diseases associated with the nuclear envelope affect muscle. To determine whether muscle has unique nuclear envelope proteins, rat skeletal muscle nuclear envelopes were prepared and analyzed by multidimensional protein identification technology. Many novel muscle-specific proteins were identified that did not appear in previous nuclear envelope data sets. Nuclear envelope residence was confirmed for 11 of these by expression of fusion proteins and by antibody staining of muscle tissue cryosections. Moreover, transcript levels for several of the newly identified nuclear envelope transmembrane proteins increased during muscle differentiation using mouse and human in vitro model systems. Some of these proteins tracked with microtubules at the nuclear surface in interphase cells and accumulated at the base of the microtubule spindle in mitotic cells, suggesting they may associate with complexes that connect the nucleus to the cytoskeleton. The finding of tissue-specific proteins in the skeletal muscle nuclear envelope proteome argues the importance of analyzing nuclear envelopes from all tissues linked to disease and suggests that general investigation of tissue differences in organellar proteomes might yield critical insights. PMID:20876400

  12. Several Novel Nuclear Envelope Transmembrane Proteins Identified in Skeletal Muscle Have Cytoskeletal Associations*

    PubMed Central

    Wilkie, Gavin S.; Korfali, Nadia; Swanson, Selene K.; Malik, Poonam; Srsen, Vlastimil; Batrakou, Dzmitry G.; de las Heras, Jose; Zuleger, Nikolaj; Kerr, Alastair R. W.; Florens, Laurence; Schirmer, Eric C.

    2011-01-01

    Nuclear envelopes from liver and a neuroblastoma cell line have previously been analyzed by proteomics; however, most diseases associated with the nuclear envelope affect muscle. To determine whether muscle has unique nuclear envelope proteins, rat skeletal muscle nuclear envelopes were prepared and analyzed by multidimensional protein identification technology. Many novel muscle-specific proteins were identified that did not appear in previous nuclear envelope data sets. Nuclear envelope residence was confirmed for 11 of these by expression of fusion proteins and by antibody staining of muscle tissue cryosections. Moreover, transcript levels for several of the newly identified nuclear envelope transmembrane proteins increased during muscle differentiation using mouse and human in vitro model systems. Some of these proteins tracked with microtubules at the nuclear surface in interphase cells and accumulated at the base of the microtubule spindle in mitotic cells, suggesting they may associate with complexes that connect the nucleus to the cytoskeleton. The finding of tissue-specific proteins in the skeletal muscle nuclear envelope proteome argues the importance of analyzing nuclear envelopes from all tissues linked to disease and suggests that general investigation of tissue differences in organellar proteomes might yield critical insights. PMID:20876400

  13. Identification of Biofilm Matrix-Associated Proteins from an Acid Mine Drainage Microbial Community ▿ †

    PubMed Central

    Jiao, Yongqin; D'haeseleer, Patrik; Dill, Brian D.; Shah, Manesh; VerBerkmoes, Nathan C.; Hettich, Robert L.; Banfield, Jillian F.; Thelen, Michael P.

    2011-01-01

    In microbial communities, extracellular polymeric substances (EPS), also called the extracellular matrix, provide the spatial organization and structural stability during biofilm development. One of the major components of EPS is protein, but it is not clear what specific functions these proteins contribute to the extracellular matrix or to microbial physiology. To investigate this in biofilms from an extremely acidic environment, we used shotgun proteomics analyses to identify proteins associated with EPS in biofilms at two developmental stages, designated DS1 and DS2. The proteome composition of the EPS was significantly different from that of the cell fraction, with more than 80% of the cellular proteins underrepresented or undetectable in EPS. In contrast, predicted periplasmic, outer membrane, and extracellular proteins were overrepresented by 3- to 7-fold in EPS. Also, EPS proteins were more basic by ∼2 pH units on average and about half the length. When categorized by predicted function, proteins involved in motility, defense, cell envelope, and unknown functions were enriched in EPS. Chaperones, such as histone-like DNA binding protein and cold shock protein, were overrepresented in EPS. Enzymes, such as protein peptidases, disulfide-isomerases, and those associated with cell wall and polysaccharide metabolism, were also detected. Two of these enzymes, identified as β-N-acetylhexosaminidase and cellulase, were confirmed in the EPS fraction by enzymatic activity assays. Compared to the differences between EPS and cellular fractions, the relative differences in the EPS proteomes between DS1 and DS2 were smaller and consistent with expected physiological changes during biofilm development. PMID:21685158

  14. Hsp56: A novel heat shock protein associated with untransformed steroid receptor complexes

    SciTech Connect

    Sanchez, E.R. )

    1990-12-25

    The recently-described p59 protein has been shown to be associated with untransformed steroid receptors present in rabbit uterus and rat liver cytosols while a smaller version of this protein (p56) interacts with glucocorticoid receptors in human IM-9 cell cytosols. In addition to interacting with glucocorticoid receptors, the p56 protein of IM-9 cell cytosol is also found as part of a large heteromeric complex that contains both the 70-kDa and 90-kDa heat shock proteins (hsp70 and hsp90, respectively). Given this association of p56 with the two major stress proteins, I have speculated that p56 may itself be a heat shock protein. In this paper, the effect of heat stress on the rate of synthesis of p56 is determined. Intact IM-9 cells were exposed to 37 or 43 degrees C for 4 h, followed by pulse-labeling with (35S)methionine. Analysis of whole cytosolic extracts by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography reveal an increased rate of radiolabeling for hsp70, hsp90, hsp100, ad hsp110, but no heat-inducible protein of smaller relative molecular mass is detected. However, immune-purification of p56 from normal and heat-stressed cytosols with the EC1 monoclonal antibody results in the presence of a 56-kDa protein that exhibits an increased rate of synthesis in response to heat stress. The results of two-dimensional gel Western blots employing the EC1 antibody demonstrate that this heat-inducible protein is indeed the EC1-reactive p56 protein and that the induction effect is not due to unequal yields of p56 during immune-purification.

  15. Serum Proteins Alteration in Association with Body Mass Index in Human Volunteers

    PubMed Central

    Madhuvanthi, M.

    2016-01-01

    Introduction Serum proteins are an important indicator of the nutritional status in an individual. There is a worldwide prevalence of both undernourishment and obesity. It has been suggested that low Body Mass Index (BMI) is associated with a decrease in serum protein levels predisposing them to other illnesses. Overweight and obese individuals carry risk for various other non-communicable diseases. Aim To compare the serum protein levels in underweight, overweight and obese individuals with that of normal body mass index individuals. Materials and Methods This prospective study was conducted in subjects who attended the master health checkup clinic of PSG hospitals. Subjects in the age group of 20-50 years were selected. Their serum proteins and BMI was measured. Twenty subjects each of underweight, normal, overweight and obese individuals were selected, categorized and compared. Results The serum protein level of normal individuals (Group I) was compared with underweight (Group II), overweight (Group III) and obese subjects (Group IV) by one-way ANOVA analysis. The mean serum total proteins in gm/dl in group I controls was 7.555±0.37 compared to Group II (underweight) which was 7.295±0.419. Low BMI was found to be associated with a decrease in serum protein level which was not statistically significant. Elevated BMI as in overweight and obese subjects showed no significant alterations in serum protein levels with p >0.05 and the changes were found to be independent of the body mass index. Conclusion Underweight individuals showed a decrease in serum protein levels whereas there were no significant changes in the serum protein levels in overweight and obese individuals. PMID:27504281

  16. Identification of Biofilm Matrix-Associated Proteins from an Acid Mine Drainage Microbial Community

    SciTech Connect

    Jiao, Yongqin; D'Haeseleer, Patrik M; Dill, Brian; Shah, Manesh B; Verberkmoes, Nathan C; Hettich, Robert {Bob} L; Banfield, Jillian F.; Thelen, Michael P.

    2011-01-01

    In microbial communities, extracellular polymeric substances (EPS), also called the extracellular matrix, provide the spatial organization and structural stability during biofilm development. One of the major components of EPS is protein, but it is not clear what specific functions these proteins contribute to the extracellular matrix or to microbial physiology. To investigate this in biofilms from an extremely acidic environment, we used shotgun proteomics analyses to identify proteins associated with EPS in biofilms at two developmental stages, designated DS1 and DS2. The proteome composition of the EPS was significantly different from that of the cell fraction, with more than 80% of the cellular proteins underrepresented or undetectable in EPS. In contrast, predicted periplasmic, outer membrane, and extracellular proteins were overrepresented by 3- to 7-fold in EPS. Also, EPS proteins were more basic by 2 pH units on average and about half the length. When categorized by predicted function, proteins involved in motility, defense, cell envelope, and unknown functions were enriched in EPS. Chaperones, such as histone-like DNA binding protein and cold shock protein, were overrepresented in EPS. Enzymes, such as protein peptidases, disulfide-isomerases, and those associated with cell wall and polysaccharide metabolism, were also detected. Two of these enzymes, identified as -N-acetylhexosaminidase and cellulase, were confirmed in the EPS fraction by enzymatic activity assays. Compared to the differences between EPS and cellular fractions, the relative differences in the EPS proteomes between DS1 and DS2 were smaller and consistent with expected physiological changes during biofilm development.

  17. Endobrevin, a novel synaptobrevin/VAMP-like protein preferentially associated with the early endosome.

    PubMed

    Wong, S H; Zhang, T; Xu, Y; Subramaniam, V N; Griffiths, G; Hong, W

    1998-06-01

    Synaptobrevins/vesicle-associated membrane proteins (VAMPs) together with syntaxins and a synaptosome-associated protein of 25 kDa (SNAP-25) are the main components of a protein complex involved in the docking and/or fusion of synaptic vesicles with the presynaptic membrane. We report here the molecular, biochemical, and cell biological characterization of a novel member of the synaptobrevin/VAMP family. The amino acid sequence of endobrevin has 32, 33, and 31% identity to those of synaptobrevin/VAMP-1, synaptobrevin/VAMP-2, and cellubrevin, respectively. Membrane fractionation studies demonstrate that endobrevin is enriched in membrane fractions that are also enriched in the asialoglycoprotein receptor. Indirect immunofluorescence microscopy establishes that endobrevin is primarily associated with the perinuclear vesicular structures of the early endocytic compartment. The preferential association of endobrevin with the early endosome was further established by electron microscopy (EM) immunogold labeling. In vitro binding assays show that endobrevin interacts with immobilized recombinant alpha-SNAP fused to glutathione S-transferase (GST). Our results highlight the general importance of members of the synaptobrevin/VAMP protein family in membrane traffic and provide new avenues for future functional and mechanistic studies of this protein as well as the endocytotic pathway. PMID:9614193

  18. Structure of the virulence-associated protein VapD from the intracellular pathogen Rhodococcus equi

    PubMed Central

    Whittingham, Jean L.; Blagova, Elena V.; Finn, Ciaran E.; Luo, Haixia; Miranda-CasoLuengo, Raúl; Turkenburg, Johan P.; Leech, Andrew P.; Walton, Paul H.; Murzin, Alexey G.; Meijer, Wim G.; Wilkinson, Anthony J.

    2014-01-01

    Rhodococcus equi is a multi-host pathogen that infects a range of animals as well as immune-compromised humans. Equine and porcine isolates harbour a virulence plasmid encoding a homologous family of virulence-associated proteins associated with the capacity of R. equi to divert the normal processes of endosomal maturation, enabling bacterial survival and proliferation in alveolar macrophages. To provide a basis for probing the function of the Vap proteins in virulence, the crystal structure of VapD was determined. VapD is a monomer as determined by multi-angle laser light scattering. The structure reveals an elliptical, compact eight-stranded β-barrel with a novel strand topology and pseudo-twofold symmetry, suggesting evolution from an ancestral dimer. Surface-associated octyl-β-d-glucoside molecules may provide clues to function. Circular-dichroism spectroscopic analysis suggests that the β-barrel structure is preceded by a natively disordered region at the N-terminus. Sequence comparisons indicate that the core folds of the other plasmid-encoded virulence-associated proteins from R. equi strains are similar to that of VapD. It is further shown that sequences encoding putative R. equi Vap-like proteins occur in diverse bacterial species. Finally, the functional implications of the structure are discussed in the light of the unique structural features of VapD and its partial structural similarity to other β-barrel proteins. PMID:25084333

  19. Different Transmembrane Domains Associate with Distinct Endoplasmic Reticulum Components during Membrane Integration of a Polytopic Protein

    PubMed Central

    Meacock, Suzanna L.; Lecomte, Fabienne J.L.; Crawshaw, Samuel G.; High, Stephen

    2002-01-01

    We have been studying the insertion of the seven transmembrane domain (TM) protein opsin to gain insights into how the multiple TMs of polytopic proteins are integrated at the endoplasmic reticulum (ER). We find that the ER components associated with the first and second TMs of the nascent opsin polypeptide chain are clearly distinct. The first TM (TM1) is adjacent to the α and β subunits of the Sec61 complex, and a novel component, a protein associated with the ER translocon of 10 kDa (PAT-10). The most striking characteristic of PAT-10 is that it remains adjacent to TM1 throughout the biogenesis and membrane integration of the full-length opsin polypeptide. TM2 is also found to be adjacent to Sec61α and Sec61β during its membrane integration. However, TM2 does not form any adducts with PAT-10; rather, a transient association with the TRAM protein is observed. We show that the association of PAT-10 with opsin TM1 does not require the N-glycosylation of the nascent chain and occurs irrespective of the amino acid sequence and transmembrane topology of TM1. We conclude that the precise makeup of the ER membrane insertion site can be distinct for the different transmembrane domains of a polytopic protein. We find that the environment of a particular TM can be influenced by both the “stage” of nascent chain biosynthesis reached, and the TM's relative location within the polypeptide. PMID:12475939

  20. Isolation and solubilization of gram-positive bacterial cell wall-associated proteins.

    PubMed

    Cole, Jason N; Djordjevic, Steven P; Walker, Mark J

    2008-01-01

    This chapter describes a simple, rapid and reproducible method to prepare bacterial cell wall extracts for two-dimensional gel electrophoresis (2DE). The extraction process uses mutanolysin, an N-acetylmuramidase, to gently solubilize cell wall-associated proteins from Gram-positive prokaryotes. The cells are first washed with buffer and resuspended in a solution containing mutanolysin. Following incubation at 37 degrees C, the sample is centrifuged and the supernatant containing the soluble cell wall-associated proteins is harvested. Following a brief precipitation step, the pellet is solubilized in sample buffer ready for isoelectric focusing and 2DE analysis. PMID:18369905

  1. Detection of gene annotations and protein-protein interaction associated disorders through transitive relationships between integrated annotations

    PubMed Central

    2015-01-01

    Background Increasingly high amounts of heterogeneous and valuable controlled biomolecular annotations are available, but far from exhaustive and scattered in many databases. Several annotation integration and prediction approaches have been proposed, but these issues are still unsolved. We previously created a Genomic and Proteomic Knowledge Base (GPKB) that efficiently integrates many distributed biomolecular annotation and interaction data of several organisms, including 32,956,102 gene annotations, 273,522,470 protein annotations and 277,095 protein-protein interactions (PPIs). Results By comprehensively leveraging transitive relationships defined by the numerous association data integrated in GPKB, we developed a software procedure that effectively detects and supplement consistent biomolecular annotations not present in the integrated sources. According to some defined logic rules, it does so only when the semantic type of data and of their relationships, as well as the cardinality of the relationships, allow identifying molecular biology compliant annotations. Thanks to controlled consistency and quality enforced on data integrated in GPKB, and to the procedures used to avoid error propagation during their automatic processing, we could reliably identify many annotations, which we integrated in GPKB. They comprise 3,144 gene to pathway and 21,942 gene to biological function annotations of many organisms, and 1,027 candidate associations between 317 genetic disorders and 782 human PPIs. Overall estimated recall and precision of our approach were 90.56 % and 96.61 %, respectively. Co-functional evaluation of genes with known function showed high functional similarity between genes with new detected and known annotation to the same pathway; considering also the new detected gene functional annotations enhanced such functional similarity, which resembled the one existing between genes known to be annotated to the same pathway. Strong evidence was also found in

  2. Protein association of the neurotoxin and non-protein amino acid BMAA (β-N-methylamino-L-alanine) in the liver and brain following neonatal administration in rats.

    PubMed

    Karlsson, Oskar; Jiang, Liying; Andersson, Marie; Ilag, Leopold L; Brittebo, Eva B

    2014-04-01

    The environmental neurotoxin β-N-methylamino-L-alanine (BMAA) is not an amino acid that is normally found in proteins. Our previous autoradiographic study of (3)H-labeled BMAA in adult mice unexpectedly revealed a tissue distribution similar to that of protein amino acids. The aim of this study was to characterize the distribution of free and protein-bound BMAA in neonatal rat tissues following a short exposure using autoradiographic imaging and ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). The autoradiographic imaging of (14)C-L-BMAA demonstrated a distinct uptake of radioactivity that was retained following acid extraction in tissues with a high rate of cell turnover and/or protein synthesis. The UHPLC-MS/MS analysis conclusively demonstrated a dose-dependent increase of protein-associated BMAA in neonatal rat tissues. The level of protein-associated BMAA in the liver was more than 10 times higher than that in brain regions not fully protected by the blood-brain barrier which may be due to the higher rate of protein synthesis in the liver. In conclusion, this study demonstrated that BMAA was associated with rat proteins suggesting that BMAA may be misincorporated into proteins. However, protein-associated BMAA seemed to be cleared over time, as none of the samples from adult rats had any detectable free or protein-associated BMAA. PMID:24472610

  3. Identification of a novel contactin-associated transmembrane receptor with multiple domains implicated in protein-protein interactions.

    PubMed Central

    Peles, E; Nativ, M; Lustig, M; Grumet, M; Schilling, J; Martinez, R; Plowman, G D; Schlessinger, J

    1997-01-01

    Receptor protein tyrosine phosphatase beta (RPTPbeta) expressed on the surface of glial cells binds to the glycosylphosphatidylinositol (GPI)-anchored recognition molecule contactin on neuronal cells leading to neurite outgrowth. We describe the cloning of a novel contactin-associated transmembrane receptor (p190/Caspr) containing a mosaic of domains implicated in protein-protein interactions. The extracellular domain of Caspr contains a neurophilin/coagulation factor homology domain, a region related to fibrinogen beta/gamma, epidermal growth factor-like repeats, neurexin motifs as well as unique PGY repeats found in a molluscan adhesive protein. The cytoplasmic domain of Caspr contains a proline-rich sequence capable of binding to a subclass of SH3 domains of signaling molecules. Caspr and contactin exist as a complex in rat brain and are bound to each other by means of lateral (cis) interactions in the plasma membrane. We propose that Caspr may function as a signaling component of contactin, enabling recruitment and activation of intracellular signaling pathways in neurons. The binding of RPTPbeta to the contactin-Caspr complex could provide a mechanism for cell-cell communication between glial cells and neurons during development. PMID:9118959

  4. Uncovering RNA binding proteins associated with age and gender during liver maturation

    PubMed Central

    Chaturvedi, Praneet; Neelamraju, Yaseswini; Arif, Waqar; Kalsotra, Auinash; Janga, Sarath Chandra

    2015-01-01

    In the present study, we perform an association analysis focusing on the expression changes of 1344 RNA Binding proteins (RBPs) as a function of age and gender in human liver. We identify 88 and 45 RBPs to be significantly associated with age and gender respectively. Experimental verification of several of the predicted associations in mice confirmed our findings. Our results suggest that a small fraction of the gender-associated RBPs (~40%) are expressed higher in males than females. Altogether, these observations show that several of these RBPs are important and conserved regulators in maintaining liver function. Further analysis of the protein interaction network of RBPs associated with age and gender based on the centrality measures like degree, betweenness and closeness revealed that several of these RBPs might be prominent players in aging liver and impart gender specific alterations in gene expression via the formation of protein complexes. Indeed, both age and gender-associated RBPs in liver were found to show significantly higher clustering coefficients and network centrality measures compared to non-associated RBPs. The compendium of RBPs and this study will help us gain insight into the role of post-transcriptional regulatory molecules in aging and gender specific expression of genes. PMID:25824884

  5. Anomalous self-diffusion and sticky Rouse dynamics in associative protein hydrogels.

    PubMed

    Tang, Shengchang; Wang, Muzhou; Olsen, Bradley D

    2015-03-25

    Natural and synthetic materials based on associating polymers possess diverse mechanical behavior, transport properties and responsiveness to external stimuli. Although much is known about their dynamics on the molecular and macroscopic level, knowledge of self-diffusive dynamics of the network-forming constituents remains limited. Using forced Rayleigh scattering, anomalous self-diffusion is observed in model associating protein hydrogels originating from the interconversion between species that diffuse in both the molecular and associated state. The diffusion can be quantitatively modeled using a two-state model for polymers in the gel, where diffusivity in the associated state is critical to the super diffusive behavior. The dissociation time from bulk rheology measurements was 2-3 orders of magnitude smaller than the one measured by diffusion, because the former characterizes submolecular dissociation dynamics, whereas the latter depicts single protein molecules completely disengaging from the network. Rheological data also show a sticky Rouse-like relaxation at long times due to collective relaxation of large groups of proteins, suggesting mobility of associated molecules. This study experimentally demonstrates a hierarchy of relaxation processes in associating polymer networks, and it is anticipated that the results can be generalized to other associative systems to better understand the relationship of dynamics among sticky bonds, single molecules, and the entire network. PMID:25764061

  6. Cytoskeletal proteins in cortical development and disease: actin associated proteins in periventricular heterotopia

    PubMed Central

    Lian, Gewei; Sheen, Volney L.

    2015-01-01

    The actin cytoskeleton regulates many important cellular processes in the brain, including cell division and proliferation, migration, and cytokinesis and differentiation. These developmental processes can be regulated through actin dependent vesicle and organelle movement, cell signaling, and the establishment and maintenance of cell junctions and cell shape. Many of these processes are mediated by extensive and intimate interactions of actin with cellular membranes and proteins. Disruption in the actin cytoskeleton in the brain gives rise to periventricular heterotopia (PH), a malformation of cortical development, characterized by abnormal neurons clustered deep in the brain along the lateral ventricles. This disorder can give rise to seizures, dyslexia and psychiatric disturbances. Anatomically, PH is characterized by a smaller brain (impaired proliferation), heterotopia (impaired initial migration) and disruption along the neuroependymal lining (impaired cell-cell adhesion). Genes causal for PH have also been implicated in actin-dependent processes. The current review provides mechanistic insight into actin cytoskeletal regulation of cortical development in the context of this malformation of cortical development. PMID:25883548

  7. Conformational Transitions in Protein-Protein Association: Binding of Fasciculin-2 to Acetylcholinesterase

    PubMed Central

    Bui, Jennifer M.; Radic, Zoran; Taylor, Palmer; McCammon, J. Andrew

    2006-01-01

    The neurotoxin fasciculin-2 (FAS2) is a picomolar inhibitor of synaptic acetylcholinesterase (AChE). The dynamics of binding between FAS2 and AChE is influenced by conformational fluctuations both before and after protein encounter. Submicrosecond molecular dynamics trajectories of apo forms of fasciculin, corresponding to different conformational substates, are reported here with reference to the conformational changes of loop I of this three-fingered toxin. This highly flexible loop exhibits an ensemble of conformations within each substate corresponding to its functions. The high energy barrier found between the two major substates leads to transitions that are slow on the timescale of the diffusional encounter of noninteracting FAS2 and AChE. The more stable of the two apo substates may not be the one observed in the complex with AChE. It seems likely that the more stable apo form binds rapidly to AChE and conformational readjustments then occur in the resulting encounter complex. PMID:16473897

  8. Bactericidal Permeability Increasing Protein Gene Polymorphism is Associated with Inflammatory Bowel Diseases in the Turkish Population

    PubMed Central

    Can, Güray; Akın, Hakan; Özdemir, Filiz T.; Can, Hatice; Yılmaz, Bülent; Eren, Fatih; Atuğ, Özlen; Ünsal, Belkıs; Hamzaoğlu, Hülya O.

    2015-01-01

    Background/Aims: Inflammatory bowel disease, a chronic inflammatory disease with unknown etiology, affects the small and large bowel at different levels. It is increasingly considered that innate immune system may have a central position in the pathogenesis of the disease. As a part of the innate immune system, bactericidal permeability increasing protein has an important role in the recognition and neutralization of gram-negative bacteria. The aim of our study was to investigate the involvement of bactericidal permeability increasing protein gene polymorphism (bactericidal permeability increasing protein Lys216Glu) in inflammatory bowel disease in a large group of Turkish patients. Patients and Methods: The present study included 528 inflammatory bowel disease patients, 224 with Crohn's disease and 304 with ulcerative colitis, and 339 healthy controls. Results: Bactericidal permeability increasing protein Lys216Glu polymorphism was found to be associated with both Crohn's disease and ulcerative colitis (P = 0.0001). The frequency of the Glu/Glu genotype was significantly lower in patients using steroids and in those with steroid dependence (P = 0.012, OR, 0.80; 95% confidence interval [CI]: 0.68-0.94; P = 0.0286, OR, 0.75; 95% CI: 0.66-0.86, respectively). There was no other association between bactericidal permeability increasing protein gene polymorphism and phenotypes of inflammatory bowel disease. Conclusions: Bactericidal permeability increasing protein Lys216Glu polymorphism is associated with both Crohn's disease and ulcerative colitis. This is the first study reporting the association of bactericidal permeability increasing protein gene polymorphism with steroid use and dependence in Crohn's disease. PMID:26228368

  9. Analysis of proteins associated with growth of Bacteroides ovatus on the branched galactomannan guar gum.

    PubMed Central

    Valentine, P J; Salyers, A A

    1992-01-01

    Bacteroides ovatus, a gram-negative obligate anaerobe from the human colon, can ferment the branched galactomannan guar gum. Previously, three enzymes involved in guar gum breakdown were characterized. The expression of these enzymes appeared to be regulated; i.e., specific activities were higher in extracts from bacteria grown on guar gum than in extracts from bacteria grown on the monosaccharide constituents of guar gum, mannose and galactose. In the present study, we used two-dimensional gel analysis to determine the total number of B. ovatus proteins enhanced during growth on guar gum. Twelve soluble proteins and 20 membrane proteins were expressed at higher levels in guar gum-grown cells than in galactose-grown cells. An unexpected finding was that the expression of the two galactomannanases was induced by glucose as well as guar gum. Three other proteins, one membrane protein and two soluble proteins, had this same expression pattern. The remainder of the guar gum-associated proteins seen on two-dimensional gels and the guar gum-associated alpha-galactosidase were induced in cells grown on guar gum but not in cells grown on glucose. Two transposon-generated mutants (M-5 and M-7) that could not grow on guar gum were isolated. Both mutants still expressed the galactomannanases and the alpha-galactosidase. They also still expressed all of the guar gum-associated proteins that could be detected in two-dimensional gels of glucose-grown or galactose-grown cells. A second transposon insertion that suppressed the guar gum-negative phenotype of M-5 was isolated and characterized. The characteristics of this suppressor mutant indicated that the original transposon insertion was probably in a regulatory locus. Images PMID:1622222

  10. Protein O-Mannosyltransferases Associate with the Translocon to Modify Translocating Polypeptide Chains*

    PubMed Central

    Loibl, Martin; Wunderle, Lina; Hutzler, Johannes; Schulz, Benjamin L.; Aebi, Markus; Strahl, Sabine

    2014-01-01

    O-Mannosylation and N-glycosylation are essential protein modifications that are initiated in the endoplasmic reticulum (ER). Protein translocation across the ER membrane and N-glycosylation are highly coordinated processes that take place at the translocon-oligosaccharyltransferase (OST) complex. In analogy, it was assumed that protein O-mannosyltransferases (PMTs) also act at the translocon, however, in recent years it turned out that prolonged ER residence allows O-mannosylation of un-/misfolded proteins or slow folding intermediates by Pmt1-Pmt2 complexes. Here, we reinvestigate protein O-mannosylation in the context of protein translocation. We demonstrate the association of Pmt1-Pmt2 with the OST, the trimeric Sec61, and the tetrameric Sec63 complex in vivo by co-immunoprecipitation. The coordinated interplay between PMTs and OST in vivo is further shown by a comprehensive mass spectrometry-based analysis of N-glycosylation site occupancy in pmtΔ mutants. In addition, we established a microsomal translation/translocation/O-mannosylation system. Using the serine/threonine-rich cell wall protein Ccw5 as a model, we show that PMTs efficiently mannosylate proteins during their translocation into microsomes. This in vitro system will help to unravel mechanistic differences between co- and post-translocational O-mannosylation. PMID:24519942

  11. Mutational analysis of the adeno-associated virus type 2 Rep68 protein helicase motifs.

    PubMed

    Walker, S L; Wonderling, R S; Owens, R A

    1997-09-01

    The adeno-associated virus type 2 (AAV) Rep78 and Rep68 proteins are required for viral replication. These proteins are encoded by unspliced and spliced transcripts, respectively, from the p5 promoter of AAV and therefore have overlapping amino acid sequences. The Rep78 and Rep68 proteins share a variety of activities including endonuclease, helicase, and ATPase activities and the ability to bind AAV hairpin DNA. The part of the amino acid sequence which is identical in Rep78 and Rep68 contains consensus helicase motifs that are conserved among the parvovirus replication proteins. In the present study, we mutated highly conserved amino acids within these helicase motifs. The mutant proteins were synthesized as maltose binding protein-Rep68 fusions in Escherichia coli cells and affinity purified on amylose resin. The fusion proteins were assayed in vitro, and their activities were directly compared to those of the fusion protein MBP-Rep68 delta, which contains most of the amino acid sequences common to Rep78 and Rep68 and was demonstrated previously to have all of the in vitro activities of wild-type Rep78 and Rep68. Our analysis showed that almost all mutations in the putative helicase motifs severely reduced or abolished helicase activity in vitro. Most mutants also had ATPase activity less than one-eighth of the wild-type levels and lacked endonuclease activity. PMID:9261429

  12. Mining the surface proteome of tomato (Solanum lycopersicum) fruit for proteins associated with cuticle biogenesis

    PubMed Central

    Yeats, Trevor H.; Howe, Kevin J.; Matas, Antonio J.; Buda, Gregory J.; Thannhauser, Theodore W.; Rose, Jocelyn K. C.

    2010-01-01

    The aerial organs of plants are covered by the cuticle, a polyester matrix of cutin and organic solvent-soluble waxes that is contiguous with the polysaccharide cell wall of the epidermis. The cuticle is an important surface barrier between a plant and its environment, providing protection against desiccation, disease, and pests. However, many aspects of the mechanisms of cuticle biosynthesis, assembly, and restructuring are entirely unknown. To identify candidate proteins with a role in cuticle biogenesis, a surface protein extract was obtained from tomato (Solanum lycopersicum) fruits by dipping in an organic solvent and the constituent proteins were identified by several complementary fractionation strategies and two mass spectrometry techniques. Of the ∼200 proteins that were identified, a subset is potentially involved in the transport, deposition, or modification of the cuticle, such as those with predicted lipid-associated protein domains. These include several lipid-transfer proteins, GDSL-motif lipase/hydrolase family proteins, and an MD-2-related lipid recognition domain-containing protein. The epidermal-specific transcript accumulation of several of these candidates was confirmed by laser-capture microdissection and quantitative reverse transcription-PCR (qRT-PCR), together with their expression during various stages of fruit development. This indicated a complex pattern of cuticle deposition, and models for cuticle biogenesis and restructuring are discussed. PMID:20571035

  13. Identification of a nonhistone chromosomal protein associated with heterochromatin in Drosophila melanogaster and its gene.

    PubMed Central

    James, T C; Elgin, S C

    1986-01-01

    Monoclonal antibodies were prepared against a fraction of nuclear proteins of Drosophila melanogaster identified as tightly binding to DNA. Four of these antibodies were directed against a 19-kilodalton nuclear protein; immunofluorescence staining of the polytene chromosomes localized the antigen to the alpha, beta, and intercalary heterochromatic regions. Screening of a lambda gt11 cDNA expression library with one of the monoclonal antibodies identified a recombinant DNA phage clone that produced a fusion protein immunologically similar to the heterochromatin-associated protein. Polyclonal sera directed against the bacterial lacZ fusion protein recognized the same nuclear protein on Western blots. A full-length cDNA clone was isolated from a lambda gt10 library, and its DNA sequence was obtained. Analysis of the open reading frame revealed an 18,101-dalton protein encoded by this cDNA. Two overlapping genomic DNA clones were isolated from a Charon 4 library of D. melanogaster with the cDNA clone, and a restriction map was obtained. In situ hybridization with these probes indicated that the gene maps to a single chromosome location at 29A on the 2L chromosome. This general strategy should be effective for cloning the genes and identifying the genetic loci of chromosomal proteins which cannot be readily assayed by other means. Images PMID:3099166

  14. Parkinsonism-associated protein DJ-1/Park7 is a major protein deglycase that repairs methylglyoxal- and glyoxal-glycated cysteine, arginine, and lysine residues.

    PubMed

    Richarme, Gilbert; Mihoub, Mouadh; Dairou, Julien; Bui, Linh Chi; Leger, Thibaut; Lamouri, Aazdine

    2015-01-16

    Glycation is an inevitable nonenzymatic covalent reaction between proteins and endogenous reducing sugars or dicarbonyls (methylglyoxal, glyoxal) that results in protein inactivation. DJ-1 was reported to be a multifunctional oxidative stress response protein with poorly defined function. Here, we show that human DJ-1 is a protein deglycase that repairs methylglyoxal- and glyoxal-glycated amino acids and proteins by acting on early glycation intermediates and releases repaired proteins and lactate or glycolate, respectively. DJ-1 deglycates cysteines, arginines, and lysines (the three major glycated amino acids) of serum albumin, glyceraldehyde-3-phosphate dehydrogenase, aldolase, and aspartate aminotransferase and thus reactivates these proteins. DJ-1 prevented protein glycation in an Escherichia coli mutant deficient in the DJ-1 homolog YajL and restored cell viability in glucose-containing media. These results suggest that DJ-1-associated Parkinsonism results from excessive protein glycation and establishes DJ-1 as a major anti-glycation and anti-aging protein. PMID:25416785

  15. Parkinsonism-associated Protein DJ-1/Park7 Is a Major Protein Deglycase That Repairs Methylglyoxal- and Glyoxal-glycated Cysteine, Arginine, and Lysine Residues

    PubMed Central

    Richarme, Gilbert; Mihoub, Mouadh; Dairou, Julien; Bui, Linh Chi; Leger, Thibaut; Lamouri, Aazdine

    2015-01-01

    Glycation is an inevitable nonenzymatic covalent reaction between proteins and endogenous reducing sugars or dicarbonyls (methylglyoxal, glyoxal) that results in protein inactivation. DJ-1 was reported to be a multifunctional oxidative stress response protein with poorly defined function. Here, we show that human DJ-1 is a protein deglycase that repairs methylglyoxal- and glyoxal-glycated amino acids and proteins by acting on early glycation intermediates and releases repaired proteins and lactate or glycolate, respectively. DJ-1 deglycates cysteines, arginines, and lysines (the three major glycated amino acids) of serum albumin, glyceraldehyde-3-phosphate dehydrogenase, aldolase, and aspartate aminotransferase and thus reactivates these proteins. DJ-1 prevented protein glycation in an Escherichia coli mutant deficient in the DJ-1 homolog YajL and restored cell viability in glucose-containing media. These results suggest that DJ-1-associated Parkinsonism results from excessive protein glycation and establishes DJ-1 as a major anti-glycation and anti-aging protein. PMID:25416785

  16. Regulation of starch accumulation by granule-associated plant 14-3-3 proteins.

    PubMed

    Sehnke, P C; Chung, H J; Wu, K; Ferl, R J

    2001-01-16

    In higher plants the production of starch is orchestrated by chloroplast-localized biosynthetic enzymes, namely starch synthases, ADP-glucose pyrophosphorylase, and starch branching and debranching enzymes. Diurnal regulation of these enzymes, as well as starch-degrading enzymes, influences both the levels and composition of starch, and is dependent in some instances upon phosphorylation-linked regulation. The phosphoserine/threonine-binding 14-3-3 proteins participate in environmentally responsive phosphorylation-related regulatory functions in plants, and as such are potentially involved in starch regulation. We report here that reduction of the epsilon subgroup of Arabidopsis 14-3-3 proteins by antisense technology resulted in a 2- to 4-fold increase in leaf starch accumulation. Dark-governed starch breakdown was unaffected in these "antisense plants," indicating an unaltered starch-degradation pathway and suggesting a role for 14-3-3 proteins in regulation of starch synthesis. Absorption spectra and gelatinization properties indicate that the starch from the antisense plants has an altered branched glucan composition. Biochemical characterization of protease-treated starch granules from both Arabidopsis leaves and maize endosperm showed that 14-3-3 proteins are internal intrinsic granule proteins. These data suggest a direct role for 14-3-3 proteins in starch accumulation. The starch synthase III family is a possible target for 14-3-3 protein regulation because, uniquely among plastid-localized starch metabolic enzymes, all members of the family contain the conserved 14-3-3 protein phosphoserine/threonine-binding consensus motif. This possibility is strengthened by immunocapture using antibodies to DU1, a maize starch synthase III family member, and direct interaction with biotinylated 14-3-3 protein, both of which demonstrated an association between 14-3-3 proteins and DU1 or DU1-like proteins. PMID:11149942

  17. Promyelocytic leukemia protein interacts with the apoptosis-associated speck-like protein to limit inflammasome activation.

    PubMed

    Dowling, Jennifer K; Becker, Christine E; Bourke, Nollaig M; Corr, Sinead C; Connolly, Dympna J; Quinn, Susan R; Pandolfi, Paolo P; Mansell, Ashley; O'Neill, Luke A J

    2014-03-01

    The apoptosis-associated speck-like protein containing a caspase-activating recruitment domain (ASC) is an essential component of several inflammasomes, multiprotein complexes that regulate caspase-1 activation and inflammation. We report here an interaction between promyelocytic leukemia protein (PML) and ASC. We observed enhanced formation of ASC dimers in PML-deficient macrophages. These macrophages also display enhanced levels of ASC in the cytosol. Furthermore, IL-1β production was markedly enhanced in these macrophages in response to both NLRP3 and AIM2 inflammasome activation and following bone marrow-derived macrophage infection with herpes simplex virus-1 (HSV-1) and Salmonella typhimurium. Collectively, our data indicate that PML limits ASC function, retaining ASC in the nucleus. PMID:24407287

  18. Structure of the virulence-associated protein VapD from the intracellular pathogen Rhodococcus equi

    SciTech Connect

    Whittingham, Jean L.; Blagova, Elena V.; Finn, Ciaran E.; Luo, Haixia; Miranda-CasoLuengo, Raúl; Turkenburg, Johan P.; Leech, Andrew P.; Walton, Paul H.; Murzin, Alexey G.; Meijer, Wim G.; Wilkinson, Anthony J.

    2014-08-01

    VapD is one of a set of highly homologous virulence-associated proteins from the multi-host pathogen Rhodococcus equi. The crystal structure reveals an eight-stranded β-barrel with a novel fold and a glycine rich ‘bald’ surface. Rhodococcus equi is a multi-host pathogen that infects a range of animals as well as immune-compromised humans. Equine and porcine isolates harbour a virulence plasmid encoding a homologous family of virulence-associated proteins associated with the capacity of R. equi to divert the normal processes of endosomal maturation, enabling bacterial survival and proliferation in alveolar macrophages. To provide a basis for probing the function of the Vap proteins in virulence, the crystal structure of VapD was determined. VapD is a monomer as determined by multi-angle laser light scattering. The structure reveals an elliptical, compact eight-stranded β-barrel with a novel strand topology and pseudo-twofold symmetry, suggesting evolution from an ancestral dimer. Surface-associated octyl-β-d-glucoside molecules may provide clues to function. Circular-dichroism spectroscopic analysis suggests that the β-barrel structure is preceded by a natively disordered region at the N-terminus. Sequence comparisons indicate that the core folds of the other plasmid-encoded virulence-associated proteins from R. equi strains are similar to that of VapD. It is further shown that sequences encoding putative R. equi Vap-like proteins occur in diverse bacterial species. Finally, the functional implications of the structure are discussed in the light of the unique structural features of VapD and its partial structural similarity to other β-barrel proteins.

  19. The plant cytoskeleton: recent advances in the study of the plant microtubule-associated proteins MAP-65, MAP-190 and the Xenopus MAP215-like protein, MOR1.

    PubMed

    Hussey, Patrick J; Hawkins, Timothy J; Igarashi, Hisako; Kaloriti, Despina; Smertenko, Andrei

    2002-12-01

    The microtubule cytoskeleton is a dynamic filamentous structure involved in many key processes in plant cell morphogenesis including nuclear and cell division, deposition of cell wall, cell expansion, organelle movement and secretion. The principal microtubule protein is tubulin, which associates to form the wall of the tubule. In addition, various associated proteins bind microtubules either to anchor, cross-link or regulate the microtubule network within cells. Biochemical, molecular biological and genetic approaches are being successfully used to identify these microtubule-associated proteins (MAPs) in plants, and we describe recent progress on three of these proteins. PMID:12516862

  20. Purification of the M. magneticum strain AMB-1 magnetosome associated protein MamADelta41.

    PubMed

    Zeytuni, Natalie; Zarivach, Raz

    2010-01-01

    Magnetotactic bacteria comprise a diverse group of aquatic microorganisms that are able to orientate themselves along geomagnetic fields. This behavior is believed to aid their search for suitable environments (1). This capability is conferred by the magnetosome, a subcellular organelle that consists of a linear-chain assembly of lipid vesicles each able to biomineralize and enclose a ~50-nm crystal of magnetite or greigite. A principle component of the magnetosome that was shown to be required for the formation of functional vesicles is MamA. MamA is a highly abundant magnetosome-associated protein which is one of the most characterized magnetosome-associated proteins in vivo (2-6). This article focuses on the purification of MamA, which despite being studied in vivo, no clear functional or structural details have been identified for it. Bioinformatics analysis suggested that MamA is a tetra-tricopeptide repeat (TPR) containing protein. TPR is a structural motif found as such or forming part of a bigger fold in a wide range of proteins, it serves as a template for protein-protein interactions and mediates multi-protein complexes (7). TPRs are involved in many crucial tasks in eukaryotic cell organelle processes and many bacterial pathways (8-14). In order to understand MamA, a unique TPR containing protein, highly purified protein is required as a first step. In this article, we present the purification protocol for a stable MamA deletion mutant (MamADelta41) from M. magneticum AMB-1. PMID:20339346

  1. The ciliopathy-associated CPLANE proteins direct basal body recruitment of intraflagellar transport machinery.

    PubMed

    Toriyama, Michinori; Lee, Chanjae; Taylor, S Paige; Duran, Ivan; Cohn, Daniel H; Bruel, Ange-Line; Tabler, Jacqueline M; Drew, Kevin; Kelly, Marcus R; Kim, Sukyoung; Park, Tae Joo; Braun, Daniela A; Pierquin, Ghislaine; Biver, Armand; Wagner, Kerstin; Malfroot, Anne; Panigrahi, Inusha; Franco, Brunella; Al-Lami, Hadeel Adel; Yeung, Yvonne; Choi, Yeon Ja; Duffourd, Yannis; Faivre, Laurence; Rivière, Jean-Baptiste; Chen, Jiang; Liu, Karen J; Marcotte, Edward M; Hildebrandt, Friedhelm; Thauvin-Robinet, Christel; Krakow, Deborah; Jackson, Peter K; Wallingford, John B

    2016-06-01

    Cilia use microtubule-based intraflagellar transport (IFT) to organize intercellular signaling. Ciliopathies are a spectrum of human diseases resulting from defects in cilia structure or function. The mechanisms regulating the assembly of ciliary multiprotein complexes and the transport of these complexes to the base of cilia remain largely unknown. Combining proteomics, in vivo imaging and genetic analysis of proteins linked to planar cell polarity (Inturned, Fuzzy and Wdpcp), we identified and characterized a new genetic module, which we term CPLANE (ciliogenesis and planar polarity effector), and an extensive associated protein network. CPLANE proteins physically and functionally interact with the poorly understood ciliopathy-associated protein Jbts17 at basal bodies, where they act to recruit a specific subset of IFT-A proteins. In the absence of CPLANE, defective IFT-A particles enter the axoneme and IFT-B trafficking is severely perturbed. Accordingly, mutation of CPLANE genes elicits specific ciliopathy phenotypes in mouse models and is associated with ciliopathies in human patients. PMID:27158779

  2. Kebab: Kinetochore and EB1 Associated Basic Protein That Dynamically Changes Its Localisation during Drosophila Mitosis

    PubMed Central

    Meireles, Ana M.; Dzhindzhev, Nikola S.; Ohkura, Hiroyuki

    2011-01-01

    Microtubule plus ends are dynamic ends that interact with other cellular structures. Microtubule plus end tracking proteins are considered to play important roles in the regulation of microtubule plus ends. Recent studies revealed that EB1 is the central regulator for microtubule plus end tracking proteins by recruiting them to microtubule plus ends through direct interaction. Here we report the identification of a novel Drosophila protein, which we call Kebab (kinetochore and EB1 associated basic protein), through in vitro expression screening for EB1-interacting proteins. Kebab fused to GFP shows a novel pattern of dynamic localisation in mitosis. It localises to kinetochores weakly in metaphase and accumulates progressively during anaphase. In telophase, it associates with microtubules in central-spindle and centrosomal regions. The localisation to kinetochores depends on microtubules. The protein has a domain most similar to the atypical CH domain of Ndc80, and a coiled-coil domain. The interaction with EB1 is mediated by two SxIP motifs but is not required for the localisation. Depletion of Kebab in cultured cells by RNA interference did not show obvious defects in mitotic progression or microtubule organisation. Generation of mutants lacking the kebab gene indicated that Kebab is dispensable for viability and fertility. PMID:21912673

  3. RBM45 homo-oligomerization mediates association with ALS-linked proteins and stress granules

    PubMed Central

    Li, Yang; Collins, Mahlon; Geiser, Rachel; Bakkar, Nadine; Riascos, David; Bowser, Robert

    2015-01-01

    The aggregation of RNA-binding proteins is a pathological hallmark of amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). RBM45 is an RNA-binding protein that forms cytoplasmic inclusions in neurons and glia in ALS and FTLD. To explore the role of RBM45 in ALS and FTLD, we examined the contribution of the protein’s domains to its function, subcellular localization, and interaction with itself and ALS-linked proteins. We find that RBM45 forms homo-oligomers and physically associates with the ALS-linked proteins TDP-43 and FUS in the nucleus. Nuclear localization of RBM45 is mediated by a bipartite nuclear-localization sequence (NLS) located at the C-terminus. RBM45 mutants that lack a functional NLS accumulate in the cytoplasm and form TDP-43 positive stress granules. Moreover, we identify a novel structural element, termed the homo-oligomer assembly (HOA) domain, that is highly conserved across species and promote homo-oligomerization of RBM45. RBM45 mutants that fail to form homo-oligomers exhibit significantly reduced association with ALS-linked proteins and inclusion into stress granules. These results show that RMB45 may function as a homo-oligomer and that its oligomerization contributes to ALS/FTLD RNA-binding protein aggregation. PMID:26391765

  4. Rho-associated protein kinase modulates neurite extension by regulating microtubule remodeling and vinculin distribution

    PubMed Central

    Chen, Ke’en; Zhang, Wenbin; Chen, Jing; Li, Sumei; Guo, Guoqing

    2013-01-01

    Rho-associated protein kinase is an essential regulator of cytoskeletal dynamics during the process of neurite extension. However, whether Rho kinase regulates microtubule remodeling or the distribution of adhesive proteins to mediate neurite outgrowth remains unclear. By specifically modulating Rho kinase activity with pharmacological agents, we studied the morpho-dynamics of neurite outgrowth. We found that lysophosphatidic acid, an activator of Rho kinase, inhibited neurite outgrowth, which could be reversed by Y-27632, an inhibitor of Rho kinase. Meanwhile, reorganization of microtubules was noticed during these processes, as indicated by their significant changes in the soma and growth cone. In addition, exposure to lysophosphatidic acid led to a decreased membrane distribution of vinculin, a focal adhesion protein in neurons, whereas Y-27632 recruited vinculin to the membrane. Taken together, our data suggest that Rho kinase regulates rat hippocampal neurite growth and microtubule formation via a mechanism associated with the redistribution of vinculin. PMID:25206623

  5. Phase transition of spindle-associated protein regulate spindle apparatus assembly.

    PubMed

    Jiang, Hao; Wang, Shusheng; Huang, Yuejia; He, Xiaonan; Cui, Honggang; Zhu, Xueliang; Zheng, Yixian

    2015-09-24

    Spindle assembly required during mitosis depends on microtubule polymerization. We demonstrate that the evolutionarily conserved low-complexity protein, BuGZ, undergoes phase transition or coacervation to promote assembly of both spindles and their associated components. BuGZ forms temperature-dependent liquid droplets alone or on microtubules in physiological buffers. Coacervation in vitro or in spindle and spindle matrix depends on hydrophobic residues in BuGZ. BuGZ coacervation and its binding to microtubules and tubulin are required to promote assembly of spindle and spindle matrix in Xenopus egg extract and in mammalian cells. Since several previously identified spindle-associated components also contain low-complexity regions, we propose that coacervating proteins may be a hallmark of proteins that comprise a spindle matrix that functions to promote assembly of spindles by concentrating its building blocks. PMID:26388440

  6. Association of intestinal peptide transport with a protein related to the cadherin superfamily.

    PubMed

    Dantzig, A H; Hoskins, J A; Tabas, L B; Bright, S; Shepard, R L; Jenkins, I L; Duckworth, D C; Sportsman, J R; Mackensen, D; Rosteck, P R

    1994-04-15

    The first step in oral absorption of many medically important peptide-based drugs is mediated by an intestinal proton-dependent peptide transporter. This transporter facilitates the oral absorption of beta-lactam antibiotics and angiotensin-converting enzyme inhibitors from the intestine into enterocytes lining the luminal wall. A monoclonal antibody that blocked uptake of cephalexin was used to identify and clone a gene that encodes an approximately 92-kilodalton membrane protein that was associated with the acquisition of peptide transport activity by transport-deficient cells. The amino acid sequence deduced from the complementary DNA sequence of the cloned gene indicated that this transport-associated protein shares several conserved structural elements with the cadherin superfamily of calcium-dependent, cell-cell adhesion proteins. PMID:8153632

  7. The evolutionarily conserved Krueppel-associated box domain defines a subfamily of eukaryotic multifingered proteins

    SciTech Connect

    Bellefroid, E.J.; Poncelet, D.A.; Lecocq, P.J.; Revelant, O.; Martial, J.A. )

    1991-05-01

    The authors have previously shown that the human genome includes hundreds of genes coding for putative factors related to the Krueppel zinc-finger protein, which regulates Drosophila segmentation. They report herein that about one-third of these genes code for proteins that share a very conserved region of about 75 amino acids in their N-terminal nonfinger portion. Homologous regions are found in a number of previously described finger proteins, including mouse Zfp-1 and Xenopus Xfin. They named this region the Krueppel-associated box (KRAB). This domain has the potential to form two amphipathic {alpha}-helices. Southern blot analysis of zoo blots suggests that the Krueppel-associated box is highly conserved during evolution. Northern blot analysis shows that these genes are expressed in most adult tissues and are down-regulated during in vitro terminal differentiation of human myeloid cells.

  8. Detection of the disease associated form of the prion protein in biological samples

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Transmissible spongiform encephalopathies (TSEs) or prion diseases are neurodegenerative diseases that occur in a variety of mammals. In these diseases, a chromosomally encoded protein (PrP**c) undergoes a conformational change to the disease associated form (PrP**d), and PrP**d is capable inducing ...

  9. Calving traits of crossbred Brahman Cows are Associated with Heat Shock Protein 70 Genetic Polymorphisms

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Objectives were to: 1) identify single nucleotide polymorphisms (SNP) located in the promoter region of the bovine heat shock protein 70 gene, and 2) evaluate associations between Hsp70 SNP and calving rates of Brahman-influenced cows. Specific primers were designed for PCR amplification of a 539 b...

  10. Challenges associated with heterologous expression of Flavobacterium psychrophilum proteins in Escherichia coli

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A two-parameter statistical model was used to predict the solubility of 96 putative virulence associated genes of Flavobacterium psychrophilum (CSF259-93) upon over expression in E. coli. This analysis indicated that 88.5% of the F. psychrophilum proteins would be expressed as insoluble aggregates c...

  11. Identification of a new class of lipid droplet-associated proteins in plants

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Lipid droplets in plants (also known as oil bodies, lipid bodies or oleosomes) are well characterized in seeds, and oleosins, the major proteins associated with their surface, were shown to be important for stabilizing lipid droplets during seed desiccation and rehydration. However, lipid droplets ...

  12. Lamina-associated Polypeptide 1: Protein Interactions and Tissue-selective Functions

    PubMed Central

    Shin, Ji-Yeon; Dauer, William T.; Worman, Howard J.

    2014-01-01

    Mutations in genes encoding widely expressed nuclear envelope proteins often lead to diseases that manifest in specific tissues. Lamina-associated polypeptide 1 (LAP1) is an integral protein of the inner nuclear membrane that is expressed in most cells and tissues. Within the nuclear envelope, LAP1 interacts physically with lamins, torsinA and emerin, suggesting it may serve as a key node for transducing signals across the inner nuclear membrane. Indeed, recent in vivo studies in genetically modified mice strongly support functional links between LAP1 and both torsinA (in neurons) and emerin (in muscle). These studies suggest that tissue-selective diseases caused by mutations in genes encoding nuclear envelope proteins may result, at least in part, from the selective disruption of discrete nuclear envelope protein complexes. PMID:24508913

  13. Senescence-Associated Vacuoles, a Specific Lytic Compartment for Degradation of Chloroplast Proteins?

    PubMed Central

    Carrión, Cristian A.; Martínez, Dana E.; Costa, M. Lorenza; Guiamet, Juan José

    2014-01-01

    Degradation of chloroplasts and chloroplast components is a distinctive feature of leaf senescence. In spite of its importance in the nutrient economy of plants, knowledge about the mechanism(s) involved in the breakdown of chloroplast proteins is incomplete. A novel class of vacuoles, “senescence-associated vacuoles” (SAVs), characterized by intense proteolytic activity appear during senescence in chloroplast-containing cells of leaves. Since SAVs contain some chloroplast proteins, they are candidate organelles to participate in chloroplast breakdown. In this review we discuss the characteristics of SAVs, and their possible involvement in the degradation of Rubisco, the most abundant chloroplast protein. Finally, SAVs are compared with other extra-plastidial protein degradation pathways operating in senescing leaves. PMID:27135516

  14. Nutrient-dependent methylation of a membrane-associated protein of Escherichia coli

    SciTech Connect

    Young, C.C.; Alvarez, J.D.; Bernlohr, R.W. )

    1990-09-01

    Starvation of a mid-log-phase culture of Escherichia coli B/r for nitrogen, phosphate, or carbon resulted in methylation of a membrane-associated protein of about 43,000 daltons (P-43) in the presence of chloramphenicol and (methyl-3H)methionine. The in vivo methylation reaction occurred with a doubling time of 2 to 5 min and was followed by a slower demethylation process. Addition of the missing nutrient to a starving culture immediately prevented further methylation of P-43. P-43 methylation is not related to the methylated chemotaxis proteins because P-43 is methylated in response to a different spectrum of nutrients and because P-43 is methylated on lysine residues. The characteristics of P-43 are similar to those of a methylated protein previously described in Bacillus subtilis and B. licheniformis and are consistent with the proposal that methylation of this protein functions in nutrient sensing.

  15. Molecular docking of fisetin with AD associated AChE, ABAD and BACE1 proteins

    PubMed Central

    Dash, Raju; Emran, Talha Bin; Uddin, Mir Muhammad Nasir; Islam, Ashekul; Junaid, Md

    2014-01-01

    Alzheimer׳s disease (AD) is one of the most common dementias showing slow progressive cognitive decline. Progression of intracerebral accumulation of beta amyloid (Aβ) peptides by the action of amyloid binding alcohol dehydrogenase (ABAD), a mitochondrial enzyme and β-site amyloid precursor protein cleaving enzyme 1 (BACE1) and the degradation of Acetylcholinesterase (AChE) the main pathological characteristics of AD. Therefore, it is of interest to evaluate the importance of fisetin (a flavonol that belongs to the flavonoid group of polyphenols) binding with AChE, ABAD and BACE1 proteins. Docking experiment of fisetin with these proteins using two different tools namely iGEMDOCK and FlexX show significant binding with acceptable binding values. Thus, the potential inhibitory role of fisetin with AD associated proteins is documented. PMID:25352723

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

    PubMed

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

    2015-01-01

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

  17. Transmembrane receptor DCC associates with protein synthesis machinery and regulates translation

    PubMed Central

    Tcherkezian, Joseph; Brittis, Perry A.; Thomas, Franziska; Roux, Philippe P.; Flanagan, John G.

    2010-01-01

    Summary Extracellular signals regulate protein translation in many cell functions. A key advantage of control at the translational level is the opportunity to regulate protein synthesis within specific cellular subregions. However, little is known about mechanisms that may link extracellular cues to translation with spatial precision. Here we show that a transmembrane receptor, DCC, forms a binding complex containing multiple translation components, including eukaryotic initiation factors, ribosomal large and small subunits, and monosomes. In neuronal axons and dendrites DCC colocalizes in particles with translation machinery, and newly synthesized protein. The extracellular ligand netrin promoted DCC-mediated translation and disassociation of translation components. The functional and physical association of a cell surface receptor with the translation machinery leads to a generalizable model for localization and extracellular regulation of protein synthesis, based on a transmembrane translation regulation complex. PMID:20434207

  18. Nucleolar localization of DGCR8 and identification of eleven DGCR8-associated proteins.

    PubMed

    Shiohama, Aiko; Sasaki, Takashi; Noda, Setsuko; Minoshima, Shinsei; Shimizu, Nobuyoshi

    2007-12-10

    We identified 11 proteins that are associated with DGCR8 by immunoprecipitation assay and mass spectrometry. These proteins included Nucleolin, ILF3 and others, most of which appeared to be involved in the RNA processing or RNA transportation. We detected at least four kinds of protein complex, such as DROSHA/DGCR8, DGCR8/Nucleolin, DGCR8/ILF3 and ILF3/XPO5, by co-immunoprecipitation. The complex formation of DGCR8 with Nucleolin was dependent on RNA. Subcellular localization analysis by the immunofluorescent microscopy and immunoelectron microscopy indicated that DGCR8 locates at the nucleolus and small foci adjacent to splicing speckles in the nucleoplasm. Furthermore, the localization of DGCR8 at the nucleolus was changed by the inhibition of RNA transcription. Thus, our studies provided additional new evidence for the involvement of various protein complexes in the molecular mechanisms of apparently complex innate RNA interference machinery. PMID:17765891

  19. Tissue-specific alternative RNA splicing of rat vesicle-associated membrane protein-1 (VAMP-1).

    PubMed

    Mandic, R; Trimble, W S; Lowe, A W

    1997-10-15

    The vesicle-associated membrane protein (VAMP) family is essential to vesicle-mediated protein transport. Three mammalian isoforms, VAMP-1, VAMP-2, and cellubrevin, play a role in protein transport to the plasma membrane. In this study, we describe a new rat VAMP-1 isoform produced by alternative pre-mRNA splicing. Only one VAMP-1 isoform dominates in each tissue. Analysis of the nucleotide sequence for the newly discovered isoform, VAMP-1b, reveals that its expression is determined by whether an intron is retained or removed. The predicted amino acid sequences for the VAMP-1 isoforms differ at the carboxy-terminal end of the protein. A similar process has been described for VAMPs in Drosophila melanogaster and suggests a conserved function for the carboxy-terminal domain that can be modulated. PMID:9358054

  20. Modulation of Kaposi's Sarcoma-Associated Herpesvirus Interleukin-6 Function by Hypoxia-Upregulated Protein 1

    PubMed Central

    Giffin, Louise; Yan, Feng; Major, M. Ben

    2014-01-01

    ABSTRACT Kaposi's sarcoma-associated herpesvirus (KSHV, also called human herpesvirus 8) is linked to the development of Kaposi's sarcoma (KS), primary effusion lymphoma (PEL), and multicentric Castleman's disease (MCD). KSHV expresses several proteins that modulate host cell signaling pathways. One of these proteins is viral interleukin-6 (vIL-6), which is a homolog of human IL-6 (hIL-6). vIL-6 is able to prevent apoptosis and promote proinflammatory signaling, angiogenesis, and cell proliferation. Although it can be secreted, vIL-6 is mainly an intracellular protein that is retained in the endoplasmic reticulum (ER). We performed affinity purification and mass spectrometry to identify novel vIL-6 binding partners and found that a cellular ER chaperone, hypoxia-upregulated protein 1 (HYOU1), interacts with vIL-6. Immunohistochemical staining reveals that both PEL and KS tumor tissues express significant amounts of HYOU1. We also show that HYOU1 increases endogenous vIL-6 protein levels and that HYOU1 facilitates vIL-6-induced JAK/STAT signaling, migration, and survival in endothelial cells. Furthermore, our data suggest that HYOU1 also modulates vIL-6's ability to induce CCL2, a chemokine involved in cell migration. Finally, we investigated the impact of HYOU1 on cellular hIL-6 signaling. Collectively, our data indicate that HYOU1 is important for vIL-6 function and may play a role in the pathogenesis of KSHV-associated cancers. IMPORTANCE KSHV vIL-6 is detectable in all KSHV-associated malignancies and promotes tumorigenesis and inflammation. We identified a cellular protein, called hypoxia-upregulated protein 1 (HYOU1), that interacts with KSHV vIL-6 and is present in KSHV-infected tumors. Our data suggest that HYOU1 facilitates the vIL-6-induced signaling, migration, and survival of endothelial cells. PMID:24920810

  1. Identification of a common protein association region in the neuronal Cdk5 activator.

    PubMed

    Wang, X; Ching, Y P; Lam, W H; Qi, Z; Zhang, M; Wang, J H

    2000-10-13

    Cyclin-dependent protein kinase 5 (Cdk5) depends on the association with neuronal Cdk5 activator (Nck5a) for kinase activity. A variety of cellular proteins have been shown to undergo high affinity association with Nck5a, including three novel proteins, C42, C48, and C53 found by a yeast two-hybrid screen (Ching, Y. P., Qi, Z., and Wang, J. H. (2000) Gene 242, 285-294). The three proteins show competitive binding to Nck5a suggesting that they bind at a common site. The binding site has been mapped to a region of 26 amino acid residues (residues 145 to 170) at the N-terminal boundary of the kinase activation domain of Nck5a. This region of Nck5a contains an amphipathic alpha-helix whose hydrophobic face is involved in Cdk5 activation (Chin, K. T., Ohki, S, Tang, D., Cheng, H. C., Wang, J. H. , and Zhang, M. (1999) J. Biol. Chem. 274, 7120-7127). Several lines of evidence suggest that Nck5a interacts with the binding proteins at the hydrophilic face of the amphipathic alpha-helix. First, the Nck5a-(145-170) peptide can bind Cdk5 and Nck5a-binding proteins simultaneously. Second, the association of Nck5a-(145-170) to C48 can be markedly reduced by high ionic strength whereas the interaction between Nck5a and Cdk5 is not affected. Third, substitution of Glu(157) by glutamine in Nck5a-(145-170) abolishes the peptide's ability to bind to the three Nck5a-binding proteins without diminishing its Cdk5 binding activity. PMID:10915792

  2. High intakes of protein and processed meat associate with increased incidence of type 2 diabetes.

    PubMed

    Ericson, Ulrika; Sonestedt, Emily; Gullberg, Bo; Hellstrand, Sophie; Hindy, George; Wirfält, Elisabet; Orho-Melander, Marju

    2013-03-28

    Diets high in protein have shown positive effects on short-term weight reduction and glycaemic control. However, the understanding of how dietary macronutrient composition relates to long-term risk of type 2 diabetes is limited. The aim of the present study was to examine intakes of macronutrients, fibre and protein sources in relation to incident type 2 diabetes. In total, 27 140 individuals, aged 45-74 years, from the population-based Malmö Diet and Cancer cohort, were included. Dietary data were collected with a modified diet history method, including registration of cooked meals. During 12 years of follow-up, 1709 incident type 2 diabetes cases were identified. High protein intake was associated with increased risk of type 2 diabetes (hazard ratio (HR) 1.27 for highest compared with lowest quintile; 95 % CI 1.08, 1.49; P for trend = 0.01). When protein consumption increased by 5 % of energy at the expense of carbohydrates (HR 1.20; 95 % CI 1.09, 1.33) or fat (HR 1.21; 95 % CI 1.09, 1.33), increased diabetes risk was observed. Intakes in the highest quintiles of processed meat (HR 1.16; 95 % CI 1.00, 1.36; P for trend = 0.01) and eggs (HR 1.21; 95 % CI 1.04, 1.41; P for trend = 0.02) were associated with increased risk. Intake of fibre-rich bread and cereals was inversely associated with type 2 diabetes (HR 0.84; 95 % CI 0.73, 0.98; P for trend = 0.004). In conclusion, results from the present large population-based prospective study indicate that high protein intake is associated with increased risk of type 2 diabetes. Replacing protein with carbohydrates may be favourable, especially if fibre-rich breads and cereals are chosen as carbohydrate sources. PMID:22850191

  3. Rapamycin-binding FKBP25 associates with diverse proteins that form large intracellular entities

    SciTech Connect

    Galat, Andrzej Thai, Robert

    2014-08-08

    Highlights: • The hFKBP25 interacts with diverse components of macromolecular entities. • We show that the endogenous human FKBP25 is bound to polyribosomes. • The endogenous hFKBP25 co-immunoprecipitated with nucleosomal proteins. • FKBP25 could induce conformational switch in macromolecular complexes. - Abstract: In this paper, we show some evidence that a member of the FK506-binding proteins, FKBP25 is associated to diverse components that are part of several different intracellular large-molecular mass entities. The FKBP25 is a high-affinity rapamycin-binding immunophilin, which has nuclear translocation signals present in its PPIase domain but it was detected both in the cytoplasm compartment and in the nuclear proteome. Analyses of antiFKBP25-immunoprecipitated proteins have revealed that the endogenous FKBP25 is associated to the core histones of the nucleosome, and with several proteins forming spliceosomal complexes and ribosomal subunits. Using polyclonal antiFKBP25 we have detected FKBP25 associated with polyribosomes. Added RNAs or 0.5 M NaCl release FKBP25 that was associated with the polyribosomes indicating that the immunophilin has an intrinsic capacity to form complexes with polyribonucleotides via its charged surface patches. Rapamycin or FK506 treatments of the polyribosomes isolated from porcine brain, HeLa and K568 cells caused a residual release of the endogenous FKBP25, which suggests that the immunophilin also binds to some proteins via its PPIase cavity. Our proteomics study indicates that the nuclear pool of the FKBP25 targets various nuclear proteins that are crucial for packaging of DNA, chromatin remodeling and pre-mRNA splicing whereas the cytosolic pool of this immunophilin is bound to some components of the ribosome.

  4. Mapping the LINE1 ORF1 protein interactome reveals associated inhibitors of human retrotransposition

    PubMed Central

    Goodier, John L.; Cheung, Ling E.; Kazazian, Haig H.

    2013-01-01

    LINE1s occupy 17% of the human genome and are its only active autonomous mobile DNA. L1s are also responsible for genomic insertion of processed pseudogenes and >1 million non-autonomous retrotransposons (Alus and SVAs). These elements have significant effects on gene organization and expression. Despite the importance of retrotransposons for genome evolution, much about their biology remains unknown, including cellular factors involved in the complex processes of retrotransposition and forming and transporting L1 ribonucleoprotein particles. By co-immunoprecipitation of tagged L1 constructs and mass spectrometry, we identified proteins associated with the L1 ORF1 protein and its ribonucleoprotein. These include RNA transport proteins, gene expression regulators, post-translational modifiers, helicases and splicing factors. Many cellular proteins co-localize with L1 ORF1 protein in cytoplasmic granules. We also assayed the effects of these proteins on cell culture retrotransposition and found strong inhibiting proteins, including some that control HIV and other retroviruses. These data suggest candidate cofactors that interact with the L1 to modulate its activity and increase our understanding of the means by which the cell coexists with these genomic ‘parasites’. PMID:23749060

  5. THE DELICATE BALANCE BETWEEN SECRETED PROTEIN FOLDING AND ENDOPLASMIC RETICULUM-ASSOCIATED DEGRADATION IN HUMAN PHYSIOLOGY

    PubMed Central

    Guerriero, Christopher J.; Brodsky, Jeffrey L.

    2014-01-01

    Protein folding is a complex, error-prone process that often results in an irreparable protein by-product. These by-products can be recognized by cellular quality control machineries and targeted for proteasome-dependent degradation. The folding of proteins in the secretory pathway adds another layer to the protein folding “problem,” as the endoplasmic reticulum maintains a unique chemical environment within the cell. In fact, a growing number of diseases are attributed to defects in secretory protein folding, and many of these by-products are targeted for a process known as endoplasmic reticulum-associated degradation (ERAD). Since its discovery, research on the mechanisms underlying the ERAD pathway has provided new insights into how ERAD contributes to human health during both normal and diseases states. Links between ERAD and disease are evidenced from the loss of protein function as a result of degradation, chronic cellular stress when ERAD fails to keep up with misfolded protein production, and the ability of some pathogens to coopt the ERAD pathway. The growing number of ERAD substrates has also illuminated the differences in the machineries used to recognize and degrade a vast array of potential clients for this pathway. Despite all that is known about ERAD, many questions remain, and new paradigms will likely emerge. Clearly, the key to successful disease treatment lies within defining the molecular details of the ERAD pathway and in understanding how this conserved pathway selects and degrades an innumerable cast of substrates. PMID:22535891

  6. Identification of Dynamic Changes in Proteins Associated with the Cellular Cytoskeleton after Exposure to Okadaic Acid

    PubMed Central

    Opsahl, Jill A.; Ljostveit, Sonja; Solstad, Therese; Risa, Kristin; Roepstorff, Peter; Fladmark, Kari E.

    2013-01-01

    Exposure of cells to the diarrhetic shellfish poison, okadaic acid, leads to a dramatic reorganization of cytoskeletal architecture and loss of cell-cell contact. When cells are exposed to high concentrations of okadaic acid (100–500 nM), the morphological rearrangement is followed by apoptotic cell death. Okadaic acid inhibits the broad acting Ser/Thr protein phosphatases 1 and 2A, which results in hyperphosphorylation of a large number of proteins. Some of these hyperphosphorylated proteins are most likely key players in the reorganization of the cell morphology induced by okadaic acid. We wanted to identify these phosphoproteins and searched for them in the cellular lipid rafts, which have been found to contain proteins that regulate cytoskeletal dynamics and cell adhesion. By using stable isotope labeling by amino acids in cell culture cells treated with okadaic acid (400 nM) could 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, microtubules and cell adhesion structures. A large number of these okadaic acid-regulated proteins have previously also been shown to be similarly regulated prior to cell proliferation and migration. Our results suggest that okadaic acid activates general cell signaling pathways that induce breakdown of the cortical actin cytoskeleton and cell detachment. PMID:23708184

  7. What determines the strength of noncovalent association of ligands to proteins in aqueous solution?

    PubMed Central

    Miyamoto, S; Kollman, P A

    1993-01-01

    Free energy perturbation methods using molecular dynamics have been used to calculate the absolute free energy of association of two ligand-protein complexes. The calculations reproduce the significantly more negative free energy of association of biotin to streptavidin, compared to N-L-acetyltryptophanamide/alpha-chymotrypsin. This difference in free energy of association is due to van der Waals/dispersion effects in the nearly ideally performed cavity that streptavidin presents to biotin, which involves four tryptophan residues. Images Fig. 2 PMID:8378312

  8. Identification of a Putative Protein Profile Associated with Tamoxifen Therapy Resistance in Breast Cancer*S⃞

    PubMed Central

    Umar, Arzu; Kang, Hyuk; Timmermans, Annemieke M.; Look, Maxime P.; Meijer-van Gelder, Marion E.; den Bakker, Michael A.; Jaitly, Navdeep; Martens, John W. M.; Luider, Theo M.; Foekens, John A.; Paša-Tolić, Ljiljana

    2009-01-01

    Tamoxifen resistance is a major cause of death in patients with recurrent breast cancer. Current clinical factors can correctly predict therapy response in only half of the treated patients. Identification of proteins that are associated with tamoxifen resistance is a first step toward better response prediction and tailored treatment of patients. In the present study we intended to identify putative protein biomarkers indicative of tamoxifen therapy resistance in breast cancer using nano-LC coupled with FTICR MS. Comparative proteome analysis was performed on ∼5,500 pooled tumor cells (corresponding to ∼550 ng of protein lysate/analysis) obtained through laser capture microdissection (LCM) from two independently processed data sets (n = 24 and n = 27) containing both tamoxifen therapy-sensitive and therapy-resistant tumors. Peptides and proteins were identified by matching mass and elution time of newly acquired LC-MS features to information in previously generated accurate mass and time tag reference databases. A total of 17,263 unique peptides were identified that corresponded to 2,556 non-redundant proteins identified with ≥2 peptides. 1,713 overlapping proteins between the two data sets were used for further analysis. Comparative proteome analysis revealed 100 putatively differentially abundant proteins between tamoxifen-sensitive and tamoxifen-resistant tumors. The presence and relative abundance for 47 differentially abundant proteins were verified by targeted nano-LC-MS/MS in a selection of unpooled, non-microdissected discovery set tumor tissue extracts. ENPP1, EIF3E, and GNB4 were significantly associated with progression-free survival upon tamoxifen treatment for recurrent disease. Differential abundance of our top discriminating protein, extracellular matrix metalloproteinase inducer, was validated by tissue microarray in an independent patient cohort (n = 156). Extracellular matrix metalloproteinase inducer levels were higher in therapy

  9. A Genome-Wide Association Study Identifies Protein Quantitative Trait Loci (pQTLs)

    PubMed Central

    Corsi, Anna-Maria; Stevens, Kara; Rafferty, Ian; Lauretani, Fulvio; Murray, Anna; Gibbs, J. Raphael; Paolisso, Giuseppe; Rafiq, Sajjad; Simon-Sanchez, Javier; Lango, Hana; Scholz, Sonja; Weedon, Michael N.; Arepalli, Sampath; Rice, Neil; Washecka, Nicole; Hurst, Alison; Britton, Angela; Henley, William; van de Leemput, Joyce; Li, Rongling; Newman, Anne B.; Tranah, Greg; Harris, Tamara; Panicker, Vijay; Dayan, Colin; Bennett, Amanda; McCarthy, Mark I.; Ruokonen, Aimo; Jarvelin, Marjo-Riitta; Guralnik, Jack; Bandinelli, Stefania; Frayling, Timothy M.; Singleton, Andrew; Ferrucci, Luigi

    2008-01-01

    There is considerable evidence that human genetic variation influences gene expression. Genome-wide studies have revealed that mRNA levels are associated with genetic variation in or close to the gene coding for those mRNA transcripts – cis effects, and elsewhere in the genome – trans effects. The role of genetic variation in determining protein levels has not been systematically assessed. Using a genome-wide association approach we show that common genetic variation influences levels of clinically relevant proteins in human serum and plasma. We evaluated the role of 496,032 polymorphisms on levels of 42 proteins measured in 1200 fasting individuals from the population based InCHIANTI study. Proteins included insulin, several interleukins, adipokines, chemokines, and liver function markers that are implicated in many common diseases including metabolic, inflammatory, and infectious conditions. We identified eight Cis effects, including variants in or near the IL6R (p = 1.8×10−57), CCL4L1 (p = 3.9×10−21), IL18 (p = 6.8×10−13), LPA (p = 4.4×10−10), GGT1 (p = 1.5×10−7), SHBG (p = 3.1×10−7), CRP (p = 6.4×10−6) and IL1RN (p = 7.3×10−6) genes, all associated with their respective protein products with effect sizes ranging from 0.19 to 0.69 standard deviations per allele. Mechanisms implicated include altered rates of cleavage of bound to unbound soluble receptor (IL6R), altered secretion rates of different sized proteins (LPA), variation in gene copy number (CCL4L1) and altered transcription (GGT1). We identified one novel trans effect that was an association between ABO blood group and tumour necrosis factor alpha (TNF-alpha) levels (p = 6.8×10−40), but this finding was not present when TNF-alpha was measured using a different assay , or in a second study, suggesting an assay-specific association. Our results show that protein levels share some of the features of the genetics of gene expression. These

  10. EBNA-LP Associates with Cellular Proteins Including DNA-PK and HA95

    PubMed Central

    Han, Innoc; Harada, Shizuko; Weaver, David; Xue, Yong; Lane, William; Orstavik, Sigurd; Skalhegg, Bjorn; Kieff, Elliott

    2001-01-01

    EBNA-LP-associated proteins were identified by sequencing proteins that immunoprecipitated with Flag epitope-tagged EBNA-LP (FLP) from lymphoblasts in which FLP was stably expressed. The association of EBNA-LP with Hsp70 (72/73) was confirmed, and sequences of DNA-PK catalytic subunit (DNA-PKcs), HA95, Hsp27, prolyl 4-hydroxylase α-1 subunit, α-tubulin, and β-tubulin were identified. The fraction of total cellular HA95 that associated with FLP was very high, while progressively lower fractions of the total DNA-PKcs, Hsp70, Hsp 27, α-tubulin, and β-tubulin specifically associated with EBNA-LP as determined by immunoblotting with antibodies to these proteins. EBNA-LP bound to two domains in the DNA-PKcs C terminus and DNA-PKcs associated with the EBNA-LP repeat domain. DNA-PKcs that was bound to EBNA-LP phosphorylated p53 or EBNA-LP in vitro, and the phosphorylation of EBNA-LP was inhibited by Wortmannin, a specific in vitro inhibitor of DNA-PKcs. PMID:11160753

  11. Homologous Transcription Factors DUX4 and DUX4c Associate with Cytoplasmic Proteins during Muscle Differentiation

    PubMed Central

    Ansseau, Eugénie; Matteotti, Christel; Yip, Cassandre; Liu, Jian; Leroy, Baptiste; Hubeau, Céline; Gerbaux, Cécile; Cloet, Samuel; Wauters, Armelle; Zorbo, Sabrina; Meyer, Pierre; Pirson, Isabelle; Laoudj-Chenivesse, Dalila; Wattiez, Ruddy; Harper, Scott Q.; Belayew, Alexandra; Coppée, Frédérique

    2016-01-01

    Hundreds of double homeobox (DUX) genes map within 3.3-kb repeated elements dispersed in the human genome and encode DNA-binding proteins. Among these, we identified DUX4, a potent transcription factor that causes facioscapulohumeral muscular dystrophy (FSHD). In the present study, we performed yeast two-hybrid screens and protein co-purifications with HaloTag-DUX fusions or GST-DUX4 pull-down to identify protein partners of DUX4, DUX4c (which is identical to DUX4 except for the end of the carboxyl terminal domain) and DUX1 (which is limited to the double homeodomain). Unexpectedly, we identified and validated (by co-immunoprecipitation, GST pull-down, co-immunofluorescence and in situ Proximal Ligation Assay) the interaction of DUX4, DUX4c and DUX1 with type III intermediate filament protein desmin in the cytoplasm and at the nuclear periphery. Desmin filaments link adjacent sarcomere at the Z-discs, connect them to sarcolemma proteins and interact with mitochondria. These intermediate filament also contact the nuclear lamina and contribute to positioning of the nuclei. Another Z-disc protein, LMCD1 that contains a LIM domain was also validated as a DUX4 partner. The functionality of DUX4 or DUX4c interactions with cytoplasmic proteins is underscored by the cytoplasmic detection of DUX4/DUX4c upon myoblast fusion. In addition, we identified and validated (by co-immunoprecipitation, co-immunofluorescence and in situ Proximal Ligation Assay) as DUX4/4c partners several RNA-binding proteins such as C1QBP, SRSF9, RBM3, FUS/TLS and SFPQ that are involved in mRNA splicing and translation. FUS and SFPQ are nuclear proteins, however their cytoplasmic translocation was reported in neuronal cells where they associated with ribonucleoparticles (RNPs). Several other validated or identified DUX4/DUX4c partners are also contained in mRNP granules, and the co-localizations with cytoplasmic DAPI-positive spots is in keeping with such an association. Large muscle RNPs were

  12. Homologous Transcription Factors DUX4 and DUX4c Associate with Cytoplasmic Proteins during Muscle Differentiation.

    PubMed

    Ansseau, Eugénie; Eidahl, Jocelyn O; Lancelot, Céline; Tassin, Alexandra; Matteotti, Christel; Yip, Cassandre; Liu, Jian; Leroy, Baptiste; Hubeau, Céline; Gerbaux, Cécile; Cloet, Samuel; Wauters, Armelle; Zorbo, Sabrina; Meyer, Pierre; Pirson, Isabelle; Laoudj-Chenivesse, Dalila; Wattiez, Ruddy; Harper, Scott Q; Belayew, Alexandra; Coppée, Frédérique

    2016-01-01

    Hundreds of double homeobox (DUX) genes map within 3.3-kb repeated elements dispersed in the human genome and encode DNA-binding proteins. Among these, we identified DUX4, a potent transcription factor that causes facioscapulohumeral muscular dystrophy (FSHD). In the present study, we performed yeast two-hybrid screens and protein co-purifications with HaloTag-DUX fusions or GST-DUX4 pull-down to identify protein partners of DUX4, DUX4c (which is identical to DUX4 except for the end of the carboxyl terminal domain) and DUX1 (which is limited to the double homeodomain). Unexpectedly, we identified and validated (by co-immunoprecipitation, GST pull-down, co-immunofluorescence and in situ Proximal Ligation Assay) the interaction of DUX4, DUX4c and DUX1 with type III intermediate filament protein desmin in the cytoplasm and at the nuclear periphery. Desmin filaments link adjacent sarcomere at the Z-discs, connect them to sarcolemma proteins and interact with mitochondria. These intermediate filament also contact the nuclear lamina and contribute to positioning of the nuclei. Another Z-disc protein, LMCD1 that contains a LIM domain was also validated as a DUX4 partner. The functionality of DUX4 or DUX4c interactions with cytoplasmic proteins is underscored by the cytoplasmic detection of DUX4/DUX4c upon myoblast fusion. In addition, we identified and validated (by co-immunoprecipitation, co-immunofluorescence and in situ Proximal Ligation Assay) as DUX4/4c partners several RNA-binding proteins such as C1QBP, SRSF9, RBM3, FUS/TLS and SFPQ that are involved in mRNA splicing and translation. FUS and SFPQ are nuclear proteins, however their cytoplasmic translocation was reported in neuronal cells where they associated with ribonucleoparticles (RNPs). Several other validated or identified DUX4/DUX4c partners are also contained in mRNP granules, and the co-localizations with cytoplasmic DAPI-positive spots is in keeping with such an association. Large muscle RNPs were

  13. A genome-wide association study of seed protein and oil content in soybean

    PubMed Central

    2014-01-01

    Background Association analysis is an alternative to conventional family-based methods to detect the location of gene(s) or quantitative trait loci (QTL) and provides relatively high resolution in terms of defining the genome position of a gene or QTL. Seed protein and oil concentration are quantitative traits which are determined by the interaction among many genes with small to moderate genetic effects and their interaction with the environment. In this study, a genome-wide association study (GWAS) was performed to identify quantitative trait loci (QTL) controlling seed protein and oil concentration in 298 soybean germplasm accessions exhibiting a wide range of seed protein and oil content. Results A total of 55,159 single nucleotide polymorphisms (SNPs) were genotyped using various methods including Illumina Infinium and GoldenGate assays and 31,954 markers with minor allele frequency >0.10 were used to estimate linkage disequilibrium (LD) in heterochromatic and euchromatic regions. In euchromatic regions, the mean LD (r 2 ) rapidly declined to 0.2 within 360 Kbp, whereas the mean LD declined to 0.2 at 9,600 Kbp in heterochromatic regions. The GWAS results identified 40 SNPs in 17 different genomic regions significantly associated with seed protein. Of these, the five SNPs with the highest associations and seven adjacent SNPs were located in the 27.6-30.0 Mbp region of Gm20. A major seed protein QTL has been previously mapped to the same location and potential candidate genes have recently been identified in this region. The GWAS results also detected 25 SNPs in 13 different genomic regions associated with seed oil. Of these markers, seven SNPs had a significant association with both protein and oil. Conclusions This research indicated that GWAS not only identified most of the previously reported QTL controlling seed protein and oil, but also resulted in narrower genomic regions than the regions reported as containing these QTL. The narrower GWAS-defined genome

  14. Correcting for the study bias associated with protein–protein interaction measurements reveals differences between protein degree distributions from different cancer types

    PubMed Central

    Schaefer, Martin H.; Serrano, Luis; Andrade-Navarro, Miguel A.

    2015-01-01

    Protein–protein interaction (PPI) networks are associated with multiple types of biases partly rooted in technical limitations of the experimental techniques. Another source of bias are the different frequencies with which proteins have been studied for interaction partners. It is generally believed that proteins with a large number of interaction partners tend to be essential, evolutionarily conserved, and involved in disease. It has been repeatedly reported that proteins driving tumor formation have a higher number of PPI partners. However, it has been noticed before that the degree distribution of PPI networks is biased toward disease proteins, which tend to have been studied more often than non-disease proteins. At the same time, for many poorly characterized proteins no interactions have been reported yet. It is unclear to which extent this study bias affects the observation that cancer proteins tend to have more PPI partners. Here, we show that the degree of a protein is a function of the number of times it has been screened for interaction partners. We present a randomization-based method that controls for this bias to decide whether a group of proteins is associated with significantly more PPI partners than the proteomic background. We apply our method to cancer proteins and observe, in contrast to previous studies, no conclusive evidence for a significantly higher degree distribution associated with cancer proteins as compared to non-cancer proteins when we compare them to proteins that have been equally often studied as bait proteins. Comparing proteins from different tumor types, a more complex picture emerges in which proteins of certain cancer classes have significantly more interaction partners while others are associated with a smaller degree. For example, proteins of several hematological cancers tend to be associated with a higher number of interaction partners as expected by chance. Solid tumors, in contrast, are usually associated with a degree

  15. Isolation of a crystal matrix protein associated with calcium oxalate precipitation in vacuoles of specialized cells.

    PubMed

    Li, Xingxiang; Zhang, Dianzhong; Lynch-Holm, Valerie J; Okita, Thomas W; Franceschi, Vincent R

    2003-10-01

    The formation of calcium (Ca) oxalate crystals is considered to be a high-capacity mechanism for regulating Ca in many plants. Ca oxalate precipitation is not a stochastic process, suggesting the involvement of specific biochemical and cellular mechanisms. Microautoradiography of water lettuce (Pistia stratiotes) tissue exposed to 3H-glutamate showed incorporation into developing crystals, indicating potential acidic proteins associated with the crystals. Dissolution of crystals leaves behind a crystal-shaped matrix "ghost" that is capable of precipitation of Ca oxalate in the original crystal morphology. To assess whether this matrix has a protein component, purified crystals were isolated and analyzed for internal protein. Polyacrylamide gel electrophoresis revealed the presence of one major polypeptide of about 55 kD and two minor species of 60 and 63 kD. Amino acid analysis indicates the matrix protein is relatively high in acidic amino acids, a feature consistent with its solubility in formic acid but not at neutral pH. 45Ca-binding assays demonstrated the matrix protein has a strong affinity for Ca. Immunocytochemical localization using antibody raised to the isolated protein showed that the matrix protein is specific to crystal-forming cells. Within the vacuole, the surface and internal structures of two morphologically distinct Ca oxalate crystals, raphide and druse, were labeled by the antimatrix protein serum, as were the surfaces of isolated crystals. These results demonstrate that a specific Ca-binding protein exists as an integral component of Ca oxalate crystals, which holds important implications with respect to regulation of crystal formation. PMID:14555781

  16. No Evidence for Association of Autism with Rare Heterozygous Point Mutations in Contactin-Associated Protein-Like 2 (CNTNAP2), or in Other Contactin-Associated Proteins or Contactins

    PubMed Central

    Murdoch, John D.; Gupta, Abha R.; Sanders, Stephan J.; Walker, Michael F.; Keaney, John; Fernandez, Thomas V.; Murtha, Michael T.; Anyanwu, Samuel; Ober, Gordon T.; Raubeson, Melanie J.; DiLullo, Nicholas M.; Villa, Natalie; Waqar, Zainabdul; Sullivan, Catherine; Gonzalez, Luis; Willsey, A. Jeremy; Choe, So-Yeon; Neale, Benjamin M.; Daly, Mark J.; State, Matthew W.

    2015-01-01

    Contactins and Contactin-Associated Proteins, and Contactin-Associated Protein-Like 2 (CNTNAP2) in particular, have been widely cited as autism risk genes based on findings from homozygosity mapping, molecular cytogenetics, copy number variation analyses, and both common and rare single nucleotide association studies. However, data specifically with regard to the contribution of heterozygous single nucleotide variants (SNVs) have been inconsistent. In an effort to clarify the role of rare point mutations in CNTNAP2 and related gene families, we have conducted targeted next-generation sequencing and evaluated existing sequence data in cohorts totaling 2704 cases and 2747 controls. We find no evidence for statistically significant association of rare heterozygous mutations in any of the CNTN or CNTNAP genes, including CNTNAP2, placing marked limits on the scale of their plausible contribution to risk. PMID:25621974

  17. Association of the GTP-binding protein Rab3A with bovine adrenal chromaffin granules

    SciTech Connect

    Darchen, F.; Hammel, F.; Monteils, M.P.; Scherman, D. ); Zahraoui, A.; Tavitian, A. )

    1990-08-01

    The Rab3A protein belongs to a large family of small GTP-binding proteins that are present in eukaryotic cells and that share amino acid identities with the Ras proteins (products of the ras protooncogenes). Rab3A, which is specifically located in nervous and endocrine tissues, is suspected to play a key role in secretion. Its localization was investigated in bovine adrenal gland by using a polyclonal antibody. Rab3A was detected in adrenal medulla but not in adrenal cortex. In cultured adrenal medulla cells, Rab3A was specifically expressed in the catecholamine-secreting chromaffin cells. Subcellular fractionation suggested that Rab3A is about 30% cytosolic and that particulate Rab3A is associated with the membrane of chromaffin granules (the catecholamine storage organelles) and with a second compartment likely to be the plasma membrane. The Rab3A localization on chromaffin granule membranes was confirmed by immunoadsorption with an antibody against dopamine {beta}-hydroxylase. Rab3A was not extracted from this membrane by NaCl or KBr but was partially extracted by urea and totally solubilized by Triton X-100, suggesting either an interaction with an intrinsic protein or a membrane association through fatty acid acylation. This study suggests that Rab3A, which may also be located on other secretory vesicles containing noncharacterized small GTP-binding proteins, is involved in their biogenesis or in the regulated secretion process.

  18. Caveolin-1 regulates expression of junction-associated proteins in brain microvascular endothelial cells

    PubMed Central

    Song, Li; Ge, Shujun; Pachter, Joel S.

    2007-01-01

    Recent evidence from this laboratory indicated that reduced expression of caveolin-1 accompanied the diminished expression of tight junction (TJ)–associated proteins occludin and zonula occludens-1 (ZO-1) following stimulation of brain microvascular endothelial cells (BMECs) with the chemokine CCL2 (formerly called MCP-1). Because attenuated caveolin-1 levels have also been correlated with heightened permeability of other endothelia, the objective of this study was to test the hypothesis that reduced caveolin-1 expression is causally linked to the action of CCL2 on BMEC junctional protein expression and barrier integrity. This was achieved using adenovirus to nondestructively deliver caveolin-1 siRNA (Ad-siCav-1) to BMEC monolayers, which model the blood-brain barrier (BBB). Treatment with siRNA reduced the caveolin-1 protein level as well as occludin and ZO-1. Additionally, occludin exhibited dissociation from the cytoskeletal framework. These changes were attended by comparable alterations in adherens junction (AJ)–associated proteins, VE-cadherin and β-catenin, increased BMEC paracellular permeability, and facilitated the ability of CCL2 to stimulate monocytic transendothelial migration. Furthermore, treating BMECs with cavtratin, a synthetic cell-permeable peptide encoding the caveolin-1 scaffolding domain, antagonized effects of both Ad-siCav-1 and CCL2. These results collectively highlight caveolin-1 loss as a critical step in CCL2-induced modulation of BMEC junctional protein expression and integrity, and possibly serve a crucial role in regulating inflammation at the BBB. PMID:17023578

  19. HIV1-viral protein R (Vpr) mutations: associated phenotypes and relevance for clinical pathologies.

    PubMed

    Soares, Rui; Rocha, Graça; Meliço-Silvestre, António; Gonçalves, Teresa

    2016-09-01

    Over the last 30 years, research into HIV has advanced the knowledge of virus genetics and the development of efficient therapeutic strategies. HIV-1 viral protein R (Vpr) is a specialized and multifunctional protein that plays important roles at multiple stages of the HIV-1 viral life cycle. This protein interacts with a number of cellular and viral proteins and with multiple activities including nuclear transport of the pre-integration complex (PIC) to the nucleus, transcriptional activation, cell cycle arrest at G2/M transition phase and induction of cell death via apoptosis. Specifically, Vpr has been shown to control many host cell functions through a variety of biological processes and by interaction with several cellular pathways. The different functions of Vpr may enhance viral replication and impair the immune system in HIV-1 infected patients. Importantly, functional defects induced by mutations in the Vpr protein correlate with slow disease progression of HIV-infected patients. Vpr is also associated with other concomitant pathologies developed by these patients, which may lead it to be considered as a potential novel therapeutic target. This review will focus on HIV-1 Vpr, mainly on the importance of its structural mutations on the progression of HIV infection, associated phenotypes and relevance for clinical pathologies. Copyright © 2016 John Wiley & Sons, Ltd. PMID:27264019

  20. The selective biotin tagging and thermolysin proteolysis of chloroplast outer envelope proteins reveals information on protein topology and association into complexes

    PubMed Central

    Hardré, Hélène; Kuhn, Lauriane; Albrieux, Catherine; Jouhet, Juliette; Michaud, Morgane; Seigneurin-Berny, Daphné; Falconet, Denis; Block, Maryse A.; Maréchal, Eric

    2014-01-01

    The understanding of chloroplast function requires the precise localization of proteins in each of its sub-compartments. High-sensitivity mass spectrometry has allowed the inventory of proteins in thylakoid, stroma, and envelope fractions. Concerning membrane association, proteins can be either integral or peripheral or even soluble proteins bound transiently to a membrane complex. We sought a method providing information at the surface of the outer envelope membrane (OEM), based on specific tagging with biotin or proteolysis using thermolysin, a non-membrane permeable protease. To evaluate this method, envelope, thylakoid, and stroma proteins were separated by two-dimensional electrophoresis and analyzed by immunostaining and mass spectrometry. A short selection of proteins associated to the chloroplast envelope fraction was checked after superficial treatments of intact chloroplasts. We showed that this method could allow the characterization of OEM embedded proteins facing the cytosol, as well as peripheral and soluble proteins associated via tight or lose interactions. Some stromal proteins were associated with biotinylated spots and analyzes are still needed to determine whether polypeptides were tagged prior import or if they co-migrated with OEM proteins. This method also suggests that some proteins associated with the inner envelope membrane (IEM) might need the integrity of a trans-envelope (IEM–OEM) protein complex (e.g., division ring-forming components) or at least an intact OEM partner. Following this evaluation, proteomic analyzes should be refined and the putative role of inter-membrane space components stabilizing trans-envelope complexes demonstrated. For future comprehensive studies, perspectives include the dynamic analyses of OEM proteins and IEM–OEM complexes in various physiological contexts and using virtually any other purified membrane organelle. PMID:24999344

  1. AAA ATPases regulate membrane association of yeast oxysterol binding proteins and sterol metabolism.

    PubMed

    Wang, Penghua; Zhang, Yong; Li, Hongzhe; Chieu, Hai Kee; Munn, Alan L; Yang, Hongyuan

    2005-09-01

    The yeast genome encodes seven oxysterol binding protein homologs, Osh1p-Osh7p, which have been implicated in regulating intracellular lipid and vesicular transport. Here, we show that both Osh6p and Osh7p interact with Vps4p, a member of the AAA (ATPases associated with a variety of cellular activities) family. The coiled-coil domain of Osh7p was found to interact with Vps4p in a yeast two-hybrid screen and the interaction between Osh7p and Vps4p appears to be regulated by ergosterol. Deletion of VPS4 induced a dramatic increase in the membrane-associated pools of Osh6p and Osh7p and also caused a decrease in sterol esterification, which was suppressed by overexpression of OSH7. Lastly, overexpression of the coiled-coil domain of Osh7p (Osh7pCC) resulted in a multivesicular body sorting defect, suggesting a dominant negative role of Osh7pCC possibly through inhibiting Vps4p function. Our data suggest that a common mechanism may exist for AAA proteins to regulate the membrane association of yeast OSBP proteins and that these two protein families may function together to control subcellular lipid transport. PMID:16096648

  2. Involvement of Bcl-2-associated athanogene (BAG)-family proteins in the neuroprotection by rasagiline

    PubMed Central

    Guo, Ji-Feng; He, Shuang; Kang, Ji-Feng; Xu, Qian; Hu, Ya-Cen; Zhang, Hai-Nan; Wang, Chun-Yu; Yan, Xin-Xiang; Tang, Bei-Sha

    2015-01-01

    Rasagiline, a novel monoamine oxidase (MAO)-B inhibitor, has a mild to moderate effect in relieving Parkinson’s disease (PD) symptoms as well as unique neuroprotective effects. Previous studies demonstrated rasagiline protect neurons by regulating Bcl-2 family proteins. Our study aimed to study whether Bcl-2-associated athanogene (BAG)-family proteins, which were reported closely associated with neurodegenerative disease, were involved in the neuroprotective effect of rasagiline. We found that after the administration of 1-methy1-4-phenvl-1,2,3,6-tetrahvdropvridine (MPTP), BAG2 and BAG5 proteins were up-regulated in the substantia nigra dopaminergic neurons of PD mouse model. A further increase of BAG2 and BAG5 was detected after intragastric administration of rasagiline to post-MPTP lesioned mice. Thus, the current study proved the association of BAG family proteins with PD, and suggested the involvement and a positive role of BAG2, BAG5 in the neuroprotection of rasagiline. These preliminary results implicate a novel pathway for further study on neuroprotection of rasagiline. PMID:26770414

  3. Epidemic spreading model to characterize misfolded proteins propagation in aging and associated neurodegenerative disorders.

    PubMed

    Iturria-Medina, Yasser; Sotero, Roberto C; Toussaint, Paule J; Evans, Alan C

    2014-11-01

    Misfolded proteins (MP) are a key component in aging and associated neurodegenerative disorders. For example, misfolded Amyloid-ß (Aß) and tau proteins are two neuropathogenic hallmarks of Alzheimer's disease. Mechanisms underlying intra-brain MP propagation/deposition remain essentially uncharacterized. Here, is introduced an epidemic spreading model (ESM) for MP dynamics that considers propagation-like interactions between MP agents and the brain's clearance response across the structural connectome. The ESM reproduces advanced Aß deposition patterns in the human brain (explaining 46∼56% of the variance in regional Aß loads, in 733 subjects from the ADNI database). Furthermore, this model strongly supports a) the leading role of Aß clearance deficiency and early Aß onset age during Alzheimer's disease progression, b) that effective anatomical distance from Aß outbreak region explains regional Aß arrival time and Aß deposition likelihood, c) the multi-factorial impact of APOE e4 genotype, gender and educational level on lifetime intra-brain Aß propagation, and d) the modulatory impact of Aß propagation history on tau proteins concentrations, supporting the hypothesis of an interrelated pathway between Aß pathophysiology and tauopathy. To our knowledge, the ESM is the first computational model highlighting the direct link between structural brain networks, production/clearance of pathogenic proteins and associated intercellular transfer mechanisms, individual genetic/demographic properties and clinical states in health and disease. In sum, the proposed ESM constitutes a promising framework to clarify intra-brain region to region transference mechanisms associated with aging and neurodegenerative disorders. PMID:25412207

  4. The HSV-1 tegument protein pUL46 associates with cellular membranes and viral capsids

    SciTech Connect

    Murphy, Michael A.; Bucks, Michelle A.; O'Regan, Kevin J.; Courtney, Richard J.

    2008-07-05

    The molecular mechanisms responsible for the addition of tegument proteins into nascent herpesvirus particles are poorly understood. To better understand the tegumentation process of herpes simplex virus type 1 (HSV-1) virions, we initiated studies that showed the tegument protein pUL46 (VP11/12) has a similar cellular localization to the membrane-associated tegument protein VP22. Using membrane flotation analysis we found that pUL46 associates with membranes in both the presence and absence of other HSV-1 proteins. However, when purified virions were stripped of their envelope, the majority of pUL46 was found to associate with the capsid fraction. This strong affinity of pUL46 for capsids was confirmed by an in vitro capsid pull-down assay in which purified pUL46-GST was able to interact specifically with capsids purified from the nuclear fraction of HSV-1 infected cells. These results suggest that pUL46 displays a dynamic interaction between cellular membranes and capsids.

  5. Using Disease-Associated Coding Sequence Variation to Investigate Functional Compensation by Human Paralogous Proteins

    PubMed Central

    Miura, Sayaka; Tate, Stephanie; Kumar, Sudhir

    2015-01-01

    Gene duplication enables the functional diversification in species. It is thought that duplicated genes may be able to compensate if the function of one of the gene copies is disrupted. This possibility is extensively debated with some studies reporting proteome-wide compensation, whereas others suggest functional compensation among only recent gene duplicates or no compensation at all. We report results from a systematic molecular evolutionary analysis to test the predictions of the functional compensation hypothesis. We contrasted the density of Mendelian disease-associated single nucleotide variants (dSNVs) in proteins with no discernable paralogs (singletons) with the dSNV density in proteins found in multigene families. Under the functional compensation hypothesis, we expected to find greater numbers of dSNVs in singletons due to the lack of any compensating partners. Our analyses produced an opposite pattern; paralogs have over 35% higher dSNV density than singletons. We found that these patterns are concordant with similar differences in the rates of amino acid evolution (ie, functional constraints), as the proteins with paralogs have evolved 33% slower than singletons. Our evolutionary constraint explanation is robust to differences in family sizes, ages (young vs. old duplicates), and degrees of amino acid sequence similarities among paralogs. Therefore, disease-associated human variation does not exhibit significant signals of functional compensation among paralogous proteins, but rather an evolutionary constraint hypothesis provides a better explanation for the observed patterns of disease-associated and neutral polymorphisms in the human genome. PMID:26604664

  6. Epidemic Spreading Model to Characterize Misfolded Proteins Propagation in Aging and Associated Neurodegenerative Disorders

    PubMed Central

    Iturria-Medina, Yasser; Sotero, Roberto C.; Toussaint, Paule J.; Evans, Alan C.

    2014-01-01

    Misfolded proteins (MP) are a key component in aging and associated neurodegenerative disorders. For example, misfolded Amyloid-ß (Aß) and tau proteins are two neuropathogenic hallmarks of Alzheimer's disease. Mechanisms underlying intra-brain MP propagation/deposition remain essentially uncharacterized. Here, is introduced an epidemic spreading model (ESM) for MP dynamics that considers propagation-like interactions between MP agents and the brain's clearance response across the structural connectome. The ESM reproduces advanced Aß deposition patterns in the human brain (explaining 46∼56% of the variance in regional Aß loads, in 733 subjects from the ADNI database). Furthermore, this model strongly supports a) the leading role of Aß clearance deficiency and early Aß onset age during Alzheimer's disease progression, b) that effective anatomical distance from Aß outbreak region explains regional Aß arrival time and Aß deposition likelihood, c) the multi-factorial impact of APOE e4 genotype, gender and educational level on lifetime intra-brain Aß propagation, and d) the modulatory impact of Aß propagation history on tau proteins concentrations, supporting the hypothesis of an interrelated pathway between Aß pathophysiology and tauopathy. To our knowledge, the ESM is the first computational model highlighting the direct link between structural brain networks, production/clearance of pathogenic proteins and associated intercellular transfer mechanisms, individual genetic/demographic properties and clinical states in health and disease. In sum, the proposed ESM constitutes a promising framework to clarify intra-brain region to region transference mechanisms associated with aging and neurodegenerative disorders. PMID:25412207

  7. Non-Microtubular Localizations of Microtubule-Associated Protein 6 (MAP6)

    PubMed Central

    Gory-Fauré, Sylvie; Windscheid, Vanessa; Brocard, Jacques; Montessuit, Sylvie; Tsutsumi, Ryouhei; Denarier, Eric; Fukata, Yuko; Bosc, Christophe; Delaroche, Julie; Collomb, Nora; Fukata, Masaki; Martinou, Jean-Claude; Pernet-Gallay, Karin; Andrieux, Annie

    2014-01-01

    MAP6 proteins (MAP6s), which include MAP6-N (also called Stable Tubule Only Polypeptide, or STOP) and MAP6d1 (MAP6 domain-containing protein 1, also called STOP-Like protein 21 kD, or SL21), bind to and stabilize microtubules. MAP6 deletion in mice severely alters integrated brain functions and is associated with synaptic defects, suggesting that MAP6s may also have alternative cellular roles. MAP6s reportedly associate with the Golgi apparatus through palmitoylation of their N-terminal domain, and specific isoforms have been shown to bind actin. Here, we use heterologous systems to investigate several biochemical properties of MAP6 proteins. We demonstrate that the three N-terminal cysteines of MAP6d1 are palmitoylated by a subset of DHHC-type palmitoylating enzymes. Analysis of the subcellular localization of palmitoylated MAP6d1, including electron microscopic analysis, reveals possible localization to the Golgi and the plasma membrane but no association with the endoplasmic reticulum. Moreover, we observed localization of MAP6d1 to mitochondria, which requires the N-terminus of the protein but does not require palmitoylation. We show that endogenous MAP6d1 localized at mitochondria in mature mice neurons as well as at the outer membrane and in the intermembrane space of purified mouse mitochondria. Last, we found that MAP6d1 can multimerize via a microtubule-binding module. Interestingly, most of these properties of MAP6d1 are shared by MAP6-N. Together, these results describe several properties of MAP6 proteins, including their intercellular localization and multimerization activity, which may be relevant to neuronal differentiation and synaptic functions. PMID:25526643

  8. Regulation of membrane associated protein kinase C activity by guanine nucleotide in rabbit peritoneal neutrophils

    SciTech Connect

    Huang, C.K.; Devanney, J.F.

    1986-03-05

    Addition of phorbol myristate acetate (PMA) (0.1 ..mu..g/ml) or guanosine-5'-0-(3-thiotriphosphate) (GTP..gamma..S) (10..mu..M) to the membrane fraction from rabbit peritoneal neutrophils results in an increase of phosphorylation of several membrane proteins. To test whether membrane associated protein kinase C is involved in the activation, histone is added to the membrane as a substrate for protein kinase C. Phosphorylation of histone is determined by counting the gel pieces containing histone IIIS after separation from other membrane components by SDS-gel electrophoresis. In the presence of CaC12 (20 ..mu..M), GTP..gamma..S (10 ..mu..M) or PMA (0.1 ..mu..g/ml) stimulates the phosphorylation of histone IIIS (40% to 70% increase). To achieve this effect calcium is required for GTP..gamma..S but not for PMA. The effect of GTP..gamma..S but not PMA is inhibited in membranes obtained from cells pretreated with pertussis toxin. Membrane protein kinase C is solubilized with Triton X-100 (1%) and then applied to a DEAE-52 cellulose column chromatography. Two peaks of protein kinase C activity are observed. Peak one is eluted at 40 mM NaCl, peak two is eluted at 140 mM NaCl. The activity of peak one is stimulated with phosphatidylserine (PS) and PMA but not with PS and calcium. The activity of peak two is stimulated with either PS and PMA or PS and calcium. The results suggest that GTP binding protein is involved in the activation of membrane associated protein kinase C and the kinase may exist in two forms, calcium sensitive and calcium insensitive.

  9. Disease-associated mutations of TDP-43 promote turnover of the protein through the proteasomal pathway.

    PubMed

    Araki, Wataru; Minegishi, Seiji; Motoki, Kazumi; Kume, Hideaki; Hohjoh, Hirohiko; Araki, Yumiko M; Tamaoka, Akira

    2014-12-01

    TAR DNA-binding protein (TDP-43) is a major component of most ubiquitin-positive neuronal and glial inclusions of amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). A number of missense mutations in the TARDBP gene have been identified in patients with familial and sporadic ALS, as well as familial FTLD with ALS. In the diseased states, TDP-43 proteins exhibit characteristic alterations, including truncation, abnormal phosphorylation, and altered subcellular distribution. However, the mechanisms by which TDP-43 mutations induce neurodegeneration remain unclear at present. In the current study, we analyzed protein turnover and subcellular distribution of wild-type TDP-43 and two disease-associated mutants (G298S and A382T) in human neuroblastoma SH-SY5Y cells stably expressing TDP-43 with a C-terminal tag. Cycloheximide chase experiments revealed more rapid turnover of TDP-43 mutant proteins than their wild-type counterpart. The decrease in the TDP-43 level after cycloheximide treatment was partially recovered upon co-treatment with the proteasome inhibitor, epoxomicin, but not the lysosomotropic agent, chloroquine, suggesting involvement of the proteasomal pathway in TDP-43 degradation. Analysis of the subcellular distribution of TDP-43 revealed predominant localization in the nuclear fraction, whereas the relative level in the cytoplasm remained unaltered in cells expressing either mutant protein, compared with wild-type protein. Our results suggest that higher turnover of disease-associated mutant TDP-43 proteins through the ubiquitin proteasome system is pathogenetically relevant and highlight the significance of proteolysis in the pathogenetic mechanism of TDP-43 proteinopathy. PMID:24477737

  10. Proteomics Characterization of Cytoplasmic and Lipid-Associated Membrane Proteins of Human Pathogen Mycoplasma fermentans M64

    PubMed Central

    Hu, Wensi S.; Ng, Wailap Victor; Lu, Chi-Yu; Huang, Tsung-Yen; Shu, Hung-Wei; Hsiao, Kwang-Jen; Tsai, Shih-Feng; Chang, Chuan-Hsiung; Lin, Chao-Hsiung

    2012-01-01

    Mycoplasma fermentans is a potent human pathogen which has been implicated in several diseases. Notably, its lipid-associated membrane proteins (LAMPs) play a role in immunomodulation and development of infection-associated inflammatory diseases. However, the systematic protein identification of pathogenic M. fermentans has not been reported. From our recent sequencing results of M. fermentans M64 isolated from human respiratory tract, its genome is around 1.1 Mb and encodes 1050 predicted protein-coding genes. In the present study, soluble proteome of M. fermentans was resolved and analyzed using two-dimensional gel electrophoresis. In addition, Triton X-114 extraction was carried out to enrich amphiphilic proteins including putative lipoproteins and membrane proteins. Subsequent mass spectrometric analyses of these proteins had identified a total of 181 M. fermentans ORFs. Further bioinformatics analysis of these ORFs encoding proteins with known or so far unknown orthologues among bacteria revealed that a total of 131 proteins are homologous to known proteins, 11 proteins are conserved hypothetical proteins, and the remaining 39 proteins are likely M. fermentans-specific proteins. Moreover, Triton X-114-enriched fraction was shown to activate NF-kB activity of raw264.7 macrophage and a total of 21 lipoproteins with predicted signal peptide were identified therefrom. Together, our work provides the first proteome reference map of M. fermentans as well as several putative virulence-associated proteins as diagnostic markers or vaccine candidates for further functional study of this human pathogen. PMID:22536369

  11. Identification of indicator proteins associated with flooding injury in soybean seedlings using label-free quantitative proteomics.

    PubMed

    Nanjo, Yohei; Nakamura, Takuji; Komatsu, Setsuko

    2013-11-01

    Flooding injury is one of the abiotic constraints on soybean growth. An experimental system established for evaluating flooding injury in soybean seedlings indicated that the degree of injury is dependent on seedling density in floodwater. Dissolved oxygen levels in the floodwater were decreased by the seedlings and correlated with the degree of injury. To understand the molecular mechanism responsible for the injury, proteomic alterations in soybean seedlings that correlated with severity of stress were analyzed using label-free quantitative proteomics. The analysis showed that the abundance of proteins involved in cell wall modification, such as polygalacturonase inhibitor-like and expansin-like B1-like proteins, which may be associated with the defense system, increased dependence on stress at both the protein and mRNA levels in all organs during flooding. The manner of alteration in abundance of these proteins was distinct from those of other responsive proteins. Furthermore, proteins also showing specific changes in abundance in the root tip included protein phosphatase 2A subunit-like proteins, which are possibly involved in flooding-induced root tip cell death. Additionally, decreases in abundance of cell wall synthesis-related proteins, such as cinnamyl-alcohol dehydrogenase and cellulose synthase-interactive protein-like proteins, were identified in hypocotyls of seedlings grown for 3 days after flooding, and these proteins may be associated with suppression of growth after flooding. These flooding injury-associated proteins can be defined as indicator proteins for severity of flooding stress in soybean. PMID:23659366

  12. Z-scan Fluorescence Profile Deconvolution of Cytosolic and Membrane-associated Protein Populations

    PubMed Central

    Smith, Elizabeth M.; Hennen, Jared; Chen, Yan; Mueller, Joachim D.

    2015-01-01

    This study introduces a technique that characterizes the spatial distribution of peripheral membrane proteins that associate reversibly with the plasma membrane. An axial scan through the cell generates a z-scan intensity profile of a fluorescently labeled peripheral membrane protein. This profile is analytically separated into membrane and cytoplasmic components by accounting for both the cell geometry and the point spread function. We experimentally validated the technique and characterized both the resolvability and stability of z-scan measurements. Further, using the cellular brightness of green fluorescent protein, we were able to convert the fluorescence intensities into concentrations at the membrane and in the cytoplasm. We applied the technique to study the translocation of the pleckstrin homology domain of phospholipase C-delta1 labeled with green fluorescent protein upon ionomycin treatment. Analysis of the z-scan fluorescence profiles revealed protein-specific cell height changes and allowed for comparison between the observed fluorescence changes and predictions based on the cellular surface area to volume ratio. The quantitative capability of z-scan fluorescence profile deconvolution offers opportunities for investigating peripheral membrane proteins in the living cell that were previously not accessible. PMID:25862080

  13. Protein Network Signatures Associated with Exogenous Biofuels Treatments in Cyanobacterium Synechocystis sp. PCC 6803

    PubMed Central

    Pei, Guangsheng; Chen, Lei; Wang, Jiangxin; Qiao, Jianjun; Zhang, Weiwen

    2014-01-01

    Although recognized as a promising microbial cell factory for producing biofuels, current productivity in cyanobacterial systems is low. To make the processes economically feasible, one of the hurdles, which need to be overcome is the low tolerance of hosts to toxic biofuels. Meanwhile, little information is available regarding the cellular responses to biofuels stress in cyanobacteria, which makes it challenging for tolerance engineering. Using large proteomic datasets of Synechocystis under various biofuels stress and environmental perturbation, a protein co-expression network was first constructed and then combined with the experimentally determined protein–protein interaction network. Proteins with statistically higher topological overlap in the integrated network were identified as common responsive proteins to both biofuels stress and environmental perturbations. In addition, a weighted gene co-expression network analysis was performed to distinguish unique responses to biofuels from those to environmental perturbations and to uncover metabolic modules and proteins uniquely associated with biofuels stress. The results showed that biofuel-specific proteins and modules were enriched in several functional categories, including photosynthesis, carbon fixation, and amino acid metabolism, which may represent potential key signatures for biofuels stress responses in Synechocystis. Network-based analysis allowed determination of the responses specifically related to biofuels stress, and the results constituted an important knowledge foundation for tolerance engineering against biofuels in Synechocystis. PMID:25405149

  14. Vigilant keratinocytes trigger pathogen-associated molecular pattern signaling in response to streptococcal M1 protein.

    PubMed

    Persson, Sandra T; Wilk, Laura; Mörgelin, Matthias; Herwald, Heiko

    2015-12-01

    The human skin exerts many functions in order to maintain its barrier integrity and protect the host from invading microorganisms. One such pathogen is Streptococcus pyogenes, which can cause a variety of superficial skin wounds that may eventually progress into invasive deep soft tissue infections. Here we show that keratinocytes recognize soluble M1 protein, a streptococcal virulence factor, as a pathogen-associated molecular pattern to release alarming inflammatory responses. We found that this interaction initiates an inflammatory intracellular signaling cascade involving the activation of the mitogen-activated protein kinases extracellular signal-regulated kinase (ERK), p38, and Jun N-terminal protein kinase and the subsequent induction and mobilization of the transcription factors NF-κB and AP-1. We also determined the imprint of the inflammatory mediators released, such as interleukin-8 (IL-8), growth-related oncogene alpha, migration inhibitory factor, extracellular matrix metalloproteinase inducer, IL-1α, IL-1 receptor a, and ST2, in response to streptococcal M1 protein. The expression of IL-8 is dependent on Toll-like receptor 2 activity and subsequent activation of the mitogen-activated protein kinases ERK and p38. Notably, this signaling seems to be distinct for IL-8 release, and it is not shared with the other inflammatory mediators. We conclude that keratinocytes participate in a proinflammatory manner in streptococcal pattern recognition and that expression of the chemoattractant IL-8 by keratinocytes constitutes an important protective mechanism against streptococcal M1 protein. PMID:26416902

  15. Identification of proteins associated with adhesive prints from Holothuria dofleinii Cuvierian tubules.

    PubMed

    Peng, Yong Y; Glattauer, Veronica; Skewes, Timothy D; McDevitt, Andrew; Elvin, Christopher M; Werkmeister, Jerome A; Graham, Lloyd D; Ramshaw, John A M

    2014-12-01

    Cuvierian tubules are expelled as a defence mechanism against predators by various species within the family Holothuridae. When the tubules are expelled, they become sticky almost immediately and ensnare the predator. The mechanism of this rapid adhesion is not clear, but proteins on the surface of the expelled tubules are widely believed to be involved. This study has examined such proteins from Holothuria dofleinii, sourced from adhesive prints left on glass after the removal of adhered tubules. Gel electrophoresis showed that seven strongly staining protein bands were consistently present in all samples, with molecular masses ranging from 89 to 17 kDa. N-terminal sequence data was obtained from two bands, while others seemed blocked. Tandem mass spectrometry-based sequencing of tryptic peptides derived from individual protein bands indicated that the proteins were unlikely to be homopolymers. PCR primers designed using the peptide sequences enabled us to amplify, clone and sequence cDNA segments relating to four gel bands; for each, the predicted translation product contained other peptide sequences observed for that band that had not been used in primer design. Database searches using the peptide and cDNA-encoded sequences suggest that two of the seven proteins are novel and one is a C-type lectin, while-surprisingly-at least three of the other four are closely related to enzymes associated with the pentose phosphate cycle and glycolysis. We discuss precedents in which lectins and metabolic enzymes are involved in attachment and adhesion phenomena. PMID:25086572

  16. Vigilant Keratinocytes Trigger Pathogen-Associated Molecular Pattern Signaling in Response to Streptococcal M1 Protein

    PubMed Central

    Wilk, Laura; Mörgelin, Matthias; Herwald, Heiko

    2015-01-01

    The human skin exerts many functions in order to maintain its barrier integrity and protect the host from invading microorganisms. One such pathogen is Streptococcus pyogenes, which can cause a variety of superficial skin wounds that may eventually progress into invasive deep soft tissue infections. Here we show that keratinocytes recognize soluble M1 protein, a streptococcal virulence factor, as a pathogen-associated molecular pattern to release alarming inflammatory responses. We found that this interaction initiates an inflammatory intracellular signaling cascade involving the activation of the mitogen-activated protein kinases extracellular signal-regulated kinase (ERK), p38, and Jun N-terminal protein kinase and the subsequent induction and mobilization of the transcription factors NF-κB and AP-1. We also determined the imprint of the inflammatory mediators released, such as interleukin-8 (IL-8), growth-related oncogene alpha, migration inhibitory factor, extracellular matrix metalloproteinase inducer, IL-1α, IL-1 receptor a, and ST2, in response to streptococcal M1 protein. The expression of IL-8 is dependent on Toll-like receptor 2 activity and subsequent activation of the mitogen-activated protein kinases ERK and p38. Notably, this signaling seems to be distinct for IL-8 release, and it is not shared with the other inflammatory mediators. We conclude that keratinocytes participate in a proinflammatory manner in streptococcal pattern recognition and that expression of the chemoattractant IL-8 by keratinocytes constitutes an important protective mechanism against streptococcal M1 protein. PMID:26416902

  17. Identification of ZASP, a novel protein associated to Zona occludens-2

    SciTech Connect

    Lechuga, Susana; Alarcon, Lourdes; Solano, Jesus; Huerta, Miriam; Lopez-Bayghen, Esther; Gonzalez-Mariscal, Lorenza

    2010-11-15

    With the aim of discovering new molecular interactions of the tight junction protein ZO-2, a two-hybrid screen was performed on a human kidney cDNA library using as bait the middle segment of ZO-2. Through this assay we identified a 24-kDa novel protein herein named ZASP for ZO-2 associated speckle protein. ZO-2/ZASP interaction further confirmed by pull down and immunoprecipitation experiments, requires the presence of the intact PDZ binding motif SQV of ZASP and the third PDZ domain of ZO-2. ZASP mRNA and protein are present in the kidney and in several epithelial cell lines. Endogenous ZASP is expressed primarily in nuclear speckles in co-localization with splicing factor SC-35. Nocodazole treatment and wash out reveals that ZASP disappears from the nucleus during mitosis in accordance with speckle disassembly during metaphase. ZASP amino acid sequence exhibits a canonical nuclear exportation signal and in agreement the protein exits the nucleus through a process mediated by exportin/CRM1. ZASP over-expression blocks the inhibitory activity of ZO-2 on cyclin D1 gene transcription and protein expression. The identification of ZASP helps to unfold the complex nuclear molecular arrays that form on ZO-2 scaffolds.

  18. Subcellular localization and membrane association of the replicase protein of grapevine rupestris stem pitting-associated virus, family Betaflexiviridae.

    PubMed

    Prosser, Sean W; Xiao, Huogen; Li, Caihong; Nelson, Richard S; Meng, Baozhong

    2015-04-01

    As a member of the newly established Betaflexiviridae family, grapevine rupestris stem pitting-associated virus (GRSPaV) has an RNA genome containing five ORFs. ORF1 encodes a putative replicase polyprotein typical of the alphavirus superfamily of positive-strand ssRNA viruses. Several viruses of this superfamily have been demonstrated to replicate in structures designated viral replication complexes associated with intracellular membranes. However, structure and cellular localization of the replicase complex have not been studied for members of Betaflexiviridae, a family of mostly woody plant viruses. As a first step towards the elucidation of the replication complex of GRSPaV, we investigated the subcellular localization of full-length and truncated versions of its replicase polyprotein via fluorescent tagging, followed by fluorescence microscopy. We found that the replicase polyprotein formed distinctive punctate bodies in both Nicotiana benthamiana leaf cells and tobacco protoplasts. We further mapped a region of 76 amino acids in the methyl-transferase domain responsible for the formation of these punctate structures. The punctate structures are distributed in close proximity to the endoplasmic reticulum network. Membrane flotation and biochemical analyses demonstrate that the N-terminal region responsible for punctate structure formation associated with cellular membrane is likely through an amphipathic α helix serving as an in-plane anchor. The identity of this membrane is yet to be determined. This is, to our knowledge, the first report on the localization and membrane association of the replicase proteins of a member of the family Betaflexiviridae. PMID:25502653

  19. Downregulation of the Yes-Associated Protein Is Associated with Extracellular Matrix Disorders in Ascending Aortic Aneurysms

    PubMed Central

    Li, Haiyang; Jiang, Wenjian; Ren, Weihong; Guo, Dong; Guo, Jialong; Wang, Xiaolong; Liu, Yuyong; Lan, Feng; Du, Jie; Zhang, Hongjia

    2016-01-01

    Previous studies indicate that extracellular matrix (ECM) disorders lead to the apoptosis of Vascular Smooth Muscle Cells (VSMCs), which impairs the aortic wall by reducing the generation of elastic fibers, and ultimately result in ascending aortic aneurysm. The critical role of the Yes-associated protein (YAP) has been elucidated in cardiac/SMC proliferation during cardiovascular development. However, the association of YAP expression and extracellular matrix disorders in ascending aortic aneurysms is not clear. Here, we present for the first time that the downregulation of YAP in VSMCs is associated with ECM disorders of the media in ascending aortic aneurysms. We found that aortic ECM deteriorated with increased apoptotic VSMCs. Moreover, expression of YAP was dramatically reduced in the aortic walls of patients with ascending aortic aneurysms, while the normal aortic samples exhibited abundant YAP in the VSMCs. These results suggest that downregulation of YAP leads to apoptosis of VSMCs, which are essential for the homeostasis of the aortic wall. The resultant ECM disorders affect aortic structure and function and contribute to the development of ascending aortic aneurysms. In summary, through assessment of clinical samples, we revealed the association between downregulation of YAP in VSMCs and the development of ascending aortic aneurysms, providing new insight into the pathogenesis of this disease. PMID:26904131

  20. Polymorphisms in Fatty Acid Binding Protein 5 Show Association with Type 2 Diabetes

    PubMed Central

    Bu, Liming; Salto, Lorena M.; De Leon, Kevin J; De Leon, Marino

    2011-01-01

    Genes for the fatty acid binding protein (FABP) family encode small 14–15 kDa cytosolic proteins and can be regulated during type 2 diabetes mellitus (T2DM) and obesity. This study compared association of single nucleotide polymorphisms (SNPs) in FABP1-5 with T2DM in different ethnic groups. Associations with T2DM of SNPs in these proteins were assessed in African American (AA), non-Hispanic White (NHW), and Hispanic American (HA) individuals. A total of 650 DNA samples were genotyped; control samples were obtained from Coriell’s North American Human Variation Panel Repository (NAVP) of apparently healthy individuals and T2DM cases were taken from the American Diabetes Association GENNID Study. The rs454550 SNP of FABP5 showed a significant association with T2DM in NHW (OR: 9.03, 95% CI: 1.13–71.73, p=0.014). Our analysis also identified a new FABP5 SNP (nSNP) that showed a significant association with T2DM in NHW (OR: 0.44, 95% CI: 0.19–0.99, p=0.045) and AA (OR: 0.17, 95% CI: 0.03–0.80, p=0.016). The Ala54Thr FABP2 polymorphism was significantly associated with T2DM in HA individuals only (OR: 1.85, 95% CI: 1.05–3.27, p=0.032). All other FABP SNPs did not show association with T2DM. These findings suggest a potential distinct role of SNPs in FABP5, 2 genes in T2DM in different populations. PMID:21288588

  1. Bioinformatic analysis of functional proteins involved in obesity associated with diabetes.

    PubMed

    Rao, Allam Appa; Tayaru, N Manga; Thota, Hanuman; Changalasetty, Suresh Babu; Thota, Lalitha Saroja; Gedela, Srinubabu

    2008-03-01

    The twin epidemic of diabetes and obesity pose daunting challenges worldwide. The dramatic rise in obesity-associated diabetes resulted in an alarming increase in the incidence and prevalence of obesity an important complication of diabetes. Differences among individuals in their susceptibility to both these conditions probably reflect their genetic constitutions. The dramatic improvements in genomic and bioinformatic resources are accelerating the pace of gene discovery. It is tempting to speculate the key susceptible genes/proteins that bridges diabetes mellitus and obesity. In this regard, we evaluated the role of several genes/proteins that are believed to be involved in the evolution of obesity associated diabetes by employing multiple sequence alignment using ClustalW tool and constructed a phylogram tree using functional protein sequences extracted from NCBI. Phylogram was constructed using Neighbor-Joining Algorithm a bioinformatic tool. Our bioinformatic analysis reports resistin gene as ominous link with obesity associated diabetes. This bioinformatic study will be useful for future studies towards therapeutic inventions of obesity associated type 2 diabetes. PMID:23675069

  2. Targeting Toxoplasma Tubules: Tubulin, Microtubules, and Associated Proteins in a Human Pathogen

    PubMed Central

    2014-01-01

    Toxoplasma gondii is an obligate intracellular parasite that causes serious opportunistic infections, birth defects, and blindness in humans. Microtubules are critically important components of diverse structures that are used throughout the Toxoplasma life cycle. As in other eukaryotes, spindle microtubules are required for chromosome segregation during replication. Additionally, a set of membrane-associated microtubules is essential for the elongated shape of invasive “zoites,” and motility follows a spiral trajectory that reflects the path of these microtubules. Toxoplasma zoites also construct an intricate, tubulin-based apical structure, termed the conoid, which is important for host cell invasion and associates with proteins typically found in the flagellar apparatus. Last, microgametes specifically construct a microtubule-containing flagellar axoneme in order to fertilize macrogametes, permitting genetic recombination. The specialized roles of these microtubule populations are mediated by distinct sets of associated proteins. This review summarizes our current understanding of the role of tubulin, microtubule populations, and associated proteins in Toxoplasma; these components are used for both novel and broadly conserved processes that are essential for parasite survival. PMID:25380753

  3. The SAP, a new family of proteins, associate and function positively with the SIT4 phosphatase.

    PubMed Central

    Luke, M M; Della Seta, F; Di Como, C J; Sugimoto, H; Kobayashi, R; Arndt, K T

    1996-01-01

    SIT4 is the catalytic subunit of a type 2A-related protein phosphatase in Saccharomyces cerevisiae that is required for G1 cyclin transcription and for bud formation. SIT4 associates with several high-molecular-mass proteins in a cell cycle-dependent fashion. We purified two SIT4-associated proteins, SAP155 and SAP190, and cloned the corresponding genes. By sequence homology, we isolated two additional SAP genes, SAP185 and SAP4. Through such an association is not yet proven for SAP4, each of SAP155, SAP185, and SAP190 physically associates with SIT4 in separate complexes. The SAPs function positively with SIT4, and by several criteria, the loss of all four SAPs is equivalent to the loss of SIT4. The data suggest that the SAPs are not functional in the absence of SIT4 and likewise that SIT4 is not functional in the absence of the SAPs. The SAPs are hyperphoshorylated in cells lacking SIT4, raising the possibility that the SAPs are substrates of SIT4. By sequence similarity, the SAPs fall into two groups, the SAP4/SAP155 group and the SAP185/SAP190 group. Overexpression of a SAP from one group does not suppress the defects due to the loss of the other group. These findings and others indicate that the SAPs have distinct functions. PMID:8649382

  4. Protein self-association induced by macromolecular crowding: a quantitative analysis by magnetic relaxation dispersion.

    PubMed

    Snoussi, Karim; Halle, Bertil

    2005-04-01

    In the presence of high concentrations of inert macromolecules, the self-association of proteins is strongly enhanced through an entropic, excluded-volume effect variously called macromolecular crowding or depletion attraction. Despite the predicted large magnitude of this universal effect and its far-reaching biological implications, few experimental studies of macromolecular crowding have been reported. Here, we introduce a powerful new technique, fast field-cycling magnetic relaxation dispersion, for investigating crowding effects on protein self-association equilibria. By recording the solvent proton spin relaxation rate over a wide range of magnetic field strengths, we determine the populations of coexisting monomers and decamers of bovine pancreatic trypsin inhibitor in the presence of dextran up to a macromolecular volume fraction of 27%. Already at a dextran volume fraction of 14%, we find a 30-fold increase of the decamer population and 510(5)-fold increase of the association constant. The analysis of these results, in terms of a statistical-mechanical model that incorporates polymer flexibility as well as the excluded volume of the protein, shows that the dramatic enhancement of bovine pancreatic trypsin inhibitor self-association can be quantitatively rationalized in terms of hard repulsive interactions. PMID:15665132

  5. Exploring the association between interleukin-1β and its interacting proteins in Alzheimer’s disease

    PubMed Central

    XIE, LUSHUANG; LAI, YU; LEI, FANG; LIU, SUJUAN; LIU, RAN; WANG, TINGHUA

    2015-01-01

    Alzheimer’s disease (AD) is an age-associated progressive neurodegenerative disorder which is of clinical concern. The association between the nervous and immune system is defined as an neuroimmunological theory that supports a model of pathology or treatment for AD. Interleukin (IL)-1β has a pro-inflammatory function in AD; however, the mechanism of its dysregulation in AD remains unknown. It is therefore of significance to understand the molecular mechanisms of IL-1β and how it may regulate AD. Proteins, which have been previously reported to be associated with IL-1β in AD, have been used in the present study as nodes to illustrate a net of protein interaction in Cytoscape. The Kyoto Encyclopedia of Genes and Genomes was used to further analyze the association of these proteins with the pathology of AD. The present study identified and subsequently compared two AD and six IL-1β pathways with the network produced in Cytoscape. The present study identified important mechanisms in the pathology of AD and constructed two novel networks using Cytoscape. PMID:25585621

  6. Systematic Identification and Characterization of Novel Human Skin-Associated Genes Encoding Membrane and Secreted Proteins

    PubMed Central

    Buhren, Bettina Alexandra; Martinez, Cynthia; Schrumpf, Holger; Gasis, Marcia; Grether-Beck, Susanne; Krutmann, Jean

    2013-01-01

    Through bioinformatics analyses of a human gene expression database representing 105 different tissues and cell types, we identified 687 skin-associated genes that are selectively and highly expressed in human skin. Over 50 of these represent uncharacterized genes not previously associated with skin and include a subset that encode novel secreted and plasma membrane proteins. The high levels of skin-associated expression for eight of these novel therapeutic target genes were confirmed by semi-quantitative real time PCR, western blot and immunohistochemical analyses of normal skin and skin-derived cell lines. Four of these are expressed specifically by epidermal keratinocytes; two that encode G-protein-coupled receptors (GPR87 and GPR115), and two that encode secreted proteins (WFDC5 and SERPINB7). Further analyses using cytokine-activated and terminally differentiated human primary keratinocytes or a panel of common inflammatory, autoimmune or malignant skin diseases revealed distinct patterns of regulation as well as disease associations that point to important roles in cutaneous homeostasis and disease. Some of these novel uncharacterized skin genes may represent potential biomarkers or drug targets for the development of future diagnostics or therapeutics. PMID:23840300

  7. Differential proteomic analysis of Clostridium perfringens ATCC13124; identification of dominant, surface and structure associated proteins

    PubMed Central

    2009-01-01

    Background Clostridium perfringens is a medically important clostridial pathogen causing diseases in man and animals. To invade, multiply and colonize tissues of the host, a pathogen must be able to evade host immune system, and obtain nutrients essential for growth. The factors involved in these complex processes are largely unknown and of crucial importance to understanding microbial pathogenesis. Many of the virulence determinants and putative vaccine candidates for bacterial pathogens are known to be surface localized. Results Using 2-DE mass spectrometry strategy, we identified major surface (22) and cell envelope (10) proteins from Clostridium perfringens ATCC13124 and those differentially expressed (11) in cells grown on cooked meat medium (CMM) in comparison with cells grown in reference state (tryptose-yeast extract-glucose medium). Riboflavin biosynthesis protein, ornithine carbamoyltransferase, cystathionine beta-lyase, and threonine dehydratase were the predominant proteins that exhibited 2.19 to 8.5 fold increase in the expression level in cells growing on CMM. Conclusion Ornithine carbamoyltransferase and cystathionine beta-lyase were over-expressed in cells grown on cooked meat medium and also identified in the surface protein fraction and the former was immunogenic; making them potential vaccine candidates. Based upon bioinformatic analysis; choloylglycine hydrolase family protein, cell wall-associated serine proteinase, and rhomboid family protein were predicted as surface protein markers for specific detection of C. perfringens from the environment and food. Most of the proteins over-expressed in CMM were shown to have putative function in metabolism, of which seven were involved in amino acid transport and metabolism or lipid metabolism. PMID:19664283

  8. Topogenesis of a nucleolar protein: determination of molecular segments directing nucleolar association.

    PubMed

    Zirwes, R F; Kouzmenko, A P; Peters, J M; Franke, W W; Schmidt-Zachmann, M S

    1997-02-01

    To identify the element(s) in nucleolar proteins which determine nucleolus-specific topogenesis, we have used different kinds of cDNA constructs encoding various chimeric combinations of mutants of the constitutive nucleolar protein NO38 (B23): 1) with an amino terminally placed short "myc tag"; 2) with two different carboxyl terminally attached large alpha-helical coiled coil structures, the lamin A rod domain or the rod domain of vimentin; 3) with the sequence-related nucleoplasmic histone-binding protein nucleo-plasmin; and 4) with the soluble cytoplasmic protein pyruvate kinase. To avoid the problem of formation of complexes with endogenous wild-type (wt) molecules and "piggyback" localization, special care was taken to secure that the mutants and chimeras used did not oligomerize as is typical of protein NO38 (B23). Using microinjection and transfection of cultured cells, we found that the segment comprising the amino-terminal 123 amino acids (aa) alone was sufficient to effect nucleolar accumulation of the construct molecules, including the chimeras with the entire rod domains of lamin A and vimentin. However, when the amino-terminal 109 aa were deleted, the molecules still associated with the nucleolus. The results of further deletion experiments and of domain swaps with nucleoplasmin all point to the topogenic importance of two independent molecular regions located at both the amino- and carboxyl-terminal end. Our definition of dominant elements determining the nucleolar localization of protein NO38 (B23) as well as of diverse nonnucleolar proteins will help to identify its local binding partner(s) and functions, the construction of probes examining other proteins or sequence elements within the nucleolar microenvironment, and the generation of cells with an altered nuclear architecture. PMID:9190204

  9. Identification of proteins associated with the yeast mitochondrial RNA polymerase by tandem affinity purification

    PubMed Central

    Markov, Dmitriy A; Savkina, Maria; Anikin, Michael; Del Campo, Mark; Ecker, Karen; Lambowitz, Alan M; De Gnore, Jon P; McAllister, William T

    2009-01-01

    The abundance of mitochondrial (mt) transcripts varies under different conditions, and is thought to depend upon rates of transcription initiation, transcription termination/attenuation and RNA processing/degradation. The requirement to maintain the balance between RNA synthesis and processing may involve coordination between these processes; however, little is known about factors that regulate the activity of mtRNA polymerase (mtRNAP). Recent attempts to identify mtRNAP–protein interactions in yeast by means of a generalized tandem affinity purification (TAP) protocol were not successful, most likely because they involved a C-terminal mtRNAP–TAP fusion (which is incompatible with mtRNAP function) and because of the use of whole-cell solubilization protocols that did not preserve the integrity of mt protein complexes. Based upon the structure of T7 RNAP (to which mtRNAPs show high sequence similarity), we identified positions in yeast mtRNAP that allow insertion of a small affinity tag, confirmed the mature N-terminus, constructed a functional N-terminal TAP–mtRNAP fusion, pulled down associated proteins, and identified them by LC–MS–MS. Among the proteins found in the pull-down were a DEAD-box protein (Mss116p) and an RNA-binding protein (Pet127p). Previous genetic experiments suggested a role for these proteins in linking transcription and RNA degradation, in that a defect in the mt degradadosome could be suppressed by overexpression of either of these proteins or, independently, by mutations in either mtRNAP or its initiation factor Mtf1p. Further, we found that Mss116p inhibits transcription by mtRNAP in vitro in a steady-state reaction. Our results support the hypothesis that Mss116p and Pet127p are involved in modulation of mtRNAP activity. Copyright © 2009 John Wiley & Sons, Ltd. PMID:19536766

  10. Age- and Hypertension-Associated Protein Aggregates in Mouse Heart Have Similar Proteomic Profiles.

    PubMed

    Ayyadevara, Srinivas; Mercanti, Federico; Wang, Xianwei; Mackintosh, Samuel G; Tackett, Alan J; Prayaga, Sastry V S; Romeo, Francesco; Shmookler Reis, Robert J; Mehta, Jawahar L

    2016-05-01

    Neurodegenerative diseases are largely defined by protein aggregates in affected tissues. Aggregates contain some shared components as well as proteins thought to be specific for each disease. Aggregation has not previously been reported in the normal, aging heart or the hypertensive heart. Detergent-insoluble protein aggregates were isolated from mouse heart and characterized on 2-dimensional gels. Their levels increased markedly and significantly with aging and after sustained angiotensin II-induced hypertension. Of the aggregate components identified by high-resolution proteomics, half changed in abundance with age (392/787) or with sustained hypertension (459/824), whereas 30% (273/901) changed concordantly in both, each P<0.05. One fifth of these proteins were previously associated with age-progressive neurodegenerative or cardiovascular diseases, or both (eg, ApoE, ApoJ, ApoAIV, clusterin, complement C3, and others involved in stress-response and protein-homeostasis pathways). Because fibrosis is a characteristic of both aged and hypertensive hearts, we posited that aging of fibroblasts may contribute to the aggregates observed in cardiac tissue. Indeed, as cardiac myofibroblasts "senesced" (approached their replicative limit) in vitro, they accrued aggregates with many of the same constituent proteins observed in vivo during natural aging or sustained hypertension. In summary, we have shown for the first time that compact (detergent-insoluble) protein aggregates accumulate during natural aging, chronic hypertension, and in vitro myofibroblast senescence, sharing many common proteins. Thus, aggregates that arise from disparate causes (aging, hypertension, and replicative senescence) may have common underlying mechanisms of accrual. PMID:26975704

  11. Structural proteins of Kaposi's sarcoma-associated herpesvirus antagonize p53-mediated apoptosis.

    PubMed

    Chudasama, P; Konrad, A; Jochmann, R; Lausen, B; Holz, P; Naschberger, E; Neipel, F; Britzen-Laurent, N; Stürzl, M

    2015-01-29

    The tumor suppressor p53 is a central regulatory molecule of apoptosis and is commonly mutated in tumors. Kaposi's sarcoma-associated herpesvirus (KSHV)-related malignancies express wild-type p53. Accordingly, KSHV encodes proteins that counteract the cell death-inducing effects of p53. Here, the effects of all KSHV genes on the p53 signaling pathway were systematically analyzed using the reversely transfected cell microarray technology. With this approach we detected eight KSHV-encoded genes with potent p53 inhibiting activity in addition to the previously described inhibitory effects of KSHV genes ORF50, K10 and K10.5. Interestingly, the three most potent newly identified inhibitors were KSHV structural proteins, namely ORF22 (glycoprotein H), ORF25 (major capsid protein) and ORF64 (tegument protein). Validation of these results with a classical transfection approach showed that these proteins inhibited p53 signaling in a dose-dependent manner and that this effect could be reversed by small interfering RNA-mediated knockdown of the respective viral gene. All three genes inhibited p53-mediated apoptosis in response to Nutlin-3 treatment in non-infected and KSHV-infected cells. Addressing putative mechanisms, we could show that these proteins could also inhibit the transactivation of the promoters of apoptotic mediators of p53 such as BAX and PIG3. Altogether, we demonstrate for the first time that structural proteins of KSHV can counteract p53-induced apoptosis. These proteins are expressed in the late lytic phase of the viral life cycle and are incorporated into the KSHV virion. Accordingly, these genes may inhibit cell death in the productive and in the early entrance phase of KSHV infection. PMID:24469037

  12. Association of brominated proteins and changes in protein expression in the rat kidney with subcarcinogenic to carcinogenic doses of bromate

    SciTech Connect

    Kolisetty, Narendrababu; Bull, Richard J.; Muralidhara, Srinivasa; Costyn, Leah J.; Delker, Don A.; Guo, Zhongxian; Cotruvo, Joseph A.; Fisher, Jeffrey W.; Cummings, Brian S.

    2013-10-15

    The water disinfection byproduct bromate (BrO{sub 3}{sup −}) produces cytotoxic and carcinogenic effects in rat kidneys. Our previous studies demonstrated that BrO{sub 3}{sup −} caused sex-dependent differences in renal gene and protein expression in rats and the elimination of brominated organic carbon in their urine. The present study examined changes in renal cell apoptosis and protein expression in male and female F344 rats treated with BrO{sub 3}{sup −} and associated these changes with accumulation of 3-bromotyrosine (3-BT)-modified proteins. Rats were treated with 0, 11.5, 46 and 308 mg/L BrO{sub 3}{sup −} in drinking water for 28 days and renal sections were prepared and examined for apoptosis (TUNEL-staining), 8-oxo-deoxyguanosine (8-oxoG), 3-BT, osteopontin, Kim-1, clusterin, and p-21 expression. TUNEL-staining in renal proximal tubules increased in a dose-related manner beginning at 11.5 mg BrO{sub 3}{sup −}/L in female rats and 46 mg/L in males. Increased 8-oxoG staining was observed at doses as low as 46 mg/L. Osteopontin expression also increased in a dose-related manner after treatment with 46 mg/L, in males only. In contrast, Kim-1 expression increased in a dose-related manner in both sexes, although to a greater extent in females at the highest dose. Clusterin and p21 expression also increased in a dose-related manner in both sexes. The expression of 3-BT-modified proteins only increased in male rats, following a pattern previously reported for accumulation of α-2{sub u}-globulin. Increases in apoptosis in renal proximal tubules of male and female rats at the lowest doses suggest a common mode of action for renal carcinogenesis for the two sexes that is independent of α-2{sub u}-globulin nephropathy. - Highlights: • Bromate induced nephrotoxicity in both male and female rats by similar mechanisms. • Apoptosis was seen in both male and female rats at the lowest doses tested. • Bromate-induced apoptosis correlated to 8-oxo

  13. Genetic Variation and Association Mapping of Protein Concentration in Brown Rice Using a Diverse Rice Germplasm Collection

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Protein is the second most abundant constituent in the rice grain next to starch. Association analysis for protein concentration in brown rice was performed using a “Mini-Core” collection, which represents the germplasm diversity found in the USDA world rice collection. Protein concentration was det...

  14. Quantification of DNA-associated proteins inside eukaryotic cells using single-molecule localization microscopy

    PubMed Central

    Etheridge, Thomas J.; Boulineau, Rémi L.; Herbert, Alex; Watson, Adam T.; Daigaku, Yasukazu; Tucker, Jem; George, Sophie; Jönsson, Peter; Palayret, Matthieu; Lando, David; Laue, Ernest; Osborne, Mark A.; Klenerman, David; Lee, Steven F.; Carr, Antony M.

    2014-01-01

    Development of single-molecule localization microscopy techniques has allowed nanometre scale localization accuracy inside cells, permitting the resolution of ultra-fine cell structure and the elucidation of crucial molecular mechanisms. Application of these methodologies to understanding processes underlying DNA replication and repair has been limited to defined in vitro biochemical analysis and prokaryotic cells. In order to expand these techniques to eukaryotic systems, we have further developed a photo-activated localization microscopy-based method to directly visualize DNA-associated proteins in unfixed eukaryotic cells. We demonstrate that motion blurring of fluorescence due to protein diffusivity can be used to selectively image the DNA-bound population of proteins. We designed and tested a simple methodology and show that it can be used to detect changes in DNA binding of a replicative helicase subunit, Mcm4, and the replication sliding clamp, PCNA, between different stages of the cell cycle and between distinct genetic backgrounds. PMID:25106872

  15. Hook Proteins: Association with Alzheimer Pathology and Regulatory Role of Hook3 in Amyloid Beta Generation

    PubMed Central

    Arsalan-Werner, Annika; Hilbrich, Isabel; Jäger, Carsten; Flach, Katharina; Suttkus, Anne; Lachmann, Ingolf; Arendt, Thomas; Holzer, Max

    2015-01-01

    Defects in intracellular transport are implicated in the pathogenesis of Alzheimer’s disease (AD). Hook proteins are a family of cytoplasmic linker proteins that participate in endosomal transport. In this study we show that Hook1 and Hook3 are expressed in neurons while Hook2 is predominantly expressed in astrocytes. Furthermore, Hook proteins are associated with pathological hallmarks in AD; Hook1 and Hook3 are localized to tau aggregates and Hook2 to glial components within amyloid plaques. Additionally, the expression of Hook3 is reduced in AD. Modelling of Hook3 deficiency in cultured cells leads to slowing of endosomal transport and increases β-amyloid production. We propose that Hook3 plays a role in pathogenic events exacerbating AD. PMID:25799409

  16. Antibodies to myelin basic protein are associated with cognitive decline after stroke.

    PubMed

    Becker, Kyra J; Tanzi, Patricia; Zierath, Dannielle; Buckwalter, Marion S

    2016-06-15

    B lymphocytes cause post-stroke cognitive decline in mice. We therefore evaluated the association between autoantibodies and post-stroke cognitive decline in a prospectively collected human cohort. The mini-mental state exam (MMSE) was administered 30, 90, 180, and 365days after stroke. Antibody titers to myelin basic protein (MBP), proteolipid protein, and several non-specific proteins were determined. Among 58 subjects with initial MMSE≥20 and at least 2 MMSE examinations in the year after stroke, cognitive decline (MMSE decrease ≥2) occurred in 10 (17%) subjects. In multivariate analysis, MBP antibody titers were the only independent predictor of cognitive decline (OR=9.02 [1.18, 68.90]; P=0.03). PMID:27235342

  17. Evaluation of molecular weight distribution of unreduced wheat gluten proteins associated with noodle quality.

    PubMed

    Chaudhary, Nisha; Dangi, Priya; Khatkar, B S

    2016-06-01

    Unreduced gluten proteins of Indian wheat varieties viz.C306, DBW16, HI977 and HW2004 were separated using size-exclusion chromatography (SEC). Statistical correlation of area % of eluted peaks with properties of flour, dough and noodles was elucidated. Chromatograms of gluten proteins were classified primarily into five peaks in decreasing molecular size range and relative proportion were expressed in terms of area % of individual peaks which depicts the quantitative variation in protein eluted at different retention times. Cooking time and cooked weight of noodles depicted positive correlation with peak I and negative correlation with peak II which predominantly composed of glutenins and gliadins, respectively. Oil uptake and cooking loss were negatively association with peak I and positively with peak II. Noodle hardness, springiness, cohesiveness and chewiness were positively correlated with peak I and negatively to peak II, though adhesiveness was unaffected by SEC eluted peaks statistically. PMID:27478225

  18. The sequence, and its evolutionary implications, of a Thermococcus celer protein associated with transcription

    NASA Technical Reports Server (NTRS)

    Kaine, B. P.; Mehr, I. J.; Woese, C. R.

    1994-01-01

    Through random search, a gene from Thermococcus celer has been identified and sequenced that appears to encode a transcription-associated protein (110 amino acid residues). The sequence has clear homology to approximately the last half of an open reading frame reported previously for Sulfolobus acidocaldarius [Langer, D. & Zillig, W. (1993) Nucleic Acids Res. 21, 2251]. The protein translations of these two archaeal genes in turn are homologs of a small subunit found in eukaryotic RNA polymerase I (A12.2) and the counterpart of this from RNA polymerase II (B12.6). Homology is also seen with the eukaryotic transcription factor TFIIS, but it involves only the terminal 45 amino acids of the archaeal proteins. Evolutionary implications of these homologies are discussed.

  19. Molecular cloning, genomic organization, and chromosomal localization of the human pancreatitis-associated protein (PAP) gene

    SciTech Connect

    Dusetti, N.J.; Frigerio, J.M.; Dagorn, J.C.; Iovanna, J.L. ); Fox, M.F.; Swallow, D.M. )

    1994-01-01

    Pancreatitis-associated protein (PAP) is a secretory pancreatic protein present in small amounts in normal pancreas and overexpressed during the acute phase of pancreatitis. In this paper, the authors describe the cloning, characterization, and chromosomal mapping of the human PAP gene. The gene spans 2748 bp and contains six exons interrupted by five introns. The gene has a typical promoter containing the sequences TATAAA and CCAAT 28 and 52 bp upstream of the cap site, respectively. They found striking similarities in genomic organization as well as in the promoter sequences between the human and rat PAP genes. The human PAP gene was mapped to chromosome 2p12 using rodent-human hybrid cells and in situ chromosomal hybridization. This localization coincides with that of the reg/lithostathine gene, which encodes a pancreatic secretory protein structurally related to PAP, suggesting that both genes derived from the same ancestral gene by duplication. 35 refs., 4 figs., 1 tab.

  20. Heterotypic and homotypic associations between ezrin and moesin, two putative membrane-cytoskeletal linking proteins.

    PubMed Central

    Gary, R; Bretscher, A

    1993-01-01

    Ezrin and moesin are components of actin-rich cell surface structures that are thought to function as membrane-cytoskeletal linking proteins. Here we show that a stable complex of ezrin and moesin can be isolated from cultured cells by immunoprecipitation with specific antibodies. The capacity of these two proteins to interact directly was confirmed with a blot-overlay procedure in which biotin-tagged proteins in solution were incubated with immobilized binding partners. In addition to the heterotypic association of ezrin and moesin, homotypic binding of ezrin to ezrin and of moesin to moesin was also demonstrated in vitro. These results suggest mechanisms by which ezrin and moesin might participate in dynamic aspects of cortical cytoskeletal structure. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 PMID:8248180

  1. Heat-shock protein dysregulation is associated with functional and pathological TDP-43 aggregation

    NASA Astrophysics Data System (ADS)

    Chang, Hsiang-Yu; Hou, Shin-Chen; Way, Tzong-Der; Wong, Chi-Huey; Wang, I.-Fan

    2013-11-01

    Conformational disorders are involved in various neurodegenerative diseases. Reactive oxygen species (ROS) are the major contributors to neurodegenerative disease; however, ROS that affect the structural changes in misfolded disease proteins have yet to be well characterized. Here we demonstrate that the intrinsic propensity of TDP-43 to aggregate drives the assembly of TDP-43-positive stress granules and soluble toxic TDP-43 oligomers in response to a ROS insult via a disulfide crosslinking-independent mechanism. Notably, ROS-induced TDP-43 protein assembly correlates with the dynamics of certain TDP-43-associated chaperones. The heat-shock protein (HSP)-90 inhibitor 17-AAG prevents ROS-induced TDP-43 aggregation, alters the type of TDP-43 multimers and reduces the severity of pathological TDP-43 inclusions. In summary, our study suggests that a common mechanism could be involved in the pathogenesis of conformational diseases that result from HSP dysregulation.

  2. Operon structure and cotranslational subunit association direct protein assembly in bacteria.

    PubMed

    Shieh, Yu-Wei; Minguez, Pablo; Bork, Peer; Auburger, Josef J; Guilbride, D Lys; Kramer, Günter; Bukau, Bernd

    2015-11-01

    Assembly of protein complexes is considered a posttranslational process involving random collision of subunits. We show that within the Escherichia coli cytosol, bacterial luciferase subunits LuxA and LuxB assemble into complexes close to the site of subunit synthesis. Assembly efficiency decreases markedly if subunits are synthesized on separate messenger RNAs from genes integrated at distant chromosomal sites. Subunit assembly initiates cotranslationally on nascent LuxB in vivo. The ribosome-associated chaperone trigger factor delays the onset of cotranslational interactions until the LuxB dimer interface is fully exposed. Protein assembly is thus directly coupled to the translation process and involves spatially confined, actively chaperoned cotranslational subunit interactions. Bacterial gene organization into operons therefore reflects a fundamental cotranslational mechanism for spatial and temporal regulation that is vital to effective assembly of protein complexes. PMID:26405228

  3. Exploring systems affected by the heat shock response in Plasmodium falciparum via protein association networks

    PubMed Central

    Lilburn, Timothy G.; Cai, Hong; Gu, Jianying; Zhou, Zhan; Wang, Yufeng

    2015-01-01

    The heat shock response is a general mechanism by which organisms deal with physical insults such as sudden changes in temperature, osmotic and oxidative stresses, and exposure to toxic substances. Plasmodium falciparum is exposed to drastic temperature changes as a part of its life cycle and maintains an extensive repertoire of heat shock response-related proteins. As these proteins serve to maintain the parasite in the face of anti-malarial drugs as well, better understanding of the heat shock-related systems in the malaria parasite will lead to therapeutic approaches that frustrate these systems, leading to more effective use of anti-malarials. Here we use protein association networks to broaden our understanding of the systems impacted by and/or implicated in the heat shock response. PMID:25539848

  4. Differential label-free quantitative proteomic analysis of avian eggshell matrix and uterine fluid proteins associated with eggshell mechanical property.

    PubMed

    Sun, Congjiao; Xu, Guiyun; Yang, Ning

    2013-12-01

    Eggshell strength is a crucial economic trait for table egg production. During the process of eggshell formation, uncalcified eggs are bathed in uterine fluid that plays regulatory roles in eggshell calcification. In this study, a label-free MS-based protein quantification technology was used to detect differences in protein abundance between eggshell matrix from strong and weak eggs (shell matrix protein from strong eggshells and shell matrix protein from weak eggshells) and between the corresponding uterine fluids bathing strong and weak eggs (uterine fluid bathing strong eggs and uterine fluid bathing weak eggs) in a chicken population. Here, we reported the first global proteomic analysis of uterine fluid. A total of 577 and 466 proteins were identified in uterine fluid and eggshell matrix, respectively. Of 447 identified proteins in uterine fluid bathing strong eggs, up to 357 (80%) proteins were in common with proteins in uterine fluid bathing weak eggs. Similarly, up to 83% (328/396) of the proteins in shell matrix protein from strong eggshells were in common with the proteins in shell matrix protein from weak eggshells. The large amount of common proteins indicated that the difference in protein abundance should play essential roles in influencing eggshell strength. Ultimately, 15 proteins mainly relating to eggshell matrix specific proteins, calcium binding and transportation, protein folding and sorting, bone development or diseases, and thyroid hormone activity were considered to have closer association with the formation of strong eggshell. PMID:24151251

  5. Identification of Membrane Protein Associated with Sucrose Transport Into Cells of Developing Soybean Cotyledons 1

    PubMed Central

    Ripp, Kevin G.; Viitanen, Paul V.; Hitz, William D.; Franceschi, Vincent R.

    1988-01-01

    The photolyzable sucrose derivative 6′-deoxy-6′-(4-azido-2-hydroxy)-benzamidosucrose (6′-HABS), competitively inhibited the influx of [14C] sucrose into protoplasts from developing soybean (Glycine max L. Merr cv Wye) cotyledons. Photolysis of 125I-labeled 6′-HABS in the presence of 10 millimolar dithiothreitol and microsomal preparations from developing soybean cotyledons led to label incorporation into a moderately abundant membrane protein with an apparent molecular mass of about 62 kilodalton (kD) by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The 62 kD protein was partially protected from labeling by the inclusion of 100 millimolar sucrose in the photolysis medium and also by the inclusion of 10 millimolar phenyl α-d-thioglucopyranoside. Glucose, raffinose, or phenyl α-d-3-deoxy-3-fluoroglucopyranoside did not afford even partial protection from labeling. When the photolyzable moiety of 6′-HABS was attached to 6-deoxy-6-aminoglucose and 125I labeled, the resulting photoprobe did not label the 62 kD protein above background. The labeled protein at 62 kD is therefore apparently a specific, sucrose binding protein. Sucrose influx into cotlyedons of less than 25 milligrams fresh weight (approximately 10 days after flowering) occurred by passive processes, but metabolically dependent uptake became dominant over the next 5 to 7 days of development. Both the Coomassie staining protein at 62 kD and label incorporation at that position in analysis of membrane proteins appeared concomitant with the onset of active sucrose influx. Polyclonal antibodies to the purified 62 kD protein bound specifically to a protein in the plasmalemma of thin sections prepared from cotyledons and density stained with colloidal gold-protein A. The results suggest that the 62 kD membrane protein is associated with sucrose transport and may be the plasmalemma sucrose transporter. Images Fig. 2 Fig. 5 Fig. 6 Fig. 7 Fig. 8 Fig. 9 Fig. 10 Fig. 11 Fig. 12 Fig. 13 Fig. 14

  6. Neuroacanthocytosis associated with a defect of the 4.1R membrane protein

    PubMed Central

    Orlacchio, Antonio; Calabresi, Paolo; Rum, Adriana; Tarzia, Anna; Salvati, Anna Maria; Kawarai, Toshitaka; Stefani, Alessandro; Pisani, Antonio; Bernardi, Giorgio; Cianciulli, Paolo; Caprari, Patrizia

    2007-01-01

    Background Neuroacanthocytosis (NA) denotes a heterogeneous group of diseases that are characterized by nervous system abnormalities in association with acanthocytosis in the patients' blood. The 4.1R protein of the erythrocyte membrane is critical for the membrane-associated cytoskeleton structure and in central neurons it regulates the stabilization of AMPA receptors on the neuronal surface at the postsynaptic density. We report clinical, biochemical, and genetic features in four patients from four unrelated families with NA in order to explain the cause of morphological abnormalities and the relationship with neurodegenerative processes. Case presentation All patients were characterised by atypical NA with a novel alteration of the erythrocyte membrane: a 4.1R protein deficiency. The 4.1R protein content was significantly lower in patients (3.40 ± 0.42) than in controls (4.41 ± 0.40, P < 0.0001), reflecting weakened interactions of the cytoskeleton with the membrane. In patients IV:1 (RM23), IV:3 (RM15), and IV:6 (RM16) the 4.1 deficiency seemed to affect the horizontal interactions of spectrin and an impairment of the dimer self-association into tetramers was detected. In patient IV:1 (RM16) the 4.1 deficiency seemed to affect the skeletal attachment to membrane and the protein band 3 was partially reduced. Conclusion A decreased expression pattern of the 4.1R protein was observed in the erythrocytes from patients with atypical NA, which might reflect the expression pattern in the central nervous system, especially basal ganglia, and might lead to dysfunction of AMPA-mediated glutamate transmission. PMID:17298666

  7. DENSE: efficient and prior knowledge-driven discovery of phenotype-associated protein functional modules

    PubMed Central

    2011-01-01

    Background Identifying cellular subsystems that are involved in the expression of a target phenotype has been a very active research area for the past several years. In this paper, cellular subsystem refers to a group of genes (or proteins) that interact and carry out a common function in the cell. Most studies identify genes associated with a phenotype on the basis of some statistical bias, others have extended these statistical methods to analyze functional modules and biological pathways for phenotype-relatedness. However, a biologist might often have a specific question in mind while performing such analysis and most of the resulting subsystems obtained by the existing methods might be largely irrelevant to the question in hand. Arguably, it would be valuable to incorporate biologist's knowledge about the phenotype into the algorithm. This way, it is anticipated that the resulting subsytems would not only be related to the target phenotype but also contain information that the biologist is likely to be interested in. Results In this paper we introduce a fast and theoretically guranteed method called DENSE (Dense and ENriched Subgraph Enumeration) that can take in as input a biologist's prior knowledge as a set of query proteins and identify all the dense functional modules in a biological network that contain some part of the query vertices. The density (in terms of the number of network egdes) and the enrichment (the number of query proteins in the resulting functional module) can be manipulated via two parameters γ and μ, respectively. Conclusion This algorithm has been applied to the protein functional association network of Clostridium acetobutylicum ATCC 824, a hydrogen producing, acid-tolerant organism. The algorithm was able to verify relationships known to exist in literature and also some previously unknown relationships including those with regulatory and signaling functions. Additionally, we were also able to hypothesize that some uncharacterized

  8. Hippocampal dysregulation of synaptic plasticity-associated proteins with age-related cognitive decline

    PubMed Central

    VanGuilder, Heather D.; Farley, Julie A.; Yan, Han; Van Kirk, Colleen A.; Mitschelen, Matthew; Sonntag, William E.; Freeman, Willard M.

    2011-01-01

    Age-related cognitive decline occurs without frank neurodegeneration and is the most common cause of memory impairment in aging individuals. With increasing longevity, cognitive deficits, especially in hippocampus-dependent memory processes, are increasing in prevalence. Nevertheless, the neurobiological basis of age-related cognitive decline remains unknown. While concerted efforts have led to the identification of neurobiological changes with aging, few age-related alterations have been definitively correlated to behavioral measures of cognitive decline. In this work, adult (12 Months) and aged (28 months) rats were categorized by Morris water maze performance as Adult cognitively Intact, Aged cognitively Intact or Aged cognitively Impaired, and protein expression was examined in hippocampal synaptosome preparations. Previously described differences in synaptic expression of neurotransmission-associated proteins (Dnm1, Hpca, Stx1, Syn1, Syn2, Syp, SNAP25, VAMP2 and 14-3-3 eta, gamma, and zeta) were confirmed between Adult and Aged rats, with no further dysregulation associated with cognitive impairment. Proteins related to synaptic structural stability (MAP2, drebrin, Nogo-A) and activity-dependent signaling (PSD-95, 14-3-3θ, CaMKIIα) were up- and down-regulated, respectively, with cognitive impairment but were not altered with increasing age. Localization of MAP2, PSD-95, and CaMKIIα demonstrated protein expression alterations throughout the hippocampus. The altered expression of activity- and structural stability-associated proteins suggests that impaired synaptic plasticity is a distinct phenomenon that occurs with age-related cognitive decline, and demonstrates that cognitive decline is not simply an exacerbation of the aging phenotype. PMID:21440628

  9. Lymphocyte protein synthesis is increased with the progression of HIV-associated disease to AIDS.

    PubMed

    Caso, G; Garlick, P J; Gelato, M C; McNurlan, M A

    2001-12-01

    HIV infection has been shown to affect lymphocyte function and to reduce lymphocyte responsiveness in vitro to mitogenic stimulation, but little is known about lymphocyte metabolism in vivo and how it is affected during the course of the disease. This study investigated the metabolic activity of lymphocytes in vivo through the progression of HIV-associated disease. Lymphocyte protein synthesis was measured with L-[(2)H(5)]phenylalanine (45 mg/kg body weight) in healthy volunteers (n=7), in patients who were HIV-positive (n=7) but asymptomatic, and in patients with AIDS (n=8). The rates of lymphocyte protein synthesis [expressed as a percentage of lymphocyte protein, i.e. fractional synthesis rate (FSR)] were not altered in HIV-positive patients compared with healthy controls (7.9+/-1.28% and 9.1+/-0.53%/day respectively), but were significantly elevated in AIDS patients (14.0+/-1.16%/day; P<0.05). The serum concentration of the cytokine tumour necrosis factor-alpha (TNF-alpha) increased with the progression of the disease, and TNF-alpha levels were significantly higher in AIDS patients (6.81+/-0.88 ng/l) than in healthy controls (3.09+/-0.27 ng/l; P<0.05). Lymphocyte protein FSR was positively correlated with serum TNF-alpha concentration (r=0.55, P=0.009) and negatively correlated with CD4(+) lymphocyte count (r=-0.70, P=0.004). The elevation of lymphocyte protein synthesis in AIDS patients suggests a higher rate of turnover of lymphocytes. This may be associated with a generalized activation of the immune system, which is also reflected by the elevated serum TNF-alpha concentration in the late stages of HIV-associated disease. PMID:11724643

  10. Identification of extracellular surface-layer associated proteins in Lactobacillus acidophilus NCFM

    PubMed Central

    Johnson, Brant; Selle, Kurt; O’Flaherty, Sarah; Goh, Yong Jun

    2013-01-01

    Bacterial surface (S-) layers are crystalline arrays of self-assembling, proteinaceous subunits called S-layer proteins (Slps), with molecular masses ranging from 40 to 200 kDa. The S-layer-forming bacterium Lactobacillus acidophilus NCFM expresses three major Slps: SlpA (46 kDa), SlpB (47 kDa) and SlpX (51 kDa). SlpA has a demonstrated role in adhesion to Caco-2 intestinal epithelial cells in vitro, and has been shown to modulate dendritic cell (DC) and T-cell functionalities with murine DCs. In this study, a modification of a standard lithium chloride S-layer extraction revealed 37 proteins were solubilized from the S-layer wash fraction. Of these, 30 have predicted cleavage sites for secretion, 24 are predicted to be extracellular, six are lipid-anchored, three have N-terminal hydrophobic membrane spanning regions and four are intracellular, potentially moonlighting proteins. Some of these proteins, designated S-layer associated proteins (SLAPs), may be loosely associated with or embedded within the bacterial S-layer complex. Lba-1029, a putative SLAP gene, was deleted from the chromosome of L. acidophilus. Phenotypic characterization of the deletion mutant demonstrated that the SLAP LBA1029 contributes to a pro-inflammatory TNF-α response from murine DCs. This study identified extracellular proteins and putative SLAPs of L. acidophilus NCFM using LC-MS/MS. SLAPs appear to impart important surface display features and immunological properties to microbes that are coated by S-layers. PMID:24002751

  11. A FRET-based method for monitoring septin polymerization and binding of septin-associated proteins.

    PubMed

    Booth, E A; Thorner, J

    2016-01-01

    Much about septin function has been inferred from in vivo studies using mainly genetic methods, and much of what we know about septin organization has been obtained through examination of static structures in vitro primarily by electron microscopy. Deeper mechanistic insight requires real-time analysis of the dynamics of the assembly of septin-based structures and how other proteins associate with them. We describe here a Förster resonance energy transfer (FRET)-based approach for measuring in vitro the rate and extent of filament formation from septin complexes, binding of other proteins to septin structures, and the apparent affinities of these interactions. FRET is particularly well suited for interrogating protein-protein interactions, especially on a rapid timescale; the spectral change provides an unambiguous indication of whether two elements within the system under study are associating and serves as a molecular-level "ruler" because it is very sensitive to the separation between the donor and acceptor fluorophores over biologically relevant distances (≤10nm). The necessary procedures involve generation of appropriate cysteine-less and single cysteine-containing septin variants, expression and purification of the heterooctameric complexes containing them, efficient labeling of the purified complexes with desired fluorophores, fluorimetric measurement of FRET, and appropriate safeguards and controls in data acquisition and analysis. Our methods can be used to interrogate the effects of buffer conditions, small molecules, and septin-binding proteins on septin filament assembly or stability; determine the effect of alternative septin subunits, mutational alterations, or posttranslational modifications on assembly; and, delineate the location of septin-binding proteins. PMID:27473902

  12. Identification of extracellular surface-layer associated proteins in Lactobacillus acidophilus NCFM.

    PubMed

    Johnson, Brant; Selle, Kurt; O'Flaherty, Sarah; Goh, Yong Jun; Klaenhammer, Todd

    2013-11-01

    Bacterial surface (S-) layers are crystalline arrays of self-assembling, proteinaceous subunits called S-layer proteins (Slps), with molecular masses ranging from 40 to 200 kDa. The S-layer-forming bacterium Lactobacillus acidophilus NCFM expresses three major Slps: SlpA (46 kDa), SlpB (47 kDa) and SlpX (51 kDa). SlpA has a demonstrated role in adhesion to Caco-2 intestinal epithelial cells in vitro, and has been shown to modulate dendritic cell (DC) and T-cell functionalities with murine DCs. In this study, a modification of a standard lithium chloride S-layer extraction revealed 37 proteins were solubilized from the S-layer wash fraction. Of these, 30 have predicted cleavage sites for secretion, 24 are predicted to be extracellular, six are lipid-anchored, three have N-terminal hydrophobic membrane spanning regions and four are intracellular, potentially moonlighting proteins. Some of these proteins, designated S-layer associated proteins (SLAPs), may be loosely associated with or embedded within the bacterial S-layer complex. Lba-1029, a putative SLAP gene, was deleted from the chromosome of L. acidophilus. Phenotypic characterization of the deletion mutant demonstrated that the SLAP LBA1029 contributes to a pro-inflammatory TNF-α response from murine DCs. This study identified extracellular proteins and putative SLAPs of L. acidophilus NCFM using LC-MS/MS. SLAPs appear to impart important surface display features and immunological properties to microbes that are coated by S-layers. PMID:24002751

  13. Dietary protein intake is associated with better physical function and muscle strength among elderly women.

    PubMed

    Isanejad, Masoud; Mursu, Jaakko; Sirola, Joonas; Kröger, Heikki; Rikkonen, Toni; Tuppurainen, Marjo; Erkkilä, Arja T

    2016-04-14

    Dietary protein intake might be beneficial to physical function (PF) in the elderly. We examined the cross-sectional and prospective associations of protein intake of g/kg body weight (BW), fat mass (FM) and lean mass (LM) with PF in 554 women aged 65·3-71·6 years belonging to the Osteoporosis Risk Factor and Prevention Fracture Prevention Study. Participants filled a questionnaire on lifestyle factors and 3-d food record in 2002. Body composition was measured by dual-energy X-ray absorptiometry, and PF measures were performed at baseline and at 3-year follow-up. Sarcopaenia was defined using European Working Group on Sarcopenia in Older People criteria. At the baseline, women with higher protein intake (≥ 1·2 g/kg BW) had better performance in hand-grip strength/body mass (GS/BM) (P=0·001), knee extension/BM (P=0·003), one-leg stance (P=0·047), chair rise (P=0·043), squat (P=0·019), squat to the ground (P=0·001), faster walking speed for 10 m (P=0·005) and higher short physical performance battery score (P=0·004) compared with those with moderate and lower intakes (0·81-1·19 and ≤ 0·8 g/kg BW, respectively). In follow-up results, higher protein intake was associated with less decline in GS/BM, one-leg stance and tandem walk for 6 m over 3 years. Overall, results were no longer significant after controlling for FM. Associations were detected between protein intake and PF in non-sarcopaenic women but not in sarcopaenic women, except for change of GS (P=0·037). Further, FM but not LM was negatively associated with PF measures (P<0·050). This study suggests that higher protein intake and lower FM might be positively associated with PF in elderly women. PMID:26857389

  14. The Association between Total Protein and Vegetable Protein Intake and Low Muscle Mass among the Community-Dwelling Elderly Population in Northern Taiwan.

    PubMed

    Huang, Ru-Yi; Yang, Kuen-Cheh; Chang, Hao-Hsiang; Lee, Long-Teng; Lu, Chia-Wen; Huang, Kuo-Chin

    2016-01-01

    Sarcopenia, highly linked with fall, frailty, and disease burden, is an emerging problem in aging society. Higher protein intake has been suggested to maintain nitrogen balance. Our objective was to investigate whether pre-sarcopenia status was associated with lower protein intake. A total of 327 community-dwelling elderly people were recruited for a cross-sectional study. We adopted the multivariate nutrient density model to identify associations between low muscle mass and dietary protein intake. The general linear regression models were applied to estimate skeletal muscle mass index across the quartiles of total protein and vegetable protein density. Participants with diets in the lowest quartile of total protein density (<13.2%) were at a higher risk for low muscle mass (odds ratio (OR) 3.03, 95% confidence interval (CI) 1.37-6.72) than those with diets in the highest quartile (≥17.2%). Similarly, participants with diets in the lowest quartile of vegetable protein density (<5.8%) were at a higher risk for low muscle mass (OR 2.34, 95% CI 1.14-4.83) than those with diets in the highest quartile (≥9.4%). Furthermore, the estimated skeletal muscle mass index increased significantly across the quartiles of total protein density (p = 0.023) and vegetable protein density (p = 0.025). Increasing daily intakes of total protein and vegetable protein densities appears to confer protection against pre-sarcopenia status. PMID:27322317

  15. The Association between Total Protein and Vegetable Protein Intake and Low Muscle Mass among the Community-Dwelling Elderly Population in Northern Taiwan

    PubMed Central

    Huang, Ru-Yi; Yang, Kuen-Cheh; Chang, Hao-Hsiang; Lee, Long-Teng; Lu, Chia-Wen; Huang, Kuo-Chin

    2016-01-01

    Sarcopenia, highly linked with fall, frailty, and disease burden, is an emerging problem in aging society. Higher protein intake has been suggested to maintain nitrogen balance. Our objective was to investigate whether pre-sarcopenia status was associated with lower protein intake. A total of 327 community-dwelling elderly people were recruited for a cross-sectional study. We adopted the multivariate nutrient density model to identify associations between low muscle mass and dietary protein intake. The general linear regression models were applied to estimate skeletal muscle mass index across the quartiles of total protein and vegetable protein density. Participants with diets in the lowest quartile of total protein density (<13.2%) were at a higher risk for low muscle mass (odds ratio (OR) 3.03, 95% confidence interval (CI) 1.37–6.72) than those with diets in the highest quartile (≥17.2%). Similarly, participants with diets in the lowest quartile of vegetable protein density (<5.8%) were at a higher risk for low muscle mass (OR 2.34, 95% CI 1.14–4.83) than those with diets in the highest quartile (≥9.4%). Furthermore, the estimated skeletal muscle mass index increased significantly across the quartiles of total protein density (p = 0.023) and vegetable protein density (p = 0.025). Increasing daily intakes of total protein and vegetable protein densities appears to confer protection against pre-sarcopenia status. PMID:27322317

  16. TATA-binding protein and associated factors in polymerase II and polymerase III transcription.

    PubMed Central

    Meyers, R E; Sharp, P A

    1993-01-01

    Transcription by RNA polymerase I (pol I), pol II, and pol III requires the TATA-binding protein (TBP). This protein functions in association with distinct TBP-associated factors (TAFs) which may specify the nature of the polymerase selected for initiation at a promoter site. In the pol III transcription system, the TBP-TAF complex is a component of the TFIIIB factor. This factor has been resolved into a TBP-TAF complex and another component, both of which are required for reconstitution of transcription by pol III. Neither the TBP-TAF complexes B-TFIID and D-TFIID, which were previously characterized as active for pol II transcription, nor TBP alone can complement pol III transcription reactions that are dependent upon the TBP-TAF subcomponent of TFIIIB. Surprisingly, the TBP-TAF subcomponent of TFIIIB is active in reconstitution of pol II transcription. Images PMID:8247010

  17. Restoration of dystrophin-associated proteins in skeletal muscle of mdx mice transgenic for dystrophin gene.

    PubMed

    Matsumura, K; Lee, C C; Caskey, C T; Campbell, K P

    1993-04-12

    Duchenne muscular dystrophy (DMD) patients and mdx mice are characterized by the absence of dystrophin, a membrane cytoskeletal protein. Dystrophin is associated with a large oligomeric complex of sarcolemmal glycoproteins, including dystroglycan which provides a linkage to the extracellular matrix component, laminin. The finding that all of the dystrophin-associated proteins (DAPs) are drastically reduced in DMD and mdx skeletal muscle supports the primary function of dystrophin as an anchor of the sarcolemmal glycoprotein complex to the subsarcolemmal cytoskeleton. These findings indicate that the efficacy of dystrophin gene therapy will depend not only on replacing dystrophin but also on restoring all of the DAPs in the sarcolemma. Here we have investigated the status of the DAPs in the skeletal muscle of mdx mice transgenic for the dystrophin gene. Our results demonstrate that transfer of dystrophin gene restores all of the DAPs together with dystrophin, suggesting that dystrophin gene therapy should be effective in restoring the entire dystrophin-glycoprotein complex. PMID:8462697

  18. Role of EBNA-3 Family Proteins in EBV Associated B-cell Lymphomagenesis

    PubMed Central

    Bhattacharjee, Shaoni; Ghosh Roy, Shatadru; Bose, Priyanka; Saha, Abhik

    2016-01-01

    Epstein-Barr virus (EBV) is highly ubiquitous in human population and establishes a lifelong asymptomatic infection within the infected host unless the immune system is compromised. Following initial infection in the oropharyngeal epithelial cells, EBV primarily infects naive B-lymphocytes and develops a number of B-cell lymphomas particularly in immune-deficient individuals. In vitro, EBV can also infect and subsequently transform quiescent B-lymphocytes into continuously proliferating lymphoblastoid cell lines (LCLs) resembling EBV-induced lymphoproliferative disorders in which a subset of latent transcripts are detected. Genetic studies revealed that EBNA-3 family comprising of three adjacent genes in the viral genome—EBNA-3A and -3C, but not -3B, are critical for B-cell transformation. Nevertheless, all three proteins appear to significantly contribute to maintain the overall proliferation and viability of transformed cells, suggesting a critical role in lymphoma development. Apart from functioning as important viral transcriptional regulators, EBNA-3 proteins associate with many cellular proteins in different signaling networks, providing a suitable platform for lifelong survival of the virus and concurrent lymphoma development in the infected host. The chapter describes the function of each these EBV nuclear antigen 3 proteins employed by the virus as a means to understand viral pathogenesis of several EBV-associated B-cell malignancies. PMID:27092119

  19. Stochastic detection of Pim protein kinases reveals electrostatically enhanced association of a peptide substrate

    PubMed Central

    Harrington, Leon; Cheley, Stephen; Alexander, Leila T.; Knapp, Stefan; Bayley, Hagan

    2013-01-01

    In stochastic sensing, the association and dissociation of analyte molecules is observed as the modulation of an ionic current flowing through a single engineered protein pore, enabling the label-free determination of rate and equilibrium constants with respect to a specific binding site. We engineered sensors based on the staphylococcal α-hemolysin pore to allow the single-molecule detection and characterization of protein kinase–peptide interactions. We enhanced this approach by using site-specific proteolysis to generate pores bearing a single peptide sensor element attached by an N-terminal peptide bond to the trans mouth of the pore. Kinetics and affinities for the Pim protein kinases (Pim-1, Pim-2, and Pim-3) and cAMP-dependent protein kinase were measured and found to be independent of membrane potential and in good agreement with previously reported data. Kinase binding exhibited a distinct current noise behavior that forms a basis for analyte discrimination. Finally, we observed unusually high association rate constants for the interaction of Pim kinases with their consensus substrate Pimtide (∼107 to 108 M–1⋅s–1), the result of electrostatic enhancement, and propose a cellular role for this phenomenon. PMID:24194548

  20. Differential pattern of lipid droplet-associated proteins and de novo perilipin expression in hepatocyte steatogenesis.

    PubMed

    Straub, Beate Katharina; Stoeffel, Pamela; Heid, Hans; Zimbelmann, Ralf; Schirmacher, Peter

    2008-06-01

    Fatty change (steatosis) is the most frequent liver pathology in western countries and is caused by a broad range of disorders such as alcohol abuse and metabolic syndrome. The surface layer of lipid droplets (LDs) contains members of a protein family that share homologous sequences and domains, the so-called PAT proteins, named after their constituents, perilipin, adipophilin, and TIP47. We characterized the LD-associated proteins in normal and diseased liver connected with LD accumulation. Adipophilin and TIP47 are expressed in LDs of vitamin A-storing hepatic stellate cells and additionally in LDs of steatotic hepatocytes. Perilipin, which was thought to be characteristic for LDs of adipocytes and steroidogenic cells, becomes de novo expressed in hepatocytes of human steatotic liver. Perilipin splice variant A was found in human steatotic hepatocytes by biochemical, molecular biological, and immunohistochemical methods. Its association with LDs is different from TIP47 and adipophilin, and depends on size and localization of the LDs, suggesting that the different PAT proteins play specific roles during maturation of LDs. PMID:18393390

  1. Association between segments of zonula occludens proteins: live-cell FRET and mass spectrometric analysis.

    PubMed

    Rueckert, Christine; Castro, Victor; Gagell, Corinna; Dabrowski, Sebastian; Schümann, Michael; Krause, Eberhard; Blasig, Ingolf E; Haseloff, Reiner F

    2012-06-01

    The tight junction protein ZO-1 (zonula occludens protein 1) has recruiting/scaffolding functions in the junctional complex of epithelial and endothelial cells. Homodimerization was proposed to be crucial for ZO-1 function. Here, we investigated the ability of ZO-1 domains to mediate self-interaction in living cells. We expressed ZO-1 truncation mutants as fusions with derivatives of green fluorescent protein in tight junction-free HEK-293 cells and determined self-association by means of fluorescence resonance energy transfer measurements using live-cell imaging. We show that both an SH3-hinge-GuK fusion protein and the PDZ2 domain self-associate in our test system. The recombinant PDZ2 domain also binds to ZO-1 and ZO-2 in tight junction-forming HT29/B6 cell lysates, as demonstrated by coprecipitation. Both interaction types are of relevance for the function of ZO-1 in the regulation of the junctional complex in polar cells. PMID:22671591

  2. Association of Copper to Riboflavin Binding Protein; Characterization by EPR and XAS

    SciTech Connect

    Smith,S.; Bencze, K.; Wasiukanis, K.; Stemmler, T.; Benore-Parsons, M.

    2008-01-01

    The association of copper to Riboflavin Binding Protein (RBP) from egg white has been studied by electron paramagnetic resonance (EPR) and X-ray absorption (XAS) spectroscopies. The type II site contains a mix of copper I and II in an oxygen rich environment. The association of copper to Riboflavin Binding Protein (RBP) from egg white has been studied by electron paramagnetic resonance (EPR) and X-ray absorption (XAS) spectroscopies in order to provide insight into how this essential protein may transport and store copper in avian embryos. Riboflavin Binding Protein, RBP, purified from avian egg white, has been shown to bind copper in a 1:1 molar ratio when dialyzed against copper(II) [1]. While the egg is a unique environment and quite rich in copper, the mechanisms by which this copper is delivered during development and stored for eventual use remain unclear [2]. Since RBP is already identified in the active transport of the cofactor riboflavin to the egg, evidence of its copper binding ability may suggest an additional role for RBP in the transport and storage of copper.

  3. Expression of Babesia bovis rhoptry-associated protein 1 (RAP1) in Brucella abortus S19.

    PubMed

    Sabio y García, Julia V; Farber, Marisa; Carrica, Mariela; Cravero, Silvio; Macedo, Gilson C; Bigi, Fabiana; Oliveira, Sergio C; Rossetti, Osvaldo; Campos, Eleonora

    2008-05-01

    Brucella abortus strain 19 (live vaccine) induces a strong humoral and cellular immune response and therefore, it is an attractive vector for the delivery of heterologous antigens. The objective of the present study was to express the rhoptry-associated protein (RAP1) of Babesia bovis in B. abortus S19, as a model for heterologous expression of immunostimulatory antigens from veterinary pathogens. A plasmid for the expression of recombinant proteins fused to the aminoterminal of the outer membrane lipoprotein OMP19 was created, pursuing the objective of increasing the immunogenicity of the recombinant antigen being expressed by its association to a lipid moiety. Recombinant strains of B. abortus S19 expressing RAP1 as a fusion protein either with the first amino acids of beta-galactosidase (S19pBB-RAP1) or B. abortus OMP19 (S19pBB19-RAP1) were generated. Plasmid stability and the immunogenicity of the heterologous proteins were analyzed. Mice immunized with S19pBB-RAP1 or S19pBB19-RAP1 developed specific humoral immune response to RAP1, IgG2a being the predominant antibody isotype. Furthermore, a specific cellular immune response to recombinant RAP1 was elicited in vitro by lymphocytes from mice immunized with both strains. Therefore, we concluded that B. abortus S19 expressing RAP1 is immunostimulatory and may provide the basis for combined heterologous vaccines for babesiosis and brucellosis. PMID:18462974

  4. Regulation of Endoplasmic Reticulum-Associated Protein Degradation (ERAD) by Ubiquitin

    PubMed Central

    Lemus, Leticia; Goder, Veit

    2014-01-01

    Quality control of protein folding inside the endoplasmic reticulum (ER) includes chaperone-mediated assistance in folding and the selective targeting of terminally misfolded species to a pathway called ER-associated protein degradation, or simply ERAD. Once selected for ERAD, substrates will be transported (back) into the cytosol, a step called retrotranslocation. Although still ill defined, retrotranslocation likely involves a protein conducting channel that is in part formed by specific membrane-embedded E3 ubiquitin ligases. Early during retrotranslocation, reversible self-ubiquitination of these ligases is thought to aid in initiation of substrate transfer across the membrane. Once being at least partially exposed to the cytosol, substrates will become ubiquitinated on the cytosolic side of the ER membrane by the same E3 ubiquitin ligases. Ubiquitin on substrates was originally thought to be a permanent modification that (1) promotes late steps of retrotranslocation by recruiting the energy-providing ATPase Cdc48p/p97 via binding to its associated adaptor proteins and that (2) serves to target substrates to the proteasome. Recently it became evident, however, that the poly-ubiquitin chains (PUCs) on ERAD substrates are often subject to extensive remodeling, or processing, at several stages during ERAD. This review recapitulates the current knowledge and recent findings about PUC processing on ERAD substrates and ubiquitination of ERAD machinery components and discusses their functional consequences. PMID:25100021

  5. A Phosphotyrosine Switch Controls the Association of Histone Mark Readers with Methylated Proteins.

    PubMed

    Irving-Hooper, Bronwyn Kate; Binda, Olivier

    2016-03-22

    Although histone post-translational modifications play a paramount role in controlling access to genetic information, our understanding of the precise mechanisms regulating chromatin signaling remains superficial. For instance, histone H3 trimethylated on lysine 9 (H3K9(me3)) favors the association of chromodomain proteins such as heterochromatin protein 1α (HP1α) with chromatin. However, HP1α and other such chromatin proteins are not covering all specific histone marks at all times. Thus, how are these reader-histone interactions regulated? We propose tyrosine phosphorylation within the aromatic cage of histone mark readers as a molecular switch that can either turn ON or OFF and even alter the specificity of reader-histone interactions. We have identified tyrosine phosphorylation events on the chromatin proteins HP1α and M-phase phosphoprotein 8 that regulate their association with methylated histones in vitro (synthetic peptides, calf thymus purified histones, and nucleosomes), but also in cells, thus controlling access to genetic information. PMID:26562627

  6. RING1A and BMI1 bookmark active genes via ubiquitination of chromatin-associated proteins

    PubMed Central

    Arora, Mansi; Packard, Colin Z.; Banerjee, Tapahsama; Parvin, Jeffrey D.

    2016-01-01

    During mitosis the chromatin undergoes dramatic architectural changes with the halting of the transcriptional processes and evacuation of nearly all transcription associated machinery from genes and promoters. Molecular bookmarking of genes during mitosis is a mechanism of faithfully transmitting cell-specific transcription patterns through cell division. We previously discovered chromatin ubiquitination at active promoters as a potential mitotic bookmark. In this study, we identify the enzymes involved in the deposition of ubiquitin before mitosis. We find that the polycomb complex proteins BMI1 and RING1A regulate the ubiquitination of chromatin associated proteins bound to promoters, and this modification is necessary for the expression of marked genes once the cells enter G1. Depletion of RING1A, and thus inactivation of mitotic bookmarking by ubiquitination, is deleterious to progression through G1, cell survival and proliferation. Though the polycomb complex proteins are thought to primarily regulate gene expression by transcriptional repression, in this study, we discover that these two polycomb proteins regulate the transcription of active genes during the mitosis to G1 transition. PMID:26578590

  7. Activity-Regulated Cytoskeleton-Associated Protein Controls AMPAR Endocytosis through a Direct Interaction with Clathrin-Adaptor Protein 2123

    PubMed Central

    Wall, Mark J.; P. de Almeida, Luciana; Wauters, Sandrine C.; Januário, Yunan C.; Müller, Jürgen

    2016-01-01

    Abstract The activity-regulated cytoskeleton-associated (Arc) protein controls synaptic strength by facilitating AMPA receptor (AMPAR) endocytosis. Here we demonstrate that Arc targets AMPAR to be internalized through a direct interaction with the clathrin-adaptor protein 2 (AP-2). We show that Arc overexpression in dissociated hippocampal neurons obtained from C57BL/6 mouse reduces the density of AMPAR GluA1 subunits at the cell surface and reduces the amplitude and rectification of AMPAR-mediated miniature-EPSCs (mEPSCs). Mutations of Arc, that prevent the AP-2 interaction reduce Arc-mediated endocytosis of GluA1 and abolish the reduction in AMPAR-mediated mEPSC amplitude and rectification. Depletion of the AP-2 subunit µ2 blocks the Arc-mediated reduction in mEPSC amplitude, an effect that is restored by reintroducing µ2. The Arc–AP-2 interaction plays an important role in homeostatic synaptic scaling as the Arc-dependent decrease in mEPSC amplitude, induced by a chronic increase in neuronal activity, is inhibited by AP-2 depletion. These data provide a mechanism to explain how activity-dependent expression of Arc decisively controls the fate of AMPAR at the cell surface and modulates synaptic strength, via the direct interaction with the endocytic clathrin adaptor AP-2. PMID:27257628

  8. Golgi complex localization of the Punta Toro virus G2 protein requires its association with the G1 protein.

    PubMed

    Chen, S Y; Matsuoka, Y; Compans, R W

    1991-07-01

    The glycoproteins of bunyaviruses accumulate in membranes of the Golgi complex, where virus maturation occurs by budding. In this study we have constructed a series of full length or truncated mutants of the G2 glycoprotein of Punta Toro virus (PTV), a member of the Phlebovirus genus of the Bunyaviridae, and investigated their transport properties. The results indicate that the hydrophobic domain preceding the G2 glycoprotein can function as a translocational signal peptide, and that the hydrophobic domain near the C-terminus serves as a membrane anchor. A G2 glycoprotein construct with an extra hydrophobic sequence derived from the N-terminal NSM region was stably retained in the ER, and was unable to be transported to the Golgi complex. The full-length G2 glycoprotein, when expressed on its own, was transported out of the ER and expressed on the cell surface, whereas the G1 and G2 proteins when expressed together are retained in the Golgi complex. A truncated anchor-minus form of the G2 glycoprotein was found to be secreted into the culture medium, but was retained in the Golgi complex when coexpressed with the G1 glycoprotein. These results indicate that the G2 membrane glycoprotein is a class I membrane protein which does not contain a signal sufficient for Golgi retention, and suggest that its Golgi localization is a result of association with the G1 glycoprotein. PMID:1905078

  9. Sustained downregulation of YY1-associated protein-related protein gene expression in rat hippocampus induced by repeated electroconvulsive shock.

    PubMed

    Ohtomo, Takayuki; Kanamatsu, Tomoyuki; Fujita, Mariko; Takagi, Mitsuhiro; Yamada, Junji

    2011-01-01

    YY1AP-related protein (YARP) is a structural homolog of YY1-associated protein (YY1AP), which has a YY1-binding domain. During perinatal development, YARP mRNA expression is increased at a late stage of embryonic neurogenesis. It is not known whether YARP expression is regulated during adult neurogenesis. Electroconvulsive shock (ECS), a model for a highly effective depression treatment, is known to induce hippocampal neurogenesis after repeated treatment, so we employed ECS to measure the expression of YARP mRNA. Northern blots revealed significantly decreased expression of the YARP gene after repeated ECS but not single ECS. In situ hybridization clearly demonstrated a reduction of YARP mRNA expression in the CA (CA1, CA2, and CA3) subfields. Although clonic-tonic seizure was induced not only by ECS but also by injection of kainic acid to the striatum, the regulation of YARP mRNA expression was different between ECS and kainic acid. YARP mRNA was decreased only by the ECS method, suggesting that YARP expression is different at embryonic and adult neurogenic stage. PMID:21415536

  10. Lysosomal-Associated Protein Multispanning Transmembrane 5 Gene (LAPTM5) Is Associated with Spontaneous Regression of Neuroblastomas

    PubMed Central

    Inoue, Jun; Misawa, Akiko; Tanaka, Yukichi; Ichinose, Shizuko; Sugino, Yuriko; Hosoi, Hajime; Sugimoto, Tohru; Imoto, Issei; Inazawa, Johji

    2009-01-01

    Background Neuroblastoma (NB) is the most frequently occurring solid tumor in children, and shows heterogeneous clinical behavior. Favorable tumors, which are usually detected by mass screening based on increased levels of catecholamines in urine, regress spontaneously via programmed cell death (PCD) or mature through differentiation into benign ganglioneuroma (GN). In contrast, advanced-type NB tumors often grow aggressively, despite intensive chemotherapy. Understanding the molecular mechanisms of PCD during spontaneous regression in favorable NB tumors, as well as identifying genes with a pro-death role, is a matter of urgency for developing novel approaches to the treatment of advanced-type NB tumors. Principal Findings We found that the expression of lysosomal associated protein multispanning transmembrane 5 (LAPTM5) was usually down-regulated due to DNA methylation in an NB cell-specific manner, but up-regulated in degenerating NB cells within locally regressing areas of favorable tumors detected by mass-screening. Experiments in vitro showed that not only a restoration of its expression but also the accumulation of LAPTM5 protein, was required to induce non-apoptotic cell death with autophagic vacuoles and lysosomal destabilization with lysosomal-membrane permeabilization (LMP) in a caspase-independent manner. While autophagy is a membrane-trafficking pathway to degrade the proteins in lysosomes, the LAPTM5-mediated lysosomal destabilization with LMP leads to an interruption of autophagic flux, resulting in the accumulation of immature autophagic vacuoles, p62/SQSTM1, and ubiqitinated proteins as substrates of autophagic degradation. In addition, ubiquitin-positive inclusion bodies appeared in degenerating NB cells. Conclusions We propose a novel molecular mechanism for PCD with the accumulation of autophagic vacuoles due to LAPTM5-mediated lysosomal destabilization. LAPTM5-induced cell death is lysosomal cell death with impaired autophagy, not cell death by

  11. The structure of SAV1646 from Staphylococcus aureus belonging to a new `ribosome-associated' subfamily of bacterial proteins.

    PubMed

    Chirgadze, Yuri N; Clarke, Teresa E; Romanov, Vladimir; Kisselman, Gera; Wu-Brown, Jean; Soloveychik, Maria; Chan, Tiffany S Y; Gordon, Roni D; Battaile, Kevin P; Pai, Emil F; Chirgadze, Nickolay Y

    2015-02-01

    The crystal structure of the SAV1646 protein from the pathogenic microorganism Staphylococcus aureus has been determined at 1.7 Å resolution. The 106-amino-acid protein forms a two-layer sandwich with α/β topology. The protein molecules associate as dimers in the crystal and in solution, with the monomers related by a pseudo-twofold rotation axis. A sequence-homology search identified the protein as a member of a new subfamily of yet uncharacterized bacterial `ribosome-associated' proteins with at least 13 members to date. A detailed analysis of the crystal protein structure along with the genomic structure of the operon containing the sav1646 gene allowed a tentative functional model of this protein to be proposed. The SAV1646 dimer is assumed to form a complex with ribosomal proteins L21 and L27 which could help to complete the assembly of the large subunit of the ribosome. PMID:25664743

  12. Death Associated Protein Kinase 1 (DAPK1): A Regulator of Apoptosis and Autophagy.

    PubMed

    Singh, Pratibha; Ravanan, Palaniyandi; Talwar, Priti

    2016-01-01

    Death-Associated Protein Kinase 1 (DAPK1) belongs to a family of five serine/threonine (Ser/Thr) kinases that possess tumor suppressive function and also mediate a wide range of cellular processes, including apoptosis and autophagy. The loss and gain-of-function of DAPK1 is associated with various cancer and neurodegenerative diseases respectively. In recent years, mechanistic studies have broadened our knowledge of the molecular mechanisms involved in DAPK1-mediated autophagy/apoptosis. In the present review, we have discussed the structural information and various cellular functions of DAPK1 in a comprehensive manner. PMID:27445685

  13. Membrane association of sucrose synthase: changes during the graviresponse and possible control by protein phosphorylation

    NASA Technical Reports Server (NTRS)

    Winter, H.; Huber, J. L.; Huber, S. C.; Davies, E. (Principal Investigator)

    1997-01-01

    Sucrose synthase (SuSy) plays an important role in sucrose degradation and occurs both as a soluble and as a membrane-associated enzyme in higher plants. We show that membrane association can vary in vivo in response to gravistimulation, apparently involving SuSy dephosphorylation, and is a reversible process in vitro. Phosphorylation of SuSy has little effect on its activity but decreases its surface hydrophobicity as reported with the fluorescent probe bis-ANS. We postulate that phosphorylation of SuSy (and perhaps other membrane proteins) is involved in the release of the membrane-bound enzyme in part as a result of decreased surface hydrophobicity.

  14. Death Associated Protein Kinase 1 (DAPK1): A Regulator of Apoptosis and Autophagy

    PubMed Central

    Singh, Pratibha; Ravanan, Palaniyandi; Talwar, Priti

    2016-01-01

    Death-Associated Protein Kinase 1 (DAPK1) belongs to a family of five serine/threonine (Ser/Thr) kinases that possess tumor suppressive function and also mediate a wide range of cellular processes, including apoptosis and autophagy. The loss and gain-of–function of DAPK1 is associated with various cancer and neurodegenerative diseases respectively. In recent years, mechanistic studies have broadened our knowledge of the molecular mechanisms involved in DAPK1-mediated autophagy/apoptosis. In the present review, we have discussed the structural information and various cellular functions of DAPK1 in a comprehensive manner. PMID:27445685

  15. Protein kinase A associates with cystic fibrosis transmembrane conductance regulator via an interaction with ezrin.

    PubMed

    Sun, F; Hug, M J; Bradbury, N A; Frizzell, R A

    2000-05-12

    The cystic fibrosis transmembrane conductance regulator (CFTR) is an epithelial Cl(-) channel whose activity is controlled by cAMP-dependent protein kinase (PKA)-mediated phosphorylation. We found that CFTR immunoprecipitates from Calu-3 airway cells contain endogenous PKA, which is capable of phosphorylating CFTR. This phosphorylation is stimulated by cAMP and inhibited by the PKA inhibitory peptide. The endogenous PKA that co-precipitates with CFTR could also phosphorylate the PKA substrate peptide, Leu-Arg-Arg-Ala-Ser-Leu-Gly (kemptide). Both the catalytic and type II regulatory subunits of PKA are identified by immunoblotting CFTR immunoprecipitates, demonstrating that the endogenous kinase associated with CFTR is PKA, type II (PKA II). Phosphorylation reactions mediated by CFTR-associated PKA II are inhibited by Ht31 peptide but not by the control peptide Ht31P, indicating that a protein kinase A anchoring protein (AKAP) is responsible for the association between PKA and CFTR. Ezrin may function as this AKAP, since it is expressed in Calu-3 and T84 epithelia, ezrin binds RII in overlay assays, and RII is immunoprecipitated with ezrin from Calu-3 cells. Whole-cell patch clamp of Calu-3 cells shows that Ht31 peptide reduces cAMP-stimulated CFTR Cl(-) current, but Ht31P does not. Taken together, these data demonstrate that PKA II is linked physically and functionally to CFTR by an AKAP interaction, and they suggest that ezrin serves as an AKAP for PKA-mediated phosphorylation of CFTR. PMID:10799517

  16. Functional Analysis of an S-Layer-Associated Fibronectin-Binding Protein in Lactobacillus acidophilus NCFM

    PubMed Central

    Hymes, Jeffrey P.; Johnson, Brant R.; Barrangou, Rodolphe

    2016-01-01

    Bacterial surface layers (S-layers) are crystalline arrays of self-assembling proteinaceous subunits called S-layer proteins (Slps) that comprise the outermost layer of the cell envelope. Many additional proteins that are associated with or embedded within the S-layer have been identified in Lactobacillus acidophilus NCFM, an S-layer-forming bacterium that is widely used in fermented dairy products and probiotic supplements. One putative S-layer-associated protein (SLAP), LBA0191, was predicted to mediate adhesion to fibronectin based on the in silico detection of a fibronectin-binding domain. Fibronectin is a major component of the extracellular matrix (ECM) of intestinal epithelial cells. Adhesion to intestinal epithelial cells is considered an important trait for probiotic microorganisms during transit and potential association with the intestinal mucosa. To investigate the functional role of LBA0191 (designated FbpB) in L. acidophilus NCFM, an fbpB-deficient strain was constructed. The L. acidophilus mutant with a deletion of fbpB lost the ability to adhere to mucin and fibronectin in vitro. Homologues of fbpB were identified in five additional putative S-layer-forming species, but no homologues were detected in species outside the L. acidophilus homology group. PMID:26921419

  17. Effects of motor vehicle exhaust on male reproductive function and associated proteins.

    PubMed

    Rengaraj, Deivendran; Kwon, Woo-Sung; Pang, Myung-Geol

    2015-01-01

    Air pollution is consistently associated with various diseases and subsequent death among children, adult, and elderly people worldwide. Motor vehicle exhaust contributes to a large proportion of the air pollution present. The motor vehicle exhaust systems emit a variety of toxic components, including carbon monoxide, nitrogen oxides, volatile organic compounds, ozone, particulate matter, and polycyclic aromatic hydrocarbons. Several epidemiological studies and laboratory studies have demonstrated that these components are potentially mutagenic, carcinogenic, and endocrine disrupting agents. However, their impact on male reproductive function and associated proteins is not very clear. Therefore, a comprehensive review on the effects of motor vehicle exhaust on male reproductive function and associated proteins is needed to better understand the risks of exhaust exposure for men. We found that motor vehicle exhaust can cause harmful effects on male reproductive functions by altering organ weights, reducing the spermatozoa qualities, and inducing oxidative stress. Remarkably, motor vehicle exhaust exposure causes significant changes in the expression patterns of proteins that are key components involved in spermatogenesis and testosterone synthesis. In conclusion, this review helps to describe the risks of vehicle exhaust exposure and its relationship to potential adverse effects on the male reproduction system. PMID:25329744

  18. Mutational scanning reveals the determinants of protein insertion and association energetics in the plasma membrane

    PubMed Central

    Elazar, Assaf; Weinstein, Jonathan; Biran, Ido; Fridman, Yearit; Bibi, Eitan; Fleishman, Sarel Jacob

    2016-01-01

    Insertion of helix-forming segments into the membrane and their association determines the structure, function, and expression levels of all plasma membrane proteins. However, systematic and reliable quantification of membrane-protein energetics has been challenging. We developed a deep mutational scanning method to monitor the effects of hundreds of point mutations on helix insertion and self-association within the bacterial inner membrane. The assay quantifies insertion energetics for all natural amino acids at 27 positions across the membrane, revealing that the hydrophobicity of biological membranes is significantly higher than appreciated. We further quantitate the contributions to membrane-protein insertion from positively charged residues at the cytoplasm-membrane interface and reveal large and unanticipated differences among these residues. Finally, we derive comprehensive mutational landscapes in the membrane domains of Glycophorin A and the ErbB2 oncogene, and find that insertion and self-association are strongly coupled in receptor homodimers. DOI: http://dx.doi.org/10.7554/eLife.12125.001 PMID:26824389

  19. Severe Injury Is Associated With Insulin Resistance, Endoplasmic Reticulum Stress Response, and Unfolded Protein Response

    PubMed Central

    Jeschke, Marc G.; Finnerty, Celeste C.; Herndon, David N.; Song, Juquan; Boehning, Darren; Tompkins, Ronald G.; Baker, Henry V.; Gauglitz, Gerd G.

    2012-01-01

    Objective We determined whether postburn hyperglycemia and insulin resistance are associated with endoplasmic reticulum (ER) stress/unfolded protein response (UPR) activation leading to impaired insulin receptor signaling. Background Inflammation and cellular stress, hallmarks of severely burned and critically ill patients, have been causally linked to insulin resistance in type 2 diabetes via induction of ER stress and the UPR. Methods Twenty severely burned pediatric patients were compared with 36 nonburned children. Clinical markers, protein, and GeneChip analysis were used to identify transcriptional changes in ER stress and UPR and insulin resistance–related signaling cascades in peripheral blood leukocytes, fat, and muscle at admission and up to 466 days postburn. Results Burn-induced inflammatory and stress responses are accompanied by profound insulin resistance and hyperglycemia. Genomic and protein analysis revealed that burn injury was associated with alterations in the signaling pathways that affect insulin resistance, ER/sarcoplasmic reticulum stress, inflammation, and cell growth/apoptosis up to 466 days postburn. Conclusion Burn-induced insulin resistance is associated with persistent ER/sarcoplasmic reticulum stress/UPR and subsequent suppressed insulin receptor signaling over a prolonged period of time. PMID:22241293

  20. Differential Expression in Phanerochaete chrysosporium of Membrane-Associated Proteins Relevant to Lignin Degradation

    SciTech Connect

    Shary, Semarjit; Kapich, Alexander N.; Panisko, Ellen A.; Magnuson, Jon K.; Cullen, Dan; Hammel, Ken

    2008-10-02

    Fungal lignin-degrading systems must include membrane-associated proteins that participate in diverse processes such as uptake and oxidation of lignin fragments, secretion of ligninolytic secondary metabolites, and defense of the mycelium against ligninolytic oxidants. Despite their importance, little is known about the nature or regulation of these membrane-associated components. We grew the white rot basidiomycete Phanerochaete chrysosporium on cellulose or glucose as the carbon source and monitored the mineralization of a 14C-labeled synthetic lignin by these cultures to assess their ligninolytic competence. The results showed that the cellulose-grown cultures were ligninolytic, whereas the glucose-grown ones were not. We isolated microsomal membrane fractions from both types of culture and analyzed tryptic digests of them by shotgun liquid chromatography/tandem mass spectrometry. Comparison of the results against the predicted P. chrysosporium proteome showed that a catalase (Joint Genome Institute P. chrysosporium protein I.D. 124398), an alcohol oxidase (126879), two transporters (137220 and 132234), and two cytochrome P450s (5011 and 8912) were up-regulated under ligninolytic conditions. Real time reverse transcription polymerase chain reaction assays showed that RNA transcripts encoding all of these proteins were also up-regulated in ligninolytic cultures. Catalase 124398, alcohol oxidase 126879, and transporter 137220 were found in a proteomic analysis of partially purified plasma membranes from ligninolytic P. chrysosporium, and are therefore most likely associated with the outer envelope of the fungus.

  1. Distinct roles of the RasGAP family proteins in C. elegans associative learning and memory

    PubMed Central

    Gyurkó, M. Dávid; Csermely, Péter; Sőti, Csaba; Steták, Attila

    2015-01-01

    The Ras GTPase activating proteins (RasGAPs) are regulators of the conserved Ras/MAPK pathway. Various roles of some of the RasGAPs in learning and memory have been reported in different model systems, yet, there is no comprehensive study to characterize all gap genes in any organism. Here, using reverse genetics and neurobehavioural tests, we studied the role of all known genes of the rasgap family in C. elegans in associative learning and memory. We demonstrated that their proteins are implicated in different parts of the learning and memory processes. We show that gap-1 contribute redundantly with gap-3 to the chemosensation of volatile compounds, gap-1 plays a major role in associative learning, while gap-2 and gap-3 are predominantly required for short- and long-term associative memory. Our results also suggest that the C. elegans Ras orthologue let-60 is involved in multiple processes during learning and memory. Thus, we show that the different classes of RasGAP proteins are all involved in cognitive function and their complex interplay ensures the proper formation and storage of novel information in C. elegans. PMID:26469632

  2. Exploring the Plant-Microbe Interface by Profiling the Surface-Associated Proteins of Barley Grains.

    PubMed

    Sultan, Abida; Andersen, Birgit; Svensson, Birte; Finnie, Christine

    2016-04-01

    Cereal grains are colonized by a microbial community that actively interacts with the plant via secretion of various enzymes, hormones, and metabolites. Microorganisms decompose plant tissues by a collection of depolymerizing enzymes, including β-1,4-xylanases, that are in turn inhibited by plant xylanase inhibitors. To gain insight into the importance of the microbial consortia and their interaction with barley grains, we used a combined gel-based (2-DE coupled to MALDI-TOF-TOF MS) and gel-free (LC-MS/MS) proteomics approach complemented with enzyme activity assays to profile the surface-associated proteins and xylanolytic activities of two barley cultivars. The surface-associated proteome was dominated by plant proteins with roles in defense and stress-responses, while the relatively less abundant microbial (bacterial and fungal) proteins were involved in cell-wall and polysaccharide degradation and included xylanases. The surface-associated proteomes showed elevated xylanolytic activity and contained several xylanases. Integration of proteomics with enzyme assays is a powerful tool for analysis and characterization of the interaction between microbial consortia and plants in their natural environment. PMID:26928395

  3. Functional Analysis of an S-Layer-Associated Fibronectin-Binding Protein in Lactobacillus acidophilus NCFM.

    PubMed

    Hymes, Jeffrey P; Johnson, Brant R; Barrangou, Rodolphe; Klaenhammer, Todd R

    2016-05-01

    Bacterial surface layers (S-layers) are crystalline arrays of self-assembling proteinaceous subunits called S-layer proteins (Slps) that comprise the outermost layer of the cell envelope. Many additional proteins that are associated with or embedded within the S-layer have been identified inLactobacillus acidophilusNCFM, an S-layer-forming bacterium that is widely used in fermented dairy products and probiotic supplements. One putative S-layer-associated protein (SLAP), LBA0191, was predicted to mediate adhesion to fibronectin based on thein silicodetection of a fibronectin-binding domain. Fibronectin is a major component of the extracellular matrix (ECM) of intestinal epithelial cells. Adhesion to intestinal epithelial cells is considered an important trait for probiotic microorganisms during transit and potential association with the intestinal mucosa. To investigate the functional role of LBA0191 (designated FbpB) inL. acidophilusNCFM, anfbpB-deficient strain was constructed. TheL. acidophilusmutant with a deletion offbpBlost the ability to adhere to mucin and fibronectinin vitro Homologues offbpBwere identified in five additional putative S-layer-forming species, but no homologues were detected in species outside theL. acidophilushomology group. PMID:26921419

  4. Genomic structure, gene expression, and promoter analysis of human multidrug resistance-associated protein 7

    SciTech Connect

    Kao, Hsin-Hsin; Chang, Ming-Shi; Cheng, Jan-Fang; Huang, Jin-Ding

    2002-03-15

    The multidrug resistance-associated protein (MRP) subfamily transporters associated with anticancer drug efflux are attributed to the multidrug-resistance of cancer cells. The genomic organization of human multidrug resistance-associated protein 7 (MRP7) was identified. The human MRP7 gene, consisting of 22 exons and 21 introns, greatly differs from other members of the human MRP subfamily. A splicing variant of human MRP7, MRP7A, expressed in most human tissues, was also characterized. The 1.93-kb promoter region of MRP7 was isolated and shown to support luciferase activity at a level 4- to 5-fold greater than that of the SV40 promoter. Basal MRP7 gene expression was regulated by 2 regions in the 5-flanking region at 1,780 1,287 bp, and at 611 to 208 bp. In Madin-Darby canine kidney (MDCK) cells, MRP7 promoter activity was increased by 226 percent by genotoxic 2-acetylaminofluorene and 347 percent by the histone deacetylase inhibitor, trichostatin A. The protein was expressed in the membrane fraction of transfected MDCK cells.

  5. Identification of a Novel Determinant for Membrane Association in Hepatitis C Virus Nonstructural Protein 4B▿

    PubMed Central

    Gouttenoire, Jérôme; Castet, Valérie; Montserret, Roland; Arora, Naveen; Raussens, Vincent; Ruysschaert, Jean-Marie; Diesis, Eric; Blum, Hubert E.; Penin, François; Moradpour, Darius

    2009-01-01

    Nonstructural protein 4B (NS4B) plays an essential role in the formation of the hepatitis C virus (HCV) replication complex. It is a relatively poorly characterized integral membrane protein predicted to comprise four transmembrane segments in its central portion. Here, we describe a novel determinant for membrane association represented by amino acids (aa) 40 to 69 in the N-terminal portion of NS4B. This segment was sufficient to target and tightly anchor the green fluorescent protein to cellular membranes, as assessed by fluorescence microscopy as well as membrane extraction and flotation analyses. Circular dichroism and nuclear magnetic resonance structural analyses showed that this segment comprises an amphipathic α-helix extending from aa 42 to 66. Attenuated total reflection infrared spectroscopy and glycosylation acceptor site tagging revealed that this amphipathic α-helix has the potential to traverse the phospholipid bilayer as a transmembrane segment, likely upon oligomerization. Alanine substitution of the fully conserved aromatic residues on the hydrophobic helix side abrogated membrane association of the segment comprising aa 40 to 69 and disrupted the formation of a functional replication complex. These results provide the first atomic resolution structure of an essential membrane-associated determinant of HCV NS4B. PMID:19357161

  6. Microfluidic screening and whole-genome sequencing identifies mutations associated with improved protein secretion by yeast

    PubMed Central

    Huang, Mingtao; Bai, Yunpeng; Sjostrom, Staffan L.; Hallström, Björn M.; Liu, Zihe; Petranovic, Dina; Uhlén, Mathias; Joensson, Haakan N.; Andersson-Svahn, Helene; Nielsen, Jens

    2015-01-01

    There is an increasing demand for biotech-based production of recombinant proteins for use as pharmaceuticals in the food and feed industry and in industrial applications. Yeast Saccharomyces cerevisiae is among preferred cell factories for recombinant protein production, and there is increasing interest in improving its protein secretion capacity. Due to the complexity of the secretory machinery in eukaryotic cells, it is difficult to apply rational engineering for construction of improved strains. Here we used high-throughput microfluidics for the screening of yeast libraries, generated by UV mutagenesis. Several screening and sorting rounds resulted in the selection of eight yeast clones with significantly improved secretion of recombinant α-amylase. Efficient secretion was genetically stable in the selected clones. We performed whole-genome sequencing of the eight clones and identified 330 mutations in total. Gene ontology analysis of mutated genes revealed many biological processes, including some that have not been identified before in the context of protein secretion. Mutated genes identified in this study can be potentially used for reverse metabolic engineering, with the objective to construct efficient cell factories for protein secretion. The combined use of microfluidics screening and whole-genome sequencing to map the mutations associated with the improved phenotype can easily be adapted for other products and cell types to identify novel engineering targets, and this approach could broadly facilitate design of novel cell factories. PMID:26261321

  7. Cloning a cDNA encoding an alternatively spliced protein of BRCA2-associated factor 35.

    PubMed

    Wang, Chiang; McCarty, Ida M; Balazs, Louisa; Li, Yi; Steiner, Mitchell S

    2002-07-01

    Inheritance of mutations in the breast cancer susceptibility gene, BRCA2, predisposes humans to breast and ovarian cancers. Inherited mutations in the BRCA2 gene are also known to cause susceptibility to prostate cancer. BRCA2 protein exists in a large multi-protein complex from which a novel structural DNA binding protein BRCA2-associated factor 35 (BRAF35) has been isolated. We have cloned a novel cDNA encoding an alternatively spliced protein of BRAF35, designated as BRAF25. BRAF25 transcript is present in various human cells. We have precisely mapped the BRAF25 cDNA sequence to the genomic chromosome 19 sequence. Analysis of the predicted sequence of BRAF25 identified a protein of 215 amino acids. BRAF25 contains a truncated high mobility group domain, a kinesin-like coiled-coil domain and multiple Src homology 2 (SH2) motifs. Western blot analysis using antibodies specific for BRAF25 revealed the presence of BRAF25 in human prostate cancer cells. PMID:12083779

  8. Expression of 300-kilodalton intermediate filament-associated protein distinguishes human glioma cells from normal astrocytes.

    PubMed Central

    Yang, H Y; Lieska, N; Glick, R; Shao, D; Pappas, G D

    1993-01-01

    The availability of biochemical markers to distinguish glioma cells from normal astrocytes would have enormous diagnostic value. Such markers also may be of value in studying the basic biology of human astrocytomas. The vimentin-binding, 300-kDa intermediate filament (IF)-associated protein (IFAP-300kDa) has recently been shown to be developmentally expressed in radial glia of the central nervous system of the rat. It is not detected in the normal or reactive astrocytes of the adult rat nor in neonatal rat brain astrocytes in primary culture. In the present study, double-label immunofluorescence microscopy using antibodies to IFAP-300kDa and glial fibrillary acidic protein (GFAP, an astrocyte-specific IF structural protein) identifies this IFAP in GFAP-containing tumor cells from examples of all three major types of human astrocytomas (i.e., well-differentiated, anaplastic, and glioblastoma multiforme). Astrocytoma cells in primary cultures prepared from all three astrocytomas also express this protein. It is not detectable in normal adult brain tissue. Immunoblot analyses using the IFAP-300kDa antibody confirm the presence of a 300-kDa polypeptide in fresh astrocytoma preparations enriched for IF proteins. These results suggest the utility of IFAP-300kDa as a marker for identification of human glioma cells both in vitro and in situ. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 PMID:8378327

  9. Expression of Yes Associated Protein, YAP, Modulates Survivin Expression in Primary Liver Malignancies

    PubMed Central

    Bai, Haibo; Gayyed, Mariana F.; Lam-Himlin, Dora M.; Klein, Alison P.; Nayar, Suresh K.; Xu, Yang; Khan, Mehtab; Argani, Pedram; Pan, Duojia; Anders, Robert A.

    2012-01-01

    Hepatocellular carcinoma (HCC) and intrahepatic cholangiocarcinoma (ICC) account for 95% of primary liver cancer. For each of these malignancies the outcome is dismal; incidence is rapidly increasing and mechanistic understanding is limited. We observed abnormal proliferation of both biliary epithelium and hepatocytes in mice following genetic manipulation of Yes associated protein (YAP), a transcription co-activator. Here we comprehensively documented YAP protein expression in the human liver and primary liver cancers. We showed that nuclear YAP expression is significantly increased in human ICC and HCC. We found that increased YAP protein levels in HCC are due to multiple mechanisms including gene amplification, transcriptional and posttranscriptional regulation. Survivin, a member of the inhibitors-of-apoptosis protein (IAPs) family, has been reported as an independent prognostic factor for poor survival in both HCC and ICC. We found nuclear YAP expression correlates significantly with nuclear Survivin expression for both ICC and HCC. Furthermore, using mice engineered to conditionally overexpress YAP in the liver, we found Survivin mRNA expression depends upon YAP protein levels. Our findings suggested that YAP contributes to primary liver tumorigenesis and likely mediates its oncogenic effects through modulating Survivin expression. PMID:22436626

  10. The Roles of HIV-1 Proteins and Antiretroviral Drug Therapy in HIV-1-Associated Endothelial Dysfunction

    PubMed Central

    Kline, Erik R.; Sutliff, Roy L.

    2008-01-01

    Since the emergence of highly active antiretroviral therapy (HAART), human immunodeficiency virus-1 (HIV-1)-infected patients have demonstrated dramatic decreases in viral burden and opportunistic infections, and an overall increase in life expectancy. Despite these positive HAART-associated outcomes, it has become increasingly clear that HIV-1 patients have an enhanced risk of developing cardiovascular disease over time. Clinical studies are instrumental in our understanding of vascular dysfunction in the context of HIV-1 infection. However, most clinical studies often do not distinguish whether HIV-1 proteins, HAART, or a combination of these 2 factors cause cardiovascular complications. This review seeks to address the roles of both HIV-1 proteins and antiretroviral drugs in the development of endothelial dysfunction because endothelial dysfunction is the hallmark initial step of many cardiovascular diseases. We analyze recent in vitro and in vivo studies examining endothelial toxicity in response to HIV-1 proteins or in response to the various classes of antiretroviral drugs. Furthermore, we discuss the multiple mechanisms by which HIV-1 proteins and HAART injure the vascular endothelium in HIV-1 patients. By understanding the molecular mechanisms of HIV-1 protein- and antiretroviral-induced cardiovascular disease, we may ultimately improve the quality of life of HIV-1 patients through better drug design and the discovery of new pharmacological targets. PMID:18525451

  11. A network analysis of cofactor-protein interactions for analyzing associations between human nutrition and diseases.

    PubMed

    Scott-Boyer, Marie Pier; Lacroix, Sébastien; Scotti, Marco; Morine, Melissa J; Kaput, Jim; Priami, Corrado

    2016-01-01

    The involvement of vitamins and other micronutrients in intermediary metabolism was elucidated in the mid 1900's at the level of individual biochemical reactions. Biochemical pathways remain the foundational knowledgebase for understanding how micronutrient adequacy modulates health in all life stages. Current daily recommended intakes were usually established on the basis of the association of a single nutrient to a single, most sensitive adverse effect and thus neglect interdependent and pleiotropic effects of micronutrients on biological systems. Hence, the understanding of the impact of overt or sub-clinical nutrient deficiencies on biological processes remains incomplete. Developing a more complete view of the role of micronutrients and their metabolic products in protein-mediated reactions is of importance. We thus integrated and represented cofactor-protein interaction data from multiple and diverse sources into a multi-layer network representation that links cofactors, cofactor-interacting proteins, biological processes, and diseases. Network representation of this information is a key feature of the present analysis and enables the integration of data from individual biochemical reactions and protein-protein interactions into a systems view, which may guide strategies for targeted nutritional interventions aimed at improving health and preventing diseases. PMID:26777674

  12. Identification of 14-3-3zeta associated protein networks in oral cancer.

    PubMed

    Matta, Ajay; Masui, Olena; Siu, K W Michael; Ralhan, Ranju

    2016-04-01

    Advancements in genomics, proteomics, and bioinformatics have improved our understanding of gene/protein networks involved in intra- and intercellular communication and tumor-host interactions. Using proteomics integrated with bioinformatics, previously we reported overexpression of 14-3-3ζ in premalignant oral lesions and oral squamous cell carcinoma tissues in comparison with normal oral epithelium. 14-3-3ζ emerged as a novel molecular target for therapeutics and a potential prognostic marker in oral squamous cell carcinoma patients. However, the role of 14-3-3ζ in development and progression of oral cancer is not known yet. This study aimed to identify the 14-3-3ζ associated protein networks in oral cancer cell lines using IP-MS/MS and bioinformatics. A total of 287 binding partners of 14-3-3ζ were identified in metastatic (MDA1986) and nonmetastatic (SCC4) oral cancer cell lines including other 14-3-3 isoforms (2%), proteins involved in apoptosis (2%), cytoskeleton (9%), metabolism (16%), and maintenance of redox potential (2%). Our bioinformatics analysis revealed involvement of 14-3-3ζ in protein networks regulating cell cycle, proliferation, apoptosis, cellular trafficking, and endocytosis in oral cancer. In conclusion, our data revealed several novel protein interaction networks involving 14-3-3ζ in oral cancer progression and metastasis. PMID:26857332

  13. Deletion of conserved protein phosphatases reverses defects associated with mitochondrial DNA damage in Saccharomyces cerevisiae.

    PubMed

    Garipler, Görkem; Mutlu, Nebibe; Lack, Nathan A; Dunn, Cory D

    2014-01-28

    Mitochondrial biogenesis is regulated by signaling pathways sensitive to extracellular conditions and to the internal environment of the cell. Therefore, treatments for disease caused by mutation of mtDNA may emerge from studies of how signal transduction pathways command mitochondrial function. We have examined the role of phosphatases under the control of the conserved α4/Tap42 protein in cells lacking a mitochondrial genome. We found that deletion of protein phosphatase 2A (PP2A) or of protein phosphatase 6 (PP6) protects cells from the reduced proliferation, mitochondrial protein import defects, lower mitochondrial electrochemical potential, and nuclear transcriptional response associated with mtDNA damage. Moreover, PP2A or PP6 deletion allows viability of a sensitized yeast strain after mtDNA loss. Interestingly, the Saccharomyces cerevisiae ortholog of the mammalian AMP-activated protein kinase was required for the full benefits of PP6 deletion and also for proliferation of otherwise wild-type cells lacking mtDNA. Our work highlights the important role that nutrient-responsive signaling pathways can play in determining the response to mitochondrial dysfunction. PMID:24474773

  14. Mutations in SOD1 associated with amyotrophic lateral sclerosis cause novel protein interactions.

    PubMed

    Kunst, C B; Mezey, E; Brownstein, M J; Patterson, D

    1997-01-01

    A subset of familial and sporadic amyotrophic lateral sclerosis (ALS-a fatal disorder characterised by progressive motor neuron degeneration) cases are due to mutations in the gene encoding Cu,Zn superoxide dismutase (SOD1). Two mutations which have been successfully used to generate transgenic mice that develop an ALS-like syndrome are glycine 85 to arginine (G85R) and glycine 93 to alanine (G93A) with the mutant SOD1 allele overexpressed in a normal mouse genetic background. No ALS-like phenotype is observed in mice overexpressing wild-type SOD1 or mice without any SOD1 activity. These dominant mutations, which do not necessarily decrease SOD1 activity, may confer a gain of function that is selectively lethal to motor neurons. The yeast interaction trap system allowed us to determine whether these mutations in SOD1 caused novel protein interactions not observed with wild-type SOD1 and which might participate in the generation of the ALS phenotype. Two proteins, lysyl-tRNA synthetase and translocon-associated protein delta, interact with mutant forms of SOD1 but not with wild-type SOD1. The specificity of the interactions was confirmed by the coimmunoprecipitation of mutant SOD1 and the expressed proteins. These proteins are expressed in ventral cord, lending support to the relevance of this interaction to motor neuron disease. PMID:8988176

  15. A network analysis of cofactor-protein interactions for analyzing associations between human nutrition and diseases

    PubMed Central

    Scott-Boyer, Marie Pier; Lacroix, Sébastien; Scotti, Marco; Morine, Melissa J.; Kaput, Jim; Priami, Corrado

    2016-01-01

    The involvement of vitamins and other micronutrients in intermediary metabolism was elucidated in the mid 1900’s at the level of individual biochemical reactions. Biochemical pathways remain the foundational knowledgebase for understanding how micronutrient adequacy modulates health in all life stages. Current daily recommended intakes were usually established on the basis of the association of a single nutrient to a single, most sensitive adverse effect and thus neglect interdependent and pleiotropic effects of micronutrients on biological systems. Hence, the understanding of the impact of overt or sub-clinical nutrient deficiencies on biological processes remains incomplete. Developing a more complete view of the role of micronutrients and their metabolic products in protein-mediated reactions is of importance. We thus integrated and represented cofactor-protein interaction data from multiple and diverse sources into a multi-layer network representation that links cofactors, cofactor-interacting proteins, biological processes, and diseases. Network representation of this information is a key feature of the present analysis and enables the integration of data from individual biochemical reactions and protein-protein interactions into a systems view, which may guide strategies for targeted nutritional interventions aimed at improving health and preventing diseases. PMID:26777674

  16. Characterization and Evolution of the Cell Cycle-Associated Mob Domain-Containing Proteins in Eukaryotes

    PubMed Central

    Vitulo, Nicola; Vezzi, Alessandro; Galla, Giulio; Citterio, Sandra; Marino, Giada; Ruperti, Benedetto; Zermiani, Monica; Albertini, Emidio; Valle, Giorgio; Barcaccia, Gianni

    2007-01-01

    The MOB family includes a group of cell cycle-associated proteins highly conserved throughout eukaryotes, whose founding members are implicated in mitotic exit and co-ordination of cell cycle progression with cell polarity and morphogenesis. Here we report the characterization and evolution of the MOB domain-containing proteins as inferred from the 43 eukaryotic genomes so far sequenced. We show that genes for Mob-like proteins are present in at least 41 of these genomes, confirming the universal distribution of this protein family and suggesting its prominent biological function. The phylogenetic analysis reveals five distinct MOB domain classes, showing a progressive expansion of this family from unicellular to multicellular organisms, reaching the highest number in mammals. Plant Mob genes appear to have evolved from a single ancestor, most likely after the loss of one or more genes during the early stage of Viridiplantae evolutionary history. Three of the Mob classes are widespread among most of the analyzed organisms. The possible biological and molecular function of Mob proteins and their role in conserved signaling pathways related to cell proliferation, cell death and cell polarity are also presented and critically discussed. PMID:19468312

  17. Identification of embryo proteins associated with seed germination and seedling establishment in germinating rice seeds.

    PubMed

    Liu, Shu-Jun; Xu, Heng-Heng; Wang, Wei-Qing; Li, Ni; Wang, Wei-Ping; Lu, Zhuang; Møller, Ian Max; Song, Song-Quan

    2016-06-01

    Seed germination is a critical phase in the plant life cycle, but the mechanism of seed germination is still poorly understood. In the present study, rice (Oryza sativa L. cv. Peiai 64S) seeds were sampled individually when they reached different germination stages, quiescent, germinated sensu stricto, germinated completely and seedling, and were used to study the changes in the embryo proteome. A total of 88 protein spots showed a significant change in abundance during germination in water, and the results showed an activation of metabolic processes. Cell division, cell wall synthesis, and secondary metabolism were activated at late seed germination and during preparation for subsequent seedling establishment. Cycloheximide (CHX) at 70μM inhibited seedling establishment without an apparent negative effect on seed germination, while CHX at 500μM completely blocked seed germination. We used this observation to identify the potentially important proteins involved in seed germination (coleoptile protrusion) and seedling establishment (coleoptile and radicle protrusion). Twenty-six protein spots, mainly associated with sugar/polysaccharide metabolism and energy production, showed a significant difference in abundance during seed germination. Forty-nine protein spots, mainly involved in cell wall biosynthesis, proteolysis as well as cell defense and rescue, were required for seedling establishment. The results help improve our understanding of the key events (proteins) involved in germination and seedling development. PMID:27085178

  18. Activation of Neutrophils via IP3 Pathway Following Exposure to Demodex-Associated Bacterial Proteins.

    PubMed

    McMahon, Fred; Banville, Nessa; Bergin, David A; Smedman, Christian; Paulie, Staffan; Reeves, Emer; Kavanagh, Kevin

    2016-02-01

    Rosacea is a chronic inflammatory condition that predominantly affects the skin of the face. Sera from rosacea patients display elevated reactivity to proteins from a bacterium (Bacillus oleronius) originally isolated from a Demodex mite from a rosacea patient suggesting a possible role for bacteria in the induction and persistence of this condition. This work investigated the ability of B. oleronius proteins to activate neutrophils and demonstrated activation via the IP3 pathway. Activated neutrophils displayed increased levels of IP1 production, F-actin formation, chemotaxis, and production of the pro-inflammatory cytokines IL-1β and IL-6 following stimulation by pure and crude B. oleronius protein preparations (2 μg/ml), respectively. In addition, neutrophils exposed to pure and crude B. oleronius proteins (2 μg/ml) demonstrated increased release of internally stored calcium (Ca(2+)), a hallmark of the IP3 pathway of neutrophil activation. Neutrophils play a significant role in the inflammation associated with rosacea, and this work demonstrates how B. oleronius proteins can induce neutrophil recruitment and activation. PMID:26433579

  19. Protein biomarkers associated with acute renal failure and chronic kidney disease.

    PubMed

    Perco, P; Pleban, C; Kainz, A; Lukas, A; Mayer, G; Mayer, B; Oberbauer, R

    2006-11-01

    Acute renal failure (ARF) as well as chronic kidney disease (CKD) are currently categorized according to serum creatinine concentrations. Serum creatinine, however, has shortcomings because of its low predictive values. The need for novel markers for the early diagnosis and prognosis of renal diseases is imminent, particularly for markers reflecting intrinsic organ injury in stages when glomerular filtration is not impaired. This review summarizes protein markers discussed in the context of ARF as well as CKD, and provides an overview on currently available discovery results following 'omics' techniques. The identified set of candidate marker proteins is discussed in their cellular and functional context. The systematic review of proteomics and genomics studies revealed 56 genes to be associated with acute or chronic kidney disease. Context analysis, i.e. correlation of biological processes and molecular functions of reported kidney markers, revealed that 15 genes on the candidate list were assigned to the most significant ontology groups: immunity and defence. Other significantly enriched groups were cell communication (14 genes), signal transduction (22 genes) and apoptosis (seven genes). Among 24 candidate protein markers, nine proteins were also identified by gene expression studies. Next generation candidate marker proteins with improved diagnostic and prognostic values for kidney diseases will be derived from whole genome scans and protemics approaches. Prospective validation still remains elusive for all proposed candidates. PMID:17032342

  20. The role of macropinocytosis in the propagation of protein aggregation associated with neurodegenerative diseases

    PubMed Central

    Zeineddine, Rafaa; Yerbury, Justin J.

    2015-01-01

    With the onset of the rapidly aging population, the impact of age related neurodegenerative diseases is becoming a predominant health and economic concern. Neurodegenerative diseases such as Alzheimer's disease, Creutzfeldt-Jakob disease (CJD), Parkinson's disease, Huntington's disease, frontotemporal dementia (FTD), and amyotrophic lateral sclerosis (ALS) result from the loss of a specific subsets of neurons, which is closely associated with accumulation and deposition of specific protein aggregates. Protein aggregation, or fibril formation, is a well-studied phenomenon that occurs in a nucleation-dependent growth reaction. Recently, there has been a swell of literature implicating protein aggregation and its ability to propagate cell-to-cell in the rapid progression of these diseases. In order for protein aggregation to be kindled in recipient cells it is a requisite that aggregates must be able to be released from one cell and then taken up by others. In this article we will explore the relationship between protein aggregates, their propagation and the role of macropinocytosis in their uptake. We highlight the ability of neurons to undergo stimulated macropinocytosis and identify potential therapeutic targets. PMID:26528186

  1. Villin: The major microfilament-associated protein of the intestinal microvillus

    PubMed Central

    Bretscher, Anthony; Weber, Klaus

    1979-01-01

    The major protein associated with actin in the microfilament core of intestinal microvilli has been purified. This protein, for which we propose the name villin, has a polypeptide molecular weight of approximately 95,000. Two arguments suggest that villin may be the microvillus crossfilament protein that links the microfilament core laterally down its length to the cytoplasmic side of the plasma membrane. First, electron microscopy shows that crossfilaments stay attached to isolated membrane-free microvillus cores. Calculation of the expected abundance of the crossfilament protein shows that only villin is present in sufficient quantity to account for these structures. Second, decoration of microvillus cores by antibodies to either actin or villin, followed by ferritin-labeled second antibody in a sandwich procedure, results in specific labeling of the cores in both cases. The antivillin decoration, however, gives rise to a greater increase in diameter, in agreement with a model in which villin projects from the F-actin microfilament core. Villin is distinct from α-actinin, a protein suggested to be involved in membrane anchorage of microfilaments in nonmuscle cells. The two proteins differ in molecular weight. Specific antibodies against villin and α-actinin show no immunological crossreactivity. Immunofluorescence microscopy reveals that villin is located in the microvilli of the brush border whereas α-actinin is absent from the microvilli but is found in the terminal web. In addition, villin is not found in microfilament bundles of tissue culture cells, which are rich in α-actinin. Thus, villin and α-actinin appear to be immunologically and functionally different proteins. Images PMID:287075

  2. Comparative interactomics analysis of different ALS-associated proteins identifies converging molecular pathways.

    PubMed

    Blokhuis, Anna M; Koppers, Max; Groen, Ewout J N; van den Heuvel, Dianne M A; Dini Modigliani, Stefano; Anink, Jasper J; Fumoto, Katsumi; van Diggelen, Femke; Snelting, Anne; Sodaar, Peter; Verheijen, Bert M; Demmers, Jeroen A A; Veldink, Jan H; Aronica, Eleonora; Bozzoni, Irene; den Hertog, Jeroen; van den Berg, Leonard H; Pasterkamp, R Jeroen

    2016-08-01

    Amyotrophic lateral sclerosis (ALS) is a devastating neurological disease with no effective treatment available. An increasing number of genetic causes of ALS are being identified, but how these genetic defects lead to motor neuron degeneration and to which extent they affect common cellular pathways remains incompletely understood. To address these questions, we performed an interactomic analysis to identify binding partners of wild-type (WT) and ALS-associated mutant versions of ATXN2, C9orf72, FUS, OPTN, TDP-43 and UBQLN2 in neuronal cells. This analysis identified several known but also many novel binding partners of these proteins. Interactomes of WT and mutant ALS proteins were very similar except for OPTN and UBQLN2, in which mutations caused loss or gain of protein interactions. Several of the identified interactomes showed a high degree of overlap: shared binding partners of ATXN2, FUS and TDP-43 had roles in RNA metabolism; OPTN- and UBQLN2-interacting proteins were related to protein degradation and protein transport, and C9orf72 interactors function in mitochondria. To confirm that this overlap is important for ALS pathogenesis, we studied fragile X mental retardation protein (FMRP), one of the common interactors of ATXN2, FUS and TDP-43, in more detail in in vitro and in vivo model systems for FUS ALS. FMRP localized to mutant FUS-containing aggregates in spinal motor neurons and bound endogenous FUS in a direct and RNA-sensitive manner. Furthermore, defects in synaptic FMRP mRNA target expression, neuromuscular junction integrity, and motor behavior caused by mutant FUS in zebrafish embryos, could be rescued by exogenous FMRP expression. Together, these results show that interactomics analysis can provide crucial insight into ALS disease mechanisms and they link FMRP to motor neuron dysfunction caused by FUS mutations. PMID:27164932

  3. EP4 Receptor-Associated Protein in Microglia Promotes Inflammation in the Brain.

    PubMed

    Fujikawa, Risako; Higuchi, Sei; Nakatsuji, Masato; Yasui, Mika; Ikedo, Taichi; Nagata, Manabu; Yokode, Masayuki; Minami, Manabu

    2016-08-01

    Microglial cells play a key role in neuronal damage in neurodegenerative disorders. Overactivated microglia induce detrimental neurotoxic effects through the excess production of proinflammatory cytokines. However, the mechanisms of microglial activation are poorly understood. We focused on prostaglandin E2 type 4 receptor-associated protein (EPRAP), which suppresses macrophage activation. We demonstrated that EPRAP exists in microglia in the brain. Furthermore, EPRAP-deficient mice displayed less microglial accumulation, and intraperitoneal administration of lipopolysaccharide (LPS) led to reduced expression of tumor necrosis factor-α and monocyte chemoattractant protein-1 mRNA in the brains of EPRAP-deficient mice. Consistently, EPRAP-deficient microglia showed a marked decrease in the production of tumor necrosis factor-α and monocyte chemoattractant protein-1 induced by LPS treatment compared with wild-type controls. In addition, EPRAP deficiency decreased microglial activation and neuronal cell death induced by intraventricular injection of kainic acid. EPRAP deficiency impaired the LPS-induced phosphorylation of c-jun N-terminal kinase and p38 mitogen-activated protein kinase in microglia. The phosphorylation levels of mitogen-activated protein kinase kinase 4-which phosphorylates c-jun N-terminal kinase and p38 mitogen-activated protein kinase-were also decreased in EPRAP-deficient microglia after LPS stimulation. Although EPRAP in macrophages plays a role in the attenuation of inflammation, EPRAP promotes proinflammatory activation of microglia through mitogen-activated protein kinase kinase 4-mediated signaling and may be key to the deteriorating neuronal damage brought on by brain inflammation. PMID:27315781

  4. Synthesis of a growth-associated protein by embryonic rat cerebrocortical neurons in vitro.

    PubMed

    Perrone-Bizzozero, N I; Finklestein, S P; Benowitz, L I

    1986-12-01

    Proteins synthesized by embryonic rat cortical cultures were studied under conditions that were either permissive or nonpermissive to neurite outgrowth. Freshly dissected cortex from embryonic day 17 rat pups was mechanically dissociated and plated on poly(L-lysine) substrate in the presence of (1) serum-free media, which allowed neuronal survival but no outgrowth; (2) serum, which allowed survival of both neurons and glia as well as neurite outgrowth; or (3) a hormone-supplemented defined media, which allowed preferential survival and outgrowth of neurons. In addition, postnatal tissue was cultured as a source of glia. Cultures were pulse-labeled with 35S-methionine 48 hr after plating and the protein synthesis patterns examined by 2-dimensional gel electrophoresis followed by fluorography. The expression of an acidic 50 kDa protein, associated with the particulate fraction of cells, was found to be a prominent correlate of neurite outgrowth. This protein was synthesized in serum- or hormone-treated embryonic cultures showing neurite outgrowth but was undetectable in embryonic cultures without outgrowth or in postnatal glial cultures. By virtue of its migration position on 2-dimensional gels, its presence in a light membrane fraction, and its cleavage products after Staphylococcus aureus protease treatment, the 50 kDa protein appears to be identical to an acidic 43-49 kDa protein that has been identified in several developing and regenerating neural pathways, as well as to the B-50 phosphoprotein. These findings lend support for a critical role of this protein in neural development and demonstrate the feasibility of using primary CNS cell cultures to study its biosynthesis and function. PMID:2947982

  5. Proteins of the exocytotic core complex mediate platelet alpha-granule secretion. Roles of vesicle-associated membrane protein, SNAP-23, and syntaxin 4.

    PubMed

    Flaumenhaft, R; Croce, K; Chen, E; Furie, B; Furie, B C

    1999-01-22

    To understand the molecular basis of granule release from platelets, we examined the role of vesicle-associated membrane protein, SNAP-23, and syntaxin 4 in alpha-granule secretion. A vesicle-associated membrane protein, SNAP-23, and syntaxin 4 were detected in platelet lysate. These proteins form a SDS-resistant complex that disassembles upon platelet activation. To determine whether these proteins are involved in alpha-granule secretion, we developed a streptolysin O-permeabilized platelet model of alpha-granule secretion. Streptolysin O-permeabilized platelets released alpha-granules, as measured by surface expression of P-selectin, in response to Ca2+ up to 120 min after permeabilization. Incubation of streptolysin O-permeabilized platelets with an antibody directed against vesicle-associated membrane protein completely inhibited Ca2+-induced alpha-granule release. Tetanus toxin cleaved platelet vesicle-associated membrane protein and inhibited Ca2+-induced alpha-granule secretion from streptolysin O-permeabilized platelets. An antibody to syntaxin 4 also inhibited Ca2+-induced alpha-granule release by approximately 75% in this system. These results show that vesicle-associated membrane protein, SNAP-23, and syntaxin 4 form a heterotrimeric complex in platelets that disassembles with activation and demonstrate that alpha-granule release is dependent on vesicle SNAP receptor-target SNAP receptor (vSNARE-tSNARE) interactions. PMID:9891020

  6. Psb27, a transiently associated protein, binds to the chlorophyll binding protein CP43 in photosystem II assembly intermediates

    PubMed Central

    Liu, Haijun; Huang, Richard Y.-C.; Chen, Jiawei; Gross, Michael L.; Pakrasi, Himadri B.

    2011-01-01

    Photosystem II (PSII), a large multisubunit pigment–protein complex localized in the thylakoid membrane of cyanobacteria and chloroplasts, mediates light-driven evolution of oxygen from water. Recently, a high-resolution X-ray structure of the mature PSII complex has become available. Two PSII polypeptides, D1 and CP43, provide many of the ligands to an inorganic Mn4Ca center that is essential for water oxidation. Because of its unusual redox chemistry, PSII often undergoes degradation followed by stepwise assembly. Psb27, a small luminal polypeptide, functions as an important accessory factor in this elaborate assembly pathway. However, the structural location of Psb27 within PSII assembly intermediates has remained elusive. Here we report that Psb27 binds to CP43 in such assembly intermediates. We treated purified genetically tagged PSII assembly intermediate complexes from the cyanobacterium Synechocystis 6803 with chemical cross-linkers to examine intermolecular interactions between Psb27 and various PSII proteins. First, the water-soluble 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) was used to cross-link proteins with complementary charged groups in close association to one another. In the His27△ctpAPSII preparation, a 58-kDa cross-linked species containing Psb27 and CP43 was identified. This species was not formed in the HT3△ctpA△psb27PSII complex in which Psb27 was absent. Second, the homobifunctional thiol-cleavable cross-linker 3,3′-dithiobis(sulfosuccinimidylpropionate) (DTSSP) was used to reversibly cross-link Psb27 to CP43 in His27△ctpAPSII preparations, which allowed the use of liquid chromatography/tandem MS to map the cross-linking sites as Psb27K63↔CP43D321 (trypsin) and CP43K215↔Psb27D58AGGLK63↔CP43D321 (chymotrypsin), respectively. Our data suggest that Psb27 acts as an important regulatory protein during PSII assembly through specific interactions with the luminal domain of CP43. PMID:22031695

  7. Psb27, a transiently associated protein, binds to the chlorophyll binding protein CP43 in photosystem II assembly intermediates.

    PubMed

    Liu, Haijun; Huang, Richard Y-C; Chen, Jiawei; Gross, Michael L; Pakrasi, Himadri B

    2011-11-01

    Photosystem II (PSII), a large multisubunit pigment-protein complex localized in the thylakoid membrane of cyanobacteria and chloroplasts, mediates light-driven evolution of oxygen from water. Recently, a high-resolution X-ray structure of the mature PSII complex has become available. Two PSII polypeptides, D1 and CP43, provide many of the ligands to an inorganic Mn(4)Ca center that is essential for water oxidation. Because of its unusual redox chemistry, PSII often undergoes degradation followed by stepwise assembly. Psb27, a small luminal polypeptide, functions as an important accessory factor in this elaborate assembly pathway. However, the structural location of Psb27 within PSII assembly intermediates has remained elusive. Here we report that Psb27 binds to CP43 in such assembly intermediates. We treated purified genetically tagged PSII assembly intermediate complexes from the cyanobacterium Synechocystis 6803 with chemical cross-linkers to examine intermolecular interactions between Psb27 and various PSII proteins. First, the water-soluble 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) was used to cross-link proteins with complementary charged groups in close association to one another. In the His27△ctpAPSII preparation, a 58-kDa cross-linked species containing Psb27 and CP43 was identified. This species was not formed in the HT3△ctpA△psb27PSII complex in which Psb27 was absent. Second, the homobifunctional thiol-cleavable cross-linker 3,3'-dithiobis(sulfosuccinimidylpropionate) (DTSSP) was used to reversibly cross-link Psb27 to CP43 in His27△ctpAPSII preparations, which allowed the use of liquid chromatography/tandem MS to map the cross-linking sites as Psb27K(63)↔CP43D(321) (trypsin) and CP43K(215)↔Psb27D(58)AGGLK(63)↔CP43D(321) (chymotrypsin), respectively. Our data suggest that Psb27 acts as an important regulatory protein during PSII assembly through specific interactions with the luminal domain of CP43. PMID:22031695

  8. Interaction of structural core protein of Classical Swine Fever Virus with endoplasmic reticulum-associated degradation pathway protein OS9

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Classical Swine Fever Virus (CSFV) Core protein is involved in virus RNA protection, transcription regulation and virus virulence. To discover additional Core protein functions a yeast two-hybrid system was used to identify host proteins that interact with Core. Among the identified host proteins, t...

  9. ALS-associated peripherin spliced transcripts form distinct protein inclusions that are neuroprotective against oxidative stress.

    PubMed

    McLean, Jesse R; Smith, Gaynor A; Rocha, Emily M; Osborn, Teresia M; Dib, Samar; Hayes, Melissa A; Beagan, Jonathan A; Brown, Tana B; Lawson, Tristan F S; Hallett, Penelope J; Robertson, Janice; Isacson, Ole

    2014-11-01

    Intracellular proteinaceous inclusions are well-documented hallmarks of the fatal motor neuron disorder amyotrophic lateral sclerosis (ALS). The pathological significance of these inclusions remains unknown. Peripherin, a type III intermediate filament protein, is upregulated in ALS and identified as a component within different types of ALS inclusions. The formation of these inclusions may be associated with abnormal peripherin splicing, whereby an increase in mRNA retaining introns 3 and 4 (Per-3,4) leads to the generation of an aggregation-prone isoform, Per-28. During the course of evaluating peripherin filament assembly in SW-13 cells, we identified that expression of both Per-3,4 and Per-28 transcripts formed inclusions with categorically distinct morphology: Per-3,4 was associated with cytoplasmic condensed/bundled filaments, small inclusions (<10μM), or large inclusions (≥10μM); while Per-28 was associated with punctate inclusions in the nucleus and/or cytoplasm. We found temporal and spatial changes in inclusion morphology between 12 and 48h post-transfected cells, which were accompanied by unique immunofluorescent and biochemical changes of other ALS-relevant proteins, including TDP-43 and ubiquitin. Despite mild cytotoxicity associated with peripherin transfection, Per-3,4 and Per-28 expression increased cell viability during H2O2-mediated oxidative stress in BE(2)-M17 neuroblastoma cells. Taken together, this study shows that ALS-associated peripherin isoforms form dynamic cytoplasmic and intranuclear inclusions, effect changes in local endogenous protein expression, and afford cytoprotection against oxidative stress. These findings may have important relevance to understanding the pathophysiological role of inclusions in ALS. PMID:24907400

  10. Dynamic Protein Associations Define Two Phases of IL-1β Transcriptional Activation

    PubMed Central

    Zhang, Yue; Saccani, Simona; Shin, Hyunjin; Nikolajczyk, Barbara S.

    2010-01-01

    IL-1β is a key proinflammatory cytokine with roles in multiple diseases. Monocytes package the IL-1β promoter into a “poised architecture” characterized by a histone-free transcription start site and constitutive transcription factor associations. Upon LPS stimulation, multiple proteins inducibly associate with the IL-1β gene. To understand how the complex combination of constitutive and inducible transcription factors activate the IL-1β gene from a poised structure, we measured temporal changes in NF-κB and IFN regulatory factor (IRF) association with IL-1β regulatory elements. Association of the p65 subunit of NF-κB peaks 30–60 min post-monocyte stimulation, and it shortly precedes IRF-4 recruitment to the IL-1β enhancer and maximal mRNA production. In contrast, IRF-8/enhancer association decreases poststimulation. To test the importance of delayed IRF-4/enhancer association, we introduced a mutated PU.1 protein shown to prevent PU.1-mediated IRF-4 recruitment to the enhancer sequence. Mutated PU.1 initially increased IL-1β mRNA followed by decreased mRNA levels 2–3 h poststimulation. Taken together, these data support a dynamic model of IL-1β transcriptional activation in which a combination of IRF-8 and p65 drives the initial phase of IL-1β transcription, while PU.1-mediated IRF-4 recruitment to the enhancer is important for the second phase. We further demonstrate that activation of both NF-κB and IRF-4 depends on CK2 kinase activity. Because IRF-4/enhancer association requires CK2 but not p65 activation, we conclude that CK2 triggers the IRF-4 and p65 pathways independently to serve as a master regulator of IL-1β transcription. PMID:18566416

  11. A FRET-based method for monitoring septin polymerization and binding of septin-associated proteins

    PubMed Central

    Booth, E.A.; Thorner, J.

    2016-01-01

    Much about septin function has been inferred from in vivo studies using mainly genetic methods, and much of what we know about septin organization has been obtained through examination of static structures in vitro primarily by electron microscopy. Deeper mechanistic insight requires real-time analysis of the dynamics of the assembly of septin-based structures and how other proteins associate with them. We describe here a Förster resonance energy transfer (FRET)-based approach for measuring in vitro the rate and extent of filament formation from septin complexes, binding of other proteins to septin structures, and the apparent affinities of these interactions. FRET is particularly well suited for interrogating protein–protein interactions, especially on a rapid timescale; the spectral change provides an unambiguous indication of whether two elements within the system under study are associating and serves as a molecular-level “ruler” because it is very sensitive to the separation between the donor and acceptor fluorophores over biologically relevant distances (≤ 10 nm). The necessary procedures involve generation of appropriate cysteine-less and single cysteine-containing septin variants, expression and purification of the heterooctameric complexes containing them, efficient labeling of the purified complexes with desired fluorophores, fluorimetric measurement of FRET, and appropriate safeguards and controls in data acquisition and analysis. Our methods can be used to interrogate the effects of buffer conditions, small molecules, and septin-binding proteins on septin filament assembly or stability; determine the effect of alternative septin subunits, mutational alterations, or posttranslational modifications on assembly; and, delineate the location of septin-binding proteins. PMID:27473902

  12. Monoclonal antibodies against a specific nonhistone chromosomal protein of Drosophila associated with active genes.

    PubMed

    Howard, G C; Abmayr, S M; Shinefeld, L A; Sato, V L; Elgin, S C

    1981-01-01

    Hybridomas secreting monoclonal antibodies have been produced by fusion of NS-1 mouse myeloma cells with the spleen cells of mice inoculated with a 60-65,000-mol wt fraction of proteins released from Drosophila embryo nuclei treated with DNase I. The antibodies secreted by the hybridomas were examined with polytene chromosomes of formaldehyde-fixed salivary gland squashes by an immunofluorescence assay. Most of the clonal antibodies obtained resulted in specific staining of the chromosomes relative to the cytoplasmic debris. In the case of clone 28, the antibodies showed a preferential association with sites of gene activity, both puffs and loci identified as puffing at some time during the third instar and prepupal period. In larvae that were heat shocked (exposed to 35 degrees C for 15 min before removal and fixation of the glands), the antibodies of clone 28 stained preferentially the induced heat-shock loci while continuing to stain most of the normal set of loci. The antigen for clone 28 was identified as a single protein of approximately 62,000 mol wt by using the antibodies followed by 125I-rabbit anti-mouse Ig to stain nitrocellulose replicas of SDS polyacrylamide gels of total chromosomal proteins. This study demonstrates that monoclonal antibodies can be used successfully in immunofluorescence staining of formaldehyde-fixed polytene chromosomes. The results verify the hypothesis that a specific nonhistone chromosomal protein is preferentially associated with the set of loci that includes both active sites and those scheduled to be active at some time in this developmental program. Such proteins may play a general role in the mechanisms of cell determination and gene activation. PMID:6782108

  13. A Small Protein Associated with Fungal Energy Metabolism Affects the Virulence of Cryptococcus neoformans in Mammals.

    PubMed

    McClelland, Erin E; Ramagopal, Udupi A; Rivera, Johanna; Cox, James; Nakouzi, Antonio; Prabu, Moses M; Almo, Steven C; Casadevall, Arturo

    2016-09-01

    The pathogenic yeast Cryptococcus neoformans causes cryptococcosis, a life-threatening fungal disease. C. neoformans has multiple virulence mechanisms that are non-host specific, induce damage and interfere with immune clearance. Microarray analysis of C. neoformans strains serially passaged in mice associated a small gene (CNAG_02591) with virulence. This gene, hereafter identified as HVA1 (hypervirulence-associated protein 1), encodes a protein that has homologs of unknown function in plant and animal fungi, consistent with a conserved mechanism. Expression of HVA1 was negatively correlated with virulence and was reduced in vitro and in vivo in both mouse- and Galleria-passaged strains of C. neoformans. Phenotypic analysis in hva1Δ and hva1Δ+HVA1 strains revealed no significant differences in established virulence factors. Mice infected intravenously with the hva1Δ strain had higher fungal burden in the spleen and brain, but lower fungal burden in the lungs, and died faster than mice infected with H99W or the hva1Δ+HVA1 strain. Metabolomics analysis demonstrated a general increase in all amino acids measured in the disrupted strain and a block in the TCA cycle at isocitrate dehydrogenase, possibly due to alterations in the nicotinamide cofactor pool. Macrophage fungal burden experiments recapitulated the mouse hypervirulent phenotype of the hva1Δ strain only in the presence of exogenous NADPH. The crystal structure of the Hva1 protein was solved, and a comparison of structurally similar proteins correlated with the metabolomics data and potential interactions with NADPH. We report a new gene that modulates virulence through a mechanism associated with changes in fungal metabolism. PMID:27583447

  14. The retinoblastoma protein physically associates with the human cdc2 kinase.

    PubMed Central

    Hu, Q J; Lees, J A; Buchkovich, K J; Harlow, E

    1992-01-01

    The protein product (pRB) of the retinoblastoma susceptibility gene functions as a negative regulator of cell proliferation, and its activity appears to be modulated by phosphorylation. Using a new panel of anti-human pRB monoclonal antibodies, we have investigated the biochemical properties of this protein. These antibodies have allowed us to detect a pRB-associated kinase that has been identified as the cell cycle-regulating kinase p34cdc2 or a closely related enzyme. Since this associated kinase phosphorylates pRB at most of the sites used in vivo, these results suggest that this kinase is one of the major regulators of pRB. The associated kinase activity follows the pattern of phosphorylation seen for pRB in vivo. The associated kinase activity is not seen in the G1 phase but appears in the S phase, and the levels continue to increase throughout the remainder of the cell cycle. Images PMID:1545827

  15. Translationally optimal codons associate with aggregation-prone sites in proteins.

    PubMed

    Lee, Yaelim; Zhou, Tong; Tartaglia, Gian Gaetano; Vendruscolo, Michele; Wilke, Claus O

    2010-12-01

    We analyze the relationship between codon usage bias and residue aggregation propensity in the genomes of four model organisms, Escherichia coli, yeast, fly, and mouse, as well as the archaeon Halobacterium species NRC-1. Using the Mantel-Haenszel procedure, we find that translationally optimal codons associate with aggregation-prone residues. Our results are qualitatively and quantitatively similar to those of an earlier study where we found an association between translationally optimal codons and buried residues. We also combine the aggregation-propensity data with solvent-accessibility data. Although the resulting data set is small, and hence statistical power low, results indicate that the association between optimal codons and aggregation-prone residues exists both at buried and at exposed sites. By comparing codon usage at different combinations of sites (exposed, aggregation-prone sites versus buried, non-aggregation-prone sites; buried, aggregation-prone sites versus exposed, non-aggregation-prone sites), we find that aggregation propensity and solvent accessibility seem to have independent effects of (on average) comparable magnitude on codon usage. Finally, in fly, we assess whether optimal codons associate with sites at which amino acid substitutions lead to an increase in aggregation propensity, and find only a very weak effect. These results suggest that optimal codons may be required to reduce the frequency of translation errors at aggregation-prone sites that coincide with certain functional sites, such as protein-protein interfaces. Alternatively, optimal codons may be required for rapid translation of aggregation-prone regions. PMID:21046618

  16. Clusterin expression in follicular dendritic cells associated with prion protein accumulation.

    PubMed

    Sasaki, K; Doh-ura, K; Ironside, Jw; Mabbott, N; Iwaki, T

    2006-08-01

    Peripheral accumulation of abnormal prion protein (PrP) in variant Creutzfeldt-Jakob disease and some animal models of transmissible spongiform encephalopathies (TSEs) may occur in the lymphoreticular system. Within the lymphoid tissues, abnormal PrP accumulation occurs on follicular dendritic cells (FDCs). Clusterin (apolipoprotein J) has been recognized as one of the molecules associated with PrP in TSEs, and clusterin expression is increased in the central nervous system where abnormal PrP deposition has occurred. We therefore examined peripheral clusterin expression in the context of PrP accumulation on FDCs in a range of human and experimental TSEs. PrP was detected immunohistochemically on tissue sections using a novel highly sensitive method involving detergent autoclaving pretreatment. A dendritic network pattern of clusterin immunoreactivity in lymphoid follicles was observed in association with the abnormal PrP on FDCs. The increased clusterin immunoreactivity appeared to correlate with the extent of PrP deposition, irrespective of the pathogen strains, host mouse strains or various immune modifications. The observed co-localization and correlative expression of these proteins suggested that clusterin might be directly associated with abnormal PrP. Indeed, clusterin immunoreactivity in association with PrP was retained after FDC depletion. Together these data suggest that clusterin may act as a chaperone-like molecule for PrP and play an important role in TSE pathogenesis. PMID:16767691

  17. Microtubule-associated proteins: a monoclonal antibody to MAP2 binds to differentiated neurons.

    PubMed

    Izant, J G; McIntosh, J R

    1980-08-01

    Hybridomas that secret IgG reacting specifically with the brain microtubule-associated protein MAP2 have been prepared with speen cells from BALB/c mice hyperimmunized with high molecular weight neurotubule-associated proteins. Immunofluorecence microscopy using dual fluorochrome labeling of tubulin and MAP2 antigens revealed identical patterns of interphase fiber networks in cells from explants of newborn mouse brain. The anti-MAP2 antibody did not stain primary mouse kidney cells or CHO, 3T3, HeLa, or PtK1 cell lines. Immunoprecipitation and antibody gel staining techniques failed to demonstrate any crossreacting antigen in these cells. MAP2 antigen was not seen in association with the mitotic spindle in any of the cells examined. Radioimmunoassay showed species crossreactivity of the anti-MAP2 antibody with mammalian but not avian neural cell extracts. Glial cells and some neuroblastoma cell lines did not appear to contain MAP2. However, in the B104 rat neuroblastoma cell line the MAP2 antigen appeared to be associated with the cytoskeleton concomitant with differentiation induced by dibutyryl cyclic AMP. In disagreement with most previously published reports, our data suggest that MAP2 is found only in differentiated neuronal cells and raises the possibility that MAP2 is involved in neuronal differentiation or neuron-specific processes. PMID:7001466

  18. Reduced Tau protein expression is associated with frontotemporal degeneration with progranulin mutation.

    PubMed

    Papegaey, Anthony; Eddarkaoui, Sabiha; Deramecourt, Vincent; Fernandez-Gomez, Francisco-Jose; Pantano, Pierre; Obriot, Hélène; Machala, Camille; Anquetil, Vincent; Camuzat, Agnès; Brice, Alexis; Maurage, Claude-Alain; Le Ber, Isabelle; Duyckaerts, Charles; Buée, Luc; Sergeant, Nicolas; Buée-Scherrer, Valérie

    2016-01-01

    Reduction of Tau protein expression was described in 2003 by Zhukareva et al. in a variant of frontotemporal lobar degeneration (FTLD) referred to as diagnosis of dementia lacking distinctive histopathology, then re-classified as FTLD with ubiquitin inclusions. However, the analysis of Tau expression in FTLD has not been reconsidered since then. Knowledge of the molecular basis of protein aggregates and genes that are mutated in the FTLD spectrum would enable to determine whether the "Tau-less" is a separate pathological entity or if it belongs to an existing subclass of FTLD. To address this question, we have analyzed Tau expression in the frontal brain areas from control, Alzheimer's disease and FTLD cases, including FTLD- Tau (MAPT), FTLD-TDP (sporadic, FTLD-TDP-GRN, FTLD-TDP-C9ORF72) and sporadic FTLD-FUS, using western blot and 2D-DIGE (Two-Dimensional fluorescence Difference Gel Electrophoresis) approaches. Surprisingly, we found that most of the FTLD-TDP-GRN brains are characterized by a huge reduction of Tau protein expression without any decrease in Tau mRNA levels. Interestingly, only cases affected by point mutations, rather than cases with total deletion of one GRN allele, seem to be affected by this reduction of Tau protein expression. Moreover, proteomic analysis highlighted correlations between reduced Tau protein level, synaptic impairment and massive reactive astrogliosis in these FTLD-GRN cases. Consistent with a recent study, our data also bring new insights regarding the role of progranulin in neurodegeneration by suggesting its involvement in lysosome and synaptic regulation. Together, our results demonstrate a strong association between progranulin deficiency and reduction of Tau protein expression that could lead to severe neuronal and glial dysfunctions. Our study also indicates that this FTLD-TDP-GRN subgroup could be part as a distinct entity of FTLD classification. PMID:27435172

  19. Osmotic compression of anisotropic proteins: interaction properties and associated structures in wheat gliadin dispersions.

    PubMed

    Boire, Adeline; Menut, Paul; Morel, Marie-Hélène; Sanchez, Christian

    2015-04-30

    In this Article, we investigated the interaction properties of wheat gliadins, properties that are at the basis of their functionality in wheat grain and in food matrixes. We established the equation of state of our isolate by osmotic compression and characterized the concentration-induced structural transitions, from the secondary structure of proteins to the rheological properties. We evidenced three thermodynamical regimes corresponding to several structuring regimes. First, for Φ < 0.03, gliadins behave as repulsive colloids, with a positive second virial coefficient, arising presumably from their surface charge density and/or their steric repulsion. No intermolecular interaction was detected by FT-IR, suggesting that proteins form a stable dispersion. In the second regime, the system becomes more easily compressible, i.e., less repulsive and/or more attractive. It is associated with the disappearance of β-sheet intramolecular structures of the proteins in favor of random coils/α-helix and intermolecular β-sheet interactions. This coincides with the appearance of elasticity and the increase of the apparent viscosity. Finally, in the last regime, for Φ > 0.16, FT-IR spectra show that proteins are strongly interacting via intermolecular interactions. A correlation peak develops in SAXS, revealing a global order in the dispersion. Interestingly, the osmotic pressure applied to extract the solvent is higher than expected from a hard-sphere-like protein and we highlighted a liquid-like state at very high concentration (>450 g L(-1)) which is in contrast with most proteins that form gel or glass at such concentration. In the discussion, we questioned the existence of supramolecular assemblies and the role of the solvation that would lead to this specific behavior. PMID:25839358

  20. Melatonin decreases breast cancer metastasis by modulating Rho-associated kinase protein-1 expression

    PubMed Central

    Borin, Thaiz Ferraz; Arbab, Ali Syed; Gelaleti, Gabriela Bottaro; Ferreira, Lívia Carvalho; Moschetta, Marina Gobbe; Jardim-Perassi, Bruna Victorasso; Iskander, ASM; Varma, Nadimpalli Ravi S.; Shankar, Adarsh; Coimbra, Verena Benedick; Fabri, Vanessa Alves; de Oliveira, Juliana Garcia; de Campos Zuccari, Debora Aparecida Pires

    2016-01-01

    The occurrence of metastasis, an important breast cancer prognostic factor, depends on cell migration/invasion mechanisms, which can be controlled by regulatory and effector molecules such as Rho-associated kinase protein (ROCK-1). Increased expression of this protein promotes tumor growth and metastasis, which can be restricted by ROCK-1 inhibitors. Melatonin has shown oncostatic, antimetastatic, and anti-angiogenic effects and can modulate ROCK-1 expression. Metastatic and nonmetastatic breast cancer cell lines were treated with melatonin as well as with specific ROCK-1 inhibitor (Y27632). Cell viability, cell migration/invasion, and ROCK-1 gene expression and protein expression were determined in vitro. In vivo lung metastasis study was performed using female athymic nude mice treated with either melatonin or Y27832 for 2 and 5 wk. The metastases were evaluated by X-ray computed tomography and single photon emission computed tomography (SPECT) and by immunohistochemistry for ROCK-1 and cytokeratin proteins. Melatonin and Y27632 treatments reduced cell viability and invasion/migration of both cell lines and decreased ROCK-1 gene expression in metastatic cells and protein expression in nonmetastatic cell line. The numbers of ‘hot’ spots (lung metastasis) identified by SPECT images were significantly lower in treated groups. ROCK-1 protein expression also was decreased in metastatic foci of treated groups. Melatonin has shown to be effective in controlling metastatic breast cancer in vitro and in vivo, not only via inhibition of the proliferation of tumor cells but also through direct antagonism of metastatic mechanism of cells rendered by ROCK-1 inhibition. When Y27632 was used, the effects were similar to those found with melatonin treatment. PMID:26292662

  1. Drosophila Heterochromatin Protein 1 (HP1)/Origin Recognition Complex (ORC) Protein Is Associated with HP1 and ORC and Functions in Heterochromatin-induced Silencing

    PubMed Central

    Shareef, Mohammed Momin; King, Chadwick; Damaj, Mona; Badagu, RamaKrishna; Huang, Da Wei; Kellum, Rebecca

    2001-01-01

    Heterochromatin protein 1 (HP1) is a conserved component of the highly compact chromatin of higher eukaryotic centromeres and telomeres. Cytogenetic experiments in Drosophila have shown that HP1 localization into this chromatin is perturbed in mutants for the origin recognition complex (ORC) 2 subunit. ORC has a multisubunit DNA-binding activity that binds origins of DNA replication where it is required for origin firing. The DNA-binding activity of ORC is also used in the recruitment of the Sir1 protein to silence nucleation sites flanking silent copies of the mating-type genes in Saccharomyces cerevisiae. A fraction of HP1 in the maternally loaded cytoplasm of the early Drosophila embryo is associated with a multiprotein complex containing Drosophila melanogaster ORC subunits. This complex appears to be poised to function in heterochromatin assembly later in embryonic development. Here we report the identification of a novel component of this complex, the HP1/ORC-associated protein. This protein contains similarity to DNA sequence-specific HMG proteins and is shown to bind specific satellite sequences and the telomere-associated sequence in vitro. The protein is shown to have heterochromatic localization in both diploid interphase and mitotic chromosomes and polytene chromosomes. Moreover, the gene encoding HP1/ORC-associated protein was found to display reciprocal dose-dependent variegation modifier phenotypes, similar to those for mutants in HP1 and the ORC 2 subunit. PMID:11408576

  2. Identification of a human cDNA encoding a protein that is structurally and functionally related to the yeast adenylyl cyclase-associated CAP proteins

    SciTech Connect

    Matviw, Yu, G.; Young, D. )

    1992-11-01

    The adenylyl cyclases of both Saccharomyces cerevisiae and Schizosaccharomyces pombe are associated with related proteins named CAP. In S. cerevisiae, CAP is required for cellular responses mediated by the RAS/cyclic AMP pathway. Both yeast CAPs appear to be bifunctional proteins: The N-terminal domains are required for the proper function of adenylyl cyclase, while loss of the C-terminal domains results in morphological and nutritional defects that appear to be unrelated to the cAMP pathways. Expression of either yeast CAP in the heterologous yeast suppresses phenotypes associated with loss of the C-terminal domain of the endogenous CAP but does not suppress loss of the N-terminal domain. On the basis of the homology between the two yeast CAP proteins, we have designed degenerate oligonucleotides that we used to detect, by the polymerase chain reaction method, a human cDNA fragment encoding a CAP-related peptide. Using the polymerase chain reaction fragment as a probe, we isolated a human cDNA clone encoding a 475-amino-acid protein that is homologous to the yeast CAP proteins. Expressions of the human CAP protein in S. cerevisiae suppresses the phenotypes associated with loss of the C-terminal domain of CAP but does not suppress phenotypes associated with loss of the N-terminal domain. Thus, CAP proteins have been structurally and, to some extent, functionally conserved in evolution between yeasts and mammals. 42 refs., 5 figs.

  3. Telomere-associated proteins add deoxynucleotides to terminal proteins during replication of the telomeres of linear chromosomes and plasmids in Streptomyces

    PubMed Central

    Yang, Chien-Chin; Tseng, Shu-Min; Chen, Carton W.

    2015-01-01

    Typical telomeres of linear chromosomes and plasmids of soil bacteria Streptomyces consist of tightly packed palindromic sequences with a terminal protein (‘TP’) covalently attached to the 5′ end of the DNA. Replication of these linear replicons is initiated internally and proceeds bidirectionally toward the telomeres, which leaves single-strand overhangs at the 3′ ends. These overhangs are filled by DNA synthesis using the TPs as the primers (‘end patching’). The gene encoding for typical TP, tpg, forms an operon with tap, encoding an essential telomere-associated protein, which binds TP and the secondary structures formed by the 3′ overhangs. Previously one of the two translesion synthesis DNA polymerases, DinB1 or DinB2, was proposed to catalyze the protein-primed synthesis. However, using an in vitro end-patching system, we discovered that Tpg and Tap alone could carry out the protein-primed synthesis to a length of 13 nt. Similarly, an ‘atypical’ terminal protein, Tpc, and its cognate telomere-associated protein, Tac, of SCP1 plasmid, were sufficient to achieve protein-primed synthesis in the absence of additional polymerase. These results indicate that these two telomere-associated proteins possess polymerase activities alone or in complex with the cognate TPs. PMID:25883134

  4. On the confocal images and the rheology of whey protein isolated and modified pectins associated complex.

    PubMed

    Lutz, Rachel; Aserin, Abraham; Portnoy, Yariv; Gottlieb, Moshe; Garti, Nissim

    2009-02-15

    The conditions necessary to form an associated complex between whey protein isolate (WPI) and enzymatically modified pectin in water, at pH values above the isoelectric point of the protein, have been documented. The existence of the complex is not easily verified and its characterization in solution is even more complicated, since the structure is an intermediate entity between the non-interacting, incompatible aqueous soluble mixture of the biopolymers, and a strongly interacting coacervated precipitating complex. Evidence for the formation of this associated complex is provided from confocal laser scanning microscope images and rheological behavior of the aqueous mixtures. The associated complex is characterized by small fluorescent "patches" interpreted as small aggregates. The viscosity of this solution is greater than that of its individual biopolymer constituents, indicating a synergy of attractive interactions that occurs in the solution. While individually, the pectin and the WPI solutions at the studied range of concentrations exhibit moderately non-Newtonian behavior, at specific weight ratios, mixtures of the two behave either as highly entangled polymeric structures or as weak gels. The values of the storage modulus G' are equal to or greater than those of the loss modulus G''. We conclude that the associated complexes are formed at pH 6, and at 4 wt% WPI with a pectin concentration ranging from 0.1 to 0.75 wt%. The influence of the charge distribution (degree of order of the carboxylic groups) of pectin on the associated complex was also investigated, and it was found that the more "ordered" pectin (U63) favors the formation of the associated soluble complex. PMID:19070469

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

    PubMed Central

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

    2016-01-01

    Chromosomal translocations involving the nucleoporin NUP98 have been described in several hematopoietic malignancies, in particular acute myeloid leukemia (AML). In the resulting chimeric proteins, Nup98's N-terminal region is fused to the C-terminal region of about 30 different partners, including homeodomain (HD) transcription factors. While transcriptional targets of distinct Nup98 chimeras related to immortalization are relatively well described, little is known about other potential cellular effects of these fusion proteins. By comparing the sub-nuclear localization of a large number of Nup98 fusions with HD and non-HD partners throughout the cell cycle we found that while all Nup98 chimeras were nuclear during interphase, only Nup98-HD fusion proteins exhibited a characteristic speckled appearance. During mitosis, only Nup98-HD fusions were concentrated on chromosomes. Despite the difference in localization, all tested Nup98 chimera provoked morphological alterations in the nuclear envelope (NE), in particular affecting the nuclear lamina and the lamina-associated polypeptide 2α (LAP2α). Importantly, such aberrations were not only observed in transiently transfected HeLa cells but also in mouse bone marrow cells immortalized by Nup98 fusions and in cells derived from leukemia patients harboring Nup98 fusions. Our findings unravel Nup98 fusion-associated NE alterations that may contribute to leukemogenesis. PMID:27031510

  6. The epitope recognized by a monoclonal antibody in the myelin-associated protein CNP.

    PubMed

    Stricker, R; Kalbacher, H; Reiser, G

    1997-08-18

    The epitope recognized by a monoclonal antibody (MAb-46-1) directed against the myelin-associated protein CNP (2',3'-cyclic nucleotide 3'-phosphodiesterase; EC 3.1.4.37) from several species was characterized. MAb-46-1 can be employed for immunoprecipitation, immunostaining in Western blots and in immunohistochemistry. Short peptides derived from the human CNP1 peptide sequence were synthesized and used in enzyme linked immunosorbent assays to test the reactivity of MAb-46-1. Coarse screening experiments enabled us to localize the epitope recognized by MAb-46-1 to the amino acid residues 9 to 19 close to the N-terminus. Further investigations using shorter peptides comprising this part of the protein allowed us to identify a 9 amino acid residue long peptide (amino acids 11 to 19: ELQFPFLQD) which represents the minimal epitope recognized by MAb-46-1, probably through a 3-dimensional structure and less likely a straight linear peptide. The epitope seems to be stabilized also by the attached amino acids 7 to 10 (KDKP). The peptide sequence 9-19 is conserved in all CNP sequences described so far. Thus, MAb-46-1 might be of general usefulness for further studies of the not yet identified function of the myelin-associated protein CNP. PMID:9268698

  7. A BEN-domain-containing protein associates with heterochromatin and represses transcription.

    PubMed

    Sathyan, Kizhakke M; Shen, Zhen; Tripathi, Vidisha; Prasanth, Kannanganattu V; Prasanth, Supriya G

    2011-09-15

    In eukaryotes, higher order chromatin structure governs crucial cellular processes including DNA replication, transcription and post-transcriptional gene regulation. Specific chromatin-interacting proteins play vital roles in the maintenance of chromatin structure. We have identified BEND3, a quadruple BEN domain-containing protein that is highly conserved amongst vertebrates. BEND3 colocalizes with HP1 and H3 trimethylated at K9 at heterochromatic regions in mammalian cells. Using an in vivo gene locus, we have been able to demonstrate that BEND3 associates with the locus only when it is heterochromatic and dissociates upon activation of transcription. Furthermore, tethering BEND3 inhibits transcription from the locus, indicating that BEND3 is involved in transcriptional repression through its interaction with histone deacetylases and Sall4, a transcription repressor. We further demonstrate that BEND3 is SUMOylated and that such modifications are essential for its role in transcriptional repression. Finally, overexpression of BEND3 causes premature chromatin condensation and extensive heterochromatinization, resulting in cell cycle arrest. Taken together, our data demonstrate the role of a novel heterochromatin-associated protein in transcriptional repression. PMID:21914818

  8. The Microtubule-Associated Protein Tau and Its Relevance for Pancreatic Beta Cells

    PubMed Central

    Maj, Magdalena; Hoermann, Gregor; Rasul, Sazan; Base, Wolfgang; Wagner, Ludwig; Attems, Johannes

    2016-01-01

    Structural and biochemical alterations of the microtubule-associated protein tau (MAPT) are associated with degenerative disorders referred to as tauopathies. We have previously shown that MAPT is present in human islets of Langerhans, human insulinomas, and pancreatic beta-cell line models, with biophysical similarities to the pathological MAPT in the brain. Here, we further studied MAPT in pancreatic endocrine tissue to better understand the mechanisms that lead to functional dysregulation of pancreatic beta cells. We found upregulation of MAPT protein expression in human insulinomas when compared to human pancreatic islets of Langerhans and an imbalance between MAPT isoforms in insulinomas tissue. We cloned one 3-repeat domain MAPT and transduced this into a beta-cell derived rodent cell line Rin-5F. Proliferation experiments showed higher growth rates and metabolic activities of cells overexpressing MAPT protein. We observed that a MAPT overexpressing cell line demonstrates altered insulin transcription, translation, and insulin secretion rates. We found the relative insulin secretion rates were significantly decreased in a MAPT overexpressing cell line and these findings could be confirmed using partial MAPT knock-down cell lines. Our findings support that MAPT may play an important role in insulin granule trafficking and indicate the importance of balanced MAPT phosphorylation and dephosphorylation for adequate insulin release. PMID:26824039

  9. No association between lck gene polymorphisms and protein level in type 1 diabetes.

    PubMed

    Nervi, Solange; Nicodeme, Sandra; Gartioux, Corinne; Atlan, Catherine; Lathrop, Marc; Reviron, Denis; Naquet, Philippe; Matsuda, Fumihiko; Imbert, Jean; Vialettes, Bernard

    2002-11-01

    We previously described a reduced expression of the protein tyrosine kinase Lck in T-cells from type 1 diabetic patients, the origin of which is still unknown. The human lck gene, located on chromosome 1p35-34.3, was evaluated as a candidate susceptibility gene for type 1 diabetes. A molecular scan of the sequence variations in the coding, the relevant promoter, and most of the intronic sequences of the lck gene (representing a total of 10.5 kb fragment) was performed in 187 Caucasian subjects including 91 type 1 diabetic patients and 96 normoglycemic control subjects. We identified 35 sequence variations, including one deletion and 34 single nucleotide polymorphisms (SNPs), 33 of them being new. Four variants were frequent but not significantly associated with diabetes or Lck protein level. Of the SNP variants, 11 were only found within the diabetic population and some were associated with low Lck protein levels. The low frequency of these polymorphisms did not permit any statistically significant correlations with the disease status, suggesting that the lck gene probably does not contribute to genetic susceptibility to type 1 diabetes. PMID:12401726

  10. A VAMP-associated protein, PVA31 is involved in leaf senescence in Arabidopsis

    PubMed Central

    Ichikawa, Mie; Nakai, Yusuke; Arima, Keita; Nishiyama, Sayo; Hirano, Tomoko; Sato, Masa H

    2015-01-01

    VAMP-associated proteins (VAPs) are highly conserved among eukaryotes. Here, we report a functional analysis of one of the VAPs, PVA31, and demonstrate its novel function on leaf senescence in Arabidopsis. The expression of PVA31 is highly induced in senescence leaves, and localizes to the plasma membrane as well as the ARA7-positive endosomes. Yeast two-hybrid analysis demonstrates that PVA31 is interacted with the plasma membrane localized-VAMP proteins, VAMP721/722/724 but not with the endosome-localized VAMPs, VAMP711 and VAMP727, indicating that PVA31 is associated with VAMP721/722/724 on the plasma membrane. Strong constitutive expression of PVA31 under the control of the Cauliflower mosaic virus 35S promoter induces the typical symptom of leaf senescence earlier than WT in normal growth and an artificially induced senescence conditions. In addition, the marker genes for the SA-mediated signaling pathways, PR-1, is promptly expressed with elicitor application. These data indicate that PVA31-overexpressing plants exhibit the early senescence phenotype in their leaves, and suggest that PVA31 is involved in the SA-mediated programmed cell death process during leaf senescence and PR-protein secretion during pathogen infection in Arabidopsis. PMID:25897470

  11. Selection and characterization of human antibody fragments specific for psoriasin - a cancer associated protein.

    PubMed

    Cyranka-Czaja, Anna; Wulhfard, Sarah; Neri, Dario; Otlewski, Jacek

    2012-03-01

    S100A7 (psoriasin) is a calcium-binding protein that is upregulated in many types of cancer and often associated with poor prognosis. Its role in carcinogenesis has been associated with the stimulation of VEGF and EGF activity. The recent research showed that psoriasin directly interacts with αvβ6 integrin, a protein related to the invasive phenotype of cancer. Moreover, this interaction promotes the αvβ6-dependent invasive activity. The important function of S100A7 in carcinoma development determines a great need for valuable tools enabling its detection, quantification and also activity inhibition. Here, we show the selection of S100A7 specific antibody fragments from the human scFv phage library ETH-2 Gold. We have selected antibody fragments specific for psoriasin, purified them and analyzed by BIAcore affinity measurements. The best clone was subjected to affinity maturation procedure yielding molecule with a subnanomolar affinity towards human S100A7 protein. Selected clone was expressed in a bivalent format and applied for immunostaining analysis, which confirmed the ability of the antigen recognition in physiological conditions. We therefore propose that obtained antibody, that is the first phage display-derived human antibody against psoriasin, can serve as a useful psoriasin binding platform in research, diagnostics and therapy of cancer. PMID:22342672

  12. Annotation of human cancers with EGFR signaling-associated protein complexes using proximity ligation assays

    PubMed Central

    Smith, Matthew A.; Hall, Richard; Fisher, Kate; Haake, Scott M.; Khalil, Farah; Schabath, Matthew B.; Vuaroqueaux, Vincent; Fiebig, Heinz-Herbert; Altiok, Soner; Chen, Y. Ann; Haura, Eric B.

    2015-01-01

    Strategies to measure functional signaling-associated protein complexes have the potential to augment current molecular biomarker assays, such as genotyping and expression profiling, used to annotate diseases. Aberrant activation of epidermal growth factor receptor (EGFR) signaling contributes to diverse cancers. Here, we used a proximity ligation assay (PLA) to detect EGFR in a complex with growth factor receptor-bound protein 2 (GRB2), the major signaling adaptor for EGFR. We used multiple lung cancer cell lines to develop and characterize EGFR:GRB2 PLA and correlated this assay with established biochemical measures of EGFR signaling. In a panel of patient-derived xenografts in mice, the intensity of EGFR:GRB2 PLA correlated with the reduction in tumor size in response to the EGFR inhibitor cetuximab. In tumor biopsies from three cohorts of lung cancer patients, positive EGFR:GRB2 PLA was observed in patients with and without EGFR mutations and the intensity of EGFR:GRB2 PLA was predictive of overall survival in an EGFR inhibitor-treated cohort. Thus, we established the feasibility of using PLA to measure EGFR signaling-associated protein complexes in patient-based materials, suggesting the potential for similar assays for a broader array of receptor tyrosine kinases and other key signaling molecules. PMID:25587191

  13. Membrane-Associated Transporter Protein (MATP) Regulates Melanosomal pH and Influences Tyrosinase Activity.

    PubMed

    Bin, Bum-Ho; Bhin, Jinhyuk; Yang, Seung Ha; Shin, Misun; Nam, Yeon-Ju; Choi, Dong-Hwa; Shin, Dong Wook; Lee, Ai-Young; Hwang, Daehee; Cho, Eun-Gyung; Lee, Tae Ryong

    2015-01-01

    The SLC45A2 gene encodes a Membrane-Associated Transporter Protein (MATP). Mutations of this gene cause oculocutaneous albinism type 4 (OCA4). However, the molecular mechanism of its action in melanogenesis has not been elucidated. Here, we discuss the role of MATP in melanin production. The SLC45A2 gene is highly enriched in human melanocytes and melanoma cell lines, and its protein, MATP, is located in melanosomes. The knockdown of MATP using siRNAs reduced melanin content and tyrosinase activity without any morphological change in melanosomes or the expression of melanogenesis-related proteins. Interestingly, the knockdown of MATP significantly lowered the melanosomal pH, as verified through DAMP analysis, suggesting that MATP regulates melanosomal pH and therefore affects tyrosinase activity. Finally, we found that the reduction of tyrosinase activity associated with the knockdown of MATP was readily recovered by copper treatment in the in vitro L-DOPA oxidase activity assay of tyrosinase. Considering that copper is an important element for tyrosinase activity and that its binding to tyrosinase depends on melanosomal pH, MATP may play an important role in regulating tyrosinase activity via controlling melanosomal pH. PMID:26057890

  14. Changes in reflectin protein phosphorylation are associated with dynamic iridescence in squid

    PubMed Central

    Izumi, Michi; Sweeney, Alison M.; DeMartini, Daniel; Weaver, James C.; Powers, Meghan L.; Tao, Andrea; Silvas, Tania V.; Kramer, Ryan M.; Crookes-Goodson, Wendy J.; Mäthger, Lydia M.; Naik, Rajesh R.; Hanlon, Roger T.; Morse, Daniel E.

    2010-01-01

    Many cephalopods exhibit remarkable dermal iridescence, a component of their complex, dynamic camouflage and communication. In the species Euprymna scolopes, the light-organ iridescence is static and is due to reflectin protein-based platelets assembled into lamellar thin-film reflectors called iridosomes, contained within iridescent cells called iridocytes. Squid in the family Loliginidae appear to be unique in which the dermis possesses a dynamic iridescent component with reflective, coloured structures that are assembled and disassembled under the control of the muscarinic cholinergic system and the associated neurotransmitter acetylcholine (ACh). Here we present the sequences and characterization of three new members of the reflectin family associated with the dynamically changeable iridescence in Loligo and not found in static Euprymna iridophores. In addition, we show that application of genistein, a protein tyrosine kinase inhibitor, suppresses ACh- and calcium-induced iridescence in Loligo. We further demonstrate that two of these novel reflectins are extensively phosphorylated in concert with the activation of iridescence by exogenous ACh. This phosphorylation and the correlated iridescence can be blocked with genistein. Our results suggest that tyrosine phosphorylation of reflectin proteins is involved in the regulation of dynamic iridescence in Loligo. PMID:19776150

  15. The Leishmania donovani lipophosphoglycan T lymphocyte-reactive component is a tightly associated protein complex.

    PubMed

    Jardim, A; Tolson, D L; Turco, S J; Pearson, T W; Olafson, R W

    1991-11-15

    Lymphocytes from mice immunized with Leishmania donovani (LPG) were specifically stimulated to proliferate in vitro by purified LPG or its delipidated congener, phosphoglycan. The response was dose dependent and required prior immunization with either LPG or phosphoglycan. Proliferation was eliminated by specific depletion of Thy-1+ cells with antisera and C and the proliferating T cell subset was shown to be CD4+CD8-. Tests of various LPG fragments indicated that the T cell stimulation was associated with the core structure of LPG rather than the lipid or phosphoglycan repeat structure. However, amino acid analysis of LPG and active LPG fragments, after acid hydrolysis, showed the presence of amino acids in peptide linkage. Specific hydrolysis of the glycosidic linkages in LPG with trifluoromethanesulfonic acid provided polypeptide material reactive with two mAb previously believed to be LPG carbohydrate core specific. The protein was separated from LPG by reverse phase chromatography and shown to be a complex of proteins with common epitopes recognized by the two mAb. The dominant species isolated from LPG was a set of small, approximately 11,000 Mr, molecules. Subsequent T cell proliferation studies showed that the lymphocyte stimulation was associated with the protein component of LPG and not the glycan. PMID:1940354

  16. Microtubule-associated STOP protein deletion triggers restricted changes in dopaminergic neurotransmission.

    PubMed

    Bouvrais-Veret, Caroline; Weiss, Stéphanie; Hanoun, Naima; Andrieux, Annie; Schweitzer, Annie; Job, Didier; Hamon, Michel; Giros, Bruno; Martres, Marie-Pascale

    2008-02-01

    The microtubule-associated stable tubule only polypeptide (STOP) protein plays a key-role in neuron architecture and synaptic plasticity. Recent studies suggest that schizophrenia is associated with alterations in the synaptic connectivity. Mice invalidated for the STOP gene display phenotype reminiscent of some schizophrenic-like symptoms, such as behavioral disturbances, dopamine (DA) hyper-reactivity, and possible hypoglutamatergia, partly improved by antipsychotic treatment. In the present work, we examined potential alterations in some DAergic key proteins and behaviors in STOP knockout mice. Whereas the densities of the DA transporter, the vesicular monoamine transporter and the D(1) receptor were not modified, the densities of the D(2) and D(3) receptors were decreased in some DAergic regions in mutant versus wild-type mice. Endogenous DA levels were selectively decreased in DAergic terminals areas, although the in vivo DA synthesis was diminished both in cell bodies and terminal areas. The DA uptake was decreased in accumbic synaptosomes, but not significantly altered in striatal synaptosomes. Finally, STOP knockout mice were hypersensitive to acute and subchronic locomotor effects of cocaine, although the drug equally inhibited DA uptake in mutant and wild-type mice. Altogether, these data showed that deletion of the ubiquitous STOP protein elicited restricted alterations in DAergic neurotransmission, preferentially in the meso-limbic pathway. PMID:18199119

  17. Membrane-Associated Transporter Protein (MATP) Regulates Melanosomal pH and Influences Tyrosinase Activity

    PubMed Central

    Bin, Bum-Ho; Bhin, Jinhyuk; Yang, Seung Ha; Shin, Misun; Nam, Yeon-Ju; Choi, Dong-Hwa; Shin, Dong Wook; Lee, Ai-Young; Hwang, Daehee; Cho, Eun-Gyung; Lee, Tae Ryong

    2015-01-01

    The SLC45A2 gene encodes a Membrane-Associated Transporter Protein (MATP). Mutations of this gene cause oculocutaneous albinism type 4 (OCA4). However, the molecular mechanism of its action in melanogenesis has not been elucidated. Here, we discuss the role of MATP in melanin production. The SLC45A2 gene is highly enriched in human melanocytes and melanoma cell lines, and its protein, MATP, is located in melanosomes. The knockdown of MATP using siRNAs reduced melanin content and tyrosinase activity without any morphological change in melanosomes or the expression of melanogenesis-related proteins. Interestingly, the knockdown of MATP significantly lowered the melanosomal pH, as verified through DAMP analysis, suggesting that MATP regulates melanosomal pH and therefore affects tyrosinase activity. Finally, we found that the reduction of tyrosinase activity associated with the knockdown of MATP was readily recovered by copper treatment in the in vitro L-DOPA oxidase activity assay of tyrosinase. Considering that copper is an important element for tyrosinase activity and that its binding to tyrosinase depends on melanosomal pH, MATP may play an important role in regulating tyrosinase activity via controlling melanosomal pH. PMID:26057890

  18. Vesicle-associated membrane protein 7 is expressed in intestinal ER.

    PubMed

    Siddiqi, Shadab A; Mahan, James; Siddiqi, Shahzad; Gorelick, Fred S; Mansbach, Charles M

    2006-03-01

    Intestinal dietary triacylglycerol absorption is a multi-step process. Triacylglycerol exit from the endoplasmic reticulum (ER) is the rate-limiting step in the progress of the lipid from its apical absorption to its basolateral membrane export. Triacylglycerol is transported from the ER to the cis Golgi in a specialized vesicle, the pre-chylomicron transport vesicle (PCTV). The vesicle-associated membrane protein 7 (VAMP7) was found to be more concentrated on PCTVs compared with ER membranes. VAMP7 has been previously identified associated with post-Golgi sites in eukaryotes. To examine the potential role of VAMP7 in PCTV trafficking, antibodies were generated that identified a 25 kDa band consistent with VAMP7 but did not crossreact with VAMP1,2. VAMP7 was concentrated on intestinal ER by immunofluorescence microscopy. Immunoelectron microscopy showed that the ER proteins Sar1 and rBet1 were present on PCTVs and colocalized with VAMP7. Iodixanol gradient centrifugation showed VAMP7 to be isodense with ER and endosomes. Although VAMP7 localized to intestinal ER, it was not present in the ER of liver and kidney. Anti-VAMP7 antibodies reduced the transfer of triacylglycerol, but not newly synthesized proteins, from the ER to the Golgi by 85%. We conclude that VAMP7 is enriched in intestinal ER and that it plays a functional role in the delivery of triacylglycerol from the ER to the Golgi. PMID:16495485

  19. Coronary Plaque Type and Burden By Computed Tomography Angiography Without Association to C-Reactive Protein

    PubMed Central

    Navaravong, Leenhapong; Steenson, Carol; Sigurdsson, Gardar

    2014-01-01

    Background: Contrast-enhanced computed tomography angiography (CTA) of the coronaries allows identification of plaques. Limited data exists on the relationship between C-reactive protein (CRP) and the plaque type or plaque burden detected by CTA. Aims: We studied relationship between CRP and coronary atherosclerosis. Materials and Methods: 92 patients without history of coronary disease underwent coronary CTA for chest pain. Coronary arteries were evaluated with each detected plaque labeled as calcified, noncalcified or mixed. Logarithmic transformation was done on CRP values for statistical analysis. Results: 1380 coronary segments were evaluated. The average age was 57 years (SE 1.0) and basal metabolic index (BMI) 28.9 kg/m2 (SE 0.5). Median CRP level was 2.75 mg/L (range 0.17-16.98). No association was found between CRP quartiles and plaque type. In stepwise multivariate analysis, only diabetes was associated with noncalcified plaque (P < 0.001). When calcified and mixed plaques were added to the model, age (P < 0.001), diabetes (P < 0.02), and statin use (P < 0.05) were associated with an increased number of plaques per subject. No association was found between log-CRP for any type of plaque. Conclusion: There was no association between CRP and plaque type by CTA. Lack of association is likely due to limited spatial resolution and underestimation of noncalcified plaque burden by CTA. PMID:25006560

  20. NonO enhances the association of many DNA-binding proteins to their targets.

    PubMed

    Yang, Y S; Yang, M C; Tucker, P W; Capra, J D

    1997-06-15

    NonO is an unusual nucleic acid binding protein not only in that it binds both DNA and RNA but that it does so via functionally separable domains. Here we document that NonO enhances the binding of some (E47, OTF-1 and OTF-2) but not all (PEA3) conventional sequence-specific transcription factors to their recognition sites in artificial substrates as well as in an immunoglobulin VHpromoter. We also show that NonO induces the binding of the Ku complex to DNA ends. Ku has no known DNA sequence specificity. These enhancement of binding effects are NonO concentration dependent. Using the E box activity of E47 as a model, kinetic studies demonstrate that the association rate of the protein-DNA complex increases in the presence of NonO while the dissociation rate remains the same, thereby increasing the sum total of the interaction. Oligo competition experiments indicate that NonO does not contact the target DNA in order to enhance the binding activity of DNA binding proteins. Rather, methylation interference analysis reveals that the induced E47 binding-activity has the same DNA-binding sequence specificity as the normal binding. This result suggests that one of the effects of NonO is to induce a true protein-DNA interaction. In this way, it might be possible for NonO to play a crucial role in gene regulation. PMID:9171077

  1. Proteomic profiling of human plasma exosomes identifies PPAR{gamma} as an exosome-associated protein

    SciTech Connect

    Looze, Christopher; Yui, David; Leung, Lester; Ingham, Matthew; Kaler, Maryann; Yao, Xianglan; Wu, Wells W.; Shen Rongfong; Daniels, Mathew P.; Levine, Stewart J.

    2009-01-16

    Exosomes are nanovesicles that are released from cells as a mechanism of cell-free intercellular communication. Only a limited number of proteins have been identified from the plasma exosome proteome. Here, we developed a multi-step fractionation scheme incorporating gel exclusion chromatography, rate zonal centrifugation through continuous sucrose gradients, and high-speed centrifugation to purify exosomes from human plasma. Exosome-associated proteins were separated by SDS-PAGE and 66 proteins were identified by LC-MS/MS, which included both cellular and extracellular proteins. Furthermore, we identified and characterized peroxisome proliferator-activated receptor-{gamma} (PPAR{gamma}), a nuclear receptor that regulates adipocyte differentiation and proliferation, as well as immune and inflammatory cell functions, as a novel component of plasma-derived exosomes. Given the important role of exosomes as intercellular messengers, the discovery of PPAR{gamma} as a component of human plasma exosomes identifies a potential new pathway for the paracrine transfer of nuclear receptors.

  2. Seizures Associated With Hypocalcemia in a Yorkshire Terrier With Protein-Losing Enteropathy.

    PubMed

    Whitehead, Jim; Quimby, Jessica; Bayliss, Danielle

    2015-01-01

    A 7 yr old, male, castrated, Yorkshire terrier was presented on emergency for an acute onset of seizure activity. The owner also reported that the dog had previously exhibited other symptoms, including intermittent vomiting, diarrhea, and anorexia for several yr. The initial workup revealed a marked decrease in ionized calcium and total protein. Further diagnostics revealed decreases in magnesium, 25 hydroxyvitamin D, albumin, and globulins, and an increased parathyroid hormone level. Intestinal biopsies revealed inflammatory bowel disease and lymphangiectasia. The dog received intravenous calcium gluconate for treatment of hypocalcemia followed by oral calcium and vitamin D supplementation. Seizure activity ceased once calcium levels approached the normal range. Medical and dietary therapy for lymphangiectasia and inflammatory bowel disease consisted of prednisone, rutin, and a low-fat diet. Decreased serum total ionized calcium levels have been reported previously in dogs with protein-losing enteropathies. Typically, the hypocalcemia is not associated with clinical signs. Severe clinical signs of hypocalcemia are rarely reported in dogs with protein-losing enteropathy, but seizures, facial twitching, and tremors can occur. When presented with a dog with a history of seizure activity, panhypoproteinemia, and hypocalcemia, protein-losing enteropathy should be included on the list of differential diagnoses. PMID:26535456

  3. Expression of occludin, tight-junction-associated protein, in human digestive tract.

    PubMed Central

    Kimura, Y.; Shiozaki, H.; Hirao, M.; Maeno, Y.; Doki, Y.; Inoue, M.; Monden, T.; Ando-Akatsuka, Y.; Furuse, M.; Tsukita, S.; Monden, M.

    1997-01-01

    The tight junction seals cells together at a subapical location and functionally separates the plasma membrane into an apical and a basolateral domain. This junction is one of the most characteristic structural markers of the polarized epithelial cell. Recently, occludin has been identified as an integral transmembrane protein localizing at the tight junction and directly associated with ZO-1, an undercoat-constitutive cytoplasmic protein. We have investigated occludin expression in conjunction with ZO-1 in normal epithelia and cancers of human digestive tract by immunostaining with a new antibody raised against human occludin. In the normal simple columnar epithelium, occludin was expressed together with ZO-1 as a single line at the apical cell border. However, in the esophagus, which has a stratified squamous epithelium, no occludin expression could be detected, but ZO-1 was expressed in the spinous layer. As for cancers, both occludin and ZO-1 showed the same expression in differentiated adenocarcinoma cells as in normal epithelium, but in poorly differentiated adenocarcinomas, the expression of these two proteins was reduced. There was significant correlation between tumor differentiation and expression of these proteins. These results suggest that occludin, together with ZO-1, is involved in the formation of gland-like structures. In addition, occludin expression can serve as a histopathological indicator for differentiation in gastrointestinal adenocarcinomas. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 PMID:9212730

  4. From invagination to navigation: The story of magnetosome-associated proteins in magnetotactic bacteria.

    PubMed

    Barber-Zucker, Shiran; Keren-Khadmy, Noa; Zarivach, Raz

    2016-02-01

    Magnetotactic bacteria (MTB) are a group of Gram-negative microorganisms that are able to sense and change their orientation in accordance with the geomagnetic field. This unique capability is due to the presence of a special suborganelle called the magnetosome, composed of either a magnetite or gregite crystal surrounded by a lipid membrane. MTB were first detected in 1975 and since then numerous efforts have been made to clarify the special mechanism of magnetosome formation at the molecular level. Magnetosome formation can be divided into several steps, beginning with vesicle invagination from the cell membrane, through protein sorting, followed by the combined steps of iron transportation, biomineralization, and the alignment of magnetosomes into a chain. The magnetosome-chain enables the sensing of the magnetic field, and thus, allows the MTB to navigate. It is known that magnetosome formation is tightly controlled by a distinctive set of magnetosome-associated proteins that are encoded mainly in a genomically conserved region within MTB called the magnetosome island (MAI). Most of these proteins were shown to have an impact on the magnetism of MTB. Here, we describe the process in which the magnetosome is formed with an emphasis on the different proteins that participate in each stage of the magnetosome formation scheme. PMID:26457474

  5. Identification and Characterization of the RouenBd1987 Babesia divergens Rhopty-Associated Protein 1

    PubMed Central

    Rodriguez, Marilis; Alhassan, Andy; Ord, Rosalynn L.; Cursino-Santos, Jeny R.; Singh, Manpreet; Gray, Jeremy; Lobo, Cheryl A.

    2014-01-01

    Human babesiosis is caused by one of several babesial species transmitted by ixodid ticks that have distinct geographical distributions based on the presence of competent animal hosts. The pathology of babesiosis, like malaria, is a consequence of the parasitaemia which develops through the cyclical replication of Babesia parasites in a patient's red blood cells, though symptoms typically are nonspecific. We have identified the gene encoding Rhoptry-Associated Protein −1 (RAP-1) from a human isolate of B. divergens, Rouen1987 and characterized its protein product at the molecular and cellular level. Consistent with other Babesia RAP-1 homologues, BdRAP-1 is expressed as a 46 kDa protein in the parasite rhoptries, suggesting a possible role in red cell invasion. Native BdRAP-1 binds to an unidentified red cell receptor(s) that appears to be non-sialylated and non-proteinacious in nature, but we do not find significant reduction in growth with anti-rRAP1 antibodies in vitro, highlighting the possibility the B. divergens is able to use alternative pathways for invasion, or there is an alternative, complementary, role for BdRAP-1 during the invasion process. As it is the parasite's ability to recognize and then invade host cells which is central to clinical disease, characterising and understanding the role of Babesia-derived proteins involved in these steps are of great interest for the development of an effective prophylaxis. PMID:25226276

  6. Ultraviolet-ozone treatment reduces levels of disease-associated prion protein and prion infectivity

    USGS Publications Warehouse

    Johnson, C.J.; Gilbert, P.; McKenzie, D.; Pedersen, J.A.; Aiken, Judd M.

    2009-01-01

    Background. Transmissible spongiform encephalopathies (TSEs) are a group of fatal neurodegenerative diseases caused by novel infectious agents referred to as prions. Prions appear to be composed primarily, if not exclusively, of a misfolded isoform of the cellular prion protein. TSE infectivity is remarkably stable and can resist many aggressive decontamination procedures, increasing human, livestock and wildlife exposure to TSEs. Findings. We tested the hypothesis that UV-ozone treatment reduces levels of the pathogenic prion protein and inactivates the infectious agent. We found that UV-ozone treatment decreased the carbon and prion protein content in infected brain homogenate to levels undetectable by dry-ashing carbon analysis or immunoblotting, respectively. After 8 weeks of ashing, UV-ozone treatment reduced the infectious titer of treated material by a factor of at least 105. A small amount of infectivity, however, persisted despite UV-ozone treatment. When bound to either montmorillonite clay or quartz surfaces, PrPTSE was still susceptible to degradation by UV-ozone. Conclusion. Our findings strongly suggest that UV-ozone treatment can degrade pathogenic prion protein and inactivate prions, even when the agent is associated with surfaces. Using larger UV-ozone doses or combining UV-ozone treatment with other decontaminant methods may allow the sterilization of TSE-contaminated materials. ?? 2009 Aiken et al; licensee BioMed Central Ltd.

  7. High SEPT9_i1 Protein Expression Is Associated with High-Grade Prostate Cancers

    PubMed Central

    Gilad, Roni; Meir, Karen; Stein, Ilan; German, Larissa; Pikarsky, Eli; Mabjeesh, Nicola J.

    2015-01-01

    Septins are a family of GTP-binding cytoskeleton proteins expressed in many solid tumors. Septin 9 (SEPT9) in particular was found overexpressed in diverse carcinomas. Herein, we studied the expression of SEPT9 isoform 1 protein (SEPT9_i1) in human prostate cancer specimens. We utilized immunohistochemical staining to study the expression of SEPT9_i1 protein. Staining level was analyzed in association with clinical characteristics and the pathological Gleason grade and score. Fifty human prostate cancer specimens (42 primary tumors and 8 metastatic lesions) were stained by SEPT9_i1 antibody and analyzed. SEPT9_i1 protein was expressed in prostate cancer cells but absent in normal epithelial cells. The intensity of staining was correlated proportionally to pretreatment prostate-specific antigen (PSA) blood levels and Gleason score (P < 0.05). SEPT9_i1 was highly expressed in all metastatic lesions. A significant assocation between SEPT9_i1 expression and high Gleason score on multivariate linear regression analysis was found. We conclude that SEPT9_i1 is expressed in high-grade prostate tumors suggesting it has a significant role in prostate tumorigenesis and that it could serve as a molecular marker for prostate tumor progression. PMID:25898316

  8. Escherichia coli Response to Uranyl Exposure at Low pH and Associated Protein Regulations

    PubMed Central

    Khemiri, Arbia; Carrière, Marie; Bremond, Nicolas; Ben Mlouka, Mohamed Amine; Coquet, Laurent; Llorens, Isabelle; Chapon, Virginie; Jouenne, Thierry; Cosette, Pascal; Berthomieu, Catherine

    2014-01-01

    Better understanding of uranyl toxicity in bacteria is necessary to optimize strains for bioremediation purposes or for using bacteria as biodetectors for bioavailable uranyl. In this study, after different steps of optimization, Escherichia colicells were exposed to uranyl at low pH to minimize uranyl precipitation and to increase its bioavailability. Bacteria were adapted to mid acidic pH before exposure to 50 or 80 µM uranyl acetate for two hours at pH≈3. To evaluate the impact of uranium, growth in these conditions were compared and the same rates of cells survival were observed in control and uranyl exposed cultures. Additionally, this impact was analyzedby two-dimensional differential gel electrophoresis proteomics to discover protein actors specifically present or accumulated in contact with uranium.Exposure to uranium resulted in differential accumulation of proteins associated with oxidative stress and in the accumulation of the NADH/quinone oxidoreductase WrbA. This FMN dependent protein performs obligate two-electron reduction of quinones, and may be involved in cells response to oxidative stress. Interestingly, this WrbA protein presents similarities with the chromate reductase from E. coli, which was shown to reduce uranyl in vitro. PMID:24587082

  9. The focal adhesion protein PINCH-1 associates with EPLIN at integrin adhesion sites

    PubMed Central

    Karaköse, Esra; Geiger, Tamar; Flynn, Kevin; Lorenz-Baath, Katrin; Zent, Roy; Mann, Matthias; Fässler, Reinhard

    2015-01-01

    ABSTRACT PINCH-1 is a LIM-only domain protein that forms a ternary complex with integrin-linked kinase (ILK) and parvin (to form the IPP complex) downstream of integrins. Here, we demonstrate that PINCH-1 (also known as Lims1) gene ablation in the epidermis of mice caused epidermal detachment from the basement membrane, epidermal hyperthickening and progressive hair loss. PINCH-1-deficient keratinocytes also displayed profound adhesion, spreading and migration defects in vitro that were substantially more severe than those of ILK-deficient keratinocytes indicating that PINCH-1 also exerts functions in an ILK-independent manner. By isolating the PINCH-1 interactome, the LIM-domain-containing and actin-binding protein epithelial protein lost in neoplasm (EPLIN, also known as LIMA1) was identified as a new PINCH-1-associated protein. EPLIN localized, in a PINCH-1-dependent manner, to integrin adhesion sites of keratinocytes in vivo and in vitro and its depletion severely attenuated keratinocyte spreading and migration on collagen and fibronectin without affecting PINCH-1 levels in focal adhesions. Given that the low PINCH-1 levels in ILK-deficient keratinocytes were sufficient to recruit EPLIN to integrin adhesions, our findings suggest that PINCH-1 regulates integrin-mediated adhesion of keratinocytes through the interactions with ILK as well as EPLIN. PMID:25609703

  10. Nutrient-dependent methylation of a membrane-associated protein of Escherichia coli.

    PubMed Central

    Young, C C; Alvarez, J D; Bernlohr, R W

    1990-01-01

    Starvation of a mid-log-phase culture of Escherichia coli B/r for nitrogen, phosphate, or carbon resulted in methylation of a membrane-associated protein of about 43,000 daltons (P-43) in the presence of chloramphenicol and [methyl-3H]methionine. The in vivo methylation reaction occurred with a doubling time of 2 to 5 min and was followed by a slower demethylation process. Addition of the missing nutrient to a starving culture immediately prevented further methylation of P-43. P-43 methylation is not related to the methylated chemotaxis proteins because P-43 is methylated in response to a different spectrum of nutrients and because P-43 is methylated on lysine residues. The characteristics of P-43 are similar to those of a methylated protein previously described in Bacillus subtilis and B. licheniformis (R. W. Bernlohr, A. L. Saha, C. C. Young, B. R. Toth, and K. J. Golden, J. Bacteriol. 170:4113-4118, 1988; K. J. Golden and R. W. Bernlohr, Mol. Gen. Genet. 220:1-7, 1989) and are consistent with the proposal that methylation of this protein functions in nutrient sensing. Images PMID:2203742

  11. Human malignant melanoma-derived progestagen-associated endometrial protein immunosuppresses T lymphocytes in vitro.

    PubMed

    Ren, Suping; Chai, Lina; Wang, Chunyan; Li, Changlan; Ren, Qiquan; Yang, Lihua; Wang, Fumei; Qiao, Zhixin; Li, Weijing; He, Min; Riker, Adam I; Han, Ying; Yu, Qun

    2015-01-01

    Progestagen-associated endometrial protein (PAEP) is a glycoprotein of the lipocalin family that acts as a negative regulator of T cell receptor-mediated activation. However, the function of tumor-derived PAEP on the human immune system in the tumor microenvironment is unknown. PAEP is highly expressed in intermediate and thick primary melanomas (Breslow's 2.5mm or greater) and metastatic melanomas, correlating with its expression in daughter cell lines established in vitro. The current study investigates the role of melanoma cell-secreted PAEP protein in regulating T cell function. Upon the enrichment of CD3+, CD4+ and CD8+ T cells from human peripheral blood mononuclear cells, each subset was then mixed with either melanoma-derived PAEP protein or PAEP-poor supernatant of gene-silenced tumor cells. IL-2 and IFN-γ secretion of CD4+ T cells significantly decreased with the addition of PAEP-rich supernatant. And the addition of PAEP-positive cell supernatant to activated lymphocytes significantly inhibited lymphocyte proliferation and cytotoxic T cell activity, while increasing lymphocyte apoptosis. Our result suggests that melanoma cell-secreted PAEP protein immunosuppresses the activation, proliferation and cytotoxicity of T lymphocytes, which might partially explain the mechanism of immune tolerance induced by melanoma cells within the tumor microenvironment. PMID:25785839

  12. Differential expression of dystrophin, utrophin, and dystrophin-associated proteins in human muscle culture.

    PubMed

    Radojevic, V; Lin, S; Burgunder, J M

    2000-06-01

    The dystrophin-associated protein complex (DAP) plays an important role in sarcolemmal function. Mutations of DAP elements lead to diverse forms of muscular dystrophies, among them Duchenne muscular dystrophy, one of the most severe neuromuscular diseases. Strategies in gene therapy are being assessed to restore DAP stability. However, the relationship between DAP elements and time-course of the DAP formation are still not known in detail. In order to better understand the relationship among DAP proteins, we therefore studied their expression during development in human muscle culture in comparison with developmentally regulated muscle proteins. Desmin immunoreactivity (IR) was detected by 3 days in vitro (DIV3), IR for developmental heavy-chain myosin, vimentin, utrophin, and beta-dystroglycan, as well as alpha-, beta-, and gamma-sarcoglycan, a day later. delta-Sarcoglycan was found by DIV7; dystrophin could be detected only by DIV11. In general, DAP proteins were first located in the perinuclear region, later diffusely in the cytoplasm, and finally exclusively at the membrane. This sequence of events during muscle development gives further support to our suggestion that utrophin could be a precursor of dystrophin during development and regeneration. These data also suggest that utrophin alone is sufficient to anchor the complex, which is important when utrophin replacement strategies are being investigated for the treatment of dystrophinopathies. In this study we demonstrated the establishment of a culture technique that should allow the close study of DAP expression in diseased muscle, including its use after gene modulatory strategies. PMID:10928275

  13. Changes in conformational dynamics of basic side chains upon protein-DNA association.

    PubMed

    Esadze, Alexandre; Chen, Chuanying; Zandarashvili, Levani; Roy, Sourav; Pettitt, B Montgometry; Iwahara, Junji

    2016-08-19

    Basic side chains play major roles in recognition of nucleic acids by proteins. However, dynamic properties of these positively charged side chains are not well understood. In this work, we studied changes in conformational dynamics of basic side chains upon protein-DNA association for the zinc-finger protein Egr-1. By nuclear magnetic resonance (NMR) spectroscopy, we characterized the dynamics of all side-chain cationic groups in the free protein and in the complex with target DNA. Our NMR order parameters indicate that the arginine guanidino groups interacting with DNA bases are strongly immobilized, forming rigid interfaces. Despite the strong short-range electrostatic interactions, the majority of the basic side chains interacting with the DNA phosphates exhibited high mobility, forming dynamic interfaces. In particular, the lysine side-chain amino groups exhibited only small changes in the order parameters upon DNA-binding. We found a similar trend in the molecular dynamics (MD) simulations for the free Egr-1 and the Egr-1-DNA complex. Using the MD trajectories, we also analyzed side-chain conformational entropy. The interfacial arginine side chains exhibited substantial entropic loss upon binding to DNA, whereas the interfacial lysine side chains showed relatively small changes in conformational entropy. These data illustrate different dynamic characteristics of the interfacial arginine and lysine side chains. PMID:27288446

  14. RIPSeeker: a statistical package for identifying protein-associated transcripts from RIP-seq experiments