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Sample records for reversible protein filament

  1. Collaborative protein filaments.

    PubMed

    Ghosal, Debnath; Löwe, Jan

    2015-09-14

    It is now well established that prokaryotic cells assemble diverse proteins into dynamic cytoskeletal filaments that perform essential cellular functions. Although most of the filaments assemble on their own to form higher order structures, growing evidence suggests that there are a number of prokaryotic proteins that polymerise only in the presence of a matrix such as DNA, lipid membrane or even another filament. Matrix-assisted filament systems are frequently nucleotide dependent and cytomotive but rarely considered as part of the bacterial cytoskeleton. Here, we categorise this family of filament-forming systems as collaborative filaments and introduce a simple nomenclature. Collaborative filaments are frequent in both eukaryotes and prokaryotes and are involved in vital cellular processes including chromosome segregation, DNA repair and maintenance, gene silencing and cytokinesis to mention a few. In this review, we highlight common principles underlying collaborative filaments and correlate these with known functions.

  2. Surface manipulation of protein filaments

    NASA Astrophysics Data System (ADS)

    Kreplak, Laurent; Staple, Douglas; Loparic, Marko; Kreuzer, Hans-Juergen

    2009-03-01

    Within mammalian tissues, cells move by actively remodeling a dense network of collagen fibrils. In order to study this situation, we analyze the force response of two types of filamentous protein structures, desmin intermediate filaments 12 nm in diameter and collagen fibrils 80 nm in diameter. Both types of filaments were adsorbed at a solid-liquid interface and locally moved with an AFM tip at constant velocity against surface friction in the interfacial plane. In the case of collagen fibrils, that have an extensibility below 30% extension, we observed that microns long fibrils could be moved by the tip and deformed into shapes that could not be explain by the linear elastic theory for a stiff rod. In the case of desmin filaments that can be stretched up to 3.5 times there length, we observed local stretching of the filaments and discreet steps in the torsional force measured with the cantilever. In order to describe both types of filaments' behaviors, we described the protein filaments as a chain of beads of mass m linked together by a mass-less polymer linker. By solving the Newtonian equations of motions for the coupled beads in the presence of a point load and a viscous drag due to the surface-filament interactions we were able to reproduced our experimental data and extract information on friction.

  3. Quantifying protein diffusion and capture on filaments.

    PubMed

    Reithmann, Emanuel; Reese, Louis; Frey, Erwin

    2015-02-17

    The functional relevance of regulating proteins is often limited to specific binding sites such as the ends of microtubules or actin-filaments. A localization of proteins on these functional sites is of great importance. We present a quantitative theory for a diffusion and capture process, where proteins diffuse on a filament and stop diffusing when reaching the filament's end. It is found that end-association after one-dimensional diffusion is the main source for tip-localization of such proteins. As a consequence, diffusion and capture is highly efficient in enhancing the reaction velocity of enzymatic reactions, where proteins and filament ends are to each other as enzyme and substrate. We show that the reaction velocity can effectively be described within a Michaelis-Menten framework. Together, one-dimensional diffusion and capture beats the (three-dimensional) Smoluchowski diffusion limit for the rate of protein association to filament ends.

  4. Order and disorder in intermediate filament proteins.

    PubMed

    Kornreich, Micha; Avinery, Ram; Malka-Gibor, Eti; Laser-Azogui, Adi; Beck, Roy

    2015-09-14

    Intermediate filaments (IFs), important components of the cytoskeleton, provide a versatile, tunable network of self-assembled proteins. IF proteins contain three distinct domains: an α-helical structured rod domain, flanked by intrinsically disordered head and tail domains. Recent studies demonstrated the functional importance of the disordered domains, which differ in length and amino-acid sequence among the 70 different human IF genes. Here, we investigate the biophysical properties of the disordered domains, and review recent findings on the interactions between them. Our analysis highlights key components governing IF functional roles in the cytoskeleton, where the intrinsically disordered domains dictate protein-protein interactions, supramolecular assembly, and macro-scale order.

  5. Heterologous protein production by filamentous fungi.

    PubMed

    Jeenes, D J; Mackenzie, D A; Roberts, I N; Archer, D B

    1991-01-01

    There are clearly many facets to successful production of heterologous proteins from filamentous fungi. The objectives are to exploit the natural ability of some species to secrete high levels of protein. The heterologous target proteins produced in a fungal host must be acceptable to the public and be economic to produce, i.e. the targets must be authentic (in structure and activity) and be produced in high yield to necessary levels of purity. The appearance of heterologous products from fungi on the market is testament to some success but, equally, there are considerable limitations in our ability to produce desired yields of many target proteins. We endorse the view of van den Hondel, Punt and van Gorcom (1991) that for the commercial production of heterologous proteins from filamentous fungi more information is required on transcriptional control, introns, mRNA stability and processing, translational efficiency, protein secretion, glycosylation and proteolysis. In addition, there is scope for yield improvement based on a better understanding of the physiology of growth/product secretion coupled to appropriate bioreactor operation. The authenticity of product is an aspect which will assume increasing importance, particularly for therapeutic proteins. The level at which the structures and functional activity of heterologous proteins are assessed will ultimately be determined by legislation. The analytical methods currently available are not always sufficient, for example, to reveal folded structures, and most proteins are not amenable to analysis by two-dimensional NMR. The authenticity of target heterologous proteins will also need to be assessed in relation to the glycosylation level and pattern. This is not easily done and explains the paucity of detailed information published to date on glycosylation of fungal proteins. Novel engineered proteins are already being produced from filamentous fungi where expression is an aid to investigation of structure

  6. Mechanical properties of intermediate filament proteins

    PubMed Central

    Charrier, Elisabeth E.; Janmey, Paul A.

    2016-01-01

    Purified intermediate filament proteins can be reassembled in vitro to produce polymers closely resembling those found in cells, and these filament form viscoelastic gels. The crosslinks holding IFs together in the network include specific bonds between polypeptides extending from the filament surface and ionic interactions mediated by divalent cations. IF networks exhibit striking non-linear elasticity with stiffness, as quantified by shear modulus, increasing an order of magnitude as the networks are deformed to large stains resembling those that soft tissues undergo in vivo. Individual Ifs can be stretched to more than 2 or 3 times their resting length without breaking. At least ten different rheometric methods have been used to quantify the viscoelasticity of IF networks over a wide range of timescales and strain magnitudes. The mechanical roles of different classes of IF on mesenchymal and epithelial cells in culture have also been studied by an even wider range of microrheological methods. These studies have documented the effects on cell mechanics when IFs are genetically or pharmacologically disrupted or when normal or mutant IF proteins are exogenously expressed in cells. Consistent with in vitro rheology, the mechanical role of IFs is more apparent as cells are subjected to larger and more frequent deformations. PMID:26795466

  7. Engineering of filamentous bacteriophage for protein sensing

    NASA Astrophysics Data System (ADS)

    Brasino, Michael

    Methods of high throughput, sensitive and cost effective quantification of proteins enables personalized medicine by allowing healthcare professionals to better monitor patient condition and response to treatment. My doctoral research has attempted to advance these methods through the use of filamentous bacteriophage (phage). These bacterial viruses are particularly amenable to both genetic and chemical engineering and can be produced efficiently in large amounts. Here, I discuss several strategies for modifying phage for use in protein sensing assays. These include the expression of bio-orthogonal conjugation handles on the phage coat, the incorporation of specific recognition sequences within the phage genome, and the creation of antibody-phage conjugates via a photo-crosslinking non-canonical amino acid. The physical and chemical characterization of these engineered phage and the results of their use in modified protein sensing assays will be presented.

  8. Molecular phylogeny of metazoan intermediate filament proteins.

    PubMed

    Erber, A; Riemer, D; Bovenschulte, M; Weber, K

    1998-12-01

    We have cloned cytoplasmic intermediate filament (IF) proteins from a large number of invertebrate phyla using cDNA probes, the monoclonal antibody IFA, peptide sequence information, and various RT-PCR procedures. Novel IF protein sequences reported here include the urochordata and nine protostomic phyla, i.e., Annelida, Brachiopoda, Chaetognatha, Echiura, Nematomorpha, Nemertea, Platyhelminthes, Phoronida, and Sipuncula. Taken together with the wealth of data on IF proteins of vertebrates and the results on IF proteins of Cephalochordata, Mollusca, Annelida, and Nematoda, two IF prototypes emerge. The L-type, which includes 35 sequences from 11 protostomic phyla, shares with the nuclear lamins the long version of the coil 1b subdomain and, in most cases, a homology segment of some 120 residues in the carboxyterminal tail domain. The S-type, which includes all four subfamilies (types I to IV) of vertebrate IF proteins, lacks 42 residues in the coil 1b subdomain and the carboxyterminal lamin homology segment. Since IF proteins from all three phyla of the chordates have the 42-residue deletion, this deletion arose in a progenitor prior to the divergence of the chordates into the urochordate, cephalochordate, and vertebrate lineages, possibly already at the origin of the deuterostomic branch. Four phyla recently placed into the protostomia on grounds of their 18S rDNA sequences (Brachiopoda, Nemertea, Phoronida, and Platyhelminthes) show IF proteins of the L-type and fit by sequence identity criteria into the lophotrochozoic branch of the protostomia.

  9. Physical principles of filamentous protein self-assembly kinetics

    NASA Astrophysics Data System (ADS)

    Michaels, Thomas C. T.; Liu, Lucie X.; Meisl, Georg; Knowles, Tuomas P. J.

    2017-04-01

    The polymerization of proteins and peptides into filamentous supramolecular structures is an elementary form of self-organization of key importance to the functioning biological systems, as in the case of actin biofilaments that compose the cellular cytoskeleton. Aberrant filamentous protein self-assembly, however, is associated with undesired effects and severe clinical disorders, such as Alzheimer’s and Parkinson’s diseases, which, at the molecular level, are associated with the formation of certain forms of filamentous protein aggregates known as amyloids. Moreover, due to their unique physicochemical properties, protein filaments are finding extensive applications as biomaterials for nanotechnology. With all these different factors at play, the field of filamentous protein self-assembly has experienced tremendous activity in recent years. A key question in this area has been to elucidate the microscopic mechanisms through which filamentous aggregates emerge from dispersed proteins with the goal of uncovering the underlying physical principles. With the latest developments in the mathematical modeling of protein aggregation kinetics as well as the improvement of the available experimental techniques it is now possible to tackle many of these complex systems and carry out detailed analyses of the underlying microscopic steps involved in protein filament formation. In this paper, we review some classical and modern kinetic theories of protein filament formation, highlighting their use as a general strategy for quantifying the molecular-level mechanisms and transition states involved in these processes.

  10. Physical principles of filamentous protein self-assembly kinetics.

    PubMed

    Michaels, Thomas; Liu, Xiaoxuan; Meisl, Georg; Knowles, Tuomas P J

    2017-02-07

    The polymerization of proteins and peptides into filamentous supramolecular structures is an elementary form of self-organization of key importance to the functioning biological systems, as in the case of actin biofilaments that compose the cellular cytoskeleton. Aberrant filamentous protein self-assembly, however, is associated with undesired effects and severe clinical disorders, such as Alzheimer's and Parkinson's diseases, which, at the molecular level, are associated with the formation of certain forms of filamentous protein aggregates known as amyloids. Moreover, due to their unique physicochemical properties, protein filaments are finding extensive applications as biomaterials for nanotechnology. With all these different factors at play, the field of filamentous protein self-assembly has experienced tremendous activity in recent years. A key question in this area has been to elucidate the microscopic mechanisms through which filamentous aggregates emerge from dispersed proteins with the goal of uncovering the underlying physical principles. With the latest developments in the mathematical modeling of protein aggregation kinetics as well as the improvement of the available experimental techniques it is now possible to tackle many of these complex systems and carry out detailed analyses of the underlying microscopic steps involved in protein filament formation. In this paper, we review some classical and modern kinetic theories of protein filament formation, highlighting their use as a general strategy for quantifying the molecular-level mechanisms and transition states involved in these processes.

  11. Spontaneous oscillatory contraction without regulatory proteins in actin filament-reconstituted fibers.

    PubMed

    Fujita, H; Ishiwata, S

    1998-09-01

    Skinned skeletal and cardiac muscle fibers exhibits spontaneous oscillatory contraction (SPOC) in the presence of MgATP, MgADP, and inorganic phosphate (Pi)1 but the molecular mechanism underlying this phenomenon is not yet clear. We have investigated the role of regulatory proteins in SPOC using cardiac muscle fibers of which the actin filaments had been reconstituted without tropomyosin and troponin, according to a previously reported method (Fujita et al., 1996. Biophys. J. 71:2307-2318). That is, thin filaments in glycerinated cardiac muscle fibers were selectively removed by treatment with gelsolin. Then, by adding exogenous actin to these thin filament-free cardiac muscle fibers under polymerizing conditions, actin filaments were reconstituted. The actin filament-reconstituted cardiac muscle fibers generated active tension in a Ca(2+)-insensitive manner because of the lack of regulatory proteins. Herein we have developed a new solvent condition under which SPOC occurs, even in actin filament-reconstituted fibers: the coexistence of 2,3-butanedione 2-monoxime (BDM), a reversible inhibitor of actomyosin interactions, with MgATP, MgADP and Pi. The role of BDM in the mechanism of SPOC in the actin filament-reconstituted fibers was analogous to that of the inhibitory function of the tropomyosin-troponin complex (-Ca2+) in the control fibers. The present results suggest that SPOC is a phenomenon that is intrinsic to the actomyosin motor itself.

  12. Spontaneous oscillatory contraction without regulatory proteins in actin filament-reconstituted fibers.

    PubMed Central

    Fujita, H; Ishiwata, S

    1998-01-01

    Skinned skeletal and cardiac muscle fibers exhibits spontaneous oscillatory contraction (SPOC) in the presence of MgATP, MgADP, and inorganic phosphate (Pi)1 but the molecular mechanism underlying this phenomenon is not yet clear. We have investigated the role of regulatory proteins in SPOC using cardiac muscle fibers of which the actin filaments had been reconstituted without tropomyosin and troponin, according to a previously reported method (Fujita et al., 1996. Biophys. J. 71:2307-2318). That is, thin filaments in glycerinated cardiac muscle fibers were selectively removed by treatment with gelsolin. Then, by adding exogenous actin to these thin filament-free cardiac muscle fibers under polymerizing conditions, actin filaments were reconstituted. The actin filament-reconstituted cardiac muscle fibers generated active tension in a Ca(2+)-insensitive manner because of the lack of regulatory proteins. Herein we have developed a new solvent condition under which SPOC occurs, even in actin filament-reconstituted fibers: the coexistence of 2,3-butanedione 2-monoxime (BDM), a reversible inhibitor of actomyosin interactions, with MgATP, MgADP and Pi. The role of BDM in the mechanism of SPOC in the actin filament-reconstituted fibers was analogous to that of the inhibitory function of the tropomyosin-troponin complex (-Ca2+) in the control fibers. The present results suggest that SPOC is a phenomenon that is intrinsic to the actomyosin motor itself. PMID:9726945

  13. Mammalian CARMIL Inhibits Actin Filament Capping by Capping Protein

    PubMed Central

    Yang, Changsong; Pring, Martin; Wear, Martin A.; Huang, Minzhou; Cooper, John A.; Svitkina, Tatyana M.; Zigmond, Sally H.

    2009-01-01

    Summary Actin polymerization in cells occurs via filament elongation at the barbed end. Proteins that cap the barbed end terminate this elongation. Heterodimeric capping protein (CP) is an abundant and ubiquitous protein that caps the barbed end. We find that the mouse homolog of the adaptor protein CARMIL (mCARMIL) binds CP with high affinity and decreases its affinity for the barbed end. Addition of mCARMIL to cell extracts increases the rate and extent of Arp2/3 or spectrin-actin seed-induced polymerization. In cells, GFP-mCARMIL concentrates in lamellipodia and increases the fraction of cells with large lamellipodia. Decreasing mCARMIL levels by siRNA transfection lowers theF-actin level and slows cell migration through a mechanism that includes decreased lamellipodia protrusion. This phenotype is reversed by full-length mCARMIL but not mCARMIL lacking the domain that binds CP. Thus, mCARMIL is a key regulator of CP and has profound effects on cell behavior. PMID:16054028

  14. Identification of trichoplein, a novel keratin filament-binding protein.

    PubMed

    Nishizawa, Miwako; Izawa, Ichiro; Inoko, Akihito; Hayashi, Yuko; Nagata, Koh-ichi; Yokoyama, Tomoya; Usukura, Jiro; Inagaki, Masaki

    2005-03-01

    Keratins 8 and 18 (K8/18) are major components of the intermediate filaments (IFs) of simple epithelia. We report here the identification of a novel protein termed trichoplein. This protein shows a low degree of sequence similarity to trichohyalin, plectin and myosin heavy chain, and is a K8/18-binding protein. Among interactions between trichoplein and various IF proteins that we tested using two-hybrid methods, trichoplein interacted significantly with K16 and K18, and to some extent with K5, K6a, K8 and K14. In in vitro co-sedimentation assays, trichoplein directly binds to K8/18, but not with vimentin, desmin, actin filaments or microtubules. An antibody raised against trichoplein specifically recognized a polypeptide with a relative molecular mass of 61 kDa in cell lysates. Trichoplein was immunoprecipitated using this antibody in a complex with K8/18 and immunostaining revealed that trichoplein colocalized with K8/18 filaments in HeLa cells. In polarized Caco-2 cells, trichoplein colocalized not only with K8/18 filaments in the apical region but also with desmoplakin, a constituent of desmosomes. In the absorptive cells of the small intestine, trichoplein colocalized with K8/18 filaments at the apical cortical region, and was also concentrated at desmosomes. Taken together, these results suggest that trichoplein is a keratin-binding protein that may be involved in the organization of the apical network of keratin filaments and desmosomes in simple epithelial cells.

  15. Phosphorylated tyrosine in the flagellum filament protein of Pseudomonas aeruginosa

    SciTech Connect

    Kelly-Wintenberg, K.; Anderson, T.; Montie, T.C. )

    1990-09-01

    Purified flagella from two strains of {sup 32}P-labeled Pseudomonas aeruginosa were shown to be phosphorylated. This was confirmed by autoradiography of flagellin protein in polyacrylamide gels. Thin-layer electrophoresis and autoradiography of flagellin partial hydrolysates indicated that phosphotyrosine was the major phosphorylated amino acid. High-pressure liquid chromatographic analysis confirmed the presence of phosphotyrosine in flagellum filament protein. Preliminary data indicated that less than one tyrosine per subunit was phosphorylated. No evidence was found for phosphorylation of serine or threonine. A function related to tyrosine phosphorylation has not been determined.

  16. Soluble and filamentous proteins in Arabidopsis sieve elements.

    PubMed

    Batailler, Brigitte; Lemaître, Thomas; Vilaine, Françoise; Sanchez, Christian; Renard, Denis; Cayla, Thibaud; Beneteau, Julie; Dinant, Sylvie

    2012-07-01

    Phloem sieve elements are highly differentiated cells involved in the long-distance transport of photoassimilates. These cells contain both aggregated phloem-proteins (P-proteins) and soluble proteins, which are also translocated by mass flow. We used liquid chromatography-tandem mass spectrometry (LC-MS/MS) to carry out a proteomic survey of the phloem exudate of Arabidopsis thaliana, collected by the ethylenediaminetetraacetic acid (EDTA)-facilitated method. We identified 287 proteins, a large proportion of which were enzymes involved in the metabolic precursor generation and amino acid synthesis, suggesting that sieve tubes display high levels of metabolic activity. RNA-binding proteins, defence proteins and lectins were also found. No putative P-proteins were detected in the EDTA-exudate fraction, indicating a lack of long-distance translocation of such proteins in Arabidopsis. In parallel, we investigated the organization of P-proteins, by high-resolution transmission electron microscopy, and the localization of the phloem lectin PP2, a putative P-protein component, by immunolocalization with antibodies against PP2-A1. Transmission electron microscopy observations of P-proteins revealed bundles of filaments resembling strings of beads. PP2-A1 was found weakly associated with these structures in the sieve elements and bound to plastids. These observations suggest that PP2-A1 is anchored to P-proteins and organelles rather than being a structural component of P-proteins.

  17. Major coat protein and single-stranded DNA-binding protein of filamentous virus Pf3.

    PubMed Central

    Putterman, D G; Casadevall, A; Boyle, P D; Yang, H L; Frangione, B; Day, L A

    1984-01-01

    The region of the Pf3 virus genome encoding its major coat protein and its single-stranded DNA-binding protein is organized somewhat like the corresponding region of the fd (M13, f1) genome. Nevertheless, the major coat protein is unique among the major coat proteins of fd and the other filamentous phages studied in that it lacks a signal sequence and appears to be a direct translation product and in that it has fewer basic amino acid residues than its equivalent of DNA phosphates in the virion. These features are relevant to considerations of both protein insertion into membranes and DNA structure in filamentous viruses. The single-stranded DNA-binding protein also has a sequence that is different from the sequences of single-stranded DNA-binding proteins from other filamentous viruses. Images PMID:6422463

  18. Kinetics of fragmentation and dissociation of two-strand protein filaments: Coarse-grained simulations and experiments

    NASA Astrophysics Data System (ADS)

    Zaccone, A.; Terentjev, I.; Herling, T. W.; Knowles, T. P. J.; Aleksandrova, A.; Terentjev, E. M.

    2016-09-01

    While a significant body of investigations have been focused on the process of protein self-assembly, much less is understood about the reverse process of a filament breaking due to thermal motion into smaller fragments, or depolymerization of subunits from the filament ends. Indirect evidence for actin and amyloid filament fragmentation has been reported, although the phenomenon has never been directly observed either experimentally or in simulations. Here we report the direct observation of filament depolymerization and breakup in a minimal, calibrated model of coarse-grained molecular simulation. We quantify the orders of magnitude by which the depolymerization rate from the filament ends koff is larger than fragmentation rate k- and establish the law koff/k- = exp[(ɛ‖ - ɛ⊥)/kBT] = exp[0.5ɛ/kBT], which accounts for the topology and energy of bonds holding the filament together. This mechanism and the order-of-magnitude predictions are well supported by direct experimental measurements of depolymerization of insulin amyloid filaments.

  19. Functional synergy of actin filament cross-linking proteins.

    PubMed

    Tseng, Yiider; Schafer, Benjamin W; Almo, Steven C; Wirtz, Denis

    2002-07-12

    The organization of filamentous actin (F-actin) in resilient networks is coordinated by various F-actin cross-linking proteins. The relative tolerance of cells to null mutations of genes that code for a single actin cross-linking protein suggests that the functions of those proteins are highly redundant. This apparent functional redundancy may, however, reflect the limited resolution of available assays in assessing the mechanical role of F-actin cross-linking/bundling proteins. Using reconstituted F-actin networks and rheological methods, we demonstrate how alpha-actinin and fascin, two F-actin cross-linking/bundling proteins that co-localize along stress fibers and in lamellipodia, could synergistically enhance the resilience of F-actin networks in vitro. These two proteins can generate microfilament arrays that "yield" at a strain amplitude that is much larger than each one of the proteins separately. F-actin/alpha-actinin/fascin networks display strain-induced hardening, whereby the network "stiffens" under shear deformations, a phenomenon that is non-existent in F-actin/fascin networks and much weaker in F-actin/alpha-actinin networks. Strain-hardening is further enhanced at high rates of deformation and high concentrations of actin cross-linking proteins. A simplified model suggests that the optimum results of the competition between the increased stiffness of bundles and their decreased density of cross-links. Our studies support a re-evaluation of the notion of functional redundancy among cytoskeletal regulatory proteins.

  20. Kelch Domain of Gigaxonin Interacts with Intermediate Filament Proteins Affected in Giant Axonal Neuropathy

    PubMed Central

    Johnson-Kerner, Bethany L.; Garcia Diaz, Alejandro; Ekins, Sean; Wichterle, Hynek

    2015-01-01

    Patients with giant axonal neuropathy (GAN) show progressive loss of motor and sensory function starting in childhood and typically live for less than 30 years. GAN is caused by autosomal recessive mutations leading to low levels of gigaxonin (GIG), a ubiquitously-expressed BTB/Kelch cytoplasmic protein believed to be an E3 ligase substrate adaptor. GAN pathology is characterized by aggregates of intermediate filaments (IFs) in multiple tissues. To delineate the molecular pathway between GIG deficiency and IF pathology, we undertook a proteomic screen to identify the normal binding partners of GIG. Prominent among them were several classes of IFs, including the neurofilament subunits whose accumulation leads to the axonal swellings for which GAN is named. We showed these interactions were dependent on the Kelch domain of GIG. Furthermore, we identified the E3 ligase MYCBP2 and the heat shock proteins HSP90AA1/AB1 as interactors with the BTB domain that may result in the ubiquitination and subsequent degradation of intermediate filaments. Our open-ended proteomic screen provides support to GIG’s role as an adaptor protein, linking IF proteins through its Kelch domain to the ubiquitin pathway proteins via its BTB domain, and points to future approaches for reversing the phenotype in human patients. PMID:26460568

  1. Reversible Oxygenation of Oxygen Transport Proteins.

    ERIC Educational Resources Information Center

    Drain, C. M.; Corden, Barry B.

    1987-01-01

    Describes a lecture demonstration which illustrates changes in the visible spectra of oxygen transport proteins upon reversible oxygen binding. Provides a comparison of the physical characteristics of oxygen storage and transport proteins. Reviews essentials for preparation of the materials. (ML)

  2. Unfolded protein response in filamentous fungi-implications in biotechnology.

    PubMed

    Heimel, Kai

    2015-01-01

    The unfolded protein response (UPR) represents a mechanism to preserve endoplasmic reticulum (ER) homeostasis that is conserved in eukaryotes. ER stress caused by the accumulation of potentially toxic un- or misfolded proteins in the ER triggers UPR activation and the induction of genes important for protein folding in the ER, ER expansion, and transport from and to the ER. Along with this adaptation, the overall capacity for protein secretion is markedly increased by the UPR. In filamentous fungi, various approaches to employ the UPR for improved production of homologous and heterologous proteins have been investigated. As the effects on protein production were strongly dependent on the expressed protein, generally applicable strategies have to be developed. A combination of transcriptomic approaches monitoring secretion stress and basic research on the UPR mechanism provided novel and important insight into the complex regulatory cross-connections between UPR signalling, cellular physiology, and developmental processes. It will be discussed how this increasing knowledge on the UPR might stimulate the development of novel strategies for using the UPR as a tool in biotechnology.

  3. Filamentous-haemagglutinin-like protein genes encoded on a plasmid of Moraxella bovis.

    PubMed

    Kakuda, Tsutomu; Sarataphan, Nopporn; Tanaka, Tetsuya; Takai, Shinji

    2006-11-26

    The complete nucleotide sequence of a plasmid, pMBO-1, from Moraxella bovis strain Epp63 was determined. We identified 30 open reading frames (ORFs) encoded by the 44,215bp molecule. Two large ORFs, flpA and flpB, encoding proteins with similarity to Bordetella pertussis filamentous haemagglutinin (FHA), were identified on the same plasmid. The gene for a specific accessory protein (Fap), which may play a role in the secretion of Flp protein, was also identified. Reverse transcriptase PCR analysis of total RNA isolated from M. bovis Epp63 indicated that the flpA, flpB, and fap genes are all transcribed. Southern blot analysis indicated that the flp and fap genes are present in other clinical isolates of geographically diverse M. bovis.

  4. Two Drosophila melanogaster proteins related to intermediate filament proteins of vertebrate cells

    PubMed Central

    1981-01-01

    Monoclonal antibodies were prepared against a 46,000 mol wt major cytoplasmic protein from Drosophila melanogaster Kc cells. These antibodies reacted with the 46,000 and a 40,000 mol wt protein from Kc cells. Some antibodies showed cross-reaction with 55,000 (vimentin) and 52,000 mol wt (desmin) proteins from baby hamster kidney (BHK) cells that form intermediate sized filaments in vertebrate cells. In indirect immunofluorescence, the group of cross reacting antibodies stained a filamentous meshwork in the cytoplasm of vertebrate cells. In Kc cells the fluorescence seemed to be localized in a filamentous meshwork that became more obvious after the cells had flattened out on a surface. These cytoskeletal structures are heat-labile; the proteins in Kc or BHK cells rearrange after a brief heat shock, forming juxtanuclear cap structures. PMID:6795212

  5. Intermolecular forces between the motor protein and the filament.

    PubMed

    Suda, H; Taylor, T W

    1993-03-07

    Intermolecular forces between motor proteins and filaments were evaluated on the basis of the experimental data of an in vitro motility assay by considering the molecular friction in the movement system. The molecular friction was caused by a different mechanism from that of the hydrodynamic drag. However, the molecular frictional forces apparently gave the same expression as the hydrodynamic frictional forces. The resulting equation was very effective in examining the physical properties of the weak interaction in the dynein-microtubules system from basic experiments carried out by Vale et al. (1989). From careful analysis of their experimental data, it was concluded that the hydrodynamic friction was not dominant, even in the weak binding state. The electrostatic interaction between dynein-heads and microtubules in the weak binding state was analyzed by applying the DLVO (Derjaguin-Landau-Verway-Overbeek) theory in colloid science through the ionic dependence of one-dimensional diffusion. The interacting distance between charges which took part in the weak adhesion was estimated to be 3 nm. In the present study, the molecular mechanism of the sliding velocity was also investigated for the myosin-actin filaments and the kinesin-microtubules systems by fitting the ATP-dependence and the ionic dependence in ATP-driven active sliding.

  6. The Saccharomyces cerevisiae Fin1 protein forms cell cycle-specific filaments between spindle pole bodies.

    PubMed

    van Hemert, Martijn J; Lamers, Gerda E M; Klein, Dionne C G; Oosterkamp, Tjerk H; Steensma, H Yde; van Heusden, G Paul H

    2002-04-16

    The FIN1 gene from the yeast Saccharomyces cerevisiae encodes a basic protein with putative coiled-coil regions. Here we show that in large-budded cells a green fluorescent protein-Fin1 fusion protein is visible as a filament between the two spindle pole bodies. In resting cells the protein is undetectable, and in small-budded cells it is localized in the nucleus. During late mitosis it localizes on the spindle pole bodies. Filaments of cyano fluorescent protein-tagged Fin1 colocalize with filaments of green fluorescent protein-tagged Tub1 only in large-budded cells. By electron and atomic force microscopy we showed that purified recombinant Fin1p self-assembles into filaments with a diameter of approximately 10 nm. Our results indicate that the Fin1 protein forms a cell cycle-specific filament, additional to the microtubules, between the spindle pole bodies of dividing yeast cells.

  7. The Saccharomyces cerevisiae Fin1 protein forms cell cycle-specific filaments between spindle pole bodies

    PubMed Central

    van Hemert, Martijn J.; Lamers, Gerda E. M.; Klein, Dionne C. G.; Oosterkamp, Tjerk H.; Steensma, H. Yde; van Heusden, G. Paul H.

    2002-01-01

    The FIN1 gene from the yeast Saccharomyces cerevisiae encodes a basic protein with putative coiled-coil regions. Here we show that in large-budded cells a green fluorescent protein-Fin1 fusion protein is visible as a filament between the two spindle pole bodies. In resting cells the protein is undetectable, and in small-budded cells it is localized in the nucleus. During late mitosis it localizes on the spindle pole bodies. Filaments of cyano fluorescent protein-tagged Fin1 colocalize with filaments of green fluorescent protein-tagged Tub1 only in large-budded cells. By electron and atomic force microscopy we showed that purified recombinant Fin1p self-assembles into filaments with a diameter of ≈10 nm. Our results indicate that the Fin1 protein forms a cell cycle-specific filament, additional to the microtubules, between the spindle pole bodies of dividing yeast cells. PMID:11929974

  8. Self-assembly of a filament by curvature-inducing proteins

    NASA Astrophysics Data System (ADS)

    Kwiecinski, James; Chapman, S. Jonathan; Goriely, Alain

    2017-04-01

    We explore a simplified macroscopic model of membrane shaping by means of curvature-sensing BAR proteins. Equations describing the interplay between the shape of a freely floating filament in a fluid and the adhesion kinetics of proteins are derived from mechanical principles. The constant curvature solutions that arise from this system are studied using weakly nonlinear analysis. We show that the stability of the filament's shape is completely characterized by the parameters associated with protein recruitment and establish that in the bistable regime, proteins aggregate on the filament forming regions of high and low curvatures. This pattern formation is then followed by phase-coarsening that resolves on a time-scale dependent on protein diffusion and drift across the filament, which contend to smooth and maintain the pattern respectively. The model is generalized for multiple species of BAR proteins and we show that the stability of the assembled shape is determined by a competition between proteins attaching on opposing sides.

  9. Bypass of Candida albicans Filamentation/Biofilm Regulators through Diminished Expression of Protein Kinase Cak1

    PubMed Central

    Xu, Wenjie; Aleynikov, Tatyana; Cullen, Paul J.; Lanni, Frederick; Andes, David R.

    2016-01-01

    Biofilm formation on implanted medical devices is a major source of lethal invasive infection by Candida albicans. Filamentous growth of this fungus is tied to biofilm formation because many filamentation-associated genes are required for surface adherence. Cell cycle or cell growth defects can induce filamentation, but we have limited information about the coupling between filamentation and filamentation-associated gene expression after cell cycle/cell growth inhibition. Here we identified the CDK activating protein kinase Cak1 as a determinant of filamentation and filamentation-associated gene expression through a screen of mutations that diminish expression of protein kinase-related genes implicated in cell cycle/cell growth control. A cak1 diminished expression (DX) strain displays filamentous growth and expresses filamentation-associated genes in the absence of typical inducing signals. In a wild-type background, expression of filamentation-associated genes depends upon the transcription factors Bcr1, Brg1, Efg1, Tec1, and Ume6. In the cak1 DX background, the dependence of filamentation-associated gene expression on each transcription factor is substantially relieved. The unexpected bypass of filamentation-associated gene expression activators has the functional consequence of enabling biofilm formation in the absence of Bcr1, Brg1, Tec1, Ume6, or in the absence of both Brg1 and Ume6. It also enables filamentous cell morphogenesis, though not biofilm formation, in the absence of Efg1. Because these transcription factors are known to have shared target genes, we suggest that cell cycle/cell growth limitation leads to activation of several transcription factors, thus relieving dependence on any one. PMID:27935965

  10. Respiratory Syncytial Virus Assembles into Structured Filamentous Virion Particles Independently of Host Cytoskeleton and Related Proteins

    PubMed Central

    Shaikh, Fyza Y.; Utley, Thomas J.; Craven, Ryan E.; Rogers, Meredith C.; Lapierre, Lynne A.; Goldenring, James R.; Crowe, James E.

    2012-01-01

    Respiratory syncytial virus (RSV) is a single-stranded RNA virus that assembles into viral filaments at the cell surface. Virus assembly often depends on the ability of a virus to use host proteins to accomplish viral tasks. Since the fusion protein cytoplasmic tail (FCT) is critical for viral filamentous assembly, we hypothesized that host proteins important for viral assembly may be recruited by the FCT. Using a yeast two-hybrid screen, we found that filamin A interacted with FCT, and mammalian cell experiments showed it localized to viral filaments but did not affect viral replication. Furthermore, we found that a number of actin-associated proteins also were excluded from viral filaments. Actin or tubulin cytoskeletal rearrangement was not necessary for F trafficking to the cell surface or for viral assembly into filaments, but was necessary for optimal viral replication and may be important for anchoring viral filaments. These findings suggest that RSV assembly into filaments occurs independently of actin polymerization and that viral proteins are the principal drivers for the mechanical tasks involved with formation of complex, structured RSV filaments at the host cell plasma membrane. PMID:22808269

  11. The role of gigaxonin in the degradation of the glial-specific intermediate filament protein GFAP

    PubMed Central

    Lin, Ni-Hsuan; Huang, Yu-Shan; Opal, Puneet; Goldman, Robert D.; Messing, Albee; Perng, Ming-Der

    2016-01-01

    Alexander disease (AxD) is a primary genetic disorder of astrocytes caused by dominant mutations in the gene encoding the intermediate filament (IF) protein GFAP. This disease is characterized by excessive accumulation of GFAP, known as Rosenthal fibers, within astrocytes. Abnormal GFAP aggregation also occurs in giant axon neuropathy (GAN), which is caused by recessive mutations in the gene encoding gigaxonin. Given that one of the functions of gigaxonin is to facilitate proteasomal degradation of several IF proteins, we sought to determine whether gigaxonin is involved in the degradation of GFAP. Using a lentiviral transduction system, we demonstrated that gigaxonin levels influence the degradation of GFAP in primary astrocytes and in cell lines that express this IF protein. Gigaxonin was similarly involved in the degradation of some but not all AxD-associated GFAP mutants. In addition, gigaxonin directly bound to GFAP, and inhibition of proteasome reversed the clearance of GFAP in cells achieved by overexpressing gigaxonin. These studies identify gigaxonin as an important factor that targets GFAP for degradation through the proteasome pathway. Our findings provide a critical foundation for future studies aimed at reducing or reversing pathological accumulation of GFAP as a potential therapeutic strategy for AxD and related diseases. PMID:27798231

  12. Depolymerization dynamics of individual filaments of bacterial cytoskeletal protein FtsZ.

    PubMed

    Mateos-Gil, Pablo; Paez, Alfonso; Hörger, Ines; Rivas, Germán; Vicente, Miguel; Tarazona, Pedro; Vélez, Marisela

    2012-05-22

    We report observation and analysis of the depolymerization filaments of the bacterial cytoskeletal protein FtsZ (filament temperature-sensitive Z) formed on a mica surface. At low concentration, proteins adsorbed on the surface polymerize forming curved filaments that close into rings that remain stable for some time before opening irreversibly and fully depolymerizing. The distribution of ring lifetimes (T) as a function of length (N), shows that the rate of ring aperture correlates with filament length. If this ring lifetime is expressed as a bond survival time, (T(b) ≡ NT), this correlation is abolished, indicating that these rupture events occur randomly and independently at each monomer interface. After rings open irreversibly, depolymerization of the remaining filaments is fast, but can be slowed down and followed using a nonhydrolyzing GTP analogue. The histogram of depolymerization velocities of individual filaments has an asymmetric distribution that can be fit with a computer model that assumes two rupture rates, a slow one similar to the one observed for ring aperture, affecting monomers in the central part of the filaments, and a faster one affecting monomers closer to the open ends. From the quantitative analysis, we conclude that the depolymerization rate is affected both by nucleotide hydrolysis rate and by its exchange along the filament, that all monomer interfaces are equally competent for hydrolysis, although depolymerization is faster at the open ends than in central filament regions, and that all monomer-monomer interactions, regardless of the nucleotide present, can adopt a curved configuration.

  13. Depolymerization dynamics of individual filaments of bacterial cytoskeletal protein FtsZ

    PubMed Central

    Mateos-Gil, Pablo; Paez, Alfonso; Hörger, Ines; Rivas, Germán; Vicente, Miguel; Tarazona, Pedro; Vélez, Marisela

    2012-01-01

    We report observation and analysis of the depolymerization filaments of the bacterial cytoskeletal protein FtsZ (filament temperature-sensitive Z) formed on a mica surface. At low concentration, proteins adsorbed on the surface polymerize forming curved filaments that close into rings that remain stable for some time before opening irreversibly and fully depolymerizing. The distribution of ring lifetimes (T) as a function of length (N), shows that the rate of ring aperture correlates with filament length. If this ring lifetime is expressed as a bond survival time, (Tb ≡ NT), this correlation is abolished, indicating that these rupture events occur randomly and independently at each monomer interface. After rings open irreversibly, depolymerization of the remaining filaments is fast, but can be slowed down and followed using a nonhydrolyzing GTP analogue. The histogram of depolymerization velocities of individual filaments has an asymmetric distribution that can be fit with a computer model that assumes two rupture rates, a slow one similar to the one observed for ring aperture, affecting monomers in the central part of the filaments, and a faster one affecting monomers closer to the open ends. From the quantitative analysis, we conclude that the depolymerization rate is affected both by nucleotide hydrolysis rate and by its exchange along the filament, that all monomer interfaces are equally competent for hydrolysis, although depolymerization is faster at the open ends than in central filament regions, and that all monomer–monomer interactions, regardless of the nucleotide present, can adopt a curved configuration. PMID:22566654

  14. Arabidopsis microtubule-destabilizing protein 25 functions in pollen tube growth by severing actin filaments.

    PubMed

    Qin, Tao; Liu, Xiaomin; Li, Jiejie; Sun, Jingbo; Song, Leina; Mao, Tonglin

    2014-01-01

    The formation of distinct actin filament arrays in the subapical region of pollen tubes is crucial for pollen tube growth. However, the molecular mechanisms underlying the organization and dynamics of the actin filaments in this region remain to be determined. This study shows that Arabidopsis thaliana MICROTUBULE-DESTABILIZING PROTEIN25 (MDP25) has the actin filament-severing activity of an actin binding protein. This protein negatively regulated pollen tube growth by modulating the organization and dynamics of actin filaments in the subapical region of pollen tubes. MDP25 loss of function resulted in enhanced pollen tube elongation and inefficient fertilization. MDP25 bound directly to actin filaments and severed individual actin filaments, in a manner that was dramatically enhanced by Ca(2+), in vitro. Analysis of a mutant that bears a point mutation at the Ca(2+) binding sites demonstrated that the subcellular localization of MDP25 was determined by cytosolic Ca(2+) level in the subapical region of pollen tubes, where MDP25 was disassociated from the plasma membrane and moved into the cytosol. Time-lapse analysis showed that the F-actin-severing frequency significantly decreased and a high density of actin filaments was observed in the subapical region of mdp25-1 pollen tubes. This study reveals a mechanism whereby calcium enhances the actin filament-severing activity of MDP25 in the subapical region of pollen tubes to modulate pollen tube growth.

  15. High resolution characterization of myosin IIC protein tailpiece and its effect on filament assembly.

    PubMed

    Rosenberg, Masha M; Ronen, Daniel; Lahav, Noa; Nazirov, Elvira; Ravid, Shoshana; Friedler, Assaf

    2013-04-05

    The motor protein nonmuscle myosin II (NMII) must undergo dynamic oligomerization into filaments to perform its cellular functions. A small nonhelical region at the tail of the long coiled-coil region (tailpiece) is a common feature of all dynamically assembling myosin II proteins. This tailpiece is a key regulatory domain affecting NMII filament assembly properties and is subject to phosphorylation in vivo. We previously demonstrated that the positively charged region of the tailpiece binds to assembly-incompetent NMII-C fragments, inducing filament assembly. In the current study, we investigated the molecular mechanisms by which the tailpiece regulates NMII-C self-assembly. Using alanine scan, we found that specific positive and aromatic residues within the positively charged region of the tailpiece are important for inducing NMII-C filament assembly and for filament elongation. Combining peptide arrays with deletion studies allowed us to identify the tailpiece binding sites in the coiled-coil rod. Elucidation of the mechanism by which the tailpiece induces filament assembly permitted us further investigation into the role of tailpiece phosphorylation. Sedimentation and CD spectroscopy identified that phosphorylation of Thr(1957) or Thr(1960) inhibited the ability of the tailpiece to bind the coiled-coil rod and to induce NMII-C filament formation. This study provides molecular insight into the role of specific residues within the NMII-C tailpiece that are responsible for shifting the oligomeric equilibrium of NMII-C toward filament assembly and determining its morphology.

  16. Viral infectivity and intracellular distribution of matrix (M) protein of canine distemper virus are affected by actin filaments.

    PubMed

    Klauschies, F; Gützkow, T; Hinkelmann, S; von Messling, V; Vaske, B; Herrler, G; Haas, L

    2010-09-01

    To investigate the role of cytoskeletal components in canine distemper virus (CDV) replication, various agents were used that interfere with turnover of actin filaments and microtubules. Only inhibition of actin filaments significantly reduced viral infectivity. Analysis of the intracellular localization of the viral matrix (M) protein revealed that it aligned along actin filaments. Treatment with actin filament-disrupting drugs led to a marked intracellular redistribution of M protein during infection as well as transfection. In contrast, the localization of the CDV fusion (F) protein was not significantly changed during transfection. Thus, a M protein-actin filament interaction appears to be important for generation of infectious CDV.

  17. Self-assembly enhances the strength of fibers made from vimentin intermediate filament proteins.

    PubMed

    Pinto, Nicole; Yang, Fei-Chi; Negishi, Atsuko; Rheinstädter, Maikel C; Gillis, Todd E; Fudge, Douglas S

    2014-02-10

    Hagfish slime threads were recently established as a promising biomimetic model for efforts to produce ecofriendly alternatives to petroleum polymers. Initial attempts to make fibers from solubilized slime thread proteins fell short of achieving the outstanding mechanics of native slime threads. Here we tested the hypothesis that the high strength and toughness of slime threads arise from the ability of constituent intermediate filaments to undergo a stress-induced α-to-β transition. To do this, we made fibers from human vimentin proteins that were first allowed to self-assemble into 10 nm intermediate filaments. Fibers made from assembled vimentin hydrogels underwent an α-to-β transition when strained and exhibited improved mechanical performance. Our data demonstrate that it is possible to make materials from intermediate filament hydrogels and that mimicking the secondary structure of native hagfish slime threads using intermediate filament self-assembly is a promising strategy for improving the mechanical performance of biomimetic protein materials.

  18. Thin filament proteins and thin filament-linked regulation of vertebrate muscle contraction.

    PubMed

    Leavis, P C; Gergely, J

    1984-01-01

    Recent developments in the field of myofibrillar proteins will be reviewed. Consideration will be given to the proteins that participate in the contractile process itself as well as to those involved in Ca-dependent regulation of striated (skeletal and cardiac) and smooth muscle. The relation of protein structure to function will be emphasized and the relation of various physiologically and histochemically defined fiber types to the proteins found in them will be discussed.

  19. ATP-dependent regulation of actin monomer-filament equilibrium by cyclase-associated protein and ADF/cofilin.

    PubMed

    Nomura, Kazumi; Ono, Shoichiro

    2013-07-15

    CAP (cyclase-associated protein) is a conserved regulator of actin filament dynamics. In the nematode Caenorhabditis elegans, CAS-1 is an isoform of CAP that is expressed in striated muscle and regulates sarcomeric actin assembly. In the present study, we report that CAS-2, a second CAP isoform in C. elegans, attenuates the actin-monomer-sequestering effect of ADF (actin depolymerizing factor)/cofilin to increase the steady-state levels of actin filaments in an ATP-dependent manner. CAS-2 binds to actin monomers without a strong preference for either ATP- or ADP-actin. CAS-2 strongly enhances the exchange of actin-bound nucleotides even in the presence of UNC-60A, a C. elegans ADF/cofilin that inhibits nucleotide exchange. UNC-60A induces the depolymerization of actin filaments and sequesters actin monomers, whereas CAS-2 reverses the monomer-sequestering effect of UNC-60A in the presence of ATP, but not in the presence of only ADP or the absence of ATP or ADP. A 1:100 molar ratio of CAS-2 to UNC-60A is sufficient to increase actin filaments. CAS-2 has two independent actin-binding sites in its N- and C-terminal halves, and the C-terminal half is necessary and sufficient for the observed activities of the full-length CAS-2. These results suggest that CAS-2 (CAP) and UNC-60A (ADF/cofilin) are important in the ATP-dependent regulation of the actin monomer-filament equilibrium.

  20. Involvement of Candida albicans pyruvate dehydrogenase complex protein X (Pdx1) in filamentation

    PubMed Central

    F.Vellucci, Vincent; Gygax, Scott; Hostetter, Margaret K.

    2007-01-01

    For 50 years, physiologic studies in Candida albicans have associated fermentation with filamentation and respiration with yeast morphology. Analysis of the mitochondrial proteome of a C. albicans NDH51 mutant, known to be defective in filamentation, identified increased expression of several proteins in the respiratory pathway. Most notable was a 15-fold increase in pyruvate dehydrogenase complex protein X (Pdx1), an essential component of the pyruvate dehydrogenase complex. In basal salts medium with 100 mM glucose as carbon source, two independent pdx1 mutants displayed a filamentation defect identical to ndh51; reintegration of one PDX1 allele restored filamentation. Concentrations of glucose ≤100 mM did not correct the filamentation defect. Expanding on previous work, these studies suggest that increased expression of proteins extraneous to the electron transport chain compensates for defects in the respiratory pathway to maintain yeast morphology. Mitochondrial proteomics can aid in the identification of C. albicans genes not previously implicated in filamentation. PMID:17254815

  1. The primary structure of component 8c-1, a subunit protein of intermediate filaments in wool keratin. Relationships with proteins from other intermediate filaments.

    PubMed Central

    Dowling, L M; Crewther, W G; Inglis, A S

    1986-01-01

    Component 8c-1, one of four highly homologous component-8 subunit proteins present in the microfibrils of wool, was isolated as its S-carboxymethyl derivative and its amino acid sequence was determined. Large peptides were isolated after cleaving the protein chemically or enzymically and the sequence of each was determined with an automatic Sequenator. The peptides were ordered by sequence overlaps and, in some instances, by homology with known sequences from other component-8 subunits. The C-terminal residues were identified by three procedures. Full details of the various procedures used have been deposited as Supplementary Publication SUP 50133 (4 pp.) at the British Library Lending Division, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1986) 233, 5. The result showed that the protein comprises 412 residues and has an Mr, including the N-terminal acetyl group, of 48,300. The sequence of residues 98-200 of component 8c-1 was found to correspond to the partial or complete sequences of four homologous type I helical segments previously isolated from helical fragments recovered from chymotryptic digests of microfibrillar proteins of wool [Crewther & Dowling (1971) Appl. Polym. Symp. 18, 1-20; Crewther, Gough, Inglis & McKern (1978) Text. Res. J. 48, 160-162; Gough, Inglis & Crewther (1978) Biochem. J. 173, 385]. Considered in relation to amino acid sequences of other intermediate-filament proteins, the sequence is in accord with the view that keratin filament proteins are of two types [Hanukoglu & Fuchs (1983) Cell (Cambridge, Mass.) 33, 915-924]. Filament proteins from non-keratinous tissues, such as desmin, vimentin, neurofilament proteins and the glial fibrillary acidic protein, which form monocomponent filaments, constitute a third type. It is suggested that as a whole the proteins from intermediate filaments be classed as filamentins, the three types at present identified forming

  2. Bioprocessing strategies to improve heterologous protein production in filamentous fungal fermentations.

    PubMed

    Wang, Liping; Ridgway, Darin; Gu, Tingyue; Moo-Young, Murray

    2005-03-01

    Filamentous fungi have long been used for the production of metabolites and enzymes. With developments in genetic engineering and molecular biology, filamentous fungi have also achieved increased attention as hosts for recombinant DNA. However, the production levels of non-fungal proteins are usually low. Despite the achievements obtained using molecular tools, the heterologous protein loss caused by extracellular fungal protease degradation persists. This review provides an overview of the potential bioprocessing strategies that can be applied to inhibit protease activity thereby enhancing heterologous protein production.

  3. A network of 2-4 nm filaments found in sea urchin smooth muscle. Protein constituents and in situ localization.

    PubMed

    Pureur, R P; Coffe, G; Soyer-Gobillard, M O; de Billy, F; Pudles, J

    1986-01-01

    In this report the coisolation of two proteins from sea urchin smooth muscle of apparent molecular weights (Mr) 54 and 56 kD respectively, as determined on SDS-PAGE, is described. Like the intermediate filament proteins, these two proteins are insoluble in high ionic strength buffer solution. On two-dimensional gel electrophoresis and by immunological methods it is shown that these proteins are not related (by these criteria) to rat smooth muscle desmin (54 kD) or vimentin (56 kD). Furthermore, in conditions where both desmin and vimentin assemble in vitro into 10 nm filaments, the sea urchin smooth muscle proteins do not assemble into filaments. Ultrastructural studies on the sea urchin smooth muscle cell show that the thin and thick filaments organization resembles that described in the vertebrate smooth muscle. However, instead of 10 nm filaments, a network of filaments, 2-4 nm in diameter, is revealed, upon removal of the thin and thick filaments by 0.6 M KCl treatment. By indirect immunofluorescence microscopy, and in particular by immunocytochemical electron microscopy studies on the sea urchin smooth muscle cell, it is shown that the antibodies raised against both 54 and 56 kD proteins appear to specifically label these 2-4 nm filaments. These findings indicate that both the 54 and 56 kD proteins might be constituents of this category of filaments. The possible significance of this new cytoskeletal element, that we have named echinonematin filaments, is discussed.

  4. RecA-ssDNA filaments supercoil in the presence of single-stranded DNA-binding protein

    SciTech Connect

    Shi Weixian; Larson, Ronald G. . E-mail: rlarson@umich.edu

    2007-06-08

    Using atomic force microscopy (AFM), we find that RecA-single-stranded DNA (RecA-ssDNA) filaments, in the presence of single-stranded DNA-binding (SSB) protein, organize into left-handed bundles, which differ from the previously reported disordered aggregates formed when SSB is excluded from the reaction. In addition, we see both left- and right-handedness on bundles of two filaments. These two-filament supercoils, individual filaments, and other smaller bundles further organize into more complicated bundles, showing overall left-handedness which cannot be explained by earlier arguments that presumed supercoiling is absent in RecA-ssDNA filaments. This novel finding and our previous results regarding supercoiling of RecA-double-stranded DNA (RecA-dsDNA) filaments are, however, consistent with each other and can possibly be explained by the intrinsic tendency of RecA-DNA filaments, in their fully coated form, to order themselves into helical bundles, independent of the DNA inside the filaments (ssDNA or dsDNA). RecA-RecA interactions may dominate the bundling process, while the original conformation of DNA inside filaments and other factors (mechanical properties of filaments, concentration of filaments, and Mg{sup 2+} concentration) could contribute to the variation in the appearance and pitch of supercoils. The tendency of RecA-DNA filaments to form ordered supercoils and their presence during strand exchange suggest a possible biological importance of supercoiled filaments.

  5. Dynamic Filament Formation by a Divergent Bacterial Actin-Like ParM Protein

    PubMed Central

    Brzoska, Anthony J.; Jensen, Slade O.; Barton, Deborah A.; Davies, Danielle S.; Overall, Robyn L.; Skurray, Ronald A.; Firth, Neville

    2016-01-01

    Actin-like proteins (Alps) are a diverse family of proteins whose genes are abundant in the chromosomes and mobile genetic elements of many bacteria. The low-copy-number staphylococcal multiresistance plasmid pSK41 encodes ParM, an Alp involved in efficient plasmid partitioning. pSK41 ParM has previously been shown to form filaments in vitro that are structurally dissimilar to those formed by other bacterial Alps. The mechanistic implications of these differences are not known. In order to gain insights into the properties and behavior of the pSK41 ParM Alp in vivo, we reconstituted the parMRC system in the ectopic rod-shaped host, E. coli, which is larger and more genetically amenable than the native host, Staphylococcus aureus. Fluorescence microscopy showed a functional fusion protein, ParM-YFP, formed straight filaments in vivo when expressed in isolation. Strikingly, however, in the presence of ParR and parC, ParM-YFP adopted a dramatically different structure, instead forming axial curved filaments. Time-lapse imaging and selective photobleaching experiments revealed that, in the presence of all components of the parMRC system, ParM-YFP filaments were dynamic in nature. Finally, molecular dissection of the parMRC operon revealed that all components of the system are essential for the generation of dynamic filaments. PMID:27310470

  6. Assembly and exchange of intermediate filament proteins of neurons: neurofilaments are dynamic structures

    PubMed Central

    1989-01-01

    We have explored the dynamics of intermediate filament assembly and subunit exchange using fluorescently labeled neurofilament proteins and a fluorescence resonance energy transfer assay. Neurofilaments (NFs) are assembled from three highly phosphorylated proteins with molecular masses of 180 (NF-H), 130 (NF-M), and 66 kD (NF-L) of which NF-L forms the structural core. The core component, NF-L, was stoichiometrically labeled at cysteine 321 with fluorescein, coumarin, or biotin-maleimide to produce assembly-competent fluorescent or biotinylated derivatives, respectively. Using coumarin-labeled NF-L as fluorescence donor and fluorescein-labeled NF-L as the fluorescence acceptor, assembly of NF filaments was induced by rapidly raising the NaCl concentration to 170 mM, and the kinetics was followed by the decrease in the donor fluorescence. Assembly of NF-L subunits into filaments does not require nucleotide binding or hydrolysis but is strongly dependent on ionic strength, pH, and temperature. The critical concentration of NF-L, that concentration that remains unassembled at equilibrium with fully formed filaments, is 38 micrograms/ml or 0.6 microM. Under physiological salt conditions NF-L filaments also undergo extensive subunit exchange. Kinetic analysis and evaluation of several possible mechanisms indicate that subunit exchange is preceded by dissociation of subunits from the filament and generation of a kinetically active pool of soluble subunits. Given the concentration of NF-L found in nerve cells and the possibility of regulating this pool, these results provide the first information that intermediate filaments are dynamic structures and that NF-L within the NF complex is in dynamic equilibrium with a small but kinetically active pool of unassembled NF-L units. PMID:2925792

  7. Direct interaction of actin filaments with F-BAR protein pacsin2

    PubMed Central

    Kostan, Julius; Salzer, Ulrich; Orlova, Albina; Törö, Imre; Hodnik, Vesna; Senju, Yosuke; Zou, Juan; Schreiner, Claudia; Steiner, Julia; Meriläinen, Jari; Nikki, Marko; Virtanen, Ismo; Carugo, Oliviero; Rappsilber, Juri; Lappalainen, Pekka; Lehto, Veli-Pekka; Anderluh, Gregor; Egelman, Edward H; Djinović-Carugo, Kristina

    2014-01-01

    Two mechanisms have emerged as major regulators of membrane shape: BAR domain-containing proteins, which induce invaginations and protrusions, and nuclear promoting factors, which cause generation of branched actin filaments that exert mechanical forces on membranes. While a large body of information exists on interactions of BAR proteins with membranes and regulatory proteins of the cytoskeleton, little is known about connections between these two processes. Here, we show that the F-BAR domain protein pacsin2 is able to associate with actin filaments using the same concave surface employed to bind to membranes, while some other tested N-BAR and F-BAR proteins (endophilin, CIP4 and FCHO2) do not associate with actin. This finding reveals a new level of complexity in membrane remodeling processes. PMID:25216944

  8. Expression of Cardamom mosaic virus coat protein in Escherichia coli and its assembly into filamentous aggregates.

    PubMed

    Jacob, Thomas; Usha, R

    2002-06-01

    Cardamom mosaic virus (CdMV), a member of the genus Macluravirus of Potyviridae, causes a mosaic disease in cardamom. A polyclonal antiserum was raised against the purified virus and IgG was prepared. Electron microscopic studies on the purified virus showed flexuous filamentous particles of approximately 800 nm in length, typical of members of Potyviridae. The coat protein (CP) encoding sequence of the virus was expressed in Escherichia coli and the protein purified by affinity chromatography under denaturing conditions. The viral nature of the expressed CP was confirmed by positive reaction with anti CdMV IgG in a Western blot. The expressed CP aggregated irreversibly upon renaturation at concentrations above 0.07 mg/ml. The expression of the CP led to the formation of filamentous aggregates in E. coli as observed by immuno-gold electron microscopy. The filamentous aggregates were of 100-150 nm in length. Immuno-capture RT-PCR confirmed the absence of coat protein mRNA in the filamentous aggregates. Deletion mutations, which were expected to inhibit virus assembly, were introduced in the core region of the coat protein. However, these mutations did not improve the solubility of the CP in non-denaturing buffers.

  9. Lamellipodin promotes actin assembly by clustering Ena/VASP proteins and tethering them to actin filaments

    PubMed Central

    Hansen, Scott D; Mullins, R Dyche

    2015-01-01

    Enabled/Vasodilator (Ena/VASP) proteins promote actin filament assembly at multiple locations, including: leading edge membranes, focal adhesions, and the surface of intracellular pathogens. One important Ena/VASP regulator is the mig-10/Lamellipodin/RIAM family of adaptors that promote lamellipod formation in fibroblasts and drive neurite outgrowth and axon guidance in neurons. To better understand how MRL proteins promote actin network formation we studied the interactions between Lamellipodin (Lpd), actin, and VASP, both in vivo and in vitro. We find that Lpd binds directly to actin filaments and that this interaction regulates its subcellular localization and enhances its effect on VASP polymerase activity. We propose that Lpd delivers Ena/VASP proteins to growing barbed ends and increases their polymerase activity by tethering them to filaments. This interaction represents one more pathway by which growing actin filaments produce positive feedback to control localization and activity of proteins that regulate their assembly. DOI: http://dx.doi.org/10.7554/eLife.06585.001 PMID:26295568

  10. Active force generation in cross-linked filament bundles without motor proteins.

    PubMed

    Walcott, Sam; Sun, Sean X

    2010-11-01

    Cytoskeletal filaments often interact laterally through cross-linking proteins, contributing to passive cellular viscoelasticity and, perhaps surprisingly, active force generation. We present a theory, based on the formation and rupture of cross-linker bonds, that relates molecular properties of those interactions to the macroscale mechanics of filament bundles. Computing the force-velocity relation for such a bundle, we demonstrate significant contractile forces in the absence of molecular motors. This theory provides insight into cytokinesis, cytoskeletal mechanics, and stress-fiber contraction.

  11. Interaction of surfactant protein A with the intermediate filaments desmin and vimentin.

    PubMed

    Garcia-Verdugo, Ignacio; Synguelakis, Monique; Degrouard, Jeril; Franco, Claudio-Areias; Valot, Benoit; Zivy, Michel; Chaby, Richard; Tanfin, Zahra

    2008-05-06

    Surfactant protein A (SP-A), a member of the collectin family that modulates innate immunity, has recently been involved in the physiology of reproduction. Consistent with the activation of ERK-1/2 and COX-2 induced by SP-A in myometrial cells, we reported previously the presence of two major proteins recognized by SP-A in these cells. Here we identify by mass spectrometry one of these SP-A targets as the intermediate filament (IF) desmin. In myometrial preparations derived from desmin-deficient mice, the absence of binding of SP-A to any 50 kDa protein confirmed the identity of this SP-A-binding site as desmin. Our data based on partial chymotrypsin digestion of pure desmin suggested that SP-A recognizes especially its rod domain, which is known to play an important role during the assembly of desmin into filaments. In line with that, electron microscopy experiments showed that SP-A inhibits in vitro the polymerization of desmin filaments. SP-A also recognized in vitro polymerized filaments in a calcium-dependent manner at a physiological ionic strength but not the C1q receptor gC1qR. Furthermore, Texas Red-labeled SP-A colocalized with desmin filaments in myometrial cells. Interestingly, vimentin, the IF characteristic of leukocytes, is one of the major proteins recognized by SP-A in protein extracts of U937 cells after PMA-induced differentiation of this monocytic cell line. Interaction of SP-A with vimentin was further confirmed using recombinant vimentin in solid-phase binding assays. The ability of SP-A to interact with desmin and vimentin, and to prevent polymerization of desmin monomers, shed light on unexpected and wider biological roles of this collectin.

  12. Direct observation of dendritic actin filament networks nucleated by Arp2/3 complex and WASP/Scar proteins.

    PubMed

    Blanchoin, L; Amann, K J; Higgs, H N; Marchand, J B; Kaiser, D A; Pollard, T D

    2000-04-27

    Most nucleated cells crawl about by extending a pseudopod that is driven by the polymerization of actin filaments in the cytoplasm behind the leading edge of the plasma membrane. These actin filaments are linked into a network by Y-branches, with the pointed end of each filament attached to the side of another filament and the rapidly growing barbed end facing forward. Because Arp2/3 complex nucleates actin polymerization and links the pointed end to the side of another filament in vitro, a dendritic nucleation model has been proposed in which Arp2/3 complex initiates filaments from the sides of older filaments. Here we report, by using a light microscopy assay, many new features of the mechanism. Branching occurs during, rather than after, nucleation by Arp2/3 complex activated by the Wiskott-Aldrich syndrome protein (WASP) or Scar protein; capping protein and profilin act synergistically with Arp2/3 complex to favour branched nucleation; phosphate release from aged actin filaments favours dissociation of Arp2/3 complex from the pointed ends of filaments; and branches created by Arp2/3 complex are relatively rigid. These properties result in the automatic assembly of the branched actin network after activation by proteins of the WASP/Scar family and favour the selective disassembly of proximal regions of the network.

  13. Structure, sarcomeric organization, and thin filament binding of cardiac myosin-binding protein-C.

    PubMed

    Craig, Roger; Lee, Kyoung Hwan; Mun, Ji Young; Torre, Iratxe; Luther, Pradeep K

    2014-03-01

    Myosin-binding protein-C (MyBP-C) is an accessory protein of the myosin filaments of vertebrate striated muscle. In the heart, it plays a key role in modulating contractility in response to β-adrenergic stimulation. Mutations in the cardiac isoform (cMyBP-C) are a leading cause of inherited hypertrophic cardiomyopathy. Understanding cMyBP-C function and its role in disease requires knowledge of the structure of the molecule, its organization in the sarcomere, and its interactions with other sarcomeric proteins. Here we review the main structural features of this modular, elongated molecule and the properties of some of its key domains. We describe observations suggesting that the bulk of the molecule extends perpendicular to the thick filament, enabling it to reach neighboring thin filaments in the sarcomere. We review structural and functional evidence for interaction of its N-terminal domains with actin and how this may modulate thin filament activation. We also discuss the effects that phosphorylation of cMyBP-C has on some of these structural features and how this might relate to cMyBP-C function in the beating heart.

  14. Three-dimensional structure of actin filaments and of an actin gel made with actin-binding protein.

    PubMed

    Niederman, R; Amrein, P C; Hartwig, J

    1983-05-01

    Purified muscle actin and mixtures of actin and actin-binding protein were examined in the transmission electron microscope after fixation, critical point drying, and rotary shadowing. The three-dimensional structure of the protein assemblies was analyzed by a computer-assisted graphic analysis applicable to generalized filament networks. This analysis yielded information concerning the frequency of filament intersections, the filament length between these intersections, the angle at which filaments branch at these intersections, and the concentration of filaments within a defined volume. Purified actin at a concentration of 1 mg/ml assembled into a uniform mass of long filaments which overlap at random angles between 0 degrees and 90 degrees. Actin in the presence of macrophage actin-binding protein assembled into short, straight filaments, organized in a perpendicular branching network. The distance between branch points was inversely related to the molar ratio of actin-binding protein to actin. This distance was what would be predicted if actin filaments grew at right angles off of nucleation sites on the two ends of actin-binding protein dimers, and then annealed. The results suggest that actin in combination with actin-binding protein self-assembles to form a three-dimensional network resembling the peripheral cytoskeleton of motile cells.

  15. KLHL40 deficiency destabilizes thin filament proteins and promotes nemaline myopathy

    PubMed Central

    Garg, Ankit; O’Rourke, Jason; Long, Chengzu; Doering, Jonathan; Ravenscroft, Gianina; Bezprozvannaya, Svetlana; Nelson, Benjamin R.; Beetz, Nadine; Li, Lin; Chen, She; Laing, Nigel G.; Grange, Robert W.; Bassel-Duby, Rhonda; Olson, Eric N.

    2014-01-01

    Nemaline myopathy (NM) is a congenital myopathy that can result in lethal muscle dysfunction and is thought to be a disease of the sarcomere thin filament. Recently, several proteins of unknown function have been implicated in NM, but the mechanistic basis of their contribution to disease remains unresolved. Here, we demonstrated that loss of a muscle-specific protein, kelch-like family member 40 (KLHL40), results in a nemaline-like myopathy in mice that closely phenocopies muscle abnormalities observed in KLHL40-deficient patients. We determined that KLHL40 localizes to the sarcomere I band and A band and binds to nebulin (NEB), a protein frequently implicated in NM, as well as a putative thin filament protein, leiomodin 3 (LMOD3). KLHL40 belongs to the BTB-BACK-kelch (BBK) family of proteins, some of which have been shown to promote degradation of their substrates. In contrast, we found that KLHL40 promotes stability of NEB and LMOD3 and blocks LMOD3 ubiquitination. Accordingly, NEB and LMOD3 were reduced in skeletal muscle of both Klhl40–/– mice and KLHL40-deficient patients. Loss of sarcomere thin filament proteins is a frequent cause of NM; therefore, our data that KLHL40 stabilizes NEB and LMOD3 provide a potential basis for the development of NM in KLHL40-deficient patients. PMID:24960163

  16. MFP1, a novel plant filament-like protein with affinity for matrix attachment region DNA.

    PubMed Central

    Meier, I; Phelan, T; Gruissem, W; Spiker, S; Schneider, D

    1996-01-01

    The interaction of chromatin with the nuclear matrix via matrix attachment regions (MARs) on the DNA is considered to be of fundamental importance for higher order chromatin organization and regulation of gene expression. Here, we report a novel nuclear matrix-localized MAR DNA binding protein, designated MAR binding filament-like protein 1 (MFP1), from tomato. In contrast to the few animal MAR DNA binding proteins thus far identified, MFP1 contains a predicted N-terminal transmembrane domain and a long filament-like alpha-helical domain that is similar to diverse nuclear and cytoplasmic filament proteins from animals and yeast. DNA binding assays established that MFP1 can discriminate between animal and plant MAR DNAs and non-MAR DNA fragments of similar size and AT content. Deletion mutants of MFP1 revealed a novel, discrete DNA binding domain near the C terminus of the protein. MFP1 is an in vitro substrate for casein kinase II, a nuclear matrix-associated protein kinase. Its structure, MAR DNA binding activity, and nuclear matrix localization suggest that MFP1 is likely to participate in nuclear architecture by connecting chromatin with the nuclear matrix and potentially with the nuclear envelope. PMID:8953774

  17. The Actin Filament-Binding Protein Coronin Regulates Motility in Plasmodium Sporozoites

    PubMed Central

    Bane, Kartik S.; Singer, Mirko; Reinig, Miriam; Klug, Dennis; Heiss, Kirsten; Baum, Jake; Mueller, Ann-Kristin; Frischknecht, Friedrich

    2016-01-01

    Parasites causing malaria need to migrate in order to penetrate tissue barriers and enter host cells. Here we show that the actin filament-binding protein coronin regulates gliding motility in Plasmodium berghei sporozoites, the highly motile forms of a rodent malaria-causing parasite transmitted by mosquitoes. Parasites lacking coronin show motility defects that impair colonization of the mosquito salivary glands but not migration in the skin, yet result in decreased transmission efficiency. In non-motile sporozoites low calcium concentrations mediate actin-independent coronin localization to the periphery. Engagement of extracellular ligands triggers an intracellular calcium release followed by the actin-dependent relocalization of coronin to the rear and initiation of motility. Mutational analysis and imaging suggest that coronin organizes actin filaments for productive motility. Using coronin-mCherry as a marker for the presence of actin filaments we found that protein kinase A contributes to actin filament disassembly. We finally speculate that calcium and cAMP-mediated signaling regulate a switch from rapid parasite motility to host cell invasion by differentially influencing actin dynamics. PMID:27409081

  18. Cyclic AMP-modulated phosphorylation of intermediate filament proteins in cultured avian myogenic cells.

    PubMed Central

    Gard, D L; Lazarides, E

    1982-01-01

    The intermediate filament proteins desmin and vimentin and the muscle tropomyosins were the major protein phosphate acceptors in 8-day-old myotubes incubated for 4 h in medium containing radiolabeled phosphate. The addition of isoproterenol or 8-bromo-cyclic AMP (BrcAMP) resulted in a two- to threefold increase in incorporation of 32PO4 into both desmin and vimentin, whereas no changes in the incorporation of 32PO4 into tropomyosin or other cellular proteins were observed. The BrcAMP- or hormonally induced increase in 32PO4 incorporation into desmin and vimentin was independent of protein synthesis and was not caused by stimulation of protein phosphate turnover. In addition, BrcAMP did not induce significant changes in the specific activity of the cellular ATP pool. These data suggest that the observed increase in 32PO4 incorporation represented an actual increase in phosphorylation of the intermediate filament proteins desmin and vimentin. Two-dimensional tryptic analysis of desmin from 8-day-old myotubes revealed five phosphopeptides of which two showed a 7- to 10-fold increase in 32PO4 incorporation in BrcAMP-treated myotubes. Four of the phosphopeptides identified in desmin labeled in vivo were also observed in desmin phosphorylated in vitro by bovine heart cAMP-dependent protein kinase. Although phosphorylation of desmin and vimentin was apparent in myogenic cells at all stages of differentiation, BrcAMP- and isoproterenol-induced increases in phosphorylation of these proteins were restricted to mature myotubes. These data strongly suggest that in vivo phosphorylation of the intermediate filament proteins desmin and vimentin is catalyzed by the cAMP-dependent protein kinases and that such phosphorylation may be regulated during muscle differentiation. Images PMID:6294504

  19. How Do Filamentous Pathogens Deliver Effector Proteins into Plant Cells?

    PubMed Central

    Petre, Benjamin; Kamoun, Sophien

    2014-01-01

    Fungal and oomycete plant parasites are among the most devastating pathogens of food crops. These microbes secrete effector proteins inside plant cells to manipulate host processes and facilitate colonization. How these effectors reach the host cytoplasm remains an unclear and debated area of plant research. In this article, we examine recent conflicting findings that have generated discussion in the field. We also highlight promising approaches based on studies of both parasite and host during infection. Ultimately, this knowledge may inform future broad spectrum strategies for protecting crops from such pathogens. PMID:24586116

  20. Actin-crosslinking protein regulation of filament movement in motility assays: a theoretical model.

    PubMed Central

    Janson, L W; Taylor, D L

    1994-01-01

    The interaction of single actin filaments on a myosin-coated coverslip has been modeled by several authors. One model adds a component of "frictional drag" by myosin heads that oppose movement of the actin filaments. We have extended this concept by including the resistive drag from actin crosslinking proteins to understand better the relationship among crosslinking number, actin-myosin force generation, and motility. The validity of this model is supported by agreement with the experimental results from a previous study in which crosslinking proteins were added with myosin molecules under otherwise standard motility assay conditions. The theoretical relationship provides a means to determine many physical parameters that characterize the interaction between a single actin filament and a single actin-crosslinking molecule (various types). In particular, the force constant of a single filamin molecule is calculated as 1.105 pN, approximately 3 times less than a driving myosin head (3.4 pN). Knowledge of this parameter and others derived from this model allows a better understanding of the interaction between myosin and the actin/actin-binding protein cytoskeleton and the role of actin-binding proteins in the regulation and modulation of motility. PMID:7811954

  1. Making recombinant proteins in filamentous fungi- are we expecting too much?

    PubMed Central

    Nevalainen, Helena; Peterson, Robyn

    2014-01-01

    Hosts used for the production of recombinant proteins are typically high-protein secreting mutant strains that have been selected for a specific purpose, such as efficient production of cellulose-degrading enzymes. Somewhat surprisingly, sequencing of the genomes of a series of mutant strains of the cellulolytic Trichoderma reesei, widely used as an expression host for recombinant gene products, has shed very little light on the nature of changes that boost high-level protein secretion. While it is generally agreed and shown that protein secretion in filamentous fungi occurs mainly through the hyphal tip, there is growing evidence that secretion of proteins also takes place in sub-apical regions. Attempts to increase correct folding and thereby the yields of heterologous proteins in fungal hosts by co-expression of cellular chaperones and foldases have resulted in variable success; underlying reasons have been explored mainly at the transcriptional level. The observed physiological changes in fungal strains experiencing increasing stress through protein overexpression under strong gene promoters also reflect the challenge the host organisms are experiencing. It is evident, that as with other eukaryotes, fungal endoplasmic reticulum is a highly dynamic structure. Considering the above, there is an emerging body of work exploring the use of weaker expression promoters to avoid undue stress. Filamentous fungi have been hailed as candidates for the production of pharmaceutically relevant proteins for therapeutic use. One of the biggest challenges in terms of fungally produced heterologous gene products is their mode of glycosylation; fungi lack the functionally important terminal sialylation of the glycans that occurs in mammalian cells. Finally, exploration of the metabolic pathways and fluxes together with the development of sophisticated fermentation protocols may result in new strategies to produce recombinant proteins in filamentous fungi. PMID:24578701

  2. Sedimentation Patterns of Rapidly Reversible Protein Interactions

    PubMed Central

    Schuck, Peter

    2010-01-01

    Abstract The transport behavior of macromolecular mixtures with rapidly reversible complex formation is of great interest in the study of protein interactions by many different methods. Complicated transport patterns arise even for simple bimolecular reactions, when all species exhibit different migration velocities. Although partial differential equations are available to describe the spatial and temporal evolution of the interacting system given particular initial conditions, a general overview of the phase behavior of the systems in parameter space has not yet been reported. In the case of sedimentation of two-component mixtures, this study presents simple analytical solutions that solve the underlying equations in the diffusion-free limit previously subject to Gilbert-Jenkins theory. The new expressions describe, with high precision, the average sedimentation coefficients and composition of each boundary, which allow the examination of features of the whole parameter space at once, and may be used for experimental design and robust analysis of experimental boundary patterns to derive the stoichiometry and affinity of the complex. This study finds previously unrecognized features, including a phase transition between boundary patterns. The model reveals that the time-average velocities of all components in the reaction mixture must match—a condition that suggests an intuitive physical picture of an effective particle of the coupled cosedimentation of an interacting system. Adding to the existing numerical solutions of the relevant partial differential equations, the effective particle model provides physical insights into the relationships of the parameters that govern sedimentation patterns. PMID:20441765

  3. Molecular mechanics of cardiac myosin-binding protein C in native thick filaments.

    PubMed

    Previs, M J; Beck Previs, S; Gulick, J; Robbins, J; Warshaw, D M

    2012-09-07

    The heart's pumping capacity results from highly regulated interactions of actomyosin molecular motors. Mutations in the gene for a potential regulator of these motors, cardiac myosin-binding protein C (cMyBP-C), cause hypertrophic cardiomyopathy. However, cMyBP-C's ability to modulate cardiac contractility is not well understood. Using single-particle fluorescence imaging techniques, transgenic protein expression, proteomics, and modeling, we found that cMyBP-C slowed actomyosin motion generation in native cardiac thick filaments. This mechanical effect was localized to where cMyBP-C resides within the thick filament (i.e., the C-zones) and was modulated by phosphorylation and site-specific proteolytic degradation. These results provide molecular insight into why cMyBP-C should be considered a member of a tripartite complex with actin and myosin that allows fine tuning of cardiac muscle contraction.

  4. The production of antibody fragments and antibody fusion proteins by yeasts and filamentous fungi

    PubMed Central

    Joosten, Vivi; Lokman, Christien; van den Hondel, Cees AMJJ; Punt, Peter J

    2003-01-01

    In this review we will focus on the current status and views concerning the production of antibody fragments and antibody fusion proteins by yeasts and filamentous fungi. We will focus on single-chain antibody fragment production (scFv and VHH) by these lower eukaryotes and the possible applications of these proteins. Also the coupling of fragments to relevant enzymes or other components will be discussed. As an example of the fusion protein strategy, the 'magic bullet' approach for industrial applications, will be highlighted. PMID:12605725

  5. A novel tubulin-dependent protein kinase forming a paired helical filament epitope on tau.

    PubMed

    Ishiguro, K; Ihara, Y; Uchida, T; Imahori, K

    1988-09-01

    From rat brain microtubule proteins, we purified a protein kinase that phosphorylated tau, one of microtubule-associated proteins. The electrophoretic mobility of the phosphorylated tau on SDS-polyacrylamide gel decreased. The enzyme was not activated by cyclic nucleotides, calmodulin, or phospholipids, and was inhibited by the calcium ions. The kinase bound to tau. The phosphorylation of tau was stimulated by tubulin under the condition of microtubule formation. From these results we propose an idea that the phosphorylation could occur concomitantly with microtubule formation in the brain. Human tau phosphorylated by the kinase carried an epitope of the paired helical filaments that accumulate in the brain in Alzheimer's disease.

  6. Chemical modification of contractile 3-nm-diameter filaments in Vorticella spasmoneme by diethyl-pyrocarbonate and its reversible renaturation by hydroxylamine.

    PubMed

    Fang, Jie; Zhang, Bei; Asai, Hiroshi

    2003-10-31

    A peritrich ciliate possesses a zooid and a long stalk consisting of a bundle of 3-nm-diameter filaments. Glycerinated stalks can contract in the presence of free Ca(2+) and re-extend in the absence of free Ca(2+). In the present study, we demonstrated that histidine residue(s) played a critical role in spasmoneme contraction by using glycerinated stalk of Vorticella. Concentration-dependent inhibition of spasmoneme contraction was observed in the presence of reversible histidine-modifying reagent named diethyl-pyrocarbonate (DEPC). In addition, the contractility degree of DEPC-modified spasmoneme could be partially restored by hydroxylamine treatment. The 244nm absorption of modified spasmoneme protein(s) increased with rising DEPC concentration and decreased following the addition of hydroxylamine treatment. Adding Ca(2+) before DEPC modification could prevent the spasmoneme contraction from inhibition of DEPC. Those results suggested that histidine residues were actively involved in spasmoneme contraction. Ca(2+)-binding ability of spasmin was not inhibited by DEPC modification, which suggested that the essential histidine residues were not on the calcium-binding site of spasmin.

  7. Targeting the Reversibly Oxidized Protein Tyrosine Phosphatase Superfamily

    PubMed Central

    Boivin, Benoit; Yang, Ming; Tonks, Nicholas K.

    2010-01-01

    Controlled production of reactive oxygen species leads to reversible oxidation of protein tyrosine phosphatases (PTPs) and has emerged as an important tier of regulation over phosphorylation-dependent signal transduction. We present a modified cysteinyl-labeling assay that detects reversible oxidation of members of each of the different PTP subclasses. Here, we describe the methods for enriching reversibly oxidized PTPs from complex protein extracts, illustrating the procedure in IMR90 fibroblasts. PMID:20807953

  8. A novel intermediate filament-associated protein, NAPA-73, that binds to different filament types at different stages of nervous system development.

    PubMed

    Ciment, G; Ressler, A; Letourneau, P C; Weston, J A

    1986-01-01

    The antigen recognized by the E/C8-monoclonal antibody is expressed in various avian embryonic cell types known also to express neurofilament (NF) immunoreactivity. To determine whether the E/C8-antigen corresponds to any of the known NF components, we compared their subcellular locations, immunocross-reactivities, and electrophoretic behaviors. We found that the E/C8-antibody binds to NF bundles in electron microscope preparations of neurons, but does not correspond to any of the known NF proteins by immunological or electrophoretic criteria. Immunoadsorption with the monoclonal antibody resulted in co-purification of a 73,000-D protein with one of the known NF proteins in homogenates from 20-d embryonic chick brains, but with vimentin intermediate filament protein in similarly prepared homogenates from 4-d embryonic chicks. We suggest that the E/C8-antigen is an intermediate filament-associated protein that binds to different filament types at different stages of development. We have named it NAPA-73, an acronym for neurofilament-associated protein, avian-specific, 73,000 D, on the basis of its binding specificity in mature neurons.

  9. A novel intermediate filament-associated protein, NAPA-73, that binds to different filament types at different stages of nervous system development

    PubMed Central

    1986-01-01

    The antigen recognized by the E/C8-monoclonal antibody is expressed in various avian embryonic cell types known also to express neurofilament (NF) immunoreactivity. To determine whether the E/C8-antigen corresponds to any of the known NF components, we compared their subcellular locations, immunocross-reactivities, and electrophoretic behaviors. We found that the E/C8-antibody binds to NF bundles in electron microscope preparations of neurons, but does not correspond to any of the known NF proteins by immunological or electrophoretic criteria. Immunoadsorption with the monoclonal antibody resulted in co- purification of a 73,000-D protein with one of the known NF proteins in homogenates from 20-d embryonic chick brains, but with vimentin intermediate filament protein in similarly prepared homogenates from 4- d embryonic chicks. We suggest that the E/C8-antigen is an intermediate filament-associated protein that binds to different filament types at different stages of development. We have named it NAPA-73, an acronym for neurofilament-associated protein, avian-specific, 73,000 D, on the basis of its binding specificity in mature neurons. PMID:3510220

  10. Correlated motion of protein subdomains and large-scale conformational flexibility of RecA protein filament

    NASA Astrophysics Data System (ADS)

    Yu, Garmay; A, Shvetsov; D, Karelov; D, Lebedev; A, Radulescu; M, Petukhov; V, Isaev-Ivanov

    2012-02-01

    Based on X-ray crystallographic data available at Protein Data Bank, we have built molecular dynamics (MD) models of homologous recombinases RecA from E. coli and D. radiodurans. Functional form of RecA enzyme, which is known to be a long helical filament, was approximated by a trimer, simulated in periodic water box. The MD trajectories were analyzed in terms of large-scale conformational motions that could be detectable by neutron and X-ray scattering techniques. The analysis revealed that large-scale RecA monomer dynamics can be described in terms of relative motions of 7 subdomains. Motion of C-terminal domain was the major contributor to the overall dynamics of protein. Principal component analysis (PCA) of the MD trajectories in the atom coordinate space showed that rotation of C-domain is correlated with the conformational changes in the central domain and N-terminal domain, that forms the monomer-monomer interface. Thus, even though C-terminal domain is relatively far from the interface, its orientation is correlated with large-scale filament conformation. PCA of the trajectories in the main chain dihedral angle coordinate space implicates a co-existence of a several different large-scale conformations of the modeled trimer. In order to clarify the relationship of independent domain orientation with large-scale filament conformation, we have performed analysis of independent domain motion and its implications on the filament geometry.

  11. SR-like RNA-binding protein Slr1 affects Candida albicans filamentation and virulence.

    PubMed

    Ariyachet, Chaiyaboot; Solis, Norma V; Liu, Yaoping; Prasadarao, Nemani V; Filler, Scott G; McBride, Anne E

    2013-04-01

    Candida albicans causes both mucosal and disseminated infections, and its capacity to grow as both yeast and hyphae is a key virulence factor. Hyphal formation is a type of polarized growth, and members of the SR (serine-arginine) family of RNA-binding proteins influence polarized growth of both Saccharomyces cerevisiae and Aspergillus nidulans. Therefore, we investigated whether SR-like proteins affect filamentous growth and virulence of C. albicans. BLAST searches with S. cerevisiae SR-like protein Npl3 (ScNpl3) identified two C. albicans proteins: CaNpl3, an apparent ScNpl3 ortholog, and Slr1, another SR-like RNA-binding protein with no close S. cerevisiae ortholog. Whereas ScNpl3 was critical for growth, deletion of NPL3 in C. albicans resulted in few phenotypic changes. In contrast, the slr1Δ/Δ mutant had a reduced growth rate in vitro, decreased filamentation, and impaired capacity to damage epithelial and endothelial cells in vitro. Mice infected intravenously with the slr1Δ/Δ mutant strain had significantly prolonged survival compared to that of mice infected with the wild-type or slr1Δ/Δ mutant complemented with SLR1 (slr1Δ/Δ+SLR1) strain, without a concomitant decrease in kidney fungal burden. Histopathology, however, revealed differential localization of slr1Δ/Δ hyphal and yeast morphologies within the kidney. Mice infected with slr1Δ/Δ cells also had an increased brain fungal burden, which correlated with increased invasion of brain, but not umbilical vein, endothelial cells in vitro. The enhanced brain endothelial cell invasion was likely due to the increased surface exposure of the Als3 adhesin on slr1Δ/Δ cells. Our results indicate that Slr1 is an SR-like protein that influences C. albicans growth, filamentation, host cell interactions, and virulence.

  12. Differential effects of thin and thick filament disruption on zebrafish smooth muscle regulatory proteins

    PubMed Central

    Davuluri, G.; Seiler, C.; Abrams, J.; Soriano, A. J.; Pack, M.

    2013-01-01

    Background The smooth muscle actin binding proteins Caldesmon and Tropomyosin (Tm) promote thin filament assembly by stabilizing actin polymerization, however, whether filament assembly affects either the stability or activation of these and other smooth muscle regulatory proteins is not known. Methods Measurement of smooth muscle regulatory protein levels in wild type zebrafish larvae following antisense knockdown of smooth muscle actin (Acta2) and myosin heavy chain (Myh11) proteins, and in colourless mutants that lack enteric nerves. Comparison of intestinal peristalsis in wild type and colourless larvae. Key Results Knockdown of Acta2 led to reduced levels of phospho-Caldesmon and Tm. Total Caldesmon and phospho-myosin light chain (p-Mlc) levels were unaffected. Knockdown of Myh11 had no effect on the levels of either of these proteins. Phospho-Caldesmon and p-Mlc levels were markedly reduced in colourless mutants that have intestinal motility comparable with wild type larvae. Conclusions & Inferences These in vivo findings provide new information regarding the activation and stability of smooth muscle regulatory proteins in zebrafish larvae and their role in intestinal peristalsis in this model organism. PMID:20591105

  13. A new protein complex promoting the assembly of Rad51 filaments

    PubMed Central

    Sasanuma, Hiroyuki; Tawaramoto, Maki S.; Lao, Jessica P.; Hosaka, Harumi; Sanda, Eri; Suzuki, Mamoru; Yamashita, Eiki; Hunter, Neil; Shinohara, Miki; Nakagawa, Atsushi; Shinohara, Akira

    2015-01-01

    During homologous recombination, eukaryotic RecA homologue Rad51 assembles into a nucleoprotein filament on single-stranded DNA to catalyse homologous pairing and DNA-strand exchange with a homologous template. Rad51 nucleoprotein filaments are highly dynamic and regulated via the coordinated actions of various accessory proteins including Rad51 mediators. Here, we identify a new Rad51 mediator complex. The PCSS complex, comprising budding yeast Psy3, Csm2, Shu1 and Shu2 proteins, binds to recombination sites and is required for Rad51 assembly and function during meiosis. Within the heterotetramer, Psy3-Csm2 constitutes a core sub-complex with DNA-binding activity. In vitro, purified Psy3-Csm2 stabilizes the Rad51–single-stranded DNA complex independently of nucleotide cofactor. The mechanism of Rad51 stabilization is inferred by our high-resolution crystal structure, which reveals Psy3-Csm2 to be a structural mimic of the Rad51-dimer, a fundamental unit of the Rad51-filament. Together, these results reveal a novel molecular mechanism for this class of Rad51-mediators, which includes the human Rad51 paralogues. PMID:23575680

  14. Translation elongation factor EF-Tu modulates filament formation of actin-like MreB protein in vitro.

    PubMed

    Defeu Soufo, Hervé Joël; Reimold, Christian; Breddermann, Hannes; Mannherz, Hans G; Graumann, Peter L

    2015-04-24

    EF-Tu has been shown to interact with actin-like protein MreB and to affect its localization in Escherichia coli and in Bacillus subtilis cells. We have purified YFP-MreB in an active form, which forms filaments on glass slides in vitro and was active in dynamic light-scattering assays, polymerizing in milliseconds after addition of magnesium. Purified EF-Tu enhanced the amount of MreB filaments, as seen by sedimentation assays, the speed of filament formation and the length of MreB filaments in vitro. EF-Tu had the strongest impact on MreB filaments in a 1:1 ratio, and EF-Tu co-sedimented with MreB filaments, revealing a stoichiometric interaction between both proteins. This was supported by cross-linking assays where 1:1 species were well detectable. When expressed in E. coli cells, B. subtilis MreB formed filaments and induced the formation of co-localizing B. subtilis EF-Tu structures, indicating that MreB can direct the positioning of EF-Tu structures in a heterologous cell system. Fluorescence recovery after photobleaching analysis showed that MreB filaments have a higher turnover in B. subtilis cells than in E. coli cells, indicating different filament kinetics in homologous or heterologous cell systems. The data show that MreB can direct the localization of EF-Tu in vivo, which in turn positively affects the formation and dynamics of MreB filaments. Thus, EF-Tu is a modulator of the activity of a bacterial actin-like protein.

  15. The Tumor Inhibitor and Antiangiogenic Agent Withaferin A Targets the Intermediate Filament Protein Vimentin

    PubMed Central

    Bargagna-Mohan, Paola; Hamza, Adel; Kim, Yang-eon; Ho, Yik Khuan (Abby); Mor-Vaknin, Nirit; Wendschlag, Nicole; Liu, Junjun; Evans, Robert M.; Markovitz, David M.; Zhan, Chang-Guo; Kim, Kyung Bo; Mohan, Royce

    2011-01-01

    SUMMARY The natural product withaferin A (WFA) exhibits antitumor and antiangiogenesis activity in vivo, which results from this drug’s potent growth inhibitory activities. Here, we show that WFA binds to the intermediate filament (IF) protein, vimentin, by covalently modifying its cysteine residue, which is present in the highly conserved α-helical coiled coil 2B domain. WFA induces vimentin filaments to aggregate in vitro, an activity manifested in vivo as punctate cytoplasmic aggregates that colocalize vimentin and F-actin. WFA’s potent dominant-negative effect on F-actin requires vimentin expression and induces apoptosis. Finally, we show that WFA-induced inhibition of capillary growth in a mouse model of corneal neovascularization is compromised in vimentin-deficient mice. These findings identify WFA as a chemical genetic probe of IF functions, and illuminate a potential molecular target for withanolide-based therapeutics for treating angioproliferative and malignant diseases. PMID:17584610

  16. Modulation of myosin filament organization by C-protein family members.

    PubMed

    Seiler, S H; Fischman, D A; Leinwand, L A

    1996-01-01

    We have analyzed the interactions between two types of sarcomeric proteins: myosin heavy chain (MyHC) and members of an abundant thick filament-associated protein family (myosin-binding protein; MyBP). Previous work has demonstrated that when MyHC is transiently transfected into mammalian nonmuscle COS cells, the expressed protein forms spindle-shaped structures consisting of bundles of myosin thick filaments. Co-expression of MyHC and MyBP-C or -H modulates the MyHC structures, resulting in dramatically longer cables consisting of myosin and MyBP encircling the nucleus. Immunoelectron microscopy indicates that these cable structures are more uniform in diameter than the spindle structures consisting solely of MyHC, and that the myosin filaments are compacted in the presence of MyBP. Deletion analysis of MyBP-H indicates that cable formation is dependent on the carboxy terminal 24 amino acids. Neither the MyHC spindles nor the MyHC/MyBP cables associate with the endogenous actin cytoskeleton of the COS cell. While there is no apparent co-localization between these structures and the microtubule network, colchicine treatment of the cells promotes the formation of longer assemblages, suggesting that cytoskeletal architecture may physically impede or regulate polymer formation/extension. The data presented here contribute to a greater understanding of the interactions between the MyBP family and MyHC, and provide additional evidence for functional homology between MyBP-C and MyBP-H.

  17. (PCG) Protein Crystal Growth HIV Reverse Transcriptase

    NASA Technical Reports Server (NTRS)

    1992-01-01

    HIV Reverse Transcriptase crystals grown during the USML-1 (STS-50) mission using Commercial Refrigerator/Incubator Module (CR/IM) at 4 degrees C and the Vapor Diffusion Apparatus (VDA). Reverse transcriptase is an enzyme responsible for copying the nucleic acid genome of the AIDS virus from RNA to DNA. Studies indicated that the space-grown crystals were larger and better ordered (beyond 4 angstroms) than were comparable Earth-grown crystals. Principal Investigators were Charles Bugg and Larry DeLucas.

  18. Intermediate filament-like proteins in bacteria and a cytoskeletal function in Streptomyces

    PubMed Central

    Bagchi, Sonchita; Tomenius, Henrik; Belova, Lyubov M; Ausmees, Nora

    2008-01-01

    Actin and tubulin cytoskeletons are conserved and widespread in bacteria. A strikingly intermediate filament (IF)-like cytoskeleton, composed of crescentin, is also present in Caulobacter crescentus and determines its specific cell shape. However, the broader significance of this finding remained obscure, because crescentin appeared to be unique to Caulobacter. Here we demonstrate that IF-like function is probably a more widespread phenomenon in bacteria. First, we show that 21 genomes of 26 phylogenetically diverse species encoded uncharacterized proteins with a central segmented coiled coil rod domain, which we regarded as a key structural feature of IF proteins and crescentin. Experimental studies of three in silico predicted candidates from Mycobacterium and other actinomycetes revealed a common IF-like property to spontaneously assemble into filaments in vitro. Furthermore, the IF-like protein FilP formed cytoskeletal structures in the model actinomycete Streptomyces coelicolor and was needed for normal growth and morphogenesis. Atomic force microscopy of living cells revealed that the FilP cytoskeleton contributed to mechanical fitness of the hyphae, thus closely resembling the function of metazoan IF. Together, the bioinformatic and experimental data suggest that an IF-like protein architecture is a versatile design that is generally present in bacteria and utilized to perform diverse cytoskeletal tasks. PMID:18976278

  19. CyDiv, a Conserved and Novel Filamentous Cyanobacterial Cell Division Protein Involved in Septum Localization

    PubMed Central

    Mandakovic, Dinka; Trigo, Carla; Andrade, Derly; Riquelme, Brenda; Gómez-Lillo, Gabriela; Soto-Liebe, Katia; Díez, Beatriz; Vásquez, Mónica

    2016-01-01

    Cell division in bacteria has been studied mostly in Escherichia coli and Bacillus subtilis, model organisms for Gram-negative and Gram-positive bacteria, respectively. However, cell division in filamentous cyanobacteria is poorly understood. Here, we identified a novel protein, named CyDiv (Cyanobacterial Division), encoded by the all2320 gene in Anabaena sp. PCC 7120. We show that CyDiv plays a key role during cell division. CyDiv has been previously described only as an exclusive and conserved hypothetical protein in filamentous cyanobacteria. Using polyclonal antibodies against CyDiv, we showed that it localizes at different positions depending on cell division timing: poles, septum, in both daughter cells, but also in only one of the daughter cells. The partial deletion of CyDiv gene generates partial defects in cell division, including severe membrane instability and anomalous septum localization during late division. The inability to complete knock out CyDiv strains suggests that it is an essential gene. In silico structural protein analyses and our experimental results suggest that CyDiv is an FtsB/DivIC-like protein, and could therefore, be part of an essential late divisome complex in Anabaena sp. PCC 7120. PMID:26903973

  20. CyDiv, a Conserved and Novel Filamentous Cyanobacterial Cell Division Protein Involved in Septum Localization.

    PubMed

    Mandakovic, Dinka; Trigo, Carla; Andrade, Derly; Riquelme, Brenda; Gómez-Lillo, Gabriela; Soto-Liebe, Katia; Díez, Beatriz; Vásquez, Mónica

    2016-01-01

    Cell division in bacteria has been studied mostly in Escherichia coli and Bacillus subtilis, model organisms for Gram-negative and Gram-positive bacteria, respectively. However, cell division in filamentous cyanobacteria is poorly understood. Here, we identified a novel protein, named CyDiv (Cyanobacterial Division), encoded by the all2320 gene in Anabaena sp. PCC 7120. We show that CyDiv plays a key role during cell division. CyDiv has been previously described only as an exclusive and conserved hypothetical protein in filamentous cyanobacteria. Using polyclonal antibodies against CyDiv, we showed that it localizes at different positions depending on cell division timing: poles, septum, in both daughter cells, but also in only one of the daughter cells. The partial deletion of CyDiv gene generates partial defects in cell division, including severe membrane instability and anomalous septum localization during late division. The inability to complete knock out CyDiv strains suggests that it is an essential gene. In silico structural protein analyses and our experimental results suggest that CyDiv is an FtsB/DivIC-like protein, and could therefore, be part of an essential late divisome complex in Anabaena sp. PCC 7120.

  1. Protein-Nanocrystal Conjugates Support a Single Filament Polymerization Model in R1 Plasmid Segregation

    SciTech Connect

    Choi, Charina L.; Claridge, Shelley A.; Garner, Ethan C.; Alivisatos, A. Paul; Mullins, R. Dyche

    2008-07-15

    To ensure inheritance by daughter cells, many low-copy number bacterial plasmids, including the R1 drug-resistance plasmid, encode their own DNA segregation systems. The par operon of plasmid R1 directs construction of a simple spindle structure that converts free energy of polymerization of an actin-like protein, ParM, into work required to move sister plasmids to opposite poles of rod-shaped cells. The structures of individual components have been solved, but little is known about the ultrastructure of the R1 spindle. To determine the number of ParM filaments in a minimal R1 spindle, we used DNA-gold nanocrystal conjugates as mimics of the R1 plasmid. Wefound that each end of a single polar ParM filament binds to a single ParR/parC-gold complex, consistent with the idea that ParM filaments bind in the hollow core of the ParR/parC ring complex. Our results further suggest that multifilament spindles observed in vivo are associated with clusters of plasmidssegregating as a unit.

  2. Ash1 Protein, an Asymmetrically Localized Transcriptional Regulator, Controls Filamentous Growth and Virulence of Candida albicans

    PubMed Central

    Inglis, Diane O.; Johnson, Alexander D.

    2002-01-01

    In response to a number of distinct environmental conditions, the fungal pathogen Candida albicans undergoes a morphological transition from a round, yeast form to a series of elongated, filamentous forms. This transition is believed to be critical for virulence in a mouse model of disseminated candidiasis. Here we describe the characterization of C. albicans ASH1, a gene that encodes an asymmetrically localized transcriptional regulatory protein involved in this response. We show that C. albicans ash1 mutants are defective in responding to some filament-inducing conditions. We also show that Ash1p is preferentially localized to daughter cell nuclei in the budding-yeast form of C. albicans cell growth and to the hyphal tip cells in growing filaments. Thus, Ash1p “marks” newly formed cells and presumably directs a specialized transcriptional program in these cells. Finally, we show that ASH1 is required for full virulence of C. albicans in a mouse model of disseminated candidiasis. PMID:12446785

  3. Biofilm formation in Desulfovibrio vulgaris Hildenborough is dependent upon protein filaments.

    PubMed

    Clark, Melinda E; Edelmann, Richard E; Duley, Matt L; Wall, Judy D; Fields, Matthew W

    2007-11-01

    Desulfovibrio vulgaris Hildenborough is a Gram-negative sulfate-reducing bacterium (SRB), and the physiology of SRBs can impact many anaerobic environments including radionuclide waste sites, oil reservoirs and metal pipelines. In an attempt to understand D. vulgaris as a population that can adhere to surfaces, D. vulgaris cultures were grown in a defined medium and analysed for carbohydrate production, motility and biofilm formation. Desulfovibrio vulgaris wild-type cells had increasing amounts of carbohydrate into stationary phase and approximately half of the carbohydrate remained internal. In comparison, a mutant that lacked the 200 kb megaplasmid, strain DeltaMP, produced less carbohydrate and the majority of carbohydrate remained internal of the cell proper. To assess the possibility of carbohydrate re-allocation, biofilm formation was investigated. Wild-type cells produced approximately threefold more biofilm on glass slides compared with DeltaMP; however, wild-type biofilm did not contain significant levels of exopolysaccharide. In addition, stains specific for extracellular carbohydrate did not reveal polysaccharide material within the biofilm. Desulfovibrio vulgaris wild-type biofilms contained long filaments as observed with scanning electron microscopy (SEM), and the biofilm-deficient DeltaMP strain was also deficient in motility. Biofilms grown directly on silica oxide transmission electron microscopy (TEM) grids did not contain significant levels of an exopolysaccharide matrix when viewed with TEM and SEM, and samples stained with ammonium molybdate also showed long filaments that resembled flagella. Biofilms subjected to protease treatments were degraded, and different proteases that were added at the time of inoculation inhibited biofilm formation. The data indicated that D. vulgaris did not produce an extensive exopolysaccharide matrix, used protein filaments to form biofilm between cells and silica oxide surfaces, and the filaments appeared to be

  4. Entropic potential field formed for a linear-motor protein near a filament: Statistical-mechanical analyses using simple models.

    PubMed

    Amano, Ken-Ichi; Yoshidome, Takashi; Iwaki, Mitsuhiro; Suzuki, Makoto; Kinoshita, Masahiro

    2010-07-28

    We report a new progress in elucidating the mechanism of the unidirectional movement of a linear-motor protein (e.g., myosin) along a filament (e.g., F-actin). The basic concept emphasized here is that a potential field is entropically formed for the protein on the filament immersed in solvent due to the effect of the translational displacement of solvent molecules. The entropic potential field is strongly dependent on geometric features of the protein and the filament, their overall shapes as well as details of the polyatomic structures. The features and the corresponding field are judiciously adjusted by the binding of adenosine triphosphate (ATP) to the protein, hydrolysis of ATP into adenosine diphosphate (ADP)+Pi, and release of Pi and ADP. As the first step, we propose the following physical picture: The potential field formed along the filament for the protein without the binding of ATP or ADP+Pi to it is largely different from that for the protein with the binding, and the directed movement is realized by repeated switches from one of the fields to the other. To illustrate the picture, we analyze the spatial distribution of the entropic potential between a large solute and a large body using the three-dimensional integral equation theory. The solute is modeled as a large hard sphere. Two model filaments are considered as the body: model 1 is a set of one-dimensionally connected large hard spheres and model 2 is a double helical structure formed by two sets of connected large hard spheres. The solute and the filament are immersed in small hard spheres forming the solvent. The major findings are as follows. The solute is strongly confined within a narrow space in contact with the filament. Within the space there are locations with sharply deep local potential minima along the filament, and the distance between two adjacent locations is equal to the diameter of the large spheres constituting the filament. The potential minima form a ringlike domain in model 1

  5. Formin and capping protein together embrace the actin filament in a ménage à trois

    PubMed Central

    Shekhar, Shashank; Kerleau, Mikael; Kühn, Sonja; Pernier, Julien; Romet-Lemonne, Guillaume; Jégou, Antoine; Carlier, Marie-France

    2015-01-01

    Proteins targeting actin filament barbed ends play a pivotal role in motile processes. While formins enhance filament assembly, capping protein (CP) blocks polymerization. On their own, they both bind barbed ends with high affinity and very slow dissociation. Their barbed-end binding is thought to be mutually exclusive. CP has recently been shown to be present in filopodia and controls their morphology and dynamics. Here we explore how CP and formins may functionally coregulate filament barbed-end assembly. We show, using kinetic analysis of individual filaments by microfluidics-assisted fluorescence microscopy, that CP and mDia1 formin are able to simultaneously bind barbed ends. This is further confirmed using single-molecule imaging. Their mutually weakened binding enables rapid displacement of one by the other. We show that formin FMNL2 behaves similarly, thus suggesting that this is a general property of formins. Implications in filopodia regulation and barbed-end structural regulation are discussed. PMID:26564775

  6. Restin: a novel intermediate filament-associated protein highly expressed in the Reed-Sternberg cells of Hodgkin's disease.

    PubMed Central

    Bilbe, G; Delabie, J; Brüggen, J; Richener, H; Asselbergs, F A; Cerletti, N; Sorg, C; Odink, K; Tarcsay, L; Wiesendanger, W

    1992-01-01

    We have identified a cDNA coding for a protein of 160 kDa which is expressed in in vitro cultured human peripheral blood monocytes. The predicted amino acid sequence contains an alpha-helical rod domain possessing features characteristic of intermediate filament proteins. However, the immunocytochemical staining pattern, abundance and solubility in Triton X-100/high salt buffers suggest that this protein is probably only associated with the intermediate filament network and represents a new type of intermediate filament associated protein. In a survey of normal, inflammatory and human tumour tissue samples, this protein, which we have named restin, was found to be highly expressed in Reed-Sternberg cells, the tumoral cells diagnostic for Hodgkin's disease. We suggest that restin overexpression may be a contributing factor in the progression of Hodgkin's disease. Images PMID:1600942

  7. Molecular mimicry in virus infection: crossreaction of measles virus phosphoprotein or of herpes simplex virus protein with human intermediate filaments.

    PubMed Central

    Fujinami, R S; Oldstone, M B; Wroblewska, Z; Frankel, M E; Koprowski, H

    1983-01-01

    Using monoclonal antibodies, we demonstrate that the phosphoprotein of measles virus and a protein of herpes simplex virus type 1 crossreact with an intermediate filament protein of human cells. This intermediate filament protein, probably vimentin, has a molecular weight of 52,000, whereas the molecular weights of the measles viral phosphoprotein and the herpes virus protein are 70,000 and 146,000, respectively. Crossreactivity was shown by immunofluorescent staining of infected and uninfected cells and by immunoblotting. The monoclonal antibody against measles virus phosphoprotein did not react with herpes simplex virus protein and vice versa, indicating that these monoclonal antibodies recognize different antigenic determinants on the intermediate filament molecule. The significance of these results in explaining the appearance of autoantibodies during virus infections in humans is discussed. Images PMID:6300911

  8. Alveolar rhabdomyosarcoma. Demonstration of the muscle type of intermediate filament protein, desmin, as a diagnostic aid.

    PubMed Central

    Miettinen, M.; Lehto, V. P.; Badley, R. A.; Virtanen, I.

    1982-01-01

    Three cases of soft-tissue sarcomas with the characteristic histologic features of alveolar rhabdomyosarcoma, but lacking cytoplasmic cross-striations, were studied ultrastructurally and immunohistochemically to confirm the diagnosis and evaluate the histogenesis. The results showed that it was not possible to judge the skeletal muscle derivation of the cells at the ultrastructural level. However, immunohistochemically, the results of every case were positive for desmin-the muscle type of the intermediate filament protein. The results suggest that demonstration of desmin may be a helpful adjunct tool in the diagnosis of poorly differentiated alveolar rhabdomyosarcomas. Images Figure 4 Figure 5 Figure 1 Figure 2 Figure 3 PMID:6765734

  9. On the Mechanism of Homology Search by RecA Protein Filaments.

    PubMed

    Kochugaeva, Maria P; Shvets, Alexey A; Kolomeisky, Anatoly B

    2017-03-14

    Genetic stability is a key factor in maintaining, survival, and reproduction of biological cells. It relies on many processes, but one of the most important is a homologous recombination, in which the repair of breaks in double-stranded DNA molecules is taking place with a help of several specific proteins. In bacteria, this task is accomplished by RecA proteins that are active as nucleoprotein filaments formed on single-stranded segments of DNA. A critical step in the homologous recombination is a search for a corresponding homologous region on DNA, which is called a homology search. Recent single-molecule experiments clarified some aspects of this process, but its molecular mechanisms remain not well understood. We developed a quantitative theoretical approach to analyze the homology search. It is based on a discrete-state stochastic model that takes into account the most relevant physical-chemical processes in the system. Using a method of first-passage processes, a full dynamic description of the homology search is presented. It is found that the search dynamics depends on the degree of extension of DNA molecules and on the size of RecA nucleoprotein filaments, in agreement with experimental single-molecule measurements of DNA pairing by RecA proteins. Our theoretical calculations, supported by extensive Monte Carlo computer simulations, provide a molecular description of the mechanisms of the homology search.

  10. Paired helical filaments from Alzheimer disease brain induce intracellular accumulation of Tau protein in aggresomes.

    PubMed

    Santa-Maria, Ismael; Varghese, Merina; Ksiezak-Reding, Hanna; Dzhun, Anastasiya; Wang, Jun; Pasinetti, Giulio M

    2012-06-08

    Abnormal folding of tau protein leads to the generation of paired helical filaments (PHFs) and neurofibrillary tangles, a key neuropathological feature in Alzheimer disease and tauopathies. A specific anatomical pattern of pathological changes developing in the brain suggests that once tau pathology is initiated it propagates between neighboring neuronal cells, possibly spreading along the axonal network. We studied whether PHFs released from degenerating neurons could be taken up by surrounding cells and promote spreading of tau pathology. Neuronal and non-neuronal cells overexpressing green fluorescent protein-tagged tau (GFP-Tau) were treated with isolated fractions of human Alzheimer disease-derived PHFs for 24 h. We found that cells internalized PHFs through an endocytic mechanism and developed intracellular GFP-Tau aggregates with attributes of aggresomes. This was particularly evident by the perinuclear localization of aggregates and redistribution of the vimentin intermediate filament network and retrograde motor protein dynein. Furthermore, the content of Sarkosyl-insoluble tau, a measure of abnormal tau aggregation, increased 3-fold in PHF-treated cells. An exosome-related mechanism did not appear to be involved in the release of GFP-Tau from untreated cells. The evidence that cells can internalize PHFs, leading to formation of aggresome-like bodies, opens new therapeutic avenues to prevent propagation and spreading of tau pathology.

  11. Paired Helical Filaments from Alzheimer Disease Brain Induce Intracellular Accumulation of Tau Protein in Aggresomes*

    PubMed Central

    Santa-Maria, Ismael; Varghese, Merina; Ksiȩżak-Reding, Hanna; Dzhun, Anastasiya; Wang, Jun; Pasinetti, Giulio M.

    2012-01-01

    Abnormal folding of tau protein leads to the generation of paired helical filaments (PHFs) and neurofibrillary tangles, a key neuropathological feature in Alzheimer disease and tauopathies. A specific anatomical pattern of pathological changes developing in the brain suggests that once tau pathology is initiated it propagates between neighboring neuronal cells, possibly spreading along the axonal network. We studied whether PHFs released from degenerating neurons could be taken up by surrounding cells and promote spreading of tau pathology. Neuronal and non-neuronal cells overexpressing green fluorescent protein-tagged tau (GFP-Tau) were treated with isolated fractions of human Alzheimer disease-derived PHFs for 24 h. We found that cells internalized PHFs through an endocytic mechanism and developed intracellular GFP-Tau aggregates with attributes of aggresomes. This was particularly evident by the perinuclear localization of aggregates and redistribution of the vimentin intermediate filament network and retrograde motor protein dynein. Furthermore, the content of Sarkosyl-insoluble tau, a measure of abnormal tau aggregation, increased 3-fold in PHF-treated cells. An exosome-related mechanism did not appear to be involved in the release of GFP-Tau from untreated cells. The evidence that cells can internalize PHFs, leading to formation of aggresome-like bodies, opens new therapeutic avenues to prevent propagation and spreading of tau pathology. PMID:22496370

  12. Arabidopsis Microtubule-Destabilizing Protein 25 Functions in Pollen Tube Growth by Severing Actin Filaments[W

    PubMed Central

    Qin, Tao; Liu, Xiaomin; Li, Jiejie; Sun, Jingbo; Song, Leina; Mao, Tonglin

    2014-01-01

    The formation of distinct actin filament arrays in the subapical region of pollen tubes is crucial for pollen tube growth. However, the molecular mechanisms underlying the organization and dynamics of the actin filaments in this region remain to be determined. This study shows that Arabidopsis thaliana MICROTUBULE-DESTABILIZING PROTEIN25 (MDP25) has the actin filament–severing activity of an actin binding protein. This protein negatively regulated pollen tube growth by modulating the organization and dynamics of actin filaments in the subapical region of pollen tubes. MDP25 loss of function resulted in enhanced pollen tube elongation and inefficient fertilization. MDP25 bound directly to actin filaments and severed individual actin filaments, in a manner that was dramatically enhanced by Ca2+, in vitro. Analysis of a mutant that bears a point mutation at the Ca2+ binding sites demonstrated that the subcellular localization of MDP25 was determined by cytosolic Ca2+ level in the subapical region of pollen tubes, where MDP25 was disassociated from the plasma membrane and moved into the cytosol. Time-lapse analysis showed that the F-actin-severing frequency significantly decreased and a high density of actin filaments was observed in the subapical region of mdp25-1 pollen tubes. This study reveals a mechanism whereby calcium enhances the actin filament–severing activity of MDP25 in the subapical region of pollen tubes to modulate pollen tube growth. PMID:24424096

  13. Intermediate filament proteins in choroid plexus and ependyma and their tumors.

    PubMed Central

    Miettinen, M.; Clark, R.; Virtanen, I.

    1986-01-01

    The intermediate filament protein types of normal choroid plexus and ependymal tissue and their putative tumors were investigated. In normal human choroid plexus tissue, but not in ependyma, keratin could be demonstrated immunohistochemically. By immunoblotting, keratins 8, 18, and 19 were found, but glial fibrillary acidic protein (GFAP) was absent. In mouse and rat, choroid plexus epithelium and ependymal lining cells were keratin-positive. In addition, many ependymal cells were vimentin-positive. Keratin was immunohistochemically found in three of four choroid plexus papillomas, two of two choroid plexus carcinomas, and the lining cells of three neuroepithelial cysts. GFAP-positive cells were present in some choroid plexus tumors. In contrast, none of the eight ependymomas contained keratin, but all were strongly positive for GFAP. The results show that choroid plexus lining cells and choroid plexus tumors have true epithelial characteristics in their cytoskeleton, in contrast to ependymomas, which do not show keratin positivity but show glial filaments, as would be seen in astrocytic tumors. Images Figure 8 Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 9 PMID:2422943

  14. Crystal structure of the coat protein of the flexible filamentous papaya mosaic virus.

    PubMed

    Yang, Shaoqing; Wang, Tao; Bohon, Jen; Gagné, Marie-Ève Laliberté; Bolduc, Marilène; Leclerc, Denis; Li, Huilin

    2012-09-14

    Papaya mosaic virus (PapMV) is a filamentous plant virus that belongs to the Alphaflexiviridae family. Flexible filamentous viruses have defied more than two decades of effort in fiber diffraction, and no high-resolution structure is available for any member of the Alphaflexiviridae family. Here, we report our structural characterization of PapMV by X-ray crystallography and cryo-electron microscopy three-dimensional reconstruction. We found that PapMV is 135Å in diameter with a helical symmetry of ~10 subunits per turn. Crystal structure of the C-terminal truncated PapMV coat protein (CP) reveals a novel all-helix fold with seven α-helices. Thus, the PapMVCP structure is different from the four-helix-bundle fold of tobacco mosaic virus in which helix bundling dominates the subunit interface in tobacco mosaic virus and conveys rigidity to the rod virus. PapMV CP was crystallized as an asymmetrical dimer in which one protein lassoes the other by the N-terminal peptide. Mutation of residues critical to the inter-subunit lasso interaction abolishes CP polymerization. The crystal structure suggests that PapMV may polymerize via the consecutive N-terminal loop lassoing mechanism. The structure of PapMV will be useful for rational design and engineering of the PapMV nanoparticles into innovative vaccines.

  15. Srv2/cyclase-associated protein forms hexameric shurikens that directly catalyze actin filament severing by cofilin.

    PubMed

    Chaudhry, Faisal; Breitsprecher, Dennis; Little, Kristin; Sharov, Grigory; Sokolova, Olga; Goode, Bruce L

    2013-01-01

    Actin filament severing is critical for the dynamic turnover of cellular actin networks. Cofilin severs filaments, but additional factors may be required to increase severing efficiency in vivo. Srv2/cyclase-associated protein (CAP) is a widely expressed protein with a role in binding and recycling actin monomers ascribed to domains in its C-terminus (C-Srv2). In this paper, we report a new biochemical and cellular function for Srv2/CAP in directly catalyzing cofilin-mediated severing of filaments. This function is mediated by its N-terminal half (N-Srv2), and is physically and genetically separable from C-Srv2 activities. Using dual-color total internal reflection fluorescence microscopy, we determined that N-Srv2 stimulates filament disassembly by increasing the frequency of cofilin-mediated severing without affecting cofilin binding to filaments. Structural analysis shows that N-Srv2 forms novel hexameric star-shaped structures, and disrupting oligomerization impairs N-Srv2 activities and in vivo function. Further, genetic analysis shows that the combined activities of N-Srv2 and Aip1 are essential in vivo. These observations define a novel mechanism by which the combined activities of cofilin and Srv2/CAP lead to enhanced filament severing and support an emerging view that actin disassembly is controlled not by cofilin alone, but by a more complex set of factors working in concert.

  16. Gas7b (growth arrest specific protein 7b) regulates neuronal cell morphology by enhancing microtubule and actin filament assembly.

    PubMed

    Gotoh, Aina; Hidaka, Masafumi; Hirose, Keiko; Uchida, Takafumi

    2013-11-29

    Neurons undergo several morphological changes as a part of normal neuron maturation process. Alzheimer disease is associated with increased neuroproliferation and impaired neuronal maturation. In this study, we demonstrated that Gas7b (growth arrest specific protein 7b) expression in a neuronal cell line, Neuro 2A, induces cell maturation by facilitating formation of dendrite-like processes and/or filopodia projections and that Gas7b co-localizes with neurite microtubules. Molecular analysis was performed to evaluate whether Gas7b associates with actin filaments and microtubules, and the data revealed two novel roles of Gas7b in neurite outgrowth: we showed that Gas7b enhances bundling of several microtubule filaments and connects microtubules with actin filaments. These results suggest that Gas7b governs neural cell morphogenesis by enhancing the coordination between actin filaments and microtubules. We conclude that lower neuronal Gas7b levels may impact Alzheimer disease progression.

  17. Lateral association and elongation of vimentin intermediate filament proteins: A time-resolved light-scattering study

    PubMed Central

    Saldanha, Oliva; Huber, Klaus; Köster, Sarah

    2016-01-01

    Vimentin intermediate filaments (IFs) are part of a family of proteins that constitute one of the three filament systems in the cytoskeleton, a major contributor to cell mechanics. One property that distinguishes IFs from the other cytoskeletal filament types, actin filaments and microtubules, is their highly hierarchical assembly pathway, where a lateral association step is followed by elongation. Here we present an innovative technique to follow the elongation reaction in solution and in situ by time-resolved static and dynamic light scattering, thereby precisely capturing the relevant time and length scales of seconds to minutes and 60–600 nm, respectively. We apply a quantitative model to our data and succeed in consistently describing the entire set of data, including particle mass, radius of gyration, and hydrodynamic radius during longitudinal association. PMID:27655889

  18. Dimer dynamics and filament organization of the bacterial cell division protein FtsA.

    PubMed

    Hsin, Jen; Fu, Rui; Huang, Kerwyn Casey

    2013-11-15

    FtsA is a bacterial actin homolog and one of the core proteins involved in cell division. While previous studies have demonstrated the capability of FtsA to polymerize, little is known about its polymerization state in vivo or if polymerization is necessary for FtsA function. Given that one function of FtsA is to tether FtsZ filaments to the membrane, in vivo polymerization of FtsA imposes geometric constraints and requires a specific polymer curvature direction. Here, we report a series of molecular dynamics simulations probing the structural dynamics of FtsA as a dimer and as a tetrameric single filament. We found that the FtsA polymer exhibits a preferred bending direction that would allow for its placement parallel with FtsZ polymers underneath the cytoplasmic membrane. We also identified key interfacial amino acids that mediate FtsA-FtsA interaction and propose that some amino acids play more critical roles than others. We performed in silico mutagenesis on FtsA and demonstrated that, while a moderate mutation at the polymerization interface does not significantly affect polymer properties such as bending direction and association strength, more drastic mutations change both features and could lead to non-functional FtsA.

  19. Estimating the bending modulus of a FtsZ bacterial-division protein filament

    NASA Astrophysics Data System (ADS)

    Cytrynbaum, Eric N.; Li, Yongnan Devin; Allard, Jun F.; Mehrabian, Hadi

    2012-01-01

    FtsZ, a cytoskeletal protein homologous to tubulin, is the principle constituent of the division ring in bacterial cells. It is known to have force-generating capacity in vitro and has been conjectured to be the source of the constriction force in vivo. Several models have been proposed to explain the generation of force by the Z ring. Here we re-examine data from in vitro experiments in which Z rings formed and constricted inside tubular liposomes, and we carry out image analysis on previously published data with which to better estimate important model parameters that have proven difficult to measure by direct means. We introduce a membrane-energy-based model for the dynamics of multiple Z rings moving and colliding inside a tubular liposome and a fluid model for the drag of a Z ring as it moves through the tube. Using this model, we estimate an effective membrane bending modulus of 500-700 pNnm. If we assume that FtsZ force generation is driven by hydrolysis into a highly curved conformation, we estimate the FtsZ filament bending modulus to be 310-390 pNnm2. If we assume instead that force is generated by the non-hydrolysis-dependent intermediate curvature conformation, we find that Bf>1400pNnm2. The former value sits at the lower end of the range of previously estimated values and, if correct, may raise challenges for models that rely on filament bending to generate force.

  20. The intermediate filament protein, synemin, is an AKAP in the heart.

    PubMed

    Russell, Mary A; Lund, Linda M; Haber, Roy; McKeegan, Kathleen; Cianciola, Nicholas; Bond, Meredith

    2006-12-15

    Targeting of protein kinase A (PKA) by A-kinase anchoring proteins (AKAPs) contributes to high specificity of PKA signaling pathways. PKA phosphorylation of myofilament and cytoskeletal proteins may regulate myofibrillogenesis and myocyte remodeling during heart disease; however, known cardiac AKAPs do not localize to these regions. To identify novel AKAPs which target PKA to the cytoskeleton or myofilaments, a human heart cDNA library was screened and the intermediate filament (IF) protein, synemin, was identified as a putative RII (PKA regulatory subunit type II) binding protein. A predicted RII binding region was mutated and resulted in loss of RII binding. Furthermore, synemin co-localized with RII in SW13/cl.1-vim+ cells and co-immunoprecipitated with RII from adult rat cardiomyocytes. Synemin was localized at the level of Z-lines with RII and desmin in adult hearts, however, neonatal cardiomyocytes showed differential synemin and desmin localization. Quantitative Western blots also showed significantly more synemin was present in failing human hearts. We propose that synemin provides temporal and spatial targeting of PKA in adult and neonatal cardiac myocytes.

  1. Packing of coat protein amphipathic and transmembrane helices in filamentous bacteriophage M13: role of small residues in protein oligomerization.

    PubMed

    Williams, K A; Glibowicka, M; Li, Z; Li, H; Khan, A R; Chen, Y M; Wang, J; Marvin, D A; Deber, C M

    1995-09-08

    Filamentous bacteriophage M13, an important cloning and phage display vector, is encapsulated by ca 2700 copies of its 50-residue major coat protein (gene 8). This protein occurs as a membrane protein while stably inserted into its E. coli host inner membrane, and as a coat protein upon assembly and packing onto phage DNA in the lipid-free virion. To examine the specific protein-protein interactions underlying these processes, we used a combination of randomized and saturation mutagenesis of the entire gene 8 to assess the susceptibility of each position to mutation. In the resulting library of ca 100 viable M13 mutants, "small" residues (Ala,Gly,Ser), which constitute the non-polar face of the N-terminal amphipathic helical segment, and a face of the hydrophobic (effective transmembrane) helical segment, were found to be highly conserved. These results support a model in which coat protein packing is stabilized by the presence within each protein subunit of two "oligomerization segments", i.e. specific helical regions with faces rich in small residues which function to promote the close approach of alpha-helices.

  2. Terahertz absorption spectroscopy of protein-containing reverse micellar solution

    NASA Astrophysics Data System (ADS)

    Murakami, H.; Toyota, Y.; Nishi, T.; Nashima, S.

    2012-01-01

    Terahertz time-domain spectroscopy has been carried out for AOT/isooctane reverse micellar solution with myoglobin at the water-to-surfactant molar ratios ( w0) of 0.2 and 4.4. The amplitude of the absorption spectrum increases with increasing the protein concentration at w0 = 0.2, whereas it decreases at w0 = 4.4. The molar extinction coefficients of the protein-filled reverse micelle, and the constituents, i.e., myoglobin, water, and AOT, have been derived by use of the structural parameters of the micellar solution. The experimental results are interpreted in terms of hydration onto the protein and surfactant in the reverse micelle.

  3. [Role of G-protein alpha sub-units in the morphogenic processes of filamentous Ascomycota fungi].

    PubMed

    García-Rico, Ramón O; Fierro, Francisco

    The phylum Ascomycota comprises about 75% of all the fungal species described, and includes species of medical, phytosanitary, agricultural, and biotechnological importance. The ability to spread, explore, and colonise new substrates is a feature of critical importance for this group of organisms. In this regard, basic processes such as conidial germination, the extension of hyphae and sporulation, make up the backbone of development in most filamentous fungi. These processes require specialised morphogenic machinery, coordinated and regulated by mechanisms that are still being elucidated. In recent years, substantial progress has been made in understanding the role of the signalling pathway mediated by heterotrimericG proteins in basic biological processes of many filamentous fungi. This review focuses on the role of the alpha subunits of heterotrimericG proteins in the morphogenic processes of filamentous Ascomycota.

  4. Heterocyst-specific flavodiiron protein Flv3B enables oxic diazotrophic growth of the filamentous cyanobacterium Anabaena sp. PCC 7120.

    PubMed

    Ermakova, Maria; Battchikova, Natalia; Richaud, Pierre; Leino, Hannu; Kosourov, Sergey; Isojärvi, Janne; Peltier, Gilles; Flores, Enrique; Cournac, Laurent; Allahverdiyeva, Yagut; Aro, Eva-Mari

    2014-07-29

    Flavodiiron proteins are known to have crucial and specific roles in photoprotection of photosystems I and II in cyanobacteria. The filamentous, heterocyst-forming cyanobacterium Anabaena sp. strain PCC 7120 contains, besides the four flavodiiron proteins Flv1A, Flv2, Flv3A, and Flv4 present in vegetative cells, two heterocyst-specific flavodiiron proteins, Flv1B and Flv3B. Here, we demonstrate that Flv3B is responsible for light-induced O2 uptake in heterocysts, and that the absence of the Flv3B protein severely compromises the growth of filaments in oxic, but not in microoxic, conditions. It is further demonstrated that Flv3B-mediated photosynthetic O2 uptake has a distinct role in heterocysts which cannot be substituted by respiratory O2 uptake in the protection of nitrogenase from oxidative damage and, thus, in an efficient provision of nitrogen to filaments. In line with this conclusion, the Δflv3B strain has reduced amounts of nitrogenase NifHDK subunits and shows multiple symptoms of nitrogen deficiency in the filaments. The apparent imbalance of cytosolic redox state in Δflv3B heterocysts also has a pronounced influence on the amounts of different transcripts and proteins. Therefore, an O2-related mechanism for control of gene expression is suggested to take place in heterocysts.

  5. HB-EGF affects astrocyte morphology, proliferation, differentiation, and the expression of intermediate filament proteins.

    PubMed

    Puschmann, Till B; Zandén, Carl; Lebkuechner, Isabell; Philippot, Camille; de Pablo, Yolanda; Liu, Johan; Pekny, Milos

    2014-03-01

    Heparin-binding epidermal growth factor-like growth factor (HB-EGF), a vascular-derived trophic factor, belongs to the epidermal growth factor (EGF) family of neuroprotective, hypoxia-inducible proteins released by astrocytes in CNS injuries. It was suggested that HB-EGF can replace fetal calf serum (FCS) in astrocyte cultures. We previously demonstrated that in contrast to standard 2D cell culture systems, Bioactive3D culture system, when used with FCS, minimizes the baseline activation of astrocytes and preserves their complex morphology. Here, we show that HB-EGF induced EGF receptor (EGFR) activation by Y1068 phosphorylation, Mapk/Erk pathway activation, and led to an increase in cell proliferation, more prominent in Bioactive3D than in 2D cultures. HB-EGF changed morphology of 2D and Bioactive3D cultured astrocytes toward a radial glia-like phenotype and induced the expression of intermediate filament and progenitor cell marker protein nestin. Glial fibrillary acidic protein (GFAP) and vimentin protein expression was unaffected. RT-qPCR analysis demonstrated that HB-EGF affected the expression of Notch signaling pathway genes, implying a role for the Notch signaling in HB-EGF-mediated astrocyte response. HB-EGF can be used as a FCS replacement for astrocyte expansion and in vitro experimentation both in 2D and Bioactive3D culture systems; however, caution should be exercised since it appears to induce partial de-differentiation of astrocytes.

  6. Direct observation of the uncapping of capping protein-capped actin filaments by CARMIL homology domain 3.

    PubMed

    Fujiwara, Ikuko; Remmert, Kirsten; Hammer, John A

    2010-01-22

    Bulk solution assays have shown that the isolated CARMIL homology 3 (CAH3) domain from mouse and Acanthamoeba CARMIL rapidly and potently restores actin polymerization when added to actin filaments previously capped with capping protein (CP). To demonstrate this putative uncapping activity directly, we used total internal reflection microscopy to observe single, CP-capped actin filaments before and after the addition of the CAH3 domain from mouse CARMIL-1 (mCAH3). The addition of mCAH3 rapidly restored the polymerization of individual capped filaments, consistent with uncapping. To verify uncapping, filaments were capped with recombinant mouse CP tagged with monomeric green fluorescent protein (mGFP-CP). Restoration of polymerization upon the addition of mCAH3 was immediately preceded by the complete dissociation of mGFP-CP from the filament end, confirming the CAH3-driven uncapping mechanism. Quantitative analyses showed that the percentage of capped filaments that uncapped increased as the concentration of mCAH3 was increased, reaching a maximum of approximately 90% at approximately 250 nm mCAH3. Moreover, the time interval between mCAH3 addition and uncapping decreased as the concentration of mCAH3 increased, with the half-time of CP at the barbed end decreasing from approximately 30 min without mCAH3 to approximately 10 s with a saturating amount of mCAH3. Finally, using mCAH3 tagged with mGFP, we obtained direct evidence that the complex of CP and mCAH3 has a small but measurable affinity for the barbed end, as inferred from previous studies and kinetic modeling. We conclude that the isolated CAH3 domain of CARMIL (and presumably the intact molecule as well) possesses the ability to uncap CP-capped actin filaments.

  7. Direct Observation of the Uncapping of Capping Protein-capped Actin Filaments by CARMIL Homology Domain 3*

    PubMed Central

    Fujiwara, Ikuko; Remmert, Kirsten; Hammer, John A.

    2010-01-01

    Bulk solution assays have shown that the isolated CARMIL homology 3 (CAH3) domain from mouse and Acanthamoeba CARMIL rapidly and potently restores actin polymerization when added to actin filaments previously capped with capping protein (CP). To demonstrate this putative uncapping activity directly, we used total internal reflection microscopy to observe single, CP-capped actin filaments before and after the addition of the CAH3 domain from mouse CARMIL-1 (mCAH3). The addition of mCAH3 rapidly restored the polymerization of individual capped filaments, consistent with uncapping. To verify uncapping, filaments were capped with recombinant mouse CP tagged with monomeric green fluorescent protein (mGFP-CP). Restoration of polymerization upon the addition of mCAH3 was immediately preceded by the complete dissociation of mGFP-CP from the filament end, confirming the CAH3-driven uncapping mechanism. Quantitative analyses showed that the percentage of capped filaments that uncapped increased as the concentration of mCAH3 was increased, reaching a maximum of ∼90% at ∼250 nm mCAH3. Moreover, the time interval between mCAH3 addition and uncapping decreased as the concentration of mCAH3 increased, with the half-time of CP at the barbed end decreasing from ∼30 min without mCAH3 to ∼10 s with a saturating amount of mCAH3. Finally, using mCAH3 tagged with mGFP, we obtained direct evidence that the complex of CP and mCAH3 has a small but measurable affinity for the barbed end, as inferred from previous studies and kinetic modeling. We conclude that the isolated CAH3 domain of CARMIL (and presumably the intact molecule as well) possesses the ability to uncap CP-capped actin filaments. PMID:19926785

  8. Reversals and collisions optimize protein exchange in bacterial swarms

    NASA Astrophysics Data System (ADS)

    Amiri, Aboutaleb; Harvey, Cameron; Buchmann, Amy; Christley, Scott; Shrout, Joshua D.; Aranson, Igor S.; Alber, Mark

    2017-03-01

    Swarming groups of bacteria coordinate their behavior by self-organizing as a population to move over surfaces in search of nutrients and optimal niches for colonization. Many open questions remain about the cues used by swarming bacteria to achieve this self-organization. While chemical cue signaling known as quorum sensing is well-described, swarming bacteria often act and coordinate on time scales that could not be achieved via these extracellular quorum sensing cues. Here, cell-cell contact-dependent protein exchange is explored as a mechanism of intercellular signaling for the bacterium Myxococcus xanthus. A detailed biologically calibrated computational model is used to study how M. xanthus optimizes the connection rate between cells and maximizes the spread of an extracellular protein within the population. The maximum rate of protein spreading is observed for cells that reverse direction optimally for swarming. Cells that reverse too slowly or too fast fail to spread extracellular protein efficiently. In particular, a specific range of cell reversal frequencies was observed to maximize the cell-cell connection rate and minimize the time of protein spreading. Furthermore, our findings suggest that predesigned motion reversal can be employed to enhance the collective behavior of biological synthetic active systems.

  9. SAS-4 Protein in Trypanosoma brucei Controls Life Cycle Transitions by Modulating the Length of the Flagellum Attachment Zone Filament.

    PubMed

    Hu, Huiqing; Zhou, Qing; Li, Ziyin

    2015-12-18

    The evolutionarily conserved centriole/basal body protein SAS-4 regulates centriole duplication in metazoa and basal body duplication in flagellated and ciliated organisms. Here, we report that the SAS-4 homolog in the flagellated protozoan Trypanosoma brucei, TbSAS-4, plays an unusual role in controlling life cycle transitions by regulating the length of the flagellum attachment zone (FAZ) filament, a specialized cytoskeletal structure required for flagellum adhesion and cell morphogenesis. TbSAS-4 is concentrated at the distal tip of the FAZ filament, and depletion of TbSAS-4 in the trypomastigote form disrupts the elongation of the new FAZ filament, generating cells with a shorter FAZ associated with a longer unattached flagellum and repositioned kinetoplast and basal body, reminiscent of epimastigote-like morphology. Further, we show that TbSAS-4 associates with six additional FAZ tip proteins, and depletion of TbSAS-4 disrupts the enrichment of these FAZ tip proteins at the new FAZ tip, suggesting a role of TbSAS-4 in maintaining the integrity of this FAZ tip protein complex. Together, these results uncover a novel function of TbSAS-4 in regulating the length of the FAZ filament to control basal body positioning and life cycle transitions in T. brucei.

  10. Electrostatic Interactions between Elongated Monomers Drive Filamentation of Drosophila Shrub, a Metazoan ESCRT-III Protein.

    PubMed

    McMillan, Brian J; Tibbe, Christine; Jeon, Hyesung; Drabek, Andrew A; Klein, Thomas; Blacklow, Stephen C

    2016-08-02

    The endosomal sorting complex required for transport (ESCRT) is a conserved protein complex that facilitates budding and fission of membranes. It executes a key step in many cellular events, including cytokinesis and multi-vesicular body formation. The ESCRT-III protein Shrub in flies, or its homologs in yeast (Snf7) or humans (CHMP4B), is a critical polymerizing component of ESCRT-III needed to effect membrane fission. We report the structural basis for polymerization of Shrub and define a minimal region required for filament formation. The X-ray structure of the Shrub core shows that individual monomers in the lattice interact in a staggered arrangement using complementary electrostatic surfaces. Mutations that disrupt interface salt bridges interfere with Shrub polymerization and function. Despite substantial sequence divergence and differences in packing interactions, the arrangement of Shrub subunits in the polymer resembles that of Snf7 and other family homologs, suggesting that this intermolecular packing mechanism is shared among ESCRT-III proteins.

  11. The Role of the Ubiquitin Proteasome Pathway in Keratin Intermediate Filament Protein Degradation

    PubMed Central

    Rogel, Micah R.; Jaitovich, Ariel; Ridge, Karen M.

    2010-01-01

    Lung injury, whether caused by hypoxic or mechanical stresses, elicits a variety of responses at the cellular level. Alveolar epithelial cells respond and adapt to such injurious stimuli by reorganizing the cellular cytoskeleton, mainly accomplished through modification of the intermediate filament (IF) network. The structural and mechanical integrity in epithelial cells is maintained through this adaptive reorganization response. Keratin, the predominant IF expressed in epithelial cells, displays highly dynamic properties in response to injury, sometimes in the form of degradation of the keratin IF network. Post-translational modification, such as phosphorylation, targets keratin proteins for degradation in these circumstances. As with other structural and regulatory proteins, turnover of keratin is regulated by the ubiquitin (Ub)-proteasome pathway. The degradation process begins with activation of Ub by the Ub-activating enzyme (E1), followed by the exchange of Ub to the Ub-conjugating enzyme (E2). E2 shuttles the Ub molecule to the substrate-specific Ub ligase (E3), which then delivers the Ub to the substrate protein, thereby targeting it for degradation. In some cases of injury and IF-related disease, aggresomes form in epithelial cells. The mechanisms that regulate aggresome formation are currently unknown, although proteasome overload may play a role. Therefore, a more complete understanding of keratin degradation—causes, mechanisms, and consequences—will allow for a greater understanding of epithelial cell biology and lung pathology alike. PMID:20160151

  12. Staurosporine Induces Filamentation in the Human Fungal Pathogen Candida albicans via Signaling through Cyr1 and Protein Kinase A.

    PubMed

    Xie, Jinglin L; O'Meara, Teresa R; Polvi, Elizabeth J; Robbins, Nicole; Cowen, Leah E

    2017-01-01

    Protein kinases are key regulators of signal transduction pathways that participate in diverse cellular processes. In fungal pathogens, kinases regulate signaling pathways that govern drug resistance, stress adaptation, and pathogenesis. The impact of kinases on the fungal regulatory circuitry has recently garnered considerable attention in the opportunistic fungal pathogen Candida albicans, which is a leading cause of human morbidity and mortality. Complex regulatory circuitry governs the C. albicans morphogenetic transition between yeast and filamentous growth, which is a key virulence trait. Here, we report that staurosporine, a promiscuous kinase inhibitor that abrogates fungal drug resistance, also influences C. albicans morphogenesis by inducing filamentation in the absence of any other inducing cue. We further establish that staurosporine exerts its effect via the adenylyl cyclase Cyr1 and the cyclic AMP (cAMP)-dependent protein kinase A (PKA). Strikingly, filamentation induced by staurosporine does not require the known upstream regulators of Cyr1, Ras1 or Pkc1, or effectors downstream of PKA, including Efg1. We further demonstrate that Cyr1 is capable of activating PKA to enable filamentation in response to staurosporine through a mechanism that does not require degradation of the transcriptional repressor Nrg1. We establish that staurosporine-induced filamentation is accompanied by a defect in septin ring formation, implicating cell cycle kinases as potential staurosporine targets underpinning this cellular response. Thus, we establish staurosporine as a chemical probe to elucidate the architecture of cellular signaling governing fungal morphogenesis and highlight the existence of novel circuitry through which the Cyr1 and PKA govern a key virulence trait. IMPORTANCE The impact of fungal pathogens on human health is devastating. One of the most pervasive fungal pathogens is Candida albicans, which kills ~40% of people suffering from bloodstream

  13. Staurosporine Induces Filamentation in the Human Fungal Pathogen Candida albicans via Signaling through Cyr1 and Protein Kinase A

    PubMed Central

    Xie, Jinglin L.; O’Meara, Teresa R.; Polvi, Elizabeth J.; Robbins, Nicole

    2017-01-01

    ABSTRACT Protein kinases are key regulators of signal transduction pathways that participate in diverse cellular processes. In fungal pathogens, kinases regulate signaling pathways that govern drug resistance, stress adaptation, and pathogenesis. The impact of kinases on the fungal regulatory circuitry has recently garnered considerable attention in the opportunistic fungal pathogen Candida albicans, which is a leading cause of human morbidity and mortality. Complex regulatory circuitry governs the C. albicans morphogenetic transition between yeast and filamentous growth, which is a key virulence trait. Here, we report that staurosporine, a promiscuous kinase inhibitor that abrogates fungal drug resistance, also influences C. albicans morphogenesis by inducing filamentation in the absence of any other inducing cue. We further establish that staurosporine exerts its effect via the adenylyl cyclase Cyr1 and the cyclic AMP (cAMP)-dependent protein kinase A (PKA). Strikingly, filamentation induced by staurosporine does not require the known upstream regulators of Cyr1, Ras1 or Pkc1, or effectors downstream of PKA, including Efg1. We further demonstrate that Cyr1 is capable of activating PKA to enable filamentation in response to staurosporine through a mechanism that does not require degradation of the transcriptional repressor Nrg1. We establish that staurosporine-induced filamentation is accompanied by a defect in septin ring formation, implicating cell cycle kinases as potential staurosporine targets underpinning this cellular response. Thus, we establish staurosporine as a chemical probe to elucidate the architecture of cellular signaling governing fungal morphogenesis and highlight the existence of novel circuitry through which the Cyr1 and PKA govern a key virulence trait. IMPORTANCE The impact of fungal pathogens on human health is devastating. One of the most pervasive fungal pathogens is Candida albicans, which kills ~40% of people suffering from bloodstream

  14. The nucleoid-associated protein Dan organizes chromosomal DNA through rigid nucleoprotein filament formation in E. coli during anoxia

    PubMed Central

    Lim, Ci Ji; Lee, Sin Yi; Teramoto, Jun; Ishihama, Akira; Yan, Jie

    2013-01-01

    Dan is a transcription factor that regulates the ttd operon encoding tartrate dehydratase. During anaerobic conditions, its copy number increases by 100-fold, making Dan an abundant nucleoid-associated protein. However, little is known about the mode of Dan–DNA interaction. To understand its cellular functions, we used single-molecule manipulation and imaging techniques to show that Dan binds cooperatively along DNA, resulting in formation of a rigid periodic nucleoprotein filament that strongly restricts accessibility to DNA. Furthermore, in the presence of physiologic levels of magnesium, these filaments interact with each other to cause global DNA condensation. Overall, these results shed light on the architectural role of Dan in the compaction of Escherichia coli chromosomal DNA under anaerobic conditions. Formation of the nucleoprotein filament provides a basis in understanding how Dan may play roles in both chromosomal DNA protection and gene regulation. PMID:23180762

  15. Requirement of Fra proteins for communication channels between cells in the filamentous nitrogen-fixing cyanobacterium Anabaena sp. PCC 7120.

    PubMed

    Omairi-Nasser, Amin; Mariscal, Vicente; Austin, Jotham R; Haselkorn, Robert

    2015-08-11

    The filamentous nitrogen-fixing cyanobacterium Anabaena sp. PCC 7120 differentiates specialized cells, heterocysts, that fix atmospheric nitrogen and transfer the fixed nitrogen to adjacent vegetative cells. Reciprocally, vegetative cells transfer fixed carbon to heterocysts. Several routes have been described for metabolite exchange within the filament, one of which involves communicating channels that penetrate the septum between adjacent cells. Several fra gene mutants were isolated 25 y ago on the basis of their phenotypes: inability to fix nitrogen and fragmentation of filaments upon transfer from N+ to N- media. Cryopreservation combined with electron tomography were used to investigate the role of three fra gene products in channel formation. FraC and FraG are clearly involved in channel formation, whereas FraD has a minor part. Additionally, FraG was located close to the cytoplasmic membrane and in the heterocyst neck, using immunogold labeling with antibody raised to the N-terminal domain of the FraG protein.

  16. Fast-folding α-helices as reversible strain absorbers in the muscle protein myomesin

    PubMed Central

    Berkemeier, Felix; Bertz, Morten; Xiao, Senbo; Pinotsis, Nikos; Wilmanns, Matthias; Gräter, Frauke; Rief, Matthias

    2011-01-01

    The highly oriented filamentous protein network of muscle constantly experiences significant mechanical load during muscle operation. The dimeric protein myomesin has been identified as an important M-band component supporting the mechanical integrity of the entire sarcomere. Recent structural studies have revealed a long α-helical linker between the C-terminal immunoglobulin (Ig) domains My12 and My13 of myomesin. In this paper, we have used single-molecule force spectroscopy in combination with molecular dynamics simulations to characterize the mechanics of the myomesin dimer comprising immunoglobulin domains My12–My13. We find that at forces of approximately 30 pN the α-helical linker reversibly elongates allowing the molecule to extend by more than the folded extension of a full domain. High-resolution measurements directly reveal the equilibrium folding/unfolding kinetics of the individual helix. We show that α-helix unfolding mechanically protects the molecule homodimerization from dissociation at physiologically relevant forces. As fast and reversible molecular springs the myomesin α-helical linkers are an essential component for the structural integrity of the M band. PMID:21825161

  17. The importance of connections between the cell wall integrity pathway and the unfolded protein response in filamentous fungi.

    PubMed

    Malavazi, Iran; Goldman, Gustavo Henrique; Brown, Neil Andrew

    2014-11-01

    In the external environment, or within a host organism, filamentous fungi experience sudden changes in nutrient availability, osmolality, pH, temperature and the exposure to toxic compounds. The fungal cell wall represents the first line of defense, while also performing essential roles in morphology, development and virulence. A polarized secretion system is paramount for cell wall biosynthesis, filamentous growth, nutrient acquisition and interactions with the environment. The unique ability of filamentous fungi to secrete has resulted in their industrial adoption as fungal cell factories. Protein maturation and secretion commences in the endoplasmic reticulum (ER). The unfolded protein response (UPR) maintains ER functionality during exposure to secretion and cell wall stress. UPR, therefore, influences secretion and cell wall homeostasis, which in turn impacts upon numerous fungal traits important to pathogenesis and biotechnology. Subsequently, this review describes the relevance of the cell wall and UPR systems to filamentous fungal pathogens or industrial microbes and then highlights interconnections between the two systems. Ultimately, the possible biotechnological applications of an enhanced understanding of such regulatory systems in combating fungal disease, or the removal of natural bottlenecks in protein secretion in an industrial setting, are discussed.

  18. The ALP-Enigma protein ALP-1 functions in actin filament organization to promote muscle structural integrity in Caenorhabditis elegans.

    PubMed

    Han, Hsiao-Fen; Beckerle, Mary C

    2009-05-01

    Mutations that affect the Z-disk-associated ALP-Enigma proteins have been linked to human muscular and cardiac diseases. Despite their clear physiological significance for human health, the mechanism of action of ALP-Enigma proteins is largely unknown. In Caenorhabditis elegans, the ALP-Enigma protein family is encoded by a single gene, alp-1; thus C. elegans provides an excellent model to study ALP-Enigma function. Here we present a molecular and genetic analysis of ALP-Enigma function in C. elegans. We show that ALP-1 and alpha-actinin colocalize at dense bodies where actin filaments are anchored and that the proper localization of ALP-1 at dense bodies is dependent on alpha-actinin. Our analysis of alp-1 mutants demonstrates that ALP-1 functions to maintain actin filament organization and participates in muscle stabilization during contraction. Reducing alpha-actinin activity enhances the actin filament phenotype of the alp-1 mutants, suggesting that ALP-1 and alpha-actinin function in the same cellular process. Like alpha-actinin, alp-1 also interacts genetically with a connectin/titin family member, ketn-1, to provide mechanical stability for supporting body wall muscle contraction. Taken together, our data demonstrate that ALP-1 and alpha-actinin function together to stabilize actin filaments and promote muscle structural integrity.

  19. Co-occurence of filamentation defects and impaired biofilms in Candida albicans protein kinase mutants.

    PubMed

    Konstantinidou, Nina; Morrissey, John Patrick

    2015-12-01

    Pathogenicity of Candida albicans is linked with its developmental stages, notably the capacity switch from yeast-like to hyphal growth, and to form biofilms on surfaces. To better understand the cellular processes involved in C. albicans development, a collection of 63 C. albicans protein kinase mutants was screened for biofilm formation in a microtitre plate assay. Thirty-eight mutants displayed some degree of biofilm impairment, with 20 categorised as poor biofilm formers. All the poor biofilm formers were also defective in the switch from yeast to hyphae, establishing it as a primary defect. Five genes, VPS15, IME2, PKH3, PGA43 and CEX1, encode proteins not previously reported to influence hyphal development or biofilm formation. Network analysis established that individual components of some processes, most interestingly MAP kinase pathways, are not required for biofilm formation, most likely indicating functional redundancy. Mutants were also screened for their response to bacterial supernatants and it was found that Pseudomonas aeruginosa supernatants inhibited biofilm formation in all mutants, regardless of the presence of homoserine lactones (HSLs). In contrast, Candida morphology was only affected by supernatant containing HSLs. This confirms the distinct HSL-dependent inhibition of filamentation and the HSL-independent impairment of biofilm development by P. aeruginosa.

  20. Engineering the Controlled Assembly of Filamentous Injectisomes in E. coli K-12 for Protein Translocation into Mammalian Cells.

    PubMed

    Ruano-Gallego, David; Álvarez, Beatriz; Fernández, Luis Ángel

    2015-09-18

    Bacterial pathogens containing type III protein secretion systems (T3SS) assemble large needle-like protein complexes in the bacterial envelope, called injectisomes, for translocation of protein effectors into host cells. The application of these "molecular syringes" for the injection of proteins into mammalian cells is hindered by their structural and genomic complexity, requiring multiple polypeptides encoded along with effectors in various transcriptional units (TUs) with intricate regulation. In this work, we have rationally designed the controlled expression of the filamentous injectisomes found in enteropathogenic Escherichia coli (EPEC) in the nonpathogenic strain E. coli K-12. All structural components of EPEC injectisomes, encoded in a genomic island called the locus of enterocyte effacement (LEE), were engineered in five TUs (eLEEs) excluding effectors, promoters and transcriptional regulators. These eLEEs were placed under the control of the IPTG-inducible promoter Ptac and integrated into specific chromosomal sites of E. coli K-12 using a marker-less strategy. The resulting strain, named synthetic injector E. coli (SIEC), assembles filamentous injectisomes similar to those in EPEC. SIEC injectisomes form pores in the host plasma membrane and are able to translocate T3-substrate proteins (e.g., translocated intimin receptor, Tir) into the cytoplasm of HeLa cells reproducing the phenotypes of intimate attachment and polymerization of actin-pedestals elicited by EPEC bacteria. Hence, SIEC strain allows the controlled expression of functional filamentous injectisomes for efficient translocation of proteins with T3S-signals into mammalian cells.

  1. The Respiratory Syncytial Virus Phosphoprotein, Matrix Protein, and Fusion Protein Carboxy-Terminal Domain Drive Efficient Filamentous Virus-Like Particle Formation.

    PubMed

    Meshram, Chetan D; Baviskar, Pradyumna S; Ognibene, Cherie M; Oomens, Antonius G P

    2016-12-01

    Virus-like particles (VLPs) are attractive as a vaccine concept. For human respiratory syncytial virus (hRSV), VLP assembly is poorly understood and appears inefficient. Hence, hRSV antigens are often incorporated into foreign VLP systems to generate anti-RSV vaccine candidates. To better understand the assembly, and ultimately to enable efficient production, of authentic hRSV VLPs, we examined the associated requirements and mechanisms. In a previous analysis in HEp-2 cells, the nucleoprotein (N), phosphoprotein (P), matrix protein (M), and fusion protein (F) were required for formation of filamentous VLPs, which, similar to those of wild-type virus, were associated with the cell surface. Using fluorescence and electron microscopy combined with immunogold labeling, we examined the surfaces of transfected HEp-2 cells and further dissected the process of filamentous VLP formation. Our results show that N is not required. Coexpression of P plus M plus F, but not P plus M, M plus F, or P plus F, induced both viral protein coalescence and formation of filamentous VLPs that resembled wild-type virions. Despite suboptimal coalescence in the absence of P, the M and F proteins, when coexpressed, formed cell surface-associated filaments with abnormal morphology, appearing longer and thinner than wild-type virions. For F, only the carboxy terminus (Fstem) was required, and addition of foreign protein sequences to Fstem allowed incorporation into VLPs. Together, the data show that P, M, and the F carboxy terminus are sufficient for robust viral protein coalescence and filamentous VLP formation and suggest that M-F interaction drives viral filament formation, with P acting as a type of cofactor facilitating the process and exerting control over particle morphology.

  2. Visualization of the endocytic pathway in the filamentous fungus Aspergillus oryzae using an EGFP-fused plasma membrane protein

    SciTech Connect

    Higuchi, Yujiro; Nakahama, Tomoyuki; Shoji, Jun-ya; Arioka, Manabu; Kitamoto, Katsuhiko . E-mail: akitamo@mail.ecc.u-tokyo.ac.jp

    2006-02-17

    Endocytosis is an important process for cellular activities. However, in filamentous fungi, the existence of endocytosis has been so far elusive. In this study, we used AoUapC-EGFP, the fusion protein of a putative uric acid-xanthine permease with enhanced green fluorescent protein (EGFP) in Aspergillus oryzae, to examine whether the endocytic process occurs or not. Upon the addition of ammonium into the medium the fusion protein was internalized from the plasma membrane. The internalization of AoUapC-EGFP was completely blocked by sodium azide, cold, and cytochalasin A treatments, suggesting that the internalization possesses the general features of endocytosis. These results demonstrate the occurrence of endocytosis in filamentous fungi. Moreover, we discovered that the endosomal compartments appeared upon the induction of endocytosis and moved in a microtubule-dependent manner.

  3. Drebrin-like protein DBN-1 is a sarcomere component that stabilizes actin filaments during muscle contraction.

    PubMed

    Butkevich, Eugenia; Bodensiek, Kai; Fakhri, Nikta; von Roden, Kerstin; Schaap, Iwan A T; Majoul, Irina; Schmidt, Christoph F; Klopfenstein, Dieter R

    2015-07-06

    Actin filament organization and stability in the sarcomeres of muscle cells are critical for force generation. Here we identify and functionally characterize a Caenorhabditis elegans drebrin-like protein DBN-1 as a novel constituent of the muscle contraction machinery. In vitro, DBN-1 exhibits actin filament binding and bundling activity. In vivo, DBN-1 is expressed in body wall muscles of C. elegans. During the muscle contraction cycle, DBN-1 alternates location between myosin- and actin-rich regions of the sarcomere. In contracted muscle, DBN-1 is accumulated at I-bands where it likely regulates proper spacing of α-actinin and tropomyosin and protects actin filaments from the interaction with ADF/cofilin. DBN-1 loss of function results in the partial depolymerization of F-actin during muscle contraction. Taken together, our data show that DBN-1 organizes the muscle contractile apparatus maintaining the spatial relationship between actin-binding proteins such as α-actinin, tropomyosin and ADF/cofilin and possibly strengthening actin filaments by bundling.

  4. Phosphorylation of cardiac myosin binding protein C releases myosin heads from the surface of cardiac thick filaments.

    PubMed

    Kensler, Robert W; Craig, Roger; Moss, Richard L

    2017-02-21

    Cardiac myosin binding protein C (cMyBP-C) has a key regulatory role in cardiac contraction, but the mechanism by which changes in phosphorylation of cMyBP-C accelerate cross-bridge kinetics remains unknown. In this study, we isolated thick filaments from the hearts of mice in which the three serine residues (Ser273, Ser282, and Ser302) that are phosphorylated by protein kinase A in the m-domain of cMyBP-C were replaced by either alanine or aspartic acid, mimicking the fully nonphosphorylated and the fully phosphorylated state of cMyBP-C, respectively. We found that thick filaments from the cMyBP-C phospho-deficient hearts had highly ordered cross-bridge arrays, whereas the filaments from the cMyBP-C phospho-mimetic hearts showed a strong tendency toward disorder. Our results support the hypothesis that dephosphorylation of cMyBP-C promotes or stabilizes the relaxed/superrelaxed quasi-helical ordering of the myosin heads on the filament surface, whereas phosphorylation weakens this stabilization and binding of the heads to the backbone. Such structural changes would modulate the probability of myosin binding to actin and could help explain the acceleration of cross-bridge interactions with actin when cMyBP-C is phosphorylated because of, for example, activation of β1-adrenergic receptors in myocardium.

  5. Phosphorylation of cardiac myosin binding protein C releases myosin heads from the surface of cardiac thick filaments

    PubMed Central

    Kensler, Robert W.; Craig, Roger; Moss, Richard L.

    2017-01-01

    Cardiac myosin binding protein C (cMyBP-C) has a key regulatory role in cardiac contraction, but the mechanism by which changes in phosphorylation of cMyBP-C accelerate cross-bridge kinetics remains unknown. In this study, we isolated thick filaments from the hearts of mice in which the three serine residues (Ser273, Ser282, and Ser302) that are phosphorylated by protein kinase A in the m-domain of cMyBP-C were replaced by either alanine or aspartic acid, mimicking the fully nonphosphorylated and the fully phosphorylated state of cMyBP-C, respectively. We found that thick filaments from the cMyBP-C phospho-deficient hearts had highly ordered cross-bridge arrays, whereas the filaments from the cMyBP-C phospho-mimetic hearts showed a strong tendency toward disorder. Our results support the hypothesis that dephosphorylation of cMyBP-C promotes or stabilizes the relaxed/superrelaxed quasi-helical ordering of the myosin heads on the filament surface, whereas phosphorylation weakens this stabilization and binding of the heads to the backbone. Such structural changes would modulate the probability of myosin binding to actin and could help explain the acceleration of cross-bridge interactions with actin when cMyBP-C is phosphorylated because of, for example, activation of β1-adrenergic receptors in myocardium. PMID:28167762

  6. Effect of oxidative stress on the expression of thin filament-associated proteins in gastric smooth muscle cells.

    PubMed

    Al-Shboul, Othman Abdullah; Mustafa, Ayman; Mohammad, Mukhallad; Al-Shehabat, Mustafa; Yousef, Asmaa; Al-Hashimi, Farah

    2014-09-01

    Thin filament-associated proteins such as calponin, caldesmon, and smoothelin are believed to regulate acto-myosin interaction and thus, muscle contraction. Oxidative stress has been found to affect the normal contractile behavior of smooth muscle and is involved in the pathogenesis of a number of human diseases such as diabetes mellitus, hypertension, and atherosclerosis. However, very little is known about the effect of oxidative stress on the expression of smooth muscle contractile proteins. The aim of the current study is to investigate the effect of oxidative stress on the expression of thin filament-associated proteins in rat gastric smooth muscle. Single smooth muscle cells of the stomach obtained from Sprague-Dawley rats were used. Muscle cells were treated with hydrogen peroxide (H2O2) (500 μM) for 30 min or the peroxynitrite donor 3-morpholinosydnonimine (SIN-1) (1 mM) for 90 min to induce oxidative stress. Calponin, caldesmon, and smoothelin expressions were measured via specifically designed enzyme-linked immunosorbent assay. We found that exposure to exogenous H2O2 or incubation of dispersed gastric muscle cells with SIN-1 significantly increased the expression of calponin, caldesmon, and smoothelin proteins. In conclusion: oxidative stress increases the expression of thin filament-associated proteins in gastric smooth muscle, suggesting an important role in gastrointestinal motility disorders associated with oxidative stress.

  7. Quantifying Reversible Oxidation of Protein Thiols in Photosynthetic Organisms

    NASA Astrophysics Data System (ADS)

    Slade, William O.; Werth, Emily G.; McConnell, Evan W.; Alvarez, Sophie; Hicks, Leslie M.

    2015-04-01

    Photosynthetic organisms use dynamic post-translational modifications to survive and adapt, which include reversible oxidative modifications of protein thiols that regulate protein structure, function, and activity. Efforts to quantify thiol modifications on a global scale have relied upon peptide derivatization, typically using isobaric tags such as TMT, ICAT, or iTRAQ that are more expensive, less accurate, and provide less proteome coverage than label-free approaches—suggesting the need for improved experimental designs for studies requiring maximal coverage and precision. Herein, we present the coverage and precision of resin-assisted thiol enrichment coupled to label-free quantitation for the characterization of reversible oxidative modifications on protein thiols. Using C. reinhardtii and Arabidopsis as model systems for algae and plants, we quantified 3662 and 1641 unique cysteinyl peptides, respectively, with median coefficient of variation (CV) of 13% and 16%. Further, our method is extendable for the detection of protein abundance changes and stoichiometries of cysteine oxidation. Finally, we demonstrate proof-of-principle for our method, and reveal that exogenous hydrogen peroxide treatment regulates the C. reinhardtii redox proteome by increasing or decreasing the level of oxidation of 501 or 67 peptides, respectively. As protein activity and function is controlled by oxidative modifications on protein thiols, resin-assisted thiol enrichment coupled to label-free quantitation can reveal how intracellular and environmental stimuli affect plant survival and fitness through oxidative stress.

  8. Determination of reversible protein equilibrium association coefficients using light scattering

    NASA Astrophysics Data System (ADS)

    Larkin, Michael

    2009-03-01

    The characterization in solution of reversible protein associations as well as associations between proteins and small molecules is essential in many areas of science. Understanding cellular function or developing and formulating pharmaceuticals or other biologically active materials often requires quantitation of such associations. Most pharmaceuticals have functionality due solely to association with molecules within the body, and the discovery and accurate characterization of these associations is a key element for pharmaceutical development. Unfortunately, most methods used to measure associations of proteins require either immobilizing the protein on a surface (e.g. surface plasmon resonance), which potentially alters the protein characteristics, or require considerable time and effort and large quantities of sample (e.g. analytical ultracentrifugation, isothermal titration calorimetry). Light scattering based measurements of reversible association coefficients require much less sample and may be performed much more rapidly than other free solution techniques. In this talk I describe how static and dynamic light scattering may each independently be used to measure equilibrium association coefficients between proteins in free solution, and may also be used to observe and quantitate the association of small molecules with them. I present background theory for both static and dynamic light scattering measurements of equilibrium associations, and examples of measurements made of both model systems and of systems with commercial relevance in the pharmaceutical industry.

  9. Chemoenzymatic Reversible Immobilization and Labeling of Proteins without Prior Purification

    PubMed Central

    Rashidian, Mohammad; Song, James M.; Pricer, Rachel E.; Distefano, Mark D.

    2012-01-01

    Site-specific chemical modification of proteins is important for many applications in biology and biotechnology. Recently, our laboratory and others have exploited the high specificity of the enzyme protein farnesyltransferase (PFTase) to site-specifically modify proteins through the use of alternative substrates that incorporate bioorthogonal functionality including azides and alkynes. In this study, we evaluate two aldehyde-containing molecules as substrates for PFTase and as reactants in both oxime and hydrazone formation. Using green fluorescent protein (GFP) as a model system, we demonstrate that the purified protein can be enzymatically modified with either analogue to yield aldehyde-functionalized proteins. Oxime or hydrazone formation was then employed to immobilize, fluorescently label or PEGylate the resulting aldehyde-containing proteins. Immobilization via hydrazone formation was also shown to be reversible via transoximization with a fluorescent alkoxyamine. After characterizing this labeling strategy using pure protein, the specificity of the enzymatic process was used to selectively label GFP present in crude E. coli extract followed by capture of the aldehyde-modified protein using hydrazide-agarose. Subsequent incubation of the immobilized protein using a fluorescently labeled or PEGylated alkoxyamine resulted in the release of pure GFP containing the desired site-specific covalent modifications. This procedure was also employed to produce PEGylated glucose-dependent insulinotropic polypeptide (GIP), a protein with potential therapeutic activity for diabetes. Given the specificity of the PFTase-catalyzed reaction coupled with the ability to introduce a CAAX-box recognition sequence onto almost any protein, this method shows great potential as a general approach for the selective immobilization and labeling of recombinant proteins present in crude cellular extract without prior purification. Beyond generating site-specifically modified proteins

  10. Chemoenzymatic reversible immobilization and labeling of proteins without prior purification.

    PubMed

    Rashidian, Mohammad; Song, James M; Pricer, Rachel E; Distefano, Mark D

    2012-05-23

    Site-specific chemical modification of proteins is important for many applications in biology and biotechnology. Recently, our laboratory and others have exploited the high specificity of the enzyme protein farnesyltransferase (PFTase) to site-specifically modify proteins through the use of alternative substrates that incorporate bioorthogonal functionality including azides and alkynes. In this study, we evaluate two aldehyde-containing molecules as substrates for PFTase and as reactants in both oxime and hydrazone formation. Using green fluorescent protein (GFP) as a model system, we demonstrate that the purified protein can be enzymatically modified with either analogue to yield aldehyde-functionalized proteins. Oxime or hydrazone formation was then employed to immobilize, fluorescently label, or PEGylate the resulting aldehyde-containing proteins. Immobilization via hydrazone formation was also shown to be reversible via transoximization with a fluorescent alkoxyamine. After characterizing this labeling strategy using pure protein, the specificity of the enzymatic process was used to selectively label GFP present in crude E. coli extract followed by capture of the aldehyde-modified protein using hydrazide-agarose. Subsequent incubation of the immobilized protein using a fluorescently labeled or PEGylated alkoxyamine resulted in the release of pure GFP containing the desired site-specific covalent modifications. This procedure was also employed to produce PEGylated glucose-dependent insulinotropic polypeptide (GIP), a protein with potential therapeutic activity for diabetes. Given the specificity of the PFTase-catalyzed reaction coupled with the ability to introduce a CAAX-box recognition sequence onto almost any protein, this method shows great potential as a general approach for selective immobilization and labeling of recombinant proteins present in crude cellular extract without prior purification. Beyond generating site-specifically modified proteins, this

  11. Correlation of expression of the actin filament-bundling protein espin with stereociliary bundle formation in the developing inner ear.

    PubMed

    Li, Huawei; Liu, Hong; Balt, Steve; Mann, Sabine; Corrales, C Eduardo; Heller, Stefan

    2004-01-01

    The vertebrate hair cell is named for its stereociliary bundle or hair bundle that protrudes from the cell's apical surface. Hair bundles mediate mechanosensitivity, and their highly organized structure plays a critical role in mechanoelectrical transduction and amplification. The prototypical hair bundle is composed of individual stereocilia, 50-300 in number, depending on the animal species and on the type of hair cell. The assembly of stereocilia, in particular, the formation during development of individual rows of stereocilia with descending length, has been analyzed in great morphological detail. Electron microscopic studies have demonstrated that stereocilia are filled with actin filaments that are rigidly cross-linked. The growth of individual rows of stereocilia is associated with the addition of actin filaments and with progressively increasing numbers of cross-bridges between actin filaments. Recently, a mutation in the actin filament-bundling protein espin has been shown to underlie hair bundle degeneration in the deaf jerker mouse, subsequently leading to deafness. Our study was undertaken to investigate the appearance and developmental expression of espin in chicken inner ear sensory epithelia. We found that the onset of espin expression correlates with the initiation and growth of stereocilia bundles in vestibular and cochlear hair cells. Intense espin immunolabeling of stereocilia was colocalized with actin filament staining in all types of hair cells at all developmental stages and in adult animals. Our analysis of espin as a molecular marker for actin filament cross-links in stereocilia is in full accordance with previous morphological studies and implicates espin as an important structural component of hair bundles from initiation of bundle assembly to mature chicken hair cells.

  12. Disruption of the keratin filament network during epithelial cell division.

    PubMed Central

    Lane, E B; Goodman, S L; Trejdosiewicz, L K

    1982-01-01

    The behaviour of keratin filaments during cell division was examined in a wide range of epithelial lines from several species. Almost half of them show keratin disruption as described previously: by immunofluorescence, filaments are replaced during mitosis by a 'speckled' pattern of discrete cytoplasmic dots. In the electron microscope these ' speckles ' are seen as granules around the cell periphery, just below the actin cortical mesh, with no detectable 10 nm filament structure inside them and no keratin filament bundles in the rest of the cytoplasm. A time course of the filament reorganization was constructed from double immunofluorescence data; filaments are disrupted in prophase, and the filament network is intact again by cytokinesis. The phenomenon is restricted to cells rich in keratin filaments, such as keratinocytes; it is unrelated to the co-existence of vimentin in many of these cells, and vimentin is generally maintained as filaments while the keratin is restructured. Some resistance to the effect may be conferred by an extended cycle time. Filament reorganization takes place within minutes, so that a reversible mechanism seems more likely than one involving de novo protein synthesis, at this metabolically quiet stage of the cell cycle. Images Fig. 1. Fig. 2. Fig. 3. Fig. 4. Fig. 5. Fig. 6. PMID:6202508

  13. The zinc cluster proteins Upc2 and Ecm22 promote filamentation in Saccharomyces cerevisiae by sterol biosynthesis-dependent and -independent pathways.

    PubMed

    Woods, Kelly; Höfken, Thomas

    2016-02-01

    The transition between a unicellular yeast form to multicellular filaments is crucial for budding yeast foraging and the pathogenesis of many fungal pathogens such as Candida albicans. Here, we examine the role of the related transcription factors Ecm22 and Upc2 in Saccharomyces cerevisiae filamentation. Overexpression of either ECM22 or UPC2 leads to increased filamentation, whereas cells lacking both ECM22 and UPC2 do not exhibit filamentous growth. Ecm22 and Upc2 positively control the expression of FHN1, NPR1, PRR2 and sterol biosynthesis genes. These genes all play a positive role in filamentous growth, and their expression is upregulated during filamentation in an Ecm22/Upc2-dependent manner. Furthermore, ergosterol content increases during filamentous growth. UPC2 expression also increases during filamentation and is inhibited by the transcription factors Sut1 and Sut2. The expression of SUT1 and SUT2 in turn is under negative control of the transcription factor Ste12. We suggest that during filamentation Ste12 becomes activated and reduces SUT1/SUT2 expression levels. This would result in increased UPC2 levels and as a consequence to transcriptional activation of FHN1, NPR1, PRR2 and sterol biosynthesis genes. Higher ergosterol levels in combination with the proteins Fhn1, Npr1 and Prr2 would then mediate the transition to filamentous growth.

  14. Unexpected expression of intermediate filament protein genes in human oligodendroglioma cell lines

    SciTech Connect

    Kashima, Tsuyoshi; Vinters, H.V.; Campagnoni, A.T.

    1995-01-01

    From a human oligodendroglioma cell line cDNA library, ten intermediate filament (IF) cDNA clones were isolated. Five clones corresponded to vimentin mRNA, two corresponded to cytokeratin K7 mRNA, and two corresponded to cytokeratin K8 mRNA. One clone encoded a novel IF mRNA. The expression of these and other IF protein genes was examined in five cell lines derived from human oligodendroglioma, astrocytoma and neuroblastoma tumors. Vimentin mRNA and K18 mRNA were expressed in all the cell lines. The K7 and K8 genes were expressed only in the oligodendroglioma cell lines. Surprisingly, nestin mRNA was expressed in the astrocytoma lines and the neuroblastoma line, but was not expressed in the oligodendroglioma lines. These results indicate that oligodendroglioma cell lines express Types I and II cytokeratin genes. This pattern of IF gene expression was different from that of the astrocytoma and neuroblastoma cell lines, which expressed IF genes usually associated with the mature cell types or with differentiating fetal neural precursor cells, i.e. GFAP and neurofilament-L. The results also suggest that the oligodendroglioma cell lines are more epithelial in character and do not reflect the gene expression of mature oligodendrocytes. 46 refs., 8 figs., 2 tabs.

  15. Actin filament organization in activated mast cells is regulated by heterotrimeric and small GTP-binding proteins

    PubMed Central

    1994-01-01

    Rat peritoneal mast cells, both intact and permeabilized, have been used widely as model secretory cells. GTP-binding proteins and calcium play a major role in controlling their secretory response. Here we have examined changes in the organization of actin filaments in intact mast cells after activation by compound 48/80, and in permeabilized cells after direct activation of GTP-binding proteins by GTP-gamma-S. In both cases, a centripetal redistribution of cellular F-actin was observed: the content of F-actin was reduced in the cortical region and increased in the cell interior. The overall F-actin content was increased. Using permeabilized cells, we show that AIF4-, an activator of heterotrimeric G proteins, induces the disassembly of F-actin at the cortex, while the appearance of actin filaments in the interior of the cell is dependent on two small GTPases, rho and rac. Rho was found to be responsible for de novo actin polymerization, presumably from a membrane-bound monomeric pool, while rac was required for an entrapment of the released cortical filaments. Thus, a heterotrimeric G-protein and the small GTPases, rho and rac, participate in affecting the changes in the actin cytoskeleton observed after activation of mast cells. PMID:8051203

  16. Precocious expression of NAPA-73, an intermediate filament-associated protein, during nervous system and heart development in the chicken embryo.

    PubMed

    Ciment, G

    1990-01-01

    A monoclonal antibody was generated, against early neural crest-derived cells, which recognizes an epitope present on a novel intermediate filament-associated protein. This protein has been named NAPA-73 and is expressed by progenitor cells of the nervous system and heart. Biochemical and ultrastructural studies indicate that this protein associates with bundles of intermediate filaments and therefore may play a role in the determination of cell shape.

  17. Structural and Functional Studies of H. seropedicae RecA Protein – Insights into the Polymerization of RecA Protein as Nucleoprotein Filament

    PubMed Central

    Galvão, Carolina W.; Saab, Sérgio C.; Iulek, Jorge; Etto, Rafael M.; Steffens, Maria B. R.; Chitteni-Pattu, Sindhu; Stanage, Tyler; Keck, James L.; Cox, Michael M.

    2016-01-01

    The bacterial RecA protein plays a role in the complex system of DNA damage repair. Here, we report the functional and structural characterization of the Herbaspirillum seropedicae RecA protein (HsRecA). HsRecA protein is more efficient at displacing SSB protein from ssDNA than Escherichia coli RecA protein. HsRecA also promotes DNA strand exchange more efficiently. The three dimensional structure of HsRecA-ADP/ATP complex has been solved to 1.7 Å resolution. HsRecA protein contains a small N-terminal domain, a central core ATPase domain and a large C-terminal domain, that are similar to homologous bacterial RecA proteins. Comparative structural analysis showed that the N-terminal polymerization motif of archaeal and eukaryotic RecA family proteins are also present in bacterial RecAs. Reconstruction of electrostatic potential from the hexameric structure of HsRecA-ADP/ATP revealed a high positive charge along the inner side, where ssDNA is bound inside the filament. The properties of this surface may explain the greater capacity of HsRecA protein to bind ssDNA, forming a contiguous nucleoprotein filament, displace SSB and promote DNA exchange relative to EcRecA. Our functional and structural analyses provide insight into the molecular mechanisms of polymerization of bacterial RecA as a helical nucleoprotein filament. PMID:27447485

  18. Structural and Functional Studies of H. seropedicae RecA Protein – Insights into the Polymerization of RecA Protein as Nucleoprotein Filament

    SciTech Connect

    Leite, Wellington C.; Galvão, Carolina W.; Saab, Sérgio C.; Iulek, Jorge; Etto, Rafael M.; Steffens, Maria B. R.; Chitteni-Pattu, Sindhu; Stanage, Tyler; Keck, James L.; Cox, Michael M.; Spies, Maria

    2016-07-22

    The bacterial RecA protein plays a role in the complex system of DNA damage repair. Here, we report the functional and structural characterization of the Herbaspirillum seropedicae RecA protein (HsRecA). HsRecA protein is more efficient at displacing SSB protein from ssDNA than Escherichia coli RecA protein. HsRecA also promotes DNA strand exchange more efficiently. The three dimensional structure of HsRecA-ADP/ATP complex has been solved to 1.7 Å resolution. HsRecA protein contains a small N-terminal domain, a central core ATPase domain and a large C-terminal domain, that are similar to homologous bacterial RecA proteins. Comparative structural analysis showed that the N-terminal polymerization motif of archaeal and eukaryotic RecA family proteins are also present in bacterial RecAs. Reconstruction of electrostatic potential from the hexameric structure of HsRecA-ADP/ATP revealed a high positive charge along the inner side, where ssDNA is bound inside the filament. The properties of this surface may explain the greater capacity of HsRecA protein to bind ssDNA, forming a contiguous nucleoprotein filament, displace SSB and promote DNA exchange relative to EcRecA. In conclusion, our functional and structural analyses provide insight into the molecular mechanisms of polymerization of bacterial RecA as a helical nucleoprotein filament.

  19. Expression of the intermediate filament protein synemin in myofibrillar myopathies and other muscle diseases.

    PubMed

    Olivé, Montse; Goldfarb, Lev; Dagvadorj, Ayush; Sambuughin, Nyamkhishig; Paulin, Denise; Li, Zhenlin; Goudeau, Bertrand; Vicart, Patrick; Ferrer, Isidro

    2003-07-01

    Synemin is a member of the intermediate protein superfamily. Previous studies in avian and rodent skeletal and cardiac muscles have demonstrated that synemin localises at the Z-band, where it associates with desmin and alpha-actinin. In the present study, the distribution of synemin was examined using immunohistochemistry in muscle biopsy specimens from patients suffering from myofibrillar myopathy (MM, n=6), dermatomyositis (DM, n=3), inclusion body myositis (IBM, n=5), oculopharyngeal muscular dystrophy (OPD, n=3) and denervation atrophy (DA, n=3), to investigate the possible participation of this protein in the pathogenesis of various muscular diseases. Of patients affected by MM, two showed the presence of mutations in the desmin gene; none had mutations in the alphaB-crystallin gene; and no mutations were identified in synemin or syncoilin genes of three patients. Synemin immunohistochemistry disclosed a faint staining corresponding to the Z-bands in the cytoplasm of control muscle fibres; in contrast, focal aggregates of synemin were seen in patients with MM. Increased synemin immunoreactivity was identified diffusely or in the subsarcolemmal space of scattered fibres in patients with DM, and in vacuolated fibres of patients with IBM and OPD. Strong synemin immunoreactivity was observed in target formations and atrophic fibres of patients with denervating disorders, as well as in atrophic fibres, regardless of their origin, in all patients studied. Synemin co-localised with desmin, as seen on consecutive serial sections immunostained with anti-synemin or anti-desmin antibodies. These observations demonstrate abnormal accumulations containing both synemin and desmin in muscle fibres in patients with MM, IBM, DM, OPD and DA. Considering the important role of synemin as one of intermediate filaments of skeletal and cardiac muscle, its destruction and accumulation in the intracellular debris suggest that synemin may participate in the pathogenesis of these

  20. Characterization of the Filamentous Hemagglutinin-Like Protein FhaS in Bordetella bronchiseptica

    PubMed Central

    Julio, Steven M.; Cotter, Peggy A.

    2005-01-01

    Filamentous hemagglutinin (FHA) is a large (>200 kDa), rod-shaped protein expressed by bordetellae that is both surface-associated and secreted. FHA mediates bacterial adherence to epithelial cells and macrophages in vitro and is absolutely required for tracheal colonization in vivo. The recently sequenced Bordetella bronchiseptica genome revealed the presence of a gene, fhaS, that is nearly identical to fhaB, the FHA structural gene. We show that although fhaS expression requires the BvgAS virulence control system, it is maximal only under a subset of conditions in which BvgAS is active, suggesting an additional level of regulation. We also show that, like FHA, FhaS undergoes a C-terminal proteolytic processing event and is both surface-associated and secreted and that export across the outer membrane requires the channel-forming protein FhaC. Unlike FHA, however, FhaS was unable to mediate adherence of B. bronchiseptica to epithelial cell lines in vitro and was not required for respiratory tract colonization in vivo. In a coinfection experiment, a ΔfhaS strain was out-competed by wild-type B. bronchiseptica, indicating that fhaS is expressed in vivo and that FhaS contributes to bacterial fitness in a manner revealed when the mutant must compete with wild-type bacteria. These data suggest that FHA and FhaS perform distinct functions during the Bordetella infectious cycle. A survey of various Bordetella strains revealed two distinct fhaS alleles that segregate according to pathogen host range and that B. parapertussishu most likely acquired its fhaS allele from B. pertussis horizontally, suggesting fhaS may contribute to host-species specificity. PMID:16041011

  1. Reconstitution of a prokaryotic minus end-tracking system using TubRC centromeric complexes and tubulin-like protein TubZ filaments

    PubMed Central

    Fink, Gero; Löwe, Jan

    2015-01-01

    Segregation of DNA is a fundamental process during cell division. The mechanism of prokaryotic DNA segregation is largely unknown, but several low-copy-number plasmids encode cytomotive filament systems of the actin type and tubulin type important for plasmid inheritance. Of these cytomotive filaments, only actin-like systems are mechanistically well characterized. In contrast, the mechanism by which filaments of tubulin-like TubZ protein mediate DNA motility is unknown. To understand polymer-driven DNA transport, we reconstituted the filaments of TubZ protein (TubZ filaments) from Bacillus thuringiensis pBtoxis plasmid with their centromeric TubRC complexes containing adaptor protein TubR and tubC DNA. TubZ alone assembled into polar filaments, which annealed laterally and treadmilled. Using single-molecule imaging, we show that TubRC complexes were not pushed by filament polymerization; instead, they processively tracked shrinking, depolymerizing minus ends. Additionally, the TubRC complex nucleated TubZ filaments and allowed for treadmilling. Overall, our results indicate a pulling mechanism for DNA transport by the TubZRC system. The discovered minus end-tracking property of the TubRC complex expands the mechanistic diversity of the prokaryotic cytoskeleton. PMID:25825718

  2. Heme-induced contractile dysfunction in human cardiomyocytes caused by oxidant damage to thick filament proteins.

    PubMed

    Alvarado, Gerardo; Jeney, Viktória; Tóth, Attila; Csősz, Éva; Kalló, Gergő; Huynh, An T; Hajnal, Csaba; Kalász, Judit; Pásztor, Enikő T; Édes, István; Gram, Magnus; Akerström, Bo; Smith, Ann; Eaton, John W; Balla, György; Papp, Zoltán; Balla, József

    2015-12-01

    Intracellular free heme predisposes to oxidant-mediated tissue damage. We hypothesized that free heme causes alterations in myocardial contractility via disturbed structure and/or regulation of the contractile proteins. Isometric force production and its Ca(2+)-sensitivity (pCa50) were monitored in permeabilized human ventricular cardiomyocytes. Heme exposure altered cardiomyocyte morphology and evoked robust decreases in Ca(2+)-activated maximal active force (Fo) while increasing Ca(2+)-independent passive force (F passive). Heme treatments, either alone or in combination with H2O2, did not affect pCa50. The increase in F passive started at 3 µM heme exposure and could be partially reversed by the antioxidant dithiothreitol. Protein sulfhydryl (SH) groups of thick myofilament content decreased and sulfenic acid formation increased after treatment with heme. Partial restoration in the SH group content was observed in a protein running at 140 kDa after treatment with dithiothreitol, but not in other proteins, such as filamin C, myosin heavy chain, cardiac myosin binding protein C, and α-actinin. Importantly, binding of heme to hemopexin or alpha-1-microglobulin prevented its effects on cardiomyocyte contractility, suggesting an allosteric effect. In line with this, free heme directly bound to myosin light chain 1 in human cardiomyocytes. Our observations suggest that free heme modifies cardiac contractile proteins via posttranslational protein modifications and via binding to myosin light chain 1, leading to severe contractile dysfunction. This may contribute to systolic and diastolic cardiac dysfunctions in hemolytic diseases, heart failure, and myocardial ischemia-reperfusion injury.

  3. Targeting Protein Kinases to Reverse Multidrug Resistance in Sarcoma

    PubMed Central

    Chen, Hua; Shen, Jacson; Choy, Edwin; Hornicek, Francis J.; Duan, Zhenfeng

    2015-01-01

    Sarcomas are a group of cancers that arise from transformed cells of mesenchymal origin. They can be classified into over 50 subtypes, accounting for approximately 1% of adult and 15% of pediatric cancers. Wide surgical resection, radiotherapy, and chemotherapy are the most common treatments for the majority of sarcomas. Among these therapies, chemotherapy can palliate symptoms and prolong life for some sarcoma patients. However, sarcoma cells can have intrinsic or acquired resistance after treatment with chemotherapeutics drugs, leading to the development of multidrug resistance (MDR). MDR attenuates the efficacy of anticancer drugs and results in treatment failure for sarcomas. Therefore, overcoming MDR is an unmet need for sarcoma therapy. Certain protein kinases demonstrate aberrant expression and/or activity in sarcoma cells, which have been found to be involved in the regulation of sarcoma cell progression, such as cell cycle, apoptosis, and survival. Inhibiting these protein kinases may not only decrease the proliferation and growth of sarcoma cells, but also reverse their resistance to chemotherapeutic drugs to subsequently reduce the doses of anticancer drugs and decrease drug side-effects. The discovery of novel strategies targeting protein kinases opens a door to a new area of sarcoma research and provides insight into the mechanisms of MDR in chemotherapy. This review will focus on the recent studies in targeting protein kinase to reverse chemotherapeutic drug resistance in sarcoma. PMID:26827688

  4. Reversible binding kinetics of a cytoskeletal protein at the erythrocyte submembrane.

    PubMed Central

    Stout, A. L.; Axelrod, D.

    1994-01-01

    Reversible binding among components of the cellular submembrane cytoskeleton and reversible binding of some of these components with the plasma membrane likely play a role in nonelastic morphological changes and mechanoplastic properties of cells. However, relatively few studies have been devoted to investigating directly the kinetic aspects of the interactions of individual components of the membrane skeleton with the membrane. The experiments described here investigated whether one component of the erythrocyte membrane cytoskeleton, protein 4.1, binds to its sites on the membrane reversibly and if so, whether the different 4.1-binding sites display distinct kinetic behavior. Protein 4.1 is known to stabilize the membrane and to mediate the attachment of spectrin filaments to the membrane. Protein 4.1 previously has been shown to bind to integral membrane proteins band 3, glycophorin C, and to negatively charged phospholipids. To examine the kinetic rates of dissociation of carboxymethyl fluorescein-labeled 4.1 (CF-4.1) to the cytofacial surface of erythrocyte membrane, a special preparation of hemolyzed erythrocyte ghosts was used, in which the ghosts became flattened on a glass surface and exposed their cytofacial surfaces to the solution through a membrane rip in a distinctive characteristic pattern. This preparation was examined by the microscopy technique of total internal reflection/fluorescence recovery after photobleaching (TIR/FRAP). Four different treatments were employed to help identify which membrane binding sites gave rise to the multiplicity of observed kinetic rates. The first treatment, the control, stripped off the native spectrin, actin, 4.1, and ankyrin. About 60% of the CF-4.1 bound to this control binded irreversibly (dissociation time > 20 min), but the remaining approximately 40% binded reversibly with a range of residency times averaging approximately 3 s. The second treatment subjected these stripped membranes to trypsin, which presumably

  5. Use of a fusion protein between GFP and an actin-binding domain to visualize transient filamentous-actin structures.

    PubMed

    Pang, K M; Lee, E; Knecht, D A

    1998-03-26

    Many important processes in eukaryotic cells involve changes in the quantity, location and the organization of actin filaments [1] [2] [3]. We have been able to visualize these changes in live cells using a fusion protein (GFP-ABD) comprising the green fluorescent protein (GFP) of Aequorea victoria and the 25 kDa highly conserved actin-binding domain (ABD) from the amino terminus of the actin cross-linking protein ABP-120 [4]. In live cells of the soil amoeba Dictyostelium that were expressing GFP-ABD, the three-dimensional architecture of the actin cortex was clearly visualized. The pattern of GFP-ABD fluorescence in these cells coincided with that of rhodamine-phalloidin, indicating that GFP-ABD specifically binds filamentous (F) actin. On the ventral surface of non-polarized vegetative cells, a broad ring of F actin periodically assembled and contracted, whereas in polarized cells there were transient punctate F-actin structures; cells cycled between the polarized and non-polarized morphologies. During the formation of pseudopods, an increase in fluorescence intensity coincided with the initial outward deformation of the membrane. This is consistent with the models of pseudopod extension that predict an increase in the local density of actin filaments. In conclusion, GFP-ABD specifically binds F actin and allows the visualization of F-actin dynamics and cellular behavior simultaneously.

  6. SEC/reversed-phase separation of E. coli proteins.

    PubMed

    Apffell, Alex

    2010-05-01

    Although many chromatographic modes can be coupled for the multidimensional separation of a complex mixture, a very favorable combination is that of size-exclusion chromatography (SEC) and reversed-phase chromatography. The separation mechanisms are largely orthogonal and the mobile phases are compatible. The use of a retentive second dimension allows trapping of specific fractions from the SEC separation and transferring them to the reversed-phase separation. One of the advantages of multidimensional chromatography is that it is scalable in terms of automation. In a completely manual system, fractions eluting from a first dimension can be manually collected and injected into a second separation dimension. In an automated system, fraction transfer can be accomplished through automated valving. The following protocol illustrates this approach, with both a manual method and an external column switching method integrated into an automated high-performance liquid chromatography (HPLC) method. As an illustration, soluble proteins from Escherichia coli are separated.

  7. Protein separation using affinity-based reversed micelles

    PubMed

    Sun; Gu; Tong; Bai; Ichikawa; Furusaki

    1999-05-01

    Reversed micellar two-phase extraction is a developing technique for protein separation. Introduction of an affinity ligand is considered to be an effective approach to increase the selectivity and capacity of reversed micelles. In this article, Cibacron Blue F3G-A (CB) as an affinity ligand was immobilized to reversed micelles composed of soybean lecithin by a two-phase reaction. The affinity partitioning of lysozyme and bovine serum albumin (BSA) to the CB-lecithin micelles was studied. Formation of mixed micelles by additionally introducing a nonionic surfactant, Tween 85, to the CB-lecithin micelles was effective to increase the solubilization of lysozyme due to the increase of W0 (water/surfactant molar ratio)/micellar size. The partitioning isotherms of lysozyme to the CB-lecithin micelles with and without Tween 85 were expressed by the Langmuir equation. The dissociation constants in the Langmuir equation decreased on addition of Tween 85, indicating the increase of the effectiveness of lysozyme binding to the immobilized CB. On addition of 20 g/L Tween 85 to 50 g/L lecithin/hexane micellar phase containing 0.1 mmol/L CB, the extraction capacity for lysozyme could be increased by 42%. Moreover, the CB-lecithin micelles with or without Tween 85 showed significant size exclusion for BSA due to its high molecular weight. Thus, lysozyme and BSA were separated from artificial solutions containing the two proteins. In addition, the affinity-based reversed micellar phase containing Tween 85 was recycled three times for lysozyme purification from crude egg-white solutions. Lysozyme purity increased by 16-18-fold, reaching 60-70% in the recycled use.

  8. Self-Oligomerizing Structure of the Flagellar Cap Protein FliD and Its Implication in Filament Assembly.

    PubMed

    Song, Wan Seok; Cho, So Yeon; Hong, Ho Jeong; Park, Sun Cheol; Yoon, Sung-Il

    2017-03-24

    FliD is a self-oligomerizing structural protein that caps the growing end of the bacterial flagellar filament. FliD also plays a key role in the flagellar system by continuously adding a new flagellin protein to the tip of the filament. To structurally characterize FliD oligomerization and to provide a FliD-mediated flagellin polymerization mechanism, we have determined the crystal structures of FliD proteins from Escherichia coli and Salmonella enterica serovar Typhimurium (ecFliD and stFliD, respectively). ecFliD consists of three domains (D1, D2, and D3) and forms a hexamer plate of the D2 and D3 domains that resembles a six-pointed star with legs consisting of the D1 domain. In contrast, the D2 and D3 domains of stFliD assemble into a pentamer as a five-pointed star plate. Despite their distinct oligomeric states, ecFliD and stFliD engage a common molecular surface for oligomerization. FliD also features interdomain and intersubunit flexibility, suggesting that FliD reorganizes its domains and adjacent subunits depending on the FliD binding partner. The similarity of the FliD shape to flagellin and the structural dynamics of FliD led us to propose a FliD-catalyzed filament elongation mechanism. In this model, FliD occupies a position in place of a nascent flagellin until the flagellin reaches the growing end of the filament, and then, FliD moves aside to repeat the positional replacement.

  9. Engineering the Controlled Assembly of Filamentous Injectisomes in E. coli K-12 for Protein Translocation into Mammalian Cells

    PubMed Central

    2015-01-01

    Bacterial pathogens containing type III protein secretion systems (T3SS) assemble large needle-like protein complexes in the bacterial envelope, called injectisomes, for translocation of protein effectors into host cells. The application of these “molecular syringes” for the injection of proteins into mammalian cells is hindered by their structural and genomic complexity, requiring multiple polypeptides encoded along with effectors in various transcriptional units (TUs) with intricate regulation. In this work, we have rationally designed the controlled expression of the filamentous injectisomes found in enteropathogenic Escherichia coli (EPEC) in the nonpathogenic strain E. coli K-12. All structural components of EPEC injectisomes, encoded in a genomic island called the locus of enterocyte effacement (LEE), were engineered in five TUs (eLEEs) excluding effectors, promoters and transcriptional regulators. These eLEEs were placed under the control of the IPTG-inducible promoter Ptac and integrated into specific chromosomal sites of E. coli K-12 using a marker-less strategy. The resulting strain, named synthetic injector E. coli (SIEC), assembles filamentous injectisomes similar to those in EPEC. SIEC injectisomes form pores in the host plasma membrane and are able to translocate T3-substrate proteins (e.g., translocated intimin receptor, Tir) into the cytoplasm of HeLa cells reproducing the phenotypes of intimate attachment and polymerization of actin-pedestals elicited by EPEC bacteria. Hence, SIEC strain allows the controlled expression of functional filamentous injectisomes for efficient translocation of proteins with T3S-signals into mammalian cells. PMID:26017572

  10. Reversible lysine modification on proteins by using functionalized boronic acids.

    PubMed

    Cal, Pedro M S D; Frade, Raquel F M; Cordeiro, Carlos; Gois, Pedro M P

    2015-05-26

    Iminoboronates have been utilized to successfully install azide and alkyne bioorthogonal functions on proteins, which may then be further reacted with their bioorthogonal counterparts. These constructs were also used to add polyethylene glycol (PEG) to insulin, a modification which has been shown to be reversible in the presence of fructose. Finally, iminoboronates were used to assemble a folic acid/paclitaxel small-molecule/drug conjugate in situ with an IC50  value of 20.7 nM against NCI-H460 cancer cells and negligible cytotoxicity against the CRL-1502 noncancer cells.

  11. Exosome engineering for efficient intracellular delivery of soluble proteins using optically reversible protein-protein interaction module.

    PubMed

    Yim, Nambin; Ryu, Seung-Wook; Choi, Kyungsun; Lee, Kwang Ryeol; Lee, Seunghee; Choi, Hojun; Kim, Jeongjin; Shaker, Mohammed R; Sun, Woong; Park, Ji-Ho; Kim, Daesoo; Heo, Won Do; Choi, Chulhee

    2016-07-22

    Nanoparticle-mediated delivery of functional macromolecules is a promising method for treating a variety of human diseases. Among nanoparticles, cell-derived exosomes have recently been highlighted as a new therapeutic strategy for the in vivo delivery of nucleotides and chemical drugs. Here we describe a new tool for intracellular delivery of target proteins, named 'exosomes for protein loading via optically reversible protein-protein interactions' (EXPLORs). By integrating a reversible protein-protein interaction module controlled by blue light with the endogenous process of exosome biogenesis, we are able to successfully load cargo proteins into newly generated exosomes. Treatment with protein-loaded EXPLORs is shown to significantly increase intracellular levels of cargo proteins and their function in recipient cells in vitro and in vivo. These results clearly indicate the potential of EXPLORs as a mechanism for the efficient intracellular transfer of protein-based therapeutics into recipient cells and tissues.

  12. Thermostability and reversibility of silver nanoparticle-protein binding.

    PubMed

    Wang, Bo; Seabrook, Shane A; Nedumpully-Govindan, Praveen; Chen, Pengyu; Yin, Hong; Waddington, Lynne; Epa, V Chandana; Winkler, David A; Kirby, Jason K; Ding, Feng; Ke, Pu Chun

    2015-01-21

    The interactions between nanoparticles (NPs) and proteins in living systems are a precursor to the formation of a NP-protein "corona" that underlies cellular and organism responses to nanomaterials. However, the thermodynamic properties and reversibility of NP-protein interactions have rarely been examined. Using an automated, high-throughput and temperature-controlled dynamic light scattering (DLS) technique we observed a distinct hysteresis in the hydrodynamic radius of branched polyethyleneimine (BPEI) coated-silver nanoparticles (bAgNPs) exposed to like-charged lysozyme during the processes of heating and cooling, in contrast to the irreversible interactions between bAgNPs and oppositely charged alpha lactalbumin (ALact). Our discrete molecular dynamics (DMD) simulations offered a new molecular insight into the differential structure, dynamics and thermodynamics of bAgNPs binding with the two protein homologs and further revealed the different roles of the capping agents of citrate and BPEI in NP-protein interactions. This study facilitates our understanding of the transformation of nanomaterials in living systems, whose implications range from the field study of nanotoxicology to nanomaterials synthesis, nanobiotechnology and nanomedicine.

  13. Viral Macro Domains Reverse Protein ADP-Ribosylation

    PubMed Central

    Li, Changqing; Debing, Yannick; Jankevicius, Gytis; Neyts, Johan; Ahel, Ivan

    2016-01-01

    ABSTRACT ADP-ribosylation is a posttranslational protein modification in which ADP-ribose is transferred from NAD+ to specific acceptors to regulate a wide variety of cellular processes. The macro domain is an ancient and highly evolutionarily conserved protein domain widely distributed throughout all kingdoms of life, including viruses. The human TARG1/C6orf130, MacroD1, and MacroD2 proteins can reverse ADP-ribosylation by acting on ADP-ribosylated substrates through the hydrolytic activity of their macro domains. Here, we report that the macro domain from hepatitis E virus (HEV) serves as an ADP-ribose-protein hydrolase for mono-ADP-ribose (MAR) and poly(ADP-ribose) (PAR) chain removal (de-MARylation and de-PARylation, respectively) from mono- and poly(ADP)-ribosylated proteins, respectively. The presence of the HEV helicase in cis dramatically increases the binding of the macro domain to poly(ADP-ribose) and stimulates the de-PARylation activity. Abrogation of the latter dramatically decreases replication of an HEV subgenomic replicon. The de-MARylation activity is present in all three pathogenic positive-sense, single-stranded RNA [(+)ssRNA] virus families which carry a macro domain: Coronaviridae (severe acute respiratory syndrome coronavirus and human coronavirus 229E), Togaviridae (Venezuelan equine encephalitis virus), and Hepeviridae (HEV), indicating that it might be a significant tropism and/or pathogenic determinant. IMPORTANCE Protein ADP-ribosylation is a covalent posttranslational modification regulating cellular protein activities in a dynamic fashion to modulate and coordinate a variety of cellular processes. Three viral families, Coronaviridae, Togaviridae, and Hepeviridae, possess macro domains embedded in their polyproteins. Here, we show that viral macro domains reverse cellular ADP-ribosylation, potentially cutting the signal of a viral infection in the cell. Various poly(ADP-ribose) polymerases which are notorious guardians of cellular

  14. The Bipolar Filaments Formed by Herpes Simplex Virus Type 1 SSB/Recombination Protein (ICP8) Suggest a Mechanism for DNA Annealing

    SciTech Connect

    Makhov, A.M.; Simon, M.; Sen, A.; Yu, X.; Griffith, J. D.; Egelman, E. H.

    2009-02-20

    Herpes simplex virus type 1 encodes a multifunctional protein, ICP8, which serves both as a single-strand binding protein and as a recombinase, catalyzing reactions involved in replication and recombination of the viral genome. In the presence of divalent ions and at low temperature, previous electron microscopic studies showed that ICP8 will form long left-handed helical filaments. Here, electron microscopic image reconstruction reveals that the filaments are bipolar, with an asymmetric unit containing two subunits of ICP8 that constitute a symmetrical dimer. This organization of the filament has been confirmed using scanning transmission electron microscopy. The pitch of the filaments is {approx} 250 {angstrom}, with {approx} 6.2 dimers per turn. Docking of a crystal structure of ICP8 into the reconstructed filament shows that the C-terminal domain of ICP8, attached to the body of the subunit by a flexible linker containing {approx} 10 residues, is packed into a pocket in the body of a neighboring subunit in the crystal in a similar manner as in the filament. However, the interactions between the large N-terminal domains are quite different in the filament from that observed in the crystal. A previously proposed model for ICP8 binding single-stranded DNA (ssDNA), based upon the crystal structure, leads to a model for a continuous strand of ssDNA near the filament axis. The bipolar nature of the ICP8 filaments means that a second strand of ssDNA would be running through this filament in the opposite orientation, and this provides a potential mechanism for how ICP8 anneals complementary ssDNA into double-stranded DNA, where each strand runs in opposite directions.

  15. The role of cyclase-associated protein in regulating actin filament dynamics - more than a monomer-sequestration factor.

    PubMed

    Ono, Shoichiro

    2013-08-01

    Dynamic reorganization of the actin cytoskeleton is fundamental to a number of cell biological events. A variety of actin-regulatory proteins modulate polymerization and depolymerization of actin and contribute to actin cytoskeletal reorganization. Cyclase-associated protein (CAP) is a conserved actin-monomer-binding protein that has been studied for over 20 years. Early studies have shown that CAP sequesters actin monomers; recent studies, however, have revealed more active roles of CAP in actin filament dynamics. CAP enhances the recharging of actin monomers with ATP antagonistically to ADF/cofilin, and also promotes the severing of actin filaments in cooperation with ADF/cofilin. Self-oligomerization and binding to other proteins regulate activities and localization of CAP. CAP has crucial roles in cell signaling, development, vesicle trafficking, cell migration and muscle sarcomere assembly. This Commentary discusses the recent advances in our understanding of the functions of CAP and its implications as an important regulator of actin cytoskeletal dynamics, which are involved in various cellular activities.

  16. Actin-interacting Protein 1 Promotes Disassembly of Actin-depolymerizing Factor/Cofilin-bound Actin Filaments in a pH-dependent Manner*

    PubMed Central

    Nomura, Kazumi; Hayakawa, Kimihide; Tatsumi, Hitoshi; Ono, Shoichiro

    2016-01-01

    Actin-interacting protein 1 (AIP1) is a conserved WD repeat protein that promotes disassembly of actin filaments when actin-depolymerizing factor (ADF)/cofilin is present. Although AIP1 is known to be essential for a number of cellular events involving dynamic rearrangement of the actin cytoskeleton, the regulatory mechanism of the function of AIP1 is unknown. In this study, we report that two AIP1 isoforms from the nematode Caenorhabditis elegans, known as UNC-78 and AIPL-1, are pH-sensitive in enhancement of actin filament disassembly. Both AIP1 isoforms only weakly enhance disassembly of ADF/cofilin-bound actin filaments at an acidic pH but show stronger disassembly activity at neutral and basic pH values. However, a severing-defective mutant of UNC-78 shows pH-insensitive binding to ADF/cofilin-decorated actin filaments, suggesting that the process of filament severing or disassembly, but not filament binding, is pH-dependent. His-60 of AIP1 is located near the predicted binding surface for the ADF/cofilin-actin complex, and an H60K mutation of AIP1 partially impairs its pH sensitivity, suggesting that His-60 is involved in the pH sensor for AIP1. These biochemical results suggest that pH-dependent changes in AIP1 activity might be a novel regulatory mechanism of actin filament dynamics. PMID:26747606

  17. Control of protein trafficking by reversible masking of transport signals

    PubMed Central

    Abraham, Omer; Gotliv, Karnit; Parnis, Anna; Boncompain, Gaelle; Perez, Franck; Cassel, Dan

    2016-01-01

    Systems that allow the control of protein traffic between subcellular compartments have been valuable in elucidating trafficking mechanisms. Most current approaches rely on ligand or light-controlled dimerization, which results in either retardation or enhancement of the transport of a reporter. We developed an alternative approach for trafficking regulation that we term “controlled unmasking of targeting elements” (CUTE). Regulated trafficking is achieved by reversible masking of the signal that directs the reporter to its target organelle, relying on the streptavidin–biotin system. The targeting signal is generated within or immediately after a 38–amino acid streptavidin-binding peptide (SBP) that is appended to the reporter. The binding of coexpressed streptavidin to SBP causes signal masking, whereas addition of biotin causes complex dissociation and triggers protein transport to the target organelle. We demonstrate the application of this approach to the control of nuclear and peroxisomal protein import and the generation of biotin-dependent trafficking through the endocytic and COPI systems. By simultaneous masking of COPI and endocytic signals, we were able to generate a synthetic pathway for efficient transport of a reporter from the plasma membrane to the endoplasmic reticulum. PMID:26941332

  18. The Saccharomyces cerevisiae 14-3-3 protein Bmh2 is required for regulation of the phosphorylation status of Fin1, a novel intermediate filament protein.

    PubMed Central

    Mayordomo, Isabel; Sanz, Pascual

    2002-01-01

    In order to identify proteins that interact with Bmh2, a yeast member of the 14-3-3 protein family, we performed a two-hybrid screening using LexA-Bmh2 as bait. We identified Fin1, a novel intermediate filament protein, as the protein that showed the highest degree of interaction. We also identified components of the vesicular transport machinery such as Gic2 and Msb3, proteins involved in transcriptional regulation such as Mbf1, Gcr2 and Reg2, and a variety of other different proteins (Ppt1, Lre1, Rps0A and Ylr177w). We studied the interaction between Bmh2 and Fin1 in more detail and found that Bmh2 only interacted with phosphorylated forms of Fin1. In addition, we showed that Glc7, the catalytic subunit of the protein phosphatase 1 complex, was also able to interact with Fin1. PMID:11931638

  19. Mammalian CAP (Cyclase-associated protein) in the world of cell migration: Roles in actin filament dynamics and beyond.

    PubMed

    Zhou, Guo-Lei; Zhang, Haitao; Field, Jeffrey

    2014-01-01

    Cell migration is essential for a variety of fundamental biological processes such as embryonic development, wound healing, and immune response. Aberrant cell migration also underlies pathological conditions such as cancer metastasis, in which morphological transformation promotes spreading of cancer to new sites. Cell migration is driven by actin dynamics, which is the repeated cycling of monomeric actin (G-actin) into and out of filamentous actin (F-actin). CAP (Cyclase-associated protein, also called Srv2) is a conserved actin-regulatory protein, which is implicated in cell motility and the invasiveness of human cancers. It cooperates with another actin regulatory protein, cofilin, to accelerate actin dynamics. Hence, knockdown of CAP1 slows down actin filament turnover, which in most cells leads to reduced cell motility. However, depletion of CAP1 in HeLa cells, while causing reduction in dynamics, actually led to increased cell motility. The increases in motility are likely through activation of cell adhesion signals through an inside-out signaling. The potential to activate adhesion signaling competes with the negative effect of CAP1 depletion on actin dynamics, which would reduce cell migration. In this commentary, we provide a brief overview of the roles of mammalian CAP1 in cell migration, and highlight a likely mechanism underlying the activation of cell adhesion signaling and elevated motility caused by depletion of CAP1.

  20. Divisome-dependent subcellular localization of cell-cell joining protein SepJ in the filamentous cyanobacterium Anabaena.

    PubMed

    Ramos-León, Félix; Mariscal, Vicente; Frías, José E; Flores, Enrique; Herrero, Antonia

    2015-05-01

    Heterocyst-forming cyanobacteria are multicellular organisms that grow as filaments that can be hundreds of cells long. Septal junction complexes, of which SepJ is a possible component, appear to join the cells in the filament. SepJ is a cytoplasmic membrane protein that contains a long predicted periplasmic section and localizes not only to the cell poles in the intercellular septa but also to a position similar to a Z ring when cell division starts suggesting a relation with the divisome. Here, we created a mutant of Anabaena sp. strain PCC 7120 in which the essential divisome gene ftsZ is expressed from a synthetic NtcA-dependent promoter, whose activity depends on the nitrogen source. In the presence of ammonium, low levels of FtsZ were produced, and the subcellular localization of SepJ, which was investigated by immunofluorescence, was impaired. Possible interactions of SepJ with itself and with divisome proteins FtsZ, FtsQ and FtsW were investigated using the bacterial two-hybrid system. We found SepJ self-interaction and a specific interaction with FtsQ, confirmed by co-purification and involving parts of the SepJ and FtsQ periplasmic sections. Therefore, SepJ can form multimers, and in Anabaena, the divisome has a role beyond cell division, localizing a septal protein essential for multicellularity.

  1. Characterization of the DNA binding protein encoded by the N-specific filamentous Escherichia coli phage IKe. Binding properties of the protein and nucleotide sequence of the gene.

    PubMed

    Peeters, B P; Konings, R N; Schoenmakers, J G

    1983-09-05

    A DNA binding protein encoded by the filamentous single-stranded DNA phage IKe has been isolated from IKe-infected Escherichia coli cells. Fluorescence and in vitro binding studies have shown that the protein binds co-operatively and with a high specificity to single-stranded but not to double-stranded DNA. From titration of the protein to poly(dA) it has been calculated that approximately four bases of the DNA are covered by one monomer of protein. These binding characteristics closely resemble those of gene V protein encoded by the F-specific filamentous phages M13 and fd. The nucleotide sequence of the gene specifying the IKe DNA binding protein has been established. When compared to the nucleotide sequence of gene V of phage M13 it shows an homology of 58%, indicating that these two phages are evolutionarily related. The IKe DNA binding protein is 88 amino acids long which is one amino acid residue larger than the gene V protein sequence. When the IKe DNA binding protein sequence is compared with that of gene V protein it was found that 39 amino acid residues have identical positions in both proteins. The positions of all five tyrosine residues, a number of which are known to be involved in DNA binding, are conserved. Secondary structure predictions indicate that the two proteins contain similar structural domains. It is proposed that the tyrosine residues which are involved in DNA binding are the ones in or next to a beta-turn, at positions 26, 41 and 56 in gene V protein and at positions 27, 42 and 57 in the IKe DNA binding protein.

  2. Localizing and extracting filament distributions from microscopy images.

    PubMed

    Basu, S; Liu, C; Rohde, G K

    2015-04-01

    Detailed quantitative measurements of biological filament networks represent a crucial step in understanding architecture and structure of cells and tissues, which in turn explain important biological events such as wound healing and cancer metastases. Microscopic images of biological specimens marked for different structural proteins constitute an important source for observing and measuring meaningful parameters of biological networks. Unfortunately, current efforts at quantitative estimation of architecture and orientation of biological filament networks from microscopy images are predominantly limited to visual estimation and indirect experimental inference. Here, we describe a new method for localizing and extracting filament distributions from 2D microscopy images of different modalities. The method combines a filter-based detection of pixels likely to contain a filament with a constrained reverse diffusion-based approach for localizing the filaments centrelines. We show with qualitative and quantitative experiments, using both simulated and real data, that the new method can provide more accurate centreline estimates of filament in comparison to other approaches currently available. In addition, we show the algorithm is more robust with respect to variations in the initial filter-based filament detection step often used. We demonstrate the application of the method in extracting quantitative parameters from confocal microscopy images of actin filaments and atomic force microscopy images of DNA fragments.

  3. Development of a Novel Green Fluorescent Protein-Based Binding Assay to Study the Association of Plakins with Intermediate Filament Proteins.

    PubMed

    Favre, Bertrand; Begré, Nadja; Bouameur, Jamal-Eddine; Borradori, Luca

    2016-01-01

    Protein-protein interactions are fundamental for most biological processes, such as the formation of cellular structures and enzymatic complexes or in signaling pathways. The identification and characterization of protein-protein interactions are therefore essential for understanding the mechanisms and regulation of biological systems. The organization and dynamics of the cytoskeleton, as well as its anchorage to specific sites in the plasma membrane and organelles, are regulated by the plakins. These structurally related proteins anchor different cytoskeletal networks to each other and/or to other cellular structures. The association of several plakins with intermediate filaments (IFs) is critical for maintenance of the cytoarchitecture. Pathogenic mutations in the genes encoding different plakins can lead to dramatic manifestations, occurring principally in the skin, striated muscle, and/or nervous system, due to cytoskeletal disorganization resulting in abnormal cell fragility. Nevertheless, it is still unclear how plakins bind to IFs, although some general rules are slowly emerging. We here describe in detail a recently developed protein-protein fluorescence binding assay, based on the production of recombinant proteins tagged with green fluorescent protein (GFP) and their use as fluid-phase fluorescent ligands on immobilized IF proteins. Using this method, we have been able to assess the ability of C-terminal regions of GFP-tagged plakin proteins to bind to distinct IF proteins and IF domains. This simple and sensitive technique, which is expected to facilitate further studies in this area, can also be potentially employed for any kind of protein-protein interaction studies.

  4. Branching of keratin intermediate filaments.

    PubMed

    Nafeey, Soufi; Martin, Ines; Felder, Tatiana; Walther, Paul; Felder, Edward

    2016-06-01

    Keratin intermediate filaments (IFs) are crucial to maintain mechanical stability in epithelial cells. Since little is known about the network architecture that provides this stiffness and especially about branching properties of filaments, we addressed this question with different electron microscopic (EM) methods. Using EM tomography of high pressure frozen keratinocytes, we investigated the course of several filaments in a branching of a filament bundle. Moreover we found several putative bifurcations in individual filaments. To verify our observation we also visualized the keratin network in detergent extracted keratinocytes with scanning EM. Here bifurcations of individual filaments could unambiguously be identified additionally to bundle branchings. Interestingly, identical filament bifurcations were also found in purified keratin 8/18 filaments expressed in Escherichia coli which were reassembled in vitro. This excludes that an accessory protein contributes to the branch formation. Measurements of the filament cross sectional areas showed various ratios between the three bifurcation arms. This demonstrates that intermediate filament furcation is very different from actin furcation where an entire new filament is attached to an existing filament. Instead, the architecture of intermediate filament bifurcations is less predetermined and hence consistent with the general concept of IF formation.

  5. Ras2 signals via the Cdc42/Ste20/mitogen-activated protein kinase module to induce filamentous growth in Saccharomyces cerevisiae.

    PubMed Central

    Mösch, H U; Roberts, R L; Fink, G R

    1996-01-01

    RAS2val19, a dominant activated form of Saccharomyces cerevisiae Ras2, stimulates both filamentous growth and expression of a transcriptional reporter FG(TyA)::lacZ but does not induce the mating pathway reporter FUS1::lacZ. This induction depends upon elements of the conserved mitogen-activated protein kinase (MAPK) pathway that is required for both filamentous growth and mating, two distinct morphogenetic events. Full induction requires Ste20 (homolog of mammalian p65PAK protein kinases), Ste11 [an MEK kinase (MEKK) or MAPK kinase (MEK) kinase], Ste7 (MEK or MAPK kinase), and the transcription factor Ste12. Moreover, the Rho family protein Cdc42, a conserved morphogenetic G protein, is also a potent regulator of filamentous growth and FG(TyA)::lacZ expression in S. cerevisiae. Stimulation of both filamentous growth and FG(TyA)::lacZ by Cdc42 depends upon Ste20. In addition, dominant negative CDC42Ala118 blocks RAS2val19 activation, placing Cdc42 downstream of Ras2. Our results suggest that filamentous growth in budding yeast is regulated by an evolutionarily conserved signaling pathway that controls cell morphology. Images Fig. 1 Fig. 2 Fig. 3 PMID:8643578

  6. Withaferin A Targets Intermediate Filaments Glial Fibrillary Acidic Protein and Vimentin in a Model of Retinal Gliosis*

    PubMed Central

    Bargagna-Mohan, Paola; Paranthan, Riya R.; Hamza, Adel; Dimova, Neviana; Trucchi, Beatrice; Srinivasan, Cidambi; Elliott, Gregory I.; Zhan, Chang-Guo; Lau, Daniel L.; Zhu, Haiyan; Kasahara, Kousuke; Inagaki, Masaki; Cambi, Franca; Mohan, Royce

    2010-01-01

    Gliosis is a biological process that occurs during injury repair in the central nervous system and is characterized by the overexpression of the intermediate filaments (IFs) glial fibrillary acidic protein (GFAP) and vimentin. A common thread in many retinal diseases is reactive Müller cell gliosis, an untreatable condition that leads to tissue scarring and even blindness. Here, we demonstrate that the vimentin-targeting small molecule withaferin A (WFA) is a novel chemical probe of GFAP. Using molecular modeling studies that build on the x-ray crystal structure of tetrameric vimentin rod 2B domain we reveal that the WFA binding site is conserved in the corresponding domain of tetrameric GFAP. Consequently, we demonstrate that WFA covalently binds soluble recombinant tetrameric human GFAP at cysteine 294. In cultured primary astrocytes, WFA binds to and down-regulates soluble vimentin and GFAP expression to cause cell cycle G0/G1 arrest. Exploiting a chemical injury model that overexpresses vimentin and GFAP in retinal Müller glia, we demonstrate that systemic delivery of WFA down-regulates soluble vimentin and GFAP expression in mouse retinas. This pharmacological knockdown of soluble IFs results in the impairment of GFAP filament assembly and inhibition of cell proliferative response in Müller glia. We further show that a more severe GFAP filament assembly deficit manifests in vimentin-deficient mice, which is partly rescued by WFA. These findings illustrate WFA as a chemical probe of type III IFs and illuminate this class of withanolide as a potential treatment for diverse gliosis-dependent central nervous system traumatic injury conditions and diseases, and for orphan IF-dependent pathologies. PMID:20048155

  7. Mammalian homolog of the yeast cyclase associated protein, CAP/Srv2p, regulates actin filament assembly.

    PubMed

    Freeman, N L; Field, J

    2000-02-01

    Control of cell shape and motility requires rearrangements of the actin cytoskeleton. One cytoskeletal protein that may regulate actin dynamics is CAP (cyclase associated protein; CAP/Srv2p; ASP-56). CAP was first isolated from yeast as an adenylyl cyclase associated protein required for RAS regulation of cAMP signaling. In addition, CAP also regulates the actin cytoskeleton primarily through an actin monomer binding activity. CAP homologs are found in many eukaryotes, including mammals where they also bind actin, but little is known about their biological function. We, therefore, designed experiments to address CAP1 regulation of the actin cytoskeleton. CAP1 localized to membrane ruffles and actin stress fibers in fixed cells of various types. To address localization in living cells, we constructed GFP-CAP1 fusion proteins and found that fusion proteins lacking the actin-binding region localized like the wild type protein. We also performed microinjection studies with affinity-purified anti-CAP1 antibodies in Swiss 3T3 fibroblasts and found that the antibodies attenuated serum stimulation of stress fibers. Finally, CAP1 purified from platelets through a monoclonal antibody affinity purification step stimulated the formation of stress fiber-like filaments when it was microinjected into serum-starved Swiss 3T3 cells. Taken together, these data suggest that CAP1 promotes assembly of the actin cytoskeleton.

  8. Contemporary techniques for detecting and identifying proteins susceptible to reversible thiol oxidation.

    PubMed

    Burgoyne, Joseph R; Eaton, Philip

    2011-10-01

    Elevated protein oxidation is a widely reported hallmark of most major diseases. Historically, this 'oxidative stress' has been considered causatively detrimental, as the protein oxidation events were interpreted simply as damage. However, recent advances have changed this antiquated view; sensitive methodology for detecting and identifying proteins susceptible to oxidation has revealed a fundamental role for this modification in physiological cell signalling during health. Reversible protein oxidation that is dynamically coupled with cellular reducing systems allows oxidative protein modifications to regulate protein function, analogous to phosphoregulation. However, the relatively labile nature of many reversible protein oxidation states hampers the reliable detection and identification of modified proteins. Consequently, specialized methods to stabilize protein oxidation in combination with techniques to detect specific types of modification have been developed. Here, these techniques are discussed, and their sensitivity, selectivity and ability to reliably identify reversibly oxidized proteins are critically assessed.

  9. Reversals.

    ERIC Educational Resources Information Center

    National Center on Educational Media and Materials for the Handicapped, Columbus, OH.

    Selected from the National Instructional Materials Information System (NIMIS)--a computer based on-line interactive retrieval system on special education materials--the bibliography covers nine materials for remediating reversals in handicapped students at the early childhood and elementary levels. Entries are presented in order of NIMIS accession…

  10. In-depth characterization and computational 3D reconstruction of flagellar filament protein layer structure based on in situ spectroscopic ellipsometry measurements

    NASA Astrophysics Data System (ADS)

    Kozma, Peter; Kozma, Daniel; Nemeth, Andrea; Jankovics, Hajnalka; Kurunczi, Sandor; Horvath, Robert; Vonderviszt, Ferenc; Fried, Miklos; Petrik, Peter

    2011-06-01

    In this study, we have reconstructed the statistical 3D structure of hundreds of nanometers thick surface immobilized flagellar filament protein layers in their native environment, in buffer solution. The protein deposition onto the surface activated Ta 2O 5 film was performed in a flow cell, and the immobilization process was followed by in situ spectroscopic ellipsometry. A multilayer optical model was developed, in that the protein layer was described by five effective medium sublayers. Applying this method, an in-depth analysis of the protein layer formation was performed. Based on the kinetics in the distribution of the surface mass density, the statistical properties of the filamentous film could be determined computationally as a function of the measurement time. It was also demonstrated that the 3D structure of the protein layer can be reconstructed based on the calculated in-depth mass density profile. The computational investigation revealed that the filaments can be classified into two individual groups in approximately equal ratio according to their orientation. In the first group the filaments are close to laying position, whereas in the second group they are in a standing position, resulting in a significantly denser sublayer close to the substrate than at a larger distance.

  11. Expression of the type VI intermediate filament proteins CP49 and filensin in the mouse lens epithelium

    PubMed Central

    Sun, Ning; Shibata, Brad; Hess, John F.

    2016-01-01

    Purpose The differentiated lens fiber cell assembles a filamentous cytoskeletal structure referred to as the beaded filament (BF). The BF requires CP49 (bfsp2) and filensin (bfsp1) for assembly, both of which are highly divergent members of the large intermediate filament (IF) family of proteins. Thus far, these two proteins have been reported only in the differentiated lens fiber cell. For this reason, both proteins have been considered robust markers of fiber cell differentiation. We report here that both proteins are also expressed in the mouse lens epithelium, but only after 5 weeks of age. Methods Localization of CP49 was achieved with immunocytochemical probing of wild-type, CP49 knockout, filensin knockout, and vimentin knockout mice, in sections and in the explanted lens epithelium, at the light microscope and electron microscope levels. The relationship between CP49 and other cytoskeletal elements was probed using fluorescent phalloidin, as well as with antibodies to vimentin, GFAP, and α-tubulin. The relationship between CP49 and the aggresome was probed with antibodies to γ-tubulin, ubiquitin, and HDAC6. Results CP49 and filensin were expressed in the mouse lens epithelium, but only after 5 weeks of age. At the light microscope level, these two proteins colocalize to a large tubular structure, approximately 7 × 1 μm, which was typically present at one to two copies per cell. This structure is found in the anterior and anterolateral lens epithelium, including the zone where mitosis occurs. The structure becomes smaller and largely undetectable closer to the equator where the cell exits the cell cycle and commits to fiber cell differentiation. This structure bears some resemblance to the aggresome and is reactive with antibodies to HDAC6, a marker for the aggresome. However, the structure does not colocalize with antibodies to γ-tubulin or ubiquitin, also markers for the aggresome. The structure also colocalizes with actin but appears to largely

  12. Chaperonin filaments: The archael cytoskeleton

    SciTech Connect

    Trent, J.D.; Kagawa, H.K.; Yaoi, Takuro; Olle, E.; Zaluzec, N.J.

    1997-08-01

    Chaperonins are multi-subunit double-ring complexed composed of 60-kDa proteins that are believed to mediate protein folding in vivo. The chaperonins in the hyperthermophilic archaeon Sulfolobus shibatae are composed of the organism`s two most abundant proteins, which represent 4% of its total protein and have an intracellular concentration of {ge} 3.0 mg/ml. At concentrations of 1.0 mg/ml, purified chaperonin proteins aggregate to form ordered filaments. Filament formation, which requires Mg{sup ++} and nucleotide binding (not hydrolysis), occurs at physiological temperatures under conditions suggesting filaments may exist in vivo. If the estimated 4,600 chaperonins per cell, formed filaments in vivo, they could create a matrix of filaments that would span the diameter of an average S. shibatae cell 100 times. Direct observations of unfixed, minimally treated cells by intermediate voltage electron microscopy (300 kV) revealed an intracellular network of filaments that resembles chaperonin filaments produced in vitro. The hypothesis that the intracellular network contains chaperonins is supported by immunogold analyses. The authors propose that chaperonin activity may be regulated in vivo by filament formation and that chaperonin filaments may serve a cytoskeleton-like function in archaea and perhaps in other prokaryotes.

  13. The intermediate filament network protein, vimentin, is required for parvoviral infection

    SciTech Connect

    Fay, Nikta; Panté, Nelly

    2013-09-15

    Intermediate filaments (IFs) have recently been shown to serve novel roles during infection by many viruses. Here we have begun to study the role of IFs during the early steps of infection by the parvovirus minute virus of mice (MVM). We found that during early infection with MVM, after endosomal escape, the vimentin IF network was considerably altered, yielding collapsed immunofluorescence staining near the nuclear periphery. Furthermore, we found that vimentin plays an important role in the life cycle of MVM. The number of cells, which successfully replicated MVM, was reduced in infected cells in which the vimentin network was genetically or pharmacologically modified; viral endocytosis, however, remained unaltered. Perinuclear accumulation of MVM-containing vesicles was reduced in cells lacking vimentin. Our data suggests that vimentin is required for the MVM life cycle, presenting possibly a dual role: (1) following MVM escape from endosomes and (2) during endosomal trafficking of MVM. - Highlights: • MVM infection changes the distribution of the vimentin network to perinuclear regions. • Disrupting the vimentin network with acrylamide decreases MVM replication. • MVM replication is significantly reduced in vimentin-null cells. • Distribution of MVM-containing vesicles is affected in MVM infected vimentin-null cells.

  14. A ΩXaV motif in the Rift Valley fever virus NSs protein is essential for degrading p62, forming nuclear filaments and virulence

    PubMed Central

    Cyr, Normand; de la Fuente, Cynthia; Lecoq, Lauriane; Guendel, Irene; Chabot, Philippe R.; Kehn-Hall, Kylene; Omichinski, James G.

    2015-01-01

    Rift Valley fever virus (RVFV) is a single-stranded RNA virus capable of inducing fatal hemorrhagic fever in humans. A key component of RVFV virulence is its ability to form nuclear filaments through interactions between the viral nonstructural protein NSs and the host general transcription factor TFIIH. Here, we identify an interaction between a ΩXaV motif in NSs and the p62 subunit of TFIIH. This motif in NSs is similar to ΩXaV motifs found in nucleotide excision repair (NER) factors and transcription factors known to interact with p62. Structural and biophysical studies demonstrate that NSs binds to p62 in a similar manner as these other factors. Functional studies in RVFV-infected cells show that the ΩXaV motif is required for both nuclear filament formation and degradation of p62. Consistent with the fact that the RVFV can be distinguished from other Bunyaviridae-family viruses due to its ability to form nuclear filaments in infected cells, the motif is absent in the NSs proteins of other Bunyaviridae-family viruses. Taken together, our studies demonstrate that p62 binding to NSs through the ΩXaV motif is essential for degrading p62, forming nuclear filaments and enhancing RVFV virulence. In addition, these results show how the RVFV incorporates a simple motif into the NSs protein that enables it to functionally mimic host cell proteins that bind the p62 subunit of TFIIH. PMID:25918396

  15. Caenorhabditis elegans Kettin, a Large Immunoglobulin-like Repeat Protein, Binds to Filamentous Actin and Provides Mechanical Stability to the Contractile Apparatuses in Body Wall Muscle

    PubMed Central

    Ono, Kanako; Yu, Robinson; Mohri, Kurato

    2006-01-01

    Kettin is a large actin-binding protein with immunoglobulin-like (Ig) repeats, which is associated with the thin filaments in arthropod muscles. Here, we report identification and functional characterization of kettin in the nematode Caenorhabditis elegans. We found that one of the monoclonal antibodies that were raised against C. elegans muscle proteins specifically reacts with kettin (Ce-kettin). We determined the entire cDNA sequence of Ce-kettin that encodes a protein of 472 kDa with 31 Ig repeats. Arthropod kettins are splice variants of much larger connectin/titin-related proteins. However, the gene for Ce-kettin is independent of other connectin/titin-related genes. Ce-kettin localizes to the thin filaments near the dense bodies in both striated and nonstriated muscles. The C-terminal four Ig repeats and the adjacent non-Ig region synergistically bind to actin filaments in vitro. RNA interference of Ce-kettin caused weak disorganization of the actin filaments in body wall muscle. This phenotype was suppressed by inhibiting muscle contraction by a myosin mutation, but it was enhanced by tetramisole-induced hypercontraction. Furthermore, Ce-kettin was involved in organizing the cytoplasmic portion of the dense bodies in cooperation with α-actinin. These results suggest that kettin is an important regulator of myofibrillar organization and provides mechanical stability to the myofibrils during contraction. PMID:16597697

  16. AglZ Is a Filament-Forming Coiled-Coil Protein Required for Adventurous Gliding Motility of Myxococcus xanthus

    PubMed Central

    Yang, Ruifeng; Bartle, Sarah; Otto, Rebecca; Stassinopoulos, Angela; Rogers, Matthew; Plamann, Lynda; Hartzell, Patricia

    2004-01-01

    The aglZ gene of Myxococcus xanthus was identified from a yeast two-hybrid assay in which MglA was used as bait. MglA is a 22-kDa cytoplasmic GTPase required for both adventurous and social gliding motility and sporulation. Genetic studies showed that aglZ is part of the A motility system, because disruption or deletion of aglZ abolished movement of isolated cells and aglZ sglK double mutants were nonmotile. The aglZ gene encodes a 153-kDa protein that interacts with purified MglA in vitro. The N terminus of AglZ shows similarity to the receiver domain of two-component response regulator proteins, while the C terminus contains heptad repeats characteristic of coiled-coil proteins, such as myosin. Consistent with this motif, expression of AglZ in Escherichia coli resulted in production of striated lattice structures. Similar to the myosin heavy chain, the purified C-terminal coiled-coil domain of AglZ forms filament structures in vitro. PMID:15342587

  17. AglZ is a filament-forming coiled-coil protein required for adventurous gliding motility of Myxococcus xanthus.

    PubMed

    Yang, Ruifeng; Bartle, Sarah; Otto, Rebecca; Stassinopoulos, Angela; Rogers, Matthew; Plamann, Lynda; Hartzell, Patricia

    2004-09-01

    The aglZ gene of Myxococcus xanthus was identified from a yeast two-hybrid assay in which MglA was used as bait. MglA is a 22-kDa cytoplasmic GTPase required for both adventurous and social gliding motility and sporulation. Genetic studies showed that aglZ is part of the A motility system, because disruption or deletion of aglZ abolished movement of isolated cells and aglZ sglK double mutants were nonmotile. The aglZ gene encodes a 153-kDa protein that interacts with purified MglA in vitro. The N terminus of AglZ shows similarity to the receiver domain of two-component response regulator proteins, while the C terminus contains heptad repeats characteristic of coiled-coil proteins, such as myosin. Consistent with this motif, expression of AglZ in Escherichia coli resulted in production of striated lattice structures. Similar to the myosin heavy chain, the purified C-terminal coiled-coil domain of AglZ forms filament structures in vitro.

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

    PubMed

    Kollmar, Martin

    2015-05-29

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

  19. Reactive Self-Assembly of Polymers and Proteins to Reversibly Silence a Killer Protein.

    PubMed

    Ventura, Judy; Eron, Scott J; González-Toro, Daniella C; Raghupathi, Kishore; Wang, Feng; Hardy, Jeanne A; Thayumanavan, S

    2015-10-12

    Conjugation of biologically active proteins to polymeric materials is of great interest in the treatment of cancer and other diseases of protein deficiency. The conjugation of such biomacromolecules is challenging both due to their hydrophilicity and propensity to denature under non-native conditions. We describe a novel reactive self-assembly approach to "wrap" a protein with polymers, simultaneously protecting its delicate folded state and silencing its enzymatic activity. This approach has been demonstrated using caspase-3, an apoptosis-inducing protein, as the first case study. The protein-polymer conjugation is designed to be reversed under the native conditions for caspase-3, that is, the reducing environment found in the cytosol. The current strategy allowed release and recovery of up to 86% of caspase activity and nanogel-caspase-3 conjugates induced 70-80% apoptotic cell death shortly thereafter. This approach is widely generalizable and should be applicable to the intracellular delivery of a wide range of therapeutic proteins for treatment of complex and genetic diseases.

  20. A protein ballet around the viral genome orchestrated by HIV-1 reverse transcriptase leads to an architectural switch: from nucleocapsid-condensed RNA to Vpr-bridged DNA.

    PubMed

    Lyonnais, Sébastien; Gorelick, Robert J; Heniche-Boukhalfa, Fatima; Bouaziz, Serge; Parissi, Vincent; Mouscadet, Jean-François; Restle, Tobias; Gatell, Jose Maria; Le Cam, Eric; Mirambeau, Gilles

    2013-02-01

    HIV-1 reverse transcription is achieved in the newly infected cell before viral DNA (vDNA) nuclear import. Reverse transcriptase (RT) has previously been shown to function as a molecular motor, dismantling the nucleocapsid complex that binds the viral genome as soon as plus-strand DNA synthesis initiates. We first propose a detailed model of this dismantling in close relationship with the sequential conversion from RNA to double-stranded (ds) DNA, focusing on the nucleocapsid protein (NCp7). The HIV-1 DNA-containing pre-integration complex (PIC) resulting from completion of reverse transcription is translocated through the nuclear pore. The PIC nucleoprotein architecture is poorly understood but contains at least two HIV-1 proteins initially from the virion core, namely integrase (IN) and the viral protein r (Vpr). We next present a set of electron micrographs supporting that Vpr behaves as a DNA architectural protein, initiating multiple DNA bridges over more than 500 base pairs (bp). These complexes are shown to interact with NCp7 bound to single-stranded nucleic acid regions that are thought to maintain IN binding during dsDNA synthesis, concurrently with nucleocapsid complex dismantling. This unexpected binding of Vpr conveniently leads to a compacted but filamentous folding of the vDNA that should favor its nuclear import. Finally, nucleocapsid-like aggregates engaged in dsDNA synthesis appear to efficiently bind to F-actin filaments, a property that may be involved in targeting complexes to the nuclear envelope. More generally, this article highlights unique possibilities offered by in vitro reconstitution approaches combined with macromolecular imaging to gain insights into the mechanisms that alter the nucleoprotein architecture of the HIV-1 genome, ultimately enabling its insertion into the nuclear chromatin.

  1. Protein dynamics of human RPA and RAD51 on ssDNA during assembly and disassembly of the RAD51 filament

    PubMed Central

    Ma, Chu Jian; Gibb, Bryan; Kwon, YoungHo; Sung, Patrick; Greene, Eric C.

    2017-01-01

    Homologous recombination (HR) is a crucial pathway for double-stranded DNA break (DSB) repair. During the early stages of HR, the newly generated DSB ends are processed to yield long single-stranded DNA (ssDNA) overhangs, which are quickly bound by replication protein A (RPA). RPA is then replaced by the DNA recombinase Rad51, which forms extended helical filaments on the ssDNA. The resulting nucleoprotein filament, known as the presynaptic complex, is responsible for pairing the ssDNA with homologous double-stranded DNA (dsDNA), which serves as the template to guide DSB repair. Here, we use single-molecule imaging to visualize the interplay between human RPA (hRPA) and human RAD51 during presynaptic complex assembly and disassembly. We demonstrate that ssDNA-bound hRPA can undergo facilitated exchange, enabling hRPA to undergo rapid exchange between free and ssDNA-bound states only when free hRPA is present in solution. Our results also indicate that the presence of free hRPA inhibits RAD51 filament nucleation, but has a lesser impact upon filament elongation. This finding suggests that hRPA exerts important regulatory influence over RAD51 and may in turn affect the properties of the assembled RAD51 filament. These experiments provide an important basis for further investigations into the regulation of human presynaptic complex assembly. PMID:27903895

  2. Structural changes of the regulatory proteins bound to the thin filaments in skeletal muscle contraction by X-ray fiber diffraction

    SciTech Connect

    Sugimoto, Yasunobu Takezawa, Yasunori; Matsuo, Tatsuhito; Ueno, Yutaka; Minakata, Shiho; Tanaka, Hidehiro; Wakabayashi, Katsuzo

    2008-04-25

    In order to clarify the structural changes related to the regulation mechanism in skeletal muscle contraction, the intensity changes of thin filament-based reflections were investigated by X-ray fiber diffraction. The time course and extent of intensity changes of the first to third order troponin (TN)-associated meridional reflections with a basic repeat of 38.4 nm were different for each of these reflections. The intensity of the first and second thin filament layer lines changed in a reciprocal manner both during initial activation and during the force generation process. The axial spacings of the TN-meridional reflections decreased by {approx}0.1% upon activation relative to the relaxing state and increased by {approx}0.24% in the force generation state, in line with that of the 2.7-nm reflection. Ca{sup 2+}-binding to TN triggered the shortening and a change in the helical symmetry of the thin filaments. Modeling of the structural changes using the intensities of the thin filament-based reflections suggested that the conformation of the globular core domain of TN altered upon activation, undergoing additional conformational changes at the tension plateau. The tail domain of TN moved together with tropomyosin during contraction. The results indicate that the structural changes of regulatory proteins bound to the actin filaments occur in two steps, the first in response to the Ca{sup 2+}-binding and the second induced by actomyosin interaction.

  3. Unexpected homology between inducible cell wall protein QID74 of filamentous fungi and BR3 salivary protein of the insect Chironomus

    PubMed Central

    Rey, Manuel; Ohno, Susumu; Pintor-Toro, Jose A.; Llobell, Antonio; Benitez, Tahia

    1998-01-01

    A gene, qid74, of mycoparasitic filamentous fungus Trichoderma harzianum and its allies encodes a cell wall protein that is induced by replacing glucose in the culture medium with chitin (simulated mycoparasitism conditions). Because no trace of this gene can be detected in related species such as Gibberella fujikuroi and Saccharomyces cerevisiae, the qid74 gene appears to have arisen de novo within the genus Trichoderma. Qid74 protein, 687 residues long, is now seen as highly conserved tandem repeats of the 59-residue-long unit. This unit itself, however, may have arisen as tandem repeats of the shorter 13-residue-long basic unit. Within the genus Trichoderma, the amino acid sequence of Qid74 proteins has been conserved in toto. The most striking is the fact that Qid74 shares 25.3% sequence identity with the carboxyl-terminal half of the 1,572-residue-long BR3 protein of the dipteran insect Chironomus tentans. BR3 protein is secreted by the salivary gland of each aquatic larva of Chironomus to form a tube to house itself. Furthermore, the consensus sequence derived from these 59-residue-long repeating units resembles those of epidermal growth factor-like domains found in divergent invertebrate and vertebrate proteins as to the positions of critical cysteine residues and homology of residues surrounding these cysteines. PMID:9600944

  4. Role of Intermediate Filaments in Vesicular Traffic

    PubMed Central

    Margiotta, Azzurra; Bucci, Cecilia

    2016-01-01

    Intermediate filaments are an important component of the cellular cytoskeleton. The first established role attributed to intermediate filaments was the mechanical support to cells. However, it is now clear that intermediate filaments have many different roles affecting a variety of other biological functions, such as the organization of microtubules and microfilaments, the regulation of nuclear structure and activity, the control of cell cycle and the regulation of signal transduction pathways. Furthermore, a number of intermediate filament proteins have been involved in the acquisition of tumorigenic properties. Over the last years, a strong involvement of intermediate filament proteins in the regulation of several aspects of intracellular trafficking has strongly emerged. Here, we review the functions of intermediate filaments proteins focusing mainly on the recent knowledge gained from the discovery that intermediate filaments associate with key proteins of the vesicular membrane transport machinery. In particular, we analyze the current understanding of the contribution of intermediate filaments to the endocytic pathway. PMID:27120621

  5. Modulating Endoplasmic Reticulum-Golgi Cargo Receptors for Improving Secretion of Carrier-Fused Heterologous Proteins in the Filamentous Fungus Aspergillus oryzae

    PubMed Central

    Hoang, Huy-Dung; Maruyama, Jun-ichi

    2014-01-01

    Filamentous fungi are excellent hosts for industrial protein production due to their superior secretory capacity; however, the yield of heterologous eukaryotic proteins is generally lower than that of fungal or endogenous proteins. Although activating protein folding machinery in the endoplasmic reticulum (ER) improves the yield, the importance of intracellular transport machinery for heterologous protein secretion is poorly understood. Here, using Aspergillus oryzae as a model filamentous fungus, we studied the involvement of two putative lectin-like cargo receptors, A. oryzae Vip36 (AoVip36) and AoEmp47, in the secretion of heterologous proteins expressed in fusion with the endogenous enzyme α-amylase as the carrier. Fluorescence microscopy revealed that mDsRed-tagged AoVip36 localized in the Golgi compartment, whereas AoEmp47 showed localization in both the ER and the Golgi compartment. Deletion of AoVip36 and AoEmp47 improved heterologous protein secretion, but only AoVip36 deletion had a negative effect on the secretion of α-amylase. Analysis of ER-enriched cell fractions revealed that AoVip36 and AoEmp47 were involved in the retention of heterologous proteins in the ER. However, the overexpression of each cargo receptor had a different effect on heterologous protein secretion: AoVip36 enhanced the secretion, whereas AoEmp47 promoted the intracellular retention. Taken together, our data suggest that AoVip36 and AoEmp47 hinder the secretion of heterologous proteins by promoting their retention in the ER but that AoVip36 also promotes the secretion of heterologous proteins. Moreover, we found that genetic deletion of these putative ER-Golgi cargo receptors significantly improves heterologous protein production. The present study is the first to propose that ER-Golgi transport is a bottleneck for heterologous protein production in filamentous fungi. PMID:25362068

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

    SciTech Connect

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

    2013-12-10

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

  7. Plant 115-kDa actin-filament bundling protein, P-115-ABP, is a homologue of plant villin and is widely distributed in cells.

    PubMed

    Yokota, Etsuo; Vidali, Luis; Tominaga, Motoki; Tahara, Hiroshi; Orii, Hidefumi; Morizane, Yosuke; Hepler, Peter K; Shimmen, Teruo

    2003-10-01

    In many cases, actin filaments are arranged into bundles and serve as tracks for cytoplasmic streaming in plant cells. We have isolated an actin-filament bundling protein, which is composed of 115-kDa polypeptide (P-115-ABP), from the germinating pollen of lily, Lilium longiflorum [Nakayasu et al. (1998) BIOCHEM: Biophys. Res. Commun. 249: 61]. P-115-ABP shared similar antigenicity with a plant 135-kDa actin-filament bundling protein (P-135-ABP), a plant homologue of villin. A full-length cDNA clone (ABP115; accession no. AB097407) was isolated from an expression cDNA library of lily pollen by immuno-screening using antisera against P-115-ABP and P-135-ABP. The amino acid sequence of P-115-ABP deduced from this clone showed high homology with those of P-135-ABP and four villin isoforms of Arabidopsis thaliana (AtVLN1, AtVLN2, AtVLN3 and AtVLN4), especially AtVLN4, indicating that P-115-ABP can also be classified as a plant villin. The P-115-ABP isolated biochemically from the germinating lily pollen was able to arrange F-actin filaments with uniform polarity into bundles and this bundling activity was suppressed by Ca2+-calmodulin (CaM), similar to the actin-filament bundling properties of P-135-ABP. The P-115-ABP type of plant villin was widely distributed in plant cells, from algae to land plants. In root hair cells of Hydrocharis dubia, this type of plant villin was co-localized with actin-filament bundles in the transvacuolar strands and the sub-cortical regions. Microinjection of the antiserum against P-115-ABP into living root hair cells caused the disappearance of transvaculor strands and alteration of the route of cytoplasmic streaming. In internodal cells of Chara corallina in which the P-135-ABP type of plant villin is lacking, the P-115-ABP type showed co-localization with actin-filament cables anchored on the intracellular surface of chloroplasts. These results indicated that plant villins are widely distributed and involved in the organization of actin

  8. The formation of fibrils by intertwining of filaments: model and application to amyloid Abeta protein.

    PubMed

    van Gestel, Jeroen; de Leeuw, Simon W

    2007-02-15

    We outline a model that describes the interaction of rods that form intertwined bundles. In this simple model, we compare the elastic energy penalty that arises due to the deformation of the rods to the gain in binding energy upon intertwining. We find that, for proper values of the bending Young's modulus and the binding energy, a helical pitch may be found for which the energy of intertwining is most favorable. We apply our description to the problem of Alzheimer's Abeta protein fibrillization. If we forbid configurations that exhibit steric overlap between the protofilaments that make up a protein fibril, our model predicts that fibrils consisting of three protofilaments shall form. This agrees well with experimental results. Our model can also provide an estimate for the helical pitch of suitable fibrils.

  9. The Formation of Fibrils by Intertwining of Filaments: Model and Application to Amyloid Aβ Protein

    PubMed Central

    van Gestel, Jeroen; de Leeuw, Simon W.

    2007-01-01

    We outline a model that describes the interaction of rods that form intertwined bundles. In this simple model, we compare the elastic energy penalty that arises due to the deformation of the rods to the gain in binding energy upon intertwining. We find that, for proper values of the bending Young's modulus and the binding energy, a helical pitch may be found for which the energy of intertwining is most favorable. We apply our description to the problem of Alzheimer's Aβ protein fibrillization. If we forbid configurations that exhibit steric overlap between the protofilaments that make up a protein fibril, our model predicts that fibrils consisting of three protofilaments shall form. This agrees well with experimental results. Our model can also provide an estimate for the helical pitch of suitable fibrils. PMID:17114229

  10. Hydrodynamic behavior of shaking flasks used for producing a recombinant protein by filamentous bacteria

    NASA Astrophysics Data System (ADS)

    Cordova Aguilar, Maria Soledad; Garcia, Monica; Trujillo-Roldan, Mauricio Alberto; Ascanio, Gabriel; Zenit, Roberto; Soto, Enrique

    2012-11-01

    Shake flasks are widely used for culture research. The agitation rate is one of the factors that determines the mass transfer. However, it has not been studied in detail. In this work, a comparison of the hydrodynamic performance for conventional, baffled and coiled spring Erlenmeyer flasks is presented. The velocity fields for a horizontal plane were measured by means of a Particle Image Velocimetry (PIV) technique and high speed videos were recorded to observe the behavior of the interface as a function of the agitation rate. It was observed not only that there is a strong dependence between the geometry and the hydrodynamics, but also there is a good agreement with the results obtained previously by Gamboa et al., in 2011, with the evaluation of the influence of culture conditions of S. lividans on protein O-glycosylation. The turbulence intensity increases with shaken rate. However, for the baffled geometry, it was observed a decrease for a critical speed, which is related with the in-phase and out-phase regions. These results can be an explanation for the variations in protein productivity as a function of the flask geometry and the differences in aggregation morphology and the pattern of O-glycosylation of the recombinant protein.

  11. Exosome engineering for efficient intracellular delivery of soluble proteins using optically reversible protein–protein interaction module

    PubMed Central

    Yim, Nambin; Ryu, Seung-Wook; Choi, Kyungsun; Lee, Kwang Ryeol; Lee, Seunghee; Choi, Hojun; Kim, Jeongjin; Shaker, Mohammed R.; Sun, Woong; Park, Ji-Ho; Kim, Daesoo; Do Heo, Won; Choi, Chulhee

    2016-01-01

    Nanoparticle-mediated delivery of functional macromolecules is a promising method for treating a variety of human diseases. Among nanoparticles, cell-derived exosomes have recently been highlighted as a new therapeutic strategy for the in vivo delivery of nucleotides and chemical drugs. Here we describe a new tool for intracellular delivery of target proteins, named ‘exosomes for protein loading via optically reversible protein–protein interactions' (EXPLORs). By integrating a reversible protein–protein interaction module controlled by blue light with the endogenous process of exosome biogenesis, we are able to successfully load cargo proteins into newly generated exosomes. Treatment with protein-loaded EXPLORs is shown to significantly increase intracellular levels of cargo proteins and their function in recipient cells in vitro and in vivo. These results clearly indicate the potential of EXPLORs as a mechanism for the efficient intracellular transfer of protein-based therapeutics into recipient cells and tissues. PMID:27447450

  12. Pseudomonas fluorescens Filamentous Hemagglutinin, an Iron-Regulated Protein, Is an Important Virulence Factor that Modulates Bacterial Pathogenicity

    PubMed Central

    Sun, Yuan-Yuan; Chi, Heng; Sun, Li

    2016-01-01

    Pseudomonas fluorescens is a common bacterial pathogen to a wide range of aquaculture animals including various species of fish. In this study, we employed proteomic analysis and identified filamentous hemagglutinin (FHA) as an iron-responsive protein secreted by TSS, a pathogenic P. fluorescens isolate. In vitro study showed that compared to the wild type, the fha mutant TSSfha (i) exhibited a largely similar vegetative growth profile but significantly retarded in the ability of biofilm growth and producing extracellular matrix, (ii) displayed no apparent flagella and motility, (iii) was defective in the attachment to host cells and unable to form self-aggregation, (iv) displayed markedly reduced capacity of hemagglutination and surviving in host serum. In vivo infection analysis revealed that TSSfha was significantly attenuated in the ability of dissemination in fish tissues and inducing host mortality, and that antibody blocking of the natural FHA produced by the wild type TSS impaired the infectivity of the pathogen. Furthermore, when introduced into turbot as a subunit vaccine, recombinant FHA elicited a significant protection against lethal TSS challenge. Taken together, these results indicate for the first time that P. fluorescens FHA is a key virulence factor essential to multiple biological processes associated with pathogenicity. PMID:27602029

  13. Nonequilibrium Diffusion and Capture Mechanism Ensures Tip Localization of Regulating Proteins on Dynamic Filaments.

    PubMed

    Reithmann, Emanuel; Reese, Louis; Frey, Erwin

    2016-08-12

    Diffusive motion of regulatory enzymes on biopolymers with eventual capture at a reaction site is a common feature in cell biology. Using a lattice gas model we study the impact of diffusion and capture for a microtubule polymerase and a depolymerase. Our results show that the capture mechanism localizes the proteins and creates large-scale spatial correlations. We develop an analytic approximation that globally accounts for relevant correlations and yields results that are in excellent agreement with experimental data. Our results show that diffusion and capture operates most efficiently at cellular enzyme concentrations which points to in vivo relevance.

  14. Nonequilibrium Diffusion and Capture Mechanism Ensures Tip Localization of Regulating Proteins on Dynamic Filaments

    NASA Astrophysics Data System (ADS)

    Reithmann, Emanuel; Reese, Louis; Frey, Erwin

    2016-08-01

    Diffusive motion of regulatory enzymes on biopolymers with eventual capture at a reaction site is a common feature in cell biology. Using a lattice gas model we study the impact of diffusion and capture for a microtubule polymerase and a depolymerase. Our results show that the capture mechanism localizes the proteins and creates large-scale spatial correlations. We develop an analytic approximation that globally accounts for relevant correlations and yields results that are in excellent agreement with experimental data. Our results show that diffusion and capture operates most efficiently at cellular enzyme concentrations which points to in vivo relevance.

  15. CARMIL is a potent capping protein antagonist: identification of a conserved CARMIL domain that inhibits the activity of capping protein and uncaps capped actin filaments.

    PubMed

    Uruno, Takehito; Remmert, Kirsten; Hammer, John A

    2006-04-14

    Acanthamoeba CARMIL was previously shown to co-purify with capping protein (CP) and to bind pure CP. Here we show that this interaction inhibits the barbed end-capping activity of CP. Even more strikingly, this interaction drives the uncapping of actin filaments previously capped with CP. These activities are CP-specific; CARMIL does not inhibit the capping activities of either gelsolin or CapG and does not uncap gelsolin-capped filaments. Although full-length (FL) CARMIL (residues 1-1121) possesses both anti-CP activities, C-terminal fragments like glutathione S-transferase (GST)-P (940-1121) that contain the CARMIL CP binding site are at least 10 times more active. We localized the full activities of GST-P to its C-terminal 51 residues (1071-1121). This sequence contains a stretch of 25 residues that is highly conserved in CARMIL proteins from protozoa, flies, worms, and vertebrates (CARMIL Homology domain 3; CAH3). Point mutations showed that the majority of the most highly conserved residues within CAH3 are critical for the anti-CP activity of GST-AP (862-1121). Finally, we found that GST-AP binds CP approximately 20-fold more tightly than does FL-CARMIL. This observation together with the elevated activities of C-terminal fragments relative to FL-CARMIL suggests that FL-CARMIL might exist primarily in an autoinhibited state. Consistent with this idea, proteolytic cleavage of FL-CARMIL with thrombin generated an approximately 14-kDa C-terminal fragment that expresses full anti-CP activities. We propose that, after some type of physiological activation event, FL-CARMIL could function in vivo as a potent CP antagonist. Given the pivotal role that CP plays in determining the global actin phenotype of cells, our results suggest that CARMIL may play an important role in the physiological regulation of actin assembly.

  16. The respiratory syncytial virus fusion protein targets to the perimeter of inclusion bodies and facilitates filament formation by a cytoplasmic tail-dependent mechanism.

    PubMed

    Baviskar, Pradyumna S; Hotard, Anne L; Moore, Martin L; Oomens, Antonius G P

    2013-10-01

    The human respiratory syncytial virus (HRSV) fusion (F) protein cytoplasmic tail (CT) and matrix (M) protein are key mediators of viral assembly, but the underlying mechanisms are poorly understood. A complementation assay was developed to systematically examine the role of the F protein CT in infectious virus production. The ability of F mutants with alanine substitutions in the CT to complement an F-null virus in generating infectious progeny was quantitated by flow cytometry. Two CT regions with impact on infectious progeny production were identified: residues 557 to 566 (CT-R1) and 569 to 572 (CT-R2). Substitutions in CT-R1 decreased infectivity by 40 to 85% and increased the level of F-induced cell-cell fusion but had little impact on assembly of viral surface filaments, which are believed to be virions. Substitutions in CT-R2, as well as deletion of the entire CT, abrogated infectious progeny production and impaired viral filament formation. However, CT-R2 mutations did not block but rather delayed the formation of viral filaments, which continued to form at a low rate and contained the viral M protein and nucleoprotein (N). Microscopy analysis revealed that substitutions in CT-R2 but not CT-R1 led to accumulation of M and F proteins within and at the perimeter of viral inclusion bodies (IBs), respectively. The accumulation of M and F at IBs and coincident strong decrease in filament formation and infectivity upon CT-R2 mutations suggest that F interaction with IBs is an important step in the virion assembly process and that CT residues 569 to 572 act to facilitate release of M-ribonucleoprotein complexes from IBs.

  17. Redox regulation by reversible protein S-thiolation in bacteria

    PubMed Central

    Loi, Vu Van; Rossius, Martina; Antelmann, Haike

    2015-01-01

    Low molecular weight (LMW) thiols function as thiol-redox buffers to maintain the reduced state of the cytoplasm. The best studied LMW thiol is the tripeptide glutathione (GSH) present in all eukaryotes and Gram-negative bacteria. Firmicutes bacteria, including Bacillus and Staphylococcus species utilize the redox buffer bacillithiol (BSH) while Actinomycetes produce the related redox buffer mycothiol (MSH). In eukaryotes, proteins are post-translationally modified to S-glutathionylated proteins under conditions of oxidative stress. S-glutathionylation has emerged as major redox-regulatory mechanism in eukaryotes and protects active site cysteine residues against overoxidation to sulfonic acids. First studies identified S-glutathionylated proteins also in Gram-negative bacteria. Advances in mass spectrometry have further facilitated the identification of protein S-bacillithiolations and S-mycothiolation as BSH- and MSH-mixed protein disulfides formed under oxidative stress in Firmicutes and Actinomycetes, respectively. In Bacillus subtilis, protein S-bacillithiolation controls the activities of the redox-sensing OhrR repressor and the methionine synthase MetE in vivo. In Corynebacterium glutamicum, protein S-mycothiolation was more widespread and affected the functions of the maltodextrin phosphorylase MalP and thiol peroxidase (Tpx). In addition, novel bacilliredoxins (Brx) and mycoredoxins (Mrx1) were shown to function similar to glutaredoxins in the reduction of BSH- and MSH-mixed protein disulfides. Here we review the current knowledge about the functions of the bacterial thiol-redox buffers glutathione, bacillithiol, and mycothiol and the role of protein S-thiolation in redox regulation and thiol protection in model and pathogenic bacteria. PMID:25852656

  18. The tobamovirus Turnip Vein Clearing Virus 30-kilodalton movement protein localizes to novel nuclear filaments to enhance virus infection.

    PubMed

    Levy, Amit; Zheng, Judy Y; Lazarowitz, Sondra G

    2013-06-01

    Plant viruses overcome the barrier of the plant cell wall by encoding cell-to-cell movement proteins (MPs), which direct newly replicated viral genomes to, and across, the wall. The paradigm for how a single MP regulates and coordinates these activities is the Tobacco mosaic virus (TMV) 30-kDa protein (MP(TMV)). Detailed studies demonstrate that TMV multiplies exclusively in the cytoplasm and have documented associations of MP(TMV) with endoplasmic reticulum (ER) membrane, microtubules, and plasmodesmata throughout the course of infection. As TMV poorly infects Arabidopsis thaliana, Turnip vein clearing virus (TVCV) is the tobamovirus of choice for studies in this model plant. A key problem, which has contributed to confusion in the field, is the unproven assumption that the TVCV and TMV life cycles are identical. We engineered an infectious TVCV replicon that expressed a functional fluorescence-tagged MP(TVCV) and report here the unexpected discovery that MP(TVCV), beyond localizing to ER membrane and plasmodesmata, targeted to the nucleus in a nuclear localization signal (NLS)-dependent manner, where it localized to novel F-actin-containing filaments that associated with chromatin. The MP(TVCV) NLS appeared to be conserved in the subgroup 3 tobamoviruses, and our mutational analyses showed that nuclear localization of MP(TVCV) was necessary for efficient TVCV cell-to-cell movement and systemic infection in Nicotiana benthamiana and Arabidopsis thaliana. Our studies identify a novel nuclear stage in TVCV infection and suggest that nuclear MP encoded by TVCV and other subgroup 3 tobamoviruses interacts with F-actin and chromatin to modulate host defenses or cellular physiology to favor virus movement and infection.

  19. Quantum Dots-based Reverse Phase Protein Microarray

    SciTech Connect

    Shingyoji, Masato; Gerion, Daniele; Pinkel, Dan; Gray, Joe W.; Chen, Fanqing

    2005-07-15

    CdSe nanocrystals, also called quantum dots (Qdots) are a novel class of fluorophores, which have a diameter of a few nanometers and possess high quantum yield, tunable emission wavelength and photostability. They are an attractive alternative to conventional fluorescent dyes. Quantum dots can be silanized to be soluble in aqueous solution under biological conditions, and thus be used in bio-detection. In this study, we established a novel Qdot-based technology platform that can perform accurate and reproducible quantification of protein concentration in a crude cell lysate background. Protein lysates have been spiked with a target protein, and a dilution series of the cell lysate with a dynamic range of three orders of magnitude has been used for this proof-of-concept study. The dilution series has been spotted in microarray format, and protein detection has been achieved with a sensitivity that is at least comparable to standard commercial assays, which are based on horseradish peroxidase (HRP) catalyzed diaminobenzidine (DAB) chromogenesis. The data obtained through the Qdot method has shown a close linear correlation between relative fluorescence unit and relative protein concentration. The Qdot results are in almost complete agreement with data we obtained with the well-established HRP-DAB colorimetric array (R{sup 2} = 0.986). This suggests that Qdots can be used for protein quantification in microarray format, using the platform presented here.

  20. Gelsolin, a protein that caps the barbed ends and severs actin filaments, enhances the actin-based motility of Listeria monocytogenes in host cells.

    PubMed

    Laine, R O; Phaneuf, K L; Cunningham, C C; Kwiatkowski, D; Azuma, T; Southwick, F S

    1998-08-01

    The actin-based motility of Listeria monocytogenes requires the addition of actin monomers to the barbed or plus ends of actin filaments. Immunofluorescence micrographs have demonstrated that gelsolin, a protein that both caps barbed ends and severs actin filaments, is concentrated directly behind motile bacteria at the junction between the actin filament rocket tail and the bacterium. In contrast, CapG, a protein that strictly caps actin filaments, fails to localize near intracellular Listeria. To explore the effect of increasing concentrations of gelsolin on bacterial motility, NIH 3T3 fibroblasts stably transfected with gelsolin cDNA were infected with Listeria. The C5 cell line containing 2.25 times control levels of gelsolin supported significantly higher velocities of bacterial movement than did control fibroblasts (mean +/- standard error of the mean, 0.09 +/- 0.003 micro(m)/s [n = 176] versus 0.05 +/- 0.003 micro(m)/s [n = 65]). The rate of disassembly of the Listeria-induced actin filament rocket tail was found to be independent of gelsolin content. Therefore, if increases in gelsolin content result in increases in Listeria-induced rocket tail assembly rates, a positive correlation between gelsolin content and tail length would be expected. BODIPY-phalloidin staining of four different stably transfected NIH 3T3 fibroblast cell lines confirmed this expectation (r = 0.92). Rocket tails were significantly longer in cells with a high gelsolin content. Microinjection of gelsolin 1/2 (consisting of the amino-terminal half of native gelsolin) also increased bacterial velocity by more than 2.2 times. Microinjection of CapG had no effect on bacterial movement. Cultured skin fibroblasts derived from gelsolin-null mice were capable of supporting intracellular Listeria motility at velocities comparable to those supported by wild-type skin fibroblasts. These experiments demonstrated that the surface of Listeria contains a polymerization zone that can block the barbed

  1. Reverse transcriptase-related proteins in telomeres and in certain chromosomal loci of Rhynchosciara (Diptera: Sciaridae).

    PubMed

    Gorab, Eduardo

    2003-04-01

    The localization of reverse transcriptase-related proteins in polytene chromosomes of dipterans was investigated using previously characterized antibodies to a recombinant polypeptide containing conserved motifs of insect reverse transcriptases. The immunoreactions were carried out with polytene chromosome squashes of eight sciarids, one chironomid and three Drosophila species. Telomeric staining was regularly observed on chromosomes of the sciarid Rhynchosciara americana under normal growth conditions. Five of eight chromosomal tips were labelled except for the heterochromatic ends that are occasionally found associated forming a chromocentre in the salivary gland. Reverse transcriptase-related proteins were detected at chromosomal tips of young larvae and remained bound to the telomeres throughout larval development. As in salivary gland chromosomes, five non-telocentric ends of the chromosomes from Malpighian tubules of R. americana appeared clearly stained with anti-reverse transcriptase. The occurrence of telomeric reverse transcriptase in R. americana correlates with the presence of RNA in addition to an unusual enrichment with homopolymeric dA/dT DNA associated with the telomeric heterochromatin. The antibodies also reacted with a few interstitial sites in chromosomes of four Rhynchosciara species, one band overlapping the histone gene locus of three species in the americana -like group. The results provide evidence for a reverse transcriptase-related protein as a constitutive component in telomeres of R. americana and also in certain interstitial loci of Rhynchosciara species in which RNA was immunologically detected in the form of RNA:DNA hybrids.

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

    PubMed

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

    2014-01-24

    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.

  3. 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.

  4. Tunable and reversible drug control of protein production via a self-excising degron

    PubMed Central

    Chung, Hokyung K.; Jacobs, Conor L.; Huo, Yunwen; Yang, Jin; Krumm, Stefanie A.; Plemper, Richard K.; Tsien, Roger Y.; Lin, Michael Z.

    2015-01-01

    An effective method for direct chemical control over the production of specific proteins would be widely useful. We describe Small Molecule-Assisted Shutoff (SMASh), a technique in which proteins are fused to a degron that removes itself in the absence of drug, leaving untagged protein. Clinically tested HCV protease inhibitors can then block degron removal, inducing rapid degradation of subsequently synthesized protein copies. SMASh allows reversible and dose-dependent shutoff of various proteins in multiple mammalian cell types and in yeast. We also used SMASh to confer drug responsiveness onto a RNA virus for which no licensed inhibitors exist. As SMASh does not require permanent fusion of a large domain, it should be useful when control over protein production with minimal structural modification is desired. Furthermore, as SMASh only involves a single genetic modification and does not rely on modulating protein-protein interactions, it should be easy to generalize to multiple biological contexts. PMID:26214256

  5. Image reversal for direct electron beam patterning of protein coated surfaces.

    PubMed

    Pesen, Devrim; Erlandsson, Anna; Ulfendahl, Mats; Haviland, David B

    2007-11-01

    Electron beam lithography (EBL) is used to create surfaces with protein patterns, which are characterized by immunofluorescence and atomic force microscopies. Both negative and positive image processes are realized by electron beam irradiation of proteins absorbed on a silicon surface, where image reversal is achieved by selectively binding a second species of protein to the electron beam exposed areas on the first protein layer. Biofunctionality at the cellular level was established by culturing cortical cells on patterned lines of fibronectin adsorbed on a bovine serum albumin background for 7 days in culture.

  6. Direct CRISPR spacer acquisition from RNA by a natural reverse-transcriptase-Cas1 fusion protein

    PubMed Central

    Sidote, David J.; Markham, Laura M.; Sanchez-Amat, Antonio; Bhaya, Devaki; Lambowitz, Alan M.; Fire, Andrew Z.

    2016-01-01

    CRISPR (Clustered Regularly Interspaced Short Palindromic Repeat) systems mediate adaptive immunity in diverse prokaryotes. CRISPR-associated Cas1 and Cas2 proteins have been shown to enable adaptation to new threats in Type I and II CRISPR systems by the acquisition of short segments of DNA (“spacers”) from invasive elements. In several Type III CRISPR systems, Cas1 is naturally fused to a reverse transcriptase (RT). In the marine bacterium Marinomonas mediterranea (MMB-1), we show that an RT-Cas1 fusion enables the acquisition of RNA spacers in vivo in an RT-dependent manner. In vitro, the MMB-1 RT-Cas1 and Cas2 proteins catalyze ligation of RNA segments into the CRISPR array, followed by reverse transcription. These observations outline a host-mediated mechanism for reverse information flow from RNA to DNA. PMID:26917774

  7. A reversibly photoswitchable GFP-like protein with fluorescence excitation decoupled from switching.

    PubMed

    Brakemann, Tanja; Stiel, Andre C; Weber, Gert; Andresen, Martin; Testa, Ilaria; Grotjohann, Tim; Leutenegger, Marcel; Plessmann, Uwe; Urlaub, Henning; Eggeling, Christian; Wahl, Markus C; Hell, Stefan W; Jakobs, Stefan

    2011-09-11

    Photoswitchable fluorescent proteins have enabled new approaches for imaging cells, but their utility has been limited either because they cannot be switched repeatedly or because the wavelengths for switching and fluorescence imaging are strictly coupled. We report a bright, monomeric, reversibly photoswitchable variant of GFP, Dreiklang, whose fluorescence excitation spectrum is decoupled from that for optical switching. Reversible on-and-off switching in living cells is accomplished at illumination wavelengths of ∼365 nm and ∼405 nm, respectively, whereas fluorescence is elicited at ∼515 nm. Mass spectrometry and high-resolution crystallographic analysis of the same protein crystal in the photoswitched on- and off-states demonstrate that switching is based on a reversible hydration/dehydration reaction that modifies the chromophore. The switching properties of Dreiklang enable far-field fluorescence nanoscopy in living mammalian cells using both a coordinate-targeted and a stochastic single molecule switching approach.

  8. Structural feature extraction protocol for classifying reversible membrane binding protein domains.

    PubMed

    Källberg, Morten; Lu, Hui

    2009-01-01

    Machine learning based classification protocols for automated function annotation of protein structures have in many instances proven superior to simpler sequence based procedures. Here we present an automated method for extracting features from protein structures by construction of surface patches to be used in such protocols. The utility of the developed patch-growing procedure is exemplified by its ability to identify reversible membrane binding domains from the C1, C2, and PH families.

  9. Phosphoprotein Stability in Clinical Tissue and Its Relevance for Reverse Phase Protein Microarray Technology

    PubMed Central

    Espina, Virginia; Mueller, Claudius; Liotta, Lance A.

    2013-01-01

    Phosphorylated proteins reflect the activity of specific cell signaling nodes in biological kinase protein networks. Cell signaling pathways can be either activated or deactivated depending on the phosphorylation state of the constituent proteins. The state of these kinase pathways reflects the in vivo activity of the cells and tissue at any given point in time. As such, cell signaling pathway information can be extrapolated to infer which phosphorylated proteins/pathways are driving an individual tumor’s growth. Reverse Phase Protein Microarrays (RPMA) are a sensitive and precise platform that can be applied to the quantitative measurement of hundreds of phosphorylated signal proteins from a small sample of tissue. Pre-analytical variability originating from tissue procurement and preservation may cause significant variability and bias in downstream molecular analysis. Depending on the ex vivo delay time in tissue processing, and the manner of tissue handling, protein biomarkers such as signal pathway phosphoproteins will be elevated or suppressed in a manner that does not represent the biomarker levels at the time of excision. Consequently, assessment of the state of these kinase networks requires stabilization, or preservation, of the phosphoproteins immediately post tissue procurement. We have employed reverse phase protein microarray analysis of phosphoproteins to study the factors influencing stability of phosphoproteins in tissue following procurement. Based on this analysis we have established tissue procurement guidelines for clinical research with an emphasis on quantifying phosphoproteins by RPMA. PMID:21901591

  10. Protein alkylation by the α,β-unsaturated aldehyde acrolein. A reversible mechanism of electrophile signaling?

    PubMed Central

    Randall, Matthew J.; Hristova, Milena; van der Vliet, Albert

    2013-01-01

    Acrolein, a reactive aldehyde found in cigarette smoke, is thought to induce its biological effects primarily by irreversible adduction to cellular nucleophiles such as cysteine thiols. Here, we demonstrate that acrolein rapidly inactivates the seleno-enzyme thioredoxin reductase (TrxR) in human bronchiolar epithelial HBE1 cells, which recovered over 4-8 hrs by a mechanism depending on the presence of cellular GSH and thioredoxin 1 (Trx1), and corresponding with reversal of protein-acrolein adduction. Our findings indicate that acrolein-induced protein alkylation is not necessarily a feature of irreversible protein damage, but may reflect a reversible signaling mechanism that is regulated by GSH and Trx1. PMID:24157358

  11. Regulation of protein phosphorylation of the intermediate-sized filament vimentin in the ciliary epithelium of the mammalian eye

    SciTech Connect

    Coca-Prados, M.

    1985-08-25

    The intermediate-sized filaments of vimentin-type (Mr = 57,000) have been identified biochemically and immunochemically as a major cytoskeleton component in the ciliary epithelium of the mammalian eye. When human or rabbit ciliary processes, or cultured ciliary epithelial-derived cells were incubated in serum-free medium containing (TSP)orthophosphate and any of the following agents: 1) beta-adrenergic agonists (isoproterenol or epinephrine), 2) direct activators of adenylate cyclase (cholera toxin or forskolin), 3) analogs of cyclic AMP (8-Br-cAMP), or 4) prostaglandin E1, the phosphorylation of vimentin was significantly enhanced. The maximal enhancement ranged, in vivo and in vitro, from about 3-fold in human to 5-fold in rabbit, with either 1 mM 8-Br-cAMP or 0.1 microM forskolin. Indirect immunofluorescence microscopy using a monoclonal antibody, anti-vimentin, allowed the localization of vimentin filaments in cultured ciliary epithelial cells. Treatment of these cells in culture with the catecholamine hormone, isoproterenol (1 microM), resulted in a profound reorganization of vimentin filaments. This may be correlated with the enhanced levels of phosphorylated vimentin observed upon increasing cellular cyclic AMP.

  12. Biological adhesion of the flatworm Macrostomum lignano relies on a duo-gland system and is mediated by a cell type-specific intermediate filament protein

    PubMed Central

    2014-01-01

    Background Free-living flatworms, in both marine and freshwater environments, are able to adhere to and release from a substrate several times within a second. This reversible adhesion relies on adhesive organs comprised of three cell types: an adhesive gland cell, a releasing gland cell, and an anchor cell, which is a modified epidermal cell responsible for structural support. However, nothing is currently known about the molecules that are involved in this adhesion process. Results In this study we present the detailed morphology of the adhesive organs of the free-living marine flatworm Macrostomum lignano. About 130 adhesive organs are located in a horse-shoe-shaped arc along the ventral side of the tail plate. Each organ consists of exactly three cells, an adhesive gland cell, a releasing gland cell, and an anchor cell. The necks of the two gland cells penetrate the anchor cell through a common pore. Modified microvilli of the anchor cell form a collar surrounding the necks of the adhesive- and releasing glands, jointly forming the papilla, the outer visible part of the adhesive organs. Next, we identified an intermediate filament (IF) gene, macif1, which is expressed in the anchor cells. RNA interference mediated knock-down resulted in the first experimentally induced non-adhesion phenotype in any marine animal. Specifically, the absence of intermediate filaments in the anchor cells led to papillae with open tips, a reduction of the cytoskeleton network, a decline in hemidesmosomal connections, and to shortened microvilli containing less actin. Conclusion Our findings reveal an elaborate biological adhesion system in a free-living flatworm, which permits impressively rapid temporary adhesion-release performance in the marine environment. We demonstrate that the structural integrity of the supportive cell, the anchor cell, is essential for this adhesion process: the knock-down of the anchor cell-specific intermediate filament gene resulted in the inability of

  13. Coevolutionary Analysis Identifies Protein–Protein Interaction Sites between HIV-1 Reverse Transcriptase and Integrase

    PubMed Central

    Hetti Arachchilage, Madara; Piontkivska, Helen

    2016-01-01

    The replication of human immunodeficiency virus-1 (HIV-1) requires reverse transcription of the viral RNA genome and integration of newly synthesized pro-viral DNA into the host genome. This is mediated by the viral proteins reverse transcriptase (RT) and integrase (IN). The formation and stabilization of the pre-integration complex (PIC), which is an essential step for reverse transcription, nuclear import, chromatin targeting, and subsequent integration, involves direct and indirect modes of interaction between RT and IN proteins. While epitope-based treatments targeting IN–viral DNA and IN–RT complexes appear to be a promising combination for an anti-HIV treatment, the mechanisms of IN-RT interactions within the PIC are not well understood due to the transient nature of the protein complex and the intrinsic flexibility of its components. Here, we identify potentially interacting regions between the IN and RT proteins within the PIC through the coevolutionary analysis of amino acid sequences of the two proteins. Our results show that specific regions in the two proteins have strong coevolutionary signatures, suggesting that these regions either experience direct and prolonged interactions between them that require high affinity and/or specificity or that the regions are involved in interactions mediated by dynamic conformational changes and, hence, may involve both direct and indirect interactions. Other regions were found to exhibit weak, but positive correlations, implying interactions that are likely transient and/or have low affinity. We identified a series of specific regions of potential interactions between the IN and RT proteins (e.g., specific peptide regions within the C-terminal domain of IN were identified as potentially interacting with the Connection domain of RT). Coevolutionary analysis can serve as an important step in predicting potential interactions, thus informing experimental studies. These studies can be integrated with structural data

  14. Reversibility and two state behaviour in the thermal unfolding of oligomeric TIM barrel proteins.

    PubMed

    Romero-Romero, Sergio; Costas, Miguel; Rodríguez-Romero, Adela; Alejandro Fernández-Velasco, D

    2015-08-28

    Temperature is one of the main variables that modulate protein function and stability. Thermodynamic studies of oligomeric proteins, the dominant protein natural form, have been often hampered because irreversible aggregation and/or slow reactions are common. There are no reports on the reversible equilibrium thermal unfolding of proteins composed of (β/α)8 barrel subunits, albeit this "TIM barrel" topology is one of the most abundant and versatile in nature. We studied the eponymous TIM barrel, triosephosphate isomerase (TIM), belonging to five species of different bacterial taxa. All of them were found to be catalytically efficient dimers. The three-dimensional structure of four enzymes was solved at high/medium resolution. Irreversibility and kinetic control were observed in the thermal unfolding of two TIMs, while for the other three the thermal unfolding was found to follow a two-state equilibrium reversible process. Shifts in the global stability curves of these three proteins are related to the organismal temperature range of optimal growth and modulated by variations in maximum stability temperature and in the enthalpy change at that temperature. Reversibility appears to correlate with the low isoelectric point, the absence of a residual structure in the unfolded state, small cavity volume in the native state, low conformational stability and a low melting temperature. Furthermore, the strong coupling between dimer dissociation and monomer unfolding may reduce aggregation and favour reversibility. It is therefore very thought-provoking to find that a common topological ensemble, such as the TIM barrel, can unfold/refold in the Anfinsen way, i.e. without the help of the cellular machinery.

  15. Phosphorylation of actin-binding protein (ABP-280; filamin) by tyrosine kinase p56lck modulates actin filament cross-linking.

    PubMed

    Pal Sharma, C; Goldmann, Wolfgang H

    2004-01-01

    Actin-binding protein (ABP-280; filamin) is a phosphoprotein present in the periphery of the cytoplasm where it can cross-link actin filaments, associate with lipid membranes, and bind to membrane surface receptors. Given its function and localization in the cell, we decided to investigate the possibility of whether it serves as substrate for p56lck, a lymphocyte-specific member of the src family of protein tyrosine kinases associated with cell surface glycoproteins. The interaction of p56lck with membrane glycoproteins is important for cell development and functional activation. Here, we show that purified p56lck interacts and catalyzes in vitro kinase reactions. Tyrosine phosphorylation by p56lck is restricted to a single peptide of labeled ABP-280 shown by protease digest. The addition of phorbol ester to cells results in the inhibition of phosphorylation of ABP-280 by p56lck. These results show a decrease in phosphorylation suggesting conformationally induced regulation. Dynamic light scattering confirmed increased actin filament cross-linking due to phosphorylation of ABP-280 by p56lck.

  16. FILAMENTOUS FLOWER, a meristem and organ identity gene of Arabidopsis, encodes a protein with a zinc finger and HMG-related domains.

    PubMed

    Sawa, S; Watanabe, K; Goto, K; Liu, Y G; Shibata, D; Kanaya, E; Morita, E H; Okada, K

    1999-05-01

    Distinctive from that of the animal system, the basic plan of the plant body is the continuous formation of a structural unit, composed of a stem with a meristem at the top and lateral organs continuously forming at the meristem. Therefore, mechanisms controlling the formation, maintenance, and development of a meristem will be a key to understanding the body plan of higher plants. Genetic analyses of filamentous flower (fil) mutants have indicated that FIL is required for the maintenance and growth of inflorescence and floral meristems, and of floral organs of Arabidopsis thaliana. FIL encodes a protein carrying a zinc finger and a HMG box-like domain, which is known to work as a transcription regulator. As expected, the FIL protein was shown to have a nuclear location. In situ hybridization clearly demonstrated that FIL is expressed only at the abaxial side of primordia of leaves and floral organs. Transgenic plants, ectopically expressing FIL, formed filament-like leaves with randomly arranged cells at the leaf margin. Our results indicate that cells at the abaxial side of the lateral organs are responsible for the normal development of the organs as well as for maintaining the activity of meristems.

  17. Phospholipid transfer protein deficiency in mice impairs macrophage reverse cholesterol transport in vivo

    PubMed Central

    Si, Yanhong; Zhang, Ying; Chen, Xiaofeng; Zhai, Lei; Zhou, Guanghai; Yu, Ailing; Cao, Haijun

    2016-01-01

    Phospholipid transfer protein is expressed in various cell types and secreted into plasma, where it transfers phospholipids between lipoproteins and modulates the composition of high-density lipoprotein particles. Phospholipid transfer protein deficiency in vivo can lower high-density lipoprotein cholesterol level significantly and impact the biological quality of high-density lipoprotein. Considering high-density lipoprotein was a critical determinant for reverse cholesterol transport, we investigated the role of systemic phospholipid transfer protein deficiency in macrophage reverse cholesterol transport in vivo. After the littermate phospholipid transfer protein KO and WT mice were fed high-fat diet for one month, they were injected intraperitoneally with 3H-cholesterol-labeled and acLDL-loaded macrophages. Then the appearance of 3H-tracer in plasma, liver, bile, intestinal wall, and feces over 48 h was determined. Plasma lipid analysis indicated phospholipid transfer protein deficiency lowered total cholesterol, high-density lipoprotein-C and apolipoprotein A1 levels significantly but increased triglyceride level in mice. The isotope tracing experiment showed 3H-cholesterol of plasma was decreased by 68% for male and 62% for female, and 3H-tracer of bile was decreased by 37% for male and 21% for female in phospholipid transfer protein KO mice compared with WT mice. However, there was no difference in liver, and 3H-tracer of intestinal wall was increased by 43% for male and 27% for female. Finally, 3H-tracer of fecal excretion in phospholipid transfer protein KO mice was reduced significantly by 36% for male and 43% for female during 0–24 h period, but there was no significant difference during 24–48 h period. Meanwhile, Western Blot analysis showed the expressions of reverse cholesterol transport -related protein liver X receptor α (LXRα), ATP binding cassette transporter A1, and cholesterol 7α-hydroxylase A1 were upregulated in liver of

  18. The cytokeratin filament-aggregating protein filaggrin is the target of the so-called "antikeratin antibodies," autoantibodies specific for rheumatoid arthritis.

    PubMed Central

    Simon, M; Girbal, E; Sebbag, M; Gomès-Daudrix, V; Vincent, C; Salama, G; Serre, G

    1993-01-01

    In rheumatoid arthritis (RA), the high diagnostic value of serum antibodies to the stratum corneum of rat esophagus epithelium has been widely reported. These so-called "antikeratin antibodies," detected by indirect immunofluorescence, were found to be autoantibodies since they also labeled human epidermis. Despite their name, the actual target of these autoantibodies was not known. In this study, a 40-kD protein (designated as 40K), extracted from human epidermis and specifically immunodetected by 75% of RA sera, was purified and identified as a neutral/acidic isoform of basic filaggrin, a cytokeratin filament-aggregating protein, by peptide mapping studies and by the following evidences: (a) mAbs specific for filaggrin reacted with the 40K protein; (b) the autoantibodies, affinity-purified from RA sera on the 40K protein, immunodetected purified filaggrin; (c) the reactivity of RA sera to the 40K protein was abolished after immunoadsorption with purified filaggrin; (d) the 40K protein and filaggrin had similar amino acid compositions. Furthermore, autoantibodies against the 40K protein and the so-called "antikeratin antibodies" were shown, by immunoadsorption experiments, to be largely the same. The identification of filaggrin as a RA-specific autoantigen could contribute to the understanding of the pathogenesis of this disease and, ultimately, to the development of methods for preventing the autoimmune response. Images PMID:7690781

  19. CacyBP/SIP as a novel modulator of the thin filament.

    PubMed

    Jurewicz, Ewelina; Ostrowska, Zofia; Jozwiak, Jolanta; Redowicz, Maria Jolanta; Lesniak, Wieslawa; Moraczewska, Joanna; Filipek, Anna

    2013-03-01

    The CacyBP/SIP protein interacts with several targets, including actin. Since the majority of actin filaments are associated with tropomyosin, in this work we characterized binding of CacyBP/SIP to the actin-tropomyosin complex and examined the effects of CacyBP/SIP on actin filament functions. By using reconstituted filaments composed of actin and AEDANS-labeled tropomyosin, we observed that binding of CacyBP/SIP caused an increase in tropomyosin fluorescence intensity indicating the occurrence of conformational changes within the filament. We also found that CacyBP/SIP bound directly to tropomyosin and that these proteins did not compete with each other for binding to actin. Electron microscopy showed that in the absence of tropomyosin CacyBP/SIP destabilized actin filaments, but tropomyosin reversed this effect. Actin-activated myosin S1 ATPase activity assays, performed using a colorimetric method, indicated that CacyBP/SIP reduced ATPase activity and that the presence of tropomyosin enhanced this inhibitory effect. Thus, our results suggest that CacyBP/SIP, through its interaction with both actin and tropomyosin, regulates the organization and functional properties of the thin filament.

  20. Single amino acid replacement makes Aequorea victoria fluorescent proteins reversibly photoswitchable.

    PubMed

    Bizzarri, Ranieri; Serresi, Michela; Cardarelli, Francesco; Abbruzzetti, Stefania; Campanini, Barbara; Viappiani, Cristiano; Beltram, Fabio

    2010-01-13

    Reversibly photoswitchable (i.e., photochromic) fluorescent proteins open the way to a number of advanced bioimaging techniques applicable to living-cell studies such as sequential photolabeling of distinct cellular regions, innovative FRET schemes, or nanoscopy. Owing to the relevance of fluorescent proteins from Aequorea victoria (AFPs) for cell biology, a photochromic "toolbox" constituted by several AFPs is highly desirable. Here we introduce four new photochromic AFPs whose reversible photoswitching occurs between the native bright and a dark state at low illumination power, on account of a very efficient cis-trans photoisomerization. Most remarkably, the optical bistability of these AFPs derives from the single E222Q mutation in the primary sequence. Apparently, the E222Q substitution can restore the intrinsic photochromic behavior of the isolated chromophore. The significance of these mutants for high-resolution in vivo cell imaging is shown by means of photochromic FRET experiments.

  1. Reversible stress softening of actin networks

    PubMed Central

    Chaudhuri, Ovijit; Parekh, Sapun H.; Fletcher, Daniel A.

    2011-01-01

    The mechanical properties of cells play an essential role in numerous physiological processes. Organized networks of semiflexible actin filaments determine cell stiffness and transmit force during mechanotransduction, cytokinesis, cell motility and other cellular shape changes1–3. Although numerous actin-binding proteins have been identified that organize networks, the mechanical properties of actin networks with physiological architectures and concentrations have been difficult to measure quantitatively. Studies of mechanical properties in vitro have found that crosslinked networks of actin filaments formed in solution exhibit stress stiffening arising from the entropic elasticity of individual filaments or crosslinkers resisting extension4–8. Here we report reversible stress-softening behaviour in actin networks reconstituted in vitro that suggests a critical role for filaments resisting compression. Using a modified atomic force microscope to probe dendritic actin networks (like those formed in the lamellipodia of motile cells), we observe stress stiffening followed by a regime of reversible stress softening at higher loads. This softening behaviour can be explained by elastic buckling of individual filaments under compression that avoids catastrophic fracture of the network. The observation of both stress stiffening and softening suggests a complex interplay between entropic and enthalpic elasticity in determining the mechanical properties of actin networks. PMID:17230186

  2. Realizing the promise of reverse phase protein arrays for clinical, translational, and basic research: a workshop report: the RPPA (Reverse Phase Protein Array) society.

    PubMed

    Akbani, Rehan; Becker, Karl-Friedrich; Carragher, Neil; Goldstein, Ted; de Koning, Leanne; Korf, Ulrike; Liotta, Lance; Mills, Gordon B; Nishizuka, Satoshi S; Pawlak, Michael; Petricoin, Emanuel F; Pollard, Harvey B; Serrels, Bryan; Zhu, Jingchun

    2014-07-01

    Reverse phase protein array (RPPA) technology introduced a miniaturized "antigen-down" or "dot-blot" immunoassay suitable for quantifying the relative, semi-quantitative or quantitative (if a well-accepted reference standard exists) abundance of total protein levels and post-translational modifications across a variety of biological samples including cultured cells, tissues, and body fluids. The recent evolution of RPPA combined with more sophisticated sample handling, optical detection, quality control, and better quality affinity reagents provides exquisite sensitivity and high sample throughput at a reasonable cost per sample. This facilitates large-scale multiplex analysis of multiple post-translational markers across samples from in vitro, preclinical, or clinical samples. The technical power of RPPA is stimulating the application and widespread adoption of RPPA methods within academic, clinical, and industrial research laboratories. Advances in RPPA technology now offer scientists the opportunity to quantify protein analytes with high precision, sensitivity, throughput, and robustness. As a result, adopters of RPPA technology have recognized critical success factors for useful and maximum exploitation of RPPA technologies, including the following: preservation and optimization of pre-analytical sample quality, application of validated high-affinity and specific antibody (or other protein affinity) detection reagents, dedicated informatics solutions to ensure accurate and robust quantification of protein analytes, and quality-assured procedures and data analysis workflows compatible with application within regulated clinical environments. In 2011, 2012, and 2013, the first three Global RPPA workshops were held in the United States, Europe, and Japan, respectively. These workshops provided an opportunity for RPPA laboratories, vendors, and users to share and discuss results, the latest technology platforms, best practices, and future challenges and

  3. Light-Activated Reversible Imine Isomerization: Towards a Photochromic Protein Switch

    PubMed Central

    Berbasova, Tetyana; Santos, Elizabeth M.; Nosrati, Meisam; Vasileiou, Chrysoula; Geiger, James H.; Borhan, Babak

    2016-01-01

    Mutants of cellular retinoic acid-binding protein II (CRABPII), engineered to bind all-trans-retinal as an iminium species, demonstrate photochromism upon irradiation with light at different wavelengths. UV light irradiation populates the cis-imine geometry, which has a high pKa, leading to protonation of the imine and subsequent “turn-on” of color. Yellow light irradiation yields the trans-imine isomer, which has a depressed pKa, leading to loss of color because the imine is not protonated. The protein-bound retinylidene chromophore undergoes photoinduced reversible interconversion between the colored and uncolored species, with excellent fatigue resistance. PMID:26684483

  4. Protein and water confined in nanometer-scale reverse micelles studied by near infrared, terahertz, and ultrafast visible spectroscopies.

    PubMed

    Murakami, Hiroshi

    2013-01-01

    Protein-containing reverse (PCR) micelles are suitable systems to study the properties of proteins and waters in a cell-like environment. A model for determining the structural parameters of PCR micelles, such as the aqueous cavity size and molecule number of water within the reverse micelle, is presented. The model is based on an important hypothesis that the structural parameters of the protein-unfilled reverse micelle do not change after solubilization of protein. I describe a procedure using near infrared spectroscopy of OH stretching vibration band of water to verify the hypothesis. Further, the terahertz (THz) absorption spectrum of myoglobin is derived from THz time-domain spectroscopy of the PCR micellar solution, and the states of waters in reverse micelles with and without protein are discussed on the basis of the structural parameters. The last topic is on internal dynamics of PCR micelles on timescales from femtoseconds to nanoseconds studied by femtosecond time-resolved fluorescence spectroscopy.

  5. Actin filament-associated protein 1 is required for cSrc activity and secretory activation in the lactating mammary gland

    PubMed Central

    Cunnick, Jess M; Kim, Stephanie; Hadsell, James; Collins, Stephen; Cerra, Carmine; Reiser, Patti; Flynn, Daniel C; Cho, Youngjin

    2014-01-01

    Actin filament-associated protein 1 (AFAP1) is an adaptor protein of cSrc that binds to filamentous actin and regulates the activity of this tyrosine kinase to affect changes to the organization of the actin cytoskeleton. In breast and prostate cancer cells, AFAP1 has been shown to regulate cellular responses requiring actin cytoskeletal changes such as adhesion, invadopodia formation and invasion. However, a normal physiological role for AFAP1 has remained elusive. In this study, we generated an AFAP1 knockout mouse model that establishes a novel physiological role for AFAP1 in lactation. Specifically, these animals displayed a defect in lactation that resulted in an inability to efficiently nurse. Histologically, the mammary glands of the lactating knockout mice were distinguished by the accumulation of large cytoplasmic lipid droplets in the alveolar epithelial cells. There was a reduction in lipid synthesis and the expression of lipogenic genes without a corresponding reduction in the production of beta-casein, a milk protein. Furthermore, these defects were associated with histological and biochemical signs of precocious involution. This study also demonstrated that AFAP1 responds to prolactin, a lactogenic hormone, by forming a complex with cSrc and becoming tyrosine phosphorylated. Together, these observations pointed to a defect in secretory activation. Certain characteristics of this phenotype mirrored the defect in secretory activation in the cSrc knockout mouse, but most importantly, the activity of cSrc in the mammary gland was reduced during early lactation in the AFAP1 null mouse and the localization of active cSrc at the apical surface of luminal epithelial cells during lactation was selectively lost in the absence of AFAP1. These data define, for the first time, the requirement of AFAP1 for the spatial and temporal regulation of cSrc activity in the normal breast, specifically for milk production. PMID:25043309

  6. Self-association of the spindle pole body-related intermediate filament protein Fin1p and its phosphorylation-dependent interaction with 14-3-3 proteins in yeast.

    PubMed

    van Hemert, Martijn J; Deelder, André M; Molenaar, Chris; Steensma, H Yde; van Heusden, G Paul H

    2003-04-25

    The Fin1 protein of the yeast Saccharomyces cerevisiae forms filaments between the spindle pole bodies of dividing cells. In the two-hybrid system it binds to 14-3-3 proteins, which are highly conserved proteins involved in many cellular processes and which are capable of binding to more than 120 different proteins. Here, we describe the interaction of the Fin1 protein with the 14-3-3 proteins Bmh1p and Bmh2p in more detail. Purified Fin1p interacts with recombinant yeast 14-3-3 proteins. This interaction is strongly reduced after dephosphorylation of Fin1p. Surface plasmon resonance analysis showed that Fin1p has a higher affinity for Bmh2p than for Bmh1p (K(D) 289 versus 585 nm). Sequences in both the central and C-terminal part of Fin1p are required for the interaction with Bmh2p in the two-hybrid system. In yeast strains lacking 14-3-3 proteins Fin1 filament formation was observed, indicating that the 14-3-3 proteins are not required for this process. Fin1 also interacts with itself in the two-hybrid system. For this interaction sequences at the C terminus, containing one of two putative coiled-coil regions, are sufficient. Fin1p-Fin1p interactions were demonstrated in vivo by fluorescent resonance energy transfer between cyan fluorescent protein-labeled Fin1p and yellow fluorescent protein-labeled Fin1p.

  7. Mutation-Specific Effects on Thin Filament Length in Thin Filament Myopathy

    PubMed Central

    de Winter, Josine M.; Joureau, Barbara; Lee, Eun-Jeong; Kiss, Balázs; Yuen, Michaela; Gupta, Vandana A.; Pappas, Christopher T.; Gregorio, Carol C.; Stienen, Ger J. M.; Edvardson, Simon; Wallgren-Pettersson, Carina; Lehtokari, Vilma-Lotta; Pelin, Katarina; Malfatti, Edoardo; Romero, Norma B.; van Engelen, Baziel G.; Voermans, Nicol C.; Donkervoort, Sandra; Bönnemann, C. G.; Clarke, Nigel F.; Beggs, Alan H.; Granzier, Henk; Ottenheijm, Coen A. C.

    2016-01-01

    Objective Thin filament myopathies are among the most common nondystrophic congenital muscular disorders, and are caused by mutations in genes encoding proteins that are associated with the skeletal muscle thin filament. Mechanisms underlying muscle weakness are poorly understood, but might involve the length of the thin filament, an important determinant of force generation. Methods We investigated the sarcomere length-dependence of force, a functional assay that provides insights into the contractile strength of muscle fibers as well as the length of the thin filaments, in muscle fibers from 51 patients with thin filament myopathy caused by mutations in NEB, ACTA1, TPM2, TPM3, TNNT1, KBTBD13, KLHL40, and KLHL41. Results Lower force generation was observed in muscle fibers from patients of all genotypes. In a subset of patients who harbor mutations in NEB and ACTA1, the lower force was associated with downward shifted force–sarcomere length relations, indicative of shorter thin filaments. Confocal microscopy confirmed shorter thin filaments in muscle fibers of these patients. A conditional Neb knockout mouse model, which recapitulates thin filament myopathy, revealed a compensatory mechanism; the lower force generation that was associated with shorter thin filaments was compensated for by increasing the number of sarcomeres in series. This allowed muscle fibers to operate at a shorter sarcomere length and maintain optimal thin–thick filament overlap. Interpretation These findings might provide a novel direction for the development of therapeutic strategies for thin filament myopathy patients with shortened thin filament lengths. PMID:27074222

  8. Pseudoelastic behaviour of a natural material is achieved via reversible changes in protein backbone conformation

    PubMed Central

    Harrington, Matthew J.; Wasko, S. Scott; Masic, Admir; Fischer, F. Dieter; Gupta, Himadri S.; Fratzl, Peter

    2012-01-01

    The egg capsules of marine prosobranch gastropods, commonly know as whelks, function as a protective encapsulant for whelk embryos in wave-swept marine environments. The proteinaceous sheets comprising the wall of whelk egg capsules (WEC) exhibit long-range reversible extensibility with a hysteresis of up to 50 per cent, previously suggested to result from reversible changes in the structure of the constituent protein building blocks. Here, we further investigate the structural changes of the WEC biopolymer at various hierarchical levels using several different time-resolved in situ approaches. We find strong evidence in these biological polymers for a strain-induced reversible transition from an ordered conformational phase to a largely disordered one that leads to the characteristic reversible hysteretic behaviour, which is reminiscent of the pseudoelastic behaviour in some metallic alloys. On the basis of these results, we generate a simple numerical model incorporating a worm-like chain equation to explain the phase transition behaviour of the WEC at the molecular level. PMID:22696489

  9. Generation of monomeric reversibly switchable red fluorescent proteins for far-field fluorescence nanoscopy.

    PubMed

    Stiel, Andre C; Andresen, Martin; Bock, Hannes; Hilbert, Michael; Schilde, Jessica; Schönle, Andreas; Eggeling, Christian; Egner, Alexander; Hell, Stefan W; Jakobs, Stefan

    2008-09-15

    Reversibly switchable fluorescent proteins (RSFPs) are GFP-like proteins that may be repeatedly switched by irradiation with light from a fluorescent to a nonfluorescent state, and vice versa. They can be utilized as genetically encodable probes and bear large potential for a wide array of applications, in particular for new protein tracking schemes and subdiffraction resolution microscopy. However, the currently described monomeric RSFPs emit only blue-green or green fluorescence; the spectral window for their use is thus rather limited. Using a semirational engineering approach based on the crystal structure of the monomeric nonswitchable red fluorescent protein mCherry, we generated rsCherry and rsCherryRev. These two novel red fluorescent RSFPs exhibit fluorescence emission maxima at approximately 610 nm. They display antagonistic switching modes, i.e., in rsCherry irradiation with yellow light induces the off-to-on transition and blue light the on-to-off transition, whereas in rsCherryRev the effects of the switching wavelengths are reversed. We demonstrate time-lapse live-cell subdiffraction microscopy by imaging rsCherryRev targeted to the endoplasmic reticulum utilizing the switching and localization of single molecules.

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

    PubMed Central

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

    2015-01-01

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

  11. Combining phage display with de novo protein sequencing for reverse engineering of monoclonal antibodies.

    PubMed

    Rickert, Keith W; Grinberg, Luba; Woods, Robert M; Wilson, Susan; Bowen, Michael A; Baca, Manuel

    2016-01-01

    The enormous diversity created by gene recombination and somatic hypermutation makes de novo protein sequencing of monoclonal antibodies a uniquely challenging problem. Modern mass spectrometry-based sequencing will rarely, if ever, provide a single unambiguous sequence for the variable domains. A more likely outcome is computation of an ensemble of highly similar sequences that can satisfy the experimental data. This outcome can result in the need for empirical testing of many candidate sequences, sometimes iteratively, to identity one which can replicate the activity of the parental antibody. Here we describe an improved approach to antibody protein sequencing by using phage display technology to generate a combinatorial library of sequences that satisfy the mass spectrometry data, and selecting for functional candidates that bind antigen. This approach was used to reverse engineer 2 commercially-obtained monoclonal antibodies against murine CD137. Proteomic data enabled us to assign the majority of the variable domain sequences, with the exception of 3-5% of the sequence located within or adjacent to complementarity-determining regions. To efficiently resolve the sequence in these regions, small phage-displayed libraries were generated and subjected to antigen binding selection. Following enrichment of antigen-binding clones, 2 clones were selected for each antibody and recombinantly expressed as antigen-binding fragments (Fabs). In both cases, the reverse-engineered Fabs exhibited identical antigen binding affinity, within error, as Fabs produced from the commercial IgGs. This combination of proteomic and protein engineering techniques provides a useful approach to simplifying the technically challenging process of reverse engineering monoclonal antibodies from protein material.

  12. Combining phage display with de novo protein sequencing for reverse engineering of monoclonal antibodies

    PubMed Central

    Rickert, Keith W.; Grinberg, Luba; Woods, Robert M.; Wilson, Susan; Bowen, Michael A.; Baca, Manuel

    2016-01-01

    ABSTRACT The enormous diversity created by gene recombination and somatic hypermutation makes de novo protein sequencing of monoclonal antibodies a uniquely challenging problem. Modern mass spectrometry-based sequencing will rarely, if ever, provide a single unambiguous sequence for the variable domains. A more likely outcome is computation of an ensemble of highly similar sequences that can satisfy the experimental data. This outcome can result in the need for empirical testing of many candidate sequences, sometimes iteratively, to identity one which can replicate the activity of the parental antibody. Here we describe an improved approach to antibody protein sequencing by using phage display technology to generate a combinatorial library of sequences that satisfy the mass spectrometry data, and selecting for functional candidates that bind antigen. This approach was used to reverse engineer 2 commercially-obtained monoclonal antibodies against murine CD137. Proteomic data enabled us to assign the majority of the variable domain sequences, with the exception of 3–5% of the sequence located within or adjacent to complementarity-determining regions. To efficiently resolve the sequence in these regions, small phage-displayed libraries were generated and subjected to antigen binding selection. Following enrichment of antigen-binding clones, 2 clones were selected for each antibody and recombinantly expressed as antigen-binding fragments (Fabs). In both cases, the reverse-engineered Fabs exhibited identical antigen binding affinity, within error, as Fabs produced from the commercial IgGs. This combination of proteomic and protein engineering techniques provides a useful approach to simplifying the technically challenging process of reverse engineering monoclonal antibodies from protein material. PMID:26852694

  13. Actin filament curvature biases branching direction

    NASA Astrophysics Data System (ADS)

    Wang, Evan; Risca, Viviana; Chaudhuri, Ovijit; Chia, Jia-Jun; Geissler, Phillip; Fletcher, Daniel

    2012-02-01

    Actin filaments are key components of the cellular machinery, vital for a wide range of processes ranging from cell motility to endocytosis. Actin filaments can branch, and essential in this process is a protein complex known as the Arp2/3 complex, which nucleate new ``daughter'' filaments from pre-existing ``mother'' filaments by attaching itself to the mother filament. Though much progress has been made in understanding the Arp2/3-actin junction, some very interesting questions remain. In particular, F-actin is a dynamic polymer that undergoes a wide range of fluctuations. Prior studies of the Arp2/3-actin junction provides a very static notion of Arp2/3 binding. The question we ask is how differently does the Arp2/3 complex interact with a straight filament compared to a bent filament? In this study, we used Monte Carlo simulations of a surface-tethered worm-like chain to explore possible mechanisms underlying the experimental observation that there exists preferential branch formation by the Arp2/3 complex on the convex face of a curved filament. We show that a fluctuation gating model in which Arp2/3 binding to the actin filament is dependent upon a rare high-local-curvature shape fluctuation of the filament is consistent with the experimental data.

  14. Precise and Reversible Protein-Microtubule-Like Structure with Helicity Driven by Dual Supramolecular Interactions.

    PubMed

    Yang, Guang; Zhang, Xiang; Kochovski, Zdravko; Zhang, Yufei; Dai, Bin; Sakai, Fuji; Jiang, Lin; Lu, Yan; Ballauff, Matthias; Li, Xueming; Liu, Cong; Chen, Guosong; Jiang, Ming

    2016-02-17

    Protein microtubule is a significant self-assembled architecture found in nature with crucial biological functions. However, mimicking protein microtubules with precise structure and controllable self-assembly behavior remains highly challenging. In this work, we demonstrate that by using dual supramolecular interactions from a series of well-designed ligands, i.e., protein-sugar interaction and π-π stacking, highly homogeneous protein microtubes were achieved from tetrameric soybean agglutinin without any chemical or biological modification. Using combined cryo-EM single-particle reconstruction and computational modeling, the accurate structure of protein microtube was determined. The helical protein microtube is consisted of three protofilaments, each of which features an array of soybean agglutinin tetramer linked by the designed ligands. Notably, the microtubes resemble the natural microtubules in their structural and dynamic features such as the shape and diameter and the controllable and reversible assembly behavior, among others. Furthermore, the protein microtubes showed an ability to enhance immune response, demonstrating its great potential for biological applications.

  15. Effective protein separation by coupling hydrophobic interaction and reverse phase chromatography for top-down proteomics.

    PubMed

    Xiu, Lichen; Valeja, Santosh G; Alpert, Andrew J; Jin, Song; Ge, Ying

    2014-08-05

    One of the challenges in proteomics is the proteome's complexity, which necessitates the fractionation of proteins prior to the mass spectrometry (MS) analysis. Despite recent advances in top-down proteomics, separation of intact proteins remains challenging. Hydrophobic interaction chromatography (HIC) appears to be a promising method that provides high-resolution separation of intact proteins, but unfortunately the salts conventionally used for HIC are incompatible with MS. In this study, we have identified ammonium tartrate as a MS-compatible salt for HIC with comparable separation performance as the conventionally used ammonium sulfate. Furthermore, we found that the selectivity obtained with ammonium tartrate in the HIC mobile phases is orthogonal to that of reverse phase chromatography (RPC). By coupling HIC and RPC as a novel two-dimensional chromatographic method, we have achieved effective high-resolution intact protein separation as demonstrated with standard protein mixtures and a complex cell lysate. Subsequently, the separated intact proteins were identified by high-resolution top-down MS. For the first time, these results have shown the high potential of HIC as a high-resolution protein separation method for top-down proteomics.

  16. Primary Role of the Chromophore Bond Length Alternation in Reversible Photoconversion of Red Fluorescence Proteins

    PubMed Central

    Drobizhev, Mikhail; Hughes, Thomas E.; Stepanenko, Yuriy; Wnuk, Pawel; O'Donnell, Kieran; Scott, J. Nathan; Callis, Patrik R.; Mikhaylov, Alexander; Dokken, Leslie; Rebane, Aleksander

    2012-01-01

    Rapid photobleaching of fluorescent proteins can limit their use in imaging applications. The underlying kinetics is multi-exponential and strongly depends on the local chromophore environment. The first, reversible, step may be attributed to a rotation around one of the two exocyclic C-C bonds bridging phenol and imidazolinone groups in the chromophore. However it is not clear how the protein environment controls this motion - either by steric hindrances or by modulating the electronic structure of the chromophore through electrostatic interactions. Here we study the first step of the photobleaching kinetics in 13 red fluorescent proteins (RFPs) with different chromophore environment and show that the associated rate strongly correlates with the bond length alternation (BLA) of the two bridge bonds. The sign of the BLA appears to determine which rotation is activated. Our results present experimental evidence for the dominance of electronic effects in the conformational dynamics of the RFP chromophore. PMID:23008753

  17. Use of reverse micelles for the simultaneous extraction of oil, proteins, and glucosinolates from cruciferous oilseeds.

    PubMed

    Ugolini, Luisa; De Nicola, Gina; Palmieri, Sandro

    2008-03-12

    Cruciferous oilseeds are important sources of oil, proteins, and glucosinolates (GLs), potentially available when biorefinery processes are used. The proposed extraction technology is based on the use of reverse micelles (RMs) made with cetyltrimethylammonium bromide (CTAB) dispersed in organic solvent. The physicochemical properties of this extraction system and the good water solubility of many high value compounds, such as GLs and some proteins, permit the simultaneous extraction of oil, and these products from cruciferous oilseed meals. This procedure is based on three main steps: (i) seed conditioning; (ii) solid-liquid extraction by RM solution; and (iii) back-transfer of the RM solution for recovery of the extracted compounds. The method makes it possible to simultaneously extract almost the same amount of oil as with pure organic solvents used in the current extraction plants and more than 90% of soluble proteins and GLs. It is a promising biorefinery technology alternative to traditional oil extraction processes.

  18. An improved reversibly dimerizing mutant of the FK506-binding protein FKBP

    PubMed Central

    Barrero, Juan J.; Papanikou, Effrosyni; Casler, Jason C.; Day, Kasey J.; Glick, Benjamin S.

    2016-01-01

    ABSTRACT FK506-binding protein (FKBP) is a monomer that binds to FK506, rapamycin, and related ligands. The F36M substitution, in which Phe36 in the ligand-binding pocket is changed to Met, leads to formation of antiparallel FKBP dimers, which can be dissociated into monomers by ligand binding. This FKBP(M) mutant has been employed in the mammalian secretory pathway to generate aggregates that can be dissolved by ligand addition to create cargo waves. However, when testing this approach in yeast, we found that dissolution of FKBP(M) aggregates was inefficient. An improved reversibly dimerizing FKBP formed aggregates that dissolved more readily. This FKBP(L,V) mutant carries the F36L mutation, which increases the affinity of ligand binding, and the I90V mutation, which accelerates ligand-induced dissociation of the dimers. The FKBP(L,V) mutant expands the utility of reversibly dimerizing FKBP. PMID:27738551

  19. The near-atomic cryoEM structure of a flexible filamentous plant virus shows homology of its coat protein with nucleoproteins of animal viruses.

    PubMed

    Agirrezabala, Xabier; Méndez-López, Eduardo; Lasso, Gorka; Sánchez-Pina, M Amelia; Aranda, Miguel; Valle, Mikel

    2015-12-16

    Flexible filamentous viruses include economically important plant pathogens. Their viral particles contain several hundred copies of a helically arrayed coat protein (CP) protecting a (+)ssRNA. We describe here a structure at 3.9 Å resolution, from electron cryomicroscopy, of Pepino mosaic virus (PepMV), a representative of the genus Potexvirus (family Alphaflexiviridae). Our results allow modeling of the CP and its interactions with viral RNA. The overall fold of PepMV CP resembles that of nucleoproteins (NPs) from the genus Phlebovirus (family Bunyaviridae), a group of enveloped (-)ssRNA viruses. The main difference between potexvirus CP and phlebovirus NP is in their C-terminal extensions, which appear to determine the characteristics of the distinct multimeric assemblies - a flexuous, helical rod or a loose ribonucleoprotein. The homology suggests gene transfer between eukaryotic (+) and (-)ssRNA viruses.

  20. The near-atomic cryoEM structure of a flexible filamentous plant virus shows homology of its coat protein with nucleoproteins of animal viruses

    PubMed Central

    Agirrezabala, Xabier; Méndez-López, Eduardo; Lasso, Gorka; Sánchez-Pina, M Amelia; Aranda, Miguel; Valle, Mikel

    2015-01-01

    Flexible filamentous viruses include economically important plant pathogens. Their viral particles contain several hundred copies of a helically arrayed coat protein (CP) protecting a (+)ssRNA. We describe here a structure at 3.9 Å resolution, from electron cryomicroscopy, of Pepino mosaic virus (PepMV), a representative of the genus Potexvirus (family Alphaflexiviridae). Our results allow modeling of the CP and its interactions with viral RNA. The overall fold of PepMV CP resembles that of nucleoproteins (NPs) from the genus Phlebovirus (family Bunyaviridae), a group of enveloped (-)ssRNA viruses. The main difference between potexvirus CP and phlebovirus NP is in their C-terminal extensions, which appear to determine the characteristics of the distinct multimeric assemblies – a flexuous, helical rod or a loose ribonucleoprotein. The homology suggests gene transfer between eukaryotic (+) and (-)ssRNA viruses. DOI: http://dx.doi.org/10.7554/eLife.11795.001 PMID:26673077

  1. Occurrence of autoantibodies to intermediate filament proteins in human visceral leishmaniasis and their induction by experimental polyclonal B-cell activation.

    PubMed Central

    Böhme, M W; Evans, D A; Miles, M A; Holborow, E J

    1986-01-01

    Fifteen sera of patients with visceral leishmaniasis were investigated for the occurrence of autoantibodies. They were found in high incidence and titre, and with specificity to the intermediate filament (INFIL) proteins vimentin (12 out of 15 with a titre higher than 1:10) and keratin (9 out of 15 with a titre higher than 1:10) as well as to speckled anti-nuclear antigens (ANA). Additionally, supernatants of Leishmania major and Leishmania donovani cultures containing soluble parasite-derived antigens were mitogenic to cultures of mononuclear cells (MNC) obtained from healthy donors without specific antibodies to leishmanial antigens. The activation of MNC resulted in significant immunoglobulin production, some of which demonstrated autoantibody specificity to INFIL. The co-operation of monocytes, T cells and B cells was required in order to obtain maximal stimulation. The importance of polyclonal B-cell activation for the genesis and occurrence of autoantibodies in visceral leishmaniasis is discussed. PMID:3492440

  2. Photomechanical Wave-Driven Delivery of siRNAs Targeting Intermediate Filament Proteins Promotes Functional Recovery after Spinal Cord Injury in Rats

    PubMed Central

    Ando, Takahiro; Sato, Shunichi; Toyooka, Terushige; Kobayashi, Hiroaki; Nawashiro, Hiroshi; Ashida, Hiroshi; Obara, Minoru

    2012-01-01

    The formation of glial scars after spinal cord injury (SCI) is one of the factors inhibiting axonal regeneration. Glial scars are mainly composed of reactive astrocytes overexpressing intermediate filament (IF) proteins such as glial fibrillary acidic protein (GFAP) and vimentin. In the current study, we delivered small interfering RNAs (siRNAs) targeting these IF proteins to SCI model rats using photomechanical waves (PMWs), and examined the restoration of motor function in the rats. PMWs are generated by irradiating a light-absorbing material with 532-nm nanosecond laser pulses from a Q-switched Nd:YAG laser. PMWs can site-selectively increase the permeability of the cell membrane for molecular delivery. Rat spinal cord was injured using a weight-drop device and the siRNA(s) solutions were intrathecally injected into the vicinity of the exposed SCI, to which PMWs were applied. We first confirmed the substantial uptake of fluorescence-labeled siRNA by deep glial cells; then we delivered siRNAs targeting GFAP and vimentin into the lesion. The treatment led to a significant improvement in locomotive function from five days post-injury in rats that underwent PMW-mediated siRNA delivery. This was attributable to the moderate silencing of the IF proteins and the subsequent decrease in the cavity area in the injured spinal tissue. PMID:23272155

  3. Heterologous expression of surface-active proteins from barley and filamentous fungi in Pichia pastoris and characterization of their contribution to beer gushing.

    PubMed

    Lutterschmid, Georg; Muranyi, Monika; Stübner, Matthias; Vogel, Rudi F; Niessen, Ludwig

    2011-05-14

    The spontaneous over-foaming of beer upon opening, i.e. beer gushing, is an unwanted phenomenon for the brewing industry. Currently, surface-active proteins from filamentous fungi and non-specific lipid transfer proteins (nsLTP1) from barley are discussed as gushing inducers. In our study the class I hydrophobin FcHyd3p from Fusarium culmorum, the class II hydrophobin Hfb2 from Trichoderma reesei, the alkaline foam protein A (AfpA) from F. graminearum and nsLTP1 from Hordeum vulgare cv. Marnie (barley) were heterologously expressed in Pichia pastoris and used in gushing tests. The class I hydrophobin FcHyd3p was unable to induce gushing in beer. The class II hydrophobin Hfb2 was able to induce gushing in beer, but proved to be inhibited by heat treatment as well as by the presence of enriched hop compounds. Both resulted in a reduced gushing potential. AfpA and nsLTP1 exhibited no gushing-inducing potential at the amounts added to beer. Addition of these proteins to beer or carbonated water previously treated with class II hydrophobins revealed a gushing reducing character.

  4. Centrosome detection in sea urchin eggs with a monoclonal antibody against Drosophila intermediate filament proteins: characterization of stages of the division cycle of centrosomes.

    PubMed

    Schatten, H; Walter, M; Mazia, D; Biessmann, H; Paweletz, N; Coffe, G; Schatten, G

    1987-12-01

    A mouse monoclonal antibody generated against Drosophila intermediate filament proteins (designated Ah6/5/9 and referred to herein as Ah6) is found to cross-react specifically with centrosomes in sea urchin eggs and with a 68-kDa antigen in eggs and isolated mitotic apparatus. When preparations stained with Ah6 are counterstained with a human autoimmune serum whose anti-centrosome activity has been established, the immunofluorescence images superimpose exactly. A more severe test of the specificity of the antibody demands that it display all of the stages of the centrosome cycle in the cell cycle: the flattening and spreading of the compact centrosomes followed by their division and the establishment of two compact poles. The test was made by an experimental design that uses a period of exposure of the eggs to 2-mercaptoethanol. This treatment allows observation of the stages of the centrosome cycle--separation, division, and bipolarization--while the chromosomes are arrested in metaphase. Mitosis is arrested in the presence of 0.1 M 2-mercaptoethanol. Chromosomes remain in a metaphase configuration while the centrosomes divide, producing four poles perpendicular to the original spindle axis. Microtubules are still present in the mitotic apparatus, as indicated by immunofluorescence and transmission electron microscopy. When 2-mercaptoethanol is removed, the chromosomes reorient to the poles of a tetrapolar (sometimes tripolar) mitotic apparatus. During the following cycle, the blastomeres form a monopolar mitotic apparatus. The observations of the centrosome cycle with the Ah6 antibody display very clearly all the stages that have been seen or deduced from work with other probes. The 68-kDa antigen that reacts with the Ah6 monoclonal antibody to Drosophila intermediate filament proteins must be a constant component of sea urchin centrosomes because it is present at all stages of the centrosome cycle.

  5. CAP1, an Adenylate Cyclase-Associated Protein Gene, Regulates Bud-Hypha Transitions, Filamentous Growth, and Cyclic AMP Levels and Is Required for Virulence of Candida albicans

    PubMed Central

    Bahn, Yong-Sun; Sundstrom, Paula

    2001-01-01

    In response to a wide variety of environmental stimuli, the opportunistic fungal pathogen Candida albicans exits the budding cycle, producing germ tubes and hyphae concomitant with expression of virulence genes, such as that encoding hyphal wall protein 1 (HWP1). Biochemical studies implicate cyclic AMP (cAMP) increases in promoting bud-hypha transitions, but genetic evidence relating genes that control cAMP levels to bud-hypha transitions has not been reported. Adenylate cyclase-associated proteins (CAPs) of nonpathogenic fungi interact with Ras and adenylate cyclase to increase cAMP levels under specific environmental conditions. To initiate studies on the relationship between cAMP signaling and bud-hypha transitions in C. albicans, we identified, cloned, characterized, and disrupted the C. albicans CAP1 gene. C. albicans strains with inactivated CAP1 budded in conditions that led to germ tube formation in isogenic strains with CAP1. The addition of 10 mM cAMP and dibutyryl cAMP promoted bud-hypha transitions and filamentous growth in the cap1/cap1 mutant in liquid and solid media, respectively, showing clearly that cAMP promotes hypha formation in C. albicans. Increases in cytoplasmic cAMP preceding germ tube emergence in strains having CAP1 were markedly diminished in the budding cap1/cap1 mutant. C. albicans strains with deletions of both alleles of CAP1 were avirulent in a mouse model of systemic candidiasis. The avirulence of a germ tube-deficient cap1/cap1 mutant coupled with the role of Cap1 in regulating cAMP levels shows that the Cap1-mediated cAMP signaling pathway is required for bud-hypha transitions, filamentous growth, and the pathogenesis of candidiasis. PMID:11325951

  6. The Arabidopsis microtubule-associated protein MAP65-3 supports infection by filamentous biotrophic pathogens by down-regulating salicylic acid-dependent defenses.

    PubMed

    Quentin, Michaël; Baurès, Isabelle; Hoefle, Caroline; Caillaud, Marie-Cécile; Allasia, Valérie; Panabières, Franck; Abad, Pierre; Hückelhoven, Ralph; Keller, Harald; Favery, Bruno

    2016-03-01

    The oomycete Hyaloperonospora arabidopsidis and the ascomycete Erysiphe cruciferarum are obligate biotrophic pathogens causing downy mildew and powdery mildew, respectively, on Arabidopsis. Upon infection, the filamentous pathogens induce the formation of intracellular bulbous structures called haustoria, which are required for the biotrophic lifestyle. We previously showed that the microtubule-associated protein AtMAP65-3 plays a critical role in organizing cytoskeleton microtubule arrays during mitosis and cytokinesis. This renders the protein essential for the development of giant cells, which are the feeding sites induced by root knot nematodes. Here, we show that AtMAP65-3 expression is also induced in leaves upon infection by the downy mildew oomycete and the powdery mildew fungus. Loss of AtMAP65-3 function in the map65-3 mutant dramatically reduced infection by both pathogens, predominantly at the stages of leaf penetration. Whole-transcriptome analysis showed an over-represented, constitutive activation of genes involved in salicylic acid (SA) biosynthesis, signaling, and defense execution in map65-3, whereas jasmonic acid (JA)-mediated signaling was down-regulated. Preventing SA synthesis and accumulation in map65-3 rescued plant susceptibility to pathogens, but not the developmental phenotype caused by cytoskeleton defaults. AtMAP65-3 thus has a dual role. It positively regulates cytokinesis, thus plant growth and development, and negatively interferes with plant defense against filamentous biotrophs. Our data suggest that downy mildew and powdery mildew stimulate AtMAP65-3 expression to down-regulate SA signaling for infection.

  7. CAP1, an adenylate cyclase-associated protein gene, regulates bud-hypha transitions, filamentous growth, and cyclic AMP levels and is required for virulence of Candida albicans.

    PubMed

    Bahn, Y S; Sundstrom, P

    2001-05-01

    In response to a wide variety of environmental stimuli, the opportunistic fungal pathogen Candida albicans exits the budding cycle, producing germ tubes and hyphae concomitant with expression of virulence genes, such as that encoding hyphal wall protein 1 (HWP1). Biochemical studies implicate cyclic AMP (cAMP) increases in promoting bud-hypha transitions, but genetic evidence relating genes that control cAMP levels to bud-hypha transitions has not been reported. Adenylate cyclase-associated proteins (CAPs) of nonpathogenic fungi interact with Ras and adenylate cyclase to increase cAMP levels under specific environmental conditions. To initiate studies on the relationship between cAMP signaling and bud-hypha transitions in C. albicans, we identified, cloned, characterized, and disrupted the C. albicans CAP1 gene. C. albicans strains with inactivated CAP1 budded in conditions that led to germ tube formation in isogenic strains with CAP1. The addition of 10 mM cAMP and dibutyryl cAMP promoted bud-hypha transitions and filamentous growth in the cap1/cap1 mutant in liquid and solid media, respectively, showing clearly that cAMP promotes hypha formation in C. albicans. Increases in cytoplasmic cAMP preceding germ tube emergence in strains having CAP1 were markedly diminished in the budding cap1/cap1 mutant. C. albicans strains with deletions of both alleles of CAP1 were avirulent in a mouse model of systemic candidiasis. The avirulence of a germ tube-deficient cap1/cap1 mutant coupled with the role of Cap1 in regulating cAMP levels shows that the Cap1-mediated cAMP signaling pathway is required for bud-hypha transitions, filamentous growth, and the pathogenesis of candidiasis.

  8. A novel Bacillus thuringiensis Cry-like protein from a rare filamentous strain promotes crystal localization within the exosporium.

    PubMed

    Ammons, David R; Reyna, Antonio; Granados, Jose C; Ventura-Suárez, Antonio; Rojas-Avelizapa, Luz I; Short, John D; Rampersad, Joanne N

    2013-09-01

    Mutation of a novel cry-like gene (cry256) from Bacillus thuringiensis resulted in a protein crystal, normally located within the spore's exosporium, being found predominately outside the exosporium. The cry256 gene codes for a 3-domain Cry-like protein that does not correspond to any of the known Cry protein holotypes.

  9. A filamentous hemagglutinin-like protein of Xanthomonas axonopodis pv. citri, the phytopathogen responsible for citrus canker, is involved in bacterial virulence.

    PubMed

    Gottig, Natalia; Garavaglia, Betiana S; Garofalo, Cecilia G; Orellano, Elena G; Ottado, Jorgelina

    2009-01-01

    Xanthomonas axonopodis pv. citri, the phytopathogen responsible for citrus canker has a number of protein secretion systems and among them, at least one type V protein secretion system belonging to the two-partner secretion pathway. This system is mainly associated to the translocation of large proteins such as adhesins to the outer membrane of several pathogens. Xanthomonas axonopodis pv. citri possess a filamentous hemagglutinin-like protein in close vicinity to its putative transporter protein, XacFhaB and XacFhaC, respectively. Expression analysis indicated that XacFhaB was induced in planta during plant-pathogen interaction. By mutation analysis of XacFhaB and XacFhaC genes we determined that XacFhaB is involved in virulence both in epiphytic and wound inoculations, displaying more dispersed and fewer canker lesions. Unexpectedly, the XacFhaC mutant in the transporter protein produced an intermediate virulence phenotype resembling wild type infection, suggesting that XacFhaB could be secreted by another partner different from XacFhaC. Moreover, XacFhaB mutants showed a general lack of adhesion and were affected in leaf surface attachment and biofilm formation. In agreement with the in planta phenotype, adhesin lacking cells moved faster in swarming plates. Since no hyperflagellation phenotype was observed in this bacteria, the faster movement may be attributed to the lack of cell-to-cell aggregation. Moreover, XacFhaB mutants secreted more exopolysaccharide that in turn may facilitate its motility. Our results suggest that this hemagglutinin-like protein is required for tissue colonization being mainly involved in surface attachment and biofilm formation, and that plant tissue attachment and cell-to-cell aggregation are dependent on the coordinated action of adhesin molecules and exopolysaccharides.

  10. Protein polymer hydrogels by in situ, rapid and reversible self-gelation.

    PubMed

    Asai, Daisuke; Xu, Donghua; Liu, Wenge; Garcia Quiroz, Felipe; Callahan, Daniel J; Zalutsky, Michael R; Craig, Stephen L; Chilkoti, Ashutosh

    2012-07-01

    Protein-based biomaterials are an important class of materials for applications in biotechnology and medicine. The exquisite control of their composition, stereochemistry, and chain length offers unique opportunities to engineer biofunctionality, biocompatibility, and biodegradability into these materials. Here, we report the synthesis of a thermally responsive peptide polymer-based hydrogel composed of a recombinant elastin-like polypeptide (ELP) that rapidly forms a reversibly cross-linked hydrogel by the formation of intermolecular disulfide cross-links. To do so, we designed and synthesized ELPs that incorporate periodic cysteine residues (cELPs), and show that cELPs are thermally responsive protein polymers that display rapid gelation under physiologically relevant, mild oxidative conditions. Gelation of cELPs, at concentrations as low as 2.5 wt%, occurs in ≈ 2.5 min upon addition a low concentration of hydrogen peroxide (0.3 wt%). We show the utility of these hydrogels for the sustained release of a model protein in vitro, and demonstrate the ability of this injectable biomaterial to pervade tumors to maximize tumor coverage and retention time upon intratumoral injection. cELPs represent a new class of injectable reversibly cross-linked hydrogels with properties intermediate between ELP coacervates and chemically cross-linked ELP hydrogels that will find useful applications in drug delivery and tissue engineering.

  11. Conformational transition and mass transfer in extraction of proteins by AOT--alcohol--isooctane reverse micellar systems.

    PubMed

    Hong, D P; Lee, S S; Kuboi, R

    2000-06-23

    We examined quantitatively the effect of alcohols on protein and reverse micellar structure. We used circular dichroism (CD) to compare the effects of various alcohols on the protein structure, and percolation phenomena to evaluate the effects of various alcohols on reverse micellar structure. Upon the addition of alcohols to the bulk aqueous phase, proteins were denatured significantly, depending on the alcohol species and concentration, suggesting that use of alcohol directly to the stripping solution is not effective in back-extraction processes of proteins. In the present study, a new method, a small amount of alcohol is added to the surfactant-organic solution to improve the back-extraction behaviors of proteins. Practically, in the back-extraction process, the alcohols suppressing the cluster formation of reverse micelles (high value of beta1), remarkably improved the back-extraction behavior of proteins. In addition, the same alcohol molecules showed a positive effect on the rate and fraction of protein back-extraction. From a result of the CD measurement of the back-extracted proteins, it was known that the alcohols added to reverse micellar solution allowed the proteins to back-extract safely without causing structural changes. These results show that the values of beta(t), defined by the variation of percolation processes, and the back-extraction behaviors of proteins have a good relationship, suggesting that the back-extraction processes were controlled by the micellar-micellar and protein-micellar interactions.

  12. Reversible and irreversible modifications of skeletal muscle proteins in a rat model of acute oxidative stress.

    PubMed

    Fedorova, Maria; Kuleva, Nadezhda; Hoffmann, Ralf

    2009-12-01

    Oxidative stress caused by an imbalance of the production of "reactive oxygen species" (ROS) and cellular scavenging systems is known to a play a key role in the development of various diseases and aging processes. Such elevated ROS levels can damage all components of cells, including proteins, lipids and DNA. Here, we study the influence of highly reactive ROS species on skeletal muscle proteins in a rat model of acute oxidative stress caused by X-ray irradiation at different time points. Protein preparations depleted for functional actin by polymerization were separated by gel electrophoresis in two dimensions by applying first non-reductive and then reductive conditions in SDS-PAGE. This diagonal redox SDS-PAGE revealed significant alterations to intra- and inter-molecular disulfide bridges for several proteins, but especially actin, creatine kinase and different isoforms of the myosin light chain. Though the levels of these reversible modifications were increased by oxidative stress, all proteins followed different kinetics. Moreover, a significant degree of protein was irreversibly oxidized (carbonylated), as revealed by western blot analyses performed at different time points.

  13. Light-Activated Reversible Imine Isomerization: Towards a Photochromic Protein Switch.

    PubMed

    Berbasova, Tetyana; Santos, Elizabeth M; Nosrati, Meisam; Vasileiou, Chrysoula; Geiger, James H; Borhan, Babak

    2016-03-02

    Mutants of cellular retinoic acid-binding protein II (CRABPII), engineered to bind all-trans-retinal as an iminium species, demonstrate photochromism upon irradiation with light at different wavelengths. UV light irradiation populates the cis-imine geometry, which has a high pKa , leading to protonation of the imine and subsequent "turn-on" of color. Yellow light irradiation yields the trans-imine isomer, which has a depressed pKa , leading to loss of color because the imine is not protonated. The protein-bound retinylidene chromophore undergoes photoinduced reversible interconversion between the colored and uncolored species, with excellent fatigue resistance.

  14. The intermediate filament protein keratin 8 is a novel cytoplasmic substrate for c-Jun N-terminal kinase.

    PubMed

    He, Tao; Stepulak, Andrzej; Holmström, Tim H; Omary, M Bishr; Eriksson, John E

    2002-03-29

    Keratins 8 (K8) and 18 are the primary intermediate filaments of simple epithelia. Phosphorylation of keratins at specific sites affects their organization, assembly dynamics, and their interaction with signaling molecules. A number of keratin in vitro and in vivo phosphorylation sites have been identified. One example is K8 Ser-73, which has been implicated as an important phosphorylation site during mitosis, cell stress, and apoptosis. We show that K8 is strongly phosphorylated on Ser-73 upon stimulation of the pro-apoptotic cytokine receptor Fas/CD95/Apo-1 in HT-29 cells. Kinase assays showed that c-Jun N-terminal kinase (JNK) was also activated with activation kinetics corresponding to that of K8 phosphorylation. Furthermore, K8 was also phosphorylated on Ser-73 by JNK in vitro, yielding similar phosphopeptide maps as the in vivo phosphorylated material. In addition, co-immunoprecipitation studies revealed that part of JNK is associated with K8 in vivo, correlating with decreased ability of JNK to phosphorylate the endogenous c-Jun. Taken together, K8 is a new cytoplasmic target for JNK in Fas receptor-mediated signaling. The functional significance of this phosphorylation could relate to regulation of JNK signaling and/or regulation of keratin dynamics.

  15. Covisualization in living onion cells of putative integrin, putative spectrin, actin, putative intermediate filaments, and other proteins at the cell membrane and in an endomembrane sheath

    NASA Technical Reports Server (NTRS)

    Reuzeau, C.; Doolittle, K. W.; McNally, J. G.; Pickard, B. G.; Evans, M. L. (Principal Investigator)

    1997-01-01

    Covisualizations with wide-field computational optical-sectioning microscopy of living epidermal cells of the onion bulb scale have evidenced two major new cellular features. First, a sheath of cytoskeletal elements clads the endomembrane system. Similar elements clad the inner faces of punctate plasmalemmal sites interpreted as plasmalemmal control centers. One component of the endomembrane sheath and plasmalemmal control center cladding is anti-genicity-recognized by two injected antibodies against animal spectrin. Immunoblots of separated epidermal protein also showed bands recognized by these antibodies. Injected phalloidin identified F-actin with the same cellular distribution pattern, as did antibodies against intermediate-filament protein and other cytoskeletal elements known from animal cells. Injection of general protein stains demonstrated the abundance of endomembrane sheath protein. Second, the endomembrane system, like the plasmalemmal puncta, contains antigen recognized by an anti-beta 1 integrin injected into the cytoplasm. Previously, immunoblots of separated epidermal protein were shown to have a major band recognized both by this antibody prepared against a peptide representing the cytosolic region of beta 1 integrin and an antibody against the matrix region of beta 1 integrin. The latter antiboby also identified puncta at the external face of protoplasts. It is proposed that integrin and associated transmembrane proteins secure the endomembrane sheath and transmit signals between it and the lumen or matrix of the endoplasmic reticulum and organellar matrices. This function is comparable to that proposed for such transmembrane linkers in the plasmalemmal control centers, which also appear to bind cytoskeleton and a host of related molecules and transmit signals between them and the wall matrix. It is at the plasmalemmal control centers that the endoplasmic reticulum, a major component of the endomembrane system, attaches to the plasma membrane.

  16. Mutations in proteins of the Conserved Oligomeric Golgi Complex affect polarity, cell wall structure, and glycosylation in the filamentous fungus Aspergillus nidulans.

    PubMed

    Gremillion, S K; Harris, S D; Jackson-Hayes, L; Kaminskyj, S G W; Loprete, D M; Gauthier, A C; Mercer, S; Ravita, A J; Hill, T W

    2014-12-01

    We have described two Aspergillus nidulans gene mutations, designated podB1 (polarity defective) and swoP1 (swollen cell), which cause temperature-sensitive defects during polarization. Mutant strains also displayed unevenness and abnormal thickness of cell walls. Un-polarized or poorly-polarized mutant cells were capable of establishing normal polarity after a shift to a permissive temperature, and mutant hyphae shifted from permissive to restrictive temperature show wall and polarity abnormalities in subsequent growth. The mutated genes (podB=AN8226.3; swoP=AN7462.3) were identified as homologues of COG2 and COG4, respectively, each predicted to encode a subunit of the multi-protein COG (Conserved Oligomeric Golgi) Complex involved in retrograde vesicle trafficking in the Golgi apparatus. Down-regulation of COG2 or COG4 resulted in abnormal polarization and cell wall staining. The GFP-tagged COG2 and COG4 homologues displayed punctate, Golgi-like localization. Lectin-blotting indicated that protein glycosylation was altered in the mutant strains compared to the wild type. A multicopy expression experiment showed evidence for functional interactions between the homologues COG2 and COG4 as well as between COG2 and COG3. To date, this work is the first regarding a functional role of the COG proteins in the development of a filamentous fungus.

  17. Increase of calcium levels in epithelial cells induces translocation of calcium-binding proteins migration inhibitory factor-related protein 8 (MRP8) and MRP14 to keratin intermediate filaments.

    PubMed

    Goebeler, M; Roth, J; van den Bos, C; Ader, G; Sorg, C

    1995-07-15

    Migration inhibitory factor-related protein 8 (MRP8) and MRP14, two S-100-like Ca(2+)-binding proteins, have been described in cells of the epithelial lineage where they are either expressed constitutively (e.g. by mucosal squamous epithelium) or induced during disease (e.g. in keratinocytes during the course of psoriasis). Their biological function, however, is not yet clear. Recent studies have provided evidence that S-100-like proteins may interact with cytoskeletal components; we have therefore studied the biochemical properties and subcellular distribution of MRP8 and MRP14 in epithelial cells. TR146 human squamous carcinoma cells, which were found to express MRP8 and MRP14 in Northern and Western blot studies, were chosen for analysis. Cross-linking experiments using bis(sulphosuccinimidyl)suberate followed by SDS/PAGE and Western blot analysis revealed formation of heteromeric MRP8-MRP14 complexes. On subjecting TR146 cell lysates to two-dimensional gel electrophoresis and Western blotting, four distinct MRP14 isoforms could be identified resembling those described earlier in macrophages. A differential centrifugation technique revealed a Ca(2+)-dependent translocation of MRP8-MRP14 from the cytoplasm to the membrane and the Nonidet P40-insoluble cytoskeletal fraction. Double-label immunofluorescence microscopy of Ca2+ ionophore A23187-stimulated TR146 cells and cytochalasin B and demecolcine cytoskeleton disruption studies identified these structures as keratin intermediate filaments. Ca(2+)-dependent binding of MRP8-MRP14 to keratin filaments was additionally confirmed by an in vitro binding assay. In conclusion, our data suggest that MRP8 and MRP14 may be involved in Ca(2+)-dependent reorganization of cytoskeletal filaments in epithelial cells, which could be of importance for events associated with differentiation and inflammatory activation.

  18. Superresolution Imaging of Dynamic MreB Filaments in B. subtilis—A Multiple-Motor-Driven Transport?

    PubMed Central

    Olshausen, Philipp v.; Defeu Soufo, Hervé Joël; Wicker, Kai; Heintzmann, Rainer; Graumann, Peter L.; Rohrbach, Alexander

    2013-01-01

    The cytoskeletal protein MreB is an essential component of the bacterial cell-shape generation system. Using a superresolution variant of total internal reflection microscopy with structured illumination, as well as three-dimensional stacks of deconvolved epifluorescence microscopy, we found that inside living Bacillus subtilis cells, MreB forms filamentous structures of variable lengths, typically not longer than 1 μm. These filaments move along their orientation and mainly perpendicular to the long bacterial axis, revealing a maximal velocity at an intermediate length and a decreasing velocity with increasing filament length. Filaments move along straight trajectories but can reverse or alter their direction of propagation. Based on our measurements, we provide a mechanistic model that is consistent with all observations. In this model, MreB filaments mechanically couple several motors that putatively synthesize the cell wall, whereas the filaments’ traces mirror the trajectories of the motors. On the basis of our mechanistic model, we developed a mathematical model that can explain the nonlinear velocity length dependence. We deduce that the coupling of cell wall synthesis motors determines the MreB filament transport velocity, and the filament mechanically controls a concerted synthesis of parallel peptidoglycan strands to improve cell wall stability. PMID:24010660

  19. Reversion by hypotonic medium of the shutoff of protein synthesis induced by encephalomyocarditis virus.

    PubMed Central

    Alonso, M A; Carrasco, L

    1981-01-01

    Infection of human HeLa cells by picornaviruses produces a drastic inhibition of host protein synthesis. Treatment of encephalomyocarditis virus-infected HeLa cells with hypotonic medium reversed this inhibition; no viral protein synthesis was detected. The blockade of viral translation by hypotonic conditions was observed for a wide range of multiplicities of infection. However, only with low virus-to-cell ratios did cellular protein synthesis resume. The ratio of cellular to viral mRNA translation was strongly influenced by the concentration of monovalent ions present in the culture medium: a high concentration of NaCl or KCl favored the translation of viral mRNA and strongly inhibited cellular protein synthesis, whereas the opposite was true when NaCl was omitted from the culture medium. Once viral protein synthesis had been blocked by hypotonic medium treatment, it resumed when the infected cells were placed in a normal or hypertonic medium, indicating that the viral components synthesized in the infected cells were not destroyed by this treatment. These observations reinforced the idea that ions play a role in discriminating between viral and cellular mRNA translation in virus-infected animal cells. Images PMID:6261005

  20. Boolean gates on actin filaments

    NASA Astrophysics Data System (ADS)

    Siccardi, Stefano; Tuszynski, Jack A.; Adamatzky, Andrew

    2016-01-01

    Actin is a globular protein which forms long polar filaments in the eukaryotic cytoskeleton. Actin networks play a key role in cell mechanics and cell motility. They have also been implicated in information transmission and processing, memory and learning in neuronal cells. The actin filaments have been shown to support propagation of voltage pulses. Here we apply a coupled nonlinear transmission line model of actin filaments to study interactions between voltage pulses. To represent digital information we assign a logical TRUTH value to the presence of a voltage pulse in a given location of the actin filament, and FALSE to the pulse's absence, so that information flows along the filament with pulse transmission. When two pulses, representing Boolean values of input variables, interact, then they can facilitate or inhibit further propagation of each other. We explore this phenomenon to construct Boolean logical gates and a one-bit half-adder with interacting voltage pulses. We discuss implications of these findings on cellular process and technological applications.

  1. RPPAML/RIMS: A metadata format and an information management system for reverse phase protein arrays

    PubMed Central

    Stanislaus, Romesh; Carey, Mark; Deus, Helena F; Coombes, Kevin; Hennessy, Bryan T; Mills, Gordon B; Almeida, Jonas S

    2008-01-01

    Background Reverse Phase Protein Arrays (RPPA) are convenient assay platforms to investigate the presence of biomarkers in tissue lysates. As with other high-throughput technologies, substantial amounts of analytical data are generated. Over 1000 samples may be printed on a single nitrocellulose slide. Up to 100 different proteins may be assessed using immunoperoxidase or immunoflorescence techniques in order to determine relative amounts of protein expression in the samples of interest. Results In this report an RPPA Information Management System (RIMS) is described and made available with open source software. In order to implement the proposed system, we propose a metadata format known as reverse phase protein array markup language (RPPAML). RPPAML would enable researchers to describe, document and disseminate RPPA data. The complexity of the data structure needed to describe the results and the graphic tools necessary to visualize them require a software deployment distributed between a client and a server application. This was achieved without sacrificing interoperability between individual deployments through the use of an open source semantic database, S3DB. This data service backbone is available to multiple client side applications that can also access other server side deployments. The RIMS platform was designed to interoperate with other data analysis and data visualization tools such as Cytoscape. Conclusion The proposed RPPAML data format hopes to standardize RPPA data. Standardization of data would result in diverse client applications being able to operate on the same set of data. Additionally, having data in a standard format would enable data dissemination and data analysis. PMID:19102773

  2. Titin strain contributes to the Frank–Starling law of the heart by structural rearrangements of both thin- and thick-filament proteins

    PubMed Central

    Ait-Mou, Younss; Hsu, Karen; Farman, Gerrie P.; Kumar, Mohit; Greaser, Marion L.; Irving, Thomas C.; de Tombe, Pieter P.

    2016-01-01

    The Frank–Starling mechanism of the heart is due, in part, to modulation of myofilament Ca2+ sensitivity by sarcomere length (SL) [length-dependent activation (LDA)]. The molecular mechanism(s) that underlie LDA are unknown. Recent evidence has implicated the giant protein titin in this cellular process, possibly by positioning the myosin head closer to actin. To clarify the role of titin strain in LDA, we isolated myocardium from either WT or homozygous mutant (HM) rats that express a giant splice isoform of titin, and subjected the muscles to stretch from 2.0 to 2.4 μm of SL. Upon stretch, HM compared with WT muscles displayed reduced passive force, twitch force, and myofilament LDA. Time-resolved small-angle X-ray diffraction measurements of WT twitching muscles during diastole revealed stretch-induced increases in the intensity of myosin (M2 and M6) and troponin (Tn3) reflections, as well as a reduction in cross-bridge radial spacing. Independent fluorescent probe analyses in relaxed permeabilized myocytes corroborated these findings. X-ray electron density reconstruction revealed increased mass/ordering in both thick and thin filaments. The SL-dependent changes in structure observed in WT myocardium were absent in HM myocardium. Overall, our results reveal a correlation between titin strain and the Frank–Starling mechanism. The molecular basis underlying this phenomenon appears not to involve interfilament spacing or movement of myosin toward actin but, rather, sarcomere stretch-induced simultaneous structural rearrangements within both thin and thick filaments that correlate with titin strain and myofilament LDA. PMID:26858417

  3. Titin strain contributes to the Frank-Starling law of the heart by structural rearrangements of both thin- and thick-filament proteins.

    PubMed

    Ait-Mou, Younss; Hsu, Karen; Farman, Gerrie P; Kumar, Mohit; Greaser, Marion L; Irving, Thomas C; de Tombe, Pieter P

    2016-02-23

    The Frank-Starling mechanism of the heart is due, in part, to modulation of myofilament Ca(2+) sensitivity by sarcomere length (SL) [length-dependent activation (LDA)]. The molecular mechanism(s) that underlie LDA are unknown. Recent evidence has implicated the giant protein titin in this cellular process, possibly by positioning the myosin head closer to actin. To clarify the role of titin strain in LDA, we isolated myocardium from either WT or homozygous mutant (HM) rats that express a giant splice isoform of titin, and subjected the muscles to stretch from 2.0 to 2.4 μm of SL. Upon stretch, HM compared with WT muscles displayed reduced passive force, twitch force, and myofilament LDA. Time-resolved small-angle X-ray diffraction measurements of WT twitching muscles during diastole revealed stretch-induced increases in the intensity of myosin (M2 and M6) and troponin (Tn3) reflections, as well as a reduction in cross-bridge radial spacing. Independent fluorescent probe analyses in relaxed permeabilized myocytes corroborated these findings. X-ray electron density reconstruction revealed increased mass/ordering in both thick and thin filaments. The SL-dependent changes in structure observed in WT myocardium were absent in HM myocardium. Overall, our results reveal a correlation between titin strain and the Frank-Starling mechanism. The molecular basis underlying this phenomenon appears not to involve interfilament spacing or movement of myosin toward actin but, rather, sarcomere stretch-induced simultaneous structural rearrangements within both thin and thick filaments that correlate with titin strain and myofilament LDA.

  4. Titin strain contributes to the Frank–Starling law of the heart by structural rearrangements of both thin- and thick-filament proteins

    SciTech Connect

    Ait-Mou, Younss; Hsu, Karen; Farman, Gerrie P.; Kumar, Mohit; Greaser, Marion L.; Irving, Thomas C.; de Tombe, Pieter P.

    2016-02-08

    The Frank–Starling mechanism of the heart is due, in part, to modulation of myofilament Ca2+ sensitivity by sarcomere length (SL) [length-dependent activation (LDA)]. The molecular mechanism(s) that underlie LDA are unknown. Recent evidence has implicated the giant protein titin in this cellular process, possibly by positioning the myosin head closer to actin. To clarify the role of titin strain in LDA, we isolated myocardium from either WT or homozygous mutant (HM) rats that express a giant splice isoform of titin, and subjected the muscles to stretch from 2.0 to 2.4 μm of SL. Upon stretch, HM compared with WT muscles displayed reduced passive force, twitch force, and myofilament LDA. Time-resolved small-angle X-ray diffraction measurements of WT twitching muscles during diastole revealed stretch-induced increases in the intensity of myosin (M2 and M6) and troponin (Tn3) reflections, as well as a reduction in cross-bridge radial spacing. Independent fluorescent probe analyses in relaxed permeabilized myocytes corroborated these findings. X-ray electron density reconstruction revealed increased mass/ordering in both thick and thin filaments. The SL-dependent changes in structure observed in WT myocardium were absent in HM myocardium. Overall, our results reveal a correlation between titin strain and the Frank–Starling mechanism. The molecular basis underlying this phenomenon appears not to involve interfilament spacing or movement of myosin toward actin but, rather, sarcomere stretch-induced simultaneous structural rearrangements within both thin and thick filaments that correlate with titin strain and myofilament LDA.

  5. Titin strain contributes to the Frank–Starling law of the heart by structural rearrangements of both thin- and thick-filament proteins

    DOE PAGES

    Ait-Mou, Younss; Hsu, Karen; Farman, Gerrie P.; ...

    2016-02-08

    The Frank–Starling mechanism of the heart is due, in part, to modulation of myofilament Ca2+ sensitivity by sarcomere length (SL) [length-dependent activation (LDA)]. The molecular mechanism(s) that underlie LDA are unknown. Recent evidence has implicated the giant protein titin in this cellular process, possibly by positioning the myosin head closer to actin. To clarify the role of titin strain in LDA, we isolated myocardium from either WT or homozygous mutant (HM) rats that express a giant splice isoform of titin, and subjected the muscles to stretch from 2.0 to 2.4 μm of SL. Upon stretch, HM compared with WT musclesmore » displayed reduced passive force, twitch force, and myofilament LDA. Time-resolved small-angle X-ray diffraction measurements of WT twitching muscles during diastole revealed stretch-induced increases in the intensity of myosin (M2 and M6) and troponin (Tn3) reflections, as well as a reduction in cross-bridge radial spacing. Independent fluorescent probe analyses in relaxed permeabilized myocytes corroborated these findings. X-ray electron density reconstruction revealed increased mass/ordering in both thick and thin filaments. The SL-dependent changes in structure observed in WT myocardium were absent in HM myocardium. Overall, our results reveal a correlation between titin strain and the Frank–Starling mechanism. The molecular basis underlying this phenomenon appears not to involve interfilament spacing or movement of myosin toward actin but, rather, sarcomere stretch-induced simultaneous structural rearrangements within both thin and thick filaments that correlate with titin strain and myofilament LDA.« less

  6. Titin strain contributes to the Frank-Starling law of the heart by structural rearrangements of both thin- and thick-filament proteins

    SciTech Connect

    Ait-Mou, Younss; Hsu, Karen; Farman, Gerrie P.; Kumar, Mohit; Greaser, Marion L.; Irving, Thomas C.; de Tombe, Pieter P.

    2016-04-29

    We determined that the Frank–Starling mechanism of the heart is due, in part, to modulation of myofilament Ca2+ sensitivity by sarcomere length (SL) [length-dependent activation (LDA)]. The molecular mechanism(s) that underlie LDA are unknown. Recent evidence has implicated the giant protein titin in this cellular process, possibly by positioning the myosin head closer to actin. To clarify the role of titin strain in LDA, we isolated myocardium from either WT or homozygous mutant (HM) rats that express a giant splice isoform of titin, and subjected the muscles to stretch from 2.0 to 2.4 μm of SL. Upon stretch, HM compared with WT muscles displayed reduced passive force, twitch force, and myofilament LDA. Time-resolved small-angle X-ray diffraction measurements of WT twitching muscles during diastole revealed stretch-induced increases in the intensity of myosin (M2 and M6) and troponin (Tn3) reflections, as well as a reduction in cross-bridge radial spacing. Independent fluorescent probe analyses in relaxed permeabilized myocytes corroborated these findings. X-ray electron density reconstruction revealed increased mass/ordering in both thick and thin filaments. The SL-dependent changes in structure observed in WT myocardium were absent in HM myocardium. Our results reveal a correlation between titin strain and the Frank–Starling mechanism. The molecular basis underlying this phenomenon appears not to involve interfilament spacing or movement of myosin toward actin but, rather, sarcomere stretch-induced simultaneous structural rearrangements within both thin and thick filaments that correlate with titin strain and myofilament LDA.

  7. Filamentation in Laser Wakefields

    NASA Astrophysics Data System (ADS)

    Los, Eva; Trines, Raoul; Silva, Luis; Bingham, Robert

    2016-10-01

    Laser filamentation instability is observed in plasma wakefields with sub-critical densities, and in high density inertial fusion plasmas. This leads to non-uniform acceleration or compression respectively. Here, we present simulation results on laser filamentation in plasma wakefields. The 2-D simulations are carried out using the particle-in-cell code Osiris. The filament intensity was found to increase exponentially before saturating. The maximum amplitude to which the highest intensity filament grew for a specific set of parameters was also recorded, and plotted against a corresponding parameter value. Clear, positively correlated linear trends were established between plasma density, transverse wavenumber k, laser pulse amplitude and maximum filament amplitude. Plasma density and maximum filament amplitude also showed a positive correlation, which saturated after a certain plasma density. Pulse duration and interaction length did not affect either filament intensity or transverse k value in a predictable manner. There was no discernible trend between pulse amplitude and filament width.

  8. A 45,000-mol-wt protein from unfertilized sea urchin eggs severs actin filaments in a calcium-dependent manner and increases the steady-state concentration of nonfilamentous actin

    PubMed Central

    1984-01-01

    A 45,000-mol-wt protein has been purified from unfertilized sea urchin (Strongylocentrotus purpuratus) eggs. The isolation scheme includes DEAE cellulose ion-exchange chromatography, gel filtration, and hydroxylapatite chromatography. The homogeneity of the isolated protein is greater than 90% by SDS PAGE. The 45,000-mol-wt protein reduces the viscosity of actin filaments in a Ca2+-dependent manner. The free calcium concentration required for the activity of this protein is in the micromolar range. Electron microscopic studies reveal that the formation of short filaments parallels the decrease in viscosity. Energy transfer and sedimentation experiments indicate a net disassembly of actin filaments and an increase in the steady-state nonfilamentous actin concentration in the presence of Ca2+ ions and the 45,000-mol-wt protein. The increase in the steady-state nonfilamentous actin concentration is proportional to the amount of 45,000-mol-wt protein added. The actin molecules disassembled by the addition of the 45,000-mol-wt protein are capable of polymerization. PMID:6540784

  9. Solubilization and fractionation of paired helical filaments.

    PubMed

    González, P J; Correas, I; Avila, J

    1992-09-01

    Paired helical filaments isolated from brains of two different patients with Alzheimer's disease were extensively treated with the ionic detergent, sodium dodecyl sulphate. Filaments were solubilized at different extents, depending on the brain examined, thus suggesting the existence of two types of paired helical filaments: sodium dodecyl sulphate-soluble and insoluble filaments. In the first case, the number of structures resembling paired helical filaments greatly decreased after the detergent treatment, as observed by electron microscopy. Simultaneously, a decrease in the amount of sedimentable protein was also observed upon centrifugation of the sodium dodecyl sulfate-treated paired helical filaments. A sodium dodecyl sulphate-soluble fraction was isolated as a supernatant after low-speed centrifugation of the sodium dodecyl sulphate-treated paired helical filaments. The addition of the non-ionic detergent Nonidet-P40 to this fraction resulted in the formation of paired helical filament-like structures. When the sodium dodecyl sulphate-soluble fraction was further fractionated by high-speed centrifugation, three subfractions were observed: a supernatant, a pellet and a thin layer between these two subfractions. No paired helical filaments were observed in any of these subfractions, even after addition of Nonidet P-40. However, when they were mixed back together, the treatment with Nonidet P-40 resulted in the visualization of paired helical filament-like structures. These results suggest that at least two different components are needed for the reconstitution of paired helical filaments as determined by electron microscopy. The method described here may allow the study of the components involved in the formation of paired helical filaments and the identification of possible factors capable of blocking this process.

  10. Intracellular fibril formation, calcification, and enrichment of chaperones, cytoskeletal, and intermediate filament proteins in the adult hippocampus CA1 following neonatal exposure to the nonprotein amino acid BMAA.

    PubMed

    Karlsson, Oskar; Berg, Anna-Lena; Hanrieder, Jörg; Arnerup, Gunnel; Lindström, Anna-Karin; Brittebo, Eva B

    2015-03-01

    The environmental neurotoxin β-N-methylamino-L-alanine (BMAA) has been implicated in the etiology of neurodegenerative disease, and recent studies indicate that BMAA can be misincorporated into proteins. BMAA is a developmental neurotoxicant that can induce long-term learning and memory deficits, as well as regionally restricted neuronal degeneration and mineralization in the hippocampal CA1. The aim of the study was to characterize long-term changes (2 weeks to 6 months) further in the brain of adult rats treated neonatally (postnatal days 9-10) with BMAA (460 mg/kg) using immunohistochemistry (IHC), transmission electron microscopy, and laser capture microdissection followed by LC-MS/MS for proteomic analysis. The histological examination demonstrated progressive neurodegenerative changes, astrogliosis, microglial activation, and calcification in the hippocampal CA1 3-6 months after exposure. The IHC showed an increased staining for α-synuclein and ubiquitin in the area. The ultrastructural examination revealed intracellular deposition of abundant bundles of closely packed parallel fibrils in neurons, axons, and astrocytes of the CA1. Proteomic analysis of the affected site demonstrated an enrichment of chaperones (e.g., clusterin, GRP-78), cytoskeletal and intermediate filament proteins, and proteins involved in the antioxidant defense system. Several of the most enriched proteins (plectin, glial fibrillar acidic protein, vimentin, Hsp 27, and ubiquitin) are known to form complex astrocytic inclusions, so-called Rosenthal fibers, in the neurodegenerative disorder Alexander disease. In addition, TDP-43 and the negative regulator of autophagy, GLIPR-2, were exclusively detected. The present study demonstrates that neonatal exposure to BMAA may offer a novel model for the study of hippocampal fibril formation in vivo.

  11. Clinical utility of reverse phase protein array for molecular classification of breast cancer.

    PubMed

    Negm, Ola H; Muftah, Abir A; Aleskandarany, Mohammed A; Hamed, Mohamed R; Ahmad, Dena A J; Nolan, Christopher C; Diez-Rodriguez, Maria; Tighe, Patrick J; Ellis, Ian O; Rakha, Emad A; Green, Andrew R

    2016-01-01

    Reverse Phase Protein Array (RPPA) represents a sensitive and high-throughput technique allowing simultaneous quantitation of protein expression levels in biological samples. This study aimed to confirm the ability of RPPA to classify archival formalin-fixed paraffin-embedded (FFPE) breast cancer tissues into molecular classes used in the Nottingham prognostic index plus (NPI+) determined by immunohistochemistry (IHC). Proteins were extracted from FFPE breast cancer tissues using three extraction protocols: the Q-proteome FFPE Tissue Kit (Qiagen, Hilden, Germany) and two in-house methods using Laemmli buffer with either incubation for 20 min or 2 h at 105 °C. Two preparation methods, full-face sections and macrodissection, were used to assess the yield and quality of protein extracts. Ten biomarkers used for the NPI+ (ER, PgR, HER2, Cytokeratins 5/6 and 7/8, EGFR, HER3, HER4, p53 and Mucin 1) were quantified using RPPA and compared to results determined by IHC. The Q-proteome FFPE Tissue Kit produced significantly higher protein concentration and signal intensities. The intra- and inter-array reproducibility assessment indicated that RPPA using FFPE lysates was a highly reproducible and robust technique. Expression of the biomarkers individually and in combination using RPPA was highly consistent with IHC results. Macrodissection of the invasive tumour component gave more reliable results with the majority of biomarkers determined by IHC, (80 % concordance) compared with full-face sections (60 % concordance). Our results provide evidence for the technical feasibility of RPPA for high-throughput protein expression profiling of FFPE breast cancer tissues. The sensitivity of the technique is related to the quality of extracted protein and purity of tumour tissue. RPPA could provide a quantitative technique alternative to IHC for the biomarkers used in the NPI+.

  12. Filament Eruption Onset

    NASA Technical Reports Server (NTRS)

    Sterling, Alphonse C.; Moore, Ronald L.

    2011-01-01

    We have been investigating filament eruptions in recent years. Use filament eruptions as markers of the coronal field evolution. Data from SoHO, Yohkoh, TRACE, Hinode, and other sources. We and others have observed: (1)Filaments often show slow rise, followed by fast rise, (2) Brightenings, preflares, microflares during slow rise (3) Magnetic evolution in hours prior to eruption onset. We investigated What do Hinode and SDO show for filament eruptions?

  13. Mechanistic Insights into Reversible Photoactivation in Proteins of the GFP Family

    PubMed Central

    Gayda, Susan; Nienhaus, Karin; Nienhaus, G. Ulrich

    2012-01-01

    Light-controlled modification of the fluorescence emission properties of proteins of the GFP family is of crucial importance for many imaging applications including superresolution microscopy. Here, we have studied the reversibly photoswitchable fluorescent protein mIrisGFP using optical spectroscopy. By analyzing the pH dependence of isomerization and protonation equilibria and the isomerization kinetics, we have obtained insight into the coupling of the chromophore to the surrounding protein moiety and a better understanding of the photoswitching mechanism. A different acid-base environment of the chromophore’s protonating group in its two isomeric forms, which can be inferred from the x-ray structures of IrisFP, is key to the photoswitching function and ensures that isomerization and protonation are correlated. Amino acids near the chromophore, especially Glu212, rearrange upon isomerization, and Glu212 protonation modulates the chromophore pKa. In mIrisGFP, the cis chromophore protonates in two steps, with pKcis of 5.3 and 6, which is much lower than pKtrans (>10). Based on these results, we have put forward a mechanistic scheme that explains how the combination of isomeric and acid-base properties of the chromophore in its protein environment can produce negative and positive photoswitching modes. PMID:23260054

  14. Autophagy-Associated Protein SmATG12 Is Required for Fruiting-Body Formation in the Filamentous Ascomycete Sordaria macrospora

    PubMed Central

    Werner, Antonia; Herzog, Britta; Frey, Stefan; Pöggeler, Stefanie

    2016-01-01

    In filamentous fungi, autophagy functions as a catabolic mechanism to overcome starvation and to control diverse developmental processes under normal nutritional conditions. Autophagy involves the formation of double-membrane vesicles, termed autophagosomes that engulf cellular components and bring about their degradation via fusion with vacuoles. Two ubiquitin-like (UBL) conjugation systems are essential for the expansion of the autophagosomal membrane: the UBL protein ATG8 is conjugated to the lipid phosphatidylethanolamine and the UBL protein ATG12 is coupled to ATG5. We recently showed that in the homothallic ascomycete Sordaria macrospora autophagy-related genes encoding components of the conjugation systems are required for fruiting-body development and/or are essential for viability. In the present work, we cloned and characterized the S. macrospora (Sm)atg12 gene. Two-hybrid analysis revealed that SmATG12 can interact with SmATG7 and SmATG3. To examine its role in S. macrospora, we replaced the open reading frame of Smatg12 with a hygromycin resistance cassette and generated a homokaryotic ΔSmatg12 knockout strain, which displayed slower vegetative growth under nutrient starvation conditions and was unable to form fruiting bodies. In the hyphae of S. macrospora EGFP-labeled SmATG12 was detected in the cytoplasm and as punctate structures presumed to be phagophores or phagophore assembly sites. Delivery of EGFP-labelled SmATG8 to the vacuole was entirely dependent on SmATG12. PMID:27309377

  15. Enhanced production of heterologous proteins by the filamentous fungus Trichoderma reesei via disruption of the alkaline serine protease SPW combined with a pH control strategy.

    PubMed

    Zhang, Guoxiu; Zhu, Yao; Wei, Dongzhi; Wang, Wei

    2014-01-01

    The filamentous fungus Trichoderma reesei has received attention as a host for heterologous protein production because of its high secretion capacity and eukaryotic post-translational modifications. However, the heterologous production of proteins in T. reesei is limited by its high expression of proteases. The pH control strategies have been proposed for eliminating acidic, but not alkaline, protease activity. In this study, we verified the expression of a relatively major extracellular alkaline protease (GenBank accession number: EGR49466.1, named spw in this study) from 20 candidates through real-time polymerase chain reaction. The transcriptional level of spw increased about 136 times in response to bovine serum albumin as the sole nitrogen source. Additionally, extracellular protease activity was reduced by deleting the spw gene. Therefore, using this gene expression system, we observed enhanced production and stability of the heterologous alkaline endoglucanase EGV from Humicola insolens using the Δspw strain as compared to the parental strain RUT-C30.

  16. Reversible Folding of Human Peripheral Myelin Protein 22, a Tetraspan Membrane Protein†

    PubMed Central

    Schlebach, Jonathan P.; Peng, Dungeng; Kroncke, Brett M.; Mittendorf, Kathleen F.; Narayan, Malathi; Carter, Bruce D.; Sanders, Charles R.

    2013-01-01

    Misfolding of the α-helical membrane protein peripheral myelin protein 22 (PMP22) has been implicated in the pathogenesis of the common neurodegenerative disease known as Charcot-Marie-Tooth disease (CMTD) and also several other related peripheral neuropathies. Emerging evidence suggests that the propensity of PMP22 to misfold in the cell may be due to an intrinsic lack of conformational stability. Therefore, quantitative studies of the conformational equilibrium of PMP22 are needed to gain insight into the molecular basis of CMTD. In this work, we have investigated the folding and unfolding of wild type (WT) human PMP22 in mixed micelles. Both kinetic and thermodynamic measurements demonstrate that the denaturation of PMP22 by n-lauroyl sarcosine (LS) in dodecylphosphocholine (DPC) micelles is reversible. Assessment of the conformational equilibrium indicates that a significant fraction of unfolded PMP22 persists even in the absence of the denaturing detergent. However, we find the stability of PMP22 is increased by glycerol, which facilitates quantitation of thermodynamic parameters. To our knowledge, this work represents the first report of reversible unfolding of a eukaryotic multispan membrane protein. The results indicate that WT PMP22 possesses minimal conformational stability in micelles, which parallels its poor folding efficiency in the endoplasmic reticulum. Folding equilibrium measurements for PMP22 in mixed micelles may provide an approach to assess the effects of cellular metabolites or potential therapeutic agents on its stability. Furthermore, these results pave the way for future investigation of the effects of pathogenic mutations on the conformational equilibrium of PMP22. PMID:23639031

  17. Special issue on filamentation

    NASA Astrophysics Data System (ADS)

    Li, Ruxin; Milchberg, Howard; Mysyrowicz, André

    2014-05-01

    Journal of Physics B: Atomic, Molecular and Optical Physics is delighted to announce a forthcoming special issue on filamentation, to appear in the spring of 2015, and invites you to submit a paper. This special issue will attempt to give an overview of the present status of this field in order to create synergies and foster future developments. The issue is open to papers on the following issues: Theoretical advances on filamentation. Self-focusing and collapse. Filamentation in various media. Pulse self-compression and ultrafast processes in filaments. Molecular alignment and rotation. Filamentation tailoring. Interaction between filaments. Filament weather and pollution control. Filament induced condensation and precipitation. Terahertz science with filaments. Lasing in filaments. Filament induced molecular excitation and reaction. Electric discharge and plasma. Cross-disciplinary applications. Novel concepts related to these topics are particularly welcome. Please submit your article by 1 October 2014 (expected web publication: spring 2015) using our website http://mc04.manuscriptcentral.com/jphysb-iop. Submissions received after this date will be considered for the journal, but may not be included in the special issue. The issue will be edited by Ruxin Li, Howard Milchberg and André Mysyrowicz.

  18. Highly photostable, reversibly photoswitchable fluorescent protein with high contrast ratio for live-cell superresolution microscopy

    PubMed Central

    Zhang, Xi; Zhang, Mingshu; Li, Dong; He, Wenting; Peng, Jianxin; Betzig, Eric; Xu, Pingyong

    2016-01-01

    Two long-standing problems for superresolution (SR) fluorescence microscopy are high illumination intensity and long acquisition time, which significantly hamper its application for live-cell imaging. Reversibly photoswitchable fluorescent proteins (RSFPs) have made it possible to dramatically lower the illumination intensities in saturated depletion-based SR techniques, such as saturated depletion nonlinear structured illumination microscopy (NL-SIM) and reversible saturable optical fluorescence transition microscopy. The characteristics of RSFPs most critical for SR live-cell imaging include, first, the integrated fluorescence signal across each switching cycle, which depends upon the absorption cross-section, effective quantum yield, and characteristic switching time from the fluorescent “on” to “off” state; second, the fluorescence contrast ratio of on/off states; and third, the photostability under excitation and depletion. Up to now, the RSFPs of the Dronpa and rsEGFP (reversibly switchable EGFP) families have been exploited for SR imaging. However, their limited number of switching cycles, relatively low fluorescence signal, and poor contrast ratio under physiological conditions ultimately restrict their utility in time-lapse live-cell imaging and their ability to reach the desired resolution at a reasonable signal-to-noise ratio. Here, we present a truly monomeric RSFP, Skylan-NS, whose properties are optimized for the recently developed patterned activation NL-SIM, which enables low-intensity (∼100 W/cm2) live-cell SR imaging at ∼60-nm resolution at subsecond acquisition times for tens of time points over broad field of view. PMID:27562163

  19. Intermediate filaments in nervous tissues

    PubMed Central

    Liem, RKH; Yen, S; Salomon, GD; Shelanski, ML

    1978-01-01

    Intermediate filaments have been isolated from rabbit intradural spinal nerve roots by the axonal flotation method. This method was modified to avoid exposure of axons to low ionic strength medium. The purified filaments are morphologically 75-80 percent pure. The gel electrophoretogram shows four major bands migrating at 200,000, 145,000, 68,000, and 60,000 daltons, respectively. A similar preparation from rabbit brain shows four major polypeptides with mol wt of 200,000 145,000, 68,000, and 51,000 daltons. These results indicate that the neurofilament is composed of a triplet of polypepetides with mol wt of 200,000, 145,000, and 68,000 daltons. The 51,000-dalton band that appears in brain filament preparations as the major polypeptide seems to be of glial origin. The significance of the 60,000- dalton band in the nerve root filament preparation is unclear at this time. Antibodies raised against two of the triplet proteins isolated from calf brain localize by immunofluorescence to neurons in central and peripheral nerve. On the other hand, an antibody to the 51,000-dalton polypeptide gives only glial staining in the brain, and very weak peripheral nerve staining. Prolonged exposure of axons to low ionic strength medium solubilizes almost all of the triplet polypeptides, leaving behind only the 51,000- dalton component. This would indicate that the neurofilament is soluble at low ionic strength, whereas the glial filament is not. These results indicate that neurofilaments and glial filaments are composed of different polypeptides and have different solubility characteristics. PMID:83322

  20. Epithelial Intermediate Filaments: Guardians against Microbial Infection?

    PubMed Central

    Geisler, Florian; Leube, Rudolf E.

    2016-01-01

    Intermediate filaments are abundant cytoskeletal components of epithelial tissues. They have been implicated in overall stress protection. A hitherto poorly investigated area of research is the function of intermediate filaments as a barrier to microbial infection. This review summarizes the accumulating knowledge about this interaction. It first emphasizes the unique spatial organization of the keratin intermediate filament cytoskeleton in different epithelial tissues to protect the organism against microbial insults. We then present examples of direct interaction between viral, bacterial, and parasitic proteins and the intermediate filament system and describe how this affects the microbe-host interaction by modulating the epithelial cytoskeleton, the progression of infection, and host response. These observations not only provide novel insights into the dynamics and function of intermediate filaments but also indicate future avenues to combat microbial infection. PMID:27355965

  1. Development of porous polymer monoliths for reverse-phase chromatography of proteins.

    SciTech Connect

    Shepodd, Timothy J.; Stephens, Christopher P.

    2003-09-01

    The polymers developed in this project are intended for use as a stationary phase in reverse-phase chromatography of proteins, where the mobile phase is a solution of acetonitrile and a phosphate buffer, 6.6 pH. A full library of pore sizes have been developed ranging from 0.41{micro}m to 4.09 {micro}m; these pore sizes can be determined by the solvent ratio of tetrahydrofuran:methoxyethanol during polymerization. A column that can separate proteins in an isocratic mode would be a vast improvement from the common method of separating proteins through gradient chromatography using multiple solvents. In the stationary phase, the main monomers have hydrophobic tails, lauryl acrylate and steryl acrylate. Separations of small hydrophobic molecules and peptides (trial molecules) have efficiencies of 24,000-33,000 theoretical plates m{sup -1}. The combination of a highly non-polar stationary phase and a mobile phase where the polarity can be controlled provide for excellent separation.

  2. In situ, Reversible Gating of a Mechanosensitive Ion Channel through Protein-Lipid Interactions

    PubMed Central

    Dimitrova, Anna; Walko, Martin; Hashemi Shabestari, Maryam; Kumar, Pravin; Huber, Martina; Kocer, Armagan

    2016-01-01

    Understanding the functioning of ion channels, as well as utilizing their properties for biochemical applications requires control over channel activity. Herein we report a reversible control over the functioning of a mechanosensitive ion channel by interfering with its interaction with the lipid bilayer. The mechanosensitive channel of large conductance from Escherichia coli is reconstituted into liposomes and activated to its different sub-open states by titrating lysophosphatidylcholine (LPC) into the lipid bilayer. Activated channels are closed back by the removal of LPC out of the membrane by bovine serum albumin (BSA). Electron paramagnetic resonance spectra showed the LPC-dose-dependent gradual opening of the channel pore in the form of incrementally increasing spin label mobility and decreasing spin-spin interaction. A method to reversibly open and close mechanosensitive channels to distinct sub-open conformations during their journey from the closed to the fully open state enables detailed structural studies to follow the conformational changes during channel functioning. The ability of BSA to revert the action of LPC opens new perspectives for the functional studies of other membrane proteins that are known to be activated by LPC. PMID:27708587

  3. Targeting cellular prion protein reverses early cognitive deficits and neurophysiological dysfunction in prion-infected mice.

    PubMed

    Mallucci, Giovanna R; White, Melanie D; Farmer, Michael; Dickinson, Andrew; Khatun, Husna; Powell, Andrew D; Brandner, Sebastian; Jefferys, John G R; Collinge, John

    2007-02-01

    Currently, no treatment can prevent the cognitive and motor decline associated with widespread neurodegeneration in prion disease. However, we previously showed that targeting endogenous neuronal prion protein (PrP(C)) (the precursor of its disease-associated isoform, PrP(Sc)) in mice with early prion infection reversed spongiform change and prevented clinical symptoms and neuronal loss. We now show that cognitive and behavioral deficits and impaired neurophysiological function accompany early hippocampal spongiform pathology. Remarkably, these behavioral and synaptic impairments recover when neuronal PrP(C) is depleted, in parallel with reversal of spongiosis. Thus, early functional impairments precede neuronal loss in prion disease and can be rescued. Further, they occur before extensive PrP(Sc) deposits accumulate and recover rapidly after PrP(C) depletion, supporting the concept that they are caused by a transient neurotoxic species, distinct from aggregated PrP(Sc). These data suggest that early intervention in human prion disease may lead to recovery of cognitive and behavioral symptoms.

  4. Mollusc C-reactive protein crosses species barrier and reverses hepatotoxicity of lead in rodent models.

    PubMed

    Mukherjee, Sandip; Chatterjee, Sarmishtha; Sarkar, Shuvasree; Agarwal, Soumik; Kundu, Rakesh; Maitra, Sudipta; Bhattacharya, Shelley

    2013-08-01

    Achatina fulica C-reactive protein (ACRP) reversed the toxic effects of lead nitrate both in vivo in mice and in vitro in rat hepatocytes restoring the basal level of cell viability, lipid peroxidation, reduced glutathione and superoxides. Cytotoxicity was also significantly ameliorated in rat hepatocytes by in vitro pre-treatments with individual subunits (60, 62, 90 and 110 kDa) of ACRP. Annexin V-Cy3/CFDA dual staining showed significant reduction in the number of apoptotic hepatocytes pre-treated with ACRP. ACRP induced restoration of mitochondrial membrane potential was remarkable. ACRP pre-treatment prevented Pb-induced apoptosis mediated by caspase activation. The antagonistic effect of ACRP may be due to scavenging of reactive oxygen species which maintained the homeostasis of cellular redox potential as well as reduced glutathione status. The results suggest that ACRP crosses the species barrier and it may be utilized as a viable exogenous agent of cytoprotection against heavy metal related toxicity.

  5. Pex14/17, a filamentous fungus-specific peroxin, is required for the import of peroxisomal matrix proteins and full virulence of Magnaporthe oryzae.

    PubMed

    Li, Ling; Wang, Jiaoyu; Chen, Haili; Chai, Rongyao; Zhang, Zhen; Mao, Xueqin; Qiu, Haiping; Jiang, Hua; Wang, Yanli; Sun, Guochang

    2016-08-29

    Peroxisomes are ubiquitous organelles in eukaryotic cells that fulfil a variety of biochemical functions. The biogenesis of peroxisomes requires a variety of proteins, named peroxins, which are encoded by PEX genes. Pex14/17 is a putative recently identified peroxin, specifically present in filamentous fungal species. Its function in peroxisomal biogenesis is still obscure and its roles in fungal pathogenicity have not yet been documented. Here, we demonstrate the contributions of Pex14/17 in the rice blast fungus Magnaporthe oryzae (Mopex14/17) to peroxisomal biogenesis and fungal pathogenicity by targeting gene replacement strategies. Mopex14/17 has properties of both Pex14 and Pex17 with regard to its protein sequence. Mopex14/17 is distributed at the peroxisomal membrane and is essential for efficient peroxisomal targeting of proteins containing peroxisomal targeting signal 1. MoPEX19 deletion leads to the cytoplasmic distribution of Mopex14/17, indicating that the peroxisomal import of Pex14/17 is dependent on Pex19. The knockout mutants of MoPEX14/17 show reduced fatty acid utilization, reactive oxygen species (ROS) degradation and cell wall integrity. Moreover, Δmopex14/17 mutants show delayed conidial generation and appressorial formation, and a reduction in appressorial turgor accumulation and penetration ability in host plants. These defects result in a significant reduction in the virulence of the mutant. These data indicate that MoPEX14/17 plays a crucial role in peroxisome biogenesis and contributes to fungal development and pathogenicity.

  6. Human enhancer of filamentation 1-induced colorectal cancer cell migration: Role of serine phosphorylation and interaction with the breast cancer anti-estrogen resistance 3 protein.

    PubMed

    Ibrahim, Rama; Lemoine, Antoinette; Bertoglio, Jacques; Raingeaud, Joël

    2015-07-01

    Human enhancer of filamentation 1 (HEF1) is a member of the p130Cas family of docking proteins involved in integrin-mediated cytoskeleton reorganization associated with cell migration. Elevated expression of HEF1 promotes invasion and metastasis in multiple cancer cell types. To date, little is known on its role in CRC tumor progression. HEF1 is phosphorylated on several Ser/Thr residues but the effects of these post-translational modifications on the functions of HEF1 are poorly understood. In this manuscript, we investigated the role of HEF1 in migration of colorectal adeno-carcinoma cells. First, we showed that overexpression of HEF1 in colo-carcinoma cell line HCT116 increases cell migration. Moreover, in these cells, HEF1 increases Src-mediated phosphorylation of FAK on Tyr-861 and 925. We then showed that HEF1 mutation on Ser-369 enhances HEF1-induced migration and FAK phosphorylation as a result of protein stabilization. We also, for the first time characterized a functional mutation of HEF1 on Arg-367 which mimics the effect of Ser-369 to Ala mutation. Finally through mass spectrometry experiments, we identified BCAR3 as an essential interactor and mediator of HEF1-induced migration. We demonstrated that single amino acid mutations that prevent formation of the HEF1-BCAR3 complex impair HEF1-mediated migration. Therefore, amino-acid substitutions that impede Ser-369 phosphorylation stabilize HEF1 which increases the migration of CRC cells and this latter effect requires the interaction of HEF1 with the NSP family adaptor protein BCAR3. Collectively, these data reveal the importance of HEF1 expression level in cancer cell motility and then support the utilization of HEF1 as a biomarker of tumor progression.

  7. Characterization and regulation of an additional actin-filament-binding site in large isoforms of the stereocilia actin-bundling protein espin.

    PubMed

    Zheng, Lili; Beeler, Dina M; Bartles, James R

    2014-03-15

    The espin actin-bundling proteins, which are produced as isoforms of different sizes from a single gene, are required for the growth of hair cell stereocilia. We have characterized an additional actin-filament-binding site present in the extended amino-termini of large espin isoforms. Constitutively active in espin 2, the site increased the size of actin bundles formed in vitro and inhibited actin fluorescence recovery in microvilli. In espin 1, which has an N-terminal ankyrin repeat domain, the site was autoinhibited by binding between the ankyrin repeat domain and a peptide near the actin-binding site. Deletion of this peptide from espin 1 activated its actin-binding site. The peptide resembled tail homology domain I of myosin III, a ligand of the ankyrin repeat domain localized with espin 1 at the tip of stereocilia. A myosin III tail homology domain I peptide, but not scrambled control peptides, inhibited internal binding of the ankyrin repeat domain and released the espin 1 actin-binding site from autoinhibition. Thus, this regulation could result in local activation of the additional actin-binding site of espin 1 by myosin III in stereocilia.

  8. Mechanical Heterogeneity Favors Fragmentation of Strained Actin Filaments

    PubMed Central

    De La Cruz, Enrique M.; Martiel, Jean-Louis; Blanchoin, Laurent

    2015-01-01

    We present a general model of actin filament deformation and fragmentation in response to compressive forces. The elastic free energy density along filaments is determined by their shape and mechanical properties, which were modeled in terms of bending, twisting, and twist-bend coupling elasticities. The elastic energy stored in filament deformation (i.e., strain) tilts the fragmentation-annealing reaction free-energy profile to favor fragmentation. The energy gradient introduces a local shear force that accelerates filament intersubunit bond rupture. The severing protein, cofilin, renders filaments more compliant in bending and twisting. As a result, filaments that are partially decorated with cofilin are mechanically heterogeneous (i.e., nonuniform) and display asymmetric shape deformations and energy profiles distinct from mechanically homogenous (i.e., uniform), bare actin, or saturated cofilactin filaments. The local buckling strain depends on the relative size of the compliant segment as well as the bending and twisting rigidities of flanking regions. Filaments with a single bare/cofilin-decorated boundary localize energy and force adjacent to the boundary, within the compliant cofilactin segment. Filaments with small cofilin clusters were predicted to fragment within the compliant cofilactin rather than at boundaries. Neglecting contributions from twist-bend coupling elasticity underestimates the energy density and gradients along filaments, and thus the net effects of filament strain to fragmentation. Spatial confinement causes compliant cofilactin segments and filaments to adopt higher deformation modes and store more elastic energy, thereby promoting fragmentation. The theory and simulations presented here establish a quantitative relationship between actin filament fragmentation thermodynamics and elasticity, and reveal how local discontinuities in filament mechanical properties introduced by regulatory proteins can modulate both the severing efficiency

  9. Intermediate filament protein accumulation in motor neurons derived from giant axonal neuropathy iPSCs rescued by restoration of gigaxonin

    PubMed Central

    Johnson-Kerner, Bethany L.; Ahmad, Faizzan S.; Diaz, Alejandro Garcia; Greene, John Palmer; Gray, Steven J.; Samulski, Richard Jude; Chung, Wendy K.; Van Coster, Rudy; Maertens, Paul; Noggle, Scott A.; Henderson, Christopher E.; Wichterle, Hynek

    2015-01-01

    Giant axonal neuropathy (GAN) is a progressive neurodegenerative disease caused by autosomal recessive mutations in the GAN gene resulting in a loss of a ubiquitously expressed protein, gigaxonin. Gene replacement therapy is a promising strategy for treatment of the disease; however, the effectiveness and safety of gigaxonin reintroduction have not been tested in human GAN nerve cells. Here we report the derivation of induced pluripotent stem cells (iPSCs) from three GAN patients with different GAN mutations. Motor neurons differentiated from GAN iPSCs exhibit accumulation of neurofilament (NF-L) and peripherin (PRPH) protein and formation of PRPH aggregates, the key pathological phenotypes observed in patients. Introduction of gigaxonin either using a lentiviral vector or as a stable transgene resulted in normalization of NEFL and PRPH levels in GAN neurons and disappearance of PRPH aggregates. Importantly, overexpression of gigaxonin had no adverse effect on survival of GAN neurons, supporting the feasibility of gene replacement therapy. Our findings demonstrate that GAN iPSCs provide a novel model for studying human GAN neuropathologies and for the development and testing of new therapies in relevant cell types. PMID:25398950

  10. Synthesis of surface protein-imprinted nanoparticles endowed with reversible physical cross-links.

    PubMed

    Yang, Chongchong; Yan, Xianming; Guo, Hao; Fu, Guoqi

    2016-01-15

    Researches on protein molecularly imprinted polymers have been challenged by the difficulties in facilitating biomacromolecular transfer, in particular upon the template removal step, and enhancing their recognition performance. Addressing these issues, herein we report synthesis of core–shell structured surface protein-imprinted nanoparticles with reversible physical cross-links formed in the imprinted nanoshells. The imprinted layers over nanoparticle supports are fabricated via aqueous precipitation polymerization (PP) of di(ethylene glycol) methyl ether methacrylate (MEO2MA), a thermo-responsive monomer bearing no strong H-bond donor, and other functional and cross-linking monomers. During polymerization, physical cross-links together with chemical cross-links are in site produced within the imprinted shells based on hydrophobic association among the PMEO2MA, favoring formation of high-quality imprints. While cooled appropriately below the polymerization temperature, these physical cross-links can be dissociated rapidly, thus facilitating removal of the embedded template. For proof of this concept, lysozyme-imprinted nanoparticles were synthesized at 37 °C over the nanoparticles functionalized with carboxylic and vinyl groups. The template removal from the imprinted nanoparticles was readily achieved by washing with a dilute acidic detergent solution at 4 °C. As-prepared imprinted nanoparticles showed greatly higher imprinting factor and specific rebinding than obtained with the same recipe but by solution polymerization (SP). Moreover, such imprinted nanomaterials exhibited satisfactory rebinding selectivity, kinetics and reusability.

  11. Quantitative assay of diphtherial toxin and of immunologically cross-reacting proteins by reversed passive hemagglutination.

    PubMed Central

    Holmes, R K; Perlow, R B

    1975-01-01

    A reversed passive hemagglutination (RPHA) assay for diptherial toxin has been developed. Antitoxic antibodies were isolated from commercially available equine diptherial antitoxin by immunoabsorption using highly purified diphtherial toxin covalently linked to Sepharose 4B. Formalinized, tanned sheep erythrocytes sensitized with the purified antitoxic antibodies are specifically agglutinated by diphtherial toxin but are not agglutinated by extracellular antigens of Corynebacterium diptheriae that are unrelated to toxin. The RPHA assay described can detect less than 20 pg of diphtherial toxin and is comparable in sensitivity to intracutaneous tests for toxin. The RPHA assay was shown to be at least 1,000 times more sensitive than quantitative immunological assays for diptherial toxin performed by single radial immunodiffusion or by one-dimensional double diffusion in agar gels. Fragment A prepared from purified diphtherial toxin and nontoxic mutant proteins that cross-react immunologically with toxin can be assayed directly by RPHA, but the sensitivity of the assay for these proteins is less than for native diphtherial toxin. Inhibition of RPHA was also shown to be a sensitive quantitative method for measuring diptherial antitoxin in vitro. Images PMID:54339

  12. Reversible immobilization of proteins with streptavidin affinity tags on a surface plasmon resonance biosensor chip.

    PubMed

    Li, Yong-Jin; Bi, Li-Jun; Zhang, Xian-En; Zhou, Ya-Feng; Zhang, Ji-Bin; Chen, Yuan-Yuan; Li, Wei; Zhang, Zhi-Ping

    2006-11-01

    Dissociation of biotin from streptavidin is very difficult due to their high binding affinity. The re-use of streptavidin-modified surfaces is therefore almost impossible, making devices containing them (e.g. surface plasmon resonance (SPR) sensor chips) expensive. This paper describes a new protocol for reversible and site-directed immobilization of proteins with streptavidin affinity tags on the streptavidin-coated SPR biosensor chip (SA chip). Two streptavidin affinity tags, nano-tag and streptavidin-binding peptide (SBP tag), were applied. They both can specifically interact with streptavidin but have weaker binding force compared to the biotin-streptavidin system, thus allowing association and dissociation under controlled conditions. The SA chip surface could be regenerated repeatedly without loss of activity by injection of 50 mM NaOH solution. The fusion construct of a SBP tag and a single-chain antibody to mature bovine prion protein (scFv-Z186-SBP) interacts with the SA chip, resulting in a single-chain-antibody-modified surface. The chip showed kinetic response to the prion antigen with equilibrium dissociation constant K (D) approximately equal to 4.01 x 10(-7). All results indicated that the capture activity of the SA chip has no irreversible loss after repeated immobilization and regeneration cycles. The method should be of great benefit to various biosensors, biochips and immunoassay applications based on the streptavidin capture surface.

  13. Reversible post-translational modification of proteins by nitrated fatty acids in vivo.

    PubMed

    Batthyany, Carlos; Schopfer, Francisco J; Baker, Paul R S; Durán, Rosario; Baker, Laura M S; Huang, Yingying; Cerveñansky, Carlos; Branchaud, Bruce P; Freeman, Bruce A

    2006-07-21

    Nitric oxide ((*)NO)-derived reactive species nitrate unsaturated fatty acids, yielding nitroalkene derivatives, including the clinically abundant nitrated oleic and linoleic acids. The olefinic nitro group renders these derivatives electrophilic at the carbon beta to the nitro group, thus competent for Michael addition reactions with cysteine and histidine. By using chromatographic and mass spectrometric approaches, we characterized this reactivity by using in vitro reaction systems, and we demonstrated that nitroalkene-protein and GSH adducts are present in vivo under basal conditions in healthy human red cells. Nitro-linoleic acid (9-, 10-, 12-, and 13-nitro-9,12-octadecadienoic acids) (m/z 324.2) and nitro-oleic acid (9- and 10-nitro-9-octadecaenoic acids) (m/z 326.2) reacted with GSH (m/z 306.1), yielding adducts with m/z of 631.3 and 633.3, respectively. At physiological concentrations, nitroalkenes inhibited glyceraldehyde-3-phosphate dehydrogenase (GAPDH), which contains a critical catalytic Cys (Cys-149). GAPDH inhibition displayed an IC(50) of approximately 3 microM for both nitroalkenes, an IC(50) equivalent to the potent thiol oxidant peroxynitrite (ONOO(-)) and an IC(50) 30-fold less than H(2)O(2), indicating that nitroalkenes are potent thiol-reactive species. Liquid chromatography-mass spectrometry analysis revealed covalent adducts between fatty acid nitroalkene derivatives and GAPDH, including at the catalytic Cys-149. Liquid chromatography-mass spectrometry-based proteomic analysis of human red cells confirmed that nitroalkenes readily undergo covalent, thiol-reversible post-translational modification of nucleophilic amino acids in GSH and GAPDH in vivo. The adduction of GAPDH and GSH by nitroalkenes significantly increased the hydrophobicity of these molecules, both inducing translocation to membranes and suggesting why these abundant derivatives had not been detected previously via traditional high pressure liquid chromatography analysis. The

  14. Seasonal changes in isoform composition of giant proteins of thick and thin filaments and titin (connectin) phosphorylation level in striated muscles of bears (Ursidae, Mammalia).

    PubMed

    Salmov, N N; Vikhlyantsev, I M; Ulanova, A D; Gritsyna, Yu V; Bobylev, A G; Saveljev, A P; Makariushchenko, V V; Maksudov, G Yu; Podlubnaya, Z A

    2015-03-01

    Seasonal changes in the isoform composition of thick and thin filament proteins (titin, myosin heavy chains (MyHCs), nebulin), as well as in the phosphorylation level of titin in striated muscles of brown bear (Ursus arctos) and hibernating Himalayan black bear (Ursus thibetanus ussuricus) were studied. We found that the changes that lead to skeletal muscle atrophy in bears during hibernation are not accompanied by a decrease in the content of nebulin and intact titin-1 (T1) isoforms. However, a decrease (2.1-3.4-fold) in the content of T2 fragments of titin was observed in bear skeletal muscles (m. gastrocnemius, m. longissimus dorsi, m. biceps) during hibernation. The content of the stiffer N2B titin isoform was observed to increase relative to the content of its more compliant N2BA isoform in the left ventricles of hibernating bears. At the same time, in spite of the absence of decrease in the total content of T1 in the myocardium of hibernating brown bear, the content of T2 fragments decreased ~1.6-fold. The level of titin phosphorylation only slightly increased in the cardiac muscle of hibernating brown bear. In the skeletal muscles of brown bear, the level of titin phosphorylation did not vary between seasons. However, changes in the composition of MyHCs aimed at increasing the content of slow (I) and decreasing the content of fast (IIa) isoforms of this protein during hibernation of brown bear were detected. Content of MyHCs I and IIa in the skeletal muscles of hibernating Himalayan black bear corresponded to that in the skeletal muscles of hibernating brown bear.

  15. Antibodies to intermediate filament proteins as molecular markers in clinical tumor pathology. Differentiation of carcinomas by their reaction with different cytokeratin antibodies.

    PubMed

    Krepler, R; Denk, H; Artlieb, U; Fichtinger, E; Davidovits, A

    1982-01-01

    Antibodies to human and bovine epidermal prekeratin and antibodies to mouse liver cytokeratin component D (Mr 49 000) have been applied in indirect immunofluorescence microscopy on sections of human tumors of mammary gland and liver. In non-neoplastic mammary gland all epithelial cells were stained with these antibodies. In pre-invasive and invasive ductal and lobular carcinomas a cell population was observed which was not significantly stained with antibodies to epidermal prekeratin but did strongly react with antibodies to liver cytokeratin D. In the liver, the antibodies to epidermal prekeratin as well as those directed against liver cytokeratin D strongly decorated bile duct epithelia. In contrast, significant staining of the hepatocytes was only achieved with antibodies to liver cytokeratin D. This different staining reaction was maintained in liver tumors of hepatocellular and cholangiocellular origin. Antibodies to vimentin stained mesenchymal cells and tumors of mesenchymal derivation but reacted not significantly with any of the epithelial and carcinoma cells examined. The difference is of practical importance for the discrimination between anaplastic carcinomas and sarcomas of unknown origin. Cytokeratin could also be detected by antibody staining using the peroxidase-antiperoxidase (PAP) technique in formaldehyde-fixed and paraffin-embedded material of skin, gastrointestinal, respiratory, urinary and genital tract as well as various glands, liver and kidney. Examples of positive reactions were shown in a squamous cell carcinoma, a basalioma and a pleomorphic adenoma of the parotis. It is concluded that the immunohistochemical analysis of intermediate filament proteins has diagnostic potential in clinical pathology and may help to elucidate histogenesis and differentiation of tumors and possibly also prognosis of tumor growth. It is further suggested to use antibodies recognizing different subsets of proteins of the cytokeratin family in order to

  16. Large-Scale Patterns of Filament Channels and Filaments

    NASA Astrophysics Data System (ADS)

    Mackay, Duncan

    2016-07-01

    In this review the properties and large-scale patterns of filament channels and filaments will be considered. Initially, the global formation locations of filament channels and filaments are discussed, along with their hemispheric pattern. Next, observations of the formation of filament channels and filaments are described where two opposing views are considered. Finally, the wide range of models that have been constructed to consider the formation of filament channels and filaments over long time-scales are described, along with the origin of the hemispheric pattern of filaments.

  17. Improving the electrochemical performance of carbon filaments by solvent cleansing

    SciTech Connect

    Shui, X.; Chung, D.D.L.; Frysz, C.A.

    1995-12-31

    Found inherent in the submicron-diameter vapor-grown carbon filament fabrication process was a tarry residue, which comprised polyaromatic hydrocarbons. Cyclic voltammetry conducted using carbon working electrodes and an iron cyanide electrolyte showed that the residue harmed the electrochemical performance. Removal of the residue from the filaments using a solvent resulted in increases in the electron transfer rate (to values as high as 0.2 cm/s) and reversibility of the iron cyanide redox species, increase in the packing density and decrease in the filament-filament contact electrical resistivity.

  18. Dynamics of heteromolecular filament formation

    NASA Astrophysics Data System (ADS)

    Dear, Alexander J.; Michaels, Thomas C. T.; Knowles, Tuomas P. J.

    2016-11-01

    The self-assembly of molecular building blocks into linear filaments is a common form of self-organization in nature and underlies the formation of supra-molecular polymers in a variety of contexts, including in both functional and aberrant biology. To date, attention has focused mainly on homomolecular assembly phenomena; however, it has recently become apparent that heteromolecular assemblies can be common, and, for instance, pathological protein filaments such as amyloid aggregates form in vivo in environments supporting copolymerization. Here, we present a general kinetic scheme for heteromolecular filament formation and derive closed-form analytical expressions that describe the dynamics of such systems. Our results reveal the existence of a demixing transition time controlled by the relative rates of depletion of the different aggregating species, after which predominantly homomolecular polymers are formed even when the initial solution is heteromolecular. Furthermore, these results may be applied to the analysis of experimental kinetic data on the aggregation of mixtures of proteins, to determine which fundamental reaction steps occur between unlike proteins, and to provide accurate estimates of their rate constants.

  19. A novel filamentous phage from the deep-sea bacterium Shewanella piezotolerans WP3 is induced at low temperature.

    PubMed

    Wang, Feng; Wang, Fengping; Li, Qiang; Xiao, Xiang

    2007-10-01

    Active filamentous phage particles were isolated from the deep-sea bacterium Shewanella piezotolerans WP3. A putative single-stranded DNA binding protein of the phage was found to be overexpressed at 4 degrees C compared to its expression at 25 degrees C by two-dimensional polyacrylamide gel electrophoresis. Reverse transcription quantitative PCR further revealed that the key genes of the SW1 phage were significantly induced at low temperature.

  20. Three-color FRET expands the ability to quantify the interactions of several proteins involved in actin filament nucleation

    NASA Astrophysics Data System (ADS)

    Wallrabe, Horst; Sun, Yuansheng; Fang, Xiaolan; Periasamy, Ammasi; Bloom, George

    2012-03-01

    With traditional 2-color Förster Resonance Energy Transfer (FRET) microscopy, valuable quantitative analyses can be conducted. Correlations of donor (D), acceptor (A) and their ratios (D:A) with energy transfer efficiency (E%) or distance (r) allows measurement of changes between control and experimental samples; also, clustered vs. random assembly of cellular components can be differentiated. Essentially, only the above three parameters D, A and D:A vs. E% are the basis for these deductions. 3-color FRET uses the same basic parameters, but exponentially expands the opportunities to quantify interrelationships among 3 cellular components. We investigated a number of questions based on the results of a triple combination (F1-F2-F3) of TFPNWASP/ Venus-IQGAP1/mCherry-Actin - all involved in the nucleation of actin - to apply the extensive analysis assay possible with 3-color FRET. How do changing N-WASP or IQGAP1 fluorescence levels affect actin fluorescence? What is the effect on E% of NWASP-actin by IQGAP1 or E% of IQGAP1-actin by N-WASP? These and other questions are explored in the context of all proteins of interest being in FRET distance vs. any two in the absence of the third. 4 cases are compared based on bleed-through corrected FRET: (1) all 3 interact, (2) only F1- F3 and F2-F3 [not F1-F2], (3) only F1-F2 and F2-F3 interact [not F1-F3], (4) only F1-F2 and F1-F3 interact [not F2-F3]. Other than describing the methodology in detail, several biologically relevant results are presented showing how E% (i.e. distance), fluorescence levels and ratios are affected in each of the cases. These correlations can only be observed in a 3-fluorophore combination. 3-color FRET will greatly expand the investigative range of quantitative analysis for the life-science researcher.

  1. Tungsten Filament Fire

    ERIC Educational Resources Information Center

    Ruiz, Michael J.; Perkins, James

    2016-01-01

    We safely remove the outer glass bulb from an incandescent lamp and burn up the tungsten filament after the glass is removed. This demonstration dramatically illustrates the necessity of a vacuum or inert gas for the environment surrounding the tungsten filament inside the bulb. Our approach has added historical importance since the incandescent…

  2. Tungsten filament fire

    NASA Astrophysics Data System (ADS)

    Ruiz, Michael J.; Perkins, James

    2016-05-01

    We safely remove the outer glass bulb from an incandescent lamp and burn up the tungsten filament after the glass is removed. This demonstration dramatically illustrates the necessity of a vacuum or inert gas for the environment surrounding the tungsten filament inside the bulb. Our approach has added historical importance since the incandescent light bulb is being replaced by compact fluorescent and LED lamps.

  3. Development of a reversibly switchable fluorescent protein for super-resolution optical fluctuation imaging (SOFI).

    PubMed

    Zhang, Xi; Chen, Xuanze; Zeng, Zhiping; Zhang, Mingshu; Sun, Yujie; Xi, Peng; Peng, Jianxin; Xu, Pingyong

    2015-03-24

    Reversibly switchable fluorescent proteins (RSFPs) can be effectively used for super-resolution optical fluctuation imaging (SOFI) based on the switching and fluctuation of single molecules. Several properties of RSFPs strongly influence the quality of SOFI images. These properties include (i) the averaged fluorescence intensity in the fluctuation state, (ii) the on/off contrast ratio, (iii) the photostability, and (iv) the oligomerization tendency. The first three properties determine the fluctuation range of the imaged pixels and the SOFI signal, which are of essential importance to the spatial resolution, and the last may lead to artificial aggregation of target proteins. The RSFPs that are currently used for SOFI are low in averaged fluorescence intensity in the fluctuation state, photostability, and on/off contrast ratio, thereby limiting the range of application of SOFI in biological super-resolution imaging. In this study, we developed a novel monomeric green RSFP termed Skylan-S, which features very high photostability, contrast ratio, and averaged fluorescence intensity in the fluctuation state. Taking advantage of the excellent optical properties of Skylan-S, a 4-fold improvement in the fluctuation range of the imaged pixels and higher SOFI resolution can be obtained compared with Dronpa. Furthermore, super-resolution imaging of the actin or tubulin structures and clathrin-coated pits (CCPs) in living U2OS cells labeled with Skylan-S was demonstrated using the SOFI technique. Overall, Skylan-S developed with outstanding photochemical properties is promising for long-time SOFI imaging with high spatial-temporal resolution.

  4. Quantitative capillary reversed passive latex agglutination test for C-reactive protein (CRP) in the dog.

    PubMed

    Tagata, K; Yokoyama, S; Ginbo, T; Honda, M; Okimura, T; Odakura, M; Nomura, M; Yamamoto, S

    1996-01-01

    A capillary reversed passive latex agglutination test (capillary RPLA) was developed which allows quantification of serum C-reactive protein (CRP) within approximately 15 min. The logarithmic regression line (calibration curve) obtained after measuring each CRP concentration three times in twofold dilutions of a standard canine serum containing 222 micrograms/ml of CRP was y = 6.394 + 0.030x (r = 0.995). Capillary RPLA permitted quantification of CRP in the range 6.9-222 micrograms/ml. The coefficients of variation ranged from 10.28% to 12.40%. The recovery rates (percentage recovery) of CRP by capillary RPLA were within the range 87% to 106%. On measuring the CRP concentrations in sera from 78 dogs by capillary RPLA, single radial immunodiffusion (SRID) and enzyme-linked immunosorbent assay (ELISA), close correlations were demonstrated between SRID and capillary RPLA (y = 7.250 + 1.109x, r = 0.978), between SRID and ELISA (y = 3.042 + 1.059x, r = 0.967), and between capillary RPLA and ELISA (y = 1.778 + 0.929x, r = 0.962). Capillary RPLA may be considered useful as a routine biochemical technique for measurement of serum CRP concentration in the dog.

  5. Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.

    PubMed

    Schellenberg, Matthew J; Perera, Lalith; Strom, Christina N; Waters, Crystal A; Monian, Brinda; Appel, C Denise; Vilas, Caroline K; Williams, Jason G; Ramsden, Dale A; Williams, R Scott

    2016-05-05

    Mammalian Tyrosyl-DNA phosphodiesterase 2 (Tdp2) reverses Topoisomerase 2 (Top2) DNA-protein crosslinks triggered by Top2 engagement of DNA damage or poisoning by anticancer drugs. Tdp2 deficiencies are linked to neurological disease and cellular sensitivity to Top2 poisons. Herein, we report X-ray crystal structures of ligand-free Tdp2 and Tdp2-DNA complexes with alkylated and abasic DNA that unveil a dynamic Tdp2 active site lid and deep substrate binding trench well-suited for engaging the diverse DNA damage triggers of abortive Top2 reactions. Modeling of a proposed Tdp2 reaction coordinate, combined with mutagenesis and biochemical studies support a single Mg(2+)-ion mechanism assisted by a phosphotyrosyl-arginine cation-π interface. We further identify a Tdp2 active site SNP that ablates Tdp2 Mg(2+) binding and catalytic activity, impairs Tdp2 mediated NHEJ of tyrosine blocked termini, and renders cells sensitive to the anticancer agent etoposide. Collectively, our results provide a structural mechanism for Tdp2 engagement of heterogeneous DNA damage that causes Top2 poisoning, and indicate that evaluation of Tdp2 status may be an important personalized medicine biomarker informing on individual sensitivities to chemotherapeutic Top2 poisons.

  6. Effects of protein tyrosine phosphatase-PEST are reversed by Akt in T cells.

    PubMed

    Arimura, Yutaka; Shimizu, Kazuhiko; Koyanagi, Madoka; Yagi, Junji

    2014-12-01

    T cell activation is regulated by a balance between phosphorylation and dephosphorylation that is under the control of kinases and phosphatases. Here, we examined the role of a non-receptor-type protein tyrosine phosphatase, PTP-PEST, using retrovirus-mediated gene transduction into murine T cells. Based on observations of vector markers (GFP or Thy1.1), exogenous PTP-PEST-positive CD4(+) T cells appeared within 2 days after gene transduction; the percentage of PTP-PEST-positive cells tended to decrease during a resting period in the presence of IL-2 over the next 2 days. These vector markers also showed much lower expression intensities, compared with control cells, suggesting a correlation between the percent reduction and the low marker expression intensity. A catalytically inactive PTP-PEST mutant also showed the same tendency, and stepwise deletion mutants gradually lost their ability to induce the above phenomenon. On the other hand, these PTP-PEST-transduced cells did not have an apoptotic phenotype. No difference in the total cell numbers was found in the wells of a culture plate containing VEC- and PTP-PEST-transduced T cells. Moreover, serine/threonine kinase Akt, but not the anti-apoptotic molecules Bcl-2 and Bcl-XL, reversed the phenotype induced by PTP-PEST. We discuss the novel mechanism by which Akt interferes with PTP-PEST.

  7. Reversible Conformational Change in the Plasmodium falciparum Circumsporozoite Protein Masks Its Adhesion Domains.

    PubMed

    Herrera, Raul; Anderson, Charles; Kumar, Krishan; Molina-Cruz, Alvaro; Nguyen, Vu; Burkhardt, Martin; Reiter, Karine; Shimp, Richard; Howard, Randall F; Srinivasan, Prakash; Nold, Michael J; Ragheb, Daniel; Shi, Lirong; DeCotiis, Mark; Aebig, Joan; Lambert, Lynn; Rausch, Kelly M; Muratova, Olga; Jin, Albert; Reed, Steven G; Sinnis, Photini; Barillas-Mury, Carolina; Duffy, Patrick E; MacDonald, Nicholas J; Narum, David L

    2015-10-01

    The extended rod-like Plasmodium falciparum circumsporozoite protein (CSP) is comprised of three primary domains: a charged N terminus that binds heparan sulfate proteoglycans, a central NANP repeat domain, and a C terminus containing a thrombospondin-like type I repeat (TSR) domain. Only the last two domains are incorporated in RTS,S, the leading malaria vaccine in phase 3 trials that, to date, protects about 50% of vaccinated children against clinical disease. A seroepidemiological study indicated that the N-terminal domain might improve the efficacy of a new CSP vaccine. Using a panel of CSP-specific monoclonal antibodies, well-characterized recombinant CSPs, label-free quantitative proteomics, and in vitro inhibition of sporozoite invasion, we show that native CSP is N-terminally processed in the mosquito host and undergoes a reversible conformational change to mask some epitopes in the N- and C-terminal domains until the sporozoite interacts with the liver hepatocyte. Our findings show the importance of understanding processing and the biophysical change in conformation, possibly due to a mechanical or molecular signal, and may aid in the development of a new CSP vaccine.

  8. Analysis of Reverse Phase Protein Array Data: From Experimental Design towards Targeted Biomarker Discovery

    PubMed Central

    Wachter, Astrid; Bernhardt, Stephan; Beissbarth, Tim; Korf, Ulrike

    2015-01-01

    Mastering the systematic analysis of tumor tissues on a large scale has long been a technical challenge for proteomics. In 2001, reverse phase protein arrays (RPPA) were added to the repertoire of existing immunoassays, which, for the first time, allowed a profiling of minute amounts of tumor lysates even after microdissection. A characteristic feature of RPPA is its outstanding sample capacity permitting the analysis of thousands of samples in parallel as a routine task. Until today, the RPPA approach has matured to a robust and highly sensitive high-throughput platform, which is ideally suited for biomarker discovery. Concomitant with technical advancements, new bioinformatic tools were developed for data normalization and data analysis as outlined in detail in this review. Furthermore, biomarker signatures obtained by different RPPA screens were compared with another or with that obtained by other proteomic formats, if possible. Options for overcoming the downside of RPPA, which is the need to steadily validate new antibody batches, will be discussed. Finally, a debate on using RPPA to advance personalized medicine will conclude this article. PMID:27600238

  9. Reversal of DNA damage induced Topoisomerase 2 DNA–protein crosslinks by Tdp2

    PubMed Central

    Schellenberg, Matthew J.; Perera, Lalith; Strom, Christina N.; Waters, Crystal A.; Monian, Brinda; Appel, C. Denise; Vilas, Caroline K.; Williams, Jason G.; Ramsden, Dale A.; Williams, R. Scott

    2016-01-01

    Mammalian Tyrosyl-DNA phosphodiesterase 2 (Tdp2) reverses Topoisomerase 2 (Top2) DNA–protein crosslinks triggered by Top2 engagement of DNA damage or poisoning by anticancer drugs. Tdp2 deficiencies are linked to neurological disease and cellular sensitivity to Top2 poisons. Herein, we report X-ray crystal structures of ligand-free Tdp2 and Tdp2-DNA complexes with alkylated and abasic DNA that unveil a dynamic Tdp2 active site lid and deep substrate binding trench well-suited for engaging the diverse DNA damage triggers of abortive Top2 reactions. Modeling of a proposed Tdp2 reaction coordinate, combined with mutagenesis and biochemical studies support a single Mg2+-ion mechanism assisted by a phosphotyrosyl-arginine cation-π interface. We further identify a Tdp2 active site SNP that ablates Tdp2 Mg2+ binding and catalytic activity, impairs Tdp2 mediated NHEJ of tyrosine blocked termini, and renders cells sensitive to the anticancer agent etoposide. Collectively, our results provide a structural mechanism for Tdp2 engagement of heterogeneous DNA damage that causes Top2 poisoning, and indicate that evaluation of Tdp2 status may be an important personalized medicine biomarker informing on individual sensitivities to chemotherapeutic Top2 poisons. PMID:27060144

  10. In situ ellipsometric study of surface immobilization of flagellar filaments

    NASA Astrophysics Data System (ADS)

    Kurunczi, S.; Németh, A.; Hülber, T.; Kozma, P.; Petrik, P.; Jankovics, H.; Sebestyén, A.; Vonderviszt, F.; Fried, M.; Bársony, I.

    2010-10-01

    Protein filaments composed of thousands of subunits are promising candidates as sensing elements in biosensors. In this work in situ spectroscopic ellipsometry is applied to monitor the surface immobilization of flagellar filaments. This study is the first step towards the development of layers of filamentous receptors for sensor applications. Surface activation is performed using silanization and a subsequent glutaraldehyde crosslinking. Structure of the flagellar filament layers immobilized on activated and non-activated Si wafer substrates is determined using a two-layer effective medium model that accounted for the vertical density distribution of flagellar filaments with lengths of 300-1500 nm bound to the surface. The formation of the first interface layer can be explained by the multipoint covalent attachment of the filaments, while the second layer is mainly composed of tail pinned filaments floating upwards with the free parts. As confirmed by atomic force microscopy, covalent immobilization resulted in an increased surface density compared to absorption.

  11. Overexpression of Smooth Muscle Myosin Heavy Chain Leads to Activation of the Unfolded Protein Response and Autophagic Turnover of Thick Filament-associated Proteins in Vascular Smooth Muscle Cells*

    PubMed Central

    Kwartler, Callie S.; Chen, Jiyuan; Thakur, Dhananjay; Li, Shumin; Baskin, Kedryn; Wang, Shanzhi; Wang, Zhao V.; Walker, Lori; Hill, Joseph A.; Epstein, Henry F.; Taegtmeyer, Heinrich; Milewicz, Dianna M.

    2014-01-01

    Duplications spanning nine genes at the genomic locus 16p13.1 predispose individuals to acute aortic dissections. The most likely candidate gene in this region leading to the predisposition for dissection is MYH11, which encodes smooth muscle myosin heavy chain (SM-MHC). The effects of increased expression of MYH11 on smooth muscle cell (SMC) phenotypes were explored using mouse aortic SMCs with transgenic overexpression of one isoform of SM-MHC. We found that these cells show increased expression of Myh11 and myosin filament-associated contractile genes at the message level when compared with control SMCs, but not at the protein level due to increased protein degradation. Increased expression of Myh11 resulted in endoplasmic reticulum (ER) stress in SMCs, which led to a paradoxical decrease of protein levels through increased autophagic degradation. An additional consequence of ER stress in SMCs was increased intracellular calcium ion concentration, resulting in increased contractile signaling and contraction. The increased signals for contraction further promote transcription of contractile genes, leading to a feedback loop of metabolic abnormalities in these SMCs. We suggest that overexpression of MYH11 can lead to increased ER stress and autophagy, findings that may be globally implicated in disease processes associated with genomic duplications. PMID:24711452

  12. Overexpression of smooth muscle myosin heavy chain leads to activation of the unfolded protein response and autophagic turnover of thick filament-associated proteins in vascular smooth muscle cells.

    PubMed

    Kwartler, Callie S; Chen, Jiyuan; Thakur, Dhananjay; Li, Shumin; Baskin, Kedryn; Wang, Shanzhi; Wang, Zhao V; Walker, Lori; Hill, Joseph A; Epstein, Henry F; Taegtmeyer, Heinrich; Milewicz, Dianna M

    2014-05-16

    Duplications spanning nine genes at the genomic locus 16p13.1 predispose individuals to acute aortic dissections. The most likely candidate gene in this region leading to the predisposition for dissection is MYH11, which encodes smooth muscle myosin heavy chain (SM-MHC). The effects of increased expression of MYH11 on smooth muscle cell (SMC) phenotypes were explored using mouse aortic SMCs with transgenic overexpression of one isoform of SM-MHC. We found that these cells show increased expression of Myh11 and myosin filament-associated contractile genes at the message level when compared with control SMCs, but not at the protein level due to increased protein degradation. Increased expression of Myh11 resulted in endoplasmic reticulum (ER) stress in SMCs, which led to a paradoxical decrease of protein levels through increased autophagic degradation. An additional consequence of ER stress in SMCs was increased intracellular calcium ion concentration, resulting in increased contractile signaling and contraction. The increased signals for contraction further promote transcription of contractile genes, leading to a feedback loop of metabolic abnormalities in these SMCs. We suggest that overexpression of MYH11 can lead to increased ER stress and autophagy, findings that may be globally implicated in disease processes associated with genomic duplications.

  13. Global histone modification fingerprinting in human cells using epigenetic reverse phase protein array

    PubMed Central

    Partolina, Marina; Thoms, Hazel C; MacLeod, Kenneth G; Rodriguez-Blanco, Giovanny; Clarke, Matthew N; Venkatasubramani, Anuroop V; Beesoo, Rima; Larionov, Vladimir; Neergheen-Bhujun, Vidushi S; Serrels, Bryan; Kimura, Hiroshi; Carragher, Neil O; Kagansky, Alexander

    2017-01-01

    The balance between acetylation and deacetylation of histone proteins plays a critical role in the regulation of genomic functions. Aberrations in global levels of histone modifications are linked to carcinogenesis and are currently the focus of intense scrutiny and translational research investments to develop new therapies, which can modify complex disease pathophysiology through epigenetic control. However, despite significant progress in our understanding of the molecular mechanisms of epigenetic machinery in various genomic contexts and cell types, the links between epigenetic modifications and cellular phenotypes are far from being clear. For example, enzymes controlling histone modifications utilize key cellular metabolites associated with intra- and extracellular feedback loops, adding a further layer of complexity to this process. Meanwhile, it has become increasingly evident that new assay technologies which provide robust and precise measurement of global histone modifications are required, for at least two pressing reasons: firstly, many approved drugs are known to influence histone modifications and new cancer therapies are increasingly being developed towards targeting histone deacetylases (HDACs) and other epigenetic readers and writers. Therefore, robust assays for fingerprinting the global effects of such drugs on preclinical cell, organoid and in vivo models is required; and secondly, robust histone-fingerprinting assays applicable to patient samples may afford the development of next-generation diagnostic and prognostic tools. In our study, we have used a panel of monoclonal antibodies to determine the relative changes in the global abundance of post-translational modifications on histones purified from cancer cell lines treated with HDAC inhibitors using a novel technique, called epigenetic reverse phase protein array. We observed a robust increase in acetylation levels within 2–24 h after inhibition of HDACs in different cancer cell lines

  14. Thioredoxin is required for filamentous phage assembly.

    PubMed Central

    Russel, M; Model, P

    1985-01-01

    Sequence comparisons show that the fip gene product of Escherichia coli, which is required for filamentous phage assembly, is thioredoxin. Thioredoxin serves as a cofactor for reductive processes in many cell types and is a constituent of phage T7 DNA polymerase. The fip-1 mutation makes filamentous phage and T7 growth temperature sensitive in cells that carry it. The lesion lies within a highly conserved thioredoxin active site. Thioredoxin reductase (NADPH), as well as thioredoxin, is required for efficient filamentous phage production. Mutant phages defective in phage gene I are particularly sensitive to perturbations in the fip-thioredoxin system. A speculative model is presented in which thioredoxin reductase, thioredoxin, and the gene I protein interact to drive an engine for filamentous phage assembly. Images PMID:3881756

  15. Physical properties of cytoplasmic intermediate filaments.

    PubMed

    Block, Johanna; Schroeder, Viktor; Pawelzyk, Paul; Willenbacher, Norbert; Köster, Sarah

    2015-11-01

    Intermediate filaments (IFs) constitute a sophisticated filament system in the cytoplasm of eukaryotes. They form bundles and networks with adapted viscoelastic properties and are strongly interconnected with the other filament types, microfilaments and microtubules. IFs are cell type specific and apart from biochemical functions, they act as mechanical entities to provide stability and resilience to cells and tissues. We review the physical properties of these abundant structural proteins including both in vitro studies and cell experiments. IFs are hierarchical structures and their physical properties seem to a large part be encoded in the very specific architecture of the biopolymers. Thus, we begin our review by presenting the assembly mechanism, followed by the mechanical properties of individual filaments, network and structure formation due to electrostatic interactions, and eventually the mechanics of in vitro and cellular networks. This article is part of a Special Issue entitled: Mechanobiology.

  16. Quality control of Photosystem II: reversible and irreversible protein aggregation decides the fate of Photosystem II under excessive illumination

    PubMed Central

    Yamamoto, Yasusi; Hori, Haruka; Kai, Suguru; Ishikawa, Tomomi; Ohnishi, Atsuki; Tsumura, Nodoka; Morita, Noriko

    2013-01-01

    In response to excessive light, the thylakoid membranes of higher plant chloroplasts show dynamic changes including the degradation and reassembly of proteins, a change in the distribution of proteins, and large-scale structural changes such as unstacking of the grana. Here, we examined the aggregation of light-harvesting chlorophyll-protein complexes and Photosystem II core subunits of spinach thylakoid membranes under light stress with 77K chlorophyll fluorescence; aggregation of these proteins was found to proceed with increasing light intensity. Measurement of changes in the fluidity of thylakoid membranes with fluorescence polarization of diphenylhexatriene showed that membrane fluidity increased at a light intensity of 500–1,000 μmol photons m-2 s-1, and decreased at very high light intensity (1,500 μmol photons m-2 s-1). The aggregation of light-harvesting complexes at moderately high light intensity is known to be reversible, while that of Photosystem II core subunits at extremely high light intensity is irreversible. It is likely that the reversibility of protein aggregation is closely related to membrane fluidity: increases in fluidity should stimulate reversible protein aggregation, whereas irreversible protein aggregation might decrease membrane fluidity. When spinach leaves were pre-illuminated with moderately high light intensity, the qE component of non-photochemical quenching and the optimum quantum yield of Photosystem II increased, indicating that Photosystem II/light-harvesting complexes rearranged in the thylakoid membranes to optimize Photosystem II activity. Transmission electron microscopy revealed that the thylakoids underwent partial unstacking under these light stress conditions. Thus, protein aggregation is involved in thylakoid dynamics and regulates photochemical reactions, thereby deciding the fate of Photosystem II. PMID:24194743

  17. Computational evaluation of new homologous down regulators of Translationally Controlled Tumor Protein (TCTP) targeted for tumor reversion.

    PubMed

    Nayarisseri, Anuraj; Yadav, Mukesh; Wishard, Rohan

    2013-12-01

    The Translationally Controlled Tumor Protein (TCTP) has been investigated for tumor reversion and is a target of cancer therapy. Down regulators which suppress the expression of TCTP can trigger the process of tumor reversion leading to the transformation of tumor cells into revertant cells. The present investigation is a novel protein-protein docking approach to target TCTP by a set of proteins similar to the protein: sorting nexin 6 (SNX6) which is an established down regulator of TCTP. The established down regulator along with its set of most similar proteins were modeled using the PYTHON based software - MODELLER v9.9, followed by structure validation using the Procheck Package. Further TCTP was docked with its established and prospective down regulators using the flexible docking protocol suite HADDOCK. The results were evaluated and ranked according to the RMSD values of the complex and the HADDOCK score, which is a weighted sum of van der Waal's energy, electrostatic energy, restraints violation energy and desolvation energy. Results concluded the protein sorting nexin 6 of Mus musculus to be a better down regulator of TCTP, as compared to the suggested down regulator (Homo sapiens snx6).

  18. The ionic liquid isopropylammonium formate as a mobile phase modifier to improve protein stability during reversed phase liquid chromatography.

    PubMed

    Zhou, Ling; Danielson, Neil D

    2013-12-01

    The room temperature ionic liquid isopropylammonium formate (IPAF) is studied as a reversed phase HPLC mobile phase modifier for separation of native proteins using a polymeric column and the protein stability is compared to that using acetonitrile (MeCN) as the standard organic mobile phase modifier. A variety of important proteins with different numbers of subunits are investigated, including non-subunit proteins: albumin, and amyloglucosidase (AMY); a two subunit protein: thyroglobulin (THY); and four subunit proteins: glutamate dehydrogenase (GDH) and lactate dehydrogenase (LDH). A significant enhancement in protein stability is observed in the chromatograms upon using IPAF as a mobile phase modifier. The first sharper peak at about 2min represented protein in primarily the native form and a second broader peak more retained at about 5-6min represented substantially denatured or possibly aggregated protein. The investigated proteins (except LDH) could maintain the native form within up to 50% IPAF, while a mobile phase, with as low as 10% MeCN, induced protein denaturation. The assay for pyruvate using LDH has further shown that enzymatic activity can be maintained up to 30% IPAF in water in contrast to no activity using 30% MeCN.

  19. Intermediate Filaments: Structure and Assembly.

    PubMed

    Herrmann, Harald; Aebi, Ueli

    2016-11-01

    Proteins of the intermediate filament (IF) supergene family are ubiquitous structural components that comprise, in a cell type-specific manner, the cytoskeleton proper in animal tissues. All IF proteins show a distinctly organized, extended α-helical conformation prone to form two-stranded coiled coils, which are the basic building blocks of these highly flexible, stress-resistant cytoskeletal filaments. IF proteins are highly charged, thus representing versatile polyampholytes with multiple functions. Taking vimentin, keratins, and the nuclear lamins as our prime examples, we present an overview of their molecular and structural parameters. These, in turn, document the ability of IF proteins to form distinct, highly diverse supramolecular assemblies and biomaterials found, for example, at the inner nuclear membrane, throughout the cytoplasm, and in highly complex extracellular appendages, such as hair and nails, of vertebrate organisms. Ultimately, our aim is to set the stage for a more rational understanding of the immediate effects that missense mutations in IF genes have on cellular functions and for their far-reaching impact on the development of the numerous IF diseases caused by them.

  20. Specific and reversible immobilization of proteins tagged to the affinity polypeptide C-LytA on functionalized graphite electrodes.

    PubMed

    Bello-Gil, Daniel; Maestro, Beatriz; Fonseca, Jennifer; Feliu, Juan M; Climent, Víctor; Sanz, Jesús M

    2014-01-01

    We have developed a general method for the specific and reversible immobilization of proteins fused to the choline-binding module C-LytA on functionalized graphite electrodes. Graphite electrode surfaces were modified by diazonium chemistry to introduce carboxylic groups that were subsequently used to anchor mixed self-assembled monolayers consisting of N,N-diethylethylenediamine groups, acting as choline analogs, and ethanolamine groups as spacers. The ability of the prepared electrodes to specifically bind C-LytA-tagged recombinant proteins was tested with a C-LytA-β-galactosidase fusion protein. The binding, activity and stability of the immobilized protein was evaluated by electrochemically monitoring the formation of an electroactive product in the enzymatic hydrolysis of the synthetic substrate 4-aminophenyl β-D-galactopyranoside. The hybrid protein was immobilized in an specific and reversible way, while retaining the catalytic activity. Moreover, these functionalized electrodes were shown to be highly stable and reusable. The method developed here can be envisaged as a general, immobilization procedure on the protein biosensor field.

  1. Specific and Reversible Immobilization of Proteins Tagged to the Affinity Polypeptide C-LytA on Functionalized Graphite Electrodes

    PubMed Central

    Bello-Gil, Daniel; Maestro, Beatriz; Fonseca, Jennifer; Feliu, Juan M.; Climent, Víctor; Sanz, Jesús M.

    2014-01-01

    We have developed a general method for the specific and reversible immobilization of proteins fused to the choline-binding module C-LytA on functionalized graphite electrodes. Graphite electrode surfaces were modified by diazonium chemistry to introduce carboxylic groups that were subsequently used to anchor mixed self-assembled monolayers consisting of N,N-diethylethylenediamine groups, acting as choline analogs, and ethanolamine groups as spacers. The ability of the prepared electrodes to specifically bind C-LytA-tagged recombinant proteins was tested with a C-LytA-β-galactosidase fusion protein. The binding, activity and stability of the immobilized protein was evaluated by electrochemically monitoring the formation of an electroactive product in the enzymatic hydrolysis of the synthetic substrate 4-aminophenyl β-D-galactopyranoside. The hybrid protein was immobilized in an specific and reversible way, while retaining the catalytic activity. Moreover, these functionalized electrodes were shown to be highly stable and reusable. The method developed here can be envisaged as a general, immobilization procedure on the protein biosensor field. PMID:24498237

  2. Reversible symptoms and clearance of mutant prion protein in an inducible model of a genetic prion disease in Drosophila melanogaster.

    PubMed

    Murali, A; Maue, R A; Dolph, P J

    2014-07-01

    Prion diseases are progressive disorders that affect the central nervous system leading to memory loss, personality changes, ataxia and neurodegeneration. In humans, these disorders include Creutzfeldt-Jakob disease, kuru and Gerstmann-Straüssler-Scheinker (GSS) syndrome, the latter being a dominantly inherited prion disease associated with missense mutations in the gene that codes for the prion protein. The exact mechanism by which mutant prion proteins affect the central nervous system and cause neurological disease is not well understood. We have generated an inducible model of GSS disease in Drosophila melanogaster by temporally expressing a misfolded form of the murine prion protein in cholinergic neurons. Flies accumulating this mutant protein develop motor abnormalities which are associated with electrophysiological defects in cholinergic neurons. We find that, upon blocking the expression of the mutant protein, both behavioral and electrophysiological defects can be reversed. This represents the first case of reversibility reported in a model of genetic prion disease. Additionally, we observe that endogenous mechanisms exist within Drosophila that are capable of clearing the accumulated prion protein.

  3. Snake Filament Eruption

    NASA Video Gallery

    A very long solar filament that had been snaking around the Sun erupted on Dec. 6, 2010 with a flourish. NASA's Solar Dynamics Observatory (SDO) caught the action in dramatic detail in extreme ultr...

  4. The Regulatory Subunit of Protein Kinase A (Bcy1) in Candida albicans Plays Critical Roles in Filamentation and White-Opaque Switching but Is Not Essential for Cell Growth

    PubMed Central

    Ding, Xuefen; Cao, Chengjun; Zheng, Qiushi; Huang, Guanghua

    2017-01-01

    The conserved cAMP-dependent protein kinase (PKA) is composed of the regulatory and catalytic subunits and acts as the central component of the cAMP signaling pathway. In the human fungal pathogen Candida albicans, the PKA regulatory subunit Bcy1 plays a critical role in the regulation of cell differentiation and death. It has long been considered that Bcy1 is essential for cell viability in C. albicans. In the current study, surprisingly, we found that Bcy1 is not required for cell growth, and we successfully generated a bcy1/bcy1 null mutant in C. albicans. Deletion of BCY1 leads to multiple cellular morphologies and promotes the development of filaments. Filamentous and smooth colonies are two typical morphological types of the bcy1/bcy1 mutant, which can undergo spontaneous switching between the two types. Cells of filamentous colonies grow better on a number of different culture media and have a higher survival rate than cells of smooth colonies. In addition, deletion of BCY1 significantly increased the frequency of white-to-opaque switching on N-acetylglucosamine (GlcNAc)-containing medium. The bcy1/bcy1 null mutant generated herein provides the field a new resource to study the biological functions of the cAMP signaling pathway in C. albicans. PMID:28105026

  5. Site-specific and reversible anchoring of active proteins onto cellulose using a cellulosome-like complex.

    PubMed

    Eklund, Malin; Sandström, Kristofer; Teeri, Tuula T; Nygren, Per-Ake

    2004-04-29

    Protein engineering strategies facilitating controlled and spontaneous assembly of macromolecular complexes are of great interest for the design of artificial multi-enzyme systems of pre-defined composition. Here we have combined affinity proteins from different sources to achieve specific and reversible anchoring of affinity domain-tagged reporter proteins to a cellulose-anchored fusion protein. The design principle mimics the architecture of macromolecular cellulosome complexes produced by some cellulolytic microbes. A fusion protein between a cellulose-binding module (CBM1Cel6A) of the Trichoderma reesei cellobiohydrolase Cel6A and a five-domain staphylococcal protein A (SPA) was constructed to serve as platform for docking of easily detectable reporter proteins onto cellulose surfaces. In turn, the reporter proteins were produced as fusions to two copies of a SPA-binding affinity protein (an affibody denoted Z(SPA-1)), selected from a phage display library constructed by combinatorial protein engineering. In a series of experiments, involving repeated washing and low pH elution, affinity-tagged Enhanced Green Fluorescent Protein (EGFP) and Fusarium solani pisi lipase cutinase reporter proteins were both found to be specifically directed from solution to the same region of a cellulose filter paper where SPA-CBM1Cel6A fusion protein had been previously applied. This showed that the SPA-CBM1Cel6A fusion protein had been stably anchored to the cellulose surface without loss of binding capacity and that the interaction between SPA and the Z(SPA-1) affibody domains was selective. The generality of this biospecificity-driven system for assembly applications is discussed.

  6. Probing the binding behavior and conformational states of globular proteins in reversed-phase high-performance liquid chromatography.

    PubMed

    Purcell, A W; Aguilar, M I; Hearn, M T

    1999-07-01

    Reversed-phase high-performance liquid chromatography (RP-HPLC) is a widely used technique for the separation of proteins under low pH aquo-organic solvent gradient elution conditions, typically carried out at ambient temperatures. These conditions can however induce conformational effects with proteins as evident from changes in their biological or immunological activities. By monitoring the influence of temperature on the retention and band-broadening characteristics of proteins, the role of conformational processes in these lipophilic environments can be examined. These processes can then be interpreted in terms of a two-state model involving a native (N) and a fully unfolded species (U) or more complex folding/unfolding models. In the present study, the gradient elution RP-HPLC behavior of sperm whale myoglobin (SWMYO) and hen egg white lysozyme (HEWL) has been investigated at temperatures between 5 and 85 degrees C with n-octadecyl (C18)- and n-butyl (C4)-silica reversed-phase sorbents. The interaction of these proteins with these reversed-phase sorbents has also been examined in terms of the contributions that the heme prosthetic group of SWMYO and the disulfide bonds in HEWL make to the stabilization of the native conformation of these proteins in these hydrophobic environments. The observed interconversions of multiple peak zones of SWMYO and HEWL in the presence of C18 and C4 ligands have been subsequently analyzed in terms of the unfolding processes that these proteins can undergo at low pH and at elevated temperatures. The ability of hydrocarbonaceous ligands to trap ensemblies of partially unfolded conformational intermediates of proteins in these perturbing environments has been examined. Pseudo-first-order rate constants have been derived for these processes from analysis of the dependencies on time of the concentration of the different protein species at specified temperatures. The relationship of these processes to the conformational transitions

  7. A Robust Actin Filaments Image Analysis Framework

    PubMed Central

    Alioscha-Perez, Mitchel; Benadiba, Carine; Goossens, Katty; Kasas, Sandor; Dietler, Giovanni; Willaert, Ronnie; Sahli, Hichem

    2016-01-01

    The cytoskeleton is a highly dynamical protein network that plays a central role in numerous cellular physiological processes, and is traditionally divided into three components according to its chemical composition, i.e. actin, tubulin and intermediate filament cytoskeletons. Understanding the cytoskeleton dynamics is of prime importance to unveil mechanisms involved in cell adaptation to any stress type. Fluorescence imaging of cytoskeleton structures allows analyzing the impact of mechanical stimulation in the cytoskeleton, but it also imposes additional challenges in the image processing stage, such as the presence of imaging-related artifacts and heavy blurring introduced by (high-throughput) automated scans. However, although there exists a considerable number of image-based analytical tools to address the image processing and analysis, most of them are unfit to cope with the aforementioned challenges. Filamentous structures in images can be considered as a piecewise composition of quasi-straight segments (at least in some finer or coarser scale). Based on this observation, we propose a three-steps actin filaments extraction methodology: (i) first the input image is decomposed into a ‘cartoon’ part corresponding to the filament structures in the image, and a noise/texture part, (ii) on the ‘cartoon’ image, we apply a multi-scale line detector coupled with a (iii) quasi-straight filaments merging algorithm for fiber extraction. The proposed robust actin filaments image analysis framework allows extracting individual filaments in the presence of noise, artifacts and heavy blurring. Moreover, it provides numerous parameters such as filaments orientation, position and length, useful for further analysis. Cell image decomposition is relatively under-exploited in biological images processing, and our study shows the benefits it provides when addressing such tasks. Experimental validation was conducted using publicly available datasets, and in osteoblasts

  8. Development of a reverse genetics system to generate a recombinant Ebola virus Makona expressing a green fluorescent protein

    SciTech Connect

    Albariño, César G. Wiggleton Guerrero, Lisa; Lo, Michael K.; Nichol, Stuart T.; Towner, Jonathan S.

    2015-10-15

    Previous studies have demonstrated the potential application of reverse genetics technology in studying a broad range of aspects of viral biology, including gene regulation, protein function, cell entry, and pathogenesis. Here, we describe a highly efficient reverse genetics system used to generate recombinant Ebola virus (EBOV) based on a recent isolate from a human patient infected during the 2014–2015 outbreak in Western Africa. We also rescued a recombinant EBOV expressing a fluorescent reporter protein from a cleaved VP40 protein fusion. Using this virus and an inexpensive method to quantitate the expression of the foreign gene, we demonstrate its potential usefulness as a tool for screening antiviral compounds and measuring neutralizing antibodies. - Highlights: • Recombinant Ebola virus (EBOV) derived from Makona variant was rescued. • New protocol for viral rescue allows 100% efficiency. • Modified EBOV expresses a green fluorescent protein from a VP40-fused protein. • Modified EBOV was tested as tool to screen antiviral compounds and measure neutralizing antibodies.

  9. Characterization of osmotically induced filaments of Salmonella enterica.

    PubMed

    Pratt, Zachary L; Chen, Bingming; Czuprynski, Charles J; Wong, Amy C L; Kaspar, Charles W

    2012-09-01

    Salmonella enterica forms aseptate filaments with multiple nucleoids when cultured in hyperosmotic conditions. These osmotic-induced filaments are viable and form single colonies on agar plates even though they contain multiple genomes and have the potential to divide into multiple daughter cells. Introducing filaments that are formed during osmotic stress into culture conditions without additional humectants results in the formation of septa and their division into individual cells, which could present challenges to retrospective analyses of infectious dose and risk assessments. We sought to characterize the underlying mechanisms of osmotic-induced filament formation. The concentration of proteins and chromosomal DNA in filaments and control cells was similar when standardized by biomass. Furthermore, penicillin-binding proteins in the membrane of salmonellae were active in vitro. The activity of penicillin-binding protein 2 was greater in filaments than in control cells, suggesting that it may have a role in osmotic-induced filament formation. Filaments contained more ATP than did control cells in standardized cell suspensions, though the levels of two F(0)F(1)-ATP synthase subunits were reduced. Furthermore, filaments could septate and divide within 8 h in 0.2 × Luria-Bertani broth at 23°C, while nonfilamentous control cells did not replicate. Based upon the ability of filaments to septate and divide in this diluted broth, a method was developed to enumerate by plate count the number of individual, viable cells within a population of filaments. This method could aid in retrospective analyses of infectious dose of filamented salmonellae.

  10. A library of MiMICs allows tagging of genes and reversible, spatial and temporal knockdown of proteins in Drosophila.

    PubMed

    Nagarkar-Jaiswal, Sonal; Lee, Pei-Tseng; Campbell, Megan E; Chen, Kuchuan; Anguiano-Zarate, Stephanie; Gutierrez, Manuel Cantu; Busby, Theodore; Lin, Wen-Wen; He, Yuchun; Schulze, Karen L; Booth, Benjamin W; Evans-Holm, Martha; Venken, Koen J T; Levis, Robert W; Spradling, Allan C; Hoskins, Roger A; Bellen, Hugo J

    2015-03-31

    Here, we document a collection of ∼7434 MiMIC (Minos Mediated Integration Cassette) insertions of which 2854 are inserted in coding introns. They allowed us to create a library of 400 GFP-tagged genes. We show that 72% of internally tagged proteins are functional, and that more than 90% can be imaged in unfixed tissues. Moreover, the tagged mRNAs can be knocked down by RNAi against GFP (iGFPi), and the tagged proteins can be efficiently knocked down by deGradFP technology. The phenotypes associated with RNA and protein knockdown typically correspond to severe loss of function or null mutant phenotypes. Finally, we demonstrate reversible, spatial, and temporal knockdown of tagged proteins in larvae and adult flies. This new strategy and collection of strains allows unprecedented in vivo manipulations in flies for many genes. These strategies will likely extend to vertebrates.

  11. A library of MiMICs allows tagging of genes and reversible, spatial and temporal knockdown of proteins in Drosophila

    SciTech Connect

    Nagarkar-Jaiswal, Sonal; Lee, Pei-Tseng; Campbell, Megan E.; Chen, Kuchuan; Anguiano-Zarate, Stephanie; Cantu Gutierrez, Manuel; Busby, Theodore; Lin, Wen-Wen; He, Yuchun; Schulze, Karen L.; Booth, Benjamin W.; Evans-Holm, Martha; Venken, Koen J.T.; Levis, Robert W.; Spradling, Allan C.; Hoskins, Roger A.; Bellen, Hugo J.

    2015-03-31

    Here, we document a collection of ~7434 MiMIC (Minos Mediated Integration Cassette) insertions of which 2854 are inserted in coding introns. They allowed us to create a library of 400 GFP-tagged genes. We show that 72% of internally tagged proteins are functional, and that more than 90% can be imaged in unfixed tissues. Moreover, the tagged mRNAs can be knocked down by RNAi against GFP (iGFPi), and the tagged proteins can be efficiently knocked down by deGradFP technology. The phenotypes associated with RNA and protein knockdown typically correspond to severe loss of function or null mutant phenotypes. Finally, we demonstrate reversible, spatial, and temporal knockdown of tagged proteins in larvae and adult flies. This new strategy and collection of strains allows unprecedented in vivo manipulations in flies for many genes. These strategies will likely extend to vertebrates.

  12. A library of MiMICs allows tagging of genes and reversible, spatial and temporal knockdown of proteins in Drosophila

    DOE PAGES

    Nagarkar-Jaiswal, Sonal; Lee, Pei-Tseng; Campbell, Megan E.; ...

    2015-03-31

    Here, we document a collection of ~7434 MiMIC (Minos Mediated Integration Cassette) insertions of which 2854 are inserted in coding introns. They allowed us to create a library of 400 GFP-tagged genes. We show that 72% of internally tagged proteins are functional, and that more than 90% can be imaged in unfixed tissues. Moreover, the tagged mRNAs can be knocked down by RNAi against GFP (iGFPi), and the tagged proteins can be efficiently knocked down by deGradFP technology. The phenotypes associated with RNA and protein knockdown typically correspond to severe loss of function or null mutant phenotypes. Finally, we demonstratemore » reversible, spatial, and temporal knockdown of tagged proteins in larvae and adult flies. This new strategy and collection of strains allows unprecedented in vivo manipulations in flies for many genes. These strategies will likely extend to vertebrates.« less

  13. Intrathecal injection of adenosine 2A receptor agonists reversed neuropathic allodynia through protein kinase (PK)A/PKC signaling.

    PubMed

    Loram, Lisa C; Taylor, Frederick R; Strand, Keith A; Harrison, Jacqueline A; Rzasalynn, Rachael; Sholar, Paige; Rieger, Jayson; Maier, Steven F; Watkins, Linda R

    2013-10-01

    A single intrathecal dose of adenosine 2A receptor (A2AR) agonist was previously reported to produce a multi-week reversal of allodynia in a chronic constriction injury (CCI) model of neuropathic pain. We aimed to determine if this long-term reversal was induced by A2AR agonism versus more generalized across adenosine receptor subtypes, and begin to explore the intracellular signaling cascades involved. In addition, we sought to identify whether the enduring effect could be extended to other models of neuropathic pain. We tested an A1R and A2BR agonist in CCI and found the same long duration effect with A2BR but not A1R agonism. An A2AR agonist (ATL313) produced a significant long-duration reversal of mechanical allodynia induced by long established CCI (administered 6 weeks after surgery), spinal nerve ligation and sciatic inflammatory neuropathy. To determine if ATL313 had a direct effect on glia, ATL313 was coadministered with lipopolysaccharide to neonatal microglia and astrocytes in vitro. ATL313 significantly attenuated TNFα production in both microglia and astrocytes but had no effect on LPS induced IL-10. Protein kinase C significantly reversed the ATL313 effects on TNFα in vitro in microglia and astrocytes, while a protein kinase A inhibitor only effected microglia. Both intrathecal PKA and PKC inhibitors significantly reversed the effect of the A2AR agonist on neuropathic allodynia. Therefore, A2AR agonists administered IT remain an exciting novel target for the treatment of neuropathic pain.

  14. Coexistence of two types of metal filaments in oxide memristors

    NASA Astrophysics Data System (ADS)

    Xu, D.; Shangguan, X. N.; Wang, S. M.; Cao, H. T.; Liang, L. Y.; Zhang, H. L.; Gao, J. H.; Long, W. M.; Wang, J. R.; Zhuge, F.

    2017-02-01

    One generally considers the conducting filament in ZnO-based valence change memristors (VCMs) as an aggregation of oxygen vacancies. Recently, the transmission electron microscopy observation showed the filament is composed of a Zn-dominated ZnOx. In this study, careful analysis of the temperature dependence of the ON state resistance demonstrates that the formation/rupture of a Zn filament is responsible for the resistive switching in ZnO VCMs. Cu/ZnO/Pt memristive devices can be operated in both VCM and ECM (electrochemical metallization memristor) modes by forming different metal filaments including Cu, Zn and a coexistence of these two filaments. The device operation can be reversibly switched between ECM and VCM modes. The dual mode operation capability of Cu/ZnO/Pt provides a wide choice of select devices for constructing memristive crossbar architectures.

  15. Ubiquitination and filamentous structure of cytidine triphosphate synthase

    PubMed Central

    Pai, Li-Mei; Wang, Pei-Yu; Lin, Wei-Cheng; Chakraborty, Archan; Yeh, Chau-Ting; Lin, Yu-Hung

    2016-01-01

    ABSTRACT Living organisms respond to nutrient availability by regulating the activity of metabolic enzymes. Therefore, the reversible post-translational modification of an enzyme is a common regulatory mechanism for energy conservation. Recently, cytidine-5′-triphosphate (CTP) synthase was discovered to form a filamentous structure that is evolutionarily conserved from flies to humans. Interestingly, induction of the formation of CTP synthase filament is responsive to starvation or glutamine depletion. However, the biological roles of this structure remain elusive. We have recently shown that ubiquitination regulates CTP synthase activity by promoting filament formation in Drosophila ovaries during endocycles. Intriguingly, although the ubiquitination process was required for filament formation induced by glutamine depletion, CTP synthase ubiquitination was found to be inversely correlated with filament formation in Drosophila and human cell lines. In this article, we discuss the putative dual roles of ubiquitination, as well as its physiological implications, in the regulation of CTP synthase structure. PMID:27116391

  16. INTERMEDIATE FILAMENTS IN SMOOTH MUSCLE

    PubMed Central

    Tang, Dale D.

    2008-01-01

    The intermediate filament (IF) network is one of the three cytoskeletal systems in smooth muscle. The type III IF proteins vimentin and desmin are major constituents of the network in smooth muscle cells and tissues. Lack of vimentin or desmin impairs contractile ability of various smooth muscle preparations, implying their important role for smooth muscle force development. The IF framework has long been viewed as a fixed cytostructure that solely provides mechanical integrity for the cell. However, recent studies suggest that the IF cytoskeleton is dynamic in mammalian cells in response to various external stimulation. In this review, the structure and biological properties of IF proteins in smooth muscle are summarized. The role of IF proteins in the modulation of smooth muscle force development and redistribution/translocation of signaling partners (such as p130 Crk-associated substrate, CAS) is depicted. This review also summarizes our latest understanding on how the IF network may be regulated in smooth muscle. PMID:18256275

  17. Tropomyosin - master regulator of actin filament function in the cytoskeleton.

    PubMed

    Gunning, Peter W; Hardeman, Edna C; Lappalainen, Pekka; Mulvihill, Daniel P

    2015-08-15

    Tropomyosin (Tpm) isoforms are the master regulators of the functions of individual actin filaments in fungi and metazoans. Tpms are coiled-coil parallel dimers that form a head-to-tail polymer along the length of actin filaments. Yeast only has two Tpm isoforms, whereas mammals have over 40. Each cytoskeletal actin filament contains a homopolymer of Tpm homodimers, resulting in a filament of uniform Tpm composition along its length. Evidence for this 'master regulator' role is based on four core sets of observation. First, spatially and functionally distinct actin filaments contain different Tpm isoforms, and recent data suggest that members of the formin family of actin filament nucleators can specify which Tpm isoform is added to the growing actin filament. Second, Tpms regulate whole-organism physiology in terms of morphogenesis, cell proliferation, vesicle trafficking, biomechanics, glucose metabolism and organ size in an isoform-specific manner. Third, Tpms achieve these functional outputs by regulating the interaction of actin filaments with myosin motors and actin-binding proteins in an isoform-specific manner. Last, the assembly of complex structures, such as stress fibers and podosomes involves the collaboration of multiple types of actin filament specified by their Tpm composition. This allows the cell to specify actin filament function in time and space by simply specifying their Tpm isoform composition.

  18. Growth and Division of Filamentous Forms of Escherichia coli.

    PubMed

    Adler, H I; Hardigree, A A

    1965-07-01

    Adler, Howard I. (Oak Ridge National Laboratory, Oak Ridge, Tenn.), and Alice A. Hardigree. Growth and division of filamentous forms of Escherichia coli. J. Bacteriol. 90:223-226. 1965.-Cells of certain mutant strains of Escherichia coli grow into long multinucleate filaments after exposure to radiation. Deoxyribonucleic acid, ribonucleic acid, and protein synthesis proceed, but cytokinesis does not occur. Cytokinesis (cross-septation) can be initiated by exposure of the filaments to pantoyl lactone or a temperature of 42 C. If growing filaments are treated with mitomycin C, nuclear division does not occur, and nuclear material is confined to the central region of the filament. Cytokinesis cannot be induced in mitomycin C-treated filaments by pantoyl lactone or treatment at 42 C.

  19. Characterization of HI Filaments

    NASA Astrophysics Data System (ADS)

    Lubar, Emily; Verschuur, Gerrit L.

    2017-01-01

    We characterized the properties of dramatic interstellar HI filaments to learn more about the dynamics and structure of such features. Using Gauss fitting software, we searched the Effelsburg-Bonn HI Survey data for indications of a simple twisting (toroidal) motion across these filaments. Instead, we found that the structure was more complicated than expected. Apparent angular widths of several filaments were measured using the Galactic Arecibo L-band Feed Array HI (GALFA-HI), Bonn, and Leident/Argentine/Bonn (LAB) surveys. Based on filament widths and other parameters, we conclude that magnetism is the dominant force opposing internal motion and maintaining the structure of these filaments. The apparent width as a function of beam width closely follows a relationship reported in 1993 for HI features in general. They tend to subtend an angle two times the beam width, suggesting that the features remain unresolved.The Arecibo Observatory is operated by SRI International under a cooperative agreement with the National Science Foundation (AST-1100968), and in alliance with Ana G. Méndez-Universidad Metropolitana, and the Universities Space Research Association. The Arecibo Observatory REU is funded under grant AST-1559849 to Universidad Metropolitana.

  20. Exercise partly reverses the effect of maternal separation on hippocampal proteins in 6-hydroxydopamine-lesioned rat brain.

    PubMed

    Dimatelis, J J; Hendricks, S; Hsieh, J; Vlok, N M; Bugarith, K; Daniels, W M U; Russell, V A

    2013-01-01

    Animals subjected to maternal separation stress during the early stages of development display behavioural, endocrine and growth factor abnormalities that mirror the clinical findings in anxiety/depression. In addition, maternal separation has been shown to exacerbate the behavioural deficits induced by 6-hydroxydopamine (6-OHDA) in a rat model of Parkinson's disease. In contrast, voluntary exercise reduced the detrimental effects of 6-OHDA in the rat model. The beneficial effects of exercise appeared to be largely due to compensation in the non-lesioned hemisphere. The aim of the present study was to investigate whether voluntary exercise for 3 weeks could reverse the effects of maternal separation in rats challenged with the neurotoxin 6-OHDA infused into the medial forebrain bundle after 1 week of exercise, at postnatal day 60. The rats were killed 2 weeks later, at postnatal day 74. Their brains were dissected and the hippocampus rapidly removed for proteomic analysis by isobaric tagging (iTRAQ) and quantification of peptides by matrix-assisted laser desorption/ionization tandem mass spectrometry (MALDI-MS/MS). Maternal separation upregulated hippocampal proteins functionally involved in energy metabolism (nucleoside diphosphate kinase B, enolase and triosephosphate isomerase) and synaptic plasticity (α-synuclein, tenascin-R, Ba1-667, brevican and neurocan core protein) in the non-lesioned hemisphere. Exercise reversed many of these changes by downregulating the levels of hippocampal proteins functionally associated with energy metabolism (nucleoside diphosphate kinase B, enolase and triosephosphate isomerase) and synaptic plasticity (α-synuclein, tenascin-R, Ba1-667, brevican and neurocan core protein) in the non-lesioned hemisphere of rats subjected to maternal separation. Exercise and maternal separation therefore appeared to have opposing effects on the hippocampus in the non-lesioned hemisphere of the rat brain. Exercise seemed partly to reverse the

  1. Using Drosophila for Studies of Intermediate Filaments.

    PubMed

    Bohnekamp, Jens; Cryderman, Diane E; Thiemann, Dylan A; Magin, Thomas M; Wallrath, Lori L

    2016-01-01

    Drosophila melanogaster is a useful organism for determining protein function and modeling human disease. Drosophila offers a rapid generation time and an abundance of genomic resources and genetic tools. Conservation in protein structure, signaling pathways, and developmental processes make studies performed in Drosophila relevant to other species, including humans. Drosophila models have been generated for neurodegenerative diseases, muscular dystrophy, cancer, and many other disorders. Recently, intermediate filament protein diseases have been modeled in Drosophila. These models have revealed novel mechanisms of pathology, illuminated potential new routes of therapy, and make whole organism compound screens feasible. The goal of this chapter is to outline steps to study intermediate filament function and model intermediate filament-associated diseases in Drosophila. The steps are general and can be applied to study the function of almost any protein. The protocols outlined here are for both the novice and experienced Drosophila researcher, allowing the rich developmental and cell biology that Drosophila offers to be applied to studies of intermediate filaments.

  2. Filamentous Influenza Viruses

    PubMed Central

    Badham, Matthew D.; Rossman, Jeremy S.

    2016-01-01

    Influenza A virus is a pathogen of global medical importance causing significant health and socio-economic costs every year. Influenza virus is an unusual pathogen in that it is pleomorphic, capable of forming virions ranging in shape from spherical to filamentous. Despite decades of research on the influenza virus, much remains unknown about the formation of filamentous influenza viruses and their role in the viral replication cycle. Here, we discuss what is known about influenza virus assembly and budding, focusing on the viral and host factors that are involved in the determination of viral morphology. Whilst the biological function of the filamentous morphology remains unknown, recent results suggest a role in facilitating viral spread in vivo. We discuss these results and speculate on the consequences of viral morphology during influenza virus infection of the human respiratory tract. PMID:28042529

  3. [Role of the ABC transporters A1 and G1, key reverse cholesterol transport proteins, in atherosclerosis].

    PubMed

    Demina, E P; Miroshnikova, V V; Schwarzman, A L

    2016-01-01

    Atherosclerosis is one of the most common causes of death worldwide. Epidemiology studies firmly established an inverse relationship between atherogenesis and distorted lipid metabolism, in particular, higher levels of total cholesterol, an accumulation of CH-laden macrophages (foam cells), and lower plasma levels of antiatherogenic high density lipoprotein (HDL). It is believed that the reverse cholesterol transport, a process that removes excess cholesterol from peripheral tissues/cells including macrophages to circulating HDL, is one of the main mechanisms responsible for anti-atherogenic properties of HDL. The key proteins of reverse cholesterol transport-ATP-binding cassette transporters A1 (ABCA1) and G1 (ABCG1)-mediate the cholesterol efflux from macrophages and prevent their transformation into foam cells. This review focuses on the role of ABC transporters A1 and G1 in the pathogenesis of atherosclerosis.

  4. Aerogel-supported filament

    DOEpatents

    Wuest, C.R.; Tillotson, T.M.; Johnson, C.V. III

    1995-05-16

    The present invention is a thin filament embedded in a low density aerogel for use in radiation detection instruments and incandescent lamps. The aerogel provides a supportive matrix that is thermally and electrically nonconductive, mechanically strong, highly porous, gas-permeable, and transparent to ionizing radiation over short distances. A low density, open-cell aerogel is cast around a fine filament or wire, which allows the wire to be positioned with little or no tension and keeps the wire in place in the event of breakage. The aerogel support reduces the stresses on the wire caused by vibrational, gravitational, electrical, and mechanical forces. 6 Figs.

  5. Aerogel-supported filament

    DOEpatents

    Wuest, Craig R.; Tillotson, Thomas M.; Johnson, III, Coleman V.

    1995-01-01

    The present invention is a thin filament embedded in a low density aerogel for use in radiation detection instruments and incandescent lamps. The aerogel provides a supportive matrix that is thermally and electrically nonconductive, mechanically strong, highly porous, gas-permeable, and transparent to ionizing radiation over short distances. A low density, open-cell aerogel is cast around a fine filament or wire, which allows the wire to be positioned with little or no tension and keeps the wire in place in the event of breakage. The aerogel support reduces the stresses on the wire caused by vibrational, gravitational, electrical, and mechanical forces.

  6. Structure of Flexible Filamentous Plant Viruses

    SciTech Connect

    Kendall, Amy; McDonald, Michele; Bian, Wen; Bowles, Timothy; Baumgarten, Sarah C.; Shi, Jian; Stewart, Phoebe L.; Bullitt, Esther; Gore, David; Irving, Thomas C.; Havens, Wendy M.; Ghabrial, Said A.; Wall, Joseph S.; Stubbs, Gerald

    2008-10-23

    Flexible filamentous viruses make up a large fraction of the known plant viruses, but in comparison with those of other viruses, very little is known about their structures. We have used fiber diffraction, cryo-electron microscopy, and scanning transmission electron microscopy to determine the symmetry of a potyvirus, soybean mosaic virus; to confirm the symmetry of a potexvirus, potato virus X; and to determine the low-resolution structures of both viruses. We conclude that these viruses and, by implication, most or all flexible filamentous plant viruses share a common coat protein fold and helical symmetry, with slightly less than 9 subunits per helical turn.

  7. Structure of flexible filamentous plant viruses.

    PubMed

    Kendall, Amy; McDonald, Michele; Bian, Wen; Bowles, Timothy; Baumgarten, Sarah C; Shi, Jian; Stewart, Phoebe L; Bullitt, Esther; Gore, David; Irving, Thomas C; Havens, Wendy M; Ghabrial, Said A; Wall, Joseph S; Stubbs, Gerald

    2008-10-01

    Flexible filamentous viruses make up a large fraction of the known plant viruses, but in comparison with those of other viruses, very little is known about their structures. We have used fiber diffraction, cryo-electron microscopy, and scanning transmission electron microscopy to determine the symmetry of a potyvirus, soybean mosaic virus; to confirm the symmetry of a potexvirus, potato virus X; and to determine the low-resolution structures of both viruses. We conclude that these viruses and, by implication, most or all flexible filamentous plant viruses share a common coat protein fold and helical symmetry, with slightly less than 9 subunits per helical turn.

  8. Kinetics of filamentous phage assembly

    NASA Astrophysics Data System (ADS)

    Ploss, Martin; Kuhn, Andreas

    2010-12-01

    Filamentous phages release their progeny particles by a secretory process without lysing the bacterial cell. By this process about 6 viral particles per min are secreted from each cell. We show here that when the major coat protein (gp8) is provided from a plasmid we observe a phage progeny production rate depending on the induction of gp8 by IPTG. We also show that a transfection of Escherichia coli lacking F-pili is observed using a mutant of M13 that carries an ampicillin resistance gene, and phage particles are secreted in the absence of an F-plasmid. Extruding phage was visualized by atomic force microscopy (AFM) and by transmission electron microscopy (TEM) using gold-labeled antibodies to the major coat protein.

  9. Phylogenetic Insights into the Functional Relationship between Primate Lentiviral Reverse Transcriptase and Accessory Proteins Vpx/Vpr

    PubMed Central

    Sakai, Yosuke; Doi, Naoya; Miyazaki, Yasuyuki; Adachi, Akio; Nomaguchi, Masako

    2016-01-01

    The efficiency of reverse transcription to synthesize viral DNA in infected cells greatly influences replication kinetics of retroviruses. However, viral replication in non-dividing cells such as resting T cells and terminally differentiated macrophages is potently and kinetically restricted by a host antiviral factor designated SAMHD1 (sterile alpha motif and HD-domain containing protein 1). SAMHD1 reduces cellular deoxynucleoside triphosphate (dNTP) pools and affects viral reverse transcription step. Human immunodeficiency virus type 2 (HIV-2) and some simian immunodeficiency viruses (SIVs) have Vpx or Vpr to efficiently degrade SAMHD1. Interestingly, the reverse transcriptase (RT) derived from HIV-1 that encodes no anti-SAMHD1 proteins has been previously demonstrated to uniquely exhibit a high enzymatic activity. It is thus not irrational to assume that some viruses may have acquired or lost the specific RT property to better adapt themselves to the low dNTP environments confronted in non-dividing cells. This adaptation process may probably be correlated with the SAMHD1-antagonizing ability by viruses. In this report, we asked whether such adaptive events can be inferable from Vpx/Vpr and RT phylogenetic trees overlaid with SAMHD1-degrading capacity of Vpx/Vpr and with kinetic characteristics of RT. Resultant two trees showed substantially similar clustering patterns, and therefore suggested that the properties of RT and Vpx/Vpr can be linked. In other words, HIV/SIVs may possess their own RT proteins to adequately react to various dNTP circumstances in target cells. PMID:27803699

  10. Arabidopsis ACTIN-DEPOLYMERIZING FACTOR7 Severs Actin Filaments and Regulates Actin Cable Turnover to Promote Normal Pollen Tube Growth[W

    PubMed Central

    Zheng, Yiyan; Xie, Yurong; Jiang, Yuxiang; Qu, Xiaolu; Huang, Shanjin

    2013-01-01

    Actin filaments are often arranged into higher-order structures, such as the longitudinal actin cables that generate the reverse fountain cytoplasmic streaming pattern present in pollen tubes. While several actin binding proteins have been implicated in the generation of these cables, the mechanisms that regulate their dynamic turnover remain largely unknown. Here, we show that Arabidopsis thaliana ACTIN-DEPOLYMERIZING FACTOR7 (ADF7) is required for turnover of longitudinal actin cables. In vitro biochemical analyses revealed that ADF7 is a typical ADF that prefers ADP-G-actin over ATP-G-actin. ADF7 inhibits nucleotide exchange on actin and severs filaments, but its filament severing and depolymerizing activities are less potent than those of the vegetative ADF1. ADF7 primarily decorates longitudinal actin cables in the shanks of pollen tubes. Consistent with this localization pattern, the severing frequency and depolymerization rate of filaments significantly decreased, while their maximum lifetime significantly increased, in adf7 pollen tube shanks. Furthermore, an ADF7–enhanced green fluorescent protein fusion with defective severing activity but normal G-actin binding activity could not complement adf7, providing compelling evidence that the severing activity of ADF7 is vital for its in vivo functions. These observations suggest that ADF7 evolved to promote turnover of longitudinal actin cables by severing actin filaments in pollen tubes. PMID:24058157

  11. Regulation of Protein Function by Reversible Methionine Oxidation and the Role of Selenoprotein MsrB1

    PubMed Central

    Kaya, Alaattin

    2015-01-01

    Abstract Significance: Protein structure and function can be regulated via post-translational modifications by numerous enzymatic and nonenzymatic mechanisms. Regulation involving oxidation of sulfur-containing residues emerged as a key mechanism of redox control. Unraveling the participants and principles of such regulation is necessary for understanding the biological significance of redox control of cellular processes. Recent Advances: Reversible oxidation of methionine residues by monooxygenases of the Mical family and subsequent reduction of methionine sulfoxides by a selenocysteine-containing methionine sulfoxide reductase B1 (MsrB1) was found to control the assembly and disassembly of actin in mammals, and the Mical/MsrB pair similarly regulates actin in fruit flies. This finding has opened up new avenues for understanding the use of stereospecific methionine oxidation in regulating cellular processes and the roles of MsrB1 and Micals in regulation of actin dynamics. Critical Issues: So far, Micals have been the only known partners of MsrB1, and actin is the only target. It is important to identify additional substrates of Micals and characterize other Mical-like enzymes. Future Directions: Oxidation of methionine, reviewed here, is an emerging but not well-established mechanism. Studies suggest that methionine oxidation is a form of oxidative damage of proteins, a modification that alters protein structure or function, a tool in redox signaling, and a mechanism that controls protein function. Understanding the functional impact of reversible oxidation of methionine will require identification of targets, substrates, and regulators of Micals and Msrs. Linking the biological processes, in which these proteins participate, might also lead to insights into disease conditions, which involve regulation of actin by Micals and Msrs. Antioxid. Redox Signal. 23, 814–822. PMID:26181576

  12. Reversible thermal unfolding of a yfdX protein with chaperone-like activity

    PubMed Central

    Saha, Paramita; Manna, Camelia; Chakrabarti, Jaydeb; Ghosh, Mahua

    2016-01-01

    yfdX proteins are ubiquitously present in a large number of virulent bacteria. A member of this family of protein in E. coli is known to be up-regulated by the multidrug response regulator. Their abundance in such bacteria suggests some important yet unidentified functional role of this protein. Here, we study the thermal response and stability of yfdX protein STY3178 from Salmonella Typhi using circular dichroism, steady state fluorescence, dynamic light scattering and nuclear magnetic resonance experiments. We observe the protein to be stable up to a temperature of 45 °C. It folds back to the native conformation from unfolded state at temperature as high as 80 °C. The kinetic measurements of unfolding and refolding show Arrhenius behavior where the refolding involves less activation energy barrier than that of unfolding. We propose a homology model to understand the stability of the protein. Our molecular dynamic simulation studies on this model structure at high temperature show that the structure of this protein is quite stable. Finally, we report a possible functional role of this protein as a chaperone, capable of preventing DTT induced aggregation of insulin. Our studies will have broader implication in understanding the role of yfdX proteins in bacterial function and virulence. PMID:27404435

  13. Soluble components of the flagellar export apparatus, FliI, FliJ, and FliH, do not deliver flagellin, the major filament protein, from the cytosol to the export gate.

    PubMed

    Sajó, Ráchel; Liliom, Károly; Muskotál, Adél; Klein, Agnes; Závodszky, Péter; Vonderviszt, Ferenc; Dobó, József

    2014-11-01

    Flagella, the locomotion organelles of bacteria, extend from the cytoplasm to the cell exterior. External flagellar proteins are synthesized in the cytoplasm and exported by the flagellar type III secretion system. Soluble components of the flagellar export apparatus, FliI, FliH, and FliJ, have been implicated to carry late export substrates in complex with their cognate chaperones from the cytoplasm to the export gate. The importance of the soluble components in the delivery of the three minor late substrates FlgK, FlgL (hook-filament junction) and FliD (filament-cap) has been convincingly demonstrated, but their role in the transport of the major filament component flagellin (FliC) is still unclear. We have used continuous ATPase activity measurements and quartz crystal microbalance (QCM) studies to characterize interactions between the soluble export components and flagellin or the FliC:FliS substrate-chaperone complex. As controls, interactions between soluble export component pairs were characterized providing Kd values. FliC or FliC:FliS did not influence the ATPase activity of FliI alone or in complex with FliH and/or FliJ suggesting lack of interaction in solution. Immobilized FliI, FliH, or FliJ did not interact with FliC or FliC:FliS detected by QCM. The lack of interaction in the fluid phase between FliC or FliC:FliS and the soluble export components, in particular with the ATPase FliI, suggests that cells use different mechanisms for the export of late minor substrates, and the major substrate, FliC. It seems that the abundantly produced flagellin does not require the assistance of the soluble export components to efficiently reach the export gate.

  14. Coordinate-targeted and coordinate-stochastic super-resolution microscopy with the reversibly switchable fluorescent protein Dreiklang.

    PubMed

    Jensen, Nickels A; Danzl, Johann G; Willig, Katrin I; Lavoie-Cardinal, Flavie; Brakemann, Tanja; Hell, Stefan W; Jakobs, Stefan

    2014-03-17

    Diffraction-unlimited far-field super-resolution fluorescence (nanoscopy) methods typically rely on transiently transferring fluorophores between two states, whereby this transfer is usually laid out as a switch. However, depending on whether this is induced in a spatially controlled manner using a pattern of light (coordinate-targeted) or stochastically on a single-molecule basis, specific requirements on the fluorophores are imposed. Therefore, the fluorophores are usually utilized just for one class of methods only. In this study we demonstrate that the reversibly switchable fluorescent protein Dreiklang enables live-cell recordings in both spatially controlled and stochastic modes. We show that the Dreiklang chromophore entails three different light-induced switching mechanisms, namely a reversible photochemical one, off-switching by stimulated emission, and a reversible transfer to a long-lived dark state from the S1 state, all of which can be utilized to overcome the diffraction barrier. We also find that for the single-molecule-based stochastic GSDIM approach (ground-state depletion followed by individual molecule return), Dreiklang provides a larger number of on-off localization events as compared to its progenitor Citrine. Altogether, Dreiklang is a versatile probe for essentially all popular forms of live-cell fluorescence nanoscopy.

  15. The intermediate filament protein vimentin binds specifically to a recombinant integrin {alpha}2/{beta}1 cytoplasmic tail complex and co-localizes with native {alpha}2/{beta}1 in endothelial cell focal adhesions

    SciTech Connect

    Kreis, Stephanie; Schoenfeld, Hans-Joachim; Melchior, Chantal; Steiner, Beat; Kieffer, Nelly . E-mail: kieffer@cu.lu

    2005-04-15

    Integrin receptors are crucial players in cell adhesion and migration. Identification and characterization of cellular proteins that interact with their short {alpha} and {beta} cytoplasmic tails will help to elucidate the molecular mechanisms by which integrins mediate bi-directional signaling across the plasma membrane. Integrin {alpha}2{beta}1 is a major collagen receptor but to date, only few proteins have been shown to interact with the {alpha}2 cytoplasmic tail or with the {alpha}2{beta}1 complex. In order to identify novel binding partners of a {alpha}2{beta}1cytoplasmic domain complex, we have generated recombinant GST-fusion proteins, incorporating the leucine zipper heterodimerization cassettes of Jun and Fos. To ascertain proper functionality of the recombinant proteins, interaction with natural binding partners was tested. GST-{alpha}2 and GST-Jun {alpha}2 bound His-tagged calreticulin while GST-{beta}1 and GST-Fos {beta}1 proteins bound talin. In screening assays for novel binding partners, the immobilized GST-Jun {alpha}2/GST-Fos {beta}1 heterodimeric complex, but not the single subunits, interacted specifically with endothelial cell-derived vimentin. Vimentin, an abundant intermediate filament protein, has previously been shown to co-localize with {alpha}v{beta}3-positive focal contacts. Here, we provide evidence that this interaction also occurs with {alpha}2{beta}1-enriched focal adhesions and we further show that this association is lost after prolonged adhesion of endothelial cells to collagen.

  16. Phosphatidylinositol 3-Kinase-Associated Protein (PI3KAP)/XB130 Crosslinks Actin Filaments through Its Actin Binding and Multimerization Properties In Vitro and Enhances Endocytosis in HEK293 Cells

    PubMed Central

    Yamanaka, Daisuke; Akama, Takeshi; Chida, Kazuhiro; Minami, Shiro; Ito, Koichi; Hakuno, Fumihiko; Takahashi, Shin-Ichiro

    2016-01-01

    Actin-crosslinking proteins control actin filament networks and bundles and contribute to various cellular functions including regulation of cell migration, cell morphology, and endocytosis. Phosphatidylinositol 3-kinase-associated protein (PI3KAP)/XB130 has been reported to be localized to actin filaments (F-actin) and required for cell migration in thyroid carcinoma cells. Here, we show a role for PI3KAP/XB130 as an actin-crosslinking protein. First, we found that the carboxyl terminal region of PI3KAP/XB130 containing amino acid residues 830–840 was required and sufficient for localization to F-actin in NIH3T3 cells, and this region is directly bound to F-actin in vitro. Moreover, actin-crosslinking assay revealed that recombinant PI3KAP/XB130 crosslinked F-actin. In general, actin-crosslinking proteins often multimerize to assemble multiple actin-binding sites. We then investigated whether PI3KAP/XB130 could form a multimer. Blue native-PAGE analysis showed that recombinant PI3KAP/XB130 was detected at 250–1200 kDa although the molecular mass was approximately 125 kDa, suggesting that PI3KAP/XB130 formed multimers. Furthermore, we found that the amino terminal 40 amino acids were required for this multimerization by co-immunoprecipitation assay in HEK293T cells. Deletion mutants of PI3KAP/XB130 lacking the actin-binding region or the multimerizing region did not crosslink actin filaments, indicating that actin binding and multimerization of PI3KAP/XB130 were necessary to crosslink F-actin. Finally, we examined roles of PI3KAP/XB130 on endocytosis, an actin-related biological process. Overexpression of PI3KAP/XB130 enhanced dextran uptake in HEK 293 cells. However, most of the cells transfected with the deletion mutant lacking the actin-binding region incorporated dextran to a similar extent as control cells. Taken together, these results demonstrate that PI3KAP/XB130 crosslinks F-actin through both its actin-binding region and multimerizing region and

  17. A protein with antiproliferative, antifungal and HIV-1 reverse transcriptase inhibitory activities from caper (Capparis spinosa) seeds.

    PubMed

    Lam, Sze-Kwan; Ng, Tzi-Bun

    2009-05-01

    A protein exhibiting an N-terminal amino acid sequence with some similarity to imidazoleglycerol phosphate synthase was purified from fresh Capparis spinosa melon seeds. The purification protocol entailed anion exchange chromatography on DEAE-cellulose, cation exchange chromatography on SP-Sepharose, and finally gel filtration by fast protein liquid chromatography on Superdex 75. The protein was adsorbed using 20 mM Tris-HCl buffer (pH 7.4) and desorbed using 1 M NaCl in the starting buffer from the DEAE-cellulose column and SP-Sepharose column. The protein demonstrated a molecular mass of 38 kDa in gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, indicating that it was monomeric. The protein inhibited proliferation of hepatoma HepG2 cells, colon cancer HT29 cells and breast cancer MCF-7 cells with an IC(50) of about 1, 40 and 60 microM, respectively. It inhibited HIV-1 reverse transcriptase with IC(50) of 0.23 microM. It inhibited mycelial growth in the fungus, Valsa mali. It did not exhibit hemagglutinating, ribonuclease, mitogenic or protease inhibitory activities.

  18. Dissection of Filamentous Growth by Transposon Mutagenesis in Saccharomyces Cerevisiae

    PubMed Central

    Mosch, H. U.; Fink, G. R.

    1997-01-01

    Diploid Saccharomyces cerevisiae strains starved for nitrogen undergo a developmental transition from growth as single yeast form (YF) cells to a multicellular form consisting of filaments of pseudohyphal (PH) cells. Filamentous growth is regulated by an evolutionarily conserved signaling pathway that includes the small GTP-binding proteins Ras2p and Cdc42p, the protein kinases Ste20p, Ste11p and Ste7p, and the transcription factor Ste12p. Here, we designed a genetic screen for mutant strains defective for filamentous growth (dfg) to identify novel targets of the filamentation signaling pathway, and we thereby identified 16 different genes, CDC39, STE12, TEC1, WHI3, NAB1, DBR1, CDC55, SRV2, TPM1, SPA2, BNI1, DFG5, DFG9, DFG10, BUD8 and DFG16, mutations that block filamentous growth. Phenotypic analysis of dfg mutant strains genetically dissects filamentous growth into the cellular processes of signal transduction, bud site selection, cell morphogenesis and invasive growth. Epistasis tests between dfg mutant alleles and dominant activated alleles of the RAS2 and STE11 genes, RAS2(Val19) and STE11-4, respectively, identify putative targets for the filamentation signaling pathway. Several of the genes described here have homologues in filamentous fungi, where they also regulate fungal development. PMID:9055077

  19. Myosin filament 3D structure in mammalian cardiac muscle☆

    PubMed Central

    AL-Khayat, Hind A.; Morris, Edward P.; Kensler, Robert W.; Squire, John M.

    2008-01-01

    A number of cardiac myopathies (e.g. familial hypertrophic cardiomyopathy and dilated cardiomyopathy) are linked to mutations in cardiac muscle myosin filament proteins, including myosin and myosin binding protein C (MyBP-C). To understand the myopathies it is necessary to know the normal 3D structure of these filaments. We have carried out 3D single particle analysis of electron micrograph images of negatively stained isolated myosin filaments from rabbit cardiac muscle. Single filament images were aligned and divided into segments about 2 × 430 Å long, each of which was treated as an independent ‘particle’. The resulting 40 Å resolution 3D reconstruction showed both axial and azimuthal (no radial) myosin head perturbations within the 430 Å repeat, with successive crown rotations of approximately 60°, 60° and 0°, rather than the regular 40° for an unperturbed helix. However, it is shown that the projecting density peaks appear to start at low radius from origins closer to those expected for an unperturbed helical filament, and that the azimuthal perturbation especially increases with radius. The head arrangements in rabbit cardiac myosin filaments are very similar to those in fish skeletal muscle myosin filaments, suggesting a possible general structural theme for myosin filaments in all vertebrate striated muscles (skeletal and cardiac). PMID:18472277

  20. Deletions in epidermal keratins leading to alterations in filament organization in vivo and in intermediate filament assembly in vitro

    PubMed Central

    1990-01-01

    To investigate the sequences important for assembly of keratins into 10- nm filaments, we used a combined approach of (a) transfection of mutant keratin cDNAs into epithelial cells in vivo, and (b) in vitro assembly of mutant and wild-type keratins. Keratin K14 mutants missing the nonhelical carboxy- and amino-terminal domains not only integrated without perturbation into endogenous keratin filament networks in vivo, but they also formed 10-nm filaments with K5 in vitro. Surprisingly, keratin mutants missing the highly conserved L L E G E sequence, common to all intermediate filament proteins and found at the carboxy end of the alpha-helical rod domain, also assembled into filaments with only a somewhat reduced efficiency. Even a carboxy K14 mutant missing approximately 10% of the rod assembled into filaments, although in this case filaments aggregated significantly. Despite the ability of these mutants to form filaments in vitro, they often perturbed keratin filament organization in vivo. In contrast, small truncations in the amino-terminal end of the rod domain more severely disrupted the filament assembly process in vitro as well as in vivo, and in particular restricted elongation. For both carboxy and amino rod deletions, the more extensive the deletion, the more severe the phenotype. Surprisingly, while elongation could be almost quantitatively blocked with large mutations, tetramer formation and higher ordered lateral interactions still occurred. Collectively, our in vitro data (a) provide a molecular basis for the dominance of our mutants in vivo, (b) offer new insights as to why different mutants may generate different phenotypes in vivo, and (c) delineate the limit sequences necessary for K14 to both incorporate properly into a preexisting keratin filament network in vivo and assemble efficiently into 10-nm keratin filaments in vitro. PMID:1702787

  1. Redox control of surface protein sulphhydryls in bovine spermatozoa reversibly modulates sperm adhesion to the oviductal epithelium and capacitation.

    PubMed

    Gualtieri, Roberto; Mollo, Valentina; Duma, Gennaro; Talevi, Riccardo

    2009-07-01

    Oviductal fluid molecules, such as sulphated glycosaminoglycans and disulphide-reductants, may represent periovulatory signals for the release of spermatozoa from the oviductal reservoir in the bovine species. Disulphide-reductants release spermatozoa through the reduction of sperm-surface disulphides to sulphhydryls (SH). Herein, we studied sperm-surface protein SH through labelling with maleimidylpropionyl biocytin in the initial sperm suspension, in the subpopulations able and unable to adhere to the in vitro cultured oviductal epithelium, and in spermatozoa released either through the disulphide-reductant penicillamine (PEN) or the sulphated glycosaminoglycan heparin (HEP). Adhesion assays were performed to study the ability of released spermatozoa to readhere to the oviductal epithelium. Results showed that the level of SH in sperm-surface proteins was: 1) low in adhering spermatozoa; 2) high in spermatozoa unable to adhere; and 3) markedly increased in released spermatozoa. Adhesion assays showed that: 1) PEN-released spermatozoa promptly recovered adhesion after removal of the disulphide-reductant and could be released again in response to PEN; 2) conversely, a limited number of HEP-released spermatozoa was able to readhere to the oviductal epithelium and this ability was not affected by HEP removal. Recovery of adhesion was associated to reoxidation of sperm-surface protein SH and to the reversal of capacitation. In conclusion, redox modulation of sperm-surface protein SH is involved in the release of spermatozoa adhering to the oviduct in vitro; the reversible action of disulphide-reductants might be responsible for intermittent phases of adhesions and releases; and the irreversible action of HEP indicates that it may represent a terminal releasing signal.

  2. A facile method for reversibly linking a recombinant protein to DNA.

    PubMed

    Goodman, Russell P; Erben, Christoph M; Malo, Jonathan; Ho, Wei M; McKee, Mireya L; Kapanidis, Achillefs N; Turberfield, Andrew J

    2009-06-15

    We present a facile method for linking recombinant proteins to DNA. It is based on the nickel-mediated interaction between a hexahistidine tag (His(6)-tag) and DNA functionalized with three nitrilotriacetic acid (NTA) groups. The resulting DNA-protein linkage is site-specific. It can be broken quickly and controllably by the addition of a chelating agent that binds nickel. We have used this new linker to bind proteins to a variety of DNA motifs commonly used in the fabrication of nanostructures by DNA self-assembly.

  3. Shared antigenicity between the polar filaments of myxosporeans and other Cnidaria.

    PubMed

    Ringuette, Maurice J; Koehler, Anne; Desser, Sherwin S

    2011-02-01

    Nematocysts containing coiled polar filaments are a distinguishing feature of members of the phylum Cnidaria. As a first step to characterizing the molecular structure of polar filaments, a polyclonal antiserum was raised in rabbits against a cyanogen bromide-resistant protein extract of mature cysts containing spores of Myxobolus pendula. The antiserum reacted only with proteins associated with extruded polar filaments. Western blot and whole-mount immunohistochemical analyses indicated a conservation of polar filament epitopes between M. pendula and 2 related cnidarians, i.e., the anthozoan, Nematostella vectensis, and the hydrozoan, Hydra vulgaris. This conservation of polar filament epitopes lends further support to a shared affinity between Myxozoa and cnidarians.

  4. Contractile properties of thin (actin) filament-reconstituted muscle fibers.

    PubMed

    Ishiwata, S; Funatsu, T; Fujita, H

    1998-01-01

    Selective removal and reconstitution of the components of muscle fibers (fibrils) is a useful means of examining the molecular mechanism underlying the formation of the contractile apparatus. In addition, this approach is powerful for examining the structure-function relationship of a specific component of the contractile system. In previous studies, we have achieved the partial structural and functional reconstitution of thin filaments in the skeletal contractile apparatus and full reconstitution in the cardiac contractile apparatus. First, all thin filaments other than short fragments at the Z line were removed by treatment with plasma gelsolin, an actin filament-severing protein. Under these conditions, no active tension could be generated. By incorporating exogenous actin into these thin filament-free fibers, actin filaments were reconstituted by polymerization on the short actin fragments remaining at the Z line, and active tension, which was insensitive to Ca2+, was restored. The active tension after the reconstitution of thin filaments reached as high as 30% of the original level in skeletal muscle, while it reached 140% in cardiac muscle. The augmentation of tension in cardiac muscle is mainly attributable to the elongation of reconstituted filaments, longer than the average length of thin filaments in an intact muscle. These results indicate that a muscle contractile apparatus with a high order structure and function can be constructed by the self-assembly of constituent proteins. Recently, we applied this reconstitution system to the study of the mechanism of spontaneous oscillatory contraction (SPOC) in thin (actin) filament-reconstituted cardiac muscle fibers. As a result, we found that SPOC occurs even in regulatory protein-free actin filament-reconstituted fibers (Fujita & Ishiwata, manuscript submitted), although the SPOC conditions were slightly different from the standard SPOC conditions. This result strongly suggests that spontaneous oscillation

  5. Electrical contact resistance in filaments

    NASA Astrophysics Data System (ADS)

    Wu, Xiang-Fa; Zhou, Zhengping; Zhou, Wang-Min

    2012-05-01

    Electrical contact resistance (ECR) influences the electrochemical performance of porous electrodes made of stacked discrete materials (e.g., carbon nanotubes, nanofibers, etc.) for use in supercapacitors and rechargeable batteries. This study establishes a simple elasticity-conductivity model for the ECR of filaments in adhesive contact. The elastic deformation and size of electrical contact zone of the filaments are determined by using an adhesive contact model of filaments, and the ECR of adhesive filaments is obtained in explicit form. Dependencies of the ECR upon the filament geometries, surface energy, and elasticity are examined.

  6. A reverse-phase protein microarray-based screen identifies host signaling dynamics upon Burkholderia spp. infection

    PubMed Central

    Chiang, Chih-Yuan; Uzoma, Ijeoma; Lane, Douglas J.; Memišević, Vesna; Alem, Farhang; Yao, Kuan; Kota, Krishna P.; Bavari, Sina; Wallqvist, Anders; Hakami, Ramin M.; Panchal, Rekha G.

    2015-01-01

    Burkholderia is a diverse genus of gram-negative bacteria that causes high mortality rate in humans, equines and cattle. The lack of effective therapeutic treatments poses serious public health threats. Developing insights toward host-Burkholderia spp. interaction is critical for understanding the pathogenesis of infection as well as identifying therapeutic targets for drug development. Reverse-phase protein microarray technology was previously proven to identify and characterize novel biomarkers and molecular signatures associated with infectious disease and cancer. In the present study, this technology was utilized to interrogate changes in host protein expression and phosphorylation events in macrophages infected with a collection of geographically diverse strains of Burkholderia spp. The expression or phosphorylation state of 25 proteins was altered during Burkholderia spp. infections of which eight proteins were selected for further characterization by immunoblotting. Increased phosphorylation of AMPK-α1, Src, and GSK3β suggested the importance of their roles in regulating Burkholderia spp. mediated innate immune response. Modulating the inflammatory response by perturbing their activities may provide therapeutic routes for future treatments. PMID:26284031

  7. A library of MiMICs allows tagging of genes and reversible, spatial and temporal knockdown of proteins in Drosophila

    PubMed Central

    Nagarkar-Jaiswal, Sonal; Lee, Pei-Tseng; Campbell, Megan E; Chen, Kuchuan; Anguiano-Zarate, Stephanie; Cantu Gutierrez, Manuel; Busby, Theodore; Lin, Wen-Wen; He, Yuchun; Schulze, Karen L; Booth, Benjamin W; Evans-Holm, Martha; Venken, Koen JT; Levis, Robert W; Spradling, Allan C; Hoskins, Roger A; Bellen, Hugo J

    2015-01-01

    Here, we document a collection of ∼7434 MiMIC (Minos Mediated Integration Cassette) insertions of which 2854 are inserted in coding introns. They allowed us to create a library of 400 GFP-tagged genes. We show that 72% of internally tagged proteins are functional, and that more than 90% can be imaged in unfixed tissues. Moreover, the tagged mRNAs can be knocked down by RNAi against GFP (iGFPi), and the tagged proteins can be efficiently knocked down by deGradFP technology. The phenotypes associated with RNA and protein knockdown typically correspond to severe loss of function or null mutant phenotypes. Finally, we demonstrate reversible, spatial, and temporal knockdown of tagged proteins in larvae and adult flies. This new strategy and collection of strains allows unprecedented in vivo manipulations in flies for many genes. These strategies will likely extend to vertebrates. DOI: http://dx.doi.org/10.7554/eLife.05338.001 PMID:25824290

  8. Reversed cellular polarity in primary cutaneous mucinous carcinoma: A study on tight junction protein expression in sweat gland tumors.

    PubMed

    Nagasawa, Yusuke; Ishida-Yamamoto, Akemi

    2017-04-01

    Primary cutaneous mucinous carcinoma (PCMC) is a rare sweat gland tumor characterized by the presence of abundant mucin around the tumor islands, but the molecular mechanisms for this structure are not well elucidated. Because mucin is epithelial in nature, it is likely to be produced by epithelial tumor cells, not by surrounding stromal cells. We hypothesized that the abundant mucin is a result of reversed cellular polarity of the tumor. To test this hypothesis, we conducted an immunohistological study to investigate expression of tight junction (TJ) proteins occludin and ZO-1 in PCMC, as well as in normal sweat glands and other sweat gland tumors. Dot-like or linear expression of TJ proteins was observed at ductal structures of sweat glands, and ductal or cystic structures of related tumors. In PCMC, however, TJ protein expression was clearly visible at the edges of tumor cell islands. This study provides evidence to show that the characteristic histological structure of PCMC is caused by inverse polarization of the tumor cells, and that TJ proteins are useful markers of ductal differentiation in sweat gland tumors.

  9. Improved separation of fluorogenic derivatized intact proteins with high resolution and efficiency using a reversed-phase liquid chromatographic system.

    PubMed

    Ichibangase, Tomoko; Nakata, Katsunori; Imai, Kazuhiro

    2014-06-01

    Although the efficient separation of intact protein mixtures is extremely difficult, reversed-phase chromatography is an important technique for performing quantitative, accurate and reproducible proteomics analyses. Here, we show that, despite the operating constraints of conventional high-performance liquid chromatography, such as column temperature, operating pressure and separation time, comprehensive separation of fluorogenic derivatized intact proteins could be achieved with high resolution and separation efficiency. First, amylin was chosen as a model peptide and used to estimate the separation efficiency with respect to column temperature and flow rate, as indicated by peak capacity. Then, an extract of human primary hepatocytes was used to model complex component mixtures and the separation conditions were optimized. The effects of mobile-phase pH, the separation time and the column length were also investigated. Consequently, more than 890 peaks could be separated efficiently in the extract, which is 1.5-fold greater than when using conventional conditions. Finally, it was demonstrated that both longer separation time and column length contributed greatly to the effective separation of the protein mixture. These results are expected to provide insights into the separation of intact proteins.

  10. Associations between proteins and heavy metals in urine at low environmental exposures: evidence of reverse causality.

    PubMed

    Chaumont, Agnès; Nickmilder, Marc; Dumont, Xavier; Lundh, Thomas; Skerfving, Staffan; Bernard, Alfred

    2012-05-05

    Heavy metals can cause renal effects on vulnerable populations but it is uncertain whether these metals still pose health risks at the low exposure levels now prevailing in most industrialized countries. In a cross-sectional study performed on 736 adolescents, we assessed the associations between the concentrations of cadmium and lead in blood and urine and the urinary concentrations of albumin and of low-molecular-weight (LMW) proteins, retinol-binding protein (RBP) and β(2)-microglobulin. Multiple regression analyses were tested using urinary markers normalized to urinary creatinine or specific gravity. Median metal concentrations were in blood (μg/L): lead, 15.1, cadmium, 0.18 and in urine (μg/g creatinine): cadmium, 0.09 and lead, 0.82. Multivariate analyses revealed significant associations in urine between RBP and cadmium as well as between β(2)-microglobulin and lead whereas no associations were seen with metals in blood. These associations were completely abolished in subjects with increased urinary albumin, which may be explained by the competitive inhibition of LMW protein reabsorption by albumin. Given the evidence that cadmium and lead circulate mainly bound to LMW proteins, these associations observed at low exposure might simply reflect the interindividual variations in the renal uptake of proteins sharing the same affinity for tubular binding sites.

  11. Determination of proteins by a reverse biuret method combined with the copper-bathocuproine chelate reaction.

    PubMed

    Matsushita, M; Irino, T; Komoda, T; Sakagishi, Y

    1993-07-16

    A method of protein determination has been developed which combines the biuret reaction and the copper(I)-bathocuproine chelate reaction. Protein in the specimen forms a Cu(2+)-protein chelate complex (biuret reaction) during the first step. Excess Cu2+ is reduced to Cu+ by ascrobic acid, allowing the Cu+ to form a Cu(+)-bathocuproine chelate complex during the second step. The amount of Cu(+)-bathocuproine chelate complex formed is inversely proportional to the protein concentration. The sensitivity (epsilon = 1.4 x 10(6) 1.mol-1.cm-1 against human albumin) of this method was higher than that of the original Lowry (9.8 x 10(5)), pyrogallol red (1.0 x 10(6)) and commercially available Coomassie Brilliant Blue G.250 methods (6.7 x 10(5)). The color intensities of human gamma-globulin, human globulin (fractions IV-1 and IV-4), bovine albumin, egg albumin and horse gamma-globulin against human albumin (100%) ranged from 92 to 101%. The results obtained with the present method (y) correlated well with those determined by the biuret method (r = 0.998, y = 0.98 chi - 0.002, x = 1.31, y = 1.29 g/l) in 30 diluted sera. These results confirm that this assay is similar in sensitivity to the original Lowry method, is rapid and has similar reactivity to each of the various proteins in biological fluids.

  12. Growth hormone and insulin reverse net whole body and skeletal muscle protein catabolism in cancer patients.

    PubMed Central

    Wolf, R F; Pearlstone, D B; Newman, E; Heslin, M J; Gonenne, A; Burt, M E; Brennan, M F

    1992-01-01

    The authors examined the effect of recombinant-human growth hormone (r-hGH) and insulin (INS) administration on protein kinetics in cancer patients. Twenty-eight cancer patients either received r-hGH for 3 days (GH group, n = 12, weight loss = 6 +/- 2%) or were not treated (control [CTL] group, n = 16, weight loss = 11 +/- 2%) before metabolic study. Recombinant-human growth hormone dose was 0.1 mg/kg/day (n = 6) or 0.2 mg/kg/day (n = 6). Patients then underwent measurement of baseline protein kinetics (GH/B, CTL/B) followed by a 2-hour euglycemic insulin infusion (1 mU/kg/minute) and repeat kinetic measurements (GH/INS,CTL/INS). Whole-body protein net balance (mumol leucine/kg/minute) was higher (p less than 0.05) in GH/INS (0.20 +/- 0.06) than in CTL/INS (0.06 +/- 0.03) or GH/B (-0.19 +/- 0.03). Skeletal muscle protein net balance (nmol phenylalanine/100 g/minute) in GH/INS (25 +/- 6) and CTL/INS (19 +/- 5) was higher than CTL/B (-18 +/- 3). Recombinant-human growth hormone and insulin reduce whole-body and skeletal muscle protein loss in cancer patients. Simultaneous use of these agents during nutritional therapy may benefit the cancer patient. PMID:1417177

  13. Solid friction between soft filaments.

    PubMed

    Ward, Andrew; Hilitski, Feodor; Schwenger, Walter; Welch, David; Lau, A W C; Vitelli, Vincenzo; Mahadevan, L; Dogic, Zvonimir

    2015-06-01

    Any macroscopic deformation of a filamentous bundle is necessarily accompanied by local sliding and/or stretching of the constituent filaments. Yet the nature of the sliding friction between two aligned filaments interacting through multiple contacts remains largely unexplored. Here, by directly measuring the sliding forces between two bundled F-actin filaments, we show that these frictional forces are unexpectedly large, scale logarithmically with sliding velocity as in solid-like friction, and exhibit complex dependence on the filaments' overlap length. We also show that a reduction of the frictional force by orders of magnitude, associated with a transition from solid-like friction to Stokes's drag, can be induced by coating F-actin with polymeric brushes. Furthermore, we observe similar transitions in filamentous microtubules and bacterial flagella. Our findings demonstrate how altering a filament's elasticity, structure and interactions can be used to engineer interfilament friction and thus tune the properties of fibrous composite materials.

  14. Regulation of actin filament length in erythrocytes and striated muscle.

    PubMed

    Fowler, V M

    1996-02-01

    Actin filaments polymerize in vitro to lengths which display an exponential distribution, yet in many highly differentiated cells they can be precisely maintained at uniform lengths in elaborate supramolecular structures. Recent results obtained using two classic model systems, the erythrocyte membrane cytoskeleton and the striated muscle sarcomere, reveal surprising similarities and instructive differences in the molecules and mechanisms responsible for determining and maintaining actin filament lengths in these two systems. Tropomodulin caps the slow-growing, pointed filament ends in muscle and in erythrocytes. CapZ caps the fast-growing, barbed filament ends in striated muscle, whereas a newly discovered barbed end capping protein, adducin, may cap the barbed filament ends in erythrocytes. The mechanisms responsible for specifying the characteristic filament lengths in these systems are more elusive and may include strict control of the relative amounts of actin filament capping proteins and side-binding proteins, molecular templates (e.g. tropomyosin and nebulin) and/or verniers (e.g. tropomyosin).

  15. Intermediate Filaments and Polarization in the Intestinal Epithelium

    PubMed Central

    Coch, Richard A.; Leube, Rudolf E.

    2016-01-01

    The cytoplasmic intermediate filament cytoskeleton provides a tissue-specific three-dimensional scaffolding with unique context-dependent organizational features. This is particularly apparent in the intestinal epithelium, in which the intermediate filament network is localized below the apical terminal web region and is anchored to the apical junction complex. This arrangement is conserved from the nematode Caenorhabditis elegans to humans. The review summarizes compositional, morphological and functional features of the polarized intermediate filament cytoskeleton in intestinal cells of nematodes and mammals. We emphasize the cross talk of intermediate filaments with the actin- and tubulin-based cytoskeleton. Possible links of the intermediate filament system to the distribution of apical membrane proteins and the cell polarity complex are highlighted. Finally, we discuss how these properties relate to the establishment and maintenance of polarity in the intestine. PMID:27429003

  16. Reversed-phase liquid chromatographic resolution of diastereomers of protein and non-protein amino acids prepared with newly synthesized chiral derivatizing reagents based on cyanuric chloride.

    PubMed

    Bhushan, Ravi; Agarwal, Charu

    2011-02-01

    Two new chiral monochloro-s-triazines (MCT) were synthesized [viz N-(4-chloro-6-piperidinyl-[1,3,5]-triazine-2-yl)-L-leucine amide and N-(4-chloro-6-piperidinyl-[1,3,5]-triazine-2-yl)-L-leucine) (CDR 1 and 2, respectively)] by the nucleophilic displacement of chlorine atoms in s-triazine moiety. One of the Cl atoms was replaced with piperidine, and the second Cl atom in the 6-piperidinyl derivative was replaced with amino acid amide (viz L-Leu-NH(2)) and amino acid (L-Leu). These reagents were characterized and used as CDRs for chiral separation of protein and non-protein amino acids, and were separated on a reversed-phase C(18) column. The reaction conditions were optimized for the synthesis of diastereomers using one MCT reagent. The separation method was validated for limit of detection, linearity, accuracy, precision, and recovery.

  17. Reversible phenol oxidation and reduction in the structurally well-defined 2-Mercaptophenol-α₃C protein.

    PubMed

    Tommos, Cecilia; Valentine, Kathleen G; Martínez-Rivera, Melissa C; Liang, Li; Moorman, Veronica R

    2013-02-26

    2-Mercaptophenol-α₃C serves as a biomimetic model for enzymes that use tyrosine residues in redox catalysis and multistep electron transfer. This model protein was tailored for electrochemical studies of phenol oxidation and reduction with specific emphasis on the redox-driven protonic reactions occurring at the phenol oxygen. This protein contains a covalently modified 2-mercaptophenol-cysteine residue. The radical site and the phenol compound were specifically chosen to bury the phenol OH group inside the protein. A solution nuclear magnetic resonance structural analysis (i) demonstrates that the synthetic 2-mercaptophenol-α₃C model protein behaves structurally as a natural protein, (ii) confirms the design of the radical site, (iii) reveals that the ligated phenol forms an interhelical hydrogen bond to glutamate 13 (phenol oxygen-carboxyl oxygen distance of 3.2 ± 0.5 Å), and (iv) suggests a proton-transfer pathway from the buried phenol OH (average solvent accessible surface area of 3 ± 5%) via glutamate 13 (average solvent accessible surface area of the carboxyl oxygens of 37 ± 18%) to the bulk solvent. A square-wave voltammetry analysis of 2-mercaptophenol-α₃C further demonstrates that (v) the phenol oxidation-reduction cycle is reversible, (vi) formal phenol reduction potentials can be obtained, and (vii) the phenol-O(•) state is long-lived with an estimated lifetime of ≥180 millisecond. These properties make 2-mercaptophenol-α₃C a unique system for characterizing phenol-based proton-coupled electron transfer in a low-dielectric and structured protein environment.

  18. Reversible Cysteine Acylation Regulates the Activity of Human Palmitoyl-Protein Thioesterase 1 (PPT1)

    PubMed Central

    Segal-Salto, Michal; Sapir, Tamar; Reiner, Orly

    2016-01-01

    Mutations in the depalmitoylating enzyme gene, PPT1, cause the infantile form of Neuronal Ceroid Lipofuscinosis (NCL), an early onset neurodegenerative disease. During recent years there have been different therapeutic attempts including enzyme replacement. Here we show that PPT1 is palmitoylated in vivo and is a substrate for two palmitoylating enzymes, DHHC3 and DHHC7. The palmitoylated protein is detected in both cell lysates and medium. The presence of PPT1 with palmitoylated signal peptide in the cell medium suggests that a subset of the protein is secreted by a nonconventional mechanism. Using a mutant form of PPT1, C6S, which was not palmitoylated, we further demonstrate that palmitoylation does not affect intracellular localization but rather that the unpalmitoylated form enhanced the depalmitoylation activity of the protein. The calculated Vmax of the enzyme was significantly affected by the palmitoylation, suggesting that the addition of a palmitate group is reminiscent of adding a noncompetitive inhibitor. Thus, we reveal the existence of a positive feedback loop, where palmitoylation of PPT1 results in decreased activity and subsequent elevation in the amount of palmitoylated proteins. This positive feedback loop is likely to initiate a vicious cycle, which will enhance disease progression. The understanding of this process may facilitate enzyme replacement strategies. PMID:26731412

  19. Redox Modulation of Cellular Signaling and Metabolism Through Reversible Oxidation of Methionine Sensors in Calcium Regulatory Proteins

    SciTech Connect

    Bigelow, Diana J.; Squier, Thomas C.

    2005-01-17

    Adaptive responses associated with environmental stressors are critical to cell survival. These involve the modulation of central signaling protein functions through site-specific and enzymatically reversible oxidative modifications of methionines to coordinate cellular metabolism, energy utilization, and calcium signaling. Under conditions when cellular redox and antioxidant defenses are overwhelmed, the selective oxidation of critical methionines within selected protein sensors functions to down-regulate energy metabolism and the further generation of reactive oxygen species (ROS). Mechanistically, these functional changes within protein sensors take advantage of the helix-breaking character of methionine sulfoxide. Thus, depending on either the ecological niche of the organism or the cellular milieu of different organ systems, cellular metabolism can be fine-tuned to maintain optimal function in the face of variable amounts of collateral oxidative damage. The sensitivity of several calcium regulatory proteins to oxidative modification provides cellular sensors that link oxidative stress to cellular response and recovery. Calmodulin (CaM) is one such critical calcium regulatory protein, which is functionally sensitive to methionine oxidation. Helix destabilization resulting from the oxidation of either Met{sup 144} or Met{sup 145} results in the nonproductive association between CaM and target proteins. The ability of oxidized CaM to stabilize its target proteins in an inhibited state with an affinity similar to that of native (unoxidized) CaM permits this central regulatory protein to function as a cellular rheostat that down-regulates energy metabolism in response to oxidative stress. Likewise, oxidation of a methionine within a critical switch region of the regulatory protein phospholamban is expected to destabilize the phosphorylationdependent helix formation necessary for the release of enzyme inhibition, resulting in a down-regulation of the Ca-ATPase in

  20. The pcz1 Gene, which Encodes a Zn(II)2Cys6 Protein, Is Involved in the Control of Growth, Conidiation, and Conidial Germination in the Filamentous Fungus Penicillium roqueforti

    PubMed Central

    Medina, Exequiel; Vaca, Inmaculada; García-Rico, Ramón O.; Villagrán, Sebastián; Levicán, Gloria; Chávez, Renato

    2015-01-01

    Proteins containing Zn(II)2Cys6 domains are exclusively found in fungi and yeasts. Genes encoding this class of proteins are broadly distributed in fungi, but few of them have been functionally characterized. In this work, we have characterized a gene from the filamentous fungus Penicillium roqueforti that encodes a Zn(II)2Cys6 protein, whose function to date remains unknown. We have named this gene pcz1. We showed that the expression of pcz1 is negatively regulated in a P. roqueforti strain containing a dominant active Gαi protein, suggesting that pcz1 encodes a downstream effector that is negatively controlled by Gαi. More interestingly, the silencing of pcz1 in P. roqueforti using RNAi-silencing technology resulted in decreased apical growth, the promotion of conidial germination (even in the absence of a carbon source), and the strong repression of conidiation, concomitant with the downregulation of the genes of the central conidiation pathway brlA, abaA and wetA. A model for the participation of pcz1 in these physiological processes in P. roqueforti is proposed. PMID:25811807

  1. A Structural Basis for Reversible Photoswitching of Absorbance Spectra in Red Fluorescent Protein rsTagRFP

    PubMed Central

    Pletnev, Sergei; Subach, Fedor V.; Dauter, Zbigniew; Wlodawer, Alexander; Verkhusha, Vladislav V.

    2012-01-01

    rsTagRFP the first monomeric red fluorescent protein with reversibly photoswitchable absorbance spectra. The switching is realized by irradiation of rsTagRFP with blue (440 nm) and yellow (567 nm) light, turning the protein fluorescence ON and OFF, respectively. It is perhaps the most useful probe in this color class that has yet been reported. Because of the photoswitchable absorbance, rsTagRFP can be used as an acceptor in photochromic Förster resonance energy transfer (pcFRET). Yellow fluorescent proteins YPet and mVenus have been demonstrated to be excellent pcFRET donors for the rsTagRFP acceptor in its fusion constructs. Analysis of X-ray structures has shown that photoswitching of rsTagRFP is accompanied by cis-trans isomerization and protonation/deprotonation of the chromophore, with the deprotonated cis- and protonated trans- isomers corresponding to its ON and OFF states. Unlike in other photoswitchable fluorescent proteins, both conformers of rsTagRFP chromophore are essentially coplanar. Two other peculiarities of the rsTagRFP chromophore are an essentially hydrophobic environment of its p-hydroxyphenyl site and the absence of direct hydrogen bonding between this moiety and the protein scaffold. The influence of the immediate environment on rsTagRFP chromophore was probed by site-directed mutagenesis. Residues Glu145 and His197 were found to participate in protonation/deprotonation of the chromophore accompanying the photoswitching of rsTagRFP fluorescence, whereas the residues Met160 and Leu174 were shown to spatially restrict chromophore isomerization, favoring its radiative decay. PMID:22310052

  2. Genome Report: Identification and Validation of Antigenic Proteins from Pajaroellobacter abortibovis Using De Novo Genome Sequence Assembly and Reverse Vaccinology

    PubMed Central

    Welly, Bryan T.; Miller, Michael R.; Stott, Jeffrey L.; Blanchard, Myra T.; Islas-Trejo, Alma D.; O’Rourke, Sean M.; Young, Amy E.; Medrano, Juan F.; Van Eenennaam, Alison L.

    2016-01-01

    Epizootic bovine abortion (EBA), or “foothill abortion,” is the leading cause of beef cattle abortion in California and has also been reported in Nevada and Oregon. In the 1970s, the soft-shelled tick Ornithodoros coriaceus, or “pajaroello tick,” was confirmed as the disease-transmitting vector. In 2005, a novel Deltaproteobacterium was discovered as the etiologic agent of EBA (aoEBA), recently named Pajaroellobacter abortibovis. This organism cannot be grown in culture using traditional microbiological techniques; it can only be grown in experimentally-infected severe combined immunodeficient (SCID) mice. The objectives of this study were to perform a de novo genome assembly for P. abortibovis and identify and validate potential antigenic proteins as candidates for future recombinant vaccine development. DNA and RNA were extracted from spleen tissue collected from experimentally-infected SCID mice following exposure to P. abortibovis. This combination of mouse and bacterial DNA was sequenced and aligned to the mouse genome. Mouse sequences were subtracted from the sequence pool and the remaining sequences were de novo assembled at 50x coverage into a 1.82 Mbp complete closed circular Deltaproteobacterial genome containing 2250 putative protein-coding sequences. Phylogenetic analysis of P. abortibovis predicts that this bacterium is most closely related to the organisms of the order Myxococcales, referred to as Myxobacteria. In silico prediction of vaccine candidates was performed using a reverse vaccinology approach resulting in the identification and ranking of the top 10 candidate proteins that are likely to be antigenic. Immunologic testing of these candidate proteins confirmed antigenicity of seven of the nine expressed protein candidates using serum from P. abortibovis immunized mice. PMID:28040777

  3. Reversible Inhibition of Mitochondrial Protein Synthesis during Linezolid-Related Hyperlactatemia▿

    PubMed Central

    Garrabou, Glòria; Soriano, Alejandro; López, Sònia; Guallar, Jordi P.; Giralt, Marta; Villarroya, Francesc; Martínez, Jose A.; Casademont, Jordi; Cardellach, Francesc; Mensa, Josep; Miró, Òscar

    2007-01-01

    The objective of the present study was to determine the mitochondrial toxicity mechanisms of linezolid-related hyperlactatemia. Five patients on a long-term schedule of linezolid treatment were studied during the acute phase of hyperlactatemia and after clinical recovery and lactate normalization following linezolid withdrawal. Mitochondrial studies were performed with peripheral blood mononuclear cells and consisted of measurement of mitochondrial mass, mitochondrial protein synthesis homeostasis (cytochrome c oxidase [COX] activity, COX-II subunit expression, COX-II mRNA abundance, and mitochondrial DNA [mtDNA] content), and overall mitochondrial function (mitochondrial membrane potential and intact-cell oxidative capacity). During linezolid-induced hyperlactatemia, we found extremely reduced protein expression (16% of the remaining content compared to control values [100%], P < 0.001) for the mitochondrially coded, transcribed, and translated COX-II subunit. Accordingly, COX activity was also found to be decreased (51% of the remaining activity, P < 0.05). These reductions were observed despite the numbers of COX-II mitochondrial RNA transcripts being abnormally increased (297%, P = 0.10 [not significant]) and the mitochondrial DNA content remaining stable. These abnormalities persisted even after the correction for mitochondrial mass, which was mildly decreased during the hyperlactatemic phase. Most of the mitochondrial abnormalities returned to control ranges after linezolid withdrawal, lactate normalization, and clinical recovery. Linezolid inhibits mitochondrial protein synthesis, leading to decreased mitochondrial enzymatic activity, which causes linezolid-related hyperlactatemia, which resolves upon discontinuation of linezolid treatment. PMID:17194826

  4. Genome-Wide Methylation and Gene Expression Changes in Newborn Rats following Maternal Protein Restriction and Reversal by Folic Acid

    PubMed Central

    Stupka, Elia; Clark, Adrian J. L.; Langley-Evans, Simon

    2013-01-01

    A large body of evidence from human and animal studies demonstrates that the maternal diet during pregnancy can programme physiological and metabolic functions in the developing fetus, effectively determining susceptibility to later disease. The mechanistic basis of such programming is unclear but may involve resetting of epigenetic marks and fetal gene expression. The aim of this study was to evaluate genome-wide DNA methylation and gene expression in the livers of newborn rats exposed to maternal protein restriction. On day one postnatally, there were 618 differentially expressed genes and 1183 differentially methylated regions (FDR 5%). The functional analysis of differentially expressed genes indicated a significant effect on DNA repair/cycle/maintenance functions and of lipid, amino acid metabolism and circadian functions. Enrichment for known biological functions was found to be associated with differentially methylated regions. Moreover, these epigenetically altered regions overlapped genetic loci associated with metabolic and cardiovascular diseases. Both expression changes and DNA methylation changes were largely reversed by supplementing the protein restricted diet with folic acid. Although the epigenetic and gene expression signatures appeared to underpin largely different biological processes, the gene expression profile of DNA methyl transferases was altered, providing a potential link between the two molecular signatures. The data showed that maternal protein restriction is associated with widespread differential gene expression and DNA methylation across the genome, and that folic acid is able to reset both molecular signatures. PMID:24391732

  5. Rational design of ultrastable and reversibly photoswitchable fluorescent proteins for super-resolution imaging of the bacterial periplasm

    PubMed Central

    El Khatib, Mariam; Martins, Alexandre; Bourgeois, Dominique; Colletier, Jacques-Philippe; Adam, Virgile

    2016-01-01

    Phototransformable fluorescent proteins are central to several nanoscopy approaches. As yet however, there is no available variant allowing super-resolution imaging in cell compartments that maintain oxidative conditions. Here, we report the rational design of two reversibly switchable fluorescent proteins able to fold and photoswitch in the bacterial periplasm, rsFolder and rsFolder2. rsFolder was designed by hybridisation of Superfolder-GFP with rsEGFP2, and inherited the fast folding properties of the former together with the rapid switching of the latter, but at the cost of a reduced switching contrast. Structural characterisation of the switching mechanisms of rsFolder and rsEGFP2 revealed different scenarios for chromophore cis-trans isomerisation and allowed designing rsFolder2, a variant of rsFolder that exhibits improved switching contrast and is amenable to RESOLFT nanoscopy. The rsFolders can be efficiently expressed in the E. coli periplasm, opening the door to the nanoscale investigation of proteins localised in hitherto non-observable cellular compartments. PMID:26732634

  6. NRG1, a repressor of filamentous growth in C.albicans, is down-regulated during filament induction

    PubMed Central

    Braun, Burkhard R.; Kadosh, David; Johnson, Alexander D.

    2001-01-01

    In response to a variety of external signals, the fungal pathogen Candida albicans undergoes a transition between ellipsoidal single cells (blastospores) and filaments composed of elongated cells attached end-to-end. Here we identify a DNA-binding protein, Nrg1, that represses filamentous growth in Candida probably by acting through the co-repressor Tup1. nrg1 mutant cells are predominantly filamentous under non-filament-inducing conditions and their colony morphology resembles that of tup1 mutants. We also identify two filament-specific genes, ECE1 and HWP1, whose transcription is repressed by Nrg1 under non-inducing conditions. These genes constitute a subset of those under Tup1 control, providing further evidence that Nrg1 acts by recruiting Tup1 to target genes. We show that growth in serum at 37°C, a potent inducer of filamentous growth, causes a reduction of NRG1 mRNA, suggesting that filamentous growth is induced by the down-regulation of NRG1. Consistent with this idea, expression of NRG1 from a non-regulated promoter partially blocks the induction of filamentous growth. PMID:11532939

  7. Identification of proteins in Streptococcus pneumoniae by reverse vaccinology and genetic diversity of these proteins in clinical isolates.

    PubMed

    Argondizzo, Ana Paula Corrêa; da Mota, Fabio Faria; Pestana, Cristiane Pinheiro; Reis, Joice Neves; de Miranda, Antonio Basílio; Galler, Ricardo; Medeiros, Marco Alberto

    2015-02-01

    Streptococcus pneumoniae is a major cause of morbidity and mortality worldwide. Virulence-associated proteins common and conserved among all capsular types now represent the best strategy to combat pneumococcal infections. Our aim was to identify conserved targets in pneumococci that showed positive prediction for lipoprotein and extracellular subcellular location using bioinformatics programs and verify the distribution and the degree of conservation of these targets in pneumococci. These targets can be considered potential vaccine candidate to be evaluated in the future. A set of 13 targets were analyzed and confirmed the presence in all pneumococci tested. These 13 genes were highly conserved showing around >96 % of amino acid and nucleotide identity, but they were also present and show high identity in the closely related species Streptococcus mitis, Streptococcus oralis, and Streptococcus pseudopneumoniae. S. oralis clusters away from S. pneumoniae, while S. pseudopneumoniae and S. mitis cluster closer. The divergence between the selected targets was too small to be observed consistently in phylogenetic groups between the analyzed genomes of S. pneumoniae. The proteins analyzed fulfill two of the initial criteria of a vaccine candidate: targets are present in a variety of different pneumococci strains including different serotypes and are conserved among the samples evaluated.

  8. Complex Flare Dynamics Initiated by a Filament-Filament Interaction

    NASA Astrophysics Data System (ADS)

    Zhu, Chunming; Liu, Rui; Alexander, David; Sun, Xudong; McAteer, James

    2015-04-01

    We report on a filament eruption that led to a relatively rare filament-filament interaction event. The filaments were located at different heights above the same segment of a circular polarity inversion line (PIL) around a condensed leading sunspot. The onset of the eruption of the lower of the two filaments was accompanied by a simultaneous descent of the upper filament resulting in a convergence and direct interaction of the two filaments. The interaction led to the subsequent merger of the filaments into a single magnetically complex structure that erupted to create a large solar flare and an array of complex dynamical activity. A hard X-ray coronal source and an associated enhancement of hot plasma are observed at the interface between the two interacting filaments. These phenomena are related to the production of a small C flare and the subsequent development of a much stronger M flare. Magnetic loop shrinkage and descending dark voids were observed at different locations as part of the large flare energy release giving us a unique insight into these dynamic flare phenomena.

  9. Intermediate filaments: a role in epithelial polarity

    PubMed Central

    Oriolo, Andrea S.; Wald, Flavia A.; Ramsauer, Victoria P.; Salas, Pedro J.I.

    2007-01-01

    Intermediate filaments have long been considered mechanical components of the cell that provide resistance to deformation stress. Practical experimental problems, including insolubility, lack of good pharmacological antagonists, and the paucity of powerful genetic models, have handicapped the research of other functions. In single-layered epithelial cells, keratin intermediate filaments are cortical, either apically polarized or apico-lateral. This review analyzes phenotypes of genetic manipulations of simple epithelial cell keratins in mice and C. elegans that strongly suggest a role of keratins in apico-basal polarization and membrane traffic. Published evidence that intermediate filaments can act as scaffolds for proteins involved in membrane traffic and signaling is also discussed. Such a scaffolding function would generate a highly polarized compartment within the cytoplasm of simple epithelial cells. While in most cases mechanistic explanations for the keratin-null or overexpression phenotypes are still missing, it is hoped investigators will be encouraged to study these as yet poorly understood functions of intermediate filaments. PMID:17425955

  10. Separation of peptides and intact proteins by electrostatic repulsion reversed phase liquid chromatography.

    PubMed

    Gritti, Fabrice; Guiochon, Georges

    2014-12-29

    A new brand of BEH-C18 hybrid particles chemically bonded to a leash carrying an amine group permits the implementation of electrostatic repulsive interactions chromatography. Using columns packed with this material, the influence of the concentration of positive charges bonded to the BEH-C18 surface on the overloaded band profiles of a few positively charged peptides and proteins was investigated in the gradient elution mode. Three columns packed with endcapped BEH-C18 particles bonded with three different surface-charge densities (LOW, MEDIUM and HIGH) were used and compared with those provided by a column packed with non-doped, endcapped BEH-C18 particles. The surface concentrations of fixed charges in the LOW, MEDIUM and HIGH columns were estimated at 0.029, 0.050, and 0.064μmol/m(2), for example, about two orders of magnitude smaller than the surface density of bonded C18 chains (2.1μmol/m(2)). Three different mobile phase additives (0.1% v/v of trifluoro-acetic, phosphoric, and formic acid) were used to optimize the purification levels of proteins under different loading conditions. The weak ion-pairing ions (formate and phosphate) generate smaller retention but broader, more fronting band profiles than those eluted with a stronger ion-pairing ion (trifluoroactate). This effect is worse in the presence of fixed charges at the surface of the BEH-C18 particles. This was explained by an enhanced anti-Langmuirian adsorption behavior of the charged proteins in the presence of fixed surface charges. As the protein concentration increases in the bulk, so does the internal ionic strength, the electrostatic repulsive interactions weaken, and retention increases. Band fronting is mostly eliminated by replacing weak ion-pairing acids with TFA with which the adsorption isotherm remains weakly langmuirian. Faster but still complete gradient separation of insulin and myoglobin were achieved with the HIGH column than with the reference neutral column, despite a measurable

  11. Filament wound structure and method

    DOEpatents

    Dritt, William S.; Gerth, Howard L.; Knight, Jr., Charles E.; Pardue, Robert M.

    1977-01-01

    The present invention relates to a filament wound spherical structure comprising a plurality of filament band sets disposed about the surface of a mandrel with each band of each set formed of a continuous filament circumferentially wound about the mandrel a selected number of circuits and with each circuit of filament being wound parallel to and contiguous with an immediate previously wound circuit. Each filament band in each band set is wound at the same helix angle from the axis of revolution of the mandrel and all of the bands of each set are uniformly distributed about the mandrel circumference. The pole-to-equator wall thickness taper associated with each band set, as several contiguous band sets are wound about the mandrel starting at the poles, is accumulative as the band sets are nested to provide a complete filament wound sphere of essentially uniform thickness.

  12. A Digital Compendium of Genes Mediating the Reversible Phosphorylation of Proteins in Fe-Deficient Arabidopsis Roots

    PubMed Central

    Lan, Ping; Li, Wenfeng; Schmidt, Wolfgang

    2013-01-01

    Post-translational modifications of proteins such as reversible phosphorylation provide an important but understudied regulatory network that controls important nodes in the adaptation of plants to environmental conditions. Iron (Fe) is an essential mineral nutrient for plants, but due to its low solubility often a limiting factor for optimal growth. To understand the role of protein phosphorylation in the regulation of cellular Fe homeostasis, we analyzed the expression of protein kinases (PKs) and phosphatases (PPs) in Arabidopsis roots by mining differentially expressed PK and PP genes. Transcriptome analysis using RNA-seq revealed that subsets of 203 PK and 39 PP genes were differentially expressed under Fe-deficient conditions. Functional modules of these PK and PP genes were further generated based on co-expression analysis using the MACCU toolbox on the basis of 300 publicly available root-related microarray data sets. Results revealed networks comprising 87 known or annotated PK and PP genes that could be subdivided into one large and several smaller highly co-expressed gene modules. The largest module was composed of 58 genes, most of which have been assigned to the leucine-rich repeat protein kinase superfamily and associated with the biological processes “hypotonic salinity response,” “potassium ion import,” and “cellular potassium ion homeostasis.” The comprehensive transcriptional information on PK and PP genes in iron-deficient roots provided here sets the stage for follow-up experiments and contributes to our understanding of the post-translational regulation of Fe deficiency and potassium ion homeostasis. PMID:23761801

  13. Predicting Solar Filament Eruptions with HEK Filament Metadata

    NASA Astrophysics Data System (ADS)

    Aggarwal, A.; Reeves, K.; Schanche, N.

    2015-12-01

    Solar filaments are cool, dark channels of partially-ionized plasma that lie above the chromosphere. Their structure follows the neutral line between local regions of opposite magnetic polarity. Previous research (e.g. Schmieder et al. 2013) has shown a positive correlation (80%) between the occurrence of filament eruptions and coronal mass ejections (CME's). If certain filament properties, such as length, chirality, and tilt, indicate a tendency towards filament eruptions, one may be able to further predict an oncoming CME. Towards this end, we present a novel algorithm based on spatiotemporal analysis that systematically correlates filament eruptions documented in the Heliophysics Event Knowledgebase (HEK) with HEK filaments that have been grouped together using a tracking algorithm developed at Georgia State University (e.g. Kempton et al. 2014). We also find filament tracks that are not correlated with eruptions to form a null data set in a similar fashion. Finally, we compare the metadata from erupting and non-erupting filament tracks to discover which filament properties may present signs of an eruption onset. Through statistical methods such as the two-sample Kolmogorov-Smirnov test and Random Forest Classifier, we find that a filament that is increasing in length or changing in tilt with respect to the equator may be a useful gauge to predict a filament eruption. However, the average values of length and tilt for both datasets follow similar distributions, leading us to conclude that these parameters do not indicate an eruption event. This work is supported by the NSF-REU solar physics program at SAO, grant number AGS-1263241, and NSF DIBBS grant number ACI-1443061.

  14. β-Carotene can reverse dysregulation of iron protein in an in vitro model of inflammation.

    PubMed

    Katz, Oksana; Reifen, Ram; Lerner, Aaron

    2015-02-01

    Anemia of chronic disease is frequently seen in chronic inflammatory conditions. Its hallmark is disrupted iron homeostasis, with increased uptake and retention of iron in cells of the reticuloendothelial system. Using the Caco-2 cell line as an in vitro model for iron absorption, local intestinal iron-related protein dynamics were evaluated during interleukin (IL)1β/iron-induced inflammation, confirmed by IL8 release, and following β-carotene and vitamin A supplementation. Time- and dose-dependent iron administration to the cells was then studied. The effects on heavy and light ferritin, ferroportin, transferrin receptor and intracellular iron levels were compared in inflamed Caco-2 cells with and without application of the anti-inflammatory agents β-carotene and vitamin A. IL1β treatment led to IL8 release, a surge in both ferritins' expressions and suppression of ferroportin and transferrin receptor expression. β-Carotene significantly reduced IL8 (1,306.2-253.75 pg/ml), decreased light and heavy ferritin by 77.8 and 45.8%, respectively, and increased ferroportin by 59.9% (P < 0.05). Increasing iron concentrations and incubation periods resulted in increased IL8 release. A strong correlation was found between the levels of IL8 and the ferritins. Intracellular iron sequestration was induced by IL1β and iron and alleviated by β-carotene. β-Carotene normalized the main iron-related proteins' levels, reduced IL8 production, and released intracellular trapped iron. These results highlight local mucosal control of iron regulation and suggest that by applying anti-inflammatory compounds, less iron is locked in inflamed intestinal epithelial cells, leading to its increased bioavailability. This suggests a possible approach to combating anemia associated with chronic inflammatory conditions.

  15. A Comprehensive Comparison of Normalization Methods for Loading Control and Variance Stabilization of Reverse-Phase Protein Array Data

    PubMed Central

    Liu, Wenbin; Ju, Zhenlin; Lu, Yiling; Mills, Gordon B; Akbani, Rehan

    2014-01-01

    Loading control (LC) and variance stabilization of reverse-phase protein array (RPPA) data have been challenging mainly due to the small number of proteins in an experiment and the lack of reliable inherent control markers. In this study, we compare eight different normalization methods for LC and variance stabilization. The invariant marker set concept was first applied to the normalization of high-throughput gene expression data. A set of “invariant” markers are selected to create a virtual reference sample. Then all the samples are normalized to the virtual reference. We propose a variant of this method in the context of RPPA data normalization and compare it with seven other normalization methods previously reported in the literature. The invariant marker set method performs well with respect to LC, variance stabilization and association with the immunohistochemistry/florescence in situ hybridization data for three key markers in breast tumor samples, while the other methods have inferior performance. The proposed method is a promising approach for improving the quality of RPPA data. PMID:25374453

  16. A comprehensive comparison of normalization methods for loading control and variance stabilization of reverse-phase protein array data.

    PubMed

    Liu, Wenbin; Ju, Zhenlin; Lu, Yiling; Mills, Gordon B; Akbani, Rehan

    2014-01-01

    Loading control (LC) and variance stabilization of reverse-phase protein array (RPPA) data have been challenging mainly due to the small number of proteins in an experiment and the lack of reliable inherent control markers. In this study, we compare eight different normalization methods for LC and variance stabilization. The invariant marker set concept was first applied to the normalization of high-throughput gene expression data. A set of "invariant" markers are selected to create a virtual reference sample. Then all the samples are normalized to the virtual reference. We propose a variant of this method in the context of RPPA data normalization and compare it with seven other normalization methods previously reported in the literature. The invariant marker set method performs well with respect to LC, variance stabilization and association with the immunohistochemistry/florescence in situ hybridization data for three key markers in breast tumor samples, while the other methods have inferior performance. The proposed method is a promising approach for improving the quality of RPPA data.

  17. Reovirus type 3 synthesizes proteins in interferon-treated HeLa cells without reversing the antiviral state.

    PubMed

    Feduchi, E; Esteban, M; Carrasco, L

    1988-06-01

    Treatment of HeLa cells with human lymphoblastoid interferon (IFN-alpha) does not inhibit reovirus type 3 protein synthesis during virus infection. In contrast, reovirus translation is blocked by treatment of L cells with mouse IFN-alpha. The (2'-5')A synthetase activity is induced in HeLa cells by IFN-alpha treatment and is activated after reovirus infection, since cell lysates from these cells synthesize in vitro (2'-5')A oligonucleotides. The IFN-induced protein kinase activity is also triggered in those lysates upon dsRNA addition. Thus, contrary to DNA-containing viruses, such as vaccinia virus or adenovirus, reovirus infection does not destroy or reverse the IFN-induced antiviral state. In support of this conclusion, superinfection with poliovirus or vesicular stomatitis virus of reovirus-infected HeLa cells treated with IFN leads only to a blockade of translation of the former viruses. These results provide a remarkable example where in the same cells doubly infected with two different viruses, the antiviral state induced by IFN-alpha is manifested by selectively inhibiting translation of one kind of virus (poliovirus or vesicular stomatitis virus) without affecting the translation of reovirus type 3. In addition, these results indicate that the resistance of reovirus translation to inhibition by IFN is different from the mechanism of resistance induced by DNA-containing viruses.

  18. Reverse-phase HPLC separation of hemp seed (Cannabis sativa L.) protein hydrolysate produced peptide fractions with enhanced antioxidant capacity.

    PubMed

    Girgih, Abraham T; Udenigwe, Chibuike C; Aluko, Rotimi E

    2013-03-01

    Hemp seed protein hydrolysate (HPH) was produced through simulated gastrointestinal tract (GIT) digestion of hemp seed protein isolate followed by partial purification and separation into eight peptide fractions by reverse-phase (RP)-HPLC. The peptide fractions exhibited higher oxygen radical absorbance capacity as well as scavenging of 2,2-diphenyl-1-picrylhydrazyl, superoxide and hydroxyl radicals when compared to HPH. Radical scavenging activities of the fractionated peptides increased as content of hydrophobic amino acids or elution time was increased, with the exception of hydroxyl radical scavenging that showed decreased trend. Glutathione (GSH), HPH and the RP-HPLC peptide fractions possessed low ferric ion reducing ability but all had strong (>60 %) metal chelating activities. Inhibition of linoleic acid oxidation by some of the HPH peptide fractions was higher at 1 mg/ml when compared to that observed at 0.1 mg/ml peptide concentration. Peptide separation resulted in higher concentration of some hydrophobic amino acids (especially proline, leucine and isoleucine) in the fractions (mainly F5 and F8) when compared to HPH. The elution time-dependent increased concentrations of the hydrophobic amino acids coupled with decreased levels of positively charged amino acids may have been responsible for the significantly higher (p < 0.05) antioxidant properties observed for some of the peptide fractions when compared to the unfractionated HPH. In conclusion, the antioxidant activity of HPH after simulated GIT digestion is mainly influenced by the amino acid composition of some of its peptides.

  19. Mechanism of interaction of Dictyostelium severin with actin filaments

    PubMed Central

    1982-01-01

    Severin, a 40,000-dalton protein from Dictyostelium that disassembles actin filaments in a Ca2+ -dependent manner, was purified 500-fold to greater than 99% homogeneity by modifications of the procedure reported by Brown, Yamamoto, and Spudich (1982. J. Cell Biol. 93:205-210). Severin has a Stokes radius of 29 A and consists of a single polypeptide chain. It contains a single methionyl and five cysteinyl residues. We studied the action of severin on actin filaments by electron microscopy, viscometry, sedimentation, nanosecond emission anisotropy, and fluorescence energy transfer spectroscopy. Nanosecond emission anisotropy of fluoresence-labeled severin shows that this protein changes its conformation on binding Ca2+. Actin filaments are rapidly fragmented on addition of severin and Ca2+, but severin does not interact with actin filaments in the absence of Ca2+. Fluorescence energy transfer measurements indicate that fragmentation of actin filaments by severin leads to a partial depolymerization (t1/2 approximately equal to 30 s). Depolymerization is followed by exchange of a limited number of subunits in the filament fragments with the disassembled actin pool (t1/2 approximately equal to 5 min). Disassembly and exchange are probably restricted to the ends of the filament fragments since only a few subunits in each fragment participate in the disassembly or exchange process. Steady state hydrolysis of ATP by actin in the presence of Ca2+-severin is maximal at an actin: severin molar ratio of approximately 10:1, which further supports the inference that subunit exchange is limited to the ends of actin filaments. The observation of sequential depolymerization and subunit exchange following the fragmentation of actin by severin suggests that severin may regulate site-specific disassembly and turnover of actin filament arrays in vivo. PMID:6897549

  20. Bending artificial muscle from nylon filaments

    NASA Astrophysics Data System (ADS)

    Mirvakili, Seyed M.; Hunter, Ian W.

    2016-04-01

    Highly oriented nylon and polyethylene fibers shrink in length and expand in diameter when heated. Using this property, in this work, for the first time we are introducing a type of bending artificial muscle from nylon filaments such as fishing line. Reversible radius of curvature of 0.23 mm-1 was achieved with maximum reversible bending amplitude of 115 mm for the nylon bending actuator. Peak force of up to 2040 mN was measured with a catch-state force of up to 40% of the active force. A 3 dB roll-off frequency of around 0.7 Hz was observed in the frequency response of the bending actuator in water.

  1. Current filamentation model for the Weibel/Filamentation instabilities

    NASA Astrophysics Data System (ADS)

    Ryu, Chang-Mo; Huynh, Cong Tuan; Kim, Chul Min

    2016-10-01

    A current filamentaion model for a nonrelativistic plasma with e +/e- beam has been presented together with PIC simulations, which can explain the mangetic field enhancement during the Weibel/ Filamentation instabilities. This filament model assumes the Hammer-Rostoker equilibrium. In addition, this model predicts preferential acceleration/deceleration for electron-ion plasmas depending on the injected beam to be e +/e-.

  2. Actin filament-associated protein 1 (AFAP-1) is a key mediator in inflammatory signaling-induced rapid attenuation of intrinsic P-gp function in human brain capillary endothelial cells.

    PubMed

    Hoshi, Yutaro; Uchida, Yasuo; Tachikawa, Masanori; Ohtsuki, Sumio; Terasaki, Tetsuya

    2017-01-23

    The purpose of this study was to identify regulatory molecule(s) involved in the inflammatory signaling-induced decrease in P-glycoprotein (P-gp) efflux function at the blood-brain barrier (BBB) that may occur in brain diseases. We confirmed that in vivo P-gp efflux activity at the BBB was decreased without any change in P-gp protein expression level in a mouse model of acute inflammation induced by 3 mg/kg lipopolysaccharide. In a human BBB model cell line (human brain capillary endothelial cells; hCMEC/D3), 1-h treatment with 10 ng/mL tumor necrosis factor-α (TNF-α; an inflammatory mediator) rapidly reduced P-gp efflux activity, but had no effect on P-gp protein expression level. To clarify the non-transcriptional mechanism that causes the decrease in intrinsic efflux activity of P-gp in acute inflammation, we applied comprehensive quantitative phosphoproteomics to compare hCMEC/D3 cells treated with TNF-α and vehicle (control). Actin filament-associated protein-1 (AFAP-1), MAPK1, and transcription factor AP-1 (AP-1) were significantly phosphorylated in TNF-α-treated cells, and were selected as candidate proteins. In validation experiments, knockdown of AFAP-1 expression blocked the reduction in P-gp efflux activity by TNF-α treatment, whereas inhibition of MAPK function or knockdown of AP-1 expression did not. Quantitative targeted absolute proteomics revealed that the reduction in P-gp activity by TNF-α did not require any change in P-gp protein expression levels in the plasma membrane. Our results demonstrate that AFAP-1 is a key mediator in the inflammatory signaling-induced, translocation-independent rapid attenuation of P-gp efflux activity in human brain capillary endothelial cells.

  3. Profound Reversible Hypogammaglobulinemia Caused by Celiac Disease in the Absence of Protein Losing Enteropathy.

    PubMed

    Ameratunga, Rohan; Barker, Russell William; Steele, Richard Henderson; Deo, Maneka; Woon, See-Tarn; Yeong, Mee Ling; Koopmans, Wikke

    2015-08-01

    When patients with hypogammaglobulinemia are encountered, a vigorous search should be undertaken for secondary treatable causes. Here we describe the first case of a patient with severe asymptomatic hypogammaglobulinemia where the underlying cause was undiagnosed celiac disease. A strict gluten free diet resulted in resolution of her mild long-standing abdominal symptoms and correction of her hypogammaglobulinemia. There was corresponding improvement in her duodenal histology and normalisation of her celiac serology. Protein losing enteropathy was unlikely to have been the mechanism of her profound hypogammaglobulinemia, as her albumin was within the normal range and she had a normal fecal alpha 1 antitrypsin level. Application of the Ameratunga et al. (2013) diagnostic criteria was helpful in confirming this patient did not have Common Variable Immunodeficiency Disorder (CVID). Celiac disease must now be considered in the differential diagnosis of severe hypogammaglobulinemia. There should be a low threshold for undertaking celiac serology in patients with hypogammaglobulinemia, even if they have minimal symptoms attributable to gut disease.

  4. Structure-guided optimization of protein kinase inhibitors reverses aminoglycoside antibiotic resistance

    PubMed Central

    Stogios, Peter J.; Spanogiannopoulos, Peter; Evdokimova, Elena; Egorova, Olga; Shakya, Tushar; Todorovic, Nick; Capretta, Alfredo; Wright, Gerard D.; Savchenko, Alexei

    2013-01-01

    SYNOPSIS Activity of the aminoglycoside phosphotransferase APH(3’)-Ia leads to resistance to aminoglycoside antibiotics in pathogenic Gram-negative bacteria, and contributes to the clinical obsolescence of this class of antibiotics. One strategy to rescue compromised antibiotics such as aminoglycosides is targeting the enzymes that confer resistance with small molecules. Previously we demonstrated that eukaryotic protein kinase (ePK) inhibitors could inhibit APH enzymes, due to the structural similarity between these two enzyme families. However, limited structural information of enzyme-inhibitor complexes hindered interpretation of the results. As well, cross-reactivity of compounds between APHs and ePKs represents an obstacle to their use as aminoglycoside adjuvants to rescue aminoglycoside antibiotic activity. Here, we structurally and functionally characterize inhibition of APH(3’)-Ia by three diverse chemical scaffolds – anthrapyrazolone, 4-anilinoquinazoline and pyrazolopyrimidine (PP) – and reveal distinctions in the binding mode of anthrapyrazolone and PP compounds to APH(3’)-Ia versus ePKs. Using this observation, we identify PP-derivatives that select against ePKs, attenuate APH(3’)-Ia activity and rescue aminoglycoside antibiotic activity against a resistant E. coli strain. The structures presented here and these inhibition studies provide an important opportunity for structure-based design of compounds to target aminoglycoside phosphotransferases for inhibition, potentially overcoming this form of antibiotic resistance. PMID:23758273

  5. Rapamycin reverses cellular phenotypes and enhances mutant protein clearance in Hutchinson-Gilford progeria syndrome cells.

    PubMed

    Cao, Kan; Graziotto, John J; Blair, Cecilia D; Mazzulli, Joseph R; Erdos, Michael R; Krainc, Dimitri; Collins, Francis S

    2011-06-29

    Hutchinson-Gilford progeria syndrome (HGPS) is a lethal genetic disorder characterized by premature aging. HGPS is most commonly caused by a de novo single-nucleotide substitution in the lamin A/C gene (LMNA) that partially activates a cryptic splice donor site in exon 11, producing an abnormal lamin A protein termed progerin. Accumulation of progerin in dividing cells adversely affects the integrity of the nuclear scaffold and leads to nuclear blebbing in cultured cells. Progerin is also produced in normal cells, increasing in abundance as senescence approaches. Here, we report the effect of rapamycin, a macrolide antibiotic that has been implicated in slowing cellular and organismal aging, on the cellular phenotypes of HGPS fibroblasts. Treatment with rapamycin abolished nuclear blebbing, delayed the onset of cellular senescence, and enhanced the degradation of progerin in HGPS cells. Rapamycin also decreased the formation of insoluble progerin aggregates and induced clearance through autophagic mechanisms in normal fibroblasts. Our findings suggest an additional mechanism for the beneficial effects of rapamycin on longevity and encourage the hypothesis that rapamycin treatment could provide clinical benefit for children with HGPS.

  6. Reversible dimerization of Aequorea victoria fluorescent proteins increases the dynamic range of FRET-based indicators.

    PubMed

    Kotera, Ippei; Iwasaki, Takuya; Imamura, Hiromi; Noji, Hiroyuki; Nagai, Takeharu

    2010-02-19

    Fluorescent protein (FP)-based Forster resonance energy transfer (FRET) technology is useful for development of functional indicators to visualize second messenger molecules and activation of signaling components in living cells. However, the design and construction of the functional indicators require careful optimization of their structure at the atomic level. Therefore, routine procedures for constructing FRET-based indicators currently include the adjustment of the linker length between the FPs and the sensor domain and relative dipole orientation of the FP chromophore. Here we report that, in addition to these techniques, optimization of the dimerization interface of Aequorea FPs is essential to achieve the highest possible dynamic range of signal change by FRET-based indicators. We performed spectroscopic analyses of various indicators (cameleon, TN-XL, and ATeam) and their variants. We chose variants containing mutant FPs with different dimerization properties, i.e., no, weak, or enhanced dimerization of the donor or acceptor FP. Our findings revealed that the FPs that dimerized weakly yielded high-performance FRET-based indicators with the greatest dynamic range.

  7. Structure-guided optimization of protein kinase inhibitors reverses aminoglycoside antibiotic resistance.

    PubMed

    Stogios, Peter J; Spanogiannopoulos, Peter; Evdokimova, Elena; Egorova, Olga; Shakya, Tushar; Todorovic, Nick; Capretta, Alfredo; Wright, Gerard D; Savchenko, Alexei

    2013-09-01

    Activity of the aminoglycoside phosphotransferase APH(3')-Ia leads to resistance to aminoglycoside antibiotics in pathogenic Gram-negative bacteria, and contributes to the clinical obsolescence of this class of antibiotics. One strategy to rescue compromised antibiotics such as aminoglycosides is targeting the enzymes that confer resistance with small molecules. We demonstrated previously that ePK (eukaryotic protein kinase) inhibitors could inhibit APH enzymes, owing to the structural similarity between these two enzyme families. However, limited structural information of enzyme-inhibitor complexes hindered interpretation of the results. In addition, cross-reactivity of compounds between APHs and ePKs represents an obstacle to their use as aminoglycoside adjuvants to rescue aminoglycoside antibiotic activity. In the present study, we structurally and functionally characterize inhibition of APH(3')-Ia by three diverse chemical scaffolds, anthrapyrazolone, 4-anilinoquinazoline and PP (pyrazolopyrimidine), and reveal distinctions in the binding mode of anthrapyrazolone and PP compounds to APH(3')-Ia compared with ePKs. Using this observation, we identify PP derivatives that select against ePKs, attenuate APH(3')-Ia activity and rescue aminoglycoside antibiotic activity against a resistant Escherichia coli strain. The structures described in the present paper and the inhibition studies provide an important opportunity for structure-based design of compounds to target aminoglycoside phosphotransferases for inhibition, potentially overcoming this form of antibiotic resistance.

  8. Cofilin cooperates with fascin to disassemble filopodial actin filaments

    PubMed Central

    Breitsprecher, Dennis; Koestler, Stefan A.; Chizhov, Igor; Nemethova, Maria; Mueller, Jan; Goode, Bruce L.; Small, J. Victor; Rottner, Klemens; Faix, Jan

    2011-01-01

    Cells use a large repertoire of proteins to remodel the actin cytoskeleton. Depending on the proteins involved, F-actin is organized in specialized protrusions such as lamellipodia or filopodia, which serve diverse functions in cell migration and sensing. Although factors responsible for directed filament assembly in filopodia have been extensively characterized, the mechanisms of filament disassembly in these structures are mostly unknown. We investigated how the actin-depolymerizing factor cofilin-1 affects the dynamics of fascincrosslinked actin filaments in vitro and in live cells. By multicolor total internal reflection fluorescence microscopy and fluorimetric assays, we found that cofilin-mediated severing is enhanced in fascin-crosslinked bundles compared with isolated filaments, and that fascin and cofilin act synergistically in filament severing. Immunolabeling experiments demonstrated for the first time that besides its known localization in lamellipodia and membrane ruffles, endogenous cofilin can also accumulate in the tips and shafts of filopodia. Live-cell imaging of fluorescently tagged proteins revealed that cofilin is specifically targeted to filopodia upon stalling of protrusion and during their retraction. Subsequent electron tomography established filopodial actin filament and/or bundle fragmentation to precisely correlate with cofilin accumulation. These results identify a new mechanism of filopodium disassembly involving both fascin and cofilin. PMID:21940796

  9. Tuning the Binding Affinities and Reversion Kinetics of a Light Inducible Dimer Allows Control of Transmembrane Protein Localization.

    PubMed

    Zimmerman, Seth P; Hallett, Ryan A; Bourke, Ashley M; Bear, James E; Kennedy, Matthew J; Kuhlman, Brian

    2016-09-20

    Inducible dimers are powerful tools for controlling biological processes through colocalizing signaling molecules. To be effective, an inducible system should have a dissociation constant in the "off" state that is greater (i.e., weaker affinity) than the concentrations of the molecules that are being controlled, and in the "on" state a dissociation constant that is less (i.e., stronger affinity) than the relevant protein concentrations. Here, we reengineer the interaction between the light inducible dimer, iLID, and its binding partner SspB, to better control proteins present at high effective concentrations (5-100 μM). iLID contains a light-oxygen-voltage (LOV) domain that undergoes a conformational change upon activation with blue light and exposes a peptide motif, ssrA, that binds to SspB. The new variant of the dimer system contains a single SspB point mutation (A58V), and displays a 42-fold change in binding affinity when activated with blue light (from 3 ± 2 μM to 125 ± 40 μM) and allows for light-activated colocalization of transmembrane proteins in neurons, where a higher affinity switch (0.8-47 μM) was less effective because more colocalization was seen in the dark. Additionally, with a point mutation in the LOV domain (N414L), we lengthened the reversion half-life of iLID. This expanded suite of light induced dimers increases the variety of cellular pathways that can be targeted with light.

  10. Prp8, the pivotal protein of the spliceosomal catalytic center, evolved from a retroelement-encoded reverse transcriptase

    PubMed Central

    Dlakić, Mensur; Mushegian, Arcady

    2011-01-01

    Prp8 is the largest and most highly conserved protein of the spliceosome, encoded by all sequenced eukaryotic genomes but missing from prokaryotes and viruses. Despite all evidence that Prp8 is an integral part of the spliceosomal catalytic center, much remains to be learned about its molecular functions and evolutionary origin. By analyzing sequence and structure similarities between Prp8 and other protein domains, we show that its N-terminal region contains a putative bromodomain. The central conserved domain of Prp8 is related to the catalytic domain of reverse transcriptases (RTs) and is most similar to homologous enzymes encoded by prokaryotic retroelements. However, putative catalytic residues in this RT domain are only partially conserved and may not be sufficient for the nucleotidyltransferase activity. The RT domain is followed by an uncharacterized sequence region with relatives found in fungal RT-like proteins. This part of Prp8 is predicted to adopt an α-helical structure and may be functionally equivalent to diverse maturase/X domains of retroelements and to the thumb domain of retroviral RTs. Together with a previously identified C-terminal domain that has an RNaseH-like fold, our results suggest evolutionary connections between Prp8 and ancient mobile elements. Prp8 may have evolved by acquiring nucleic acid–binding domains from inactivated retroelements, and their present-day role may be in maintaining proper conformation of the bound RNA cofactors and substrates of the splicing reaction. This is only the second example—the other one being telomerase—of the RT recruitment from a genomic parasite to serve an essential cellular function. PMID:21441348

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

    PubMed Central

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

    2017-01-01

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

  12. Treatment with the matricellular protein CCN3 blocks and/or reverses fibrosis development in obesity with diabetic nephropathy.

    PubMed

    Riser, Bruce L; Najmabadi, Feridoon; Garchow, Kendra; Barnes, Jeffrey L; Peterson, Darryl R; Sukowski, Ernest J

    2014-11-01

    Fibrosis is at the core of the high morbidity and mortality rates associated with the complications of diabetes and obesity, including diabetic nephropathy (DN), without any US Food and Drug Administration-approved drugs with this specific target. We recently provided the first evidence that the matricellular protein CCN3 (official symbol NOV) functions in a reciprocal manner, acting on the profibrotic family member CCN2 to inhibit fibrosis in a mesangial cell model of DN. Herein, we used the BT/BR ob/ob mouse as a best model of human obesity and DN progression to determine whether recombinant human CCN3 could be used therapeutically, and the mechanisms involved. Eight weeks of thrice-weekly i.p. injections (0.604 and 6.04 μg/kg of recombinant human CCN3) beginning in early-stage DN completely blocked and/or reversed the up-regulation of mRNA expression of kidney cortex fibrosis genes (CCN2, Col1a2, TGF-β1, and PAI-1) seen in placebo-treated diabetic mice. The treatment completely blocked glomerular fibrosis, as determined by altered mesangial expansion and deposition of laminin. Furthermore, it protected against, or reversed, podocyte loss and kidney function reduction (rise in plasma creatinine concentration); albuminuria was also greatly reduced. This study demonstrates the potential efficacy of recombinant human CCN3 treatment in DN and points to mechanisms operating at multiple levels or pathways, upstream (eg, protecting against cell injury) and downstream (eg, regulating CCN2 activity and extracellular matrix metabolism).

  13. TubZ filament assembly dynamics requires the flexible C-terminal tail

    PubMed Central

    Fuentes-Pérez, Maria E.; Núñez-Ramírez, Rafael; Martín-González, Alejandro; Juan-Rodríguez, David; Llorca, Oscar; Moreno-Herrero, Fernando; Oliva, Maria A.

    2017-01-01

    Cytomotive filaments are essential for the spatial organization in cells, showing a dynamic behavior based on nucleotide hydrolysis. TubZ is a tubulin-like protein that functions in extrachromosomal DNA movement within bacteria. TubZ filaments grow in a helical fashion following treadmilling or dynamic instability, although the underlying mechanism is unclear. We have unraveled the molecular basis for filament assembly and dynamics combining electron and atomic force microscopy and biochemical analyses. Our findings suggest that GTP caps retain the filament helical structure and hydrolysis triggers filament stiffening upon disassembly. We show that the TubZ C-terminal tail is an unstructured domain that fulfills multiple functions contributing to the filament helical arrangement, the polymer remodeling into tubulin-like rings and the full disassembly process. This C-terminal tail displays the binding site for partner proteins and we report how it modulates the interaction of the regulator protein TubY. PMID:28230082

  14. TubZ filament assembly dynamics requires the flexible C-terminal tail.

    PubMed

    Fuentes-Pérez, Maria E; Núñez-Ramírez, Rafael; Martín-González, Alejandro; Juan-Rodríguez, David; Llorca, Oscar; Moreno-Herrero, Fernando; Oliva, Maria A

    2017-02-23

    Cytomotive filaments are essential for the spatial organization in cells, showing a dynamic behavior based on nucleotide hydrolysis. TubZ is a tubulin-like protein that functions in extrachromosomal DNA movement within bacteria. TubZ filaments grow in a helical fashion following treadmilling or dynamic instability, although the underlying mechanism is unclear. We have unraveled the molecular basis for filament assembly and dynamics combining electron and atomic force microscopy and biochemical analyses. Our findings suggest that GTP caps retain the filament helical structure and hydrolysis triggers filament stiffening upon disassembly. We show that the TubZ C-terminal tail is an unstructured domain that fulfills multiple functions contributing to the filament helical arrangement, the polymer remodeling into tubulin-like rings and the full disassembly process. This C-terminal tail displays the binding site for partner proteins and we report how it modulates the interaction of the regulator protein TubY.

  15. Realizing the Promise of Reverse Phase Protein Arrays for Clinical, Translational, and Basic Research: A Workshop Report

    PubMed Central

    Akbani, Rehan; Becker, Karl-Friedrich; Carragher, Neil; Goldstein, Ted; de Koning, Leanne; Korf, Ulrike; Liotta, Lance; Mills, Gordon B.; Nishizuka, Satoshi S.; Pawlak, Michael; Petricoin, Emanuel F.; Pollard, Harvey B.; Serrels, Bryan; Zhu, Jingchun

    2014-01-01

    Reverse phase protein array (RPPA) technology introduced a miniaturized “antigen-down” or “dot-blot” immunoassay suitable for quantifying the relative, semi-quantitative or quantitative (if a well-accepted reference standard exists) abundance of total protein levels and post-translational modifications across a variety of biological samples including cultured cells, tissues, and body fluids. The recent evolution of RPPA combined with more sophisticated sample handling, optical detection, quality control, and better quality affinity reagents provides exquisite sensitivity and high sample throughput at a reasonable cost per sample. This facilitates large-scale multiplex analysis of multiple post-translational markers across samples from in vitro, preclinical, or clinical samples. The technical power of RPPA is stimulating the application and widespread adoption of RPPA methods within academic, clinical, and industrial research laboratories. Advances in RPPA technology now offer scientists the opportunity to quantify protein analytes with high precision, sensitivity, throughput, and robustness. As a result, adopters of RPPA technology have recognized critical success factors for useful and maximum exploitation of RPPA technologies, including the following: preservation and optimization of pre-analytical sample quality,application of validated high-affinity and specific antibody (or other protein affinity) detection reagents,dedicated informatics solutions to ensure accurate and robust quantification of protein analytes, andquality-assured procedures and data analysis workflows compatible with application within regulated clinical environments. In 2011, 2012, and 2013, the first three Global RPPA workshops were held in the United States, Europe, and Japan, respectively. These workshops provided an opportunity for RPPA laboratories, vendors, and users to share and discuss results, the latest technology platforms, best practices, and future challenges and

  16. Metabolomics protocols for filamentous fungi.

    PubMed

    Gummer, Joel P A; Krill, Christian; Du Fall, Lauren; Waters, Ormonde D C; Trengove, Robert D; Oliver, Richard P; Solomon, Peter S

    2012-01-01

    Proteomics and transcriptomics are established functional genomics tools commonly used to study filamentous fungi. Metabolomics has recently emerged as another option to complement existing techniques and provide detailed information on metabolic regulation and secondary metabolism. Here, we describe broad generic protocols that can be used to undertake metabolomics studies in filamentous fungi.

  17. Magnetic Fields in Massive Filaments

    NASA Astrophysics Data System (ADS)

    Pillai, Thushara

    Magnetic fields pervade galaxies, shaping them from the largest scales to the smallest star forming scales. A firm understanding of their role is crucial to our understanding of the physics of ISM. A dominant phase of the ISM that has received considerable attention is that of filaments which are ubiquitous and dominate the mass reservoir in molecular clouds. Enormous progress has been made recently towards understanding filament properties. The next major step should be to understand the role of magnetic fields in filaments. We propose to take advantage of HAWC+ dust emission polarimeter now available on SOFIA to launch a pilot polarization study towards three major classes of filaments: (i) Pristine (ii) Hub-Filament systems and (iii) Perturbed. HAWC+ will trace the connection between the star forming cores and the filaments enveloping them. By covering a vast range in parameter space from quiescent to active filaments, we will be constraining the initial conditions prior to star formation, during star formation and after star formation (feedback from newly formed stars on their parent clouds.) The interpretation of observations will be supported by extensive custom-made numerical simulations of magnetized clouds and subsequent dust radiative transfer with various grain alignment mechanisms, as provided by collaborators. Combined, these observations will provide the first panoramic view of the magnetized nature of massive filaments in the ISM.

  18. Magnetic Fields in Massive Filaments

    NASA Astrophysics Data System (ADS)

    Pillai, G. S. Thushara

    2015-10-01

    Magnetic fields pervade galaxies, shaping them from the largest scales to the smallest star forming scales. A firm understanding of their role is crucial to our understanding of the physics of ISM. A dominant phase of the ISM that has received considerable attention is that of filaments which are ubiquitous and dominate the mass reservoir in molecular clouds. Enormous progress has been made recently towards understanding filament properties. The next major step should be to understand the role of magnetic fields in filaments. We propose to take advantage of HAWC+ dust emission polarimeter now available on SOFIA to launch a pilot polarization study towards three major classes of filaments: (i) Pristine (ii) Hub-Filament systems and (iii) Perturbed. HAWC+ will trace the connection between the star forming cores and the filaments enveloping them. By covering a vast range in parameter space from quiescent to active filaments, we will be constraining the initial conditions prior to star formation, during star formation and after star formation (feedback from newly formed stars on their parent clouds.) The interpretation of observations will be supported by extensive custom--made numerical simulations of magnetized clouds and subsequent dust radiative transfer with various grain alignment mechanisms, as provided by collaborators. Combined, these observations will provide the first panoramic view of the magnetized nature of massive filaments in the ISM.

  19. Bundling of actin filaments by elongation factor 1 alpha inhibits polymerization at filament ends

    PubMed Central

    1996-01-01

    Elongation factor 1 alpha (EF1 alpha) is an abundant protein that binds aminoacyl-tRNA and ribosomes in a GTP-dependent manner. EF1 alpha also interacts with the cytoskeleton by binding and bundling actin filaments and microtubules. In this report, the effect of purified EF1 alpha on actin polymerization and depolymerization is examined. At molar ratios present in the cytosol, EF1 alpha significantly blocks both polymerization and depolymerization of actin filaments and increases the final extent of actin polymer, while at high molar ratios to actin, EF1 alpha nucleates actin polymerization. Although EF1 alpha binds actin monomer, this monomer-binding activity does not explain the effects of EF1 alpha on actin polymerization at physiological molar ratios. The mechanism for the inhibition of polymerization is related to the actin-bundling activity of EF1 alpha. Both ends of the actin filament are inhibited for polymerization and both bundling and the inhibition of actin polymerization are affected by pH within the same physiological range; at high pH both bundling and the inhibition of actin polymerization are reduced. Additionally, it is seen that the binding of aminoacyl-tRNA to EF1 alpha releases EF1 alpha's inhibiting effect on actin polymerization. These data demonstrate that EF1 alpha can alter the assembly of F-actin, a filamentous scaffold on which non- membrane-associated protein translation may be occurring in vivo. PMID:8947553

  20. [Impacts of filamentous bulking on treatment effect and fouling characteristics of nonwoven bioreactor].

    PubMed

    Shi, Kuan; Xue, Gang; Gao, Pin; Wu, Fan

    2014-06-01

    Sludge flocs morphology and effluent qualities of nonwoven bioreactor during filamentous bulking and normal sludge status were compared. Flux variations, analysis of filtration resistances and extracellular polymer substances (EPS) contents during filamentous bulking and normal sludge status were studied. The results showed that the average particle size of bulking sludge flocs and normal sludge flocs was 448.6 microm and 234.8 microm, respectively. During the bulking sludge status, the average COD and NH4(+) -N removal rate and effluent turbidity were 90.1%, 93.1% and 1.33 NTU, respectively, compared with 91.4% , 97.0% and 0.99 NTU during the normal sludge status. Filamentous bulking had little impact on COD removal, while it inhibited NH4(+) -N removal to a certain extent and had slight impact on effluent turbidity. Average membrane flux decay rates during the bulking sludge status and the normal sludge status were 3.29 L x (m2 x h2)(-1) and 4.87 L x (m2 x h2)(-1), respectively, and the fouling during bulking sludge status was slighter. Reversible fouling was the main fouling when sludge was bulking while irreversible fouling was the main fouling when the sludge was in normal status. The prior occurrence of reversible fouling could reduce irreversible fouling therefore slow down the flux decline. Soluble microbial products (SMP) contents in normal sludge mixed liquid and bulking sludge mixed liquid were 21.369 mg x L(-1) and 10.182 mg x L(-1), respectively, protein/polysaccharide (P/C) was 0.370 and 0.497, respectively, SMP gross was related to fouling resistance and P/C was associated with reversible fouling proportion. The relation between loosely bound EPS in mixed liquid sludge and membrane fouling was similar to SMP. Sludge with more EPS could accumulate on membrane more easily, and the EPS that accumulated on membrane was mainly protein. EPS gross in mixed liquid sludge, EPS in membrane surface sludge and their P/C all had positive correlations to

  1. Filamentation in tokamaks

    SciTech Connect

    Cardozo, N.J.; Barth, C.J.; Chu, C.C.; Lok, J.; Montvai, A.; Oomens, A.A.; Peters, M.; Pijper, F.J.; de Rover, M.; Schueller, F.C.; Steenbakkers, M.F.; RTP team

    1995-09-01

    The relevance of a nest of toroidal flux surfaces as a paradigm of the magnetic topology of a tokamak plasma is challenged. High resolution Thomson scattering measurements of electron temperature and density in RTP show several hot filaments in the plasma center and sharp gradients near the sawtooth inversion radius and structures outside the sawtooth region under central ECH. In ohmic plasmas, too, the pressure and temperature profiles show significant bumps. These measurements give evidence of a complex magnetic topology. Transport in a medium with spatially strongly varying diffusivity is considered. It is shown that macroscopic transport is determined by the microscopic structure: a transport theory must predict this structure and the diffusivity in the insulating regions, while the {open_quote}turbulent{close_quote} diffusivity is irrelevant. A numerical approach to equilibria with broken surfaces is presented. {copyright} {ital 1995 American Institute of Physics.}

  2. Solid friction between soft filaments

    NASA Astrophysics Data System (ADS)

    Ward, Andrew; Hilitski, Feodor; Schwenger, Walter; Welch, David; Lau, A. W. C.; Vitelli, Vincenzo; Mahadevan, L.; Dogic, Zvonimir

    2015-06-01

    Any macroscopic deformation of a filamentous bundle is necessarily accompanied by local sliding and/or stretching of the constituent filaments. Yet the nature of the sliding friction between two aligned filaments interacting through multiple contacts remains largely unexplored. Here, by directly measuring the sliding forces between two bundled F-actin filaments, we show that these frictional forces are unexpectedly large, scale logarithmically with sliding velocity as in solid-like friction, and exhibit complex dependence on the filaments’ overlap length. We also show that a reduction of the frictional force by orders of magnitude, associated with a transition from solid-like friction to Stokes’s drag, can be induced by coating F-actin with polymeric brushes. Furthermore, we observe similar transitions in filamentous microtubules and bacterial flagella. Our findings demonstrate how altering a filament’s elasticity, structure and interactions can be used to engineer interfilament friction and thus tune the properties of fibrous composite materials.

  3. Solid friction between soft filaments

    SciTech Connect

    Ward, Andrew; Hilitski, Feodor; Schwenger, Walter; Welch, David; Lau, A. W. C.; Vitelli, Vincenzo; Mahadevan, L.; Dogic, Zvonimir

    2015-03-02

    Any macroscopic deformation of a filamentous bundle is necessarily accompanied by local sliding and/or stretching of the constituent filaments. Yet the nature of the sliding friction between two aligned filaments interacting through multiple contacts remains largely unexplored. Here, by directly measuring the sliding forces between two bundled F-actin filaments, we show that these frictional forces are unexpectedly large, scale logarithmically with sliding velocity as in solid-like friction, and exhibit complex dependence on the filaments’ overlap length. We also show that a reduction of the frictional force by orders of magnitude, associated with a transition from solid-like friction to Stokes’s drag, can be induced by coating F-actin with polymeric brushes. Furthermore, we observe similar transitions in filamentous microtubules and bacterial flagella. In conclusion, our findings demonstrate how altering a filament’s elasticity, structure and interactions can be used to engineer interfilament friction and thus tune the properties of fibrous composite materials.

  4. How eukaryotic filamentous pathogens evade plant recognition.

    PubMed

    Oliveira-Garcia, Ely; Valent, Barbara

    2015-08-01

    Plant pathogenic fungi and oomycetes employ sophisticated mechanisms for evading host recognition. After host penetration, many fungi and oomycetes establish a biotrophic interaction. It is assumed that different strategies employed by these pathogens to avoid triggering host defence responses, including establishment of biotrophic interfacial layers between the pathogen and host, masking of invading hyphae and active suppression of host defence mechanisms, are essential for a biotrophic parasitic lifestyle. During the infection process, filamentous plant pathogens secrete various effectors, which are hypothesized to be involved in facilitating effective host infection. Live-cell imaging of fungi and oomycetes secreting fluorescently labeled effector proteins as well as functional characterization of the components of biotrophic interfaces have led to the recent progress in understanding how eukaryotic filamentous pathogens evade plant recognition.

  5. Force Generation, Polymerization Dynamics and Nucleation of Actin Filaments

    NASA Astrophysics Data System (ADS)

    Wang, Ruizhe

    We study force generation and actin filament dynamics using stochastic and deterministic methods. First, we treat force generation of bundled actin filaments by polymerization via molecular-level stochastic simulations. In the widely-used Brownian Ratchet model, actin filaments grow freely whenever the tip-obstacle gap created by thermal fluctuation exceeds the monomer size. We name this model the Perfect Brownian Ratchet (PBR) model. In the PBR model, actin monomer diffusion is treated implicitly. We perform a series of simulations based on the PBR, in which obstacle motion is treated explicitly; in most previous studies, obstacle motion has been treated implicitly. We find that the cooperativity of filaments is generally weak in the PBR model, meaning that more filaments would grow more slowly given the same force per filament. Closed-form formulas are also developed, which match the simulation results. These portable and accurate formulas provide guidance for experiments and upper and lower bounds for theoretical analyses. We also studied a variation of the PBR, called the Diffusing Brownian Ratchet (DBR) model, in which both actin monomer and obstacle diffusion are treated explicitly. We find that the growth rate of multiple filaments is even lower, compared with that in PBR. This finding challenges the widely-accepted PBR assumption and suggests that pushing the study of actin dynamics down to the sub-nanometer level yields new insights. We subsequently used a rate equation approach to model the effect of local depletion of actin monomers on the nucleation of actin filaments on biomimetic beads, and how the effect is regulated by capping protein (CP). We find that near the bead surface, a higher CP concentration increases local actin concentration, which leads to an enhanced activities of actin filaments' nucleation. Our model analysis matches the experimental results and lends support to an important but undervalued hypothesis proposed by Carlier and

  6. Septal Junctions in Filamentous Heterocyst-Forming Cyanobacteria.

    PubMed

    Flores, Enrique; Herrero, Antonia; Forchhammer, Karl; Maldener, Iris

    2016-02-01

    In the filaments of heterocyst-forming cyanobacteria, septal junctions that traverse the septal peptidoglycan join adjacent cells, allowing intercellular communication. Perforations in the septal peptidoglycan have been observed, and proteins involved in the formation of such perforations and putative protein components of the septal junctions have been identified, but their relationships are debated.

  7. Desmin filaments studied by quasi-elastic light scattering.

    PubMed Central

    Hohenadl, M; Storz, T; Kirpal, H; Kroy, K; Merkel, R

    1999-01-01

    We studied polymers of desmin, a muscle-specific type III intermediate filament protein, using quasi-elastic light scattering. Desmin was purified from chicken gizzard. Polymerization was induced either by 2 mM MgCl(2) or 150 mM NaCl. The polymer solutions were in the semidilute regime. We concluded that the persistence length of the filaments is between 0.1 and 1 microm. In all cases, we found a hydrodynamic diameter of desmin filaments of 16-18 nm. The filament dynamics exhibits a characteristic frequency in the sense that correlation functions measured on one sample but at different scattering vectors collapse onto a single master curve when time is normalized by the experimentally determined initial decay rate. PMID:10512839

  8. Broken Detailed Balance of Filament Dynamics in Active Networks

    NASA Astrophysics Data System (ADS)

    Gladrow, J.; Fakhri, N.; MacKintosh, F. C.; Schmidt, C. F.; Broedersz, C. P.

    2016-06-01

    Myosin motor proteins drive vigorous steady-state fluctuations in the actin cytoskeleton of cells. Endogenous embedded semiflexible filaments such as microtubules, or added filaments such as single-walled carbon nanotubes are used as novel tools to noninvasively track equilibrium and nonequilibrium fluctuations in such biopolymer networks. Here, we analytically calculate shape fluctuations of semiflexible probe filaments in a viscoelastic environment, driven out of equilibrium by motor activity. Transverse bending fluctuations of the probe filaments can be decomposed into dynamic normal modes. We find that these modes no longer evolve independently under nonequilibrium driving. This effective mode coupling results in nonzero circulatory currents in a conformational phase space, reflecting a violation of detailed balance. We present predictions for the characteristic frequencies associated with these currents and investigate how the temporal signatures of motor activity determine mode correlations, which we find to be consistent with recent experiments on microtubules embedded in cytoskeletal networks.

  9. Adenosine monophosphate-activated protein kinase (AMPK) activators for the prevention, treatment and potential reversal of pathological pain

    PubMed Central

    Price, Theodore J.; Das, Vaskar; Dussor, Gregory

    2015-01-01

    Pathological pain is an enormous medical problem that places a significant burden on patients and can result from an injury that has long since healed or be due to an unidentifiable cause. Although treatments exist, they often either lack efficacy or have intolerable side effects. More importantly, they do not reverse the changes in the nervous system mediating pathological pain, and thus symptoms often return when therapies are discontinued. Consequently, novel therapies are urgently needed that have both improved efficacy and disease-modifying properties. Here we highlight an emerging target for novel pain therapies, adenosine monophosphate-activated protein kinase (AMPK). AMPK is capable of regulating a variety of cellular processes including protein translation, activity of other kinases, and mitochondrial metabolism, many of which are thought to contribute to pathological pain. Consistent with these properties, preclinical studies show positive, and in some cases disease-modifying effects of either pharmacological activation or genetic regulation of AMPK in models of nerve injury, chemotherapy-induced peripheral neuropathy (CIPN), postsurgical pain, inflammatory pain, and diabetic neuropathy. Given the AMPK-activating ability of metformin, a widely prescribed and well-tolerated drug, these preclinical studies provide a strong rationale for both retrospective and prospective human pain trials with this drug. They also argue for the development of novel AMPK activators, whether orthosteric, allosteric, or modulators of events upstream of the kinase. Together, this review will present the case for AMPK as a novel therapeutic target for pain and will discuss future challenges in the path toward development of AMPK-based pain therapeutics. PMID:26521775

  10. Transcriptional Control of Tight Junction Proteins via a Protein Kinase C Signal Pathway in Human Telomerase Reverse Transcriptase-Transfected Human Pancreatic Duct Epithelial Cells

    PubMed Central

    Yamaguchi, Hiroshi; Kojima, Takashi; Ito, Tatsuya; Kimura, Yasutoshi; Imamura, Masafumi; Son, Seiichi; Koizumi, Jun-ichi; Murata, Masaki; Nagayama, Minoru; Nobuoka, Takayuki; Tanaka, Satoshi; Hirata, Koichi; Sawada, Norimasa

    2010-01-01

    In human pancreatic cancer, integral membrane proteins of tight junction claudins are abnormally regulated, making these proteins promising molecular diagnostic and therapeutic targets. However, the regulation of claudin-based tight junctions remains unknown not only in the pancreatic cancer cells but also in normal human pancreatic duct epithelial (HPDE) cells. To investigate the regulation of tight junction molecules including claudins in normal HPDE cells, we introduced the human telomerase reverse transcriptase (hTERT) gene into HPDE cells in primary culture. The hTERT-transfected HPDE (hTERT-HPDE) cells were positive for the pancreatic duct epithelial markers such as CK7, CK19, and carbonic anhydrase isozyme 2 and expressed epithelial tight junction molecules claudin-1, -4, -7 and, -18, occludin, JAM-A, ZO-1, ZO-2, and tricellulin. By treatment with fetal bovine serum or 12-O-tetradecanoylphorbol 13-acetate (TPA), the tight junction molecules were up-regulated at the transcriptional level via a protein kinase C (PKC) signal pathway. A PKC-α inhibitor, Gö6976, prevented up-regulation of claudin-4 by TPA. Furthermore, a PKC-δ inhibitor, rottlerin, prevented up-regulation of claudin-7, occludin, ZO-1, and ZO-2 by TPA. By GeneChip analysis, up-regulation of the transcription factor ELF3 was observed in both fetal bovine serum- and TPA-treated cells. Treatment with small interfering RNAs of ELF3 prevented up-regulation of claudin-7 by TPA. These data suggest that tight junctions of normal HPDE cells were at least in part regulated via a PKC signal pathway by transcriptional control. PMID:20566751

  11. The Brown Algae Pl.LSU/2 Group II Intron-Encoded Protein Has Functional Reverse Transcriptase and Maturase Activities

    PubMed Central

    Zerbato, Madeleine; Holic, Nathalie; Moniot-Frin, Sophie; Ingrao, Dina; Galy, Anne; Perea, Javier

    2013-01-01

    Group II introns are self-splicing mobile elements found in prokaryotes and eukaryotic organelles. These introns propagate by homing into precise genomic locations, following assembly of a ribonucleoprotein complex containing the intron-encoded protein (IEP) and the spliced intron RNA. Engineered group II introns are now commonly used tools for targeted genomic modifications in prokaryotes but not in eukaryotes. We speculate that the catalytic activation of currently known group II introns is limited in eukaryotic cells. The brown algae Pylaiella littoralis Pl.LSU/2 group II intron is uniquely capable of in vitro ribozyme activity at physiological level of magnesium but this intron remains poorly characterized. We purified and characterized recombinant Pl.LSU/2 IEP. Unlike most IEPs, Pl.LSU/2 IEP displayed a reverse transcriptase activity without intronic RNA. The Pl.LSU/2 intron could be engineered to splice accurately in Saccharomyces cerevisiae and splicing efficiency was increased by the maturase activity of the IEP. However, spliced transcripts were not expressed. Furthermore, intron splicing was not detected in human cells. While further tool development is needed, these data provide the first functional characterization of the PI.LSU/2 IEP and the first evidence that the Pl.LSU/2 group II intron splicing occurs in vivo in eukaryotes in an IEP-dependent manner. PMID:23505475

  12. Propofol reverses oxidative stress-attenuated glutamate transporter EAAT3 activity: evidence of protein kinase C involvement.

    PubMed

    Yun, Jung-Yeon; Park, Kum-Suk; Kim, Jin-Hee; Do, Sang-Hwan; Zuo, Zhiyi

    2007-06-22

    The authors investigated the effects of propofol on EAAT3 (excitatory amino acid transporter 3) activity under oxidative stress induced by tert-butyl hydroperoxide (t-BHP), and the mediation of these effects by protein kinase C (PKC). Rat EAAT3 was expressed in Xenopus oocytes and L-glutamate (30 microM)-induced membrane currents were measured using the two-electrode voltage clamp technique. Exposure of these oocytes to t-BHP (1-20 mM) for 10 min dose-dependently decreased EAAT3 activity, and t-BHP (5 mM) significantly decreased the Vmax, but not the Km of EAAT3 for glutamate, and propofol (1-100 microM) dose-dependently reversed this t-BHP-attenuated EAAT3 activity. Phorbol-12-myristate-13-acetate (a PKC activator), also abolished this t-BHP-induced reduction in EAAT3 activity, whereas staurosporine (a PKC inhibitor), significantly decreased EAAT3 activity. However, as compared with staurosporine or t-BHP alone, t-BHP and staurosporine in combination did not further reduce EAAT3 activity. A similar pattern was observed for chelerythrine (also a PKC inhibitor). In oocytes pretreated with combinations of t-BHP and PMA (or staurosporine), propofol failed to change EAAT3 activity. Our results suggest that propofol restores oxidative stress-reduced EAAT3 activity and that these effects of propofol may be PKC-mediated.

  13. Programmable display of DNA-protein chimeras for controlling cell-hydrogel interactions via reversible intermolecular hybridization.

    PubMed

    Zhang, Zhaoyang; Li, Shihui; Chen, Niancao; Yang, Cheng; Wang, Yong

    2013-04-08

    Extensive studies have been recently carried out to achieve dynamic control of cell-material interactions primarily through physicochemical stimulation. The purpose of this study was to apply reversible intermolecular hybridization to program cell-hydrogel interactions in physiological conditions based on DNA-antibody chimeras and complementary oligonucleotides. The results showed that DNA oligonucleotides could be captured to and released from the immobilizing DNA-functionalized hydrogels with high specificity via DNA hybridization. Accordingly, DNA-antibody chimeras were captured to the hydrogels, successfully inducing specific cell attachment. The cell attachment to the hydrogels reached the plateau at approximately half an hour after the functionalized hydrogels and the cells were incubated together. The attached cells were rapidly released from the bound hydrogels when triggering complementary oligonucleotides were introduced to the system. However, the capability of the triggering complementary oligonucleotides in releasing cells was affected by the length of intermolecular hybridization. The length needed to be at least more than 20 base pairs in the current experimental setting. Notably, because the procedure of intermolecular hybridization did not involve any harsh condition, the released cells maintained the same viability as that of the cultured cells. The functionalized hydrogels also exhibited the potential to catch and release cells repeatedly. Therefore, this study demonstrates that it is promising to regulate cell-material interactions dynamically through the DNA-programmed display of DNA-protein chimeras.

  14. The brown algae Pl.LSU/2 group II intron-encoded protein has functional reverse transcriptase and maturase activities.

    PubMed

    Zerbato, Madeleine; Holic, Nathalie; Moniot-Frin, Sophie; Ingrao, Dina; Galy, Anne; Perea, Javier

    2013-01-01

    Group II introns are self-splicing mobile elements found in prokaryotes and eukaryotic organelles. These introns propagate by homing into precise genomic locations, following assembly of a ribonucleoprotein complex containing the intron-encoded protein (IEP) and the spliced intron RNA. Engineered group II introns are now commonly used tools for targeted genomic modifications in prokaryotes but not in eukaryotes. We speculate that the catalytic activation of currently known group II introns is limited in eukaryotic cells. The brown algae Pylaiella littoralis Pl.LSU/2 group II intron is uniquely capable of in vitro ribozyme activity at physiological level of magnesium but this intron remains poorly characterized. We purified and characterized recombinant Pl.LSU/2 IEP. Unlike most IEPs, Pl.LSU/2 IEP displayed a reverse transcriptase activity without intronic RNA. The Pl.LSU/2 intron could be engineered to splice accurately in Saccharomyces cerevisiae and splicing efficiency was increased by the maturase activity of the IEP. However, spliced transcripts were not expressed. Furthermore, intron splicing was not detected in human cells. While further tool development is needed, these data provide the first functional characterization of the PI.LSU/2 IEP and the first evidence that the Pl.LSU/2 group II intron splicing occurs in vivo in eukaryotes in an IEP-dependent manner.

  15. Perturbation growth in accreting filaments

    NASA Astrophysics Data System (ADS)

    Clarke, S. D.; Whitworth, A. P.; Hubber, D. A.

    2016-05-01

    We use smoothed particle hydrodynamic simulations to investigate the growth of perturbations in infinitely long filaments as they form and grow by accretion. The growth of these perturbations leads to filament fragmentation and the formation of cores. Most previous work on this subject has been confined to the growth and fragmentation of equilibrium filaments and has found that there exists a preferential fragmentation length-scale which is roughly four times the filament's diameter. Our results show a more complicated dispersion relation with a series of peaks linking perturbation wavelength and growth rate. These are due to gravo-acoustic oscillations along the longitudinal axis during the sub-critical phase of growth. The positions of the peaks in growth rate have a strong dependence on both the mass accretion rate onto the filament and the temperature of the gas. When seeded with a multiwavelength density power spectrum, there exists a clear preferred core separation equal to the largest peak in the dispersion relation. Our results allow one to estimate a minimum age for a filament which is breaking up into regularly spaced fragments, as well as an average accretion rate. We apply the model to observations of filaments in Taurus by Tafalla & Hacar and find accretion rates consistent with those estimated by Palmeirim et al.

  16. F actin bundles in Drosophila bristles. I. Two filament cross-links are involved in bundling

    PubMed Central

    1995-01-01

    Transverse sections though Drosophila bristles reveal 7-11 nearly round, plasma membrane-associated bundles of actin filaments. These filaments are hexagonally packed and in a longitudinal section they show a 12-nm periodicity in both the 1.1 and 1.0 views. From earlier studies this periodicity is attributable to cross-links and indicates that the filaments are maximally cross-linked, singed mutants also have 7-11 bundles, but the bundles are smaller, flattened, and the filaments within the bundles are randomly packed (not hexagonal); no periodicity can be detected in longitudinal sections. Another mutant, forked (f36a), also has 7-11 bundles but even though the bundles are very small, the filaments within them are hexagonally packed and display a 12-nm periodicity in longitudinal section. The singed-forked double mutant lacks filament bundles. Thus there are at least two species of cross-links between adjacent actin filaments. Hints of why two species of cross-links are necessary can be gleaned by studying bristle formation. Bristles sprout with only microtubules within them. A little later in development actin filaments appear. At early stages the filaments in the bundles are randomly packed. Later the filaments in the bundles become hexagonally packed and maximally cross-linked. We consider that the forked proteins may be necessary early in development to tie the filaments together in a bundle so that they can be subsequently zippered together by fascin (the singed gene product). PMID:7622563

  17. Cell envelope components influencing filament length in the heterocyst-forming cyanobacterium Anabaena sp. strain PCC 7120.

    PubMed

    Burnat, Mireia; Schleiff, Enrico; Flores, Enrique

    2014-12-01

    Heterocyst-forming cyanobacteria grow as chains of cells (known as trichomes or filaments) that can be hundreds of cells long. The filament consists of individual cells surrounded by a cytoplasmic membrane and peptidoglycan layers. The cells, however, share a continuous outer membrane, and septal proteins, such as SepJ, are important for cell-cell contact and filament formation. Here, we addressed a possible role of cell envelope components in filamentation, the process of producing and maintaining filaments, in the model cyanobacterium Anabaena sp. strain PCC 7120. We studied filament length and the response of the filaments to mechanical fragmentation in a number of strains with mutations in genes encoding cell envelope components. Previously published peptidoglycan- and outer membrane-related gene mutants and strains with mutations in two genes (all5045 and alr0718) encoding class B penicillin-binding proteins isolated in this work were used. Our results show that filament length is affected in most cell envelope mutants, but the filaments of alr5045 and alr2270 gene mutants were particularly fragmented. All5045 is a dd-transpeptidase involved in peptidoglycan elongation during cell growth, and Alr2270 is an enzyme involved in the biosynthesis of lipid A, a key component of lipopolysaccharide. These results indicate that both components of the cell envelope, the murein sacculus and the outer membrane, influence filamentation. As deduced from the filament fragmentation phenotypes of their mutants, however, none of these elements is as important for filamentation as the septal protein SepJ.

  18. Direct Analysis of Reversed-Phase HPTLC Separated Tryptic Protein Digests using a Liquid Microjunction Surface Sampling Probe/ESI-MS System

    SciTech Connect

    Emory, Joshua F; Walworth, Matthew J; Van Berkel, Gary J; Schulz, Michael; Minarik, susanne

    2010-01-01

    The sampling, ionization and detection of tryptic peptides separated in one-dimension on reversed phase HPTLC plates was performed using liquid microjunction surface sampling probe electrospray ionization mass spectrometry. Tryptic digests of five proteins (cytochrome c., myoglobin, beta-casein, lysozyme, and bovine serum albumin) were spotted on reversed phase HPTLC RP-8 F254s and HPTLC RP-18 F254s plates. The plates were then developed using 70/30 methanol/water with 0.1 M ammonium acetate. A dual purpose extraction/electrospray solution containing 70/30/0.1 water/methanol/formic acid was infused through the sampling probe during analysis of the developed lanes. Both full scan mass spectra and data dependent tandem mass spectra were acquired for each development lane to detect and verify the peptide distributions. Data dependent tandem mass spectra provided both protein identification and sequence coverage information. Highest sequence coverages were achieved for cytochrome c. and myoglobin (62.5% and 58.3%, respectively) on reversed phase RP-8 plates. While the tryptic peptides were separated enough for identification, the peptide bands did show some overlap with most peptides located in the lower half of the development lane. Proteins whose peptides were more separated gave higher sequence coverage. Larger proteins such as beta-casein and BSA which were spotted in lower relative amounts gave much lower sequence coverage than the smaller proteins.

  19. [Analysis of proteins in the extracts of Physalis alkekengi L. var. franchetii (Mast.) Makino using nanoflow reversed-phase liquid chromatography-tandem mass spectrometry].

    PubMed

    Yu, Haiyang; Yan, Jiaze; Guo, Ming; Jin, Yan

    2013-04-01

    The protein was extracted from Physalis alkekengi L. var. franchetii (Mast.) Makino fruit by using water extraction and acid precipitation methods, and it consisted of 188 mg/g of protein on dry basis of the extract. Among 18 amino acids, eight essential amino acids for human account for 31% were found in this extract. Based on shotgun proteomics method, the protein extracted from Physalis fruit was analysed by nanoflow reversed-phase liquid chromatography-tandem mass spectrometry (nano-RPLC-MS/MS) system. Combined with database searches and bioinformatics analysis, the protein species and six molecular functions were identified, including with catalytic activity, antioxidant activity, enzyme regular activity, nutrient reservoir activity, transporter activity and binding activity. And three antioxidant activity-related proteins were identified. These results may lay the foundation for further study of the functional properties of the proteins in Physalis fruit.

  20. Metabolic regulation via enzyme filamentation

    PubMed Central

    Aughey, Gabriel N.; Liu, Ji-Long

    2016-01-01

    Abstract Determining the mechanisms of enzymatic regulation is central to the study of cellular metabolism. Regulation of enzyme activity via polymerization-mediated strategies has been shown to be widespread, and plays a vital role in mediating cellular homeostasis. In this review, we begin with an overview of the filamentation of CTP synthase, which forms filamentous structures termed cytoophidia. We then highlight other important examples of the phenomenon. Moreover, we discuss recent data relating to the regulation of enzyme activity by compartmentalization into cytoophidia. Finally, we hypothesize potential roles for enzyme filament formation in the regulation of metabolism, development and disease. PMID:27098510

  1. Effects of filament rigidity in myosin II-induced actin network contractility and dynamics

    NASA Astrophysics Data System (ADS)

    Weirich, Kimberly; Gardel, Margaret

    2014-03-01

    Cells change shape, deforming to move and divide. The dynamic protein scaffold that shapes the cell is the cortex, a disordered, thin network of actin filaments. Random, local stresses generated by myosin II in the network create cellular-scale deformations. Myosin induced buckling and severing of actin filaments has been shown to underlie the contractility of two-dimensional disordered actin networks. This non-linear elastic response of actin filaments is thought to be an essential symmetry breaking mechanism to produce robust contractility in disordered actomyosin networks. To test this idea, we explore the effects of an actin bundling protein fascin, a crosslinker which induces polarity specific bundling of actin filaments, to create a network of F-actin bundles. We investigate myosin-induced stresses in a network of randomly oriented actin filaments, confined to a thin sheet at a supported lipid bilayer surface through a crowding agent. We find fascin-bundled filaments are less prone to filament buckling and show increased filament sliding, causing the myosin activity to induce network reorganization rather than contraction. Thus, changes in the filament bending rigidity in motor-filament systems can drive the system between distinct states with unique dynamic and mechanical signatures.

  2. Merging of Filaments in a Dual-Filament System

    NASA Astrophysics Data System (ADS)

    Mikurda, K.; Martin, S. F.

    2006-08-01

    Introduction: The dual filament system merges to form one extended filament. Methods: The filaments were observed at Helio Research at multiple wavelengths around Hα using a tunable filter and a narrow band Fabry-Perot etalon as part of the Joint Observing Campaign (JOP 178). These observations are used to create two-dimensional Dopplergrams. The Hα images are compared with data taken onboard the SOHO (Solar and Heliospheric Observatory) spacecraft (EIT at 304 A and LASCO C2). The GONG magnetograms provide the information on photospheric magnetic fields. Results and Discussion: The filaments were observed on the solar disk between October 10 and 16, 2004. We compare the morphology of the filament system in Hα and He II line at 304 A and discuss its evolution. We investigate the photospheric magnetic flux cancellation rate during the merging process. There is no clear evidence of an eruption associated with the merging of the filaments from either EIT or LASCO in contrary to some previous findings.

  3. Use of the Chinchilla model to evaluate the vaccinogenic potential of the Moraxella catarrhalis filamentous hemagglutinin-like proteins MhaB1 and MhaB2.

    PubMed

    Shaffer, Teresa L; Balder, Rachel; Buskirk, Sean W; Hogan, Robert J; Lafontaine, Eric R

    2013-01-01

    Moraxella catarrhalis causes significant health problems, including 15-20% of otitis media cases in children and ~10% of respiratory infections in adults with chronic obstructive pulmonary disease. The lack of an efficacious vaccine, the rapid emergence of antibiotic resistance in clinical isolates, and high carriage rates reported in children are cause for concern. In addition, the effectiveness of conjugate vaccines at reducing the incidence of otitis media caused by Streptococcus pneumoniae and nontypeable Haemophilus influenzae suggest that M. catarrhalis infections may become even more prevalent. Hence, M. catarrhalis is an important and emerging cause of infectious disease for which the development of a vaccine is highly desirable. Studying the pathogenesis of M. catarrhalis and the testing of vaccine candidates have both been hindered by the lack of an animal model that mimics human colonization and infection. To address this, we intranasally infected chinchilla with M. catarrhalis to investigate colonization and examine the efficacy of a protein-based vaccine. The data reveal that infected chinchillas produce antibodies against antigens known to be major targets of the immune response in humans, thus establishing immune parallels between chinchillas and humans during M. catarrhalis infection. Our data also demonstrate that a mutant lacking expression of the adherence proteins MhaB1 and MhaB2 is impaired in its ability to colonize the chinchilla nasopharynx, and that immunization with a polypeptide shared by MhaB1 and MhaB2 elicits antibodies interfering with colonization. These findings underscore the importance of adherence proteins in colonization and emphasize the relevance of the chinchilla model to study M. catarrhalis-host interactions.

  4. Isolation and preliminary characterization of 10-nm filaments from baby hamster kidney (BHK-21) cells.

    PubMed Central

    Starger, J M; Goldman, R D

    1977-01-01

    A procedure for isolating 10-nm filaments as juxtanuclear caps from normal and colchicine-treated BHK-21 cells is described. These aggregates of 10-nm filaments retain their birefringence and their structural integrity. The major proteins comprising the filament preparations can be resolved as two bands on sodium dodecyl sulfate gels, corresponding to approximate molecular weights of 54,000 and 55,000. Images PMID:329284

  5. The relationship between truncation and phosphorylation at the C-terminus of tau protein in the paired helical filaments of Alzheimer's disease

    PubMed Central

    Flores-Rodríguez, Paola; Ontiveros-Torres, Miguel A.; Cárdenas-Aguayo, María C.; Luna-Arias, Juan P.; Meraz-Ríos, Marco A.; Viramontes-Pintos, Amparo; Harrington, Charles R.; Wischik, Claude M.; Mena, Raúl; Florán-Garduño, Benjamin; Luna-Muñoz, José

    2015-01-01

    We previously demonstrated that, in the early stages of tau processing in Alzheimer's disease, the N-terminal part of the molecule undergoes a characteristic cascade of phosphorylation and progressive misfolding of the proteins resulting in a structural conformation detected by Alz-50. In this immunohistochemical study of AD brain tissue, we have found that C-terminal truncation of tau at Asp-421 was an early event in tau aggregation and analyzed the relationship between phospho-dependent tau epitopes located at the C-terminus with truncation at Glu-391. The aim of this study was to determine whether C-terminal truncation may trigger events leading to the assembly of insoluble PHFs from soluble tau aggregates present in pre-tangle cells. Our findings suggest that there is a complex interaction between phosphorylated and truncated tau species. A model is presented here in which truncated tau protein represents an early neurotoxic species while phosphorylated tau species may provide a neuroprotective role in Alzheimer's disease. PMID:25717290

  6. Semiflexible filament networks viewed as fluctuating beam frames

    NASA Astrophysics Data System (ADS)

    Su, Tianxiang; Purohit, Prashant

    2012-02-01

    We present a new method combining structural and statistical mechanics to study the entropic elasticity of semiflexible filament networks. We view a filament network as a frame structure and use structural mechanics to determine its static equilibrium configuration under applied loads in the first step. To account for thermal motion around this static equilibrium state, we then approximate the potential energy of the deformed frame structure up to the second order in kinematic variables and obtaina deformation-dependent stiffness matrix characterizing the flexibility of the network. Using statistical mechanics, we then evaluate the partition function, free energy and thermo-mechanical properties of the network in terms of the stiffness matrix. We show that penalty methods commonly used in finite elements to account for constraints, are applicable even when statistical and structural mechanics are combined in our method. We apply our framework to understand the expansion, shear, uniaxial tension and compression behavior of some simple filament networks. We are able to capture the stress-stiffening behavior due to filament reorientation and stretching out of thermal fluctuations, as well as the reversible stress-softening behavior due to filament buckling.

  7. Collisions of Vortex Filament Pairs

    NASA Astrophysics Data System (ADS)

    Banica, Valeria; Faou, Erwan; Miot, Evelyne

    2014-12-01

    We consider the problem of collisions of vortex filaments for a model introduced by Klein et al. (J Fluid Mech 288:201-248, 1995) and Zakharov (Sov Phys Usp 31(7):672-674, 1988, Lect. Notes Phys 536:369-385, 1999) to describe the interaction of almost parallel vortex filaments in three-dimensional fluids. Since the results of Crow (AIAA J 8:2172-2179, 1970) examples of collisions are searched as perturbations of antiparallel translating pairs of filaments, with initial perturbations related to the unstable mode of the linearized problem; most results are numerical calculations. In this article, we first consider a related model for the evolution of pairs of filaments, and we display another type of initial perturbation leading to collision in finite time. Moreover, we give numerical evidence that it also leads to collision through the initial model. We finally study the self-similar solutions of the model.

  8. Type-III secretion filaments as scaffolds for inorganic nanostructures

    PubMed Central

    Azam, Anum; Tullman-Ercek, Danielle

    2016-01-01

    Nanostructured materials exhibit unique magnetic, electrical and catalytic properties. These characteristics are determined by the chemical composition, size and shape of the nanostructured components, which are challenging to modulate on such small size scales and to interface with living cells. To address this problem, we are using a self-assembling filament protein, PrgI, as a scaffold for bottom-up inorganic nanostructure synthesis. PrgI is a small protein (80 amino acids) that oligomerizes to form the type-III secretion system needle of Salmonella enterica. We demonstrate that purified PrgI monomers also spontaneously self-assemble into long filaments and that high-affinity peptide tags specific for attachment to functionalized particles can be integrated into the N-terminal region of PrgI. The resulting filaments selectively bind to gold, whether the filaments are assembled in vitro, sheared from cells or remain attached to live S. enterica cell membranes. Chemical reduction of the gold-modified PrgI variants results in structures that are several micrometres in length and which incorporate a contiguous gold surface. Mutant strains with genomically incorporated metal-binding tags retain the secretion phenotype. We anticipate that self-assembled, cell-tethered protein/metal filamentous structures have applications in sensing and energy transduction in vivo. PMID:26763334

  9. Sufficient conditions for the additivity of stall forces generated by multiple filaments or motors

    NASA Astrophysics Data System (ADS)

    Bameta, Tripti; Das, Dipjyoti; Das, Dibyendu; Padinhateeri, Ranjith; Inamdar, Mandar M.

    2017-02-01

    Molecular motors and cytoskeletal filaments work collectively most of the time under opposing forces. This opposing force may be due to cargo carried by motors or resistance coming from the cell membrane pressing against the cytoskeletal filaments. Some recent studies have shown that the collective maximum force (stall force) generated by multiple cytoskeletal filaments or molecular motors may not always be just a simple sum of the stall forces of the individual filaments or motors. To understand this excess or deficit in the collective force, we study a broad class of models of both cytoskeletal filaments and molecular motors. We argue that the stall force generated by a group of filaments or motors is additive, that is, the stall force of N number of filaments (motors) is N times the stall force of one filament (motor), when the system is reversible at stall. Conversely, we show that this additive property typically does not hold true when the system is irreversible at stall. We thus present a novel and unified understanding of the existing models exhibiting such non-addivity, and generalise our arguments by developing new models that demonstrate this phenomena. We also propose a quantity similar to thermodynamic efficiency to easily predict this deviation from stall-force additivity for filament and motor collectives.

  10. Phosphoenolpyruvate carboxylase protein kinase from developing castor oil seeds: partial purification, characterization, and reversible control by photosynthate supply.

    PubMed

    Murmu, Jhadeswar; Plaxton, William C

    2007-10-01

    Phosphoenolpyruvate carboxylase (PEPC, EC 4.1.1.31) protein kinase (PPCK) was purified approximately 1,500-fold from developing castor oil seeds (COS). Gel filtration and immunoblotting with anti-(rice PPCK2)-immune serum indicated that this Ca2+-insensitive PPCK exists as a 31-kDa monomer. COS PPCK-mediated rephosphorylation of the 107-kDa subunit (p107) of COS PEPC1 (Km = 2.2 microM) activated PEPC1 by approximately 80% when assayed under suboptimal conditions (pH 7.3, 0.2 mM PEP, and 0.125 mM malate). COS PPCK displayed remarkable selectivity for phosphorylating COS PEPC1 (relative to tobacco, sorghum, or maize PEPCs), exhibited a broad pH-activity optima of approximately pH 8.5, and at pH 7.3 was activated 40-65% by 1 mM PEP, or 10 mM Gln or Asn, but inhibited 65% by 10 mM L-malate. The possible control of COS PPCK by disulfide-dithiol interconversion was suggested by its rapid inactivation and subsequent reactivation when incubated with oxidized glutathione and then dithiothreitol. In vitro PPCK activity correlated with in vivo p107 phosphorylation status, with both peaking in mid-cotyledon to full-cotyledon developing COS. Notably, PPCK activity and p107 phosphorylation of developing COS were eliminated following pod excision or prolonged darkness of intact plants. Both effects were fully reversed 12 h following reillumination of darkened plants. These results implicate a direct relationship between the up-regulation of COS PPCK and p107 phosphorylation during the recommencement of photosynthate delivery from illuminated leaves to the non-photosynthetic COS. Overall, the results support the hypothesis that PEPC and PPCK participate in the control of photosynthate partitioning into C-skeletons needed as precursors for key biosynthetic pathways of developing COS.

  11. African swine fever virus NP868R capping enzyme promotes reovirus rescue during reverse genetics by promoting reovirus protein expression, virion assembly, and RNA incorporation into infectious virions.

    PubMed

    Eaton, Heather E; Kobayashi, Takeshi; Dermody, Terence S; Johnston, Randal N; Jais, Philippe H; Shmulevitz, Maya

    2017-03-15

    Reoviruses, like many eukaryotic viruses, contain an inverted 7-methylguanosine (m7G) cap linked to the 5' nucleotide of mRNA. Traditional functions of capping are to promote mRNA stability, protein translation, and concealment from cellular proteins that recognize foreign RNA. To address the role of mRNA capping during reovirus replication, we assessed the benefits of adding the African swine fever virus NP868R capping enzyme. C3P3, a fusion protein containing T7 RNA polymerase and NP868R, was found to increase protein expression 5 to 10-fold, as compared to T7 RNA polymerase alone, while enhancing reovirus rescue from the current reverse genetics system by 100-fold. Surprisingly, RNA stability was not increased by C3P3, suggesting a direct effect on protein translation. A time course analysis revealed that C3P3 increased protein synthesis within the first 2 days of a reverse genetics transfection. This analysis also revealed that C3P3 enhanced processing of outer capsid μ1 protein to μ1C, a previously described hallmark of reovirus assembly. Finally, to determine the rate of infectious RNA incorporation into new virions, we developed a new recombinant reovirus S1 gene that expressed the fluorescent protein UnaG. Following transfection of cells with UnaG and infection with wild-type virus, passage of UnaG through progeny was significantly enhanced by C3P3. This data suggests that capping provides non-traditional functions to reovirus, such as promoting assembly and infectious RNA incorporation.IMPORTANCE The findings in this manuscript expand our understanding of how viruses utilize capping, suggesting that capping provides non-traditional functions to reovirus such as promoting assembly and infectious RNA incorporation, in addition to enhancing protein translation. Beyond providing mechanistic insight into reovirus replication, our findings also show that reovirus reverse genetics rescue is enhanced 100-fold by the NP868R capping enzyme. Since reovirus shows

  12. Geometrical and Mechanical Properties Control Actin Filament Organization

    PubMed Central

    Ennomani, Hajer; Théry, Manuel; Nedelec, Francois; Blanchoin, Laurent

    2015-01-01

    The different actin structures governing eukaryotic cell shape and movement are not only determined by the properties of the actin filaments and associated proteins, but also by geometrical constraints. We recently demonstrated that limiting nucleation to sp