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Sample records for reversible protein filament

  1. Metal ion-dependent, reversible, protein filament formation by designed beta-roll polypeptides

    PubMed Central

    Scotter, Andrew J; Guo, Meng; Tomczak, Melanie M; Daley, Margaret E; Campbell, Robert L; Oko, Richard J; Bateman, David A; Chakrabartty, Avijit; Sykes, Brian D; Davies, Peter L

    2007-01-01

    Background A right-handed, calcium-dependent β-roll structure found in secreted proteases and repeat-in-toxin proteins was used as a template for the design of minimal, soluble, monomeric polypeptides that would fold in the presence of Ca2+. Two polypeptides were synthesised to contain two and four metal-binding sites, respectively, and exploit stacked tryptophan pairs to stabilise the fold and report on the conformational state of the polypeptide. Results Initial analysis of the two polypeptides in the presence of calcium suggested the polypeptides were disordered. The addition of lanthanum to these peptides caused aggregation. Upon further study by right angle light scattering and electron microscopy, the aggregates were identified as ordered protein filaments that required lanthanum to polymerize. These filaments could be disassembled by the addition of a chelating agent. A simple head-to-tail model is proposed for filament formation that explains the metal ion-dependency. The model is supported by the capping of one of the polypeptides with biotin, which disrupts filament formation and provides the ability to control the average length of the filaments. Conclusion Metal ion-dependent, reversible protein filament formation is demonstrated for two designed polypeptides. The polypeptides form filaments that are approximately 3 nm in diameter and several hundred nm in length. They are not amyloid-like in nature as demonstrated by their behaviour in the presence of congo red and thioflavin T. A capping strategy allows for the control of filament length and for potential applications including the "decoration" of a protein filament with various functional moieties. PMID:17908326

  2. 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

  3. Mechanical Properties of Intermediate Filament Proteins.

    PubMed

    Charrier, Elisabeth E; Janmey, Paul A

    2016-01-01

    Purified intermediate filament (IF) proteins can be reassembled in vitro to produce polymers closely resembling those found in cells, and these filaments form viscoelastic gels. The cross-links 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 nonlinear elasticity with stiffness, as quantified by shear modulus, increasing an order of magnitude as the networks are deformed to large strains resembling those that soft tissues undergo in vivo. Individual IFs can be stretched to more than two or three times their resting length without breaking. At least 10 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 cytoplasmic IFs 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

  4. Hamiltonian Dynamics of Protein Filament Formation

    NASA Astrophysics Data System (ADS)

    Michaels, Thomas C. T.; Cohen, Samuel I. A.; Vendruscolo, Michele; Dobson, Christopher M.; Knowles, Tuomas P. J.

    2016-01-01

    We establish the Hamiltonian structure of the rate equations describing the formation of protein filaments. We then show that this formalism provides a unified view of the behavior of a range of biological self-assembling systems as diverse as actin, prions, and amyloidogenic polypeptides. We further demonstrate that the time-translation symmetry of the resulting Hamiltonian leads to previously unsuggested conservation laws that connect the number and mass concentrations of fibrils and allow linear growth phenomena to be equated with autocatalytic growth processes. We finally show how these results reveal simple rate laws that provide the basis for interpreting experimental data in terms of specific mechanisms controlling the proliferation of fibrils.

  5. 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. PMID:9847417

  6. Hamiltonian Dynamics of Protein Filament Formation.

    PubMed

    Michaels, Thomas C T; Cohen, Samuel I A; Vendruscolo, Michele; Dobson, Christopher M; Knowles, Tuomas P J

    2016-01-22

    We establish the Hamiltonian structure of the rate equations describing the formation of protein filaments. We then show that this formalism provides a unified view of the behavior of a range of biological self-assembling systems as diverse as actin, prions, and amyloidogenic polypeptides. We further demonstrate that the time-translation symmetry of the resulting Hamiltonian leads to previously unsuggested conservation laws that connect the number and mass concentrations of fibrils and allow linear growth phenomena to be equated with autocatalytic growth processes. We finally show how these results reveal simple rate laws that provide the basis for interpreting experimental data in terms of specific mechanisms controlling the proliferation of fibrils. PMID:26849615

  7. 48-Kilodalton intermediate-filament-associated protein in astrocytes.

    PubMed

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

    1988-01-01

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

  8. High Throughput Screening for Drugs that Modulate Intermediate Filament Proteins

    PubMed Central

    Sun, Jingyuan; Groppi, Vincent E.; Gui, Honglian; Chen, Lu; Xie, Qing; Liu, Li

    2016-01-01

    Intermediate filament (IF) proteins have unique and complex cell and tissue distribution. Importantly, IF gene mutations cause or predispose to more than 80 human tissue-specific diseases (IF-pathies), with the most severe disease phenotypes being due to mutations at conserved residues that result in a disrupted IF network. A critical need for the entire IF-pathy field is the identification of drugs that can ameliorate or cure these diseases, particularly since all current therapies target the IF-pathy complication, such as diabetes or cardiovascular disease, rather than the mutant IF protein or gene. We describe a high throughput approach to identify drugs that can normalize disrupted IF proteins. This approach utilizes transduction of lentivirus that expresses green-fluorescent-protein-tagged keratin 18 (K18) R90C in A549 cells. The readout is drug ‘hits’ that convert the dot-like keratin filament distribution, due to the R90C mutation, to a wildtype-like filamentous array. A similar strategy can be used to screen thousands of compounds and can be utilized for practically any IF protein with a filament-disrupting mutation, and could therefore potentially target many IF-pathies. ‘Hits’ of interest require validation in cell culture then using in vivo experimental models. Approaches to study the mechanism of mutant-IF normalization by potential drugs of interest are also described. The ultimate goal of this drug screening approach is to identify effective and safe compounds that can potentially be tested for clinical efficacy in patients. PMID:26795471

  9. 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.

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

    PubMed Central

    Vincent, I J; Davies, P

    1992-01-01

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

  11. 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

  12. Single-molecule visualization of a formin-capping protein ‘decision complex' at the actin filament barbed end

    PubMed Central

    Bombardier, Jeffrey P.; Eskin, Julian A.; Jaiswal, Richa; Corrêa, Ivan R.; Xu, Ming-Qun; Goode, Bruce L.; Gelles, Jeff

    2015-01-01

    Precise control of actin filament length is essential to many cellular processes. Formins processively elongate filaments, whereas capping protein (CP) binds to barbed ends and arrests polymerization. While genetic and biochemical evidence has indicated that these two proteins function antagonistically, the mechanism underlying the antagonism has remained unresolved. Here we use multi-wavelength single-molecule fluorescence microscopy to observe the fully reversible formation of a long-lived ‘decision complex' in which a CP dimer and a dimer of the formin mDia1 simultaneously bind the barbed end. Further, mDia1 displaced from the barbed end by CP can randomly slide along the filament and later return to the barbed end to re-form the complex. Quantitative kinetic analysis reveals that the CP-mDia1 antagonism that we observe in vitro occurs through the decision complex. Our observations suggest new molecular mechanisms for the control of actin filament length and for the capture of filament barbed ends in cells. PMID:26566078

  13. Protoplast preparation and reversion to the normal filamentous growth in antibiotic-producing uncommon actinomycetes.

    PubMed

    Marcone, Giorgia Letizia; Carrano, Lucia; Marinelli, Flavia; Beltrametti, Fabrizio

    2010-02-01

    Protoplast preparation, regeneration and fusion represent essential tools for those poorly studied biotechnologically valuable microorganisms inapplicable with the current molecular biology protocols. The protoplast production and regeneration method developed for Planobispora rosea and using the combination of hen egg-white lysozyme (HEWL) and Streptomyces globisporus mutanolysin was applied to a set of antibiotic-producing filamentous actinomycetes belonging to the Streptosporangiaceae, Micromonosporaceae and Streptomycetaceae. 10(7)-10(9) protoplasts were obtained from 100 ml of culture, after incubation times in the digestion solution ranging from a few hours to 1 or 2 days depending on the strain. The efficiency of protoplast reversion to the normal filamentous growth varied from 0.1 to nearly 50%. Analysis of cell wall peptidoglycan in three representative strains (Nonomuraea sp. ATCC 39727, Actinoplanes teichomyceticus ATCC 31121 and Streptomyces coelicolor A3(2)) has evidenced structural variations in the glycan strand and in the peptide chain, which may account for the different response to cell digestion and protoplast regeneration treatments. PMID:20057514

  14. Mechanosensitive kinetic preference of actin-binding protein to actin filament

    NASA Astrophysics Data System (ADS)

    Inoue, Yasuhiro; Adachi, Taiji

    2016-04-01

    The kinetic preference of actin-binding proteins to actin filaments is altered by external forces on the filament. Such an altered kinetic preference is largely responsible for remodeling the actin cytoskeletal structure in response to intracellular forces. During remodeling, actin-binding proteins and actin filaments interact under isothermal conditions, because the cells are homeostatic. In such a temperature homeostatic state, we can rigorously and thermodynamically link the chemical potential of actin-binding proteins to stresses on the actin filaments. From this relationship, we can construct a physical model that explains the force-dependent kinetic preference of actin-binding proteins to actin filaments. To confirm the model, we have analyzed the mechanosensitive alternation of the kinetic preference of Arp2/3 and cofilin to actin filaments. We show that this model captures the qualitative responses of these actin-binding proteins to the forces, as observed experimentally. Moreover, our theoretical results demonstrate that, depending on the structural parameters of the binding region, actin-binding proteins can show different kinetic responses even to the same mechanical signal tension, in which the double-helix nature of the actin filament also plays a critical role in a stretch-twist coupling of the filament.

  15. Reversibility and efficiency in coding protein information.

    PubMed

    Tamir, Boaz; Priel, Avner

    2010-12-21

    Why the genetic code has a fixed length? Protein information is transferred by coding each amino acid using codons whose length equals 3 for all amino acids. Hence the most probable and the least probable amino acid get a codeword with an equal length. Moreover, the distributions of amino acids found in nature are not uniform and therefore the efficiency of such codes is sub-optimal. The origins of these apparently non-efficient codes are yet unclear. In this paper we propose an a priori argument for the energy efficiency of such codes resulting from their reversibility, in contrast to their time inefficiency. Such codes are reversible in the sense that a primitive processor, reading three letters in each step, can always reverse its operation, undoing its process. We examine the codes for the distributions of amino acids that exist in nature and show that they could not be both time efficient and reversible. We investigate a family of Zipf-type distributions and present their efficient (non-fixed length) prefix code, their graphs, and the condition for their reversibility. We prove that for a large family of such distributions, if the code is time efficient, it could not be reversible. In other words, if pre-biotic processes demand reversibility, the protein code could not be time efficient. The benefits of reversibility are clear: reversible processes are adiabatic, namely, they dissipate a very small amount of energy. Such processes must be done slowly enough; therefore time efficiency is non-important. It is reasonable to assume that early biochemical complexes were more prone towards energy efficiency, where forward and backward processes were almost symmetrical. PMID:20868696

  16. How capping protein enhances actin filament growth and nucleation on biomimetic beads

    NASA Astrophysics Data System (ADS)

    Wang, Ruizhe; Carlsson, Anders E.

    2015-12-01

    Capping protein (CP), which caps the growing ends of actin filaments, accelerates actin-based motility. Recent experiments on biomimetic beads have shown that CP also enhances the rate of actin filament nucleation. Proposed explanations for these phenomena include (i) the actin funneling hypothesis (AFH), in which the presence of CP increases the free-actin concentration, and (ii) the monomer gating model, in which CP binding to actin filament barbed ends makes more monomers available for filament nucleation. To establish how CP increases the rates of filament elongation and nucleation on biomimetic beads, we perform a quantitative modeling analysis of actin polymerization, using rate equations that include actin filament nucleation, polymerization and capping, as modified by monomer depletion near the surface of the bead. With one adjustable parameter, our simulation results match previously measured time courses of polymerized actin and filament number. The results support a version of the AFH where CP increases the local actin monomer concentration at the bead surface, but leaves the global free-actin concentration nearly constant. Because the rate of filament nucleation increases with the monomer concentration, the increased local monomer concentration enhances actin filament nucleation. We derive a closed-form formula for the characteristic CP concentration where the local free-actin concentration reaches half the bulk value, and find it to be comparable to the global Arp2/3 complex concentration. We also propose an experimental protocol for distinguishing branching nucleation of filaments from spontaneous nucleation.

  17. The adaptive metabolic response involves specific protein glutathionylation during the filamentation process in the pathogen Candida albicans.

    PubMed

    Gergondey, R; Garcia, C; Serre, V; Camadro, J M; Auchère, F

    2016-07-01

    Candida albicans is an opportunist pathogen responsible for a large spectrum of infections, from superficial mycosis to the systemic disease candidiasis. Its ability to adopt various morphological forms, such as unicellular yeasts, filamentous pseudohyphae and hyphae, contributes to its ability to survive within the host. It has been suggested that the antioxidant glutathione is involved in the filamentation process. We investigated S-glutathionylation, the reversible binding of glutathione to proteins, and the functional consequences on C. albicans metabolic remodeling during the yeast-to-hyphae transition. Our work provided evidence for the specific glutathionylation of mitochondrial proteins involved in bioenergetics pathways in filamentous forms and a regulation of the main enzyme of the glyoxylate cycle, isocitrate lyase, by glutathionylation. Isocitrate lyase inactivation in the hyphal forms was reversed by glutaredoxin treatment, in agreement with a glutathionylation process, which was confirmed by proteomic data showing the binding of one glutathione molecule to the enzyme (data are available via ProteomeXchange with identifier PXD003685). We also assessed the effect of alternative carbon sources on glutathione levels and isocitrate lyase activity. Changes in nutrient availability led to morphological flexibility and were related to perturbations in glutathione levels and isocitrate lyase activity, confirming the key role of the maintenance of intracellular redox status in the adaptive metabolic strategy of the pathogen. PMID:27083931

  18. Reverse Phase Protein Arrays for Compound Profiling.

    PubMed

    Moerke, Nathan; Fallahi-Sichani, Mohammad

    2016-01-01

    Reverse phase protein arrays (RPPAs), also called reverse phase lysate arrays (RPLAs), involve immobilizing cell or tissue lysates, in small spots, onto solid supports which are then probed with primary antibodies specific for proteins or post-translational modifications of interest. RPPA assays are well suited for large-scale, high-throughput measurement of protein and PTM levels in cells and tissues. RPPAs are affordable and highly multiplexable, as a large number of arrays can readily be produced in parallel and then probed separately with distinct primary antibodies. This article describes a procedure for treating cells and preparing cell lysates, as well as a procedure for generating RPPAs using these lysates. A method for probing, imaging, and analyzing RPPAs is also described. These procedures are readily adaptable to a wide range of studies of cell signaling in response to drugs and other perturbations. © 2016 by John Wiley & Sons, Inc. PMID:27622568

  19. High Resolution Characterization of Myosin IIC Protein Tailpiece and Its Effect on Filament Assembly

    PubMed Central

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

    2013-01-01

    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 Thr1957 or Thr1960 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. PMID:23426373

  20. Skelemin, a cytoskeletal M-disc periphery protein, contains motifs of adhesion/recognition and intermediate filament proteins.

    PubMed

    Price, M G; Gomer, R H

    1993-10-15

    In striated muscle, myofibrils are anchored to an interconnecting cytoskeleton of desmin intermediate filaments. Skelemin (195 kDa) may be a link between myofibrils and the intermediate filament cytoskeleton. Skelemin partitions with desmin to the insoluble cytoskeleton, and increases the thickness of reconstituted intermediate filaments. Concentrated at the M-disc periphery, skelemin may also contact myosin filaments. We used immunoscreening to isolate a mouse muscle cDNA which encodes a protein with a calculated molecular mass of 185 kDa. Anti-skelemin antibodies bound to the protein products of each of three nonoverlapping regions of the open reading frame. Antibodies directed against the protein products of each one-third of the cDNA react with a 195-kDa muscle protein and stain the M-disc indistinguishably from the original anti-skelemin antibodies, suggesting that the cDNA encodes skelemin. A single skelemin mRNA is detected in muscle but not non-muscle tissues, consistent with immunostaining results. Skelemin is a member of a family of myosin-associated proteins containing fibronectin type III and immunoglobulin superfamily C2 motifs. Skelemin is unique in this family in having intermediate filament core-like motifs, one near each terminus. We hypothesize that skelemin could interact with myosin or myosin-associated proteins through its fibronectin and/or immunoglobulin motifs, and with intermediate filaments through intermediate filament-like motifs. PMID:8408035

  1. Astrocytoma grade IV (glioblastoma multiforme) displays 3 subtypes with unique expression profiles of intermediate filament proteins.

    PubMed

    Skalli, Omar; Wilhelmsson, Ulrika; Orndahl, Charlotte; Fekete, Boglarka; Malmgren, Kristina; Rydenhag, Bertil; Pekny, Milos

    2013-10-01

    Astrocytoma grade IV (glioblastoma multiforme) is the most common and most malignant tumor of the central nervous system and is currently noncurable. Here, we have examined a population-based cohort of 47 patients with grade IV astrocytoma, who underwent tumor surgery at Sahlgrenska University Hospital in Sweden and who survived after surgery for less than 200 days (short survivors, 28 patients) and more than 500 days (long survivors, 19 patients). For each tumor, we ascertained information on patient age, sex, tumor location, oncological treatment, and survival after surgery. The analysis of the tumor volume and the extent of tumor resection (incomplete versus complete resection of the macroscopic tumor) was made retrospectively from the preoperative radiological investigations and, when available, also from postoperative radiology. We performed semiquantitative immunohistochemical evaluation of the presence of intermediate filament (nanofilament) proteins glial fibrillary acidic protein, vimentin, nestin, and synemin in tumor cells. The intermediate filament system helps cells and tissues to cope with various types of stress, and thus, it might affect the malignant potential of grade IV astrocytoma. We propose a subclassification of astrocytomas grade IV with respect to the expression of the intermediate filament proteins glial fibrillary acidic protein, vimentin, nestin, and synemin, namely, type A, B, and C. Our results suggest that the expression of the intermediate filament proteins glial fibrillary acidic protein, vimentin, nestin, and synemin is coregulated in grade IV astrocytomas. The expression patterns of the intermediate filament proteins in astrocytoma type A, B, and C might have biological and clinical significance. PMID:23791210

  2. Antagonism between Ena/VASP proteins and actin filament capping regulates fibroblast motility.

    PubMed

    Bear, James E; Svitkina, Tatyana M; Krause, Matthias; Schafer, Dorothy A; Loureiro, Joseph J; Strasser, Geraldine A; Maly, Ivan V; Chaga, Oleg Y; Cooper, John A; Borisy, Gary G; Gertler, Frank B

    2002-05-17

    Cell motility requires lamellipodial protrusion, a process driven by actin polymerization. Ena/VASP proteins accumulate in protruding lamellipodia and promote the rapid actin-driven motility of the pathogen Listeria. In contrast, Ena/VASP negatively regulate cell translocation. To resolve this paradox, we analyzed the function of Ena/VASP during lamellipodial protrusion. Ena/VASP-deficient lamellipodia protruded slower but more persistently, consistent with their increased cell translocation rates. Actin networks in Ena/VASP-deficient lamellipodia contained shorter, more highly branched filaments compared to controls. Lamellipodia with excess Ena/VASP contained longer, less branched filaments. In vitro, Ena/VASP promoted actin filament elongation by interacting with barbed ends, shielding them from capping protein. We conclude that Ena/VASP regulates cell motility by controlling the geometry of actin filament networks within lamellipodia. PMID:12086607

  3. Protein-protein interactions in reversibly assembled nanopatterns.

    PubMed

    Rakickas, Tomas; Gavutis, Martynas; Reichel, Annett; Piehler, Jacob; Liedberg, Bo; Valiokas, Ramūnas

    2008-10-01

    We describe herein a platform to study protein-protein interactions and to form functional protein complexes in nanoscopic surface domains. For this purpose, we employed multivalent chelator (MCh) templates, which were fabricated in a stepwise procedure combining dip-pen nanolithography (DPN) and molecular recognition-directed assembly. First, we demonstrated that an atomic force microscope (AFM) tip inked with an oligo(ethylene glycol) (OEG) disulfide compound bearing terminal biotin groups can be used to generate biotin patterns on gold achieving line widths below 100 nm, a generic platform for fabrication of functional nanostructures via the highly specific biotin-streptavidin recognition. Subsequently, we converted such biotin/streptavidin patterns into functional MCh patterns for reversible assembly of histidine-tagged (His-tagged) proteins via the attachment of a tris-nitriloacetic acid (trisNTA) biotin derivative. Fluorescence microscopy confirmed reversible immobilization of the receptor subunit ifnar2-His10 and its interaction with interferon-alpha2 labeled with fluorescent quantum dots in a 7 x 7 dot array consisting of trisNTA spots with a diameter of approximately 230 nm. Moreover, we carried out characterization of the specificity, stability, and reversibility as well as quantitative real-time analysis of protein-protein interactions at the fabricated nanopatterns by imaging surface plasmon resonance. Our work offers a route for construction and analysis of functional protein-based nanoarchitectures. PMID:18788824

  4. Assembly of glial intermediate filament protein is initiated in the centriolar region.

    PubMed

    Kalnins, V I; Subrahmanyan, L; Fedoroff, S

    1985-10-21

    Assembly of glial intermediate filament protein (GFP) into intermediate filaments (IF) was first detected by immunofluorescence in the perinuclear region of astrocytes differentiating in colony cultures before the rest of the cytoplasm was labeled. Double labeling with antisera specific for centrioles indicated that this site corresponds to the centriolar region. These studies suggest that the centriolar region plays an important role in the assembly of some types of IF as well as microtubules. PMID:3899284

  5. 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

  6. 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

  7. 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. PMID:3509996

  8. Structural characterization of a capping protein interaction motif defines a family of actin filament regulators

    PubMed Central

    Hernandez-Valladares, Maria; Kim, Taekyung; Kannan, Balakrishnan; Tung, Alvin; Aguda, Adeleke H; Larsson, Mårten; Cooper, John A; Robinson, Robert C

    2011-01-01

    Capping protein (CP) regulates actin dynamics by binding the barbed ends of actin filaments. Removal of CP may be one means to harness actin polymerization for processes such as cell movement and endocytosis. Here we structurally and biochemically investigated a CP interaction (CPI) motif present in the otherwise unrelated proteins CARMIL and CD2AP. The CPI motif wraps around the stalk of the mushroom-shaped CP at a site distant from the actin-binding interface, which lies on the top of the mushroom cap. We propose that the CPI motif may act as an allosteric modulator, restricting CP to a low-affinity, filament-binding conformation. Structure-based sequence alignments extend the CPI motif–containing family to include CIN85, CKIP-1, CapZIP and a relatively uncharacterized protein, WASHCAP (FAM21). Peptides comprising these CPI motifs are able to inhibit CP and to uncap CP-bound actin filaments. PMID:20357771

  9. 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.

  10. 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

  11. Kinetic theory of protein filament growth: Self-consistent methods and perturbative techniques

    NASA Astrophysics Data System (ADS)

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

    2015-12-01

    Filamentous protein structures are of high relevance for the normal functioning of the cell, where they provide the structural component for the cytoskeleton, but are also implicated in the pathogenesis of many disease states. The self-assembly of these supra-molecular structures from monomeric proteins has been studied extensively in the past 50 years and much interest has focused on elucidating the microscopic events that drive linear growth phenomena in a biological setting. Master equations have proven to be particularly fruitful in this context, allowing specific assembly mechanisms to be linked directly to experimental observations of filamentous growth. Recently, these approaches have increasingly been applied to aberrant protein polymerization, elucidating potential implications for controlling or combating the formation of pathological filamentous structures. This article reviews recent theoretical advances in the field of filamentous growth phenomena through the use of the master-equation formalism. We use perturbation and self-consistent methods for obtaining analytical solutions to the rate equations describing fibrillar growth and show how the resulting closed-form expressions can be used to shed light on the general physical laws underlying this complex phenomenon. We also present a connection between the underlying ideas of the self-consistent analysis of filamentous growth and the perturbative renormalization group.

  12. Characterization of distinct early assembly units of different intermediate filament proteins.

    PubMed

    Herrmann, H; Häner, M; Brettel, M; Ku, N O; Aebi, U

    1999-03-12

    We have determined the mass-per-length (MPL) composition of distinct early assembly products of recombinant intermediate filament (IF) proteins from the four cytoplasmic sequence homology classes, and compared these values with those of the corresponding mature filaments. After two seconds under standard assembly conditions (i.e. 25 mM Tris-HCl (pH 7.5), 50 mM NaCl, 37 degrees C), vimentin, desmin and the neurofilament triplet protein NF-L aggregated into similar types of "unit-length filaments" (ULFs), whereas cytokeratins (CKs) 8/18 already yielded long IFs at this time point, so the ionic strength had to be reduced. The number of molecules per filament cross-section, as deduced from the MPL values, was lowest for CK8/18, i.e. 16 and 25 at two seconds compared to 16 and 21 at one hour. NF-L exhibited corresponding values of 26 and 30. Vimentin ULFs yielded a pronounced heterogeneity, with major peak values of 32 and 45 at two seconds and 30, 37 and 44 after one hour. Desmin formed filaments of distinctly higher mass with 47 molecules per cross-section, at two seconds and after one hour of assembly. This indicates that individual types of IF proteins generate filaments with distinctly different numbers of molecules per cross-section. Also, the observed significant reduction of apparent filament diameter of ULFs compared to the corresponding mature IFs is the result of a "conservative" radial compaction-type reorganization within the filament, as concluded from the fact that both the immature and mature filaments contain very similar numbers of subunits per cross-section. Moreover, the MPL composition of filaments is strikingly dependent on the assembly conditions employed. For example, vimentin fibers formed in 0.7 mM phosphate (pH 7.5), 2.5 mM MgCl2, yield a significantly increased number of molecules per cross-section (56 and 84) compared to assembly under standard conditions. Temperature also strongly influences assembly: above a certain threshold temperature

  13. Drosophila quail, a villin-related protein, bundles actin filaments in apoptotic nurse cells.

    PubMed

    Matova, N; Mahajan-Miklos, S; Mooseker, M S; Cooley, L

    1999-12-01

    Drosophila Quail protein is required for the completion of fast cytoplasm transport from nurse cells to the oocyte, an event critical for the production of viable oocytes. The abundant network of cytoplasmic filamentous actin, established at the onset of fast transport, is absent in quail mutant egg chambers. Previously, we showed that Quail is a germline-specific protein with sequence homology to villin, a vertebrate actin-regulating protein. In this study, we combined biochemical experiments with observations in egg chambers to define more precisely the function of this protein in the regulation of actin-bundle assembly in nurse cells. We report that recombinant Quail can bind and bundle filamentous actin in vitro in a manner similar to villin at a physiological calcium concentration. In contrast to villin, Quail is unable to sever or cap filamentous actin, or to promote nucleation of new actin filaments at a high calcium concentration. Instead, Quail bundles the filaments regardless of the calcium concentration. In vivo, the assembly of nurse-cell actin bundles is accompanied by extensive perforation of the nurse-cell nuclear envelopes, and both of these phenomena are manifestations of nurse-cell apoptosis. To investigate whether free calcium levels are affected during apoptosis, we loaded egg chambers with the calcium indicator Indo-1. Our observations indicate a rise in free calcium in the nurse-cell cytoplasm coincident with the permeabilization of the nuclear envelopes. We also show that human villin expressed in the Drosophila germline could sense elevated cytoplasmic calcium; in nurse cells with reduced levels of Quail protein, villin interfered with actin-bundle stability. We conclude that Quail efficiently assembles actin filaments into bundles in nurse cells and maintains their stability under fluctuating free calcium levels. We also propose a developmental model for the fast phase of cytoplasm transport incorporating findings presented in this study

  14. Saturable binding of the echinoderm microtubule-associated protein (EMAP) on microtubules, but not filamentous actin or vimentin filaments.

    PubMed

    Eichenmüller, B; Ahrens, D P; Li, Q; Suprenant, K A

    2001-11-01

    The echinoderm microtubule-associated protein (EMAP) is a 75-kDa, WD-repeat protein associated with the mitotic spindle apparatus. To understand EMAP's biological role, it is important to determine its affinity for microtubules (MTs) and other cytoskeletal components. To accomplish this goal, we utilized a low-cost, bubble-column bioreactor to express EMAP as a hexahistidine fusion (6his) protein in baculovirus-infected insect cells. After optimizing cell growth conditions, up to 30 mg of EMAP was obtained in the soluble cell lysate from a 1-liter culture. EMAP was purified to homogeneity in a two-step process that included immobilized metal-affinity chromatography (IMAC) and anion-exchange chromatography. In vitro binding studies on cytoskeletal components were performed with the 6his-EMAP. EMAP bound to MTs, but not actin or vimentin filaments, with an intrinsic dissociation constant of 0.18 microM and binding stoichiometry of 0.7 mol EMAP per mol tubulin heterodimer. In addition, we show that a strong MT binding domain resides in the 137 amino acid, NH(2)-terminus of EMAP and a weaker binding site in the WD-domain. Previous work has shown that the EMAP concentration in the sea urchin egg is over 4 microM. Together, these results show that there is sufficient EMAP in the egg to regulate the assembly of a large pool of maternally stored tubulin. PMID:11807937

  15. 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

  16. In situ detection of protein-DNA interactions in filamentous fungi by in vivo footprinting.

    PubMed Central

    Wolschek, M F; Narendja, F; Karlseder, J; Kubicek, C P; Scazzocchio, C; Strauss, J

    1998-01-01

    The method described here allows the detection of protein-DNA interactions in vivo in filamentous fungi. We outline culture conditions and conditions of in vivo methylation that permit uniform modification of all cells in an apically growing, non-uniform organism, and subsequent visualization of protected areas by ligation-mediated PCR. PMID:9685506

  17. 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

  18. Frequency Factors in a Landscape Model of Filamentous Protein Aggregation

    NASA Astrophysics Data System (ADS)

    Buell, Alexander K.; Jamie R. Blundell; Dobson, Christopher M.; Welland, Mark E.; Terentjev, Eugene M.; Knowles, Tuomas P. J.

    2010-06-01

    Using quantitative measurements of protein aggregation rates, we develop a kinetic picture of protein conversion from a soluble to a fibrillar state which shows that a single free energy barrier to aggregation controls the addition of protein molecules into amyloid fibrils, while the characteristic sublinear concentration dependence emerges as a natural consequence of finite diffusion times. These findings suggest that this reaction does not follow a simple chemical mechanism, but rather operates in a way analogous to the landscape models of protein folding defined by stochastic dynamics on a characteristic energy surface.

  19. Length regulation of mechanosensitive stereocilia depends on very slow actin dynamics and filament-severing proteins.

    PubMed

    Narayanan, Praveena; Chatterton, Paul; Ikeda, Akihiro; Ikeda, Sakae; Corey, David P; Ervasti, James M; Perrin, Benjamin J

    2015-01-01

    Auditory sensory hair cells depend on stereocilia with precisely regulated lengths to detect sound. Since stereocilia are primarily composed of crosslinked, parallel actin filaments, regulated actin dynamics are essential for controlling stereocilia length. Here we assessed stereocilia actin turnover by monitoring incorporation of inducibly expressed β-actin-GFP in adult mouse hair cells in vivo and by directly measuring β-actin-GFP turnover in explants. Stereocilia actin incorporation is remarkably slow and restricted to filament barbed ends in a small tip compartment, with minimal accumulation in the rest of the actin core. Shorter rows of stereocilia, which have mechanically gated ion channels, show more variable actin turnover than the tallest stereocilia, which lack channels. Finally, the proteins ADF and AIP1, which both mediate actin filament severing, contribute to stereocilia length maintenance. Altogether, the data support a model whereby stereocilia actin cores are largely static, with dynamic regulation at the tips to maintain a critical length. PMID:25897778

  20. A Candida albicans cell wall-linked protein promotes invasive filamentation into semi-solid medium

    PubMed Central

    Zucchi, Paola C.; Davis, Talya R.; Kumamoto, Carol A.

    2011-01-01

    SUMMARY Growth of cells in contact with an abiotic or biological surface profoundly affects cellular physiology. In the opportunistic human pathogen, Candida albicans, growth on a semi-solid matrix such as agar results in invasive filamentation, a process in which cells change their morphology to highly elongated filamentous hyphae that grow into the matrix. We hypothesized that a plasma membrane receptor-type protein would sense the presence of matrix and activate a signal transduction cascade, thus promoting invasive filamentation. In this communication, we demonstrate that during growth in contact with a semi-solid surface, activation of a MAP kinase, Cek1p, is promoted, in part, by a plasma membrane protein termed Dfi1p and results in invasive filamentation. A C. albicans mutant lacking Dfi1p showed reduced virulence in a murine model of disseminated candidiasis. Dfi1p is a relatively small, integral membrane protein that localizes to the plasma membrane. Some Dfi1p molecules become cross-linked to the carbohydrate polymers of the cell wall. Thus, Dfi1p is capable of linking the cell wall to the plasma membrane and cytoplasm. PMID:20384695

  1. Capping Protein Modulates the Dynamic Behavior of Actin Filaments in Response to Phosphatidic Acid in Arabidopsis[C][W

    PubMed Central

    Li, Jiejie; Henty-Ridilla, Jessica L.; Huang, Shanjin; Wang, Xia; Blanchoin, Laurent; Staiger, Christopher J.

    2012-01-01

    Remodeling of actin filament arrays in response to biotic and abiotic stimuli is thought to require precise control over the generation and availability of filament ends. Heterodimeric capping protein (CP) is an abundant filament capper, and its activity is inhibited by membrane signaling phospholipids in vitro. How exactly CP modulates the properties of filament ends in cells and whether its activity is coordinated by phospholipids in vivo is not well understood. By observing directly the dynamic behavior of individual filament ends in the cortical array of living Arabidopsis thaliana epidermal cells, we dissected the contribution of CP to actin organization and dynamics in response to the signaling phospholipid, phosphatidic acid (PA). Here, we examined three cp knockdown mutants and found that reduced CP levels resulted in more dynamic activity at filament ends, and this significantly enhanced filament-filament annealing and filament elongation from free ends. The cp mutants also exhibited more dense actin filament arrays. Treatment of wild-type cells with exogenous PA phenocopied the actin-based defects in cp mutants, with an increase in the density of filament arrays and enhanced annealing frequency. These cytoskeletal responses to exogenous PA were completely abrogated in cp mutants. Our data provide compelling genetic evidence that the end-capping activity of CP is inhibited by membrane signaling lipids in eukaryotic cells. Specifically, CP acts as a PA biosensor and key transducer of fluxes in membrane signaling phospholipids into changes in actin cytoskeleton dynamics. PMID:22960908

  2. Characterization of the cytoplasmic filament protein gene (cfpA) of Treponema pallidum subsp. pallidum.

    PubMed Central

    You, Y; Elmore, S; Colton, L L; Mackenzie, C; Stoops, J K; Weinstock, G M; Norris, S J

    1996-01-01

    Treponema pallidum and other members of the genera Treponema, Spirochaeta, and Leptonema contain multiple cytoplasmic filaments that run the length of the organism just underneath the cytoplasmic membrane. These cytoplasmic filaments have a ribbon-like profile and consist of a major cytoplasmic filament protein subunit (CfpA, formerly called TpN83) with a relative molecular weight of approximately 80,000. Degenerate DNA primers based on N-terminal and CNBr cleavage fragment amino acid sequences of T. pallidum subsp. pallidum (Nichols) CfpA were utilized to amplify a fragment of the encoding gene (cfpA). A 6.8-kb EcoRI fragment containing all but the 5' end of cfpA was identified by hybridization with the resulting PCR product and cloned into Lambda ZAP II. The 5' region was obtained by inverse PCR, and the complete gene sequence was determined. The cfpA sequence contained a 2,034-nucleotide coding region, a putative promoter with consensus sequences (5'-TTTACA-3' for -35 and 5'-TACAAT-3' for -10) similar to the sigma70 recognition sequence of Escherichia coli and other organisms, and a putative ribosome-binding site (5'-AGGAG-3'). The deduced amino acid sequence of CfpA indicated a protein of 678 residues with a calculated molecular mass of 78.5 kDa and an estimated pI of 6.15. No significant homology to known proteins or structural motifs was found among known prokaryotic or eukaryotic sequences. Expression of a LacZ-CfpA fusion protein in E. coli was detrimental to survival and growth of the host strain and resulted in the formation of short, irregular filaments suggestive of partial self-assembly of CfpA. The cytoplasmic filaments of T. pallidum and other spirochetes appear to represent a unique form of prokaryotic intracytoplasmic inclusions. PMID:8655496

  3. The N-terminal domain of MuB protein has striking structural similarity to DNA-binding domains and mediates MuB filament-filament interactions.

    PubMed

    Dramićanin, Marija; López-Méndez, Blanca; Boskovic, Jasminka; Campos-Olivas, Ramón; Ramón-Maiques, Santiago

    2015-08-01

    MuB is an ATP-dependent DNA-binding protein that regulates the activity of MuA transposase and delivers the target DNA for transposition of phage Mu. Mechanistic insight into MuB function is limited to its AAA+ ATPase module, which upon ATP binding assembles into helical filaments around the DNA. However, the structure and function of the flexible N-terminal domain (NTD) appended to the AAA+ module remains uncharacterized. Here we report the solution structure of MuB NTD determined by NMR spectroscopy. The structure reveals a compact domain formed by four α-helices connected by short loops, and confirms the presence of a helix-turn-helix motif. High structural similarity and sequence homology with λ repressor-like DNA-binding domains suggest a possible role of MuB NTD in DNA binding. We also demonstrate that the NTD directly mediates the ability of MuB to establish filament-filament interactions. These findings lead us to a model in which the NTD interacts with the AAA+ spirals and perhaps also with the DNA bound within the filament, favoring MuB polymerization and filament clustering. We propose that the MuB NTD-dependent filament interactions might be an effective mechanism to bridge distant DNA regions during Mu transposition. PMID:26169224

  4. alpha-Internexin, a 66-kD intermediate filament-binding protein from mammalian central nervous tissues.

    PubMed

    Pachter, J S; Liem, R K

    1985-10-01

    In this paper we describe a 66-kD protein that co-purifies with intermediate filaments from rat optic nerve and spinal cord but can be separated further by ion-exchange chromatography. This protein is distinct from the 68-kD neurofilament subunit protein as judged by isoelectric focusing, immunoblotting, peptide mapping, and tests of polymerization competence. This protein is avidly recognized by the monoclonal anti-intermediate filament antigen antibody, previously demonstrated to recognize a common antigenic determinant in all five known classes of intermediate filaments. Also, when isolated this protein binds to various intermediate filament subunit proteins, which suggests an in vivo interaction with the intermediate filament cytoskeleton, and it appears to be axonally transported in the rat optic nerve. Because of this ability to bind to intermediate filaments in situ and in vitro we have named this protein alpha-internexin. A possible functional role for the protein in organizing filament assembly and distribution is discussed. PMID:2413040

  5. Comparative analysis of nanomechanics of protein filaments under lateral loading

    NASA Astrophysics Data System (ADS)

    Solar, Max; Buehler, Markus J.

    2012-02-01

    Using a combination of explicit solvent atomistic simulation and continuum theory, here we study the lateral deformation mechanics of three distinct protein structures: an amyloid fibril, a beta helix, and an alpha helix. We find that the two β-sheet rich structures - amyloid fibril and beta helix, with persistence lengths on the order of μm - are well described by continuum mechanical theory, but differ in the degree to which shear deformation affects the overall bending behavior. The alpha helical protein structure, however, with a persistence length on the order of one nanometer, does not conform to the continuum theory and its deformation is dominated by entropic elasticity due to significant fluctuations. This study provides fundamental insight into the nanomechanics of widely found protein motifs and insight into molecular-scale deformation mechanisms, as well as quantitative estimates of Young's modulus and shear modulus in agreement with experimental results.

  6. 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

  7. Reaction-diffusion waves of reversible actin filament assembly drive cell oscillations and locomotion

    NASA Astrophysics Data System (ADS)

    Vicker, Michael G.

    Excitation waves of actin filament (F-actin) polymerization and depolymerization have been visualized in fixed and in living Dictyostelium cells by confocal and fluorescence resonance energy transfer (FRET) microscopy. F-actin waves generate supramolecular F-actin patterns, typical of chemical wave systems. Scroll waves distinguishable as sphere, ring and spiral patterns propagate up to several micrometres in diameter in a few seconds at wavefront speeds measured at up to 25 µm/min. These newly identified nonlinear F-actin dynamics drive eukaryotic cell locomotion. F-actin autowaves also induce oscillatory modi of temporally variable frequency and amplitude as cell surface projections, including pseudopodia and lamellipodia, which may traverse the cell surface as waves. F-actin waves may also govern a range of cell functions and behaviours, including phagocytosis, chemotaxis, cell surface receptor activity and biological rhythms.

  8. 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

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

    PubMed

    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

  10. Target Molecular Simulations of RecA Family Protein Filaments

    PubMed Central

    Su, Zhi-Yuan; Lee, Wen-Jay; Su, Wan-Sheng; Wang, Yeng-Tseng

    2012-01-01

    Modeling of the RadA family mechanism is crucial to understanding the DNA SOS repair process. In a 2007 report, the archaeal RadA proteins function as rotary motors (linker region: I71-K88) such as shown in Figure 1. Molecular simulations approaches help to shed further light onto this phenomenon. We find 11 rotary residues (R72, T75-K81, M84, V86 and K87) and five zero rotary residues (I71, K74, E82, R83 and K88) in the simulations. Inclusion of our simulations may help to understand the RadA family mechanism. PMID:22837683

  11. Folding and Stabilization of Native-Sequence-Reversed Proteins

    PubMed Central

    Zhang, Yuanzhao; Weber, Jeffrey K; Zhou, Ruhong

    2016-01-01

    Though the problem of sequence-reversed protein folding is largely unexplored, one might speculate that reversed native protein sequences should be significantly more foldable than purely random heteropolymer sequences. In this article, we investigate how the reverse-sequences of native proteins might fold by examining a series of small proteins of increasing structural complexity (α-helix, β-hairpin, α-helix bundle, and α/β-protein). Employing a tandem protein structure prediction algorithmic and molecular dynamics simulation approach, we find that the ability of reverse sequences to adopt native-like folds is strongly influenced by protein size and the flexibility of the native hydrophobic core. For β-hairpins with reverse-sequences that fail to fold, we employ a simple mutational strategy for guiding stable hairpin formation that involves the insertion of amino acids into the β-turn region. This systematic look at reverse sequence duality sheds new light on the problem of protein sequence-structure mapping and may serve to inspire new protein design and protein structure prediction protocols. PMID:27113844

  12. Folding and Stabilization of Native-Sequence-Reversed Proteins.

    PubMed

    Zhang, Yuanzhao; Weber, Jeffrey K; Zhou, Ruhong

    2016-01-01

    Though the problem of sequence-reversed protein folding is largely unexplored, one might speculate that reversed native protein sequences should be significantly more foldable than purely random heteropolymer sequences. In this article, we investigate how the reverse-sequences of native proteins might fold by examining a series of small proteins of increasing structural complexity (α-helix, β-hairpin, α-helix bundle, and α/β-protein). Employing a tandem protein structure prediction algorithmic and molecular dynamics simulation approach, we find that the ability of reverse sequences to adopt native-like folds is strongly influenced by protein size and the flexibility of the native hydrophobic core. For β-hairpins with reverse-sequences that fail to fold, we employ a simple mutational strategy for guiding stable hairpin formation that involves the insertion of amino acids into the β-turn region. This systematic look at reverse sequence duality sheds new light on the problem of protein sequence-structure mapping and may serve to inspire new protein design and protein structure prediction protocols. PMID:27113844

  13. 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

  14. Reversible and irreversible protein glutathionylation: biological and clinical aspects

    PubMed Central

    Cooper, Arthur J L.; Pinto, John T.; Callery, Patrick S.

    2011-01-01

    Introduction Depending in part on the glutathione to glutathione disulfide ratio, reversible protein glutathionylation to a mixed disulfide may occur. Reversible glutathionylation is important in protecting proteins against oxidative stress, guiding correct protein folding, regulating protein activity, and modulating proteins critical to redox signaling. The potential also exists for irreversible protein glutathionylation via Michael addition of an -SH group to a dehydroalanyl residue, resulting in formation of a stable, non-reducible thioether linkage. Areas covered This article reviews factors contributing to reversible and irreversible protein glutathionylation and their biomedical implications. It also examines the possibility that certain drugs such as busulfan may be toxic by promoting irreversible glutathionylation. The reader will gain an appreciation of the protective nature and control of function resulting from reversible protein glutathionylation. The reader is also introduced to the recently identified phenomenon of irreversible protein glutathionylation and its possible deleterious effects. Expert opinion The process of reversible protein glutathionylation is now well established but these findings need to be substantiated at the tissue and organ levels, and also with disease state. That being said, irreversible protein glutathionylation can also occur and this has implications in disease and aging. Toxicologists should consider this when evaluating the possible side effects of certain drugs such as busulfan that may generate a glutathionylating species in vivo. PMID:21557709

  15. 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.

  16. (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.

  17. 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

  18. Glial Fibrillary acidic protein: From intermediate filament assembly and gliosis to neurobiomarker

    PubMed Central

    Yang, Zhihui; Wang, Kevin K.W.

    2015-01-01

    Glial fibrillary acidic protein (GFAP) is an intermediate filament-III protein uniquely found in astrocytes in the CNS, non-myelinating Schwann cells in the PNS and enteric glial cells. GFAP mRNA expressions are regulated by several nuclear-receptor hormones, growth factors and lipopolysaccharides. GFAP is also subjected to a number of post-translational modifications while GFAP mutations result in protein deposits known as Rosenthal fibers in Alexander disease. GFAP gene activation and protein induction appear to play a critical role in astroglia cell activation (astrogliosis) following CNS injuries and neurodegeneration. Emerging evidence also suggests that, following traumatic brain and spinal cord injuries and stroke, GFAP protein and its breakdown products are rapidly released into biofluids, making them strong candidate biomarkers for such neurological disorders. PMID:25975510

  19. Cloning and immunologic characterization of a truncated Bordetella bronchiseptica filamentous hemagglutinin fusion protein.

    PubMed

    Keil, D J; Burns, E H; Kisker, W R; Bemis, D; Fenwick, B

    1999-12-10

    Filamentous hemagglutinin (FHA) is an outer-membrane associated adhesin conserved within the genus Bordetella. FHA provides protection against B. pertussis infections in humans and is a component of acellular whooping cough vaccines. Furthermore, FHA serves as a protective antigen in several animal models of infection with B. bronchiseptica and may serve as a protective antigen of canine bordetellosis. In this study, polyclonal anti-B. pertussis FHA antiserum was used to identify an immunoreactive clone from the genomic DNA library of a canine B. bronchiseptica field isolate. The nucleotide and predicted amino acid sequences of the immunoreactive clone were compared to fhaB and FhaB from B. pertussis revealing 94% identity at the nucleic acid level, and 86% identity at the protein level. A truncated fusion protein (FHAt) was prepared which included a conserved domain homologous to the immunodominant region in the FHA of B. pertussis [Leininger E, Bowen S, Renauld-Mongen G, Rouse JH, Menozzi FD, Locht C, Heron I, Brennan MJ. Immunodominant domain present on the Bordetella pertussis vaccine component filamentous hemagglutinin. J. Infect. Dis. 1997;175:1423-1431; Wilson DR, Siebers A, Finlay BB. Antigenic analysis of Bordetella pertussis filamentous hemagglutinin with phage display libraries and rabbit anti-filamentous hemagglutinin polyclonal antibodies. Infect. Immun. 1998;66:4884-4894]. FHAt was shown to be safe and antigenic in rabbits. FHAt induced the formation of antibodies that inhibit the hemagglutination associated with full length B. pertussis FHA, and inhibit adherence of B. bronchisepitca to canine fibroblasts by as much as 65%. This information may have implications for the development of safe and efficacious subunit vaccines for the prevention of canine bordetellosis and may contribute to future acellular whooping cough vaccines. PMID:10580199

  20. Search and Analysis of Identical Reverse Octapeptides in Unrelated Proteins

    PubMed Central

    Saravanan, Konda Mani; Selvaraj, Samuel

    2013-01-01

    For the past few decades, intensive studies have been carried out in an attempt to understand how the amino acid sequences of proteins encode their three dimensional structures to perform their specific functions. In order to understand the sequence-structure relationship of proteins, several sub-sequence search studies in non-redundant sequence-structure databases have been undertaken which have given some fruitful clues. In our earlier work, we analyzed a set of 3124 non-redundant protein sequences from the Protein Data Bank (PDB) and retrieved 30 identical octapeptides having different secondary structures. These octapeptides were characterized by using different computational procedures. This prompted us to explore the presence of octapeptides with reverse sequences and to analyze whether these octapeptides would adopt similar structures as that of their parent octapeptides. Our identical reverse octapeptide search resulted in the finding of eight octapeptide pairs (octapeptide and reverse octapeptide) with similar secondary structure and 23 octapeptide pairs with different secondary structures. In the present work, the geometrical and biophysical characteristics of identical reverse octapeptides were explored and compared with unrelated octapeptide pairs by using various computational tools. We thus conclude that proteins containing identical reverse octapeptides are not very abundant and residues in the octapeptide pairs do not contribute to the stability of the protein. Furthermore, compared to unrelated octapeptides, identical reverse octapeptides do not show certain biophysical and geometrical properties. PMID:23523652

  1. Crystal structure of AFV1-102, a protein from the acidianus filamentous virus 1

    PubMed Central

    Keller, Jenny; Leulliot, Nicolas; Collinet, Bruno; Campanacci, Valerie; Cambillau, Christian; Pranghisvilli, David; van Tilbeurgh, Herman

    2009-01-01

    Viruses infecting hyperthermophilic archaea have intriguing morphologies and genomic properties. The vast majority of their genes do not have homologs other than in other hyperthermophilic viruses, and the biology of these viruses is poorly understood. As part of a structural genomics project on the proteins of these viruses, we present here the structure of a 102 amino acid protein from acidianus filamentous virus 1 (AFV1-102). The structure shows that it is made of two identical motifs that have poor sequence similarity. Although no function can be proposed from structural analysis, tight binding of the gateway tag peptide in a groove between the two motifs suggests AFV1-102 is involved in protein protein interactions. PMID:19319936

  2. 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

  3. 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.

  4. Lanosterol reverses protein aggregation in cataracts.

    PubMed

    Zhao, Ling; Chen, Xiang-Jun; Zhu, Jie; Xi, Yi-Bo; Yang, Xu; Hu, Li-Dan; Ouyang, Hong; Patel, Sherrina H; Jin, Xin; Lin, Danni; Wu, Frances; Flagg, Ken; Cai, Huimin; Li, Gen; Cao, Guiqun; Lin, Ying; Chen, Daniel; Wen, Cindy; Chung, Christopher; Wang, Yandong; Qiu, Austin; Yeh, Emily; Wang, Wenqiu; Hu, Xun; Grob, Seanna; Abagyan, Ruben; Su, Zhiguang; Tjondro, Harry Christianto; Zhao, Xi-Juan; Luo, Hongrong; Hou, Rui; Perry, J Jefferson P; Gao, Weiwei; Kozak, Igor; Granet, David; Li, Yingrui; Sun, Xiaodong; Wang, Jun; Zhang, Liangfang; Liu, Yizhi; Yan, Yong-Bin; Zhang, Kang

    2015-07-30

    The human lens is comprised largely of crystallin proteins assembled into a highly ordered, interactive macro-structure essential for lens transparency and refractive index. Any disruption of intra- or inter-protein interactions will alter this delicate structure, exposing hydrophobic surfaces, with consequent protein aggregation and cataract formation. Cataracts are the most common cause of blindness worldwide, affecting tens of millions of people, and currently the only treatment is surgical removal of cataractous lenses. The precise mechanisms by which lens proteins both prevent aggregation and maintain lens transparency are largely unknown. Lanosterol is an amphipathic molecule enriched in the lens. It is synthesized by lanosterol synthase (LSS) in a key cyclization reaction of a cholesterol synthesis pathway. Here we identify two distinct homozygous LSS missense mutations (W581R and G588S) in two families with extensive congenital cataracts. Both of these mutations affect highly conserved amino acid residues and impair key catalytic functions of LSS. Engineered expression of wild-type, but not mutant, LSS prevents intracellular protein aggregation of various cataract-causing mutant crystallins. Treatment by lanosterol, but not cholesterol, significantly decreased preformed protein aggregates both in vitro and in cell-transfection experiments. We further show that lanosterol treatment could reduce cataract severity and increase transparency in dissected rabbit cataractous lenses in vitro and cataract severity in vivo in dogs. Our study identifies lanosterol as a key molecule in the prevention of lens protein aggregation and points to a novel strategy for cataract prevention and treatment. PMID:26200341

  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. The promoter of filamentation (POF1) protein from Saccharomyces cerevisiae is an ATPase involved in the protein quality control process

    PubMed Central

    2011-01-01

    Background The gene YCL047C, which has been renamed promoter of filamentation gene (POF1), has recently been described as a cell component involved in yeast filamentous growth. The objective of this work is to understand the molecular and biological function of this gene. Results Here, we report that the protein encoded by the POF1 gene, Pof1p, is an ATPase that may be part of the Saccharomyces cerevisiae protein quality control pathway. According to the results, Δpof1 cells showed increased sensitivity to hydrogen peroxide, tert-butyl hydroperoxide, heat shock and protein unfolding agents, such as dithiothreitol and tunicamycin. Besides, the overexpression of POF1 suppressed the sensitivity of Δpct1, a strain that lacks a gene that encodes a phosphocholine cytidylyltransferase, to heat shock. In vitro analysis showed, however, that the purified Pof1p enzyme had no cytidylyltransferase activity but does have ATPase activity, with catalytic efficiency comparable to other ATPases involved in endoplasmic reticulum-associated degradation of proteins (ERAD). Supporting these findings, co-immunoprecipitation experiments showed a physical interaction between Pof1p and Ubc7p (an ubiquitin conjugating enzyme) in vivo. Conclusions Taken together, the results strongly suggest that the biological function of Pof1p is related to the regulation of protein degradation. PMID:22204397

  7. 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

  8. Reverse transcription of the pFOXC mitochondrial retroplasmids of Fusarium oxysporum is protein primed

    PubMed Central

    2011-01-01

    Background The pFOXC retroplasmids are small, autonomously replicating DNA molecules found in mitochondria of certain strains of the filamentous fungus Fusarium oxysporum and are among the first linear genetic elements shown to replicate via reverse transcription. The plasmids have a unique clothespin structure that includes a 5'-linked protein and telomere-like terminal repeats, with pFOXC2 and pFOXC3 having iterative copies of a 5 bp sequence. The plasmids contain a single large open reading frame (ORF) encoding an active reverse transcriptase (RT). The pFOXC-RT is associated with the plasmid transcript in a ribonucleoprotein (RNP) complex and can synthesize full-length (-) strand cDNA products. In reactions containing partially purified RT preparations with exogenous RNAs, the pFOXC3-RT has been shown to initiate cDNA synthesis by use of snapped-back RNAs, as well as loosely associated DNA primers. Results The complete sequence of the distantly related pFOXC1 plasmid was determined and found to terminate in 3-5 copies of a 3 bp sequence. Unexpectedly, the majority of (-) strand cDNA molecules produced from endogenous pFOXC1 transcripts were attached to protein. In vitro experiments using partially purified pFOXC3-RT preparations having a single radiolabeled deoxyribonucleotide triphosphate (dNTP) generated a nucleotide-labeled protein that migrated at the size of the pFOXC-RT. The nucleotide preference of deoxynucleotidylation differed between pFOXC3 and pFOXC1 and showed complementarity to the respective 3' terminal repeats. In reactions that include exogenous RNA templates corresponding to the 3' end of pFOXC1, a protein-linked cDNA product was generated following deoxynucleotidylation, suggesting that reverse transcription initiates with a protein primer. Conclusions The finding that reverse transcription is protein primed suggests the pFOXC retroplasmids may have an evolutionary relationship with hepadnaviruses, the only other retroelement family known to

  9. A novel multitarget tracking algorithm for Myosin VI protein molecules on actin filaments in TIRFM sequences.

    PubMed

    Li, G; Sanchez, V; Nagaraj, P C S B; Khan, S; Rajpoot, N

    2015-12-01

    We propose a novel multitarget tracking framework for Myosin VI protein molecules in total internal reflection fluorescence microscopy sequences which integrates an extended Hungarian algorithm with an interacting multiple model filter. The extended Hungarian algorithm, which is a linear assignment problem based method, helps to solve measurement assignment and spot association problems commonly encountered when dealing with multiple targets, although a two-motion model interacting multiple model filter increases the tracking accuracy by modelling the nonlinear dynamics of Myosin VI protein molecules on actin filaments. The evaluation of our tracking framework is conducted on both real and synthetic total internal reflection fluorescence microscopy sequences. The results show that the framework achieves higher tracking accuracies compared to the state-of-the-art tracking methods, especially for sequences with high spot density. PMID:26259144

  10. Higher order structure of proteins solubilized in AOT reverse micelles.

    PubMed

    Naoe, Kazumitsu; Noda, Kazuki; Kawagoe, Mikio; Imai, Masanao

    2004-11-15

    The higher order structure of proteins solubilized in an bis(2-ethylhexyl) sulfosuccinate sodium (AOT) reverse micellar system was investigated. From circular dichroic (CD) measurement, CD spectra of cytochrome c, which is solubilized at the interface of reverse micelles, markedly changed on going from buffer solution to the reverse micellar solution, and the ellipticity values in the far- and near-UV regions decreased with decreasing the water content (W0: molar ratio of water to AOT), indicating that the secondary and tertiary structures of cytochrome c changed with the water content. The ellipticity of ribonuclease A, which is solubilized in the center of micellar water pool, in the near-UV region was dependent on W0 and became minimum when W0 of ca. 8 while the ellipticity in the far-UV region was almost constant, indicating that the tertiary structure of ribonuclease A was affected by the water content, but the secondary structure was conserved. The degree of curvature of the micellar interface appears to influence the protein structure because the reverse micelle size is linearly proportional to the W0 value. As evidence of this, when the micelle size was comparable to the protein's dimensions, the structures were more affected by the water content. Judging from the dependence of the factor influencing the protein structure on the protein species, the location of solubilized protein in reverse micelles is significantly related to whether the protein structure in the system is affected by the micellar interface. In the cases of cytochrome c and lysozyme, the ellipticity against W0 was dependent on the AOT concentration. In contrast, ribonuclease A gave very similar ellipticity values whatever the AOT concentration. In the n-hexane micellar system, cytochrome c exhibited lower ellipticity values and ribonuclease A in the lower W0 range (W0protein and the micellar

  11. Expression of 300-kilodalton intermediate filament-associated protein distinguishes human glioma cells from normal astrocytes.

    PubMed Central

    Yang, H Y; Lieska, N; Glick, R; Shao, D; Pappas, G D

    1993-01-01

    The availability of biochemical markers to distinguish glioma cells from normal astrocytes would have enormous diagnostic value. Such markers also may be of value in studying the basic biology of human astrocytomas. The vimentin-binding, 300-kDa intermediate filament (IF)-associated protein (IFAP-300kDa) has recently been shown to be developmentally expressed in radial glia of the central nervous system of the rat. It is not detected in the normal or reactive astrocytes of the adult rat nor in neonatal rat brain astrocytes in primary culture. In the present study, double-label immunofluorescence microscopy using antibodies to IFAP-300kDa and glial fibrillary acidic protein (GFAP, an astrocyte-specific IF structural protein) identifies this IFAP in GFAP-containing tumor cells from examples of all three major types of human astrocytomas (i.e., well-differentiated, anaplastic, and glioblastoma multiforme). Astrocytoma cells in primary cultures prepared from all three astrocytomas also express this protein. It is not detectable in normal adult brain tissue. Immunoblot analyses using the IFAP-300kDa antibody confirm the presence of a 300-kDa polypeptide in fresh astrocytoma preparations enriched for IF proteins. These results suggest the utility of IFAP-300kDa as a marker for identification of human glioma cells both in vitro and in situ. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 PMID:8378327

  12. Small heat shock proteins, phylogeny in filamentous fungi and expression analyses in Aspergillus nidulans.

    PubMed

    Wu, Jianbing; Wang, Mingshuang; Zhou, Liting; Yu, Dongliang

    2016-01-10

    Small heat shock proteins (sHSPs) have been characterized in organisms from all three domains of life and viruses and are involved in a wide range of biological functions. However, the evolution and function of sHSP in Aspergillus species are largely unknown. In the present work, sHSPs were identified in 31 filamentous fungi, including species from Aspergillus, Penicillium, Fusarium and Magnaporthe, as well as Botrytis cinerea and Neurospora crassa. Phylogenetic analysis revealed high level of divergence of sHSPs among filamentous fungi that orthologs could be only found between very closely related species. Strikingly, duplication of shsp genes occurred in genera Penicillium and also Aspergillus nidulans was observed, which might be an important pathway of sHSPs evolution. Expression analysis of shsp genes revealed that sHSPs were involved in response of A. nidulans to various conditions, including cold/heat as well as oxidative and osmotic stresses, and that the recent duplicated sHSPs in A. nidulans had highly similar function. PMID:26403724

  13. The IQGAP1 Protein Is a Calmodulin-regulated Barbed End Capper of Actin Filaments

    PubMed Central

    Pelikan-Conchaudron, Andrea; Le Clainche, Christophe; Didry, Dominique; Carlier, Marie-France

    2011-01-01

    IQGAP1 is a large modular protein that displays multiple partnership and is thought to act as a scaffold in coupling cell signaling to the actin and microtubule cytoskeletons in cell migration, adhesion, and cytokinesis. However the molecular mechanisms underlying the activities of IQGAP1 are poorly understood in part because of its large size, poor solubility and lack of functional assays to challenge biochemical properties in various contexts. We have purified bacterially expressed recombinant human IQGAP1. The protein binds Cdc42, Rac1, and the CRIB domain of N-WASP in a calmodulin-sensitive fashion. We further show that in addition to bundling of filaments via a single N-terminal calponin-homology domain, IQGAP1 actually regulates actin assembly. It caps barbed ends, with a higher affinity for ADP-bound terminal subunits (KB = 4 nm). The barbed end capping activity is inhibited by calmodulin, consistent with calmodulin binding to IQGAP1 with a KC of 40 nm, both in the absence and presence of Ca2+ ions. The barbed end capping activity resides in the C-terminal half of IQGAP1. It is possible that the capping activity of IQGAP1 accounts for its stimulation of cell migration. We further find that bacterially expressed recombinant IQGAP1 fragments easily co-purify with nucleic acids that turn out to activate N-WASP protein to branch filaments with Arp2/3 complex. The present results open perspectives for tackling the function of IQGAP1 in more complex reconstituted systems. PMID:21730051

  14. Casein Kinase II Induced Polymerization of Soluble TDP-43 into Filaments Is Inhibited by Heat Shock Proteins

    PubMed Central

    Davis, Mary; Lin, Wen-Lang; Cook, Casey; Dunmore, Judy; Tay, William; Menkosky, Kyle; Cao, Xiangkun; Petrucelli, Leonard; DeTure, Michael

    2014-01-01

    Background Trans-activation Response DNA-binding Protein-43 (TDP-43) lesions are observed in Amyotrophic Lateral Sclerosis (ALS), Frontotemporal Lobar Degeneration with ubiquitin inclusions (FTLD-TDP) and 25–50% of Alzheimer's Disease (AD) cases. These abnormal protein inclusions are composed of either amorphous TDP-43 aggregates or highly ordered filaments. The filamentous TDP-43 accumulations typically contain clean 10–12 nm filaments though wider 18–20 nm coated filaments may be observed. The TDP-43 present within these lesions is phosphorylated, truncated and ubiquitinated, and these modifications appear to be abnormal as they are linked to both a cellular heat shock response and microglial activation. The mechanisms associated with this abnormal TDP-43 accumulation are believed to result in a loss of TDP-43 function, perhaps due to the post-translational modifications or resulting from physical sequestration of the TDP-43. The formation of TDP-43 inclusions involves cellular translocation and conversion of TDP-43 into fibrillogenic forms, but the ability of these accumulations to sequester normal TDP-43 and propagate this behavior between neurons pathologically is mostly inferred. The lack of methodology to produce soluble full length TDP-43 and recapitulate this polymerization into filaments as observed in disease has limited our understanding of these pathogenic cascades. Results The protocols described here generate soluble, full-length and untagged TDP-43 allowing for a direct assessment of the impact of various posttranslational modifications on TDP-43 function. We demonstrate that Casein Kinase II (CKII) promotes the polymerization of this soluble TDP-43 into 10 nm diameter filaments that resemble the most common TDP-43 structures observed in disease. Furthermore, these filaments are recognized as abnormal by Heat Shock Proteins (HSPs) which can inhibit TDP-43 polymerization or directly promote TDP-43 filament depolymerization. Conclusion These

  15. Reverse Phase Protein Arrays: Mapping the path towards personalized medicine

    PubMed Central

    Gallagher, Rosa I.; Espina, Virginia

    2016-01-01

    Reverse phase protein array (RPPA) technology evolved from the advent of miniaturized immunoassays and gene microarray technology. Reverse phase protein arrays provide either a low throughput or high throughput methodology for quantifying proteins and their post-translationally modified forms in both cellular and non-cellular samples. As the demand for patient tailored therapies increases so does the need for precise and sensitive technology to accurately profile the molecular circuitry driving an individual patient’s disease. RPPAs are currently utilized in clinical trials for profiling and comparing the functional state of protein signaling pathways, either temporally within tumors, between patients, or within the same patients before/after treatment. RPPAs are generally employed for quantifying large numbers of samples on one array, under identical experimental conditions. However, the goal of personalized cancer medicine is to design therapies based on the molecular portrait of a patient’s tumor, which in turn result in more efficacious treatments with less toxicity. Therefore, RPPAs are also being validated for low throughput assays of individual patient samples. This review explores reverse phase protein array technology in the cancer research field, concentrating on its role as a fundamental tool for deciphering protein signaling networks and its emerging role in personalized medicine. PMID:25358623

  16. CASEIN KINASE1-LIKE PROTEIN2 Regulates Actin Filament Stability and Stomatal Closure via Phosphorylation of Actin Depolymerizing Factor.

    PubMed

    Zhao, Shuangshuang; Jiang, Yuxiang; Zhao, Yang; Huang, Shanjin; Yuan, Ming; Zhao, Yanxiu; Guo, Yan

    2016-06-01

    The opening and closing of stomata are crucial for plant photosynthesis and transpiration. Actin filaments undergo dynamic reorganization during stomatal closure, but the underlying mechanism for this cytoskeletal reorganization remains largely unclear. In this study, we identified and characterized Arabidopsis thaliana casein kinase 1-like protein 2 (CKL2), which responds to abscisic acid (ABA) treatment and participates in ABA- and drought-induced stomatal closure. Although CKL2 does not bind to actin filaments directly and has no effect on actin assembly in vitro, it colocalizes with and stabilizes actin filaments in guard cells. Further investigation revealed that CKL2 physically interacts with and phosphorylates actin depolymerizing factor 4 (ADF4) and inhibits its activity in actin filament disassembly. During ABA-induced stomatal closure, deletion of CKL2 in Arabidopsis alters actin reorganization in stomata and renders stomatal closure less sensitive to ABA, whereas deletion of ADF4 impairs the disassembly of actin filaments and causes stomatal closure to be more sensitive to ABA Deletion of ADF4 in the ckl2 mutant partially recues its ABA-insensitive stomatal closure phenotype. Moreover, Arabidopsis ADFs from subclass I are targets of CKL2 in vitro. Thus, our results suggest that CKL2 regulates actin filament reorganization and stomatal closure mainly through phosphorylation of ADF. PMID:27268429

  17. Arabidopsis VILLIN5, an Actin Filament Bundling and Severing Protein, Is Necessary for Normal Pollen Tube Growth[W

    PubMed Central

    Zhang, Hua; Qu, Xiaolu; Bao, Chanchan; Khurana, Parul; Wang, Qiannan; Xie, Yurong; Zheng, Yiyan; Chen, Naizhi; Blanchoin, Laurent; Staiger, Christopher J.; Huang, Shanjin

    2010-01-01

    A dynamic actin cytoskeleton is essential for pollen germination and tube growth. However, the molecular mechanisms underlying the organization and turnover of the actin cytoskeleton in pollen remain poorly understood. Villin plays a key role in the formation of higher-order structures from actin filaments and in the regulation of actin dynamics in eukaryotic cells. It belongs to the villin/gelsolin/fragmin superfamily of actin binding proteins and is composed of six gelsolin-homology domains at its core and a villin headpiece domain at its C terminus. Recently, several villin family members from plants have been shown to sever, cap, and bundle actin filaments in vitro. Here, we characterized a villin isovariant, Arabidopsis thaliana VILLIN5 (VLN5), that is highly and preferentially expressed in pollen. VLN5 loss-of-function retarded pollen tube growth and sensitized actin filaments in pollen grains and tubes to latrunculin B. In vitro biochemical analyses revealed that VLN5 is a typical member of the villin family and retains a full suite of activities, including barbed-end capping, filament bundling, and calcium-dependent severing. The severing activity was confirmed with time-lapse evanescent wave microscopy of individual actin filaments in vitro. We propose that VLN5 is a major regulator of actin filament stability and turnover that functions in concert with oscillatory calcium gradients in pollen and therefore plays an integral role in pollen germination and tube growth. PMID:20807879

  18. Arabidopsis CROLIN1, a Novel Plant Actin-binding Protein, Functions in Cross-linking and Stabilizing Actin Filaments*

    PubMed Central

    Jia, Honglei; Li, Jisheng; Zhu, Jingen; Fan, Tingting; Qian, Dong; Zhou, Yuelong; Wang, Jiaojiao; Ren, Haiyun; Xiang, Yun; An, Lizhe

    2013-01-01

    Higher order actin filament structures are necessary for cytoplasmic streaming, organelle movement, and other physiological processes. However, the mechanism by which the higher order cytoskeleton is formed in plants remains unknown. In this study, we identified a novel actin-cross-linking protein family (named CROLIN) that is well conserved only in the plant kingdom. There are six isovariants of CROLIN in the Arabidopsis genome, with CROLIN1 specifically expressed in pollen. In vitro biochemical analyses showed that CROLIN1 is a novel actin-cross-linking protein with binding and stabilizing activities. Remarkably, CROLIN1 can cross-link actin bundles into actin networks. CROLIN1 loss of function induces pollen germination and pollen tube growth hypersensitive to latrunculin B. All of these results demonstrate that CROLIN1 may play an important role in stabilizing and remodeling actin filaments by binding to and cross-linking actin filaments. PMID:24072702

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

    PubMed Central

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

    2014-01-01

    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. PMID:25002499

  20. 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. PMID:25002499

  1. A preparation of Alzheimer paired helical filaments that displays distinct tau proteins by polyacrylamide gel electrophoresis.

    PubMed Central

    Greenberg, S G; Davies, P

    1990-01-01

    Paired helical filaments (PHFs) are prominent components of Alzheimer disease (AD) neurofibrillary tangles (NFTs). Rather than isolating NFTs, we selected for PHF populations that can be extracted from AD brain homogenates. About 50% of PHF immunoreactivity can be obtained in 27,200 x g supernatants following homogenization in buffers containing 0.8 M NaCl. We further enriched for PHFs by taking advantage of their insolubility in the presence of zwitterionic detergents and 2-mercaptoethanol, removal of aggregates by filtration through 0.45-microns filters, and sucrose density centrifugation. PHF-enriched fractions contained two to five proteins of 57-68 kDa that displayed the same antigenic properties as PHFs. Since the 57- to 68-kDa PHF proteins are antigenically related to tau proteins, they are similar to the tau proteins previously observed in NFTs. However, further analysis revealed that PHF-associated tau can be distinguished from normal, soluble tau by PHF antibodies that do not recognize human adult tau and by one- and two-dimensional PAGE. Images PMID:2116006

  2. Fluorescence dynamics of green fluorescent protein in AOT reversed micelles.

    PubMed

    Uskova, M A; Borst, J W; Hink, M A; van Hoek, A; Schots, A; Klyachko, N L; Visser, A J

    2000-09-15

    We have used the enhanced green fluorescent protein (EGFP) to investigate the properties of surfactant-entrapped water pools in organic solvents (reversed micelles) with steady-state and time-resolved fluorescence methods. The surfactant used was sodium bis(2-ethylhexyl)sulfosuccinate (AOT) and the organic solvents were isooctane and (the more viscous) dodecane, respectively. The water content of the water pools could be controlled through the parameter w0, which is the water-to-surfactant molar ratio. With steady-state fluorescence, it was observed that subtle fluorescence changes could be noted in reversed micelles of different water contents. EGFP can be used as a pH-indicator of the water droplets in reversed micelles. Time-resolved fluorescence methods also revealed subtle changes in fluorescence decay times when the results in bulk water were compared with those in reversed micelles. The average fluorescence lifetimes of EGFP scaled with the relative fluorescence intensities. Time-resolved fluorescence anisotropy of EGFP in aqueous solution and reversed micelles yielded single rotational correlation times. Geometrical considerations could assign the observed correlation times to dehydrated protein at low w0 and internal EGFP rotation within the droplet at the highest w0. PMID:11036971

  3. 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

    2016-08-01

    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. PMID:27452459

  4. 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

  5. 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.

  6. Quantifying reversible oxidation of protein thiols in photosynthetic organisms.

    PubMed

    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. PMID:25698223

  7. Desmin: molecular interactions and putative functions of the muscle intermediate filament protein.

    PubMed

    Costa, M L; Escaleira, R; Cataldo, A; Oliveira, F; Mermelstein, C S

    2004-12-01

    Desmin is the intermediate filament (IF) protein occurring exclusively in muscle and endothelial cells. There are other IF proteins in muscle such as nestin, peripherin, and vimentin, besides the ubiquitous lamins, but they are not unique to muscle. Desmin was purified in 1977, the desmin gene was characterized in 1989, and knock-out animals were generated in 1996. Several isoforms have been described. Desmin IFs are present throughout smooth, cardiac and skeletal muscle cells, but can be more concentrated in some particular structures, such as dense bodies, around the nuclei, around the Z-line or in costameres. Desmin is up-regulated in muscle-derived cellular adaptations, including conductive fibers in the heart, electric organs, some myopathies, and experimental treatments with drugs that induce muscle degeneration, like phorbol esters. Many molecules have been reported to associate with desmin, such as other IF proteins (including members of the membrane dystroglycan complex), nebulin, the actin and tubulin binding protein plectin, the molecular motor dynein, the gene regulatory protein MyoD, DNA, the chaperone alphaB-crystallin, and proteases such as calpain and caspase. Desmin has an important medical role, since it is used as a marker of tumors' origin. More recently, several myopathies have been described, with accumulation of desmin deposits. Yet, after almost 30 years since its identification, the function of desmin is still unclear. Suggested functions include myofibrillogenesis, mechanical support for the muscle, mitochondrial localization, gene expression regulation, and intracellular signaling. This review focuses on the biochemical interactions of desmin, with a discussion of its putative functions. PMID:15558188

  8. A Comparison of Reversible Versus Irreversible Protein Glutathionylation

    PubMed Central

    Townsend, Danyelle M.; Lushchak, Volodymyr I.; Cooper, Arthur J.L.

    2015-01-01

    Glutathionylation is generally a reversible posttranslational modification that occurs to cysteine residues that have been exposed to reactive oxygen species (P-SSG). This cyclical process can regulate various clusters of proteins, including those involved in critical cellular signaling functions. However, certain conditions can favor the formation of dehydroamino acids, such as 2,3-didehydroalanine (2,3-dehydroalanine, DHA) and 2,3-didehydrobutyrine (2,3-dehydrobutyrine), which can act as Michael acceptors. In turn, these can form Michael adducts with glutathione (GSH), resulting in the formation of a stable thioether conjugate, an irreversible process referred to as nonreducible glutathionylation. This is predicted to be prevalent in nature, particularly in more slowly turning over proteins. Such nonreducible glutathionylation can be distinguished from the more facile cycling signaling processes and is predicted to be of gerontological, toxicological, pharmacological, and oncological relevance. Here, we compare reversible and irreversible glutathionylation. PMID:24974182

  9. Elastic deformation and failure in protein filament bundles: atomistic simulations and coarse-grained modeling

    PubMed Central

    Hammond, N. A.

    2008-01-01

    The synthetic peptide RAD16-II has shown promise in tissue engineering and drug delivery. It has been studied as a vehicle for cell delivery and controlled release of IGF-1 to repair infarcted cardiac tissue, and as a scaffold to promote capillary formation for an in vitro model of angiogenesis. The structure of RAD16-II is hierarchical, with monomers forming long β-sheets that pair together to form filaments; filaments form bundles approximately 30–60 nm in diameter; branching networks of filament bundles form macroscopic gels. We investigate the mechanics of shearing between the two β-sheets constituting one filament, and between cohered filaments of RAD16-II. This shear loading is found in filament bundle bending or in tensile loading of fibers composed of partial-length filaments. Molecular dynamics simulations show that time to failure is a stochastic function of applied shear stress, and that for a given loading time behavior is elastic for sufficiently small shear loads. We propose a coarse-grained model based on Langevin dynamics that matches molecular dynamics results and facilities extending simulations in space and time. The model treats a filament as an elastic string of particles, each having potential energy that is a periodic function of its position relative to the neighboring filament. With insight from these simulations, we discuss strategies for strengthening RAD16-II and similar materials. PMID:18440063

  10. Subtyping of breast cancer using reverse phase protein arrays.

    PubMed

    Sonntag, Johanna; Schlüter, Kerstin; Bernhardt, Stephan; Korf, Ulrike

    2014-12-01

    Reverse phase protein arrays (RPPAs) present a robust and sensitive high capacity platform for targeted proteomics that relies on highly specific antibodies to obtain a quantitative readout regarding phosphorylation state and abundance of proteins of interest. This review summarizes the current state of RPPA-based proteomic profiling of breast cancer in the context of existing preanalytical strategies and sample preparation protocols. RPPA-based subtypes identified so far are compared to those obtained by other approaches such as immunohistochemistry, genomics and transcriptomics. Special attention is given to discussing the potential of RPPA for biomarker discovery and biomarker validation. PMID:25400094

  11. 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. PMID:26017572

  12. 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.

  13. Photo-convertible tagging for localization and dynamic analyses of low-expression proteins in filamentous fungi.

    PubMed

    Perez-de-Nanclares-Arregi, Elixabet; Etxebeste, Oier

    2014-09-01

    Photo-convertible fluorescent proteins (PCFPs) undergo a dramatic change in their excitation and emission spectra upon irradiation at specific wavelengths, thus rendering a different color. Dendra2 is a commercially available PCFP used to track the redistribution of proteins within cellular compartments, their life-time or interactions. Before photo-conversion Dendra2 exhibits green fluorescence, which becomes red after irradiation with either UV or blue lights. Multiple studies including Dendra2 as a molecular tool have been described in eukaryotes but not in filamentous fungi. Here we present a method to tag low-expression proteins from the filamentous fungus Aspergillus nidulans with Dendra2 and track their cellular dynamics. The regulator of asexual development FlbB was selected as control, a transcription factor that is expressed at low levels and can be used as a marker for the tip and nuclei of vegetative hyphae. This control provided us with a visual way to confirm the functionality of our genomic and plasmid constructs, since a non-functional FlbB protein renders a block in development and a characteristic aconidial phenotype. Our protocol combines standardized cloning and transformation procedures with the use of a mercury lamp microscope to convert and follow Dendra2 within cells. Hence, we present a rapid, simple and inexpensive method that makes tracking analysis of proteins that present technical difficulties to be followed feasible in filamentous fungi. PMID:25014896

  14. 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

    2015-01-01

    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. PMID:26146072

  15. Actin turnover-dependent fast dissociation of capping protein in the dendritic nucleation actin network: evidence of frequent filament severing.

    PubMed

    Miyoshi, Takushi; Tsuji, Takahiro; Higashida, Chiharu; Hertzog, Maud; Fujita, Akiko; Narumiya, Shuh; Scita, Giorgio; Watanabe, Naoki

    2006-12-18

    Actin forms the dendritic nucleation network and undergoes rapid polymerization-depolymerization cycles in lamellipodia. To elucidate the mechanism of actin disassembly, we characterized molecular kinetics of the major filament end-binding proteins Arp2/3 complex and capping protein (CP) using single-molecule speckle microscopy. We have determined the dissociation rates of Arp2/3 and CP as 0.048 and 0.58 s(-1), respectively, in lamellipodia of live XTC fibroblasts. This CP dissociation rate is three orders of magnitude faster than in vitro. CP dissociates slower from actin stress fibers than from the lamellipodial actin network, suggesting that CP dissociation correlates with actin filament dynamics. We found that jasplakinolide, an actin depolymerization inhibitor, rapidly blocked the fast CP dissociation in cells. Consistently, the coexpression of LIM kinase prolonged CP speckle lifetime in lamellipodia. These results suggest that cofilin-mediated actin disassembly triggers CP dissociation from actin filaments. We predict that filament severing and end-to-end annealing might take place fairly frequently in the dendritic nucleation actin arrays. PMID:17178911

  16. Targeting protein kinases to reverse multidrug resistance in sarcoma.

    PubMed

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

    2016-02-01

    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

  17. Reverse micellar extraction for downstream processing of proteins/enzymes.

    PubMed

    Krishna, S Hari; Srinivas, N D; Raghavarao, K S M S; Karanth, N G

    2002-01-01

    New developments in the area of downstream processing are, hopefully, to fulfill the promises of modern biotechnology. The traditional separation processes such as chromatography or electrophoresis can become prohibitively expensive unless the product is of high value. Hence, there is a need to develop efficient and cost-effective downstream processing methods. Reverse micellar extraction is one such potential and a promising liquid-liquid extraction technique, which has received immense attention for isolation and purification of proteins/enzymes in the recent times. This technique is easy to scale-up and offers continuous operation. This review, besides briefly considering important physico-chemical and biological aspects, highlights the engineering aspects including mass transfer, mathematical modeling, and technology development. It also discusses recent developments in reverse micellar extraction such as affinity based separations, enzymatic reactions in reverse micelles coupled with membrane processes, reverse micellar extraction in hollow fibers, etc. Special emphasis has been given to some recent applications of this technique. PMID:11787493

  18. 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

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

    PubMed

    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

    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

  20. Reverse footprinting to map sites of RNA-protein interactions.

    PubMed

    Nilsen, Timothy W

    2014-06-01

    Nuclease protection of a site-specifically labeled RNA by an RNA-binding protein is an extremely powerful method for determining the site of an RNA-protein interaction. If a protein binds to the region that contains the site-specific label, it will protect the label and adjoining sequences from nuclease digestion. The protected region, or "footprint," can then be characterized by extensive fragmentation and gel electrophoresis. As the name implies, reverse footprinting produces a mirror image of a conventional footprint; the footprint is revealed by the presence, not the absence, of bands. This method has a distinct advantage over conventional footprinting in that only a small fraction of the labeled RNA must be bound. PMID:24890211

  1. 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. PMID:26448198

  2. 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

  3. 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.

  4. 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

    2016-01-01

    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

  5. The Small Heat Shock Protein Hsp27 Affects Assembly Dynamics and Structure of Keratin Intermediate Filament Networks

    PubMed Central

    Kayser, Jona; Haslbeck, Martin; Dempfle, Lisa; Krause, Maike; Grashoff, Carsten; Buchner, Johannes; Herrmann, Harald; Bausch, Andreas R.

    2013-01-01

    The mechanical properties of living cells are essential for many processes. They are defined by the cytoskeleton, a composite network of protein fibers. Thus, the precise control of its architecture is of paramount importance. Our knowledge about the molecular and physical mechanisms defining the network structure remains scarce, especially for the intermediate filament cytoskeleton. Here, we investigate the effect of small heat shock proteins on the keratin 8/18 intermediate filament cytoskeleton using a well-controlled model system of reconstituted keratin networks. We demonstrate that Hsp27 severely alters the structure of such networks by changing their assembly dynamics. Furthermore, the C-terminal tail domain of keratin 8 is shown to be essential for this effect. Combining results from fluorescence and electron microscopy with data from analytical ultracentrifugation reveals the crucial role of kinetic trapping in keratin network formation. PMID:24138853

  6. 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. PMID:26409224

  7. Cloning and sequencing of the cDNA encoding a core protein of the paired helical filament of Alzheimer's disease: Identification as the microtubule-associated protein tau

    SciTech Connect

    Goedert, M.; Wischik, C.M.; Crowther, R.A.; Walker, J.E.; Klug, A. )

    1988-06-01

    Screening of cDNA libraries prepared from the frontal cortex of an Alzheimer's disease patient and from fetal human brain has led to isolation of the cDNA for a core protein of the paired helical filament of Alzheimer's disease. The partial amino acid sequence of this core protein was used to design synthetic oligonucleotide probes. The cDNA encodes a protein of 352 amino acids that contains a characteristic amino acid repeat in its carboxyl-terminal half. This protein is highly homologous to the sequence of the mouse microtubule-associated protein tau and thus constitutes the human equivalent of mouse tau. RNA blot analysis indicates the presence of two major transcripts, 6 and 2 kilobases long, with a wide distribution in normal human brain. Tau protein mRNAs were found in normal amounts in the frontal cortex from patients with Alzheimer's disease. The proof that at least part of tau protein forms a component of the paired helical filament core opens the way to understanding the mode of formation of paired helical filaments and thus, ultimately, the pathogenesis of Alzheimer's disease.

  8. A family of intermediate filament-like proteins is sequentially assembled into the cytoskeleton of Toxoplasma gondii

    PubMed Central

    Anderson-White, Brooke R.; Ivey, F. Douglas; Cheng, Katherine; Szatanek, Tomasz; Lorestani, Alexander; Beckers, Con J.; Ferguson, David J.P.; Sahoo, Nivedita; Gubbels, Marc-Jan

    2010-01-01

    Summary The intracellular protozoan parasite Toxoplasma gondii divides by a unique process of internal budding that involves the assembly of two daughter cells within the mother. The cytoskeleton of Toxoplasma, which is composed of microtubules associated with an inner membrane complex (IMC), has an important role in this process. The IMC, which is directly under the plasma membrane, contains a set of flattened membranous sacs lined on the cytoplasmic side by a network of filamentous proteins. This network contains a family of intermediate filament-like proteins or IMC proteins. In order to elucidate the division process, we have characterized a 14-member sub-family of Toxoplasma IMC proteins that share a repeat motif found in proteins associated with the cortical alveoli in all alveolates. By creating fluorescent protein fusion reporters for the family members we determined the spatio-temporal patterns of all 14 IMC proteins through tachyzoite development. This revealed several distinct distribution patterns and some provide the basis for novel structural models such as the assembly of certain family members into the basal complex. Furthermore we identified IMC15 as an early marker of budding and, lastly, the dynamic patterns observed throughout cytokinesis provide a timeline for daughter parasite development and division. PMID:20698859

  9. Reverse phase protein microarrays advance to use in clinical trials.

    PubMed

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

    2010-12-01

    Individualizing cancer therapy for molecular targeted inhibitors requires a new class of molecular profiling technology that can map the functional state of the cancer cell signal pathways containing the drug targets. Reverse phase protein microarrays (RPMA) are a technology platform designed for quantitative, multiplexed analysis of specific phosphorylated, cleaved, or total (phosphorylated and non-phosphorylated) forms of cellular proteins from a limited amount of sample. This class of microarray can be used to interrogate tissue samples, cells, serum, or body fluids. RPMA were previously a research tool; now this technology has graduated to use in research clinical trials with clinical grade sensitivity and precision. In this review we describe the application of RPMA for multiplexed signal pathway analysis in therapeutic monitoring, biomarker discovery, and evaluation of pharmaceutical targets, and conclude with a summary of the technical aspects of RPMA construction and analysis. PMID:20974554

  10. Soluble CD44 interacts with intermediate filament protein vimentin on endothelial cell surface.

    PubMed

    Päll, Taavi; Pink, Anne; Kasak, Lagle; Turkina, Marina; Anderson, Wally; Valkna, Andres; Kogerman, Priit

    2011-01-01

    CD44 is a cell surface glycoprotein that functions as hyaluronan receptor. Mouse and human serum contain substantial amounts of soluble CD44, generated either by shedding or alternative splicing. During inflammation and in cancer patients serum levels of soluble CD44 are significantly increased. Experimentally, soluble CD44 overexpression blocks cancer cell adhesion to HA. We have previously found that recombinant CD44 hyaluronan binding domain (CD44HABD) and its non-HA-binding mutant inhibited tumor xenograft growth, angiogenesis, and endothelial cell proliferation. These data suggested an additional target other than HA for CD44HABD. By using non-HA-binding CD44HABD Arg41Ala, Arg78Ser, and Tyr79Ser-triple mutant (CD443MUT) we have identified intermediate filament protein vimentin as a novel interaction partner of CD44. We found that vimentin is expressed on the cell surface of human umbilical vein endothelial cells (HUVEC). Endogenous CD44 and vimentin coprecipitate from HUVECs, and when overexpressed in vimentin-negative MCF-7 cells. By using deletion mutants, we found that CD44HABD and CD443MUT bind vimentin N-terminal head domain. CD443MUT binds vimentin in solution with a Kd in range of 12-37 nM, and immobilised vimentin with Kd of 74 nM. CD443MUT binds to HUVEC and recombinant vimentin displaces CD443MUT from its binding sites. CD44HABD and CD443MUT were internalized by wild-type endothelial cells, but not by lung endothelial cells isolated from vimentin knock-out mice. Together, these data suggest that vimentin provides a specific binding site for soluble CD44 on endothelial cells. PMID:22216242

  11. Expression patterns of keratin intermediate filament and keratin associated protein genes in wool follicles.

    PubMed

    Yu, Zhidong; Gordon, Steven W; Nixon, Allan J; Bawden, C Simon; Rogers, Michael A; Wildermoth, Janet E; Maqbool, Nauman J; Pearson, Allan J

    2009-03-01

    The catalogue of hair keratin intermediate filaments (KIFs) and keratin-associated proteins (KAPs) present in wool follicles is incomplete. The full coding sequences for three novel sheep KIFs (KRT27, KRT35 and KRT38) and one KAP (KRTAP4-3) were established in this study. Spatial expression patterns of these and other genes (KRT31, KRT85, KRTAP6-1 and trichohyalin) were determined by in situ hybridisation in wool follicles at synchronised stages of growth. Transcription proceeded in the order: trichohyalin, KRT27, KRT85, KRT35, KRT31, KRT38, KRTAP6-1 and KRTAP4-3, as determined by increasing distance of their expression zones from the germinal matrix in anagen follicles. Expression became gradually more restricted to the lower follicle during follicle regression (catagen), and ceased during dormancy (telogen). Some genes (KRT27, KRT31, KRT85 and KRTAP6-1), but not others, were expressed in cortical cells forming the brush-end, indicating specific requirements for the formation of this anchoring structure. The resumption of keratin expression was observed only in later stages of follicle reactivation (proanagen). KIF expression patterns in primary wool follicles showed general resemblance to their human homologues but with some unique features. Consistent differences in localisation between primary and secondary wool follicles were observed. Asymmetrical expression of KRT27, KRT31, KRT35, KRT85 and trichohyalin genes in secondary follicles were associated with bulb deflection and follicle curvature, suggesting a role in the determination of follicle and fibre morphology. PMID:19272529

  12. 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

  13. Characterization of the restricted component of Epstein-Barr virus early antigens as a cytoplasmic filamentous protein.

    PubMed Central

    Luka, J; Miller, G; Jörnvall, H; Pearson, G R

    1986-01-01

    Four monoclonal antibodies produced against the restricted component of the Epstein-Barr virus (EBV) early antigen (EA-R) precipitated a polypeptide with an approximate molecular weight of 85,000. Three of these antibodies prepared against the native 85,000-molecular-weight protein (85K protein) reacted by immunofluorescence with acetone-fixed smears but not methanol-fixed smears of EBV-producing cells activated with tumor-promoting agent and sodium butyrate. The fourth monoclonal antibody which was produced against the denatured 85K protein reacted with both acetone-fixed cells and methanol-fixed cells. Blocking of direct immunofluorescence by the different monoclonal antibodies established that these monoclonal antibodies were directed against three different epitopes expressed on the 85K protein. The cytoplasmic staining pattern produced by each antibody was granular during the first 24 to 28 h after induction, developed into filamentous structures about 36 h after induction, and then began to aggregate after 48 h. Similar structures were observed in human placental cells transfected by EBV DNA and stained with three of the monoclonal antibodies. These results suggest that the EA-R polypeptide is assembled into filaments during the EBV lytic cycle. The significance of this in regards to replication has yet to be determined. Biochemical characterization of this major EA-R component did not reveal any major differences in this protein isolated from different cell lines. Images PMID:2422401

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

    PubMed

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

    2016-04-15

    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

  15. Functional expression of NF1 tumor suppressor protein: association with keratin intermediate filaments during the early development of human epidermis

    PubMed Central

    Malminen, Maria; Peltonen, Sirkku; Koivunen, Jussi; Peltonen, Juha

    2002-01-01

    Background NF1 refers to type 1 neurofibromatosis syndrome, which has been linked with mutations of the large NF1 gene. NF1 tumor suppressor protein, neurofibromin, has been shown to regulate ras: the NF1 protein contains a GTPase activating protein (GAP) related domain which functions as p21rasGAP. Our studies have previously demonstrated that the NF1 protein forms a high affinity association with cytokeratin 14 during the formation of desmosomes and hemidesmosomes in cultured keratinocytes. Methods The expression of NF1 protein was studied in developing human epidermis using western transfer analysis, indirect immunofluorescence, confocal laser scanning microscopy, immunoelectron microscopy, and in situ hybridization. Results The expression of NF1 protein was noted to be highly elevated in the periderm at 8 weeks estimated gestational age (EGA) and in the basal cells at 8–14 weeks EGA. During this period, NF1 protein was associated with cytokeratin filaments terminating to desmosomes and hemidesmosomes. NF1 protein did not display colocalization with α-tubulin or actin of the cytoskeleton, or with adherens junction proteins. Conclusions These results depict an early fetal period when the NF1 tumor suppressor is abundantly expressed in epidermis and associated with cytokeratin filaments. This period is characterized by the initiation of differentiation of the basal cells, maturation of the basement membrane zone as well as accentuated formation of selected cellular junctions. NF1 tumor suppressor may function in the regulation of epidermal histogenesis via controlling the organization of the keratin cytoskeleton during the assembly of desmosomes and hemidesmosomes. PMID:12199909

  16. 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.

  17. Cucurbitacin covalent bonding to cysteine thiols: the filamentous-actin severing protein Cofilin1 as an exemplary target

    PubMed Central

    2013-01-01

    Background Cucurbitacins are a class of triterpenoid natural compounds with potent bioactivities that led to their use as traditional remedies, and which continue to attract considerable attention as chemical biology tools and potential therapeutics. One obvious target is the actin-cytoskeleton; treatment with cucurbitacins results in cytoskeletal rearrangements that impact upon motility and cell morphology. Findings Cucurbitacin reacted with protein cysteine thiols as well as dithiothreitol, and we propose that the cucurbitacin mechanism of action is through broad protein thiol modifications that could result in inhibition of numerous protein targets. An example of such a target protein is Cofilin1, whose filamentous actin severing activity is inhibited by cucurbitacin conjugation. Conclusions The implications of these results are that cucurbitacins are unlikely to be improved for selectivity by medicinal chemistry and that their use as chemical biology probes to analyse the role of specific signalling pathways should be undertaken with caution. PMID:23945128

  18. Promoter and signal sequence from filamentous fungus can drive recombinant protein production in the yeast Kluyveromyces lactis.

    PubMed

    Madhavan, Aravind; Sukumaran, Rajeev K

    2014-08-01

    Cross-recognition of promoters from filamentous fungi in yeast can have important consequences towards developing fungal expression systems, especially for the rapid evaluation of their efficacy. A truncated 510bp inducible Trichoderma reesei cellobiohydrolase I (cbh1) promoter was tested for the expression of green fluorescent protein (GFP) in Kluyveromyces lactis after disrupting its native β-galactosidase (lac4) promoter. The efficiency of the CBH1 secretion signal was also evaluated by fusing it to the lac4 promoter of the yeast, which significantly increased the secretion of recombinant protein in K. lactis compared to the native α-mating factor secretion signal. The fungal promoter is demonstrated to have potential to drive heterologous protein production in K. lactis; and the small sized T. reesei cbh1 secretion signal can mediate the protein secretion in K. lactis with high efficiency. PMID:24661814

  19. 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. PMID:26216997

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

    PubMed Central

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

    2015-01-01

    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. PMID:26216997

  1. Role of the Unfolded Protein Response in Regulating the Mucin-Dependent Filamentous-Growth Mitogen-Activated Protein Kinase Pathway

    PubMed Central

    Adhikari, Hema; Vadaie, Nadia; Chow, Jacky; Caccamise, Lauren M.; Chavel, Colin A.; Li, Boyang; Bowitch, Alexander; Stefan, Christopher J.

    2015-01-01

    Signaling mucins are evolutionarily conserved regulators of signal transduction pathways. The signaling mucin Msb2p regulates the Cdc42p-dependent mitogen-activated protein kinase (MAPK) pathway that controls filamentous growth in yeast. The cleavage and release of the glycosylated inhibitory domain of Msb2p is required for MAPK activation. We show here that proteolytic processing of Msb2p was induced by underglycosylation of its extracellular domain. Cleavage of underglycosylated Msb2p required the unfolded protein response (UPR), a quality control (QC) pathway that operates in the endoplasmic reticulum (ER). The UPR regulator Ire1p, which detects misfolded/underglycosylated proteins in the ER, controlled Msb2p cleavage by regulating transcriptional induction of Yps1p, the major protease that processes Msb2p. Accordingly, the UPR was required for differentiation to the filamentous cell type. Cleavage of Msb2p occurred in conditional trafficking mutants that trap secretory cargo in the endomembrane system. Processed Msb2p was delivered to the plasma membrane, and its turnover by the ubiquitin ligase Rsp5p and ESCRT attenuated the filamentous-growth pathway. We speculate that the QC pathways broadly regulate signaling glycoproteins and their cognate pathways by recognizing altered glycosylation patterns that can occur in response to extrinsic cues. PMID:25666509

  2. 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. PMID:25644579

  3. Preparation and Characterization of a Polyclonal Antibody against Human Actin Filament-Associated Protein-120 kD

    PubMed Central

    Chen, Yujian; Liu, Yong; Guo, Jiayu; Tang, Tao; Gao, Jian; Huang, Tao; Wang, Bin; Liu, Shaojun

    2016-01-01

    Actin filament-associated protein-120kD (AFAP-120) is an alternatively spliced isoform of actin filament-associated protein-110kD (AFAP-110) and contains an additional neuronal insert (NINS) fragment in addition to identical domains to the AFAP-110. Unlike AFAP-110 widely expressed in tissues, AFAP-120 is specifically expressed in the nervous system and plays a role in organizing dynamic actin structures during neuronal differentiation. However, anti-AFAP-120 antibody is still commercially unavailable, and this may hinder the function research for AFAP-120. In this study, we simultaneously used the ABCpred online server and the BepiPred 1.0 server to predict B-cell epitopes in the exclusive NINS sequence of human AFAP-120 protein, and found that a 16aa-peptide sequence was the consensus epitope predicted by both tools. This peptide was chemically synthesized and used as an immunogen to develop polyclonal antibody against AFAP-120 (anti-AFAP-120). The sensitivity and specificity of anti-AFAP-120 were analyzed with immunoblotting, immunoprecipitation, and immunofluorescence assays. Our results indicated that anti-AFAP-120 could react with over-expressed and endogenous human AFAP-120 protein under denatured condition, but not with human AFAP-110 protein. Moreover, native human AFAP-120 protein could also be recognized by the anti-AFAP-120 antibody. These results suggested that the prepared anit-AFAP-120 antibody would be a useful tool for studying the biochemical and biological functions of AFAP-120. PMID:27322249

  4. The N terminal region of human tau is present in Alzheimer's disease protein A68 and is incorporated into paired helical filaments.

    PubMed Central

    Crowe, A.; Ksiezak-Reding, H.; Liu, W. K.; Dickson, D. W.; Yen, S. H.

    1991-01-01

    Antibody (Ab) E-1 was raised to the amino terminus (19 to 33 amino acid residues) of human tau. It recognized Alzheimer's disease proteins A68 (MW 60, 64, 68 kd), labeled paired helical filaments, and had no reactivity with tau from rat, mouse, and bovine brains. The results indicate that the N terminus of tau is incorporated in A68 proteins and paired helical filaments and that human tau proteins contain species-specific amino acid sequences. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 PMID:1721492

  5. Crystallization and preliminary X-ray diffraction analysis of protein 14 from Sulfolobus islandicus filamentous virus (SIFV)

    SciTech Connect

    Goulet, Adeline; Spinelli, Silvia; Campanacci, Valérie; Porciero, Sophie; Blangy, Stéphanie; Garrett, Roger A.; Tilbeurgh, Herman van; Leulliot, Nicolas; Basta, Tamara; Prangishvili, David; Cambillau, Christian

    2006-09-01

    Crystals of S. islandicus filamentous virus (SIFV) protein 14 have been grown at 293 K. Crystals belong to space group P6{sub 2}22 or P6{sub 4}22 and diffract to a resolution of 2.95 Å. A large-scale programme has been embarked upon aiming towards the structural determination of conserved proteins from viruses infecting hyperthermophilic archaea. Here, the crystallization of protein 14 from the archaeal virus SIFV is reported. This protein, which contains 111 residues (MW 13 465 Da), was cloned and expressed in Escherichia coli with an N-terminal His{sub 6} tag and purified to homogeneity. The tag was subsequently cleaved and the protein was crystallized using PEG 1000 or PEG 4000 as a precipitant. Large crystals were obtained of the native and the selenomethionine-labelled protein using sitting drops of 100–300 nl. Crystals belong to space group P6{sub 2}22 or P6{sub 4}22, with unit-cell parameters a = b = 68.1, c = 132.4 Å. Diffraction data were collected to a maximum acceptable resolution of 2.95 and 3.20 Å for the SeMet-labelled and native protein, respectively.

  6. Characterization of protein expression levels with label-free detected reverse phase protein arrays.

    PubMed

    Guo, Xuexue; Deng, Yihong; Zhu, Chenggang; Cai, Junlong; Zhu, Xiangdong; Landry, James P; Zheng, Fengyun; Cheng, Xunjia; Fei, Yiyan

    2016-09-15

    In reverse-phase protein arrays (RPPA), one immobilizes complex samples (e.g., cellular lysate, tissue lysate or serum etc.) on solid supports and performs parallel reactions of antibodies with immobilized protein targets from the complex samples. In this work, we describe a label-free detection of RPPA that enables quantification of RPPA data and thus facilitates comparison of studies performed on different samples and on different solid supports. We applied this detection platform to characterization of phosphoserine aminotransferase (PSAT) expression levels in Acanthamoeba lysates treated with artemether and the results were confirmed by Western blot studies. PMID:27372609

  7. A putative protein-sequestration site involving intermediate filaments for protein degradation by autophagy. Studies with microinjected purified glycolytic enzymes in 3T3-L1 cells.

    PubMed Central

    Doherty, F J; Wassell, J A; Mayer, R J

    1987-01-01

    Several glycolytic enzymes (lactate dehydrogenase, pyruvate kinase, glyceraldehyde-3-phosphate dehydrogenase) were radiolabelled by [125I]iodination, conjugation with 125I-labelled Bolton & Hunter reagent and reductive [3H]methylation, and their degradative rates after microinjection into 3T3-L1 cells compared with that of the extracellular protein bovine serum albumin. Although the albumin remains largely cytosolic in recipient cells, the glycolytic enzymes rapidly (less than 30 min) become insoluble, as measured by detergent and salt extractions. The microinjected glycolytic enzymes appear to form disulphide-linked aggregates, are found in a cell fraction rich in vimentin-containing intermediate filaments and histones (nuclear-intermediate-filament fraction), and are degraded slowly by a lysosomal mechanism, as judged by the effects of inhibitors (NH4Cl, leupeptin, 3-methyladenine). 125I-labelled bovine serum albumin appears to be degraded rapidly and non-lysosomally. Prolonged treatment (96 h) of cultured cells with leupeptin results in the accumulation of pulse-labelled ([35S]methionine for 24 h) endogenous cell proteins in the detergent-and salt-non-extractable residue, but NH4Cl and 3-methyladenine do not have this effect. The findings are in terms of the interpretation of experiments involving microinjection of proteins to study intracellular protein protein degradation by autophagy. Images Fig. 1. Fig. 5. PMID:3593223

  8. Reconstruction of Protein Networks Using Reverse-Phase Protein Array Data.

    PubMed

    von der Heyde, Silvia; Sonntag, Johanna; Kramer, Frank; Bender, Christian; Korf, Ulrike; Beißbarth, Tim

    2016-01-01

    In this chapter, we describe an approach to reconstruct cellular signaling networks based on measurements of protein activation after different stimulation experiments. As experimental platform reverse-phase protein arrays (RPPA) are used. RPPA allow the measurement of proteins and phosphoproteins across many samples in parallel with minimal sample consumption using a panel of highly target protein-specific antibodies. Functional interactions of proteins are modeled using a Boolean network. We describe the Boolean network reconstruction approach ddepn (dynamic deterministic effects propagation networks), which uses time course data to derive protein interactions based on perturbation experiments. We explain how the method works, give a practical application example, and describe how the results can be interpreted. Furthermore prior knowledge on signaling pathways is essential for network reconstruction. Here we describe the use of our software rBiopaxParser to integrate prior knowledge on protein signaling available in public databases. All applied methods are freely available as open-source R software packages. We describe the preparation of RPPA data as well as all relevant programming steps to format the RPPA data, to infer the prior knowledge, and to reconstruct and analyze the protein signaling networks. PMID:26519181

  9. A Novel Link between Fic (Filamentation Induced by cAMP)-mediated Adenylylation/AMPylation and the Unfolded Protein Response*

    PubMed Central

    Sanyal, Anwesha; Chen, Andy J.; Nakayasu, Ernesto S.; Lazar, Cheri S.; Zbornik, Erica A.; Worby, Carolyn A.; Koller, Antonius; Mattoo, Seema

    2015-01-01

    The maintenance of endoplasmic reticulum (ER) homeostasis is a critical aspect of determining cell fate and requires a properly functioning unfolded protein response (UPR). We have discovered a previously unknown role of a post-translational modification termed adenylylation/AMPylation in regulating signal transduction events during UPR induction. A family of enzymes, defined by the presence of a Fic (filamentation induced by cAMP) domain, catalyzes this adenylylation reaction. The human genome encodes a single Fic protein, called HYPE (Huntingtin yeast interacting protein E), with adenylyltransferase activity but unknown physiological target(s). Here, we demonstrate that HYPE localizes to the lumen of the endoplasmic reticulum via its hydrophobic N terminus and adenylylates the ER molecular chaperone, BiP, at Ser-365 and Thr-366. BiP functions as a sentinel for protein misfolding and maintains ER homeostasis. We found that adenylylation enhances BiP's ATPase activity, which is required for refolding misfolded proteins while coping with ER stress. Accordingly, HYPE expression levels increase upon stress. Furthermore, siRNA-mediated knockdown of HYPE prevents the induction of an unfolded protein response. Thus, we identify HYPE as a new UPR regulator and provide the first functional data for Fic-mediated adenylylation in mammalian signaling. PMID:25601083

  10. Long-term rescue of a familial hypertrophic cardiomyopathy caused by a mutation in the thin filament protein, tropomyosin, via modulation of a calcium cycling protein.

    PubMed

    Gaffin, Robert D; Peña, James R; Alves, Marco S L; Dias, Fernando A L; Chowdhury, Shamim A K; Heinrich, Lynley S; Goldspink, Paul H; Kranias, Evangelia G; Wieczorek, David F; Wolska, Beata M

    2011-11-01

    We have recently shown that a temporary increase in sarcoplasmic reticulum (SR) cycling via adenovirus-mediated overexpression of sarcoplasmic reticulum ATPase (SERCA2) transiently improves relaxation and delays hypertrophic remodeling in a familial hypertrophic cardiomyopathy (FHC) caused by a mutation in the thin filament protein, tropomyosin (i.e., α-TmE180G or Tm180). In this study, we sought to permanently alter calcium fluxes via phospholamban (PLN) gene deletion in Tm180 mice in order to sustain long-term improvements in cardiac function and adverse cardiac remodeling/hypertrophy. While similar work has been done in FHCs resulting from mutations in thick myofilament proteins, no one has studied these effects in an FHC resulting from a thin filament protein mutation. Tm180 transgenic (TG) mice were crossbred with PLN knockout (KO) mice and four groups were studied in parallel: 1) non-TG (NTG), 2) Tm180, 3) PLNKO/NTG and 4) PLNKO/Tm180. Tm180 mice exhibit increased heart weight/body weight and hypertrophic gene markers compared to NTG mice, but levels in PLNKO/Tm180 mice were similar to NTG. Tm180 mice also displayed altered function as assessed via in situ pressure-volume analysis and echocardiography at 3-6 months and one year; however, altered function in Tm180 mice was rescued back to NTG levels in PLNKO/Tm180 mice. Collagen deposition, as assessed by Picrosirius Red staining, was increased in Tm180 mice but was similar in NTG and in PLNKO/Tm180 mice. Extracellular signal-regulated kinase (ERK1/2) phosphorylation increased in Tm180 mice while levels in PLNKO/Tm180 mice were similar to NTGs. The present study shows that by modulating SR calcium cycling, we were able to rescue many of the deleterious aspects of FHC caused by a mutation in the thin filament protein, Tm. PMID:21840315

  11. Functional Analysis of DNA Replication Fork Reversal Catalyzed by Mycobacterium tuberculosis RuvAB Proteins*

    PubMed Central

    Khanduja, Jasbeer Singh; Muniyappa, K.

    2012-01-01

    Initially discovered in Escherichia coli, RuvAB proteins are ubiquitous in bacteria and play a dual role as molecular motor proteins responsible for branch migration of the Holliday junction(s) and reversal of stalled replication forks. Despite mounting genetic evidence for a crucial role of RuvA and RuvB proteins in reversal of stalled replication forks, the mechanistic aspects of this process are still not fully understood. Here, we elucidate the ability of Mycobacterium tuberculosis RuvAB (MtRuvAB) complex to catalyze the reversal of replication forks using a range of DNA replication fork substrates. Our studies show that MtRuvAB, unlike E. coli RuvAB, is able to drive replication fork reversal via the formation of Holliday junction intermediates, suggesting that RuvAB-catalyzed fork reversal involves concerted unwinding and annealing of nascent leading and lagging strands. We also demonstrate the reversal of replication forks carrying hemi-replicated DNA, indicating that MtRuvAB complex-catalyzed fork reversal is independent of symmetry at the fork junction. The fork reversal reaction catalyzed by MtRuvAB is coupled to ATP hydrolysis, is processive, and culminates in the formation of an extended reverse DNA arm. Notably, we found that sequence heterology failed to impede the fork reversal activity of MtRuvAB. We discuss the implications of these results in the context of recognition and processing of varied types of replication fork structures by RuvAB proteins. PMID:22094465

  12. 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. PMID:27039023

  13. MamK, a bacterial actin, forms dynamic filaments in vivo that are regulated by the acidic proteins MamJ and LimJ

    PubMed Central

    Draper, Olga; Byrne, Meghan E.; Li, Zhuo; Keyhani, Sepehr; Cueto Barrozo, Joyce; Jensen, Grant; Komeili, Arash

    2011-01-01

    SUMMARY Bacterial actins, in contrast to their eukaryotic counterparts, are highly divergent proteins whose wide-ranging functions are thought to correlate with their evolutionary diversity. One clade, represented by the MamK protein of magnetotactic bacteria, is required for the subcellular organization of magnetosomes, membrane-bound organelles that aid in navigation along the earth’s magnetic field. Using a fluorescence recovery after photobleaching assay in Magnetospirillum magneticum AMB-1, we find that, like traditional actins, MamK forms dynamic filaments that require an intact NTPase motif for their turnover in vivo. We also uncover two proteins, MamJ and LimJ, which perform a redundant function to promote the dynamic behavior of MamK filaments in wildtype cells. The absence of both MamJ and LimJ leads to static filaments, a disrupted magnetosome chain, and an anomalous build-up of cytoskeletal filaments between magnetosomes. Our results suggest that MamK filaments, like eukaryotic actins, are intrinsically stable and rely on regulators for their dynamic behavior, a feature that stands in contrast to some classes of bacterial actins characterized to date. PMID:21883528

  14. Structural Dynamics of C-domain of Cardiac Troponin I Protein in Reconstituted Thin Filament*

    PubMed Central

    Zhou, Zhiqun; Li, King-Lun; Rieck, Daniel; Ouyang, Yexin; Chandra, Murali; Dong, Wen-Ji

    2012-01-01

    The regulatory function of cardiac troponin I (cTnI) involves three important contiguous regions within its C-domain: the inhibitory region (IR), the regulatory region (RR), and the mobile domain (MD). Within these regions, the dynamics of regional structure and kinetics of transitions in dynamic state are believed to facilitate regulatory signaling. This study was designed to use fluorescence anisotropy techniques to acquire steady-state and kinetic information on the dynamic state of the C-domain of cTnI in the reconstituted thin filament. A series of single cysteine cTnI mutants was generated, labeled with the fluorophore tetramethylrhodamine, and subjected to various anisotropy experiments at the thin filament level. The structure of the IR was found to be less dynamic than that of the RR and the MD, and Ca2+ binding induced minimal changes in IR dynamics: the flexibility of the RR decreased, whereas the MD became more flexible. Anisotropy stopped-flow experiments showed that the kinetics describing the transition of the MD and RR from the Ca2+-bound to the Ca2+-free dynamic states were significantly faster (53.2–116.8 s−1) than that of the IR (14.1 s−1). Our results support the fly casting mechanism, implying that an unstructured MD with rapid dynamics and kinetics plays a critical role to initiate relaxation upon Ca2+ dissociation by rapidly interacting with actin to promote the dissociation of the RR from the N-domain of cTnC. In contrast, the IR responds to Ca2+ signals with slow structural dynamics and transition kinetics. The collective findings suggested a fourth state of activation. PMID:22207765

  15. Microtubule-associated protein tau (tau) is a major antigenic component of paired helical filaments in Alzheimer disease.

    PubMed Central

    Kosik, K S; Joachim, C L; Selkoe, D J

    1986-01-01

    The detailed protein composition of the paired helical filaments (PHF) that accumulate in human neurons in aging and Alzheimer disease is unknown. However, the identity of certain components has been surmised by using immunocytochemical techniques. Whereas PHF share epitopes with neurofilament proteins and microtubule-associated protein (MAP) 2, we report evidence that the MAP tau (tau) appears to be their major antigenic component. Immunization of rabbits with NaDodSO4-extracted, partially purified PHF (free of normal cytoskeletal elements, including tau) consistently produces antibodies to tau but not, for example, to neurofilaments. Such PHF antibodies label all of the heterogeneous fetal and mature forms of tau from rat and human brain. Absorption of PHF antisera with heat-stable MAPs (rich in tau) results in almost complete loss of staining of neurofibrillary tangles (NFT) in human brain sections. An affinity-purified antibody to tau specifically labels NFT and the neurites of senile plaques in human brain sections as well as NaDodSO4-extracted NFT. tau-Immunoreactive NFT frequently extend into the apical dendrites of pyramidal neurons, suggesting an aberrant intracellular locus for this axonal protein. tau and PHF antibodies label tau proteins identically on electrophoretic transfer blots and stain the gel-excluded protein representing NaDodSO4-insoluble PHF in homogenates of human brain. The progressive accumulation of altered tau protein in neurons in Alzheimer disease may result in instability of microtubules, consequent loss of effective transport of molecules and organelles, and, ultimately, neuronal death. Images PMID:2424016

  16. Purification and characterization of caldesmon77: a calmodulin-binding protein that interacts with actin filaments from bovine adrenal medulla.

    PubMed Central

    Sobue, K; Tanaka, T; Kanda, K; Ashino, N; Kakiuchi, S

    1985-01-01

    Caldesmon150, a protein composed of the Mr 150,000/147,000 doublet, alternately binds to calmodulin and actin filaments in a Ca2+-dependent "flip-flop" fashion. In all fibroblast cell lines examined, we also found a Mr 77,000 protein that crossreacts with anti-caldesmon150 antibody by using an immunoprecipitation technique [Owada, M.K., Hakura, A., Iida, K., Yahara, I., Sobue, K. & Kakiuchi, S. (1984) Proc. Natl. Acad. Sci. USA 81, 3133-3137]. In this report, we examine the tissue distribution of caldesmon by the method of immunoblotting, using caldesmon-specific antibody. Both caldesmon150 and caldesmon77 show widespread distribution in the tissues examined. Caldesmon77 is more widely distributed than caldesmon150, and we have purified caldesmon77 from bovine adrenal medulla. Its molecular weight estimated by NaDodSO4/polyacrylamide gel electrophoresis was 77,000, and a tetramer of this polypeptide may constitute the native molecule (Mr, 300,000). Caldesmon77 possesses a number of features in common with caldesmon150, including flip-flop binding to calmodulin and actin filaments depending on the concentration of Ca2+ and crossreactivity with caldesmon150-specific antibody. Analysis of caldesmon77-F actin interaction by sedimentation and electrophoresis revealed that 0.5 mg of caldesmon77 bound to 1 mg of F actin. This indicated that the molar ratio between caldesmon77 (tetramer) and actin monomer was calculated to be 1:12-14. In addition, caldesmon77 regulated the actin-myosin interaction in Ca2+-sensitive actomyosin obtained from adrenal medulla. These results suggest that caldesmon77 might be a ubiquitous actin-linked regulator of nonmuscle contractile processes, including those in adrenal medulla. Images PMID:2991905

  17. 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

  18. Photocontrolled reversible morphology conversion of protein nanowires mediated by an azobenzene-cored dendrimer.

    PubMed

    Sun, Hongcheng; Zhao, Linlu; Wang, Tingting; An, Guo; Fu, Shuang; Li, Xiumei; Deng, Xiaoli; Liu, Junqiu

    2016-05-21

    A novel strategy to construct photocontrolled protein nanowires with reversible morphology was reported through photoisomerizable azobenzene-cored dendrimer evoked protein self-assembly. Furthermore, the curvature of the protein nanowires could be switched by alternatively irradiating with visible light and ultraviolet light. PMID:27062988

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

    PubMed

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

    2015-05-12

    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

  20. 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

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

    PubMed Central

    Kollmar, Martin

    2015-01-01

    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. PMID:26024016

  2. 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.

  3. 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.

  4. Correction: Reversibility and two state behaviour in the thermal unfolding of oligomeric TIM barrel proteins.

    PubMed

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

    2016-04-21

    Correction for 'Reversibility and two state behaviour in the thermal unfolding of oligomeric TIM barrel proteins' by Sergio Romero-Romero et al., Phys. Chem. Chem. Phys., 2015, 17, 20699-20714. PMID:27010946

  5. 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

  6. Two Modes of Binding of DinI to RecA Filament Provide a New Insight Into Regulation of SOS Response by DinI Protein

    PubMed Central

    Galkin, Vitold E.; Britt, Rachel L.; Bane, Lukas B.; Yu, Xiong; Cox, Michael M.; Egelman, Edward H.

    2011-01-01

    The RecA protein plays a principal role in the bacterial SOS response to DNA damage. The induction of the SOS response is well understood and involves the cleavage of the LexA repressor catalyzed by the RecA nucleoprotein filament. In contrast, our understanding of the regulation and termination of the SOS response is much more limited. RecX and DinI are two major regulators of RecA’s ability to promote LexA cleavage and a strand exchange reaction and are believed to modulate its activity in ongoing SOS events. DinI’s function in the SOS response remains controversial since its interaction with the RecA filament is concentration-dependent and may result in either stabilization or depolymerization of the filament. The 17 C-terminal residues of RecA modulate the interaction between DinI and RecA. We demonstrate that DinI binds to the active RecA filament in two distinct structural modes. In the first mode DinI binds to the C-terminus of a RecA protomer. In the second mode DinI resides deeply in the groove of the RecA filament with its negatively charged C-terminal helix proximal to the L2 loop of RecA. The deletion of the 17 C-terminal residues of RecA favors the second mode of binding. We suggest that the negatively charged C-terminus of RecA prevents DinI from entering the groove and protects the RecA filament from depolymerization. Polymorphic binding of DinI to RecA filaments implies an even more complex role of DinI in the bacterial SOS response. PMID:21458462

  7. Prediction of reversibly oxidized protein cysteine thiols using protein structure properties

    PubMed Central

    Sanchez, Ricardo; Riddle, Megan; Woo, Jongwook; Momand, Jamil

    2008-01-01

    Protein cysteine thiols can be divided into four groups based on their reactivities: those that form permanent structural disulfide bonds, those that coordinate with metals, those that remain in the reduced state, and those that are susceptible to reversible oxidation. Physicochemical parameters of oxidation-susceptible protein thiols were organized into a database named the Balanced Oxidation Susceptible Cysteine Thiol Database (BALOSCTdb). BALOSCTdb contains 161 cysteine thiols that undergo reversible oxidation and 161 cysteine thiols that are not susceptible to oxidation. Each cysteine was represented by a set of 12 parameters, one of which was a label (1/0) to indicate whether its thiol moiety is susceptible to oxidation. A computer program (the C4.5 decision tree classifier re-implemented as the J48 classifier) segregated cysteines into oxidation-susceptible and oxidation-non-susceptible classes. The classifier selected three parameters critical for prediction of thiol oxidation susceptibility: (1) distance to the nearest cysteine sulfur atom, (2) solvent accessibility, and (3) pKa. The classifier was optimized to correctly predict 136 of the 161 cysteine thiols susceptible to oxidation. Leave-one-out cross-validation analysis showed that the percent of correctly classified cysteines was 80.1% and that 16.1% of the oxidation-susceptible cysteine thiols were incorrectly classified. The algorithm developed from these parameters, named the Cysteine Oxidation Prediction Algorithm (COPA), is presented here. COPA prediction of oxidation-susceptible sites can be utilized to locate protein cysteines susceptible to redox-mediated regulation and identify possible enzyme catalytic sites with reactive cysteine thiols. PMID:18287280

  8. 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

  9. 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.

  10. 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.

  11. Capping Protein Increases the Rate of Actin-based Motility by Promoting Filament Nucleation by the Arp2/3 Complex

    PubMed Central

    Akin, Orkun; Mullins, R. Dyche

    2008-01-01

    Summary Capping protein is an integral component of Arp2/3-nucleated actin networks that drive amoeboid motility. Increasing the concentration of capping protein, which caps barbed ends of actin filaments and prevents elongation, increases the rate of actin-based motility in vivo and in vitro. We studied the synergy between capping protein and Arp2/3 using an in vitro actin-based motility system reconstituted from purified proteins. We find that capping protein increases the rate of motility by promoting more frequent filament nucleation by the Arp2/3 complex, and not by increasing the rate of filament elongation as previously suggested. One consequence of this coupling between capping and nucleation is that, while the rate of motility depends strongly on the concentration of capping protein and Arp2/3, the net rate of actin assembly is insensitive to changes in either factor. By reorganizing their architecture, dendritic actin networks harness the same assembly kinetics to drive different rates of motility. PMID:18510928

  12. Cucumber Mosaic Virus Movement Protein Severs Actin Filaments to Increase the Plasmodesmal Size Exclusion Limit in Tobacco[W][OA

    PubMed Central

    Su, Shengzhong; Liu, Zhaohui; Chen, Cheng; Zhang, Yan; Wang, Xu; Zhu, Lei; Miao, Long; Wang, Xue-Chen; Yuan, Ming

    2010-01-01

    Plant viral movement proteins (MPs) enable viruses to pass through cell walls by increasing the size exclusion limit (SEL) of plasmodesmata (PD). Here, we report that the ability of Cucumber mosaic virus (CMV) MP to increase the SEL of the PD could be inhibited by treatment with the actin filament (F-actin)–stabilizing agent phalloidin but not by treatment with the F-actin–destabilizing agent latrunculin A. In vitro studies showed that CMV MP bound globular and F-actin, inhibited actin polymerization, severed F-actin, and participated in plus end capping of F-actin. Analyses of two CMV MP mutants, one with and one without F-actin severing activities, demonstrated that the F-actin severing ability was required to increase the PD SEL. Furthermore, the Tobacco mosaic virus MP also exhibited F-actin severing activity, and its ability to increase the PD SEL was inhibited by treatment with phalloidin. Our data provide evidence to support the hypothesis that F-actin severing is required for MP-induced increase in the SEL of PD. This may have broad implications in the study of the mechanisms of actin dynamics that regulate cell-to-cell transport of viral and endogenous proteins. PMID:20435906

  13. The molecular organization of the beta-sheet region in Corneous beta-proteins (beta-keratins) of sauropsids explains its stability and polymerization into filaments.

    PubMed

    Calvaresi, Matteo; Eckhart, Leopold; Alibardi, Lorenzo

    2016-06-01

    The hard corneous material of avian and reptilian scales, claws, beak and feathers is mainly derived from the presence of proteins formerly known as beta-keratins but now termed Corneous beta-proteins of sauropsids to distinguish them from keratins, which are members of the intermediate filament protein family. The modeling of the conserved 34 amino acid residues long central beta-sheet region of Corneous beta-proteins using an ab initio protein folding and structure prediction algorithm indicates that this region is formed by four antiparallel beta-sheets. Molecular dynamic simulations and Molecular Mechanics/Poisson Boltzmann Surface Area (MM-PBSA) analysis showed that the disposition of polar and apolar amino acids within the beta-region gives rise to an amphipathic core whose stability is further increased, especially in an aqueous environment, by the association into a dimer due to apolar interactions and specific amino-acid interactions. The dimers in turn polymerize into a 3nm thick linear beta-filament due to van der Waals and hydrogen-bond interactions. It is suggested that once this nuclear core of anti-parallel sheets evolved in the genome of a reptilian ancestor of the extant reptiles and birds about 300 millions years ago, new properties emerged in the corneous material forming scales, claws, beaks and feathers in these amniotes based on the tendency of these unique corneous proteins to form stable filaments different from keratin intermediate filaments or sterical structures formed by other corneous proteins so far known. PMID:26965557

  14. 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

  15. Role of Intermediate Filaments in Vesicular Traffic.

    PubMed

    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

  16. 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

  17. Ion-specific modulation of protein interactions: Anion-induced, reversible oligomerization of a fusion protein

    PubMed Central

    Gokarn, Yatin R; Fesinmeyer, R Matthew; Saluja, Atul; Cao, Shawn; Dankberg, Jane; Goetze, Andrew; Remmele, Richard L; Narhi, Linda O; Brems, David N

    2009-01-01

    Ions can significantly modulate the solution interactions of proteins. We aim to demonstrate that the salt-dependent reversible heptamerization of a fusion protein called peptibody A or PbA is governed by anion-specific interactions with key arginyl and lysyl residues on its peptide arms. Peptibody A, an E. coli expressed, basic (pI = 8.8), homodimer (65.2 kDa), consisted of an IgG1-Fc with two, C-terminal peptide arms linked via penta-glycine linkers. Each peptide arm was composed of two, tandem, active sequences (SEYQGLPPQGWK) separated by a spacer (GSGSATGGSGGGASSGSGSATG). PbA was monomeric in 10 mM acetate, pH 5.0 but exhibited reversible self-association upon salt addition. The sedimentation coefficient (sw) and hydrodynamic diameter (DH) versus PbA concentration isotherms in the presence of 140 mM NaCl (A5N) displayed sharp increases in sw and DH, reaching plateau values of 9 s and 16 nm by 10 mg/mL PbA. The DH and sedimentation equilibrium data in the plateau region (>12 mg/mL) indicated the oligomeric ensemble to be monodisperse (PdI = 0.05) with a z-average molecular weight (Mz) of 433 kDa (stoichiometry = 7). There was no evidence of reversible self-association for an IgG1-Fc molecule in A5N by itself or in a mixture containing fluorescently labeled IgG1-Fc and PbA, indicative of PbA self-assembly being mediated through its peptide arms. Self-association increased with pH, NaCl concentration, and anion size (I− > Br− > Cl− > F−) but could be inhibited using soluble Trp-, Phe-, and Leu-amide salts (Trp > Phe > Leu). We propose that in the presence of salt (i) anion binding renders PbA self-association competent by neutralizing the peptidyl arginyl and lysyl amines, (ii) self-association occurs via aromatic and hydrophobic interactions between the..xx..xxx..xx.. motifs, and (iii) at >10 mg/mL, PbA predominantly exists as heptameric clusters. PMID:19177361

  18. Filament disappearances

    NASA Technical Reports Server (NTRS)

    Wagner, William J.

    1986-01-01

    The phenomenon of the sudden filament disappearance (Disparition Brusque) is a familiar one to observers at H alpha telescopes. Nevertherless, the importance in Disparition Brusques (DB) continues to grow for several reasons which are cited in the discussion. It is reported that there seems to be more interest on building and maintain filaments than in destroying them. As a consequence, this sub-group is smaller than most of the others. All the same, progress in this area of filament disapperences seems steady and assured. The importance and interest in DBs is discussed and future directions are indicated.

  19. Hydration and protein folding in water and in reverse micelles: compressibility and volume changes.

    PubMed

    Valdez, D; Le Huérou, J Y; Gindre, M; Urbach, W; Waks, M

    2001-06-01

    The partial specific volume and adiabatic compressibility of proteins reflect the hydration properties of the solvent-exposed protein surface, as well as changes in conformational states. Reverse micelles, or water-in-oil microemulsions, are protein-sized, optically-clear microassemblies in which hydration can be experimentally controlled. We explore, by densimetry and ultrasound velocimetry, three basic proteins: cytochrome c, lysozyme, and myelin basic protein in reverse micelles made of sodium bis (2-ethylhexyl) sulfosuccinate, water, and isooctane and in aqueous solvents. For comparison, we use beta-lactoglobulin (pI = 5.1) as a reference protein. We examine the partial specific volume and adiabatic compressibility of the proteins at increasing levels of micellar hydration. For the lowest water content compatible with complete solubilization, all proteins display their highest compressibility values, independent of their amino acid sequence and charge. These values lie within the range of empirical intrinsic protein compressibility estimates. In addition, we obtain volumetric data for the transition of myelin basic protein from its initially unfolded state in water free of denaturants, to a folded, compact conformation within the water-controlled microenvironment of reverse micelles. These results disclose yet another aspect of the protein structural properties observed in membrane-mimetic molecular assemblies. PMID:11371450

  20. Hydration and protein folding in water and in reverse micelles: compressibility and volume changes.

    PubMed Central

    Valdez, D; Le Huérou, J Y; Gindre, M; Urbach, W; Waks, M

    2001-01-01

    The partial specific volume and adiabatic compressibility of proteins reflect the hydration properties of the solvent-exposed protein surface, as well as changes in conformational states. Reverse micelles, or water-in-oil microemulsions, are protein-sized, optically-clear microassemblies in which hydration can be experimentally controlled. We explore, by densimetry and ultrasound velocimetry, three basic proteins: cytochrome c, lysozyme, and myelin basic protein in reverse micelles made of sodium bis (2-ethylhexyl) sulfosuccinate, water, and isooctane and in aqueous solvents. For comparison, we use beta-lactoglobulin (pI = 5.1) as a reference protein. We examine the partial specific volume and adiabatic compressibility of the proteins at increasing levels of micellar hydration. For the lowest water content compatible with complete solubilization, all proteins display their highest compressibility values, independent of their amino acid sequence and charge. These values lie within the range of empirical intrinsic protein compressibility estimates. In addition, we obtain volumetric data for the transition of myelin basic protein from its initially unfolded state in water free of denaturants, to a folded, compact conformation within the water-controlled microenvironment of reverse micelles. These results disclose yet another aspect of the protein structural properties observed in membrane-mimetic molecular assemblies. PMID:11371450

  1. 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.

  2. The cephalochordate Branchiostoma genome contains 26 intermediate filament (IF) genes: implications for evolution of chordate IF proteins.

    PubMed

    Karabinos, Anton

    2013-01-01

    We analyzed the draft genome of the cephalochordate Branchiostoma floridae (B. floridae) for genes encoding intermediate filament (IF) proteins. From 26 identified IF genes 13 were not reported before. Four of the new IF genes belong to the previously established Branchiostoma IF group A, four to the Branchiostoma IF group B, one is homologous to the type II keratin E2 while the remaining four new IF sequences N1 to N4 could not be readily classified in any of the previously established Branchiostoma IF groups. All eleven identified A and B2-type IF genes are located on the same genomic scaffold and arose due to multiple cephalochordate-specific duplications. Another IF gene cluster, identified in the B. floridae genome, contains three keratins (E1, Y1, D1), two keratin-like IF genes (C2, X1), one new IF gene (N1) and one IF unrelated gene, but does not show any similarities to the well defined vertebrate type I or type II keratin gene clusters. In addition, some type III sequence features were documented in the new IF protein N2, which, however, seems to share a common ancestry with the Branchiostoma keratins D1 and two keratin-related genes C. Thus, a few type I and type II keratin genes existed in a common ancestor of cephalochordates and vertebrates, which after separation of these two lineages gave rise to the known complexities of the vertebrate cytoplasmic type I-IV IF proteins, as well as to the multiple keratin and related IF genes in cephalochordates, due to multiple gene duplications, deletions and sequence divergences. PMID:24246581

  3. Protein extraction using the sodium bis(2-ethylhexyl) phosphate (NaDEHP) reverse micellar system.

    PubMed

    Hu, Z; Gulari, E

    1996-04-20

    The reverse micellar system of sodium bis(2-ethylhexyl) phosphate (NaDEHP)/isooctane/brine was used for liquid-liquid extraction of proteins. We investigated the solubilization of cytochrome-c and alpha-chymotrypsin into the NaDEHP reverse micellar phase by varying the pH and NaCl concentration in the aqueous phase. At neutral pH and relatively low ionic strength, the proteins are extracted into the micellar phase with high yield. By contacting the micellar phase with a divalent cation (e.g., Ca(2+)) aqueous solution, the reverse micelles are destabilized and release the protein molecules back into an aqueous solution for recovery. This method separates the proteins from the surfactant with very high overall efficiencies. PMID:18626936

  4. Emulsion-templated fully reversible protein-in-oil gels.

    PubMed

    Romoscanu, Alexandre I; Mezzenga, Raffaele

    2006-08-29

    We have developed a new method allowing us to transform low-viscous apolar fluids into elastic solids with a shear elastic modulus of the order of 10(3)-10(5) Pa. The elasticity of the elastic solid is provided by a percolating 3D network of proteins, which are originally adsorbed at the interface of an oil-in-water emulsion template. By cross-linking the protein films at the interface and upon removal of water, the template is driven into a structure resembling a dry foam where the protein interfaces constitute the walls of the foam and the air is replaced by oil confined within polyhedral, closely packed droplets. Depending on the density of the protein network, the final material consists of chemically unmodified oil in a proportion of 95 to 99.9%. The physical properties of the elastic solid obtained can be tuned by changing either the average diameter size of the emulsion template or the cross-linking process of the protein film. However, the original low-viscosity emulsion can be restored by simply rehydrating the solidified fluid. Therefore, the present procedure offers an appealing strategy to build up solid properties for hydrophobic liquids while preserving the low viscosity and ease of manufacturing. PMID:16922568

  5. Helical filaments

    NASA Astrophysics Data System (ADS)

    Barbieri, Nicholas; Hosseinimakarem, Zahra; Lim, Khan; Durand, Magali; Baudelet, Matthieu; Johnson, Eric; Richardson, Martin

    2014-06-01

    The shaping of laser-induced filamenting plasma channels into helical structures by guiding the process with a non-diffracting beam is demonstrated. This was achieved using a Bessel beam superposition to control the phase of an ultrafast laser beam possessing intensities sufficient to induce Kerr effect driven non-linear self-focusing. Several experimental methods were used to characterize the resulting beams and confirm the observed structures are laser air filaments.

  6. 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

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

    PubMed

    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

  8. Protein separation and enrichment by counter-current chromatography using reverse micelle solvent systems.

    PubMed

    Shen, Ching-Wei; Yu, Tiing

    2007-06-01

    A protein mixture consisting of myoglobin, cytochrome c, and lysozyme was separated by high-speed counter-current chromatography using a two-phase aqueous/reverse micelle-containing organic solvent system. About 50% stationary phase retention ratio was obtained in most chromatographic experiments. Separations were manipulated mainly by pH gradients that controlled the electrostatic interactions between the protein molecules and reverse micelles. Separations were further improved by incorporating an ionic strength gradient along with the pH gradient. Control of ionic strength in the aqueous solution helped fine-tune protein partitioning between the stationary and mobile phases. Although non-specific protein interactions affected baseline resolution, recovery of cytochrome c and lysozyme reached 90% and 82%. Furthermore, concentration or enrichment of these two proteins was achieved from a large-volume sample load. This technique can potentially be employed in the recovery and enrichment of proteins from large-volume aqueous solutions. PMID:17289061

  9. ATP-independent reversal of a membrane protein aggregate by a chloroplast SRP subunit

    SciTech Connect

    Jaru-Ampornpan, Peera; Shen, Kuang; Lam, Vinh Q.; Ali, Mona; Doniach, Sebastian; Jia, Tony Z.; Shan, Shu-ou

    2010-07-23

    Membrane proteins impose enormous challenges to cellular protein homeostasis during their post-translational targeting, and they require chaperones to keep them soluble and translocation competent. Here we show that a novel targeting factor in the chloroplast signal recognition particle (cpSRP), cpSRP43, is a highly specific molecular chaperone that efficiently reverses the aggregation of its substrate proteins. In contrast to 'ATPases associated with various cellular activities' (AAA{sup +}) chaperones, cpSRP43 uses specific binding interactions with its substrate to mediate its 'disaggregase' activity. This disaggregase capability can allow targeting machineries to more effectively capture their protein substrates and emphasizes a close connection between protein folding and trafficking processes. Moreover, cpSRP43 provides the first example to our knowledge of an ATP-independent disaggregase and shows that efficient reversal of protein aggregation can be attained by specific binding interactions between a chaperone and its substrate.

  10. 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.

  11. The Tobamovirus Turnip Vein Clearing Virus 30-Kilodalton Movement Protein Localizes to Novel Nuclear Filaments To Enhance Virus Infection

    PubMed Central

    Levy, Amit; Zheng, Judy Y.

    2013-01-01

    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 (MPTMV). Detailed studies demonstrate that TMV multiplies exclusively in the cytoplasm and have documented associations of MPTMV 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 MPTVCV and report here the unexpected discovery that MPTVCV, 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 MPTVCV NLS appeared to be conserved in the subgroup 3 tobamoviruses, and our mutational analyses showed that nuclear localization of MPTVCV 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. PMID:23536678

  12. Insulin-like growth factor binding protein-1 expression in baboon endometrial stromal cells: regulation by filamentous actin and requirement for de novo protein synthesis.

    PubMed

    Kim, J J; Jaffe, R C; Fazleabas, A T

    1999-02-01

    Stromal fibroblasts in the primate endometrium undergo dramatic morphological and biochemical changes in response to pregnancy. This transformation is characterized by the expression of insulin-like growth factor binding protein-1 (IGFBP-1). Stromal cells from the baboon endometrium of nonpregnant animals were cultured and subsequently treated with cytochalasin D to disrupt actin filaments. In response to cytochalasin D treatment, cells contracted and became rounded as early as 10 min after the initiation of treatment. When cytochalasin D was removed, cells reverted back to their original fibroblastic shape within 1 h. After cells were treated with cytochalasin D for 5 h, addition of (Bu)2cAMP and/or hormones (estradiol, medroxyprogesterone acetate, and relaxin) resulted in the expression of IGFBP-1 messenger RNA and protein within 24 h. Cells with an intact cytoskeleton did not express detectable levels of IGFBP-1 in response to hormones and/or (Bu)2cAMP. Furthermore, the addition of cycloheximide inhibited expression of IGFBP-1 in cytochalasin D-treated cells. Stromal cells were also isolated from early pregnant and simulated pregnant animals. Within 48 h, cells from both the pregnant and simulated pregnant animals produced IGFBP-1 in response to hormones and/or (Bu)2cAMP. In these studies, IGFBP-1 expression was also inhibited by cycloheximide. These studies suggest that induction of IGFBP-1 requires an intermediary protein and that alterations in the cytoskeleton may be involved. PMID:9927334

  13. High molecular weight polypeptides (270,000-340,000) from cultured cells are related to hog brain microtubule-associated proteins but copurify with intermediate filaments.

    PubMed Central

    Pytela, R; Wiche, G

    1980-01-01

    High molecular weight polypeptides (HMWPs) of 270,000 to 340,000 were found to be major components of intermediate filaments prepared by Triton X-100 extraction after spreading of rat glioma C6, HeLa, Chinese hamster ovary, and simian virus 40-transformed Chinese hamster lung cells. C6 HMWPs were shown to resemble high molecular weight microtubule-associated proteins from hog brain by four criteria: (i) comigration in electrophoresis on high-resolution sodium dodecyl sulfate/polyacrylamide gels, (ii) one-dimensional peptide mapping, (iii) phosphorylation in vitro with [gamma-32P]ATP, and (iv) ability to promote microtubule assembly in vitro. HMWPs were also found to be major components of one-time polymerized C6 microtubule preparations, which contained a sizable amount of intermediate filaments. The predominant part of HMWPs present in these microtubule preparations was found not to copurify with microtubules in cycles of temperature-dependent assembly/disassembly but to remain with the cold-insoluble intermediate filaments. These results provide an explanation for the low yields that have hampered attempts to purify microtubule-associated porteins, in particular HMWPs, from cultured cells in the past. Moreover, they suggest that HMWPs might have a dual role in the cell, serving not only as regulators of microtubule assembly but also as linker components between microtubules and intermediate filaments. Images PMID:6933530

  14. Gel-free proteomic methodologies to study reversible cysteine oxidation and irreversible protein carbonyl formation.

    PubMed

    Boronat, S; García-Santamarina, S; Hidalgo, E

    2015-05-01

    Oxidative modifications in proteins have been traditionally considered as hallmarks of damage by oxidative stress and aging. However, oxidants can generate a huge variety of reversible and irreversible modifications in amino acid side chains as well as in the protein backbones, and these post-translational modifications can contribute to the activation of signal transduction pathways, and also mediate the toxicity of oxidants. Among the reversible modifications, the most relevant ones are those arising from cysteine oxidation. Thus, formation of sulfenic acid or disulfide bonds is known to occur in many enzymes as part of their catalytic cycles, and it also participates in the activation of signaling cascades. Furthermore, these reversible modifications have been usually attributed with a protective role, since they may prevent the formation of irreversible damage by scavenging reactive oxygen species. Among irreversible modifications, protein carbonyl formation has been linked to damage and death, since it cannot be repaired and can lead to protein loss-of-function and to the formation of protein aggregates. This review is aimed at researchers interested on the biological consequences of oxidative stress, both at the level of signaling and toxicity. Here we are providing a concise overview on current mass-spectrometry-based methodologies to detect reversible cysteine oxidation and irreversible protein carbonyl formation in proteomes. We do not pretend to impose any of the different methodologies, but rather to provide an objective catwalk on published gel-free approaches to detect those two types of modifications, from a biologist's point of view. PMID:25782062

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

    PubMed

    Silas, Sukrit; Mohr, Georg; Sidote, David J; Markham, Laura M; Sanchez-Amat, Antonio; Bhaya, Devaki; Lambowitz, Alan M; Fire, Andrew Z

    2016-02-26

    CRISPR 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 showed that a RT-Cas1 fusion protein enables the acquisition of RNA spacers in vivo in a RT-dependent manner. In vitro, the MMB-1 RT-Cas1 and Cas2 proteins catalyze the ligation of RNA segments into the CRISPR array, which is followed by reverse transcription. These observations outline a host-mediated mechanism for reverse information flow from RNA to DNA. PMID:26917774

  16. 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.

  17. The Plant-Specific Actin Binding Protein SCAB1 Stabilizes Actin Filaments and Regulates Stomatal Movement in Arabidopsis[C][W

    PubMed Central

    Zhao, Yang; Zhao, Shuangshuang; Mao, Tonglin; Qu, Xiaolu; Cao, Wanhong; Zhang, Li; Zhang, Wei; He, Liu; Li, Sidi; Ren, Sulin; Zhao, Jinfeng; Zhu, Guoli; Huang, Shanjin; Ye, Keqiong; Yuan, Ming; Guo, Yan

    2011-01-01

    Microfilament dynamics play a critical role in regulating stomatal movement; however, the molecular mechanism underlying this process is not well understood. We report here the identification and characterization of STOMATAL CLOSURE-RELATED ACTIN BINDING PROTEIN1 (SCAB1), an Arabidopsis thaliana actin binding protein. Plants lacking SCAB1 were hypersensitive to drought stress and exhibited reduced abscisic acid-, H2O2-, and CaCl2-regulated stomatal movement. In vitro and in vivo analyses revealed that SCAB1 binds, stabilizes, and bundles actin filaments. SCAB1 shares sequence similarity only with plant proteins and contains a previously undiscovered actin binding domain. During stomatal closure, actin filaments switched from a radial orientation in open stomata to a longitudinal orientation in closed stomata. This switch took longer in scab1 plants than in wild-type plants and was correlated with the delay in stomatal closure seen in scab1 mutants in response to drought stress. Our results suggest that SCAB1 is required for the precise regulation of actin filament reorganization during stomatal closure. PMID:21719691

  18. Intermediate Filaments: A Historical Perspective

    PubMed Central

    Oshima, Robert G.

    2007-01-01

    Intracellular protein filaments intermediate in size between actin microfilaments and microtubules are composed of a surprising variety of tissue specific proteins commonly interconnected with other filamentous systems for mechanical stability and decorated by a variety of proteins that provide specialized functions. The sequence conservation of the coiled-coil, alpha-helical structure responsible for polymerization into individual 10 nm filaments defines the classification of intermediate filament proteins into a large gene family. Individual filaments further assemble into bundles and branched cytoskeletons visible in the light microscope. However, it is the diversity of the variable terminal domains that likely contributes most to different functions. The search for the functions of intermediate filament proteins has led to discoveries of roles in diseases of the skin, heart, muscle, liver, brain, adipose tissues and even premature aging. The diversity of uses of intermediate filaments as structural elements and scaffolds for organizing the distribution of decorating molecules contrasts with other cytoskeletal elements. This review is an attempt to provide some recollection of how such a diverse field emerged and changed over about 30 years. PMID:17493611

  19. Bridged filaments of histone-like nucleoid structuring protein pause RNA polymerase and aid termination in bacteria

    PubMed Central

    Kotlajich, Matthew V; Hron, Daniel R; Boudreau, Beth A; Sun, Zhiqiang; Lyubchenko, Yuri L; Landick, Robert

    2015-01-01

    Bacterial H-NS forms nucleoprotein filaments that spread on DNA and bridge distant DNA sites. H-NS filaments co-localize with sites of Rho-dependent termination in Escherichia coli, but their direct effects on transcriptional pausing and termination are untested. In this study, we report that bridged H-NS filaments strongly increase pausing by E. coli RNA polymerase at a subset of pause sites with high potential for backtracking. Bridged but not linear H-NS filaments promoted Rho-dependent termination by increasing pause dwell times and the kinetic window for Rho action. By observing single H-NS filaments and elongating RNA polymerase molecules using atomic force microscopy, we established that bridged filaments surround paused complexes. Our results favor a model in which H-NS-constrained changes in DNA supercoiling driven by transcription promote pausing at backtracking-susceptible sites. Our findings provide a mechanistic rationale for H-NS stimulation of Rho-dependent termination in horizontally transferred genes and during pervasive antisense and noncoding transcription in bacteria. DOI: http://dx.doi.org/10.7554/eLife.04970.001 PMID:25594903

  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 Central

    Laine, Roney O.; Phaneuf, Katherine L.; Cunningham, Casey C.; Kwiatkowski, David; Azuma, Toshi; Southwick, Frederick S.

    1998-01-01

    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 μm/s [n = 176] versus 0.05 ± 0.003 μm/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. 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

  2. Structure of an invertebrate gene encoding cytoplasmic intermediate filament (IF) proteins: implications for the origin and the diversification of IF proteins.

    PubMed Central

    Dodemont, H; Riemer, D; Weber, K

    1990-01-01

    The structure of the single gene encoding the cytoplasmic intermediate filament (IF) proteins in non-neuronal cells of the gastropod Helix aspersa is described. Genomic and cDNA sequences show that the gene is composed of 10 introns and 11 exons, spanning greater than 60 kb of DNA. Alternative RNA processing accounts for two mRNA families which encode two IF proteins differing only in their C-terminal sequence. The intron/exon organization of the Helix rod domain is identical to that of the vertebrate type III IF genes in spite of low overall protein sequence homology and the presence of an additional 42 residues in coil 1b of the invertebrate sequence. Intron position homology extends to the entire coding sequence comprising both the rod and tail domains when the invertebrate IF gene is compared with the nuclear lamin LIII gene of Xenopus laevis presented in the accompanying report of Döring and Stick. In contrast the intron patterns of the tail domains of the invertebrate IF and the lamin genes differ from those of the vertebrate type III genes. The combined data are in line with an evolutionary descent of cytoplasmic IF proteins from a nuclear lamin-like progenitor and suggest a mechanism for this derivation. The unique position of intron 7 in the Helix IF gene indicates that the archetype IF gene arose by the elimination of the nuclear localization sequence due to the recruitment of a novel splice site. The presumptive structural organization of the archetype IF gene allows predictions with respect to the later diversification of metazoan IF genes. Whereas models proposing a direct derivation of neurofilament genes seem unlikely, the earlier speculation of an mRNA transposition mechanism is compatible with current results. Images Fig. 1. Fig. 2. Fig. 3. Fig. 4. Fig. 6. PMID:2249666

  3. Reversible Optogenetic Control of Subcellular Protein Localization in a Live Vertebrate Embryo

    PubMed Central

    Buckley, Clare E.; Moore, Rachel E.; Reade, Anna; Goldberg, Anna R.; Weiner, Orion D.; Clarke, Jonathan D.W.

    2016-01-01

    Summary We demonstrate the utility of the phytochrome system to rapidly and reversibly recruit proteins to specific subcellular regions within specific cells in a living vertebrate embryo. Light-induced heterodimerization using the phytochrome system has previously been used as a powerful tool to dissect signaling pathways for single cells in culture but has not previously been used to reversibly manipulate the precise subcellular location of proteins in multicellular organisms. Here we report the experimental conditions necessary to use this system to manipulate proteins in vivo. As proof of principle, we demonstrate that we can manipulate the localization of the apical polarity protein Pard3 with high temporal and spatial precision in both the neural tube and the embryo’s enveloping layer epithelium. Our optimizations of optogenetic component expression and chromophore purification and delivery should significantly lower the barrier for establishing this powerful optogenetic system in other multicellular organisms. PMID:26766447

  4. Reversible Optogenetic Control of Subcellular Protein Localization in a Live Vertebrate Embryo.

    PubMed

    Buckley, Clare E; Moore, Rachel E; Reade, Anna; Goldberg, Anna R; Weiner, Orion D; Clarke, Jonathan D W

    2016-01-11

    We demonstrate the utility of the phytochrome system to rapidly and reversibly recruit proteins to specific subcellular regions within specific cells in a living vertebrate embryo. Light-induced heterodimerization using the phytochrome system has previously been used as a powerful tool to dissect signaling pathways for single cells in culture but has not previously been used to reversibly manipulate the precise subcellular location of proteins in multicellular organisms. Here we report the experimental conditions necessary to use this system to manipulate proteins in vivo. As proof of principle, we demonstrate that we can manipulate the localization of the apical polarity protein Pard3 with high temporal and spatial precision in both the neural tube and the embryo's enveloping layer epithelium. Our optimizations of optogenetic component expression and chromophore purification and delivery should significantly lower the barrier for establishing this powerful optogenetic system in other multicellular organisms. PMID:26766447

  5. Complete Reversible Refolding of a G-Protein Coupled Receptor on a Solid Support

    PubMed Central

    Di Bartolo, Natalie; Compton, Emma L. R.; Warne, Tony; Edwards, Patricia C.; Tate, Christopher G.; Schertler, Gebhard F. X.; Booth, Paula J.

    2016-01-01

    The factors defining the correct folding and stability of integral membrane proteins are poorly understood. Folding of only a few select membrane proteins has been scrutinised, leaving considerable deficiencies in knowledge for large protein families, such as G protein coupled receptors (GPCRs). Complete reversible folding, which is problematic for any membrane protein, has eluded this dominant receptor family. Moreover, attempts to recover receptors from denatured states are inefficient, yielding at best 40–70% functional protein. We present a method for the reversible unfolding of an archetypal family member, the β1-adrenergic receptor, and attain 100% recovery of the folded, functional state, in terms of ligand binding, compared to receptor which has not been subject to any unfolding and retains its original, folded structure. We exploit refolding on a solid support, which could avoid unwanted interactions and aggregation that occur in bulk solution. We determine the changes in structure and function upon unfolding and refolding. Additionally, we employ a method that is relatively new to membrane protein folding; pulse proteolysis. Complete refolding of β1-adrenergic receptor occurs in n-decyl-β-D-maltoside (DM) micelles from a urea-denatured state, as shown by regain of its original helical structure, ligand binding and protein fluorescence. The successful refolding strategy on a solid support offers a defined method for the controlled refolding and recovery of functional GPCRs and other membrane proteins that suffer from instability and irreversible denaturation once isolated from their native membranes. PMID:26982879

  6. Complete Reversible Refolding of a G-Protein Coupled Receptor on a Solid Support.

    PubMed

    Di Bartolo, Natalie; Compton, Emma L R; Warne, Tony; Edwards, Patricia C; Tate, Christopher G; Schertler, Gebhard F X; Booth, Paula J

    2016-01-01

    The factors defining the correct folding and stability of integral membrane proteins are poorly understood. Folding of only a few select membrane proteins has been scrutinised, leaving considerable deficiencies in knowledge for large protein families, such as G protein coupled receptors (GPCRs). Complete reversible folding, which is problematic for any membrane protein, has eluded this dominant receptor family. Moreover, attempts to recover receptors from denatured states are inefficient, yielding at best 40-70% functional protein. We present a method for the reversible unfolding of an archetypal family member, the β1-adrenergic receptor, and attain 100% recovery of the folded, functional state, in terms of ligand binding, compared to receptor which has not been subject to any unfolding and retains its original, folded structure. We exploit refolding on a solid support, which could avoid unwanted interactions and aggregation that occur in bulk solution. We determine the changes in structure and function upon unfolding and refolding. Additionally, we employ a method that is relatively new to membrane protein folding; pulse proteolysis. Complete refolding of β1-adrenergic receptor occurs in n-decyl-β-D-maltoside (DM) micelles from a urea-denatured state, as shown by regain of its original helical structure, ligand binding and protein fluorescence. The successful refolding strategy on a solid support offers a defined method for the controlled refolding and recovery of functional GPCRs and other membrane proteins that suffer from instability and irreversible denaturation once isolated from their native membranes. PMID:26982879

  7. Reverse micelles as a platform for dynamic nuclear polarization in solution NMR of proteins.

    PubMed

    Valentine, Kathleen G; Mathies, Guinevere; Bédard, Sabrina; Nucci, Nathaniel V; Dodevski, Igor; Stetz, Matthew A; Can, Thach V; Griffin, Robert G; Wand, A Joshua

    2014-02-19

    Despite tremendous advances in recent years, solution NMR remains fundamentally restricted due to its inherent insensitivity. Dynamic nuclear polarization (DNP) potentially offers significant improvements in this respect. The basic DNP strategy is to irradiate the EPR transitions of a stable radical and transfer this nonequilibrium polarization to the hydrogen spins of water, which will in turn transfer polarization to the hydrogens of the macromolecule. Unfortunately, these EPR transitions lie in the microwave range of the electromagnetic spectrum where bulk water absorbs strongly, often resulting in catastrophic heating. Furthermore, the residence times of water on the surface of the protein in bulk solution are generally too short for efficient transfer of polarization. Here we take advantage of the properties of solutions of encapsulated proteins dissolved in low viscosity solvents to implement DNP in liquids. Such samples are largely transparent to the microwave frequencies required and thereby avoid significant heating. Nitroxide radicals are introduced into the reverse micelle system in three ways: attached to the protein, embedded in the reverse micelle shell, and free in the aqueous core. Significant enhancements of the water resonance ranging up to ∼-93 at 0.35 T were observed. We also find that the hydration properties of encapsulated proteins allow for efficient polarization transfer from water to the protein. These and other observations suggest that merging reverse micelle encapsulation technology with DNP offers a route to a significant increase in the sensitivity of solution NMR spectroscopy of proteins and other biomolecules. PMID:24456213

  8. Filamentation of Metabolic Enzymes in Saccharomyces cerevisiae.

    PubMed

    Shen, Qing-Ji; Kassim, Hakimi; Huang, Yong; Li, Hui; Zhang, Jing; Li, Guang; Wang, Peng-Ye; Yan, Jun; Ye, Fangfu; Liu, Ji-Long

    2016-06-20

    Compartmentation via filamentation has recently emerged as a novel mechanism for metabolic regulation. In order to identify filament-forming metabolic enzymes systematically, we performed a genome-wide screening of all strains available from an open reading frame-GFP collection in Saccharomyces cerevisiae. We discovered nine novel filament-forming proteins and also confirmed those identified previously. From the 4159 strains, we found 23 proteins, mostly metabolic enzymes, which are capable of forming filaments in vivo. In silico protein-protein interaction analysis suggests that these filament-forming proteins can be clustered into several groups, including translational initiation machinery and glucose and nitrogen metabolic pathways. Using glutamine-utilising enzymes as examples, we found that the culture conditions affect the occurrence and length of the metabolic filaments. Furthermore, we found that two CTP synthases (Ura7p and Ura8p) and two asparagine synthetases (Asn1p and Asn2p) form filaments both in the cytoplasm and in the nucleus. Live imaging analyses suggest that metabolic filaments undergo sub-diffusion. Taken together, our genome-wide screening identifies additional filament-forming proteins in S. cerevisiae and suggests that filamentation of metabolic enzymes is more general than currently appreciated. PMID:27312010

  9. Rapid and reversible knockdown of endogenous proteins by peptide-directed lysosomal degradation

    PubMed Central

    Fan, Xuelai; Jin, Wu Yang; Lu, Jie; Wang, Jin; Wang, Yu Tian

    2014-01-01

    Rapid and reversible methods for altering the level of endogenous proteins are critically important for studying biological systems and developing therapeutics. Here, we describe a membrane permeable targeting peptide-based method that rapidly and reversibly knocks down endogenous proteins through chaperone-mediated autophagy in vitro and in vivo. We demonstrated the specificity, efficacy and generalizability of the method by showing efficient knockdown of various proteins including death associated protein kinase 1 (160kDa), scaffolding protein PSD-95 (95kDa) and α-synuclein (18kDa) with their respective targeting peptides in a dose-, time- and lysosomal activity-dependent manner in neuronal cultures. More significantly, we showed that when given systemically, the peptide system efficiently knocked down the targeted protein in the brain of intact rats. Our study provides a robust and convenient research tool to manipulate endogenous protein levels, and may also lead to the development of protein knockdown-based novel therapeutics for treating various human diseases. PMID:24464042

  10. 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

  11. Two proteins with reverse transcriptase activities associated with hepatitis B virus-like particles

    SciTech Connect

    Bavand, M.R.; Laub, O. )

    1988-02-01

    Recent studies suggest that hepatitis B virus (HBV), despite being a DNA virus, replicates via an RNA intermediate. The HBV life cycle is therefore a permuted version of the RNA retroviral life cycle. Sequence homology between retroviral reverse transcriptase and the putative HBV polymerase gene product suggests the presence of an HBV reverse transcriptase. As yet, there has been no direct evidence that reverse transcriptase activity is present in the viral particle. The authors used activity gel analysis to detect the in situ catalytic activities of DNA polymerases after sodium dodecyl sulfate-polyacrylamide gel electrophorsis. These studies demonstrated that HBV-like particles secreted by a differentiated human hepatoma cell line tranfected with genomic HBV DNA contain two major polymerase activities which migrate as {approximately}90- and {approximately}70-kilodalton (kDa) proteins. This demonstrated, for the first time, that HBV-like particles contain a novel DNA polymerase-reverse transcriptase activity. Furthermore, they propose that the 70-kDa reverse transcriptase may be produced by proteolytic self-cleavage of the 90-kDa precursor protein.

  12. Reverse Phase Protein Arrays—Quantitative Assessment of Multiple Biomarkers in Biopsies for Clinical Use

    PubMed Central

    Boellner, Stefanie; Becker, Karl-Friedrich

    2015-01-01

    Reverse Phase Protein Arrays (RPPA) represent a very promising sensitive and precise high-throughput technology for the quantitative measurement of hundreds of signaling proteins in biological and clinical samples. This array format allows quantification of one protein or phosphoprotein in multiple samples under the same experimental conditions at the same time. Moreover, it is suited for signal transduction profiling of small numbers of cultured cells or cells isolated from human biopsies, including formalin fixed and paraffin embedded (FFPE) tissues. Owing to the much easier sample preparation, as compared to mass spectrometry based technologies, and the extraordinary sensitivity for the detection of low-abundance signaling proteins over a large linear range, RPPA have the potential for characterization of deregulated interconnecting protein pathways and networks in limited amounts of sample material in clinical routine settings. Current aspects of RPPA technology, including dilution curves, spotting, controls, signal detection, antibody validation, and calculation of protein levels are addressed.

  13. Site-specific, reversible and fluorescent immobilization of proteins on CrAsH-modified surfaces for microarray analytics.

    PubMed

    Schulte-Zweckel, Janine; Rosi, Federica; Sreenu, Domalapally; Schröder, Hendrik; Niemeyer, Christof M; Triola, Gemma

    2014-10-28

    A novel technique for protein immobilization onto CrAsH-modified surfaces is presented. This approach enables an efficient, reversible and fluorogenic immobilization of proteins. Moreover, expressed proteins can also be directly immobilized from cellular lysates without prior purification. The immobilized proteins are suitable for protein-protein interaction studies and the fluorescence enhancement upon immobilization can be employed for the direct detection of the immobilized protein without the need for secondary detection methods. PMID:25207673

  14. Filament winding

    NASA Astrophysics Data System (ADS)

    Shibley, A. M.

    The major aspects of filament winding are discussed, emphasizing basic reinforcement and matrix materials, winding procedures, process controls, and cured composite properties. Fiberglass (E-glass and S-glass strengths are 500,000 and 665,000 psi respectively) and polyester resins are the principal reinforcement constituent materials. Graphite and aramid reinforcements are being used more frequently, primarily for the more critical pressure vessels. Matrix systems are most commonly based on epoxy as it has superior mechanical properties, fatigue behavior, and heat resistance as compard with polyesters. A fiberglass overwrap of PVC pipe is an anticipated development in on-site winding and combination winding, and the compression molding of filament wound lay-ups will be investigated. The fabrication of weight-sensitive structural components may be achieved by using such moldings.

  15. A Novel Fic (Filamentation Induced by cAMP) Protein from Clostridium difficile Reveals an Inhibitory Motif-independent Adenylylation/AMPylation Mechanism.

    PubMed

    Dedic, Emil; Alsarraf, Husam; Welner, Ditte Hededam; Østergaard, Ole; Klychnikov, Oleg I; Hensbergen, Paul J; Corver, Jeroen; van Leeuwen, Hans C; Jørgensen, René

    2016-06-17

    Filamentation induced by cAMP (Fic) domain proteins have been shown to catalyze the transfer of the AMP moiety from ATP onto a protein target. This type of post-translational modification was recently shown to play a crucial role in pathogenicity mediated by two bacterial virulence factors. Herein we characterize a novel Fic domain protein that we identified from the human pathogen Clostridium difficile The crystal structure shows that the protein adopts a classical all-helical Fic fold, which belongs to class II of Fic domain proteins characterized by an intrinsic N-terminal autoinhibitory α-helix. A conserved glutamate residue in the inhibitory helix motif was previously shown in other Fic domain proteins to prevent proper binding of the ATP γ-phosphate. However, here we demonstrate that both ATP binding and autoadenylylation activity of the C. difficile Fic domain protein are independent of the inhibitory motif. In support of this, the crystal structure of a mutant of this Fic protein in complex with ATP reveals that the γ-phosphate adopts a conformation unique among Fic domains that seems to override the effect of the inhibitory helix. These results provide important structural insight into the adenylylation reaction mechanism catalyzed by Fic domains. Our findings reveal the presence of a class II Fic domain protein in the human pathogen C. difficile that is not regulated by autoinhibition and challenge the current dogma that all class I-III Fic domain proteins are inhibited by the inhibitory α-helix. PMID:27076635

  16. Cloning of the non-neuronal intermediate filament protein of the gastropod Aplysia californica; identification of an amino acid residue essential for the IFA epitope.

    PubMed

    Riemer, D; Dodemont, H; Weber, K

    1991-12-01

    We describe the isolation and characterization of a full-length cDNA corresponding to the larger non-neuronal (nn) intermediate filament (IF) protein of the gastropod Aplysia californica. Comparison of the sequences of the nn-IF proteins from Aplysia californica and Helix aspersa shows a strong evolutionary drift. At a 72% sequence identity level, the IF proteins of Opisthobranchia and Pulmonata show a larger distance than vimentins from Xenopus and mammals. The sequence comparison of the two snail proteins provides an important step in understanding the epitope of the monoclonal antibody IFA mapped by previous studies to the consensus sequence at the carboxy-terminal end of the rod domain of IF proteins. We identify for the first time in a naturally occurring IF protein a single amino acid exchange which leads to the loss of the epitope. The consensus sequence YRKLLEGEE present in IFA-positive proteins such as the Helix IF protein is changed in the IFA-negative Aplysia protein only by the conservative substitution of the arginine (R) by a lysine (K). Thus, the IFA epitope is not a necessity of IF structure, and its presence or absence on different IF proteins reflects only small changes in an otherwise conserved consensus sequence. Consequently, lack of IFA reactivity does not exclude the presence of IF. This result predicts that IF are much more universally expressed in lower eukaryotes than currently expected from immunological results with the monoclonal antibody IFA. PMID:1724961

  17. Orthogonal site-specific protein modification by engineering reversible thiol protection mechanisms

    PubMed Central

    Smith, J. Jefferson; Conrad, David W.; Cuneo, Matthew J.; Hellinga, Homme W.

    2005-01-01

    Covalent modification is an important strategy for introducing new functions into proteins. As engineered proteins become more sophisticated, it is often desirable to introduce multiple, modifications involving several different functionalities in a site-specific manner. Such orthogonal labeling schemes require independent labeling of differentially reactive nucleophilic amino acid side chains. We have developed two protein-mediated protection schemes that permit independent labeling of multiple thiols. These schemes exploit metal coordination or disulfide bond formation to reversibly protect cysteines in a Cys2His2 zinc finger domain. We constructed a variety of N- and C-terminal fusions of these domains with maltose-binding protein, which were labeled with two or three different fluorophores. Multiple modifications were made by reacting an unprotected cysteine in MBP first, deprotecting the zinc finger, and then reacting the zinc finger cysteines. The fusion proteins were orthogonally labeled with two different fluorophores, which exhibited intramolecular fluorescene resonance energy transfer (FRET). These conjugates showed up to a threefold ratiometric change in emission intensities in response to maltose binding. We also demonstrated that the metal- and redox-mediated protection methods can be combined to produce triple independent modifications, and prepared a protein labeled with three different fluorophores that exhibited a FRET relay. Finally, labeled glucose-binding protein was covalently patterned on glass slides using thiol-mediated immobilization chemistries. Together, these experiments demonstrated that reversible thiol protection schemes provide a rapid, straightforward method for producing multiple, site-specific modifications. PMID:15576565

  18. Role of the C-terminal Extension of Formin 2 in Its Activation by Spire Protein and Processive Assembly of Actin Filaments.

    PubMed

    Montaville, Pierre; Kühn, Sonja; Compper, Christel; Carlier, Marie-France

    2016-02-12

    Formin 2 (Fmn2), a member of the FMN family of formins, plays an important role in early development. This formin cooperates with profilin and Spire, a WASP homology domain 2 (WH2) repeat protein, to stimulate assembly of a dynamic cytoplasmic actin meshwork that facilitates translocation of the meiotic spindle in asymmetric division of mouse oocytes. The kinase-like non-catalytic domain (KIND) of Spire directly interacts with the C-terminal extension of the formin homology domain 2 (FH2) domain of Fmn2, called FSI. This direct interaction is required for the synergy between the two proteins in actin assembly. We have recently demonstrated how Spire, which caps barbed ends via its WH2 domains, activates Fmn2. Fmn2 by itself associates very poorly to filament barbed ends but is rapidly recruited to Spire-capped barbed ends via the KIND domain, and it subsequently displaces Spire from the barbed end to elicit rapid processive assembly from profilin·actin. Here, we address the mechanism by which Spire and Fmn2 compete at barbed ends and the role of FSI in orchestrating this competition as well as in the processivity of Fmn2. We have combined microcalorimetric, fluorescence, and hydrodynamic binding assays, as well as bulk solution and single filament measurements of actin assembly, to show that removal of FSI converts Fmn2 into a Capping Protein. This activity is mimicked by association of KIND to Fmn2. In addition, FSI binds actin at filament barbed ends as a weak capper and plays a role in displacing the WH2 domains of Spire from actin, thus allowing the association of actin-binding regions of FH2 to the barbed end. PMID:26668326

  19. 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. PMID:26206330

  20. A tunable and reversible platform for the intracellular formation of genetically engineered protein microdomains.

    PubMed

    Pastuszka, Martha K; Janib, Siti M; Weitzhandler, Isaac; Okamoto, Curtis T; Hamm-Alvarez, Sarah; Mackay, J Andrew

    2012-11-12

    From mitochondria to the nuclear envelope, the controlled assembly of micro- and nanostructures is essential for life; however, the level at which we can deliberately engineer the assembly of microstructures within intracellular environments remains primitive. To overcome this obstacle, we present a platform to reversibly assemble genetically engineered protein microdomains (GEPMs) on the time scale of minutes within living cells. Biologically inspired from the human protein tropoelastin, these protein polymers form a secondary aqueous phase above a tunable transition temperature. This assembly process is easily manipulated to occur at or near physiological temperature by adjusting molecular weight and hydrophobicity. We fused protein polymers to green fluorescent protein (GFP) to visualize their behavior within the cytoplasm. While soluble, these polymers have a similar intracellular diffusion constant as cytosolic proteins at 7.4 μm(2)/s; however, above their phase transition temperature, the proteins form distinct microdomains (0.1-2 μm) with a reduced diffusion coefficient of 1.1 μm(2)/s. Microdomain assembly and disassembly are both rapid processes with half-lives of 3.8 and 1.0 min, respectively. Via selection of the protein polymer, the assembly temperature is tunable between 20 and 40 °C. This approach may be useful to control intracellular formation of genetically engineered proteins and protein complexes into concentrated microdomains. PMID:23088632

  1. XRCC4 and XLF form long helical protein filaments suitable for DNA end protection and alignment to facilitate DNA double strand break repair

    PubMed Central

    Mahaney, Brandi L.; Hammel, Michal; Meek, Katheryn; Tainer, John A.; Lees-Miller, Susan P.

    2013-01-01

    DNA double strand breaks (DSBs), induced by ionizing radiation (IR) and endogenous stress including replication failure, are the most cytotoxic form of DNA damage. In human cells, most IR-induced DSBs are repaired by the non-homologous end joining (NHEJ) pathway. One of the most critical steps in NHEJ is ligation of DNA ends by DNA ligase IV (LIG4), which interacts with, and is stabilized by, the scaffolding protein X-ray cross-complementing gene 4 (XRCC4). XRCC4 also interacts with XRCC4-like factor (XLF, also called Cernunnos); yet, XLF has been one of the least mechanistically understood proteins and precisely how XLF functions in NHEJ has been enigmatic. Here, we examine current combined structural and mutational findings that uncover integrated functions of XRCC4 and XLF and reveal their interactions to form long, helical protein filaments suitable to protect and align DSB ends. XLF-XRCC4 provides a global structural scaffold for ligating DSBs without requiring long complementary DNA ends, thus ensuring accurate and efficient ligation and repair. The assembly of these XRCC4-XLF filaments, providing both DNA end protection and alignment, may commit cells to NHEJ with general biological implications for NHEJ and DSB repair processes and their links to cancer predispositions and interventions. PMID:23442139

  2. XRCC4 and XLF form long helical protein filaments suitable for DNA end protection and alignment to facilitate DNA double strand break repair.

    PubMed

    Mahaney, Brandi L; Hammel, Michal; Meek, Katheryn; Tainer, John A; Lees-Miller, Susan P

    2013-02-01

    DNA double strand breaks (DSBs), induced by ionizing radiation (IR) and endogenous stress including replication failure, are the most cytotoxic form of DNA damage. In human cells, most IR-induced DSBs are repaired by the nonhomologous end joining (NHEJ) pathway. One of the most critical steps in NHEJ is ligation of DNA ends by DNA ligase IV (LIG4), which interacts with, and is stabilized by, the scaffolding protein X-ray cross-complementing gene 4 (XRCC4). XRCC4 also interacts with XRCC4-like factor (XLF, also called Cernunnos); yet, XLF has been one of the least mechanistically understood proteins and precisely how XLF functions in NHEJ has been enigmatic. Here, we examine current combined structural and mutational findings that uncover integrated functions of XRCC4 and XLF and reveal their interactions to form long, helical protein filaments suitable to protect and align DSB ends. XLF-XRCC4 provides a global structural scaffold for ligating DSBs without requiring long DNA ends, thus ensuring accurate and efficient ligation and repair. The assembly of these XRCC4-XLF filaments, providing both DNA end protection and alignment, may commit cells to NHEJ with general biological implications for NHEJ and DSB repair processes and their links to cancer predispositions and interventions. PMID:23442139

  3. Ca(2+)/calmodulin-dependent protein kinase phosphatase (CaMKP/PPM1F) interacts with neurofilament L and inhibits its filament association.

    PubMed

    Ozaki, Hana; Katoh, Tsuyoshi; Nakagawa, Ryoko; Ishihara, Yasuhiro; Sueyoshi, Noriyuki; Kameshita, Isamu; Taniguchi, Takanobu; Hirano, Tetsuo; Yamazaki, Takeshi; Ishida, Atsuhiko

    2016-09-01

    Ca(2+)/calmodulin-dependent protein kinase phosphatase (CaMKP/PPM1F) is a Ser/Thr phosphatase that belongs to the PPM family. Growing evidence suggests that PPM phosphatases including CaMKP act as a complex with other proteins to regulate cellular functions. In this study, using the two-dimensional far-western blotting technique with digoxigenin-labeled CaMKP as a probe, in conjunction with peptide mass fingerprinting analysis, we identified neurofilament L (NFL) as a CaMKP-binding protein in a Triton-insoluble fraction of rat brain. We confirmed binding of fluorescein-labeled CaMKP (F-CaMKP) to NFL in solution by fluorescence polarization. The analysis showed that the dissociation constant of F-CaMKP for NFL is 73 ± 17 nM (n = 3). Co-immunoprecipitation assay using a cytosolic fraction of NGF-differentiated PC12 cells showed that endogenous CaMKP and NFL form a complex in cells. Furthermore, the effect of CaMKP on self-assembly of NFL was examined. Electron microscopy revealed that CaMKP markedly prevented NFL from forming large filamentous aggregates, suggesting that CaMKP-binding to NFL inhibits its filament association. These findings may provide new insights into a novel mechanism for regulating network formation of neurofilaments during neuronal differentiation. PMID:27369073

  4. Prediction of Mutational Tolerance in HIV-1 Protease and Reverse Transcriptase Using Flexible Backbone Protein Design

    PubMed Central

    Varela, Rocco; Ó Conchúir, Shane; Kortemme, Tanja

    2012-01-01

    Predicting which mutations proteins tolerate while maintaining their structure and function has important applications for modeling fundamental properties of proteins and their evolution; it also drives progress in protein design. Here we develop a computational model to predict the tolerated sequence space of HIV-1 protease reachable by single mutations. We assess the model by comparison to the observed variability in more than 50,000 HIV-1 protease sequences, one of the most comprehensive datasets on tolerated sequence space. We then extend the model to a second protein, reverse transcriptase. The model integrates multiple structural and functional constraints acting on a protein and uses ensembles of protein conformations. We find the model correctly captures a considerable fraction of protease and reverse-transcriptase mutational tolerance and shows comparable accuracy using either experimentally determined or computationally generated structural ensembles. Predictions of tolerated sequence space afforded by the model provide insights into stability-function tradeoffs in the emergence of resistance mutations and into strengths and limitations of the computational model. PMID:22927804

  5. Assessing the potential of atomistic molecular dynamics simulations to probe reversible protein-protein recognition and binding

    PubMed Central

    Abriata, Luciano A.; Dal Peraro, Matteo

    2015-01-01

    Protein-protein recognition and binding are governed by diffusion, noncovalent forces and conformational flexibility, entangled in a way that only molecular dynamics simulations can dissect at high resolution. Here we exploited ubiquitin’s noncovalent dimerization equilibrium to assess the potential of atomistic simulations to reproduce reversible protein-protein binding, by running submicrosecond simulations of systems with multiple copies of the protein at millimolar concentrations. The simulations essentially fail because they lead to aggregates, yet they reproduce some specificity in the binding interfaces as observed in known covalent and noncovalent ubiquitin dimers. Following similar observations in literature we hint at electrostatics and water descriptions as the main liable force field elements, and propose that their optimization should consider observables relevant to multi-protein systems and unfolded proteins. Within limitations, analysis of binding events suggests salient features of protein-protein recognition and binding, to be retested with improved force fields. Among them, that specific configurations of relative direction and orientation seem to trigger fast binding of two molecules, even over 50 Å distances; that conformational selection can take place within surface-to-surface distances of 10 to 40 Å i.e. well before actual intermolecular contact; and that establishment of contacts between molecules further locks their conformations and relative orientations. PMID:26023027

  6. 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.

  7. 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

  8. 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

  9. 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

  10. Selective and Reversible Photochemical Derivatization of Cysteine Residues in Peptides and Proteins.

    PubMed

    Arumugam, Selvanathan; Guo, Jun; Mbua, Ngalle Eric; Friscourt, Frédéric; Lin, Nannan; Nekongo, Emmanuel; Boons, Geert-Jan; Popik, Vladimir V

    2014-04-01

    Selective derivatization of solvent-exposed cysteine residues in peptides and proteins is achieved by brief irradiation of an aqueous solution containing 3-(hydroxymethyl)-2-naphthol derivatives (NQMPs) with 350 nm fluorescent lamp. NQMP can be conjugated with various moieties, such as PEG, dyes, carbohydrates, or possess a fragment for further selective derivatization, e.g., biotin, azide, alkyne, etc. Attractive features of this labeling approach include an exceptionally fast rate of the reaction and a requirement for low equivalence of the reagent. The NQMP-thioether linkage is stable under ambient conditions, survives protein digestion and MS analysis. Irradiation of NQMP-labeled protein in a dilute solution (<40 μM) or in the presence of a vinyl ether results in a traceless release of the substrate. The reversible biotinylation of bovine serum albumin, as well as capture and release of this protein using NeutrAvidin Agarose resin beads has been demonstrated. PMID:24765521

  11. Simultaneous serum desalting and total protein determination by macroporous reversed-phase chromatography.

    PubMed

    Boichenko, Alexander; Govorukhina, Natalia; van der Zee, Ate G J; Bischoff, Rainer

    2013-04-01

    Macroporous reversed-phase (mRP) chromatography was successfully used to develop an accurate and precise method for total protein in serum. The limits of detection (0.83 μg, LOD) and quantification (2.51 μg, LOQ) for the mRP method are comparable with those of the widely used micro BCA protein assay. The mRP method can be used to determine the total protein concentration across a wide dynamic range by detecting chromatographic peaks at 215 nm and 280 nm. The method has the added advantage of desalting and denaturing proteins, leading to more complete digestion by trypsin and to better LC-MS-MS identification in shotgun proteomics experiments. PMID:23388688

  12. Construction, exploitation and evolution of a new peptide library displayed at high density by fusion to the major coat protein of filamentous phage.

    PubMed

    Iannolo, G; Minenkova, O; Gonfloni, S; Castagnoli, L; Cesareni, G

    1997-06-01

    The amino-terminus of the major coat protein (PVIII) of filamentous phage can be extended, up to 6-7 residues, without interfering with the phage life cycle. We have constructed a library of approximately ten millions different phage each displaying a different octapeptide joined to the amino-terminus of the 2700 copies of PVIII. Most of the resulting clones are able to produce infective particles. This molecular repertoire constituted by the periodic regular decoration of the phage filament surface, can be utilized to search elements that bind proteins or relatively small organic molecules like the textile dye Cibacron blue. By sequential growth cycles we have performed a library evolution experiment to select phage clones that have a growth advantage in the absence of any requirement for binding a specific target. The consensus of the best growers reveals a Pro rich sequence with large hydrophobic residues at position 7 and Asn at position 1 of the random peptide insert. We propose that the assembly secretion process is favoured in phages displaying this family of peptides since they fit the groove between two adjacent PVIII subunits by making advantageous molecular contacts on the phage surface. PMID:9224932

  13. Selective binding of proteins on functional nanoparticles via reverse charge parity model: an in vitro study

    NASA Astrophysics Data System (ADS)

    Ghosh, Goutam; Panicker, Lata; Barick, K. C.

    2014-03-01

    The conformation of proteins absorbed on nanoparticles surface plays a crucial role in applications of nanoparticles in biomedicine. The surface protein conformation depends on several factors, namely, nature of protein-nanoparticles interaction, chemical composition of the surface of nanoparticles etc. A model of the electrostatic binding of proteins on charged surface nanoparticles has been proposed earlier (Ghosh et al 2013 Colloids Surf. B 103 267). Also, the irreversible denaturation of the protein conformation due to binding of counterions was reported. In this paper, we have used this model, involving reverse charge parity, to show selective binding of proteins on charged surface iron oxide nanoparticles (IONPs). IONPs were surface functionalized with cetylpyridinium chloride (CPC), cetyl(trimethyl)ammonium bromide (CTAB) and cetylpyridinium iodide (CPI). The effect of counterions (Cl-, Br- and I-) on protein conformation has also been investigated. Several proteins such as α-lactalbumin (ALA), β-lactoglobulin (BLG), ovalbumin (OVA), bovin serum albumin (BSA) and HEWL were chosen for this investigation.

  14. Lactobacillus salivarius reverse diabetes-induced intestinal defense impairment in mice through non-defensin protein.

    PubMed

    Chung, Pei-Hsuan; Wu, Ying-Ying; Chen, Pei-Hsuan; Fung, Chang-Phone; Hsu, Ching-Mei; Chen, Lee-Wei

    2016-09-01

    Altered intestinal microbiota and subsequent endotoxemia play pathogenic roles in diabetes. We aimed to study the mechanisms of intestinal defense impairment in type 1 diabetes and the effects of Lactobacillus salivarius as well as fructooligosaccharides (FOS) supplementation on diabetes-induced bacterial translocation. Alterations in the enteric microbiome, expression of mucosal antibacterial proteins and bacteria-killing activity of the intestinal mucosa in streptozotocin (STZ)-induced diabetic mice and Ins2(Akita) mice were investigated. The effects of dead L. salivarius (2×10(8)CFU/ml) and FOS (250 mg per day) supplementation for 1 week on endotoxin levels and Klebsiella pneumoniae translocation were also examined. Finally, germ-free mice were cohoused with wild-type or Ins2(Akita) mice for 2 weeks to examine the contribution of microbiota on the antibacterial protein expression. STZ-induced diabetic mice developed intestinal defense impairment as demonstrated by decreased mucosal bacteria-killing activity; reduction of non-defensin family proteins, such as Reg3β, Reg3γ, CRP-ductin and RELMβ, but not the defensin family proteins; and increased bacterial translocation. Intestinal bacteria overgrowth, enteric dysbiosis and increased intestinal bacterial translocation, particularly pathogenic K. pneumoniae in STZ-induced diabetic mice and Ins2(Akita) mice, were noted. Treating diabetic mice with dead L. salivarius or FOS reversed enteric dysbiosis, restored mucosal antibacterial protein and lessened endotoxin levels as well as K. pneumoniae translocation. Moreover, germ-free mice cohoused with wild-type mice demonstrated more intestinal Reg3β and RELMβ expression than those cohoused with Ins2(Akita) mice. These results indicate that hyperglycemia induces enteric dysbiosis, reduction of non-defensin proteins as well as bacteria-killing activity of the intestinal mucosa and intestinal defense impairment. Reversal of enteric dysbiosis with dead L. salivarius or

  15. Lithium reverses increased rates of cerebral protein synthesis in a mouse model of fragile X syndrome

    PubMed Central

    Liu, Zhong-Hua; Huang, Tianjian; Smith, Carolyn Beebe

    2012-01-01

    Individuals with fragile X syndrome (FXS), an inherited form of cognitive disability, have a wide range of symptoms including hyperactivity, autistic behavior, seizures and learning deficits. FXS is caused by silencing of FMR1 and the consequent absence of fragile X mental retardation protein (FMRP). FMRP is an RNA-binding protein that associates with polyribosomes and negatively regulates translation. In a previous study of a mouse model of FXS (Fmr1 knockout (KO)) we demonstrated that in vivo rates of cerebral protein synthesis (rCPS) were elevated in selective brain regions suggesting that the absence of FMRP in FXS may result in dysregulation of cerebral protein synthesis. Lithium, a drug used clinically to treat bipolar disorder, has been used to improve mood dysregulation in individuals with FXS. We reported previously that in the Fmr1 KO mouse chronic dietary lithium treatment reversed or ameliorated both behavioral and morphological abnormalities. Herein we report that chronic dietary lithium treatment reversed the increased rCPS in Fmr1 KO mice with little effect on wild type mice. We also report our results of analyses of key signaling molecules involved in regulation of mRNA translation. Our analyses indicate that neither effects on the PI3K/Akt nor the MAPK/ERK 1/2 pathway fully account for the effects of lithium treatment on rCPS. Collectively our findings and those from other laboratories on the efficacy of lithium treatment in animal models support further studies in patients with FXS. PMID:22227453

  16. 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

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

    PubMed

    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

  18. 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

    2016-04-01

    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. PMID:26852694

  19. Chaperonin filaments: The archaeal cytoskeleton?

    PubMed Central

    Trent, Jonathan D.; Kagawa, Hiromi K.; Yaoi, Takuro; Olle, Eric; Zaluzec, Nestor J.

    1997-01-01

    Chaperonins are high molecular mass double-ring structures composed of 60-kDa protein subunits. In the hyperthermophilic archaeon Sulfolobus shibatae the two chaperonin proteins represent ≈4% of its total protein and have a combined intracellular concentration of >30 mg/ml. At concentrations ≥ 0.5 mg/ml purified chaperonins form filaments in the presence of Mg2+ and nucleotides. Filament formation requires nucleotide binding (not hydrolysis), and occurs at physiological temperatures in biologically relevant buffers, including a buffer made from cell extracts. These observations suggest that chaperonin filaments may exist in vivo and the estimated 4600 chaperonins per cell suggest that such filaments could form an extensive cytostructure. We observed filamentous structures in unfixed, uranyl-acetate-stained S. shibatae cells, which resemble the chaperonin filaments in size and appearance. ImmunoGold (Janssen) labeling using chaperonin antibodies indicated that many chaperonins are associated with insoluble cellular structures and these structures appear to be filamentous in some areas, although they could not be uranyl-acetate-stained. The existence of chaperonin filaments in vivo suggests a mechanism whereby their protein-folding activities can be regulated. More generally, the filaments themselves may play a cytoskeletal role in Archaea. PMID:9144246

  20. Differential Regulation of Telomerase Reverse Transcriptase Promoter Activation and Protein Degradation by Histone Deacetylase Inhibition.

    PubMed

    Qing, Hua; Aono, Jun; Findeisen, Hannes M; Jones, Karrie L; Heywood, Elizabeth B; Bruemmer, Dennis

    2016-06-01

    Telomerase reverse transcriptase (TERT) maintains telomeres and is rate limiting for replicative life span. While most somatic tissues silence TERT transcription resulting in telomere shortening, cells derived from cancer or cardiovascular diseases express TERT and activate telomerase. In the present study, we demonstrate that histone deacetylase (HDAC) inhibition induces TERT transcription and promoter activation. At the protein level in contrast, HDAC inhibition decreases TERT protein abundance through enhanced degradation, which decreases telomerase activity and induces senescence. Finally, we demonstrate that HDAC inhibition decreases TERT expression during vascular remodeling in vivo. These data illustrate a differential regulation of TERT transcription and protein stability by HDAC inhibition and suggest that TERT may constitute an important target for the anti-proliferative efficacy of HDAC inhibitors. PMID:26505494

  1. Red Light-Regulated Reversible Nuclear Localization of Proteins in Mammalian Cells and Zebrafish.

    PubMed

    Beyer, Hannes M; Juillot, Samuel; Herbst, Kathrin; Samodelov, Sophia L; Müller, Konrad; Schamel, Wolfgang W; Römer, Winfried; Schäfer, Eberhard; Nagy, Ferenc; Strähle, Uwe; Weber, Wilfried; Zurbriggen, Matias D

    2015-09-18

    Protein trafficking in and out of the nucleus represents a key step in controlling cell fate and function. Here we report the development of a red light-inducible and far-red light-reversible synthetic system for controlling nuclear localization of proteins in mammalian cells and zebrafish. First, we synthetically reconstructed and validated the red light-dependent Arabidopsis phytochrome B nuclear import mediated by phytochrome-interacting factor 3 in a nonplant environment and support current hypotheses on the import mechanism in planta. On the basis of this principle we next regulated nuclear import and activity of target proteins by the spatiotemporal projection of light patterns. A synthetic transcription factor was translocated into the nucleus of mammalian cells and zebrafish to drive transgene expression. These data demonstrate the first in vivo application of a plant phytochrome-based optogenetic tool in vertebrates and expand the repertoire of available light-regulated molecular devices. PMID:25803699

  2. 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

  3. Structural basis for reversible photobleaching of a green fluorescent protein homologue

    PubMed Central

    Henderson, J. Nathan; Ai, Hui-wang; Campbell, Robert E.; Remington, S. James

    2007-01-01

    Fluorescent protein (FP) variants that can be reversibly converted between fluorescent and nonfluorescent states have proven to be a catalyst for innovation in the field of fluorescence microscopy. However, the structural basis of the process remains poorly understood. High-resolution structures of a FP derived from Clavularia in both the fluorescent and the light-induced nonfluorescent states reveal that the rapid and complete loss of fluorescence observed upon illumination with 450-nm light results from cis–trans isomerization of the chromophore. The photoinduced change in configuration from the well ordered cis isomer to the highly nonplanar and disordered trans isomer is accompanied by a dramatic rearrangement of internal side chains. Taken together, the structures provide an explanation for the loss of fluorescence upon illumination, the slow light-independent recovery, and the rapid light-induced recovery of fluorescence. The fundamental mechanism appears to be common to all of the photoactivatable and reversibly photoswitchable FPs reported to date. PMID:17420458

  4. Angiopoietin-like protein 4 inhibition of lipoprotein lipase: evidence for reversible complex formation.

    PubMed

    Lafferty, Michael J; Bradford, Kira C; Erie, Dorothy A; Neher, Saskia B

    2013-10-01

    Elevated triglycerides are associated with an increased risk of cardiovascular disease, and lipoprotein lipase (LPL) is the rate-limiting enzyme for the hydrolysis of triglycerides from circulating lipoproteins. The N-terminal domain of angiopoietin-like protein 4 (ANGPTL4) inhibits LPL activity. ANGPTL4 was previously described as an unfolding molecular chaperone of LPL that catalytically converts active LPL dimers into inactive monomers. Our studies show that ANGPTL4 is more accurately described as a reversible, noncompetitive inhibitor of LPL. We find that inhibited LPL is in a complex with ANGPTL4, and upon dissociation, LPL regains lipase activity. Furthermore, we have generated a variant of ANGPTL4 that is dependent on divalent cations for its ability to inhibit LPL. We show that LPL inactivation by this regulatable variant of ANGPTL4 is fully reversible after treatment with a chelator. PMID:23960078

  5. Mutations in Human Immunodeficiency Virus Type 1 Nucleocapsid Protein Zinc Fingers Cause Premature Reverse Transcription ▿

    PubMed Central

    Thomas, James A.; Bosche, William J.; Shatzer, Teresa L.; Johnson, Donald G.; Gorelick, Robert J.

    2008-01-01

    Human immunodeficiency virus type 1 (HIV-1) requires that its genome be reverse transcribed into double-stranded DNA for productive infection of cells. This process requires not only reverse transcriptase but also the nucleocapsid protein (NC), which functions as a nucleic acid chaperone. Reverse transcription generally begins once the core of the virion enters the cytoplasm of a newly infected cell. However, some groups have reported the presence of low levels of viral DNA (vDNA) within particles prior to infection, the significance and function of which is controversial. We report here that several HIV-1 NC mutants, which we previously identified as being replication defective, contain abnormally high levels of intravirion DNA. These findings were further reinforced by the inability of these NC mutants to perform endogenous reverse transcription (ERT), in contrast to the readily measurable ERT activity in wild-type HIV-1. When either of the NC mutations is combined with a mutation that inactivates the viral protease, we observed a significant reduction in the amount of intravirion DNA. Interestingly, we also observed high levels of intravirion DNA in the context of wild-type NC when we delayed budding by means of a PTAP(−) (Pro-Thr-Ala-Pro) mutation. Premature reverse transcription is most probably occurring before these mutant virions bud from producer cells, but we fail to see any evidence that the NC mutations alter the timing of Pr55Gag processing. Critically, our results also suggest that the presence of intravirion vDNA could serve as a diagnostic for identifying replication-defective HIV-1. PMID:18667500

  6. Elafin Reverses Pulmonary Hypertension via Caveolin-1–Dependent Bone Morphogenetic Protein Signaling

    PubMed Central

    Nickel, Nils P.; Spiekerkoetter, Edda; Gu, Mingxia; Li, Caiyun G.; Li, Hai; Kaschwich, Mark; Diebold, Isabel; Hennigs, Jan K.; Kim, Ki-Yoon; Miyagawa, Kazuya; Wang, Lingli; Cao, Aiqin; Sa, Silin; Jiang, Xinguo; Stockstill, Raymond W.; Nicolls, Mark R.; Zamanian, Roham T.; Bland, Richard D.

    2015-01-01

    Rationale: Pulmonary arterial hypertension is characterized by endothelial dysfunction, impaired bone morphogenetic protein receptor 2 (BMPR2) signaling, and increased elastase activity. Synthetic elastase inhibitors reverse experimental pulmonary hypertension but cause hepatotoxicity in clinical studies. The endogenous elastase inhibitor elafin attenuates hypoxic pulmonary hypertension in mice, but its potential to improve endothelial function and BMPR2 signaling, and to reverse severe experimental pulmonary hypertension or vascular pathology in the human disease was unknown. Objectives: To assess elafin-mediated regression of pulmonary vascular pathology in rats and in lung explants from patients with pulmonary hypertension. To determine if elafin amplifies BMPR2 signaling in pulmonary artery endothelial cells and to elucidate the underlying mechanism. Methods: Rats with pulmonary hypertension induced by vascular endothelial growth factor receptor blockade and hypoxia (Sugen/hypoxia) as well as lung organ cultures from patients with pulmonary hypertension were used to assess elafin-mediated reversibility of pulmonary vascular disease. Pulmonary arterial endothelial cells from patients and control subjects were used to determine the efficacy and mechanism of elafin-mediated BMPR2 signaling. Measurements and Main Results: In Sugen/hypoxia rats, elafin reduced elastase activity and reversed pulmonary hypertension, judged by regression of right ventricular systolic pressure and hypertrophy and pulmonary artery occlusive changes. Elafin improved endothelial function by increasing apelin, a BMPR2 target. Elafin induced apoptosis in human pulmonary arterial smooth muscle cells and decreased neointimal lesions in lung organ culture. In normal and patient pulmonary artery endothelial cells, elafin promoted angiogenesis by increasing pSMAD-dependent and -independent BMPR2 signaling. This was linked mechanistically to augmented interaction of BMPR2 with caveolin-1 via

  7. Natural plasmids of filamentous fungi.

    PubMed Central

    Griffiths, A J

    1995-01-01

    Among eukaryotes, plasmids have been found in fungi and plants but not in animals. Most plasmids are mitochondrial. In filamentous fungi, plasmids are commonly encountered in isolates from natural populations. Individual populations may show a predominance of one type, but some plasmids have a global distribution, often crossing species boundaries. Surveys have shown that strains can contain more than one type of plasmid and that different types appear to be distributed independently. In crosses, plasmids are generally inherited maternally. Horizontal transmission is by cell contact. Circular plasmids are common only in Neurospora spp., but linear plasmids have been found in many fungi. Circular plasmids have one open reading frame (ORF) coding for a DNA polymerase or a reverse transcriptase. Linear plasmids generally have two ORFs, coding for presumptive DNA and RNA polymerases with amino acid motifs showing homology to viral polymerases. Plasmids often attain a high copy number, in excess of that of mitochondrial DNA. Linear plasmids have a protein attached to their 5' end, and this is presumed to act as a replication primer. Most plasmids are neutral passengers, but several linear plasmids integrate into mitochondrial DNA, causing death of the host culture. Inferred amino acid sequences of linear plasmid ORFs have been used to plot phylogenetic trees, which show a fair concordance with conventional trees. The circular Neurospora plasmids have replication systems that seem to be evolutionary intermediates between the RNA and the DNA worlds. PMID:8531891

  8. Mutant forms of growth factor-binding protein-2 reverse BCR-ABL-induced transformation.

    PubMed Central

    Gishizky, M L; Cortez, D; Pendergast, A M

    1995-01-01

    Growth factor-binding protein 2 (Grb2) is an adaptor protein that links tyrosine kinases to Ras. BCR-ABL is a tyrosine kinase oncoprotein that is implicated in the pathogenesis of Philadelphia chromosome (Ph1)-positive leukemias. Grb2 forms a complex with BCR-ABL and the nucleotide exchange factor Sos that leads to the activation of the Ras protooncogene. In this report we demonstrate that Grb2 mutant proteins lacking amino- or carboxyl-terminal src homology SH3 domains suppress BCR-ABL-induced Ras activation and reverse the oncogenic phenotype. The Grb2 SH3-deletion mutant proteins bind to BCR-ABL and do not impair tyrosine kinase activity. Expression of the Grb2 SH3-deletion mutant proteins in BCR-ABL-transformed Rat-1 fibroblasts and in the human Ph1-positive leukemic cell line K562 inhibits their ability to grow as foci in soft agar and form tumors in nude mice. Furthermore, expression of the Grb2 SH3-deletion mutants in K562 cells induced their differentiation. Because Ras plays an important role in signaling by receptor and nonreceptor tyrosine kinases, the use of interfering mutant Grb2 proteins may be applied to block the proliferation of other cancers that depend in part on activated tyrosine kinases for growth. Images Fig. 1 Fig. 2 Fig. 3 PMID:7479904

  9. Reversible suppression of protein synthesis in concert with polysome disaggregation during anoxia exposure in Littorina littorea.

    PubMed

    Larade, Kevin; Storey, Kenneth B

    2002-03-01

    Many marine invertebrates can live without oxygen for long periods of time, a capacity that is facilitated by the ability to suppress metabolic rate in anoxia to a value that is typically less than 10% of the normal aerobic rate. The present study demonstrates that a reduction in the rate of protein synthesis is one factor in the overall anoxia-induced metabolic suppression in the marine snail, Littorina littorea. The rate of [3H]leucine incorporation into newly translated protein in hepatopancreas isolated from 48 h anoxic snails was determined to be 49% relative to normoxic controls. However, protein concentration in hepatopancreas did not change during anoxia, suggesting a coordinated suppression of net protein turnover. Analysis of hepatopancreas samples from snails exposed to 24-72 h anoxia showed a gradual disaggregation of polysomes into monosomes. A re-aggregation of monosomes into polysomes was observed after 3 h of aerobic recovery. Analysis of fractions from the ribosome profile using radiolabeled probe to detect alpha-tubulin transcripts confirmed a general decrease in protein translation during anoxia exposure (transcript association with polysomes decreased) with a reversal during aerobic recovery. Western blotting of hepatopancreas samples from normoxic, 24 h anoxic, and 1 h aerobic recovered snails demonstrated that eIF-2alpha is substantially phosphorylated during anoxia exposure and dephosphorylated during normoxia and aerobic recovery, suggesting a decrease in translation initiation during anoxia exposure. These results suggest that metabolic suppression during anoxia exposure in L. littorea involves a decrease in protein translation. PMID:12030368

  10. Reversible oxidation of phosphatase and tensin homolog (PTEN) alters its interactions with signaling and regulatory proteins.

    PubMed

    Verrastro, Ivan; Tveen-Jensen, Karina; Woscholski, Rudiger; Spickett, Corinne M; Pitt, Andrew R

    2016-01-01

    Phosphatase and tensin homolog (PTEN) is involved in a number of different cellular processes including metabolism, apoptosis, cell proliferation and survival. It is a redox-sensitive dual-specificity protein phosphatase that acts as a tumor suppressor by negatively regulating the PI3K/Akt pathway. While direct evidence of redox regulation of PTEN downstream signaling has been reported, the effect of PTEN redox status on its protein-protein interactions is poorly understood. PTEN-GST in its reduced and a DTT-reversible H2O2-oxidized form was immobilized on a glutathione-sepharose support and incubated with cell lysate to capture interacting proteins. Captured proteins were analyzed by LC-MSMS and comparatively quantified using label-free methods. 97 Potential protein interactors were identified, including a significant number that are novel. The abundance of fourteen interactors was found to vary significantly with the redox status of PTEN. Altered binding to PTEN was confirmed by affinity pull-down and Western blotting for Prdx1, Trx, and Anxa2, while DDB1 was validated as a novel interactor with unaltered binding. These results suggest that the redox status of PTEN causes a functional variation in the PTEN interactome. The resin capture method developed had distinct advantages in that the redox status of PTEN could be directly controlled and measured. PMID:26561776

  11. Intermediate filament proteins in classic and variant types of small cell lung carcinoma cell lines: a biochemical and immunochemical analysis using a panel of monoclonal and polyclonal antibodies.

    PubMed

    Broers, J L; Carney, D N; Klein Rot, M; Schaart, G; Lane, E B; Vooijs, G P; Ramaekers, F C

    1986-07-01

    The intermediate filament protein (IFP) characteristics of a panel of lung cancer cell lines including adenocarcinoma (two cell lines) and small cell lung cancer (SCLC, three classic and three variant cell lines) were examined using one- and two-dimensional gel electrophoretic techniques, immunocytochemical techniques and immunoblotting assays. A panel of 28 monoclonal and polyclonal antibodies to the five different types of IFP were used. The results of our studies indicate that these human lung adenocarcinoma, classic SCLC and variant SCLC cell lines can be differentiated on the basis of their pattern of IFP. The main conclusions from this study can be summarized as follows. The two adenocarcinoma cell lines contain cytokeratins 7, 8, 18, and sometimes 19, next to vimentin intermediate filament (IF). The three classic-type SCLC cell lines contain only cytokeratin IFs but not vimentin IF or neurofilaments (NFs). Cytokeratin polypeptides 7, 8, 18 and 19 could be detected. All three variant-type SCLC cell lines do not contain detectable amounts of cytokeratins. In contrast, two out of three variant SCLC cell lines contain neurofilament proteins. All three variant-type SCLC cell lines contain vimentin IF. Using immunoblotting assays with monoclonal and polyclonal antibodies to defined NF proteins the presence of the 68 X 10(3) Mr and the 160 X 10(3) Mr NF polypeptide could be demonstrated in two variant SCLC cell lines. As patients with SCLC-variant phenotype have a poorer prognosis after cytotoxic therapy than patients with 'pure' SCLC, the use of antibodies to IFP in staining fresh lung tumours, especially anaplastic ones, may differentiate the two subtypes of SCLC. Such a distinction would have a major impact on therapy selections and may be of prognostic importance. PMID:2433295

  12. 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

    2016-03-01

    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

  13. Buckling of Branched Cytoskeletal Filaments

    NASA Astrophysics Data System (ADS)

    Quint, D. A.; Schwarz, J. M.

    2011-03-01

    In vitro experiments of growing dendritic actin networks demonstrate reversible stress-softening at high loads, above some critical load. The transition to the stress-softening regime has been attributed to the elastic buckling of individual actin filaments. To estimate the critical load above which softening should occur, we extend the elastic theory of buckling of individual filaments embedded in a network to include the buckling of branched filaments, a signature trait of growing dendritic actin networks. Under certain assumptions, there will be approximately a seven-fold increase in the classical critical bucking load, when compared to the unbranched filament, which is entirely due to the presence of a branch. Moreover, we go beyond the classical buckling regime to investigate the effect of entropic fluctuations. The result of compressing the filament in this case leads to an increase in these fluctuations and eventually the harmonic approximation breaks down signifying the onset of the buckling transition. We compute corrections to the classical critical buckling load near this breakdown.

  14. Filamentous Fungi.

    PubMed

    Powers-Fletcher, Margaret V; Kendall, Brian A; Griffin, Allen T; Hanson, Kimberly E

    2016-06-01

    Filamentous mycoses are often associated with significant morbidity and mortality. Prompt diagnosis and aggressive treatment are essential for good clinical outcomes in immunocompromised patients. The host immune response plays an essential role in determining the course of exposure to potential fungal pathogens. Depending on the effectiveness of immune response and the burden of organism exposure, fungi can either be cleared or infection can occur and progress to a potentially fatal invasive disease. Nonspecific cellular immunity (i.e., neutrophils, natural killer [NK] cells, and macrophages) combined with T-cell responses are the main immunologic mechanisms of protection. The most common potential mold pathogens include certain hyaline hyphomycetes, endemic fungi, the Mucorales, and some dematiaceous fungi. Laboratory diagnostics aimed at detecting and differentiating these organisms are crucial to helping clinicians make informed decisions about treatment. The purpose of this chapter is to provide an overview of the medically important fungal pathogens, as well as to discuss the patient characteristics, antifungal-therapy considerations, and laboratory tests used in current clinical practice for the immunocompromised host. PMID:27337469

  15. Gramicidin S: a peptide model for protein glycation and reversal of glycation using nucleophilic amines.

    PubMed

    Shakkottai, V G; Sudha, R; Balaram, P

    2002-08-01

    Nonenzymatic glycation of proteins has been implicated in various diabetic complications and age-related disorders. Proteins undergo glycation at the N-terminus or at the epsilon-amino group of lysine residues. Glycation of proteins proceeds through the stages of Schiff base formation, conversion to ketoamine product and advanced glycation end products. Gramicidin S, which has two ornithine residues, was used as a model system to study the various stages of glycation of proteins using electrospray ionization mass spectrometry. The proximity of two ornithine residues in the peptide favors the glycation reaction. Formation of advanced glycation end products and diglycation on ornithine residues in gramicidin S were observed. The formation of Schiff base adduct is reversible, whereas the Amadori rearrangement to the ketoamine product is irreversible. Nucleophilic amines and hydrazines can deglycate the Schiff base adduct of glucose with peptides and proteins. Hydroxylamine, isonicotinic acid hydrazide and aminoguanidine effectively removed glucose from the Schiff base adduct of gramicidin S. Hydroxylamine is more effective in deglycating the adduct compared with isonicotinic acid hydrazide and aminoguanidine. The observation that the hydrazines are effective in deglycating the Schiff base adduct even in the presence of high concentrations of glucose, may have a possible therapeutic application in preventing complications of diabetes mellitus. Hydrazines may be used to distinguish between the Schiff base and the ketoamine products formed at the initial stages of glycation. PMID:12102724

  16. 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

  17. Impact of blocking and detection chemistries on antibody performance for reverse phase protein arrays.

    PubMed

    Ambroz, Kristi

    2011-01-01

    Careful selection of well-qualified antibodies is critical for accurate data collection from reverse phase protein arrays (RPPA). The most common way to qualify antibodies for RPPA analysis is by Western blotting because the detection mechanism is based on the same immunodetection principles. Western blots of tissue or cell lysates that result in single bands and low cross-reactivity indicate appropriate antibodies for RPPA detection. Western blot conditions used to validate antibodies for RPPA experiments, including blocking and detection reagents, have significant effects on aspects of antibody performance such as cross-reactivity against other proteins in the sample. We have found that there can be a dramatic impact on antibody behavior with changes in blocking reagent and detection method, and offer an alternative method that allows detection reagents and conditions to be held constant in both antibody validation and RPPA experiments. PMID:21901590

  18. Hydrogen Bond Fluctuations Control Photochromism in a Reversibly Photo-Switchable Fluorescent Protein.

    PubMed

    Morozov, Dmitry; Groenhof, Gerrit

    2016-01-11

    Reversibly switchable fluorescent proteins (RSFPs) are essential for high-resolution microscopy of biological samples, but the reason why these proteins are photochromic is still poorly understood. To address this problem, we performed molecular dynamics simulations of the fast switching Met159Thr mutant of the RSFP Dronpa. Our simulations revealed a ground state structural heterogeneity in the chromophore pocket that consists of three populations with one, two, or three hydrogen bonds to the phenolate moiety of the chromophore. By means of non-adiabatic quantum mechanics/molecular dynamics simulations, we demonstrated that the subpopulation with a single hydrogen bond is responsible for off-switching through photo-isomerization of the chromophore, whereas two or more hydrogen bonds inhibit the isomerization and promote fluorescence instead. While rational design of new RSFPs has so far focused on structure alone, our results suggest that structural heterogeneity must be considered as well. PMID:26612709

  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

    2015-01-01

    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. PMID:26673077

  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. 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. Hyaluronan Regulates Bone Morphogenetic Protein-7-dependent Prevention and Reversal of Myofibroblast Phenotype*

    PubMed Central

    Midgley, Adam C.; Duggal, Lucy; Jenkins, Robert; Hascall, Vincent; Steadman, Robert; Phillips, Aled O.; Meran, Soma

    2015-01-01

    Hyaluronan (HA) promotes transforming growth factor (TGF)-β1-driven myofibroblast phenotype. However, HA can also have disease-limiting activity. Bone morphogenetic protein-7 (BMP7) is an antifibrotic cytokine that antagonizes TGF-β1, and isolated studies have demonstrated that HA can both mediate and modulate BMP7 responses. In this study, we investigated whether BMP7 can modulate HA in a manner that leads to prevention/reversal of TGF-β1-driven myofibroblast differentiation in human lung fibroblasts. Results demonstrated that BMP7 prevented and reversed TGF-β1-driven myofibroblast differentiation through a novel mechanism. BMP7 promoted the dissolution and internalization of cell-surface HA into cytoplasmic endosomes. Endosomal HA co-localized with the HA-degrading enzymes, hyaluronidase-1 and hyaluronidase-2 (Hyal2). Moreover, BMP7 showed differential regulation of CD44 standard and variant isoform expression, when compared with TGF-β1. In particular, BMP7 increased membrane expression of CD44v7/8. Inhibiting CD44v7/8 as well as blocking Hyal2 and the Na+/H+ exchanger-1 at the cell-surface prevented BMP7-driven HA internalization and BMP7-mediated prevention/reversal of myofibroblast phenotype. In summary, a novel mechanism of TGF-β1 antagonism by BMP7 is shown and identifies alteration in HA as critical in mediating BMP7 responses. In addition, we identify Hyal2 and CD44v7/8 as new potential targets for manipulation in prevention and reversal of fibrotic pathology. PMID:25716319

  3. Hyaluronan regulates bone morphogenetic protein-7-dependent prevention and reversal of myofibroblast phenotype.

    PubMed

    Midgley, Adam C; Duggal, Lucy; Jenkins, Robert; Hascall, Vincent; Steadman, Robert; Phillips, Aled O; Meran, Soma

    2015-05-01

    Hyaluronan (HA) promotes transforming growth factor (TGF)-β1-driven myofibroblast phenotype. However, HA can also have disease-limiting activity. Bone morphogenetic protein-7 (BMP7) is an antifibrotic cytokine that antagonizes TGF-β1, and isolated studies have demonstrated that HA can both mediate and modulate BMP7 responses. In this study, we investigated whether BMP7 can modulate HA in a manner that leads to prevention/reversal of TGF-β1-driven myofibroblast differentiation in human lung fibroblasts. Results demonstrated that BMP7 prevented and reversed TGF-β1-driven myofibroblast differentiation through a novel mechanism. BMP7 promoted the dissolution and internalization of cell-surface HA into cytoplasmic endosomes. Endosomal HA co-localized with the HA-degrading enzymes, hyaluronidase-1 and hyaluronidase-2 (Hyal2). Moreover, BMP7 showed differential regulation of CD44 standard and variant isoform expression, when compared with TGF-β1. In particular, BMP7 increased membrane expression of CD44v7/8. Inhibiting CD44v7/8 as well as blocking Hyal2 and the Na(+)/H(+) exchanger-1 at the cell-surface prevented BMP7-driven HA internalization and BMP7-mediated prevention/reversal of myofibroblast phenotype. In summary, a novel mechanism of TGF-β1 antagonism by BMP7 is shown and identifies alteration in HA as critical in mediating BMP7 responses. In addition, we identify Hyal2 and CD44v7/8 as new potential targets for manipulation in prevention and reversal of fibrotic pathology. PMID:25716319

  4. Yeast Ivy1p Is a Putative I-BAR-domain Protein with pH-sensitive Filament Forming Ability in vitro.

    PubMed

    Itoh, Yuzuru; Kida, Kazuki; Hanawa-Suetsugu, Kyoko; Suetsugu, Shiro

    2016-01-01

    Bin-Amphiphysin-Rvs161/167 (BAR) domains mold lipid bilayer membranes into tubules, by forming a spiral polymer on the membrane. Most BAR domains are thought to be involved in forming membrane invaginations through their concave membrane binding surfaces, whereas some members have convex membrane binding surfaces, and thereby mold membranes into protrusions. The BAR domains with a convex surface form a subtype called the inverse BAR (I-BAR) domain or IRSp53-MIM-homology domain (IMD). Although the mammalian I-BAR domains have been studied, those from other organisms remain elusive. Here, we found putative I-BAR domains in Fungi and animal-like unicellular organisms. The fungal protein containing the putative I-BAR-domain is known as Ivy1p in yeast, and is reportedly localized in the vacuole. The phylogenetic analysis of the I-BAR domains revealed that the fungal I-BAR-domain containing proteins comprise a distinct group from those containing IRSp53 or MIM. Importantly, Ivy1p formed a polymer with a diameter of approximately 20 nm in vitro, without a lipid membrane. The filaments were formed at neutral pH, but disassembled when pH was reverted to basic. Moreover, Ivy1p and the I-BAR domain expressed in mammalian HeLa cells was localized at a vacuole-like structure as filaments as revealed by super-resolved microscopy. These data indicate the pH-sensitive polymer forming ability and the functional conservation of Ivy1p in eukaryotic cells. PMID:26657738

  5. 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. PMID:3120191

  6. Inhibition of Wnt/β-catenin/CREB binding protein (CBP) signaling reverses pulmonary fibrosis

    PubMed Central

    Henderson, William R.; Chi, Emil Y.; Ye, Xin; Nguyen, Cu; Tien, Ying-tzang; Zhou, Beiyun; Borok, Zea; Knight, Darryl A.; Kahn, Michael

    2010-01-01

    Idiopathic pulmonary fibrosis (IPF)/usual interstitial pneumonia is a ravaging condition of progressive lung scarring and destruction. Anti-inflammatory therapies including corticosteroids have limited efficacy in this ultimately fatal disorder. An important unmet need is to identify new agents that interact with key molecular pathways involved in the pathogenesis of pulmonary fibrosis to prevent progression or reverse fibrosis in these patients. Because aberrant activation of the Wnt/β-catenin signaling cascade occurs in lungs of patients with IPF, we have targeted this pathway for intervention in pulmonary fibrosis using ICG-001, a small molecule that specifically inhibits T-cell factor/β-catenin transcription in a cyclic AMP response-element binding protein binding protein (CBP)-dependent fashion. ICG-001 selectively blocks the β-catenin/CBP interaction without interfering with the β-catenin/p300 interaction. We report here that ICG-001 (5 mg/kg per day) significantly inhibits β-catenin signaling and attenuates bleomycin-induced lung fibrosis in mice, while concurrently preserving the epithelium. Administration of ICG-001 concurrent with bleomycin prevents fibrosis, and late administration is able to reverse established fibrosis and significantly improve survival. Because no effective treatment for IPF exists, selective inhibition of Wnt/β-catenin-dependent transcription suggests a potential unique therapeutic approach for pulmonary fibrosis. PMID:20660310

  7. Ultrastructural studies on scrapie prion protein crystals obtained from reverse micellar solutions.

    PubMed Central

    Wille, H; Prusiner, S B

    1999-01-01

    The structural transition from the cellular prion protein (PrPC) that is rich in alpha-helices to the pathological form (PrPSc) that has a high beta-sheet content seems to be the fundamental event underlying the prion diseases. Determination of the structure of PrPSc and the N-terminally truncated PrP 27-30 has been complicated by their insolubility. Here we report the solubilization of PrP 27-30 through a system of reverse micelles that yields monomeric and dimeric PrP. Although solubilization of PrP 27-30 was not accompanied by any recognizable change in secondary structure as measured by FTIR spectroscopy, it did result in a loss of prion infectivity. The formation of small two- and three-dimensional crystals upon exposure to uranyl salts argues that soluble PrP 27-30 possesses considerable tertiary structure. The crystals of PrP 27-30 grown from reverse micellar solutions suggest a novel crystallization mechanism that might be applicable for other membrane proteins. A variety of different crystal lattices diffracted up to 1.85 nm by electron microscopy. Despite the lack of measurable biological activity, the structure of PrP 27-30 in these crystals may provide insight into the structural transition that occurs during PrPSc formation. PMID:9916037

  8. 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+. PMID:26661092

  9. 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.

  10. Reverse phase protein arrays in signaling pathways: a data integration perspective

    PubMed Central

    Creighton, Chad J; Huang, Shixia

    2015-01-01

    The reverse phase protein array (RPPA) data platform provides expression data for a prespecified set of proteins, across a set of tissue or cell line samples. Being able to measure either total proteins or posttranslationally modified proteins, even ones present at lower abundances, RPPA represents an excellent way to capture the state of key signaling transduction pathways in normal or diseased cells. RPPA data can be combined with those of other molecular profiling platforms, in order to obtain a more complete molecular picture of the cell. This review offers perspective on the use of RPPA as a component of integrative molecular analysis, using recent case examples from The Cancer Genome Altas consortium, showing how RPPA may provide additional insight into cancer besides what other data platforms may provide. There also exists a clear need for effective visualization approaches to RPPA-based proteomic results; this was highlighted by the recent challenge, put forth by the HPN-DREAM consortium, to develop visualization methods for a highly complex RPPA dataset involving many cancer cell lines, stimuli, and inhibitors applied over time course. In this review, we put forth a number of general guidelines for effective visualization of complex molecular datasets, namely, showing the data, ordering data elements deliberately, enabling generalization, focusing on relevant specifics, and putting things into context. We give examples of how these principles can be utilized in visualizing the intrinsic subtypes of breast cancer and in meaningfully displaying the entire HPN-DREAM RPPA dataset within a single page. PMID:26185419