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Sample records for 51-kda subunit maps

  1. Dependency Map of Proteins in the Small Ribosomal Subunit

    PubMed Central

    Hamacher, Kay; Trylska, Joanna; McCammon, J. Andrew

    2006-01-01

    The assembly of the ribosome has recently become an interesting target for antibiotics in several bacteria. In this work, we extended an analytical procedure to determine native state fluctuations and contact breaking to investigate the protein stability dependence in the 30S small ribosomal subunit of Thermus thermophilus. We determined the causal influence of the presence and absence of proteins in the 30S complex on the binding free energies of other proteins. The predicted dependencies are in overall agreement with the experimentally determined assembly map for another organism, Escherichia coli. We found that the causal influences result from two distinct mechanisms: one is pure internal energy change, the other originates from the entropy change. We discuss the implications on how to target the ribosomal assembly most effectively by suggesting six proteins as targets for mutations or other hindering of their binding. Our results show that by blocking one out of this set of proteins, the association of other proteins is eventually reduced, thus reducing the translation efficiency even more. We could additionally determine the binding dependency of THX—a peptide not present in the ribosome of E. coli—and suggest its assembly path. PMID:16485038

  2. The neuronal nicotinic acetylcholine receptor {alpha}7 subunit gene: Cloning, mapping, structure, and targeting in mouse

    SciTech Connect

    Orr-Urtreger, A.; Baldini, A.; Beaudet, A.L.

    1994-09-01

    The neuronal nicotinic acetylcholine receptor {alpha}7 subunit is a member of a family of ligand-gated ion channels, and is the only subunit know to bind {alpha}-bungarotoxin in mammalian brain. {alpha}-Bungarotoxin binding sites are known to be more abundant in the hippocampus of mouse strains that are particularly sensitive to nicotine-induced seizures. The {alpha}7 receptor is highly permeable to calcium, which could suggest a role in synaptic plasticity in the nervous system. Auditory gating deficiency, an abnormal response to a second auditory stimulus, is characteristic of schizophrenia. Mouse strains that exhibit a similar gating deficit have reduced hippocampal expression of the {alpha}7 subunit. We have cloned and sequenced the full length cDNA for the mouse {alpha}7 gene (Acra-7) and characterized its gene structure. The murine {alpha}7 shares amino acid identity of 99% and 93% with the rat and human {alpha}7 subunits, respectively. Using an interspecies backcross panel, the murine gene was mapped to chromosome 7 near the p locus, a region syntenic with human chromosome 15; the human gene (CHRNA7) was confirmed to map to 15q13-q14 by FISH. To generate a mouse {alpha}7 mutant by homologous recombination, we have constructed a replacement vector which will delete transmembrane domains II-IV and the cytoplasmic domain from the gene product. Recombinant embryonic stem (ES) cell clones were selected and used to develop mouse chimeras that are currently being bred to obtain germline transmission.

  3. Reducing a polymer to its subunits as an aid to molecular mapping

    NASA Astrophysics Data System (ADS)

    Brayshaw, Debra J.; Berry, Monica; McMaster, Terence J.

    2004-11-01

    Detailed, single-molecule AFM mapping can further structural studies of polymeric biomolecules by pinpointing discrete changes in subunits or subunit concatenation. This study explored the binding of purified (ocular) mucins, polymers composed of genetically identical subunits, to controlled surfaces. This process was followed in situ, in real time, as were the effects of the disulfide bond reducing agent dithiothreitol (DTT), a reagent routinely used to depolymerize mucins. The addition of this reagent, while mucins were bound to gold surfaces by thiol-type binding, suggested a way of assessing the strength and extent of this gold-molecule bond formation relative to other forms of mucin-substrate interactions. Real-time AFM has allowed us to visualize the cleavage of in-chain disulfide bonds in a single mucin molecule, and subsequent removal from the substrate of mucin subunits between disulfide sites. In contrast, mucins that were covalently bound via amine groups to a self-assembled succinimide monolayer were not observed to move from the point of their initial attachment to the substrate and the addition of dithiothreitol was not followed by the loss of any sections of molecules from the substrate, emphasizing the different immobilization bond types. This demonstration of the ability to follow the structural changes to a single molecule as a result of a series of chemical processes points to new approaches to single-molecule mapping, and localization of specific chemical moieties.

  4. Cytochrome oxidase subunit V gene of Neurospora crassa: DNA sequences, chromosomal mapping, and evidence that the cya-4 locus specifies the structural gene for subunit V.

    PubMed Central

    Sachs, M S; Bertrand, H; Metzenberg, R L; RajBhandary, U L

    1989-01-01

    The sequences of cDNA and genomic DNA clones for Neurospora cytochrome oxidase subunit V show that the protein is synthesized as a 171-amino-acid precursor containing a 27-amino-acid N-terminal extension. The subunit V protein sequence is 34% identical to that of Saccharomyces cerevisiae subunit V; these proteins, as well as the corresponding bovine subunit, subunit IV, contain a single hydrophobic domain which most likely spans the inner mitochondrial membrane. The Neurospora crassa subunit V gene (cox5) contains two introns, 398 and 68 nucleotides long, which share the conserved intron boundaries 5'GTRNGT...CAG3' and the internal consensus sequence ACTRACA. Two short sequences, YGCCAG and YCCGTTY, are repeated four times each in the cox5 gene upstream of the mRNA 5' termini. The cox5 mRNA 5' ends are heterogeneous, with the major mRNA 5' end located 144 to 147 nucleotides upstream from the translational start site. The mRNA contains a 3'-untranslated region of 186 to 187 nucleotides. Using restriction-fragment-length polymorphism, we mapped the cox5 gene to linkage group IIR, close to the arg-5 locus. Since one of the mutations causing cytochrome oxidase deficiency in N. crassa, cya-4-23, also maps there, we transformed the cya-4-23 strain with the wild-type cox5 gene. In contrast to cya-4-23 cells, which grow slowly, cox5 transformants grew quickly, contained cytochrome oxidase, and had 8- to 11-fold-higher levels of subunit V in their mitochondria. These data suggest (i) that the cya-4 locus in N. crassa specifies structural information for cytochrome oxidase subunit V and (ii) that, in N. crassa, as in S. cerevisiae, deficiencies in the production of nuclearly encoded cytochrome oxidase subunits result in deficiency in cytochrome oxidase activity. Finally, we show that the lower levels of subunit V in cya-4-23 cells are most likely due to substantially reduced levels of translatable subunit V mRNA. Images PMID:2540423

  5. Mapping of a conformational epitope on the cashew allergen Ana o 2: a discontinuous large subunit epitope dependent upon homologous or heterologous small subunit association.

    PubMed

    Xia, Lixin; Willison, LeAnna N; Porter, Lauren; Robotham, Jason M; Teuber, Suzanne S; Sathe, Shridhar K; Roux, Kenneth H

    2010-05-01

    The 11S globulins are members of the cupin protein superfamily and represent an important class of tree nut allergens for which a number of linear epitopes have been mapped. However, specific conformational epitopes for these allergens have yet to be described. We have recently reported a cashew Ana o 2 conformational epitope defined by murine mAb 2B5 and competitively inhibited by a subset of patient IgE antibodies. The 2B5 epitope appears to reside on the large (acidic) subunit, is dependent upon small (basic) subunit association for expression, and is highly susceptible to denaturation. Here we fine map the epitope using a combination of recombinant chimeric cashew Ana o 2-soybean Gly m 6 chimeras, deletion and point mutations, molecular modeling, and electron microscopy of 2B5-Ana o 2 immune complexes. Key residues appear confined to a 24 amino acid segment near the N-terminus of the large subunit peptide, a portion of which makes direct contact with the small subunit. These data provide an explanation for both the small subunit dependence and the structurally labile nature of the epitope. PMID:20362338

  6. Mass spectrometry reveals modularity and a complete subunit interaction map of the eukaryotic translation factor eIF3.

    PubMed

    Zhou, Min; Sandercock, Alan M; Fraser, Christopher S; Ridlova, Gabriela; Stephens, Elaine; Schenauer, Matthew R; Yokoi-Fong, Theresa; Barsky, Daniel; Leary, Julie A; Hershey, John W; Doudna, Jennifer A; Robinson, Carol V

    2008-11-25

    The eukaryotic initiation factor 3 (eIF3) plays an important role in translation initiation, acting as a docking site for several eIFs that assemble on the 40S ribosomal subunit. Here, we use mass spectrometry to probe the subunit interactions within the human eIF3 complex. Our results show that the 13-subunit complex can be maintained intact in the gas phase, enabling us to establish unambiguously its stoichiometry and its overall subunit architecture via tandem mass spectrometry and solution disruption experiments. Dissociation takes place as a function of ionic strength to form three stable modules eIF3(c:d:e:l:k), eIF3(f:h:m), and eIF3(a:b:i:g). These modules are linked by interactions between subunits eIF3b:c and eIF3c:h. We confirmed our interaction map with the homologous yeast eIF3 complex that contains the five core subunits found in the human eIF3 and supplemented our data with results from immunoprecipitation. These results, together with the 27 subcomplexes identified with increasing ionic strength, enable us to define a comprehensive interaction map for this 800-kDa species. Our interaction map allows comparison of free eIF3 with that bound to the hepatitis C virus internal ribosome entry site (HCV-IRES) RNA. We also compare our eIF3 interaction map with related complexes, containing evolutionarily conserved protein domains, and reveal the location of subunits containing RNA recognition motifs proximal to the decoding center of the 40S subunit of the ribosome. PMID:18599441

  7. Mapping of the alpha-bungarotoxin binding site within the alpha subunit of the acetylcholine receptor.

    PubMed Central

    Neumann, D; Barchan, D; Safran, A; Gershoni, J M; Fuchs, S

    1986-01-01

    Synthetic peptides and their respective antibodies have been used in order to map the alpha-bungarotoxin binding site within the alpha subunit of the acetylcholine receptor. By using antibodies to a synthetic peptide corresponding to residues 169-181 of the alpha subunit, we demonstrate that this sequence is included within the 18-kDa toxin binding fragment previously reported. Furthermore, the 18-kDa fragment was also found to bind a monoclonal antibody (5.5) directed against the cholinergic binding site. Sequential proteolysis of the acetylcholine receptor with trypsin, prior to Staphylococcus aureus V8 protease digestion, resulted in a 15-kDa toxin binding fragment that is included within the 18-kDa fragment but is shorter than it only at its carboxyl terminus. This 15-kDa fragment therefore initiates beyond Asp-152 and terminates in the region of Arg-313/Lys-314. In addition, experiments are reported that indicate that in the intact acetylcholine receptor, Cys-128 and/or Cys-142 are not crosslinked by disulfide bridges with any of the cysteines (at positions 192, 193, and 222) that reside in the 15-kDa toxin binding fragment. Finally, the synthetic dodecapeptide Lys-His-Trp-Val-Tyr-Tyr-Thr-Cys-Cys-Pro-Asp-Thr, which is present in the 15-kDa fragment (corresponding to residues 185-196 of the alpha subunit) was shown to bind alpha-bungarotoxin directly. This binding was completely inhibited by competition with d-tubocurarine. Images PMID:3458258

  8. Mapping of the alpha-bungarotoxin binding site within the alpha subunit of the acetylcholine receptor.

    PubMed

    Neumann, D; Barchan, D; Safran, A; Gershoni, J M; Fuchs, S

    1986-05-01

    Synthetic peptides and their respective antibodies have been used in order to map the alpha-bungarotoxin binding site within the alpha subunit of the acetylcholine receptor. By using antibodies to a synthetic peptide corresponding to residues 169-181 of the alpha subunit, we demonstrate that this sequence is included within the 18-kDa toxin binding fragment previously reported. Furthermore, the 18-kDa fragment was also found to bind a monoclonal antibody (5.5) directed against the cholinergic binding site. Sequential proteolysis of the acetylcholine receptor with trypsin, prior to Staphylococcus aureus V8 protease digestion, resulted in a 15-kDa toxin binding fragment that is included within the 18-kDa fragment but is shorter than it only at its carboxyl terminus. This 15-kDa fragment therefore initiates beyond Asp-152 and terminates in the region of Arg-313/Lys-314. In addition, experiments are reported that indicate that in the intact acetylcholine receptor, Cys-128 and/or Cys-142 are not crosslinked by disulfide bridges with any of the cysteines (at positions 192, 193, and 222) that reside in the 15-kDa toxin binding fragment. Finally, the synthetic dodecapeptide Lys-His-Trp-Val-Tyr-Tyr-Thr-Cys-Cys-Pro-Asp-Thr, which is present in the 15-kDa fragment (corresponding to residues 185-196 of the alpha subunit) was shown to bind alpha-bungarotoxin directly. This binding was completely inhibited by competition with d-tubocurarine. PMID:3458258

  9. Sequence analysis and mapping of a novel human mitochondrial ATP synthase subunit 9 cDNA (ATP5G3)

    SciTech Connect

    Yan, W.L.; Gusella, J.F. |; Haines, J.L. |

    1994-11-15

    The authors describe the cloning, sequence analysis, and chromosomal mapping of a novel mitochondrial ATP synthase subunit 9 cDNA, P3. Subunit 9 transports protons across the inner mitochondrial membrane to the F{sub 1}-ATPase protruding on the matrix side, resulting in the generation of ATP. Sequence analysis of the P3 cDNA reveals only 80% identity with the human subunit 9 genes P1 and P2 in the DNA sequence encoding the mature protein identical to P1 and P2. The predicted sequence of the P3 leader peptide differs from the P1 and P2 leaders, but retains the {open_quotes}RFS{close_quotes} motif critical for mitochondrial import and maturation. The P3 gene (ATP5G3) maps to chromosome 2. 8 refs., 1 fig., 1 tab.

  10. Mapping the principal interaction site of the Brf1 and Bdp1 subunits of Saccharomyces cerevisiae TFIIIB.

    PubMed

    Kassavetis, George A; Driscoll, Robert; Geiduschek, E Peter

    2006-05-19

    The Brf1 subunit of the central RNA polymerase (pol) III transcription initiation factor TFIIIB is bipartite; its N-terminal TFIIB-related half is principally responsible for recruiting pol III to the promoter and for promoter opening near the transcriptional start site, whereas its pol III-specific C-terminal half contributes most of the affinities that hold the three subunits of TFIIIB together. Here, the principal attachment site of Brf1 for the Bdp1 subunit of TFIIIB has been mapped by a combination of structure-informed, site-directed mutagenesis and photochemical protein-DNA cross-linking. A 66-amino acid segment of Brf1 is shown to serve as a two-sided adhesive surface, with the side chains projecting away from its extended interface with TATA-binding protein anchoring Bdp1 binding. An extensive collection of N-terminal, C-terminal, and internal deletion proteins has been used to demarcate the interacting Bdp1 domain to a 66-amino acid segment that includes the SANT domain of this subunit and is phylogenetically the most conserved region of Bdp1. PMID:16551611

  11. Disulfide Engineering to Map Subunit Interactions in the Proteasome and Other Macromolecular Complexes

    PubMed Central

    Hochstrasser, Mark; Funakoshi, Minoru

    2013-01-01

    Summary In studies of protein complexes for which high-resolution structural data are unavailable, it is often still possible to determine both nearest-neighbor relationships between subunits and atomic-resolution details of these interactions. The eukaryotic 26S proteasome, a ~2.5 MDa protein complex with at least 33 different subunits, is a prime example. Important information about quaternary organization and assembly of proteasomes has been gained using a combination of sequence alignments with related proteins of known tertiary structure, molecular modeling, and disulfide engineering to allow oxidative crosslinking between predicted polypeptide neighbors. Here we provide detailed protocols for engineered cysteine crosslinking of yeast proteasome subunits in whole cell extracts, in active 26S proteasome complexes first isolated by native polyacrylamide gel electrophoresis, and in subcomplexes that function as potential assembly intermediates. PMID:22350897

  12. Exon junction complex subunits are required to splice Drosophila MAP kinase, a large heterochromatic gene

    PubMed Central

    Roignant, Jean-Yves; Treisman, Jessica E.

    2010-01-01

    Summary The exon junction complex (EJC) is assembled on spliced mRNAs upstream of exon-exon junctions, and can regulate their subsequent translation, localization, or degradation. We isolated mutations in Drosophila mago nashi (mago), which encodes a core EJC subunit, based on their unexpectedly specific effects on photoreceptor differentiation. Loss of Mago prevents Epidermal growth factor receptor signaling, due to a large reduction in MAPK mRNA levels. MAPK expression also requires the EJC subunits Y14 and eIF4AIII, and EJC-associated splicing factors. Mago depletion does not affect the transcription or stability of MAPK mRNA, but alters its splicing pattern. MAPK expression from an exogenous promoter requires Mago only when the template includes introns. MAPK is the primary functional target of mago in eye development; in cultured cells, Mago knockdown disproportionately affects other large genes located in heterochromatin. These data support a nuclear role for EJC components in splicing a specific subset of introns. PMID:20946982

  13. Rat hepatic (Na/sup +/, K/sup +/)-ATPase:. cap alpha. =subunit isolation by immunoaffinity chromatography and structural analysis by peptide mapping

    SciTech Connect

    Hubert, J.J.; Schenk, D.B.; Skelly, H.; Leffert, H.L.

    1986-07-01

    The catalytic ..cap alpha..-subunit of rat hepatic (Na/sup +/, K/sup +/)-ATPase (EC 3.6.1.3) has been isolated by immunoaffinity chromatography from microsomes solubilized in n-dodecyl octaethylene glycol monoether. The procedure employs an anticatalytic mouse monoclonal antibody (9-A5) covalently linked to Sepharose 4B that specifically block phosphorylation of the sodium pump's ..cap alpha..-subunit from (..gamma..-/sup 32/P)ATP. The hepatic subunit is virtually identical with purified rat, dog, and human renal ..cap alpha..-subunits as judged by its apparent molecular weight after polyacrylamide gel electrophoresis in sodium dodecyl sulfate (M/sub r/ 92K) and its two-dimensional tryptic and chymotryptic peptide maps on cellulose-coated thin-layer plates. In contrast, the structures of authentic renal ..beta..-subunits from the three species differ significantly from each other as judged by their peptide maps; no detectable homologies are seen between their chymotryptic maps and those of putative hepatic ..beta..-subunits (M/sub r/ 50K and 55K) eluted from 9-A5-Spharose. Additional studies of ouabain-sensitive /sup 86/Rb/sup +/ uptake in primary cultures of adult rat hepatocytes reveal inhibition curves with single inflection points in the absence or presence of pump-stimulating peptides like insulin, glucagon, and epidermal growth factor. These findings indicate that rat hepatocytes express only one of two know structurally conserved forms of catalytic subunit (the renallike ..cap alpha.. form) and, if at all, structurally divergent forms of the sodium pump's ..beta..-subunit.

  14. Map of the Antelope Valley-East Kern Water Agency area, California, showing ground-water subunits and areas and well hydrographs

    USGS Publications Warehouse

    Lamb, C.E.; McIntyre, M.J.

    1978-01-01

    The U.S. Geological Survey has released to the open file a map, scale 1:125,000, showing ground-water subunits and areas, and well hydrographs in the Antelope Valley-East Kern Water Agency area, California. (Woodard-USGS)

  15. Mapping of the gene for the p60 subunit of the human chromatin assembly factor (CAF1A) to the Down syndrome region of chromosome 21

    SciTech Connect

    Blouin, J.L.; Gos, A.; Morris, M.A.; Antonarakis, S.E.

    1996-04-15

    Exon trapping was used to clone portions of genes from the Down syndrome critical region (DSCR) of human chromosome 21. One trapped sequence showed complete homology with nucleotide sequence U20980 (GenBank), which corresponds to the gene for the p60 subunit of the human chromatin assembly factor-1 (CAF1A). We mapped this gene to human chromosome 21 by fluorescence in situ hybridization, by the use of somatic cell hybrids, and by hybridization to chromosome 21-specific YACs and cosmids. The CAF1A gene localizes to YACs 745H11 and 230E8 of the Chumakov et al. YAC contig, within the DSCR on 21q22. This CAF1A, which belongs to the WD-motif family of genes and interacts with other polypeptide subunits to promote assembly of histones to replicating DNA, may contribute in a gene dosage-dependent manner to the phenotype of Down syndrome. 22 refs., 1 fig.

  16. Five subunit genes of the human muscle nicotinic acetylcholine receptor are mapped to two linkage groups on chromosome 2 and 17

    SciTech Connect

    Lobos, E.A. )

    1993-09-01

    RFLPs were detected in the five subunit genes of the human muscle nicotinic acetylcholine receptor (nAChR) using genomic DNA or cDNA probes from the homologous mouse loci. The RFLPs at the alpha-, beta-, gamma-, delta-, and epsilon-subunit gene loci were analyzed for genetic linkage in 16 families (n = 188). Significant evidence was obtained for close linkage of the [beta]- and [epsilon]-nAChR genes and much greater genetic distance between the [alpha]-nAChR gene and the [gamma]/[delta]-nAChR gene complex. The linkage analysis program CRI-MAP was used to map the positions of the [beta]- and [epsilon]-nAChR genes relative to seven markers on chromosome 17. The results indicate the [beta]- and [epsilon]-nAChR probes to a panel of human-hamster somatic cell hybrids. The [alpha]-, [gamma]-, and [delta]-nAChR genes were placed on a map of 13 chromosome 2 markers. The linkage analysis placed the nAChR genes at two sites on chromosome 2q about equidistant from the marker CRYGP1, with the [alpha]-nAChR gene about 27 cM proximal and the [gamma]/[delta]-nAChR gene complex about 31 cM distal to CRYGP1. 46 refs., 2 figs., 5 tabs.

  17. Resolving the Fluorescence Response of E. coli Carbamoyl Phosphate Synthetase: Mapping Intra- and Inter-Subunit Conformational Changes †

    PubMed Central

    Johnson, Jason L.; West, Joseph K.; Nelson, Andrew D. L.; Reinhart, Gregory D.

    2008-01-01

    Carbamoyl phosphate synthetase (CPS) from E. coli is potentially overlaid with a network of allosterism, interconnecting active sites, effector binding sites, and aggregate interfaces to control its mechanisms of catalytic synchronization, regulation, and oligomerization, respectively. To characterize these conformational changes, a tryptophan-free variant of CPS was genetically engineered by substituting six native tryptophans with tyrosines. Each tryptophan was then reinserted, singly, as a specific fluorescence probe of its corresponding microenvironment. The amino acid substitutions themselves result in little apparent disruption of the protein; variants maintain catalytic and allosteric functionality, and the fluorescence properties of each tryptophan, while unique, are additive to wild-type CPS. Whereas the collective, intrinsic fluorescence response of E. coli CPS is largely insensitive to ligand binding, changes of the individual probes in intensity, lifetime, anisotropy, and accessibility to acrylamide quenching highlight the dynamic interplay between several protein domains, as well as between subunits. W213 within the carboxy phosphate domain, for example, exhibits an almost 40% increase in intensity upon saturation with ATP; W437 of the oligomerization domain, in contrast, is essentially silent in its fluorescence to the binding of ligands. Nucleotide and bicarbonate association within the large subunit induce fluorescence changes in both W170 and W175 of the small subunit, indicative of the type of long-range interactions purportedly synchronizing the carboxy phosphate and amidotransferase domains of the enzyme to initiate catalysis. ATP and ADP engender different fluorescence responses in most tryptophans, perhaps reflecting coordinating, conformational changes accompanying the cycling of reactants and products during catalysis. PMID:17209549

  18. Temperature-Sensitive Mutants Blocked in the Folding or Subunit Assmbly of the Bacteriophage P22 Tailspike Protein. I. Fine-Structure Mapping

    PubMed Central

    Smith, Donna H.; Berget, Peter B.; King, Jonathan

    1980-01-01

    As part of a study of protein folding, we have constructed a fine-structure map of 9 existing and 29 newly isolated UV- and hydroxylamine-induced temperature-sensitive (ts) mutations in gene 9 of Salmonella bacteriophage P22. Gene 9 specifies the polypeptide chain of the multimeric tail spikes, six of which form the cell attachment organelle of the phage. The 38 ts mutants were mapped against deletion lysogens with endpoints in gene 9. They mapped in 10 of the 15 deletion intervals. Two- and three-factor crosses between mutants within each interval indicated that at least 31 ts sites are represented among the 38 mutants. To determine the distribution of ts sites within the physical map, we identified the protein fragments from infection of su- hosts with 10 gene 9 amber mutants. Their molecular weights, ranging from 13,900 to 55,000 daltons, were combined with the genetic data to yield a composite map of gene 9. The 31 ts sites were distributed through most of the gene, but were most densely clustered in the central third.—None of the ts mutant pairs tested exhibited intragenic complementation. Studies of the defective phenotypes of the ts mutants (Goldenberg and King 1981; Smith and King 1981) revealed that most do not affect the thermostability of the mature protein, but instead prevent the folding or subunit assembly of the mutant chains synthesized at restrictive temperature. Thus, many of thes ts mutations identify sites in the polypeptide chain that are critical for the folding or maturation of the tail-spike protein. PMID:7021307

  19. The genes for nicein/kalinin 125- and 100-kDa subunits, candidates for junctional epidermiolysis bullosa, map to chromosomes 1q32 and 1q25-q31

    SciTech Connect

    Vailly, J.; Ortonne, J.P.; Meneguzzi, G.; Szepetowski, P.; Pedeutour, F. ); Mattei, M.G. ); Burgeson, R. )

    1994-05-01

    Expression of nicein is specifically hampered in the severe form of junctional epidermolysis bullosa (JEB), a recessive genodermatosis characterized by blister formation of integument believed to be due to defects in hemidesmosomes. Nicein genes are therefore the prime candidates for involvement in JEB. To map the gene encoding the 125-kDa subunit of nicein, the authors used the cDNA Kal5.5C coding for the amino-terminal domain of the protein. In situ hybridization was carried out on chromosomes in phytohemagglutinin-stimulated blood lymphocytes of healthy donors. In 100 metaphases examined, 153 silver grains were found associated with chromosomes; 45 (29%) of these were located on chromosome 1, and 33 (73%) of these 45 grains mapped to region 1q32.1-q41 with a maximum in band 1q32. To confirm the regional localization of the genes for nicein subunits of 100 and 125 kDa, fluorescence in situ hybridization was performed on normal lymphocytes from two unrelated normal males and fibroblast cell lines GM00257 (karyotype 46,XX, t(1;2)(1q32;2p23)) and GM004088 (46,XY,t(1;4)(q32;p16)). It was thus confirmed that the genes for nicein 125- and 100-kDa subunits are localized at 1q32 and 1q25-q31, respectively. 9 refs., 1 fig.

  20. A new sodium channel {alpha}-subunit gene (Scn9a) from Schwann cells maps to the Scn1a, Scn2a, Scn3a cluster of mouse chromosome 2

    SciTech Connect

    Beckers, M.C.; Ernst, E.; Gros, P.

    1996-08-15

    We have used a total of 27 AXB/BXA recombinant inbred mouse strains to determine the chromosomal location of a newly identified gene encoding an {alpha}-subunit isoform of the sodium channel from Schwann cells, Scn9a. Linkage analysis established that Scn9a mapped to the proximal segment of mouse chromosome 2. The segregation of restriction fragment length polymorphisms in 145 progeny from a Mus spretus x C57BL/6J backcross indicates that Scn9a is very tightly linked to Scn1a (gene encoding the type I sodium channel {alpha}-subunit of the brain) and forms part of a cluster of four Scna genes located on mouse chromosome 2. 17 refs., 1 fig., 3 tabs.

  1. High-resolution mapping of the [gamma]-aminobutyric acid receptor subunit [beta]3 and [alpha]5 gene cluster on chromosome 15q11-q13, and localization of breakpoints in two Angelman syndrome patients

    SciTech Connect

    Sinnett, D.; Wagstaff, J.; Woolf, E. Harvard Medical School, Boston, MA ); Glatt, K. ); Kirkness, E.J. )Lalande, M. Harvard Medical School, Boston, MA Howard Hughes Medical Inst., Boston, MA )

    1993-06-01

    The [gamma]-aminobutyric acid (GABA[sub A]) receptors are a family of ligand-gated chloride channels constituting the major inhibitory neurotransmitter receptors in the nervous system. In order to determine the genomic organization of the GABA[sub A] receptor [beta]3 subunit gene (GABRB3) and [alpha]5 subunit gene (GABRA5) in chromosome 15q11-q13, the authors have constructed a high-resolution physical map using the combined techniques of field-inversion gel electrophoresis and phage genomic library screening. This map, which covers nearly 1.0 Mb, shows that GABRB3 and GABRA5 are separated by less than 100 kb and are arranged in a head-to-head configuration. GABRB3 encompasses approximately 250 kb, while GABRA5 is contained within 70 kb. This difference in size is due in large part to an intron of 150 kb within GABRB3. The authors have also identified seven putative CpG islands within a 600-kb interval. Chromosomal rearrangement breakpoints -- in one Angelman syndrome (AS) patient with an unbalanced translocation and in another patient with a submicroscopic deletion -- are located within the large GABRB3 intron. These findings will facilitate chromosomal walking strategies for cloning the regions disrupted by the DNA rearrangements in these AS patients and will be valuable for mapping new genes to the AS chromosomal region. 64 refs., 6 figs., 2 tabs.

  2. Direct interaction and functional coupling between human 5-HT6 receptor and the light chain 1 subunit of the microtubule-associated protein 1B (MAP1B-LC1).

    PubMed

    Kim, Soon-Hee; Kim, Dong Hyuk; Lee, Kang Ho; Im, Sun-Kyoung; Hur, Eun-Mi; Chung, Kwang Chul; Rhim, Hyewhon

    2014-01-01

    Serotonin (5-HT) receptors of type 6 (5-HT6R) play important roles in mood, psychosis, and eating disorders. Recently, a growing number of studies support the use of 5-HT6R-targeting compounds as promising drug candidates for treating cognitive dysfunction associated with Alzheimer's disease. However, the mechanistic linkage between 5-HT6R and such functions remains poorly understood. By using yeast two-hybrid, GST pull-down, and co-immunoprecipitation assays, here we show that human 5-HT6R interacts with the light chain 1 (LC1) subunit of MAP1B protein (MAP1B-LC1), a classical microtubule-associated protein highly expressed in the brain. Functionally, we have found that expression of MAP1B-LC1 regulates serotonin signaling in a receptor subtype-specific manner, specifically controlling the activities of 5-HT6R, but not those of 5-HT4R and 5-HT7R. In addition, we have demonstrated that MAP1B-LC1 increases the surface expression of 5-HT6R and decreases its endocytosis, suggesting that MAP1B-LC1 is involved in the desensitization and trafficking of 5-HT6R via a direct interaction. Together, we suggest that signal transduction pathways downstream of 5-HT6R are regulated by MAP1B, which might play a role in 5-HT6R-mediated signaling in the brain. PMID:24614691

  3. Direct Interaction and Functional Coupling between Human 5-HT6 Receptor and the Light Chain 1 Subunit of the Microtubule-Associated Protein 1B (MAP1B-LC1)

    PubMed Central

    Kim, Soon-Hee; Kim, Dong Hyuk; Lee, Kang Ho; Im, Sun-Kyoung; Hur, Eun-Mi; Chung, Kwang Chul; Rhim, Hyewhon

    2014-01-01

    Serotonin (5-HT) receptors of type 6 (5-HT6R) play important roles in mood, psychosis, and eating disorders. Recently, a growing number of studies support the use of 5-HT6R-targeting compounds as promising drug candidates for treating cognitive dysfunction associated with Alzheimer’s disease. However, the mechanistic linkage between 5-HT6R and such functions remains poorly understood. By using yeast two-hybrid, GST pull-down, and co-immunoprecipitation assays, here we show that human 5-HT6R interacts with the light chain 1 (LC1) subunit of MAP1B protein (MAP1B-LC1), a classical microtubule-associated protein highly expressed in the brain. Functionally, we have found that expression of MAP1B-LC1 regulates serotonin signaling in a receptor subtype-specific manner, specifically controlling the activities of 5-HT6R, but not those of 5-HT4R and 5-HT7R. In addition, we have demonstrated that MAP1B-LC1 increases the surface expression of 5-HT6R and decreases its endocytosis, suggesting that MAP1B-LC1 is involved in the desensitization and trafficking of 5-HT6R via a direct interaction. Together, we suggest that signal transduction pathways downstream of 5-HT6R are regulated by MAP1B, which might play a role in 5-HT6R-mediated signaling in the brain. PMID:24614691

  4. Mapping the domain structure of the influenza A virus polymerase acidic protein (PA) and its interaction with the basic protein 1 (PB1) subunit

    SciTech Connect

    Guu, Tom S.Y.; Dong Liping; Wittung-Stafshede, Pernilla; Tao, Yizhi J.

    2008-09-15

    The influenza A virus polymerase consists of three subunits (PA, PB1, and PB2) necessary for viral RNA synthesis. The heterotrimeric polymerase complex forms through PA interacting with PB1 and PB1 interacting with PB2. PA has been shown to play critical roles in the assembly, catalysis, and nuclear localization of the polymerase. To probe the structure of PA, we isolated recombinant PA from insect cells. Limited proteolysis revealed that PA contained two domains connected by a 20-residue linker (residues 257-276). Far-UV circular dichroism established that the two domains folded into a mixed {alpha}/{beta} structure when separately expressed. In vitro pull-down assays showed that neither individually nor cooperatively expressed PA domains, without the linker, could assure PA-PB1 interaction. Protease treatment of PA-PB1 complex indicated that its PA subunit was significantly more stable than free PA, suggesting that the linker is protected and it constitutes an essential component of the PA-PB1 interface.

  5. Mapping.

    ERIC Educational Resources Information Center

    Kinney, Douglas M.; McIntosh, Willard L.

    1979-01-01

    The area of geological mapping in the United States in 1978 increased greatly over that reported in 1977; state geological maps were added for California, Idaho, Nevada, and Alaska last year. (Author/BB)

  6. TFIIIB subunit locations on U6 gene promoter DNA mapped by site-specific protein-DNA photo-cross-linking.

    PubMed

    Kang, Jin Joo; Kang, Yoon Soon; Stumph, William E

    2016-05-01

    RNA polymerase III-transcribed U6 snRNA genes have gene-external promoters that contain TATA boxes. U6 TATA sequences are bound by TFIIIB that in Drosophila contains the three subunits TBP, Brf1, and Bdp1. The overall structure of TFIIIB is still not well understood. We have therefore studied the mode of TFIIIB binding to DNA by site-specific protein-DNA photo-cross-linking. The results indicate that a portion of Brf1 is sandwiched between Bdp1 and TBP upstream of the TATA box. Furthermore, Bdp1 traverses the DNA under the N-terminal stirrup of TBP to interact with the DNA (and very likely Brf1) downstream of the TATA sequence. PMID:27112515

  7. The gene for human U2 snRNP auxiliary factor small 35-kDa subunit (U2AF1) maps to the progressive myoclonus epilepsy (EPM1) critical region on chromosome 21q22.3

    SciTech Connect

    Lalioti, M.D.; Rossier, C.; Antonarakis, S.E.

    1996-04-15

    We used targeted exon trapping to clone portions of genes from human chromosome 21q22.3. One trapped sequence showed complete homology with the cDNA of human U2AF{sup 35} (M96982; HGM-approved nomenclature U2AF1), which encodes for the small 35-kDa subunit of the U2 snRNP auxiliary factor. Using the U2AF1 cDNA as a probe, we mapped this gene to cosmid Q15D2, a P1, and YAC 350F7 of the Chumakov et al. contig, close to the cystathionine-{beta}-synthase gene (CBS) on 21q22.3. This localization was confirmed by PCR using oligonucleotides from the 3{prime} UTR and by FISH. As U2AF1 associated with a number of different factors during mRNA splicing, overexpression in trisomy 21 individuals could contribute to some Down syndrome phenotypes by interfering with the splicing process. Furthermore, because this gene maps in the critical region for the progressive myoclonus epilepsy I locus (EPM1), mutation analysis will be carried out in patients to evaluate the potential role of U2AF1 as a candidate for EPM1. 24 refs., 1 fig.

  8. Mapping of the human cone transducin {alpha} subunit (GNAT2) gene to 1p13 and mutation analysis in patients with Stargardt`s disease

    SciTech Connect

    Magovcevic, I.; Weremowicz, S.; Morton, C.C.

    1994-09-01

    Transducin {alpha} subunits are members of a large family of G-proteins and play an important role in phototransduction in rod and cone photoreceptors. We report the localization of the human cone {alpha} transducin (GNAT2) gene using fluorescence in situ hybridization (FISH) on chromosome 1 in band p13. The recent assignment of a gene for Stargardt`s disease to the same chromosomal region by linkage analysis prompted us to investigate the possible role of GNAT2 in the pathogenesis of this disease. Stargardt`s disease is characterized by degeneration in late childhood or early adulthood of the macula of the retina, a region rich in cones. We screened patients with Stargardt`s disease, with or without peripheral cone involvement as monitored by the full-field ERG, for mutations in this gene. We investigated 66 unrelated patients including 22 with peripheral cone dysfunction for mutations in the coding region of the GNAT2 gene using polymerase chain reaction-single strand conformation polymorphism analysis (SSCP) and direct sequencing. One patient (034-16) was heterozygous for a silent change in exon VI, Asp238Asp (GAT to GAC). Two patients, one (035-005) with peripheral cone involvement and one (071-001) without peripheral cone involvement, were heterozygous for the missense change Val124Met (GTG to ATG) in exon IV. A subsequent screen of 96 unrelated, unaffected controls revealed one individual (N10) who was also heterozygous for the Val124Met alteration. We concluded that Asp238Asp and Val124Met are rare variants not causing Stargardt`s disease. Hence, no disease-specific mutations were found indicating that GNAT2 is probably not involved in the pathogenesis of most cases of Stargardt`s disease.

  9. Mapping precursor-binding site on TatC subunit of twin arginine-specific protein translocase by site-specific photo cross-linking.

    PubMed

    Zoufaly, Stefan; Fröbel, Julia; Rose, Patrick; Flecken, Tobias; Maurer, Carlo; Moser, Michael; Müller, Matthias

    2012-04-13

    A number of secreted precursor proteins of bacteria, archaea, and plant chloroplasts stand out by a conserved twin arginine-containing sequence motif in their signal peptides. Many of these precursor proteins are secreted in a completely folded conformation by specific twin arginine translocation (Tat) machineries. Tat machineries are high molecular mass complexes consisting of two types of membrane proteins, a hexahelical TatC protein, and usually one or two single-spanning membrane proteins, called TatA and TatB. TatC has previously been shown to be involved in the recognition of twin arginine signal peptides. We have performed an extensive site-specific cross-linking analysis of the Escherichia coli TatC protein under resting and translocating conditions. This strategy allowed us to map the recognition site for twin arginine signal peptides to the cytosolic N-terminal region and first cytosolic loop of TatC. In addition, discrete contact sites between TatC, TatB, and TatA were revealed. We discuss a tentative model of how a twin arginine signal sequence might be accommodated in the Tat translocase. PMID:22362773

  10. Comparison of Large Subunits of Type II DNA-dependent RNA Polymerases from Higher Plants.

    PubMed

    Kidd, G H; Link, G; Bogorad, L

    1979-10-01

    Two-dimensional tryptic mapping of (125)I-labeled polypeptides has been employed to compare the large subunits of type II DNA-dependent RNA polymerases from maize, parsley (Petroselinum sativum), and wheat. Maps of the 220 kilodalton (kd) and 140 kd subunits from wheat RNA polymerase II differ from those of the corresponding subunits from parsley enzyme II. The 180 kd subunits from maize and parsley type II enzymes also yield dissimilar tryptic maps. Thus, despite similarities in molecular mass, the large subunits of wheat, parsley, and maize type II RNA polymerases are unique to each individual plant species. PMID:16661032

  11. Gel-based chemical cross-linking analysis of 20S proteasome subunit-subunit interactions in breast cancer.

    PubMed

    Song, Hai; Xiong, Hua; Che, Jing; Xi, Qing-Song; Huang, Liu; Xiong, Hui-Hua; Zhang, Peng

    2016-08-01

    The ubiquitin-proteasome system plays a pivotal role in breast tumorigenesis by controlling transcription factors, thus promoting cell cycle growth, and degradation of tumor suppressor proteins. However, breast cancer patients have failed to benefit from proteasome inhibitor treatment partially due to proteasome heterogeneity, which is poorly understood in malignant breast neoplasm. Chemical crosslinking is an increasingly important tool for mapping protein three-dimensional structures and proteinprotein interactions. In the present study, two cross-linkers, bis (sulfosuccinimidyl) suberate (BS(3)) and its water-insoluble analog disuccinimidyl suberate (DSS), were used to map the subunit-subunit interactions in 20S proteasome core particle (CP) from MDA-MB-231 cells. Different types of gel electrophoresis technologies were used. In combination with chemical cross-linking and mass spectrometry, we applied these gel electrophoresis technologies to the study of the noncovalent interactions among 20S proteasome subunits. Firstly, the CP subunit isoforms were profiled. Subsequently, using native/SDSPAGE, it was observed that 0.5 mmol/L BS(3) was a relatively optimal cross-linking concentration for CP subunit-subunit interaction study. 2-DE analysis of the cross-linked CP revealed that α1 might preinteract with α2, and α3 might pre-interact with α4. Moreover, there were different subtypes of α1α2 and α3α4 due to proteasome heterogeneity. There was no significant difference in cross-linking pattern for CP subunits between BS(3) and DSS. Taken together, the gel-based characterization in combination with chemical cross-linking could serve as a tool for the study of subunit interactions within a multi-subunit protein complex. The heterogeneity of 20S proteasome subunit observed in breast cancer cells may provide some key information for proteasome inhibition strategy. PMID:27465334

  12. Cloning of the cDNA for the human ATP synthase OSCP subunit (ATP5O) by exon trapping and mapping to chromosome 21q22.1-q22.2

    SciTech Connect

    Chen, Haiming; Morris, M.A.; Rossier, C.

    1995-08-10

    Exon trapping was used to clone portions of potential genes from human chromosome 21. One trapped sequence showed striking homology with the bovine and rat ATP synthase OSCP (oligomycin sensitivity conferring protein) subunit. We subsequently cloned the full-length human ATP synthase OSCP cDNA (GDB/HGMW approved name ATP50) from infant brain and muscle libraries and determined its nucleotide and deduced amino acid sequence (EMBL/GenBank Accession No. X83218). The encoded polypeptide contains 213 amino acids, with more than 80% identity to bovine and murine ATPase OSCP subunits and over 35% identity to Saccharomyces cerevisiae and sweet potato sequences. The human ATP5O gene is located at 21q22.1-q22.2, just proximal to D21S17, in YACs 860G11 and 838C7 of the Chumakov et al. YAC contig. The gene is expressed in all human tissues examined, most strongly in muscle and heart. This ATP5O subunit is a key structural component of the stalk of the mitochondrial respiratory chain F{sub 1}F{sub 0}-ATP synthase and as such may contribute in a gene dosage-dependent manner to the phenotype of Down syndrome (trisomy 21). 39 refs., 5 figs.

  13. Both subunits of ADP-glucose pyrophosphorylase are regulatory.

    PubMed

    Cross, Joanna M; Clancy, Maureen; Shaw, Janine R; Greene, Thomas W; Schmidt, Robert R; Okita, Thomas W; Hannah, L Curtis

    2004-05-01

    The allosteric enzyme ADP-Glc pyrophosphorylase (AGPase) catalyzes the synthesis of ADP-Glc, a rate-limiting step in starch synthesis. Plant AGPases are heterotetramers, most of which are activated by 3-phosphoglyceric acid (3-PGA) and inhibited by phosphate. The objectives of these studies were to test a hypothesis concerning the relative roles of the two subunits and to identify regions in the subunits important in allosteric regulation. We exploited an Escherichia coli expression system and mosaic AGPases composed of potato (Solanum tuberosum) tuber and maize (Zea mays) endosperm subunit fragments to pursue this objective. Whereas potato and maize subunits have long been separated by speciation and evolution, they are sufficiently similar to form active mosaic enzymes. Potato tuber and maize endosperm AGPases exhibit radically different allosteric properties. Hence, comparing the kinetic properties of the mosaics to those of the maize endosperm and potato tuber AGPases has enabled us to identify regions important in regulation. The data herein conclusively show that both subunits are involved in the allosteric regulation of AGPase. Alterations in the small subunit condition drastically different allosteric properties. In addition, extent of 3-PGA activation and extent of 3-PGA affinity were found to be separate entities, mapping to different regions in both subunits. PMID:15122037

  14. Mapping of the {beta}{sub 2} subunit gene (GABRB2) to microdissected human chromosome 5q34-q35 defines a gene cluster for the most abundant GABA{sub A} receptor isoform

    SciTech Connect

    Russek, S.J.; Farb, D.H. |

    1994-10-01

    The {gamma}-aminobutyric acid receptor (GABA{sub A}R) is a multisubunit Cl{sup -} channel that mediates most fast inhibitory synaptic transmission in the central nervous system. Molecular evolution has given rise to many genetic variants of GABA{sub A}R subunits, including {alpha}{sub 1-6}, {beta}{sub 1-4}, {gamma}{sub 1-4}, {sigma}, and {rho}{sub 1-2}, suggesting that an enormous number of combinations of subunits are possible. Here we report that the {beta}{sub 2} gene is located on chromosome 5q34-q35, defining a cluster comprising {alpha}{sub 1}, {beta}{sub 2}, and {gamma}{sub 2} genes that together code for the most abundant GABA{sub A}R isoform. The fact that intron position is conserved in the {beta}{sub 1-3} genes, taken together with the observation that chromosomes 4 and 15 also contain distinct {alpha}-{beta}-{gamma} gene clusters, strongly suggests that an ancestral {alpha}-{beta}-{gamma} cluster was duplicated and translocated to at least two different chromosomes. This organization of GABA{sub A}R gene clusters may have been preserved as linkage provides a mechanism for facilitating coordinate gene expression. 34 refs., 5 figs., 1 tab.

  15. Mapping of the {alpha}{sub 4} subunit gene (GABRA4) to human chromosome 4 defines an {alpha}{sub 2}-{alpha}{sub 4}-{beta}{sub 1}-{gamma}{sub 1} gene cluster: Further evidence that modern GABA{sub a} receptor gene clusters are derived from an ancestral cluster

    SciTech Connect

    McLean, P.J.; Farb, D.H.; Russek, S.J.

    1995-04-10

    We demonstrated previously that an {alpha}{sub 1}-{beta}{sub 2}-{gamma}{sub 2} gene cluster of the {gamma}-aminobutyric acid (GABA{sub A}) receptor is located on human chromosome 5q34-q35 and that an ancestral {alpha}-{beta}-{gamma} gene cluster probably spawned clusters on chromosomes 4, 5, and 15. Here, we report that the {alpha}{sub 4} gene (GABRA4) maps to human chromosome 4p14-q12, defining a cluster comprising the {alpha}{sub 2}, {alpha}{sub 4}, {beta}{sub 1}, and {gamma}{sub 1} genes. The existence of an {alpha}{sub 2}-{alpha}{sub 4}-{beta}{sub 1}-{gamma}{sub 2} cluster on chromosome 4 and an {alpha}{sub 1}-{alpha}{sub 6}-{beta}{sub 2}-{gamma}{sub 2} cluster on chromosome 5 provides further evidence that the number of ancestral GABA{sub A} receptor subunit genes has been expanded by duplication within an ancestral gene cluster. Moreover, if duplication of the {alpha} gene occurred before duplication of the ancestral gene cluster, then a heretofore undiscovered subtype of a subunit should be located on human chromosome 15q11-q13 within an {alpha}{sub 5}-{alpha}{sub x}-{beta}{sub 3}-{gamma}{sub 3} gene cluster at the locus for Angelman and Prader-Willi syndromes. 34 refs., 6 figs., 1 tab.

  16. The genes for the {alpha}-type HC3 (PMSA2) and {beta}-type HC5 (PMSB1) subunits of human proteasomes map to chromosomes 6q27 and 7p12-p13 by fluorescence in situ hybridization

    SciTech Connect

    Okumura, Katsuzumi; Nogami, Masahiro; Taguchi, Hiroshi

    1995-05-20

    The authors have determined the locations of the genes for the two subunits, HC3 and HC5, by fluorescence in situ hybridization (FISH). Chromosome spreads were obtained from phytohemagglutinin-stimulated blood lymphocytes of a healthy donor after thymidine synchronization and bromodeoxyuridine incorporation by the method of Takahashi et al. Genomic DNA fragments of HC3 (4.3 kb, including exons 3, 4, and 5) and HC5 (7.5 kb including exons 1 and 2) (11) were labeled with biotin-16-dUTP by nick-translation. In situ hybridization was performed according to Lichter et al. in the presence of COT-1 DNA as a competitor. Hybridized probe was detected with FITC-conjugated avidin without further signal amplification. Comparison of the fluorescence signals and the banding patterns of the chromosomes indicated that the HC3 and HC5 genes were located on chromosome band 6q27 and 7p12-p13, respectively.

  17. Neutron Scattering and the 30 S Ribosomal Subunit of E. Coli

    DOE R&D Accomplishments Database

    Moore, P. B.; Engelman, D. M.; Langer, J. A.; Ramakrishnan, V. R.; Schindler, D. G.; Schoenborn, B. P.; Sillers, I. Y.; Yabuki, S.

    1982-06-01

    This paper reviews the progress made in the study of the internal organization of the 30 S ribosomal subunit of E. coli by neutron scattering since 1975. A map of that particle showing the position of 14 of the subunit's 21 proteins is presented, and the methods currently used for collecting and analyzing such data are discussed. Also discussed is the possibility of extending the interpretation of neutron mapping data beyond the limits practical today.

  18. Interaction of factor XIII subunits.

    PubMed

    Katona, Eva; Pénzes, Krisztina; Csapó, Andrea; Fazakas, Ferenc; Udvardy, Miklós L; Bagoly, Zsuzsa; Orosz, Zsuzsanna Z; Muszbek, László

    2014-03-13

    Coagulation factor XIII (FXIII) is a heterotetramer consisting of 2 catalytic A subunits (FXIII-A2) and 2 protective/inhibitory B subunits (FXIII-B2). FXIII-B, a mosaic protein consisting of 10 sushi domains, significantly prolongs the lifespan of catalytic subunits in the circulation and prevents their slow progressive activation in plasmatic conditions. In this study, the biochemistry of the interaction between the 2 FXIII subunits was investigated. Using a surface plasmon resonance technique and an enzyme-linked immunosorbent assay-type binding assay, the equilibrium dissociation constant (Kd) for the interaction was established in the range of 10(-10) M. Based on the measured Kd, it was calculated that in plasma approximately 1% of FXIII-A2 should be in free form. This value was confirmed experimentally by measuring FXIII-A2 in plasma samples immunodepleted of FXIII-A2B2. Free plasma FXIII-A2 is functionally active, and when activated by thrombin and Ca(2+), it can cross-link fibrin. In cerebrospinal fluid and tears with much lower FXIII subunit concentrations, >80% of FXIII-A2 existed in free form. A monoclonal anti-FXIII-B antibody that prevented the interaction between the 2 subunits reacted with the recombinant combined first and second sushi domains of FXIII-B, and its epitope was localized to the peptide spanning positions 96 to 103 in the second sushi domain. PMID:24408323

  19. Involvement of tryptophans at the catalytic and subunit-binding domains of transcarboxylase.

    PubMed

    Kumar, G K; Beegen, H; Wood, H G

    1988-08-01

    Transcarboxylase from Propionibacterium shermanii is a multisubunit enzyme. It consists of one central hexameric subunit to which six outer dimeric subunits are attached through twelve biotinyl subunits. Both the central and the outer subunits are multi-tryptophan (Trp) proteins, and each contains 5 Trps per monomer. The roles of the Trps during catalysis and assembly of the enzyme have been studied by using N-bromosuccinimide (NBS) oxidation as a probe. Modification of approximately 10 Trps of the total 90 Trps of the intact enzyme results in loss of activity. Both the substrates, viz., methylmalonyl-CoA and pyruvate, afford protection (approximately 50%) against inactivation caused by NBS. Analyses of tryptic peptide maps and intrinsic fluorescence studies have indicated that modification of 10 Trps of the whole enzyme does not cause extensive conformational changes. Therefore, the Trps appear to be essential for catalytic activity. NBS modification of the individual subunits at pH 6.5 has demonstrated differential reactivity of their Trps. Modification of the exposed/reactive Trps of either one of the subunits significantly affects the subunit assembly with the complementary unmodified subunits to form active enzyme. It is proposed that Trps are involved at the subunit-binding domains of either the central or the outer subunit of transcarboxylase, in addition to those critical for catalysis. PMID:3191102

  20. Phylogenetic analysis of ADP-glucose pyrophosphorylase subunits reveals a role of subunit interfaces in the allosteric properties of the enzyme.

    PubMed

    Georgelis, Nikolaos; Shaw, Janine R; Hannah, L Curtis

    2009-09-01

    ADP-glucose pyrophosphorylase (AGPase) catalyzes a rate-limiting step in glycogen and starch synthesis in bacteria and plants, respectively. Plant AGPase consists of two large and two small subunits that were derived by gene duplication. AGPase large subunits have functionally diverged, leading to different kinetic and allosteric properties. Amino acid changes that could account for these differences were identified previously by evolutionary analysis. In this study, these large subunit residues were mapped onto a modeled structure of the maize (Zea mays) endosperm enzyme. Surprisingly, of 29 amino acids identified via evolutionary considerations, 17 were located at subunit interfaces. Fourteen of the 29 amino acids were mutagenized in the maize endosperm large subunit (SHRUNKEN-2 [SH2]), and resulting variants were expressed in Escherichia coli with the maize endosperm small subunit (BT2). Comparisons of the amount of glycogen produced in E. coli, and the kinetic and allosteric properties of the variants with wild-type SH2/BT2, indicate that 11 variants differ from the wild type in enzyme properties or in vivo glycogen level. More interestingly, six of nine residues located at subunit interfaces exhibit altered allosteric properties. These results indicate that the interfaces between the large and small subunits are important for the allosteric properties of AGPase, and changes at these interfaces contribute to AGPase functional specialization. Our results also demonstrate that evolutionary analysis can greatly facilitate enzyme structure-function analyses. PMID:19625637

  1. Phosphorylation of the RNA polymerase II largest subunit during Xenopus laevis oocyte maturation.

    PubMed Central

    Bellier, S; Dubois, M F; Nishida, E; Almouzni, G; Bensaude, O

    1997-01-01

    Xenopus laevis oogenesis is characterized by an active transcription which ceases abruptly upon maturation. To survey changes in the characteristics of the transcriptional machinery which might contribute to this transcriptional arrest, the phosphorylation status of the RNA polymerase II largest subunit (RPB1 subunit) was analyzed during oocyte maturation. We found that the RPB1 subunit accumulates in large quantities from previtellogenic early diplotene oocytes up to fully grown oocytes. The C-terminal domain (CTD) of the RPB1 subunit was essentially hypophosphorylated in growing oocytes from Dumont stage IV to stage VI. Upon maturation, the proportion of hyperphosphorylated RPB1 subunits increased dramatically and abruptly. The hyperphosphorylated RPB1 subunits were dephosphorylated within 1 h after fertilization or heat shock of the matured oocytes. Extracts from metaphase II-arrested oocytes showed a much stronger CTD kinase activity than extracts from prophase stage VI oocytes. Most of this kinase activity was attributed to the activated Xp42 mitogen-activated protein (MAP) kinase, a MAP kinase of the ERK type. Making use of artificial maturation of the stage VI oocyte through microinjection of a recombinant stable cyclin B1, we observed a parallel activation of Xp42 MAP kinase and phosphorylation of RPB1. Both events required protein synthesis, which demonstrated that activation of p34(cdc2)off kinase was insufficient to phosphorylate RPB1 ex vivo and was consistent with a contribution of the Xp42 MAP kinase to RPB1 subunit phosphorylation. These results further support the possibility that the largest RNA polymerase II subunit is a substrate of the ERK-type MAP kinases during oocyte maturation, as previously proposed during stress or growth factor stimulation of mammalian cells. PMID:9032270

  2. The ribosomal subunit assembly line

    PubMed Central

    Dlakić, Mensur

    2005-01-01

    Recent proteomic studies in Saccharomyces cerevisiae have identified nearly 200 proteins, other than the structural ribosomal proteins, that participate in the assembly of ribosomal subunits and their transport from the nucleus. In a separate line of research, proteomic studies of mature plant ribosomes have revealed considerable variability in the protein composition of individual ribosomes. PMID:16207363

  3. Chromosomal localization of genes encoding guanine nucleotide-binding protein subunits in mouse and human.

    PubMed

    Blatt, C; Eversole-Cire, P; Cohn, V H; Zollman, S; Fournier, R E; Mohandas, L T; Nesbitt, M; Lugo, T; Jones, D T; Reed, R R

    1988-10-01

    A variety of genes have been identified that specify the synthesis of the components of guanine nucleotide-binding proteins (G proteins). Eight different guanine nucleotide-binding alpha-subunit proteins, two different beta subunits, and one gamma subunit have been described. Hybridization of cDNA clones with DNA from human-mouse somatic cell hybrids was used to assign many of these genes to human chromosomes. The retinal-specific transducin subunit genes GNAT1 and GNAT2 were on chromosomes 3 and 1; GNAI1, GNAI2, and GNAI3 were assigned to chromosomes 7, 3, and 1, respectively; GNAZ and GNAS were found on chromosomes 22 and 20. The beta subunits were also assigned--GNB1 to chromosome 1 and GNB2 to chromosome 7. Restriction fragment length polymorphisms were used to map the homologues of some of these genes in the mouse. GNAT1 and GNAI2 were found to map adjacent to each other on mouse chromosome 9 and GNAT2 was mapped on chromosome 17. The mouse GNB1 gene was assigned to chromosome 19. These mapping assignments will be useful in defining the extent of the G alpha gene family and may help in attempts to correlate specific genetic diseases with genes corresponding to G proteins. PMID:2902634

  4. Activities of complete and truncated forms of pertussis toxin subunits S1 and S2 synthesized by Escherichia coli.

    PubMed Central

    Locht, C; Cieplak, W; Marchitto, K S; Sato, H; Keith, J M

    1987-01-01

    The genes encoding the S1 and S2 subunits of pertussis toxin were expressed in Escherichia coli under lac operon transcription and translation control with pUC8 and pUC18 as the expression vectors. Various versions of the subunits were detected with anti-S1 or anti-S2 monoclonal antibodies. Recombinant S1, but not S2, subunit contained the enzymatic NAD-glycohydrolase and NAD:Gi ADP-ribosyltransferase activities. Both activities were also expressed by a truncated version of the S1 subunit in which the 48 carboxy-terminal amino acid residues, including a predicted Rossman structure and one of the two cysteines, had been deleted. The epitope for an anti-S2 monoclonal antibody was localized to the N-terminal 40-amino-acid region of the S2 subunit. Both the S1 and S2 subunits expressed in E. coli reacted with human hyperimmune serum. The full length and the truncated recombinant S1 subunit also reacted in Western blots with a neutralizing and protective monoclonal anti-S1 antibody. The different versions of S1 and S2 subunits expressed in E. coli are useful for mapping active sites, epitopes, and regions that interact with receptors or the other subunits in the holotoxin. These recombinant subunits will also facilitate the development of a safer, new-generation vaccine against whooping cough. Images PMID:3117686

  5. Arrangement of subunits in intact mammalian mitochondrial ATP synthase determined by cryo-EM

    PubMed Central

    Baker, Lindsay A.; Watt, Ian N.; Runswick, Michael J.; Walker, John E.; Rubinstein, John L.

    2012-01-01

    Mitochondrial ATP synthase is responsible for the synthesis of ATP, a universal energy currency in cells. Whereas X-ray crystallography has revealed the structure of the soluble region of the complex and the membrane-intrinsic c-subunits, little is known about the structure of the six other proteins (a, b, f, A6L, e, and g) that comprise the membrane-bound region of the complex in animal mitochondria. Here, we present the structure of intact bovine mitochondrial ATP synthase at ∼18 Å resolution by electron cryomicroscopy of single particles in amorphous ice. The map reveals that the a-subunit and c8-ring of the complex interact with a small contact area and that the b-subunit spans the membrane without contacting the c8-ring. The e- and g-subunits extend from the a-subunit density distal to the c8-ring. The map was calculated from images of a preparation of the enzyme solubilized with the detergent dodecyl maltoside, which is visible in electron cryomicroscopy maps. The structure shows that the micelle surrounding the complex is curved. The observed bend in the micelle of the detergent-solubilized complex is consistent with previous electron tomography experiments and suggests that monomers of ATP synthase are sufficient to produce curvature in lipid bilayers. PMID:22753497

  6. Further characterization of the subunits of the receptor with high affinity for immunoglobulin E

    SciTech Connect

    Alcaraz, G.; Kinet, J.P.; Liu, T.Y.; Metzger, H.

    1987-05-05

    The ..cap alpha.., ..beta.., ..gamma.. subunits of the receptor with high affinity for immunoglobulin E were isolated and their compositions assessed by direct amino acid analysis and by incorporation of radioactive precursors. The compositions show no unusual features other than a rather high content of tryptophan in the ..cap alpha.. chain as assessed from the incorporation studies. The results combined with future sequence data will permit unambiguous determination of the multiplicity of the chains in the receptor. Chymotryptic peptide maps of the extrinsically iodinated subunits show several similar peptides, particularly for ..cap alpha.. and ..beta... However, these putative homologies were not apparent when tryptic maps of the biosynthetically ((/sup 3/H)leucine) labeled subunits were analyzed.

  7. 28 CFR 51.6 - Political subunits.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 28 Judicial Administration 2 2012-07-01 2012-07-01 false Political subunits. 51.6 Section 51.6 Judicial Administration DEPARTMENT OF JUSTICE (CONTINUED) PROCEDURES FOR THE ADMINISTRATION OF SECTION 5 OF THE VOTING RIGHTS ACT OF 1965, AS AMENDED General Provisions § 51.6 Political subunits. All...

  8. 28 CFR 51.6 - Political subunits.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 28 Judicial Administration 2 2010-07-01 2010-07-01 false Political subunits. 51.6 Section 51.6 Judicial Administration DEPARTMENT OF JUSTICE (CONTINUED) PROCEDURES FOR THE ADMINISTRATION OF SECTION 5 OF THE VOTING RIGHTS ACT OF 1965, AS AMENDED General Provisions § 51.6 Political subunits. All...

  9. 28 CFR 51.6 - Political subunits.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 28 Judicial Administration 2 2011-07-01 2011-07-01 false Political subunits. 51.6 Section 51.6 Judicial Administration DEPARTMENT OF JUSTICE (CONTINUED) PROCEDURES FOR THE ADMINISTRATION OF SECTION 5 OF THE VOTING RIGHTS ACT OF 1965, AS AMENDED General Provisions § 51.6 Political subunits. All...

  10. 28 CFR 51.6 - Political subunits.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 28 Judicial Administration 2 2013-07-01 2013-07-01 false Political subunits. 51.6 Section 51.6 Judicial Administration DEPARTMENT OF JUSTICE (CONTINUED) PROCEDURES FOR THE ADMINISTRATION OF SECTION 5 OF THE VOTING RIGHTS ACT OF 1965, AS AMENDED General Provisions § 51.6 Political subunits. All...

  11. 28 CFR 51.6 - Political subunits.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 28 Judicial Administration 2 2014-07-01 2014-07-01 false Political subunits. 51.6 Section 51.6 Judicial Administration DEPARTMENT OF JUSTICE (CONTINUED) PROCEDURES FOR THE ADMINISTRATION OF SECTION 5 OF THE VOTING RIGHTS ACT OF 1965, AS AMENDED General Provisions § 51.6 Political subunits. All...

  12. Structural Characterization of Tip20p and Dsl1p, Subunits of the Dsl1p Vesicle Tethering Complex

    SciTech Connect

    Tripathi, A.; Ren, Y; Jeffrey, P; Hughson, F

    2009-01-01

    Multisubunit tethering complexes are essential for intracellular trafficking and have been proposed to mediate the initial interaction between vesicles and the membranes with which they fuse. Here we report initial structural characterization of the Dsl1p complex, whose three subunits are essential for trafficking from the Golgi apparatus to the endoplasmic reticulum (ER). Crystal structures reveal that two of the three subunits, Tip20p and Dsl1p, resemble known subunits of the exocyst complex, establishing a structural connection among several multisubunit tethering complexes and implying that many of their subunits are derived from a common progenitor. We show, moreover, that Tip20p and Dsl1p interact directly via N-terminal alpha-helices. Finally, we establish that different Dsl1p complex subunits bind independently to different ER SNARE proteins. Our results map out two alternative protein-interaction networks capable of tethering COPI-coated vesicles, via the Dsl1p complex, to ER membranes.

  13. Impact of Ancillary Subunits on Ventricular Repolarization

    PubMed Central

    Abbott, Geoffrey W.; Xu, Xianghua; Roepke, Torsten K.

    2007-01-01

    Voltage-gated potassium (Kv) channels generate the outward K+ ion currents that constitute the primary force in ventricular repolarization. Kv channels comprise tetramers of pore-forming α subunits and, in probably the majority of cases in vivo, ancillary or β subunits that help define the properties of the Kv current generated. Ancillary subunits can be broadly categorized as cytoplasmic or transmembrane, and can modify Kv channel trafficking, conductance, gating, ion selectivity, regulation and pharmacology. Because of their often profound effects on Kv channel function, studies of the molecular correlates of ventricular repolarization must take into account ancillary subunits as well as α subunits. Cytoplasmic ancillary subunits include the Kvβ subunits, which regulate a range of Kv channels and may link channel gating to redox potential; and the KChIPs, which appear most often associated with Kv4 subfamily channels that generate the ventricular Ito current. Transmembrane ancillary subunits include the MinK-related proteins (MiRPs) encoded by KCNE genes, which modulate members of most Kv α subunit subfamilies; and the putative 12-transmembrane domain KCR1 protein which modulates hERG. In some cases, such as the ventricular IKs channel complex, it is well-established that the KCNQ1 α subunit must co-assemble with the MinK (KCNE1) single transmembrane domain ancillary subunit for recapitulation of the characteristic, unusually slowly-activating IKs current. In other cases it is not so clear-cut, and in particular the roles of the other MinK-related proteins (MiRPs 1–4) in regulating cardiac Kv channels such as KCNQ1 and hERG in vivo are under debate. MiRP1 alters hERG function and pharmacology, and inherited MiRP1 mutations are associated with inherited and acquired arrhythmias, but controversy exists over the native role of MiRP1 in regulating hERG (and therefore ventricular IKr) in vivo. Some ancillary subunits may exhibit varied expression to shape

  14. Antelope Valley-East Kern Water Agency area, California, showing ground-water subunits and areas, location of wells, and lines of equal depth to water

    USGS Publications Warehouse

    Blankenbaker, G.G.

    1978-01-01

    The U.S. Geological Survey has released to the open file a map showing ground-water subunits and areas, and depth to water for spring 1978, in the Antelope Valley-East Kern Water Agency area, California.

  15. Conformational flexibility and subunit arrangement of the modular yeast Spt-Ada-Gcn5 acetyltransferase complex.

    PubMed

    Setiaputra, Dheva; Ross, James D; Lu, Shan; Cheng, Derrick T; Dong, Meng-Qiu; Yip, Calvin K

    2015-04-17

    The Spt-Ada-Gcn5 acetyltransferase (SAGA) complex is a highly conserved, 19-subunit histone acetyltransferase complex that activates transcription through acetylation and deubiquitination of nucleosomal histones in Saccharomyces cerevisiae. Because SAGA has been shown to display conformational variability, we applied gradient fixation to stabilize purified SAGA and systematically analyzed this flexibility using single-particle EM. Our two- and three-dimensional studies show that SAGA adopts three major conformations, and mutations of specific subunits affect the distribution among these. We also located the four functional modules of SAGA using electron microscopy-based labeling and transcriptional activator binding analyses and show that the acetyltransferase module is localized in the most mobile region of the complex. We further comprehensively mapped the subunit interconnectivity of SAGA using cross-linking mass spectrometry, revealing that the Spt and Taf subunits form the structural core of the complex. These results provide the necessary restraints for us to generate a model of the spatial arrangement of all SAGA subunits. According to this model, the chromatin-binding domains of SAGA are all clustered in one face of the complex that is highly flexible. Our results relate information of overall SAGA structure with detailed subunit level interactions, improving our understanding of its architecture and flexibility. PMID:25713136

  16. Conformational Flexibility and Subunit Arrangement of the Modular Yeast Spt-Ada-Gcn5 Acetyltransferase Complex*

    PubMed Central

    Setiaputra, Dheva; Ross, James D.; Lu, Shan; Cheng, Derrick T.; Dong, Meng-Qiu; Yip, Calvin K.

    2015-01-01

    The Spt-Ada-Gcn5 acetyltransferase (SAGA) complex is a highly conserved, 19-subunit histone acetyltransferase complex that activates transcription through acetylation and deubiquitination of nucleosomal histones in Saccharomyces cerevisiae. Because SAGA has been shown to display conformational variability, we applied gradient fixation to stabilize purified SAGA and systematically analyzed this flexibility using single-particle EM. Our two- and three-dimensional studies show that SAGA adopts three major conformations, and mutations of specific subunits affect the distribution among these. We also located the four functional modules of SAGA using electron microscopy-based labeling and transcriptional activator binding analyses and show that the acetyltransferase module is localized in the most mobile region of the complex. We further comprehensively mapped the subunit interconnectivity of SAGA using cross-linking mass spectrometry, revealing that the Spt and Taf subunits form the structural core of the complex. These results provide the necessary restraints for us to generate a model of the spatial arrangement of all SAGA subunits. According to this model, the chromatin-binding domains of SAGA are all clustered in one face of the complex that is highly flexible. Our results relate information of overall SAGA structure with detailed subunit level interactions, improving our understanding of its architecture and flexibility. PMID:25713136

  17. Cleft Lip Repair: The Hybrid Subunit Method.

    PubMed

    Tollefson, Travis T

    2016-04-01

    The unilateral cleft lip repair is one of the most rewarding and challenging of plastic surgery procedures. Surgeons have introduced a variety of straight line, geometric, and rotation-advancement designs, while in practice the majority of North American surgeons have been using hybrids of the rotation-advancement techniques. The anatomic subunit approach was introduced in 2005 by Fisher and has gained popularity, with early adopters of the design touting its simplicity and effectiveness. The objectives of this article are to summarize the basic tenets of respecting the philtral subunit, accurate measurement and planning, and tips for transitioning to this subunit approach. PMID:27097136

  18. Structural determinants within the subunit protein of Ty1 virus-like particles.

    PubMed

    Martin-Rendon, E; Marfany, G; Wilson, S; Ferguson, D J; Kingsman, S M; Kingsman, A J

    1996-11-01

    The Ty virus-like particles (VLPs) are functionally analogous to retroviral particles. They package the enzymes and the RNA necessary for retrotransposition, and mediate the integration of the reverse-transcription product into the genome of the host cell. Here we map three structural determinants of particle assembly in the subunit protein. We have also identified key residues in these regions that seem to be involved in subunit interaction and particle morphology. In particular, two point mutations in putative amphipathic helices have remarkable effects on VLP morphology, increasing the diameter as much as eightfold. PMID:8951814

  19. Positions of S2, S13, S16, S17, S19 and S21 in the 30 S ribosomal subunit of Escherichia coli.

    PubMed

    Capel, M S; Kjeldgaard, M; Engelman, D M; Moore, P B

    1988-03-01

    Neutron scattering distance data are presented for 33 protein pairs in the 30 S ribosomal subunit from Escherichia coli, along with the methods used for measuring distances between its exchangeable components. When combined with prior data, these new results permit the positioning of S2, S13, S16, S17, S19 and S21 in the 30 S ribosomal subunit, completing the mapping of its proteins by neutron scattering. Comparisons with other data suggest that the neutron map is a reliable guide to the quaternary structure of the 30 S subunit. PMID:3288761

  20. R-subunit Isoform Specificity in Protein Kinase A: Distinct Features of Protein Interfaces in PKA Types I and II by Amide H/2H exchange Mass Spectrometry

    PubMed Central

    Anand, Ganesh S.; Hotchko, Matthew; Brown, Simon H.J.; Ten Eyck, Lynn F.; Komives, Elizabeth A.; Taylor, Susan S.

    2009-01-01

    The two isoforms (RI and RII) of the regulatory (R) subunit of cAMP-dependent protein kinase or protein kinase A (PKA) are similar in sequence yet have different biochemical properties and physiological functions. To further understand the molecular basis for R-isoform-specificity, the interactions of the RIIβ isoform with the PKA catalytic (C) subunit were analyzed by amide H/2H exchange mass spectrometry to compare solvent accessibility of RIIβ and the C subunit in their free and complexed states. Direct mapping of the RIIβ-C interface revealed important differences between the intersubunit interfaces in the type I and type II holoenzyme complexes. These differences are seen in both the R-subunits as well as the C-subunit. Unlike the type I isoform, the type II isoform complexes require both cAMP-binding domains, and ATP is not obligatory for high affinity interactions with the C-subunit. Surprisingly, the C-subunit mediates distinct, overlapping surfaces of interaction with the two R-isoforms despite a strong homology in sequence and similarity in domain organization. Identification of a remote allosteric site on the C-subunit that is essential for interactions with RII, but not RI subunits, further highlights the considerable diversity in interfaces found in higher order protein complexes mediated by the C-subunit of PKA. PMID:17942118

  1. Subunit Organisation of In Vitro Reconstituted HOPS and CORVET Multisubunit Membrane Tethering Complexes

    PubMed Central

    Guo, Zhong; Johnston, Wayne; Kovtun, Oleksiy; Mureev, Sergey; Bröcker, Cornelia; Ungermann, Christian; Alexandrov, Kirill

    2013-01-01

    Biochemical and structural analysis of macromolecular protein assemblies remains challenging due to technical difficulties in recombinant expression, engineering and reconstitution of multisubunit complexes. Here we use a recently developed cell-free protein expression system based on the protozoan Leishmania tarentolae to produce in vitro all six subunits of the 600 kDa HOPS and CORVET membrane tethering complexes. We demonstrate that both subcomplexes and the entire HOPS complex can be reconstituted in vitro resulting in a comprehensive subunit interaction map. To our knowledge this is the largest eukaryotic protein complex in vitro reconstituted to date. Using the truncation and interaction analysis, we demonstrate that the complex is assembled through short hydrophobic sequences located in the C-terminus of the individual Vps subunits. Based on this data we propose a model of the HOPS and CORVET complex assembly that reconciles the available biochemical and structural data. PMID:24312556

  2. Regulation of the mammalian elongation cycle by subunit rolling: a eukaryotic-specific ribosome rearrangement.

    PubMed

    Budkevich, Tatyana V; Giesebrecht, Jan; Behrmann, Elmar; Loerke, Justus; Ramrath, David J F; Mielke, Thorsten; Ismer, Jochen; Hildebrand, Peter W; Tung, Chang-Shung; Nierhaus, Knud H; Sanbonmatsu, Karissa Y; Spahn, Christian M T

    2014-07-01

    The extent to which bacterial ribosomes and the significantly larger eukaryotic ribosomes share the same mechanisms of ribosomal elongation is unknown. Here, we present subnanometer resolution cryoelectron microscopy maps of the mammalian 80S ribosome in the posttranslocational state and in complex with the eukaryotic eEF1A⋅Val-tRNA⋅GMPPNP ternary complex, revealing significant differences in the elongation mechanism between bacteria and mammals. Surprisingly, and in contrast to bacterial ribosomes, a rotation of the small subunit around its long axis and orthogonal to the well-known intersubunit rotation distinguishes the posttranslocational state from the classical pretranslocational state ribosome. We term this motion "subunit rolling." Correspondingly, a mammalian decoding complex visualized in substates before and after codon recognition reveals structural distinctions from the bacterial system. These findings suggest how codon recognition leads to GTPase activation in the mammalian system and demonstrate that in mammalia subunit rolling occurs during tRNA selection. PMID:24995983

  3. Amaranth (Amaranthus hypochondriacus) vicilin subunit structure.

    PubMed

    Quiroga, Alejandra; Martínez, E Nora; Rogniaux, Hélène; Geairon, Audrey; Añón, M Cristina

    2010-12-22

    The 7S-globulin fraction is a minor component of the amaranth storage proteins. The present work provides new information about this protein. The amaranth 7S-globulin or vicilin presented a sedimentation coefficient of 8.6 ± 0.6 S and was composed of main subunits of 66, 52, 38, and 16 kDa. On the basis of mass spectrometry (MS) analysis of tryptic fragments, the 52, 38, and 16 kDa subunits presented sequence homology with sesame vicilin, whereas the 66 kDa subunit showed sequence similarity with a putative vicilin. Several characteristics of the 66 kDa subunit were similar to members of the convicilin family. Results support the hypothesis that the 7S-globulin molecules are composed of subunits coming from at least two gene families with primary products of 66 and 52 kDa, respectively. According to the present information, amaranth vicilin may be classified into the vicilin group that includes pea, broad bean, and sesame vicilins, among others. PMID:21117690

  4. Genetic exploration of interactive domains in RNA polymerase II subunits.

    PubMed Central

    Martin, C; Okamura, S; Young, R

    1990-01-01

    The two large subunits of RNA polymerase II, RPB1 and RPB2, contain regions of extensive homology to the two large subunits of Escherichia coli RNA polymerase. These homologous regions may represent separate protein domains with unique functions. We investigated whether suppressor genetics could provide evidence for interactions between specific segments of RPB1 and RPB2 in Saccharomyces cerevisiae. A plasmid shuffle method was used to screen thoroughly for mutations in RPB2 that suppress a temperature-sensitive mutation, rpb1-1, which is located in region H of RPB1. All six RPB2 mutations that suppress rpb1-1 were clustered in region I of RPB2. The location of these mutations and the observation that they were allele specific for suppression of rpb1-1 suggests an interaction between region H of RPB1 and region I of RPB2. A similar experiment was done to isolate and map mutations in RPB1 that suppress a temperature-sensitive mutation, rpb2-2, which occurs in region I of RPB2. These suppressor mutations were not clustered in a particular region. Thus, fine structure suppressor genetics can provide evidence for interactions between specific segments of two proteins, but the results of this type of analysis can depend on the conditional mutation to be suppressed. Images PMID:2183012

  5. Modulation of the skeletal muscle sodium channel alpha-subunit by the beta 1-subunit.

    PubMed

    Wallner, M; Weigl, L; Meera, P; Lotan, I

    1993-12-28

    Co-expression of cloned sodium channel beta 1-subunit with the rat skeletal muscle-subunit (alpha microI) accelerated the macroscopic current decay, enhanced the current amplitude, shifted the steady state inactivation curve to more negative potentials and decreased the time required for complete recovery from inactivation. Sodium channels expressed from skeletal muscle mRNA showed a similar behaviour to that observed from alpha microI/beta 1, indicating that beta 1 restores 'physiological' behaviour. Northern blot analysis revealed that the Na+ channel beta 1-subunit is present in high abundance (about 0.1%) in rat heart, brain and skeletal muscle, and the hybridization with untranslated region of the 'brain' beta 1 cDNA to skeletal muscle and heart mRNA indicated that the different Na+ channel alpha-subunits in brain, skeletal muscle and heart may share a common beta 1-subunit. PMID:8282123

  6. Localization of the regulatory particle subunit Sem1 in the 26S proteasome

    SciTech Connect

    Bohn, Stefan; Sakata, Eri; Beck, Florian; Pathare, Ganesh R.; Schnitger, Jérôme; Nágy, Istvan; Baumeister, Wolfgang Förster, Friedrich

    2013-05-31

    Highlights: •26S proteasome subunit Sem1 was mapped using cryo-EM and cross-linking data. •C-terminal helix of Sem1 located near winged helix motif of Rpn7. •N-terminal part of Sem1 tethers Rpn7, Rpn3 and lid helical bundle. •Sem1 binds differently to PCI-domains of proteasome subunit Rpn7 and TREX-2 subunit Thp1. -- Abstract: The ubiquitin–proteasome system is responsible for regulated protein degradation in the cell with the 26S proteasome acting as its executive arm. The molecular architecture of this 2.5 MDa complex has been established recently, with the notable exception of the small acidic subunit Sem1. Here, we localize the C-terminal helix of Sem1 binding to the PCI domain of the subunit Rpn7 using cryo-electron microscopy single particle reconstruction of proteasomes purified from yeast cells with sem1 deletion. The approximate position of the N-terminal region of Sem1 bridging the cleft between Rpn7 and Rpn3 was inferred based on site-specific cross-linking data of the 26S proteasome. Our structural studies indicate that Sem1 can assume different conformations in different contexts, which supports the idea that Sem1 functions as a molecular glue stabilizing the Rpn3/Rpn7 heterodimer.

  7. Subunit architecture of general transcription factor TFIIH.

    PubMed

    Gibbons, Brian J; Brignole, Edward J; Azubel, Maia; Murakami, Kenji; Voss, Neil R; Bushnell, David A; Asturias, Francisco J; Kornberg, Roger D

    2012-02-01

    Structures of complete 10-subunit yeast TFIIH and of a nested set of subcomplexes, containing 5, 6, and 7 subunits, have been determined by electron microscopy (EM) and 3D reconstruction. Consistency among all the structures establishes the location of the "minimal core" subunits (Ssl1, Tfb1, Tfb2, Tfb4, and Tfb5), and additional densities can be specifically attributed to Rad3, Ssl2, and the TFIIK trimer. These results can be further interpreted by placement of previous X-ray structures into the additional densities to give a preliminary picture of the RNA polymerase II preinitiation complex. In this picture, the key catalytic components of TFIIH, the Ssl2 ATPase/helicase and the Kin28 protein kinase are in proximity to their targets, downstream promoter DNA and the RNA polymerase C-terminal domain. PMID:22308316

  8. Heteromeric assembly of P2X subunits

    PubMed Central

    Saul, Anika; Hausmann, Ralf; Kless, Achim; Nicke, Annette

    2013-01-01

    Transcripts and/or proteins of P2X receptor (P2XR) subunits have been found in virtually all mammalian tissues. Generally more than one of the seven known P2X subunits have been identified in a given cell type. Six of the seven cloned P2X subunits can efficiently form functional homotrimeric ion channels in recombinant expression systems. This is in contrast to other ligand-gated ion channel families, such as the Cys-loop or glutamate receptors, where homomeric assemblies seem to represent the exception rather than the rule. P2XR mediated responses recorded from native tissues rarely match exactly the biophysical and pharmacological properties of heterologously expressed homomeric P2XRs. Heterotrimerization of P2X subunits is likely to account for this observed diversity. While the existence of heterotrimeric P2X2/3Rs and their role in physiological processes is well established, the composition of most other P2XR heteromers and/or the interplay between distinct trimeric receptor complexes in native tissues is not clear. After a description of P2XR assembly and the structure of the intersubunit ATP-binding site, this review summarizes the distribution of P2XR subunits in selected mammalian cell types and the biochemically and/or functionally characterized heteromeric P2XRs that have been observed upon heterologous co-expression of P2XR subunits. We further provide examples where the postulated heteromeric P2XRs have been suggested to occur in native tissues and an overview of the currently available pharmacological tools that have been used to discriminate between homo- and heteromeric P2XRs. PMID:24391538

  9. Oxidant regulated inter-subunit disulfide bond formation between ASIC1a subunits

    PubMed Central

    Zha, Xiang-ming; Wang, Runping; Collier, Dan M.; Snyder, Peter M.; Wemmie, John A.; Welsh, Michael J.

    2009-01-01

    The acid-sensing ion channel-1a (ASIC1a) is composed of 3 subunits and is activated by a decrease in extracellular pH. It plays an important role in diseases associated with a reduced pH and production of oxidants. Previous work showed that oxidants reduce ASIC1a currents. However, the effects on channel structure and composition are unknown. We found that ASIC1a formed inter-subunit disulfide bonds and the oxidant H2O2 increased this link between subunits. Cys-495 in the ASIC1a C terminus was particularly important for inter-subunit disulfide bond formation, although other C-terminal cysteines contributed. Inter-subunit disulfide bonds also produced some ASIC1a complexes larger than trimers. Inter-subunit disulfide bond formation reduced the proportion of ASIC1a located on the cell surface and contributed to the H2O2-induced decrease in H+-gated current. These results indicate that channel function is controlled by disulfide bond formation between intracellular residues on distinct ASIC1a subunits. They also suggest a mechanism by which the redox state can dynamically regulate membrane protein activity by forming intracellular bridges. PMID:19218436

  10. A polymorphic motif in the small subunit of ADP-glucose pyrophosphorylase modulates interactions between the small and large subunits.

    PubMed

    Cross, Joanna M; Clancy, Maureen; Shaw, Janine R; Boehlein, Susan K; Greene, Thomas W; Schmidt, Robert R; Okita, Thomas W; Hannah, L Curtis

    2005-02-01

    The heterotetrameric, allosterically regulated enzyme, adenosine-5'-diphosphoglucose pyrophosphorylase (AGPase) catalyzes the rate-limiting step in starch synthesis. Despite vast differences in allosteric properties and a long evolutionary separation, heterotetramers of potato small subunit and maize large subunit have activity comparable to either parent in an Escherichia coli expression system. In contrast, co-expression of maize small subunit with the potato large subunit produces little activity as judged by in vivo activity stain. To pinpoint the region responsible for differential activity, we expressed chimeric maize/potato small subunits in E. coli. This identified a 55-amino acid motif of the potato small subunit that is critical for glycogen production when expressed with the potato large subunit. Potato and maize small subunit sequences differ at five amino acids in this motif. Replacement experiments revealed that at least four amino acids of maize origin were required to reduce staining. An AGPase composed of a chimeric potato small subunit containing the 55-amino acid maize motif with the potato large subunit exhibited substantially less affinity for the substrates, glucose-1-phosphate and ATP and an increased Ka for the activator, 3-phosphoglyceric acid. Placement of the potato motif into the maize small subunit restored glycogen synthesis with the potato large subunit. Hence, a small polymorphic motif within the small subunit influences both catalytic and allosteric properties by modulating subunit interactions. PMID:15686515

  11. Localization of a gene for a glutamate binding subunit of a NMDA receptor (GRINA) to 8q24

    SciTech Connect

    Lewis, T.B.; DuPont, B.R.; Leach, R.

    1996-02-15

    This article reports on the localization of a gene for a glutamate binding subunit of an N-methyl-D-aspartate (NMDA) receptor, called GRINA, to human chromosome 8q24 using fluorescence in situ hybridization and radiation hybridization mapping. This gene mapped outside the critical region for benign familial neonatal convulsions (BFNC), a rare form of epilepsy; however, GRINA could be the causative genetic factor inducing idiopathic generalized epilepsy. Further studies need to be conducted. 15 refs., 2 figs.

  12. Escherichia coli rimM and yjeQ null strains accumulate immature 30S subunits of similar structure and protein complement

    PubMed Central

    Leong, Vivian; Kent, Meredith; Jomaa, Ahmad; Ortega, Joaquin

    2013-01-01

    Assembly of the Escherichia coli 30S ribosomal subunits proceeds through multiple parallel pathways. The protein factors RimM, YjeQ, RbfA, and Era work in conjunction to assist at the late stages of the maturation process of the small subunit. However, it is unclear how the functional interplay between these factors occurs in the context of multiple parallel pathways. To understand how these factors work together, we have characterized the immature 30S subunits that accumulate in ΔrimM cells and compared them with immature 30S subunits from a ΔyjeQ strain. The cryo-EM maps obtained from these particles showed that the densities representing helices 44 and 45 in the rRNA were partially missing, suggesting mobility of these motifs. These 30S subunits were also partially depleted in all tertiary ribosomal proteins, particularly those binding in the head domain. Using image classification, we identified four subpopulations of ΔrimM immature 30S subunits differing in the amount of missing density for helices 44 and 45, as well as the amount of density existing in these maps for the underrepresented proteins. The structural defects found in these immature subunits resembled those of the 30S subunits that accumulate in the ΔyjeQ strain. These findings are consistent with an “early convergency model” in which multiple parallel assembly pathways of the 30S subunit converge into a late assembly intermediate, as opposed to the mature state. Functionally related factors will bind to this intermediate to catalyze the last steps of maturation leading to the mature 30S subunit. PMID:23611982

  13. β1-subunit-induced structural rearrangements of the Ca2+- and voltage-activated K+ (BK) channel.

    PubMed

    Castillo, Juan P; Sánchez-Rodríguez, Jorge E; Hyde, H Clark; Zaelzer, Cristian A; Aguayo, Daniel; Sepúlveda, Romina V; Luk, Louis Y P; Kent, Stephen B H; Gonzalez-Nilo, Fernando D; Bezanilla, Francisco; Latorre, Ramón

    2016-06-01

    Large-conductance Ca(2+)- and voltage-activated K(+) (BK) channels are involved in a large variety of physiological processes. Regulatory β-subunits are one of the mechanisms responsible for creating BK channel diversity fundamental to the adequate function of many tissues. However, little is known about the structure of its voltage sensor domain. Here, we present the external architectural details of BK channels using lanthanide-based resonance energy transfer (LRET). We used a genetically encoded lanthanide-binding tag (LBT) to bind terbium as a LRET donor and a fluorophore-labeled iberiotoxin as the LRET acceptor for measurements of distances within the BK channel structure in a living cell. By introducing LBTs in the extracellular region of the α- or β1-subunit, we determined (i) a basic extracellular map of the BK channel, (ii) β1-subunit-induced rearrangements of the voltage sensor in α-subunits, and (iii) the relative position of the β1-subunit within the α/β1-subunit complex. PMID:27217576

  14. Molecular characterization and mutational analysis of the human B17 subunit of the mitochondrial respiratory chain complex I.

    PubMed

    Smeitink, J; Loeffen, J; Smeets, R; Triepels, R; Ruitenbeek, W; Trijbels, F; van den Heuvel, L

    1998-08-01

    Bovine NADH:ubiquinone oxidoreductase (complex 1) of the mitochondrial respiratory chain consists of about 36 nuclear-encoded subunits. We review the current knowledge of the 15 human complex I subunits cloned so far, and report the 598-bp cDNA sequence, the chromosomal localization and the tissue expression of an additional subunit, the B17 subunit. The cDNA open reading frame of B17 comprises 387 bp and encodes a protein of 128 amino acids (calculated Mr 15.5 kDa). There is 82.7% and 78.1% homology, respectively, at the cDNA and amino acid level with the bovine counterpart. The gene of the B17 subunit has been mapped to chromosome 2. Multiple-tissue dot-blots showed ubiquitous expression of the mRNA with relatively higher expression in tissues known for their high energy demand. Of these, kidney showed the highest expression. Mutational analysis of the subunit revealed no mutations or polymorphisms in 20 patients with isolated enzymatic complex I deficiency in cultured skin fibroblasts. PMID:9760212

  15. Self-association of the alpha subunit of phosphorylase kinase as determined by two-hybrid screening.

    PubMed

    Ayers, N A; Wilkinson, D A; Fitzgerald, T J; Carlson, G M

    1999-12-10

    The structural organization of the (alphabetagammadelta)(4) phosphorylase kinase complex has been studied using the yeast two-hybrid screen for the purpose of elucidating regions of alpha subunit interactions. By screening a rabbit skeletal muscle cDNA library with residues 1-1059 of the alpha subunit of phosphorylase kinase, we have isolated 16 interacting, independent, yet overlapping transcripts of the alpha subunit containing its C-terminal region. Domain mapping of binary interactions between alpha constructs revealed two regions involved in the self-association of the alpha subunit: residues 833-854, a previously unrecognized leucine zipper, and an unspecified region within residues 1015-1237. The cognate binding partner for the latter domain has been inferred to lie within the stretch from residues 864-1059. Indirect evidence from the literature suggests that the interacting domains contained within the latter two, overlapping regions may be further narrowed to the stretches from 1057 to 1237 and from 864 to 971. Cross-linking of the nonactivated holoenzyme with N-(gamma-maleimidobutyroxy)sulfosuccin-imide ester produced intramolecularly cross-linked alpha-alpha dimers, consistent with portions of two alpha subunits in the holoenyzme being in sufficient proximity to associate. This is the first report to identify potential areas of contact between the alpha subunits of phosphorylase kinase. Additionally, issues regarding the general utility of two-hybrid screening as a method for studying homodimeric interactions are discussed. PMID:10585434

  16. PKA regulatory subunit expression in tooth development.

    PubMed

    de Sousa, Sílvia Ferreira; Kawasaki, Katsushige; Kawasaki, Maiko; Volponi, Ana Angelova; Gomez, Ricardo Santiago; Gomes, Carolina Cavaliéri; Sharpe, Paul T; Ohazama, Atsushi

    2014-05-01

    Protein kinase A (PKA) plays critical roles in many biological processes including cell proliferation, cell differentiation, cellular metabolism and gene regulation. Mutation in PKA regulatory subunit, PRKAR1A has previously been identified in odontogenic myxomas, but it is unclear whether PKA is involved in tooth development. The aim of the present study was to assess the expression of alpha isoforms of PKA regulatory subunit (Prkar1a and Prkar2a) in mouse and human odontogenesis by in situ hybridization. PRKAR1A and PRKAR2A mRNA transcription was further confirmed in a human deciduous germ by qRT-PCR. Mouse Prkar1a and human PRKAR2A exhibited a dynamic spatio-temporal expression in tooth development, whereas neither human PRKAR1A nor mouse Prkar2a showed their expression in odontogenesis. These isoforms thus showed different expression pattern between human and mouse tooth germs. PMID:24755349

  17. Recent Advances in Subunit Vaccine Carriers

    PubMed Central

    Vartak, Abhishek; Sucheck, Steven J.

    2016-01-01

    The lower immunogenicity of synthetic subunit antigens, compared to live attenuated vaccines, is being addressed with improved vaccine carriers. Recent reports indicate that the physio-chemical properties of these carriers can be altered to achieve optimal antigen presentation, endosomal escape, particle bio-distribution, and cellular trafficking. The carriers can be modified with various antigens and ligands for dendritic cells targeting. They can also be modified with adjuvants, either covalently or entrapped in the matrix, to improve cellular and humoral immune responses against the antigen. As a result, these multi-functional carrier systems are being explored for use in active immunotherapy against cancer and infectious diseases. Advancing technology, improved analytical methods, and use of computational methodology have also contributed to the development of subunit vaccine carriers. This review details recent breakthroughs in the design of nano-particulate vaccine carriers, including liposomes, polymeric nanoparticles, and inorganic nanoparticles. PMID:27104575

  18. Recent Advances in Subunit Vaccine Carriers.

    PubMed

    Vartak, Abhishek; Sucheck, Steven J

    2016-01-01

    The lower immunogenicity of synthetic subunit antigens, compared to live attenuated vaccines, is being addressed with improved vaccine carriers. Recent reports indicate that the physio-chemical properties of these carriers can be altered to achieve optimal antigen presentation, endosomal escape, particle bio-distribution, and cellular trafficking. The carriers can be modified with various antigens and ligands for dendritic cells targeting. They can also be modified with adjuvants, either covalently or entrapped in the matrix, to improve cellular and humoral immune responses against the antigen. As a result, these multi-functional carrier systems are being explored for use in active immunotherapy against cancer and infectious diseases. Advancing technology, improved analytical methods, and use of computational methodology have also contributed to the development of subunit vaccine carriers. This review details recent breakthroughs in the design of nano-particulate vaccine carriers, including liposomes, polymeric nanoparticles, and inorganic nanoparticles. PMID:27104575

  19. Structural and functional consequences of succinate dehydrogenase subunit B mutations.

    PubMed

    Kim, E; Rath, E M; Tsang, V H M; Duff, A P; Robinson, B G; Church, W B; Benn, D E; Dwight, T; Clifton-Bligh, R J

    2015-06-01

    Mitochondrial dysfunction, due to mutations of the gene encoding succinate dehydrogenase (SDH), has been implicated in the development of adrenal phaeochromocytomas, sympathetic and parasympathetic paragangliomas, renal cell carcinomas, gastrointestinal stromal tumours and more recently pituitary tumours. Underlying mechanisms behind germline SDH subunit B (SDHB) mutations and their associated risk of disease are not clear. To investigate genotype-phenotype correlation of SDH subunit B (SDHB) variants, a homology model for human SDH was developed from a crystallographic structure. SDHB mutations were mapped, and biochemical effects of these mutations were predicted in silico. Results of structural modelling indicated that many mutations within SDHB are predicted to cause either failure of functional SDHB expression (p.Arg27*, p.Arg90*, c.88delC and c.311delAinsGG), or disruption of the electron path (p.Cys101Tyr, p.Pro197Arg and p.Arg242His). GFP-tagged WT SDHB and mutant SDHB constructs were transfected (HEK293) to determine biological outcomes of these mutants in vitro. According to in silico predictions, specific SDHB mutations resulted in impaired mitochondrial localisation and/or SDH enzymatic activity. These results indicated strong genotype-functional correlation for SDHB variants. This study reveals new insights into the effects of SDHB mutations and the power of structural modelling in predicting biological consequences. We predict that our functional assessment of SDHB mutations will serve to better define specific consequences for SDH activity as well as to provide a much needed assay to distinguish pathogenic mutations from benign variants. PMID:25972245

  20. Gene Expression Switching of Receptor Subunits in Human Brain Development

    PubMed Central

    Bar-Shira, Ossnat; Maor, Ronnie; Chechik, Gal

    2015-01-01

    Synaptic receptors in the human brain consist of multiple protein subunits, many of which have multiple variants, coded by different genes, and are differentially expressed across brain regions and developmental stages. The brain can tune the electrophysiological properties of synapses to regulate plasticity and information processing by switching from one protein variant to another. Such condition-dependent variant switch during development has been demonstrated in several neurotransmitter systems including NMDA and GABA. Here we systematically detect pairs of receptor-subunit variants that switch during the lifetime of the human brain by analyzing postmortem expression data collected in a population of donors at various ages and brain regions measured using microarray and RNA-seq. To further detect variant pairs that co-vary across subjects, we present a method to quantify age-corrected expression correlation in face of strong temporal trends. This is achieved by computing the correlations in the residual expression beyond a cubic-spline model of the population temporal trend, and can be seen as a nonlinear version of partial correlations. Using these methods, we detect multiple new pairs of context dependent variants. For instance, we find a switch from GLRA2 to GLRA3 that differs from the known switch in the rat. We also detect an early switch from HTR1A to HTR5A whose trends are negatively correlated and find that their age-corrected expression is strongly positively correlated. Finally, we observe that GRIN2B switch to GRIN2A occurs mostly during embryonic development, presumably earlier than observed in rodents. These results provide a systematic map of developmental switching in the neurotransmitter systems of the human brain. PMID:26636753

  1. Staggering of subunits in NMDAR channels.

    PubMed Central

    Sobolevsky, Alexander I; Rooney, LeeAnn; Wollmuth, Lonnie P

    2002-01-01

    Functional N-methyl-D-aspartate receptors (NMDARs) are heteromultimers formed by NR1 and NR2 subunits. The M3 segment, as contributed by NR1, forms the core of the extracellular vestibule, including binding sites for channel blockers, and represents a critical molecular link between ligand binding and channel opening. Taking advantage of the substituted cysteine accessibility method along with channel block and multivalent coordination, we studied the contribution of the M3 segment in NR2C to the extracellular vestibule. We find that the M3 segment in NR2C, like that in NR1, contributes to the core of the extracellular vestibule. However, the M3 segments from the two subunits are staggered relative to each other in the vertical axis of the channel. Compared to NR1, homologous positions in NR2C, including those in the highly conserved SYTANLAAF motif, are located about four amino acids more externally. The staggering of subunits may represent a key structural feature underlying the distinct functional properties of NMDARs. PMID:12496098

  2. Na Channel β Subunits: Overachievers of the Ion Channel Family.

    PubMed

    Brackenbury, William J; Isom, Lori L

    2011-01-01

    Voltage-gated Na(+) channels (VGSCs) in mammals contain a pore-forming α subunit and one or more β subunits. There are five mammalian β subunits in total: β1, β1B, β2, β3, and β4, encoded by four genes: SCN1B-SCN4B. With the exception of the SCN1B splice variant, β1B, the β subunits are type I topology transmembrane proteins. In contrast, β1B lacks a transmembrane domain and is a secreted protein. A growing body of work shows that VGSC β subunits are multifunctional. While they do not form the ion channel pore, β subunits alter gating, voltage-dependence, and kinetics of VGSCα subunits and thus regulate cellular excitability in vivo. In addition to their roles in channel modulation, β subunits are members of the immunoglobulin superfamily of cell adhesion molecules and regulate cell adhesion and migration. β subunits are also substrates for sequential proteolytic cleavage by secretases. An example of the multifunctional nature of β subunits is β1, encoded by SCN1B, that plays a critical role in neuronal migration and pathfinding during brain development, and whose function is dependent on Na(+) current and γ-secretase activity. Functional deletion of SCN1B results in Dravet Syndrome, a severe and intractable pediatric epileptic encephalopathy. β subunits are emerging as key players in a wide variety of physiopathologies, including epilepsy, cardiac arrhythmia, multiple sclerosis, Huntington's disease, neuropsychiatric disorders, neuropathic and inflammatory pain, and cancer. β subunits mediate multiple signaling pathways on different timescales, regulating electrical excitability, adhesion, migration, pathfinding, and transcription. Importantly, some β subunit functions may operate independently of α subunits. Thus, β subunits perform critical roles during development and disease. As such, they may prove useful in disease diagnosis and therapy. PMID:22007171

  3. Models for the a subunits of the Thermus thermophilus V/A-ATPase and Saccharomyces cerevisiae V-ATPase enzymes by cryo-EM and evolutionary covariance.

    PubMed

    Schep, Daniel G; Zhao, Jianhua; Rubinstein, John L

    2016-03-22

    Rotary ATPases couple ATP synthesis or hydrolysis to proton translocation across a membrane. However, understanding proton translocation has been hampered by a lack of structural information for the membrane-embedded a subunit. The V/A-ATPase from the eubacteriumThermus thermophilusis similar in structure to the eukaryotic V-ATPase but has a simpler subunit composition and functions in vivo to synthesize ATP rather than pump protons. We determined theT. thermophilusV/A-ATPase structure by cryo-EM at 6.4 Å resolution. Evolutionary covariance analysis allowed tracing of the a subunit sequence within the map, providing a complete model of the rotary ATPase. Comparing the membrane-embedded regions of theT. thermophilusV/A-ATPase and eukaryotic V-ATPase fromSaccharomyces cerevisiaeallowed identification of the α-helices that belong to the a subunit and revealed the existence of previously unknown subunits in the eukaryotic enzyme. Subsequent evolutionary covariance analysis enabled construction of a model of the a subunit in theS. cerevisaeV-ATPase that explains numerous biochemical studies of that enzyme. Comparing the two a subunit structures determined here with a structure of the distantly related a subunit from the bovine F-type ATP synthase revealed a conserved pattern of residues, suggesting a common mechanism for proton transport in all rotary ATPases. PMID:26951669

  4. How subunit coupling produces the γ-subunit rotary motion in F1-ATPase

    PubMed Central

    Pu, Jingzhi; Karplus, Martin

    2008-01-01

    FoF1-ATP synthase manufactures the energy “currency,” ATP, of living cells. The soluble F1 portion, called F1-ATPase, can act as a rotary motor, with ATP binding, hydrolysis, and product release, inducing a torque on the γ-subunit. A coarse-grained plastic network model is used to show at a residue level of detail how the conformational changes of the catalytic β-subunits act on the γ-subunit through repulsive van der Waals interactions to generate a torque that drives unidirectional rotation, as observed experimentally. The simulations suggest that the calculated 85° substep rotation is driven primarily by ATP binding and that the subsequent 35° substep rotation is produced by product release from one β-subunit and a concomitant binding pocket expansion of another β-subunit. The results of the simulation agree with single-molecule experiments [see, for example, Adachi K, et al. (2007) Cell 130:309–321] and support a tri-site rotary mechanism for F1-ATPase under physiological condition. PMID:18216260

  5. Solution nuclear magnetic resonance structure of the GATase subunit and structural basis of the interaction between GATase and ATPPase subunits in a two-subunit-type GMPS from Methanocaldococcus jannaschii.

    PubMed

    Ali, Rustam; Kumar, Sanjeev; Balaram, Hemalatha; Sarma, Siddhartha P

    2013-06-25

    The solution structure of the monomeric glutamine amidotransferase (GATase) subunit of the Methanocaldococcus janaschii (Mj) guanosine monophosphate synthetase (GMPS) has been determined using high-resolution nuclear magnetic resonance methods. Gel filtration chromatography and ¹⁵N backbone relaxation studies have shown that the Mj GATase subunit is present in solution as a 21 kDa (188-residue) monomer. The ensemble of 20 lowest-energy structures showed root-mean-square deviations of 0.35 ± 0.06 Å for backbone atoms and 0.8 ± 0.06 Å for all heavy atoms. Furthermore, 99.4% of the backbone dihedral angles are present in the allowed region of the Ramachandran map, indicating the stereochemical quality of the structure. The core of the tertiary structure of the GATase is composed of a seven-stranded mixed β-sheet that is fenced by five α-helices. The Mj GATase is similar in structure to the Pyrococcus horikoshi (Ph) GATase subunit. Nuclear magnetic resonance (NMR) chemical shift perturbations and changes in line width were monitored to identify residues on GATase that were responsible for interaction with magnesium and the ATPPase subunit, respectively. These interaction studies showed that a common surface exists for the metal ion binding as well as for the protein-protein interaction. The dissociation constant for the GATase-Mg(2+) interaction has been found to be ∼1 mM, which implies that interaction is very weak and falls in the fast chemical exchange regime. The GATase-ATPPase interaction, on the other hand, falls in the intermediate chemical exchange regime on the NMR time scale. The implication of this interaction in terms of the regulation of the GATase activity of holo GMPS is discussed. PMID:23724776

  6. Studies on chromatin. II. Isolation and characterization of chromatin subunits.

    PubMed Central

    Bakayev, V V; Melnickov, A A; Osicka, V D; Varshausky, A J

    1975-01-01

    Earlier findings /1-10/ bearing on a subunit organization of chromatin were confirmed and in some points detailed. Besides this, a large-scale isolation of chromatin subunits; their protein composition, electron microscopic appearance and CsCl banding pattern are described. Although the purified chromatin subunit contains all five histones, the relative content of histone H1 i in it is two times lower than that in the original chromatin. tit is shown that a mild digestion of chromatin with staphylococcal nuclease produced not only separate chromatin subunits and their "oligomers' but also deoxyribonucleoprotein particles which sediment more slowly than subunits. It appears that these particles and subunits are produced from different initial structures in the chromatin. Finally, a crystallization of the purified chromatin subunit as a cetyltrimethyl ammonium salt is described. Images PMID:1178523

  7. 3D Gel Map of Arabidopsis Complex I

    PubMed Central

    Peters, Katrin; Belt, Katharina; Braun, Hans-Peter

    2013-01-01

    Complex I has a unique structure in plants and includes extra subunits. Here, we present a novel study to define its protein constituents. Mitochondria were isolated from Arabidopsis thaliana cell cultures, leaves, and roots. Subunits of complex I were resolved by 3D blue-native (BN)/SDS/SDS-PAGE and identified by mass spectrometry. Overall, 55 distinct proteins were found, seven of which occur in pairs of isoforms. We present evidence that Arabidopsis complex I consists of 49 distinct types of subunits, 40 of which represent homologs of bovine complex I. The nine other subunits represent special proteins absent in the animal linage of eukaryotes, most prominently a group of subunits related to bacterial gamma-type carbonic anhydrases. A GelMap http://www.gelmap.de/arabidopsis-3d-complex-i/ is presented for promoting future complex I research in Arabidopsis thaliana. PMID:23761796

  8. Inherent conformational flexibility of F1-ATPase α-subunit.

    PubMed

    Hahn-Herrera, Otto; Salcedo, Guillermo; Barril, Xavier; García-Hernández, Enrique

    2016-09-01

    The core of F1-ATPase consists of three catalytic (β) and three noncatalytic (α) subunits, forming a hexameric ring in alternating positions. A wealth of experimental and theoretical data has provided a detailed picture of the complex role played by catalytic subunits. Although major conformational changes have only been seen in β-subunits, it is clear that α-subunits have to respond to these changes in order to be able to transmit information during the rotary mechanism. However, the conformational behavior of α-subunits has not been explored in detail. Here, we have combined unbiased molecular dynamics (MD) simulations and calorimetrically measured thermodynamic signatures to investigate the conformational flexibility of isolated α-subunits, as a step toward deepening our understanding of its function inside the α3β3 ring. The simulations indicate that the open-to-closed conformational transition of the α-subunit is essentially barrierless, which is ideal to accompany and transmit the movement of the catalytic subunits. Calorimetric measurements of the recombinant α-subunit from Geobacillus kaustophilus indicate that the isolated subunit undergoes no significant conformational changes upon nucleotide binding. Simulations confirm that the nucleotide-free and nucleotide-bound subunits show average conformations similar to that observed in the F1 crystal structure, but they reveal an increased conformational flexibility of the isolated α-subunit upon MgATP binding, which might explain the evolutionary conserved capacity of α-subunits to recognize nucleotides with considerable strength. Furthermore, we elucidate the different dependencies that α- and β-subunits show on Mg(II) for recognizing ATP. PMID:27137408

  9. Cytokine induced changes in proteasome subunit composition are concentration dependent.

    PubMed

    Stohwasser, R; Kloetzel, P M

    1996-09-01

    In eukaryotes, 20S proteasome subunit composition is controlled by the cytokine interferon-gamma (IFN-gamma). IFN-gamma induces the synthesis of the beta-subunits LMP2, LMP7 and MECL-1, which in consequence replace their constitutive subunit homologs delta, MB1 and MC14/Z in the 20S complex. By pulse labeling mouse RMA cells and immunoprecipitation of proteasome complexes with the antibody MP3, we have analysed the effect of different IFN-gamma concentrations on proteasomal subunit composition. Our experiments show that IFN-gamma concentrations as low as 5 U/ml induce subunit substitutions and that overall proteasomal subunit composition is dependent on the cytokine concentration used. An IFN-gamma concentration of 50 U/ml is sufficient for complete replacement of subunit delta by LMP2. In contrast, IFN-gamma treatment never induces a complete replacement of subunit MC14 by MECL-1. These subunits are present at an approximate 1:1 molar ratio, suggesting that both subunits coexist in the same 20S proteasome complex. Furthermore, different regulatory mechanisms have to be postulated for the synthesis and incorporation of the three IFN-gamma inducible proteasome subunits. Both IFN-gamma as well as IL-2 also seem to influence the modification state of the alpha subunit C8. Since the subunit composition is dependent on the cytokine concentration used and strongly influences the proteolytic properties of the 20S proteasome complex, our experiments represent a caveat for experiments in which IFN-gamma dependent proteasomal enzyme characteristics have been analysed without monitoring the subunit composition. PMID:9067255

  10. ParC subunit of DNA topoisomerase IV of Streptococcus pneumoniae is a primary target of fluoroquinolones and cooperates with DNA gyrase A subunit in forming resistance phenotype.

    PubMed Central

    Muñoz, R; De La Campa, A G

    1996-01-01

    The genes encoding the ParC and ParE subunits of topoisomerase IV of Streptococcus pneumoniae, together with the region encoding amino acids 46 to 172 (residue numbers are as in Escherichia coli) of the pneumococcal GyrA subunit, were partially characterized. The gyrA gene maps to a physical location distant from the gyrB and parC loci on the chromosome, whereas parC is closely linked to parE. Ciprofloxacin-resistant (Cpr) clinical isolates of S. pneumoniae had mutations affecting amino acid residues of the quinolone resistance-determining region of ParC (low-level Cpr) or in both quinolone resistance-determining regions of ParC and GyrA (high-level Cpr). Mutations were found in residue positions equivalent to the serine at position 83 and the aspartic acid at position 87 of the E. coli GyrA subunit. Transformation experiments suggest that ParC is the primary target of ciprofloxacin. Mutation in parC appears to be a prerequisite before mutations in gyrA can influence resistance levels. PMID:8891124

  11. Cloning and characterization of GABAA α subunits and GABAB subunits in Xenopus laevis during development

    PubMed Central

    Kaeser, Gwendolyn E.; Rabe, Brian A.; Saha, Margaret S.

    2011-01-01

    Gamma-aminobutyric acid (GABA), the major inhibitory neurotransmitter in the adult nervous system, acts via two classes of receptors, the ionotropic GABAA and metabotropic GABAB receptors. During the development of the nervous system GABA acts in a depolarizing, excitatory manner and plays an important role in various neural developmental processes including cell proliferation, migration, synapse formation and activity-dependent differentiation. Here we describe the spatial and temporal expression patterns of the GABAA and GABAB receptors during early development of Xenopus laevis. Using in situ hybridization and qRT-PCR, GABAA α2 was detected as a maternal mRNA. All other α-subunits were first detected by tailbud through hatching stages. Expression of the various subunits was seen in the brain, spinal cord, cranial ganglia, olfactory epithelium, pineal, and pituitary gland. Each receptor subunit showed a distinctive, unique expression pattern suggesting these receptors have specific functions and are regulated in a precise spatial and temporal manner. PMID:21384470

  12. PKA catalytic subunit mutations in adrenocortical Cushing's adenoma impair association with the regulatory subunit.

    PubMed

    Calebiro, Davide; Hannawacker, Annette; Lyga, Sandra; Bathon, Kerstin; Zabel, Ulrike; Ronchi, Cristina; Beuschlein, Felix; Reincke, Martin; Lorenz, Kristina; Allolio, Bruno; Kisker, Caroline; Fassnacht, Martin; Lohse, Martin J

    2014-01-01

    We recently identified a high prevalence of mutations affecting the catalytic (Cα) subunit of protein kinase A (PKA) in cortisol-secreting adrenocortical adenomas. The two identified mutations (Leu206Arg and Leu199_Cys200insTrp) are associated with increased PKA catalytic activity, but the underlying mechanisms are highly controversial. Here we utilize a combination of biochemical and optical assays, including fluorescence resonance energy transfer in living cells, to analyze the consequences of the two mutations with respect to the formation of the PKA holoenzyme and its regulation by cAMP. Our results indicate that neither mutant can form a stable PKA complex, due to the location of the mutations at the interface between the catalytic and the regulatory subunits. We conclude that the two mutations cause high basal catalytic activity and lack of regulation by cAMP through interference of complex formation between the regulatory and the catalytic subunits of PKA. PMID:25477193

  13. Active Mapping.

    ERIC Educational Resources Information Center

    Day, Dennis

    1994-01-01

    Explains a social studies lesson for third graders that uses KidPix, a computer software graphics program to help students make maps and map keys. Advantages to using the computer versus hand drawing maps are discussed, and an example of map requirements for the lesson is included. (LRW)

  14. Concept Mapping.

    ERIC Educational Resources Information Center

    Callison, Daniel

    2001-01-01

    Explains concept mapping as a heuristic device that is helpful in visualizing the relationships between and among ideas. Highlights include how to begin a map; brainstorming; map applications, including document or information summaries and writing composition; and mind mapping to strengthen note-taking. (LRW)

  15. The proteasomal subunit Rpn6 is a molecular clamp holding the core and regulatory subcomplexes together

    PubMed Central

    Pathare, Ganesh Ramnath; Nagy, István; Bohn, Stefan; Unverdorben, Pia; Hubert, Agnes; Körner, Roman; Nickell, Stephan; Lasker, Keren; Sali, Andrej; Tamura, Tomohiro; Nishioka, Taiki; Förster, Friedrich; Baumeister, Wolfgang; Bracher, Andreas

    2012-01-01

    Proteasomes execute the degradation of most cellular proteins. Although the 20S core particle (CP) has been studied in great detail, the structure of the 19S regulatory particle (RP), which prepares ubiquitylated substrates for degradation, has remained elusive. Here, we report the crystal structure of one of the RP subunits, Rpn6, and we describe its integration into the cryo-EM density map of the 26S holocomplex at 9.1 Å resolution. Rpn6 consists of an α-solenoid-like fold and a proteasome COP9/signalosome eIF3 (PCI) module in a right-handed suprahelical configuration. Highly conserved surface areas of Rpn6 interact with the conserved surfaces of the Pre8 (alpha2) and Rpt6 subunits from the alpha and ATPase rings, respectively. The structure suggests that Rpn6 has a pivotal role in stabilizing the otherwise weak interaction between the CP and the RP. PMID:22187461

  16. All-atom homology model of the Escherichia coli 30S ribosomal subunit.

    PubMed

    Tung, Chang-Shung; Joseph, Simpson; Sanbonmatsu, Kevin Y

    2002-10-01

    Understanding the structural basis of ribosomal function requires close comparison between biochemical and structural data. Although a large amount of biochemical data are available for the Escherichia coli ribosome, the structure has not been solved to atomic resolution. Using a new RNA homology procedure, we have modeled the all-atom structure of the E. coli 30S ribosomal subunit. We find that the tertiary structure of the ribosome core, including the A-, P- and E-sites, is highly conserved. The hypervariable regions in our structure, which differ from the structure of the 30S ribosomal subunit from Thermus thermophilus, are consistent with the cryo-EM map of the E. coli ribosome. PMID:12244297

  17. The Trypanosoma brucei protein phosphatase gene: polycistronic transcription with the RNA polymerase II largest subunit gene.

    PubMed Central

    Evers, R; Cornelissen, A W

    1990-01-01

    We have previously described the trypanosomal gene encoding the largest subunit of RNA polymerase II (RNAP II) and found that two almost identical genes are encoded within the Trypanosoma brucei genome. Here we show by Southern analyses that the 5' breakpoint between both loci is located approximately 7.5 kb upstream of the RNAP II genes. Northern analyses revealed that the 5' duplicated segment contains at least four other genes, which are transcribed in both bloodstream and procyclic trypanosomes. The gene located immediately upstream of the RNAP II gene in both loci was characterized by sequence analyses. The deduced amino acid sequences show a high degree of similarity to the catalytic subunit of protein phosphatase class 1 (PP1) genes. S1 mapping provided strong evidence in support of the fact that the PP1 and RNAP II genes belong to a single transcription unit. Images PMID:2169604

  18. Contour Mapping

    NASA Technical Reports Server (NTRS)

    1995-01-01

    In the early 1990s, the Ohio State University Center for Mapping, a NASA Center for the Commercial Development of Space (CCDS), developed a system for mobile mapping called the GPSVan. While driving, the users can map an area from the sophisticated mapping van equipped with satellite signal receivers, video cameras and computer systems for collecting and storing mapping data. George J. Igel and Company and the Ohio State University Center for Mapping advanced the technology for use in determining the contours of a construction site. The new system reduces the time required for mapping and staking, and can monitor the amount of soil moved.

  19. Structural model of the 50S subunit of E.Coli ribosomes from solution scattering

    SciTech Connect

    Svergun, D.I.; Koch, M.H.J.; Pedersen, J.S.; Serdyuk, I.N.

    1994-12-31

    The application of new methods of small-angle scattering data interpretation to a contrast variation study of the 50S ribosomal subunit of Escherichia coli in solution is described. The X-ray data from contrast variation with sucrose are analyzed in terms of the basic scattering curves from the volume inaccessible to sucrose and from the regions inside this volume occupied mainly by RNA and by proteins. From these curves models of the shape of the 50S and its RNA-rich core are evaluated and positioned so that their difference produces a scattering curve which is in good agreement with the scattering from the protein moiety. Basing on this preliminary model, the X-ray and neutron contrast variation data of the 50S subunit in aqueous solutions are interpreted in the frame of the advanced two-phase model described by the shapes of the 50S subunit and its RNA-rich core taking into account density fluctuations inside the RNA and the protein moiety. The shape of the envelope of the 50S subunit and of the RNA-rich core are evaluated with a resolution of about 40A. The shape of the envelope is in good agreement with the models of the 50S subunit obtained from electron microscopy on isolated particles. The shape of the RNA-rich core correlates well with the model of the entire particle determined by the image reconstruction from ordered sheets indicating that the latter model which is based on the subjective contouring of density maps is heavily biased towards the RNA.

  20. [Nose surgical anatomy in six aesthetic subunits].

    PubMed

    Chaput, B; Lauwers, F; Lopez, R; Saboye, J; André, A; Grolleau, J-L; Chavoin, J-P

    2013-04-01

    The nose is a complex entity, combining aesthetic and functional roles. Descriptive anatomy is a fundamental science that it can be difficult to relate directly to our daily surgical activity. Reasoning in terms of aesthetic subunits to decide on his actions appeared to us so obvious. The aim of this paper is to resume the anatomical bases relevant to our daily practice in order to fully apprehend the restorative or cosmetic procedures. We discuss the limits of the systematization of these principles in nasal oncology. PMID:22699003

  1. MspA Nanopores from Subunit Dimers

    PubMed Central

    Pavlenok, Mikhail; Derrington, Ian M.; Gundlach, Jens H.; Niederweis, Michael

    2012-01-01

    Mycobacterium smegmatis porin A (MspA) forms an octameric channel and represents the founding member of a new family of pore proteins. Control of subunit stoichiometry is important to tailor MspA for nanotechnological applications. In this study, two MspA monomers were connected by linkers ranging from 17 to 62 amino acids in length. The oligomeric pore proteins were purified from M. smegmatis and were shown to form functional channels in lipid bilayer experiments. These results indicated that the peptide linkers did not prohibit correct folding and localization of MspA. However, expression levels were reduced by 10-fold compared to wild-type MspA. MspA is ideal for nanopore sequencing due to its unique pore geometry and its robustness. To assess the usefulness of MspA made from dimeric subunits for DNA sequencing, we linked two M1-MspA monomers, whose constriction zones were modified to enable DNA translocation. Lipid bilayer experiments demonstrated that this construct also formed functional channels. Voltage gating of MspA pores made from M1 monomers and M1-M1 dimers was identical indicating similar structural and dynamic channel properties. Glucose uptake in M. smegmatis cells lacking porins was restored by expressing the dimeric mspA M1 gene indicating correct folding and localization of M1-M1 pores in their native membrane. Single-stranded DNA hairpins produced identical ionic current blockades in pores made from monomers and subunit dimers demonstrating that M1-M1 pores are suitable for DNA sequencing. This study provides the proof of principle that production of single-chain MspA pores in M. smegmatis is feasible and paves the way for generating MspA pores with altered stoichiometries. Subunit dimers enable better control of the chemical and physical properties of the constriction zone of MspA. This approach will be valuable both in understanding transport across the outer membrane in mycobacteria and in tailoring MspA for nanopore sequencing of DNA. PMID

  2. MspA nanopores from subunit dimers.

    PubMed

    Pavlenok, Mikhail; Derrington, Ian M; Gundlach, Jens H; Niederweis, Michael

    2012-01-01

    Mycobacterium smegmatis porin A (MspA) forms an octameric channel and represents the founding member of a new family of pore proteins. Control of subunit stoichiometry is important to tailor MspA for nanotechnological applications. In this study, two MspA monomers were connected by linkers ranging from 17 to 62 amino acids in length. The oligomeric pore proteins were purified from M. smegmatis and were shown to form functional channels in lipid bilayer experiments. These results indicated that the peptide linkers did not prohibit correct folding and localization of MspA. However, expression levels were reduced by 10-fold compared to wild-type MspA. MspA is ideal for nanopore sequencing due to its unique pore geometry and its robustness. To assess the usefulness of MspA made from dimeric subunits for DNA sequencing, we linked two M1-MspA monomers, whose constriction zones were modified to enable DNA translocation. Lipid bilayer experiments demonstrated that this construct also formed functional channels. Voltage gating of MspA pores made from M1 monomers and M1-M1 dimers was identical indicating similar structural and dynamic channel properties. Glucose uptake in M. smegmatis cells lacking porins was restored by expressing the dimeric mspA M1 gene indicating correct folding and localization of M1-M1 pores in their native membrane. Single-stranded DNA hairpins produced identical ionic current blockades in pores made from monomers and subunit dimers demonstrating that M1-M1 pores are suitable for DNA sequencing. This study provides the proof of principle that production of single-chain MspA pores in M. smegmatis is feasible and paves the way for generating MspA pores with altered stoichiometries. Subunit dimers enable better control of the chemical and physical properties of the constriction zone of MspA. This approach will be valuable both in understanding transport across the outer membrane in mycobacteria and in tailoring MspA for nanopore sequencing of DNA. PMID

  3. Analysis of the phosphofructokinase subunits and isoenzymes in human tissues.

    PubMed Central

    Dunaway, G A; Kasten, T P; Sebo, T; Trapp, R

    1988-01-01

    The 6-phosphofructo-1-kinase (PFK) subunits and isoenzymes were studied in human muscle, heart, brain, liver, platelets, fibroblasts, erythrocytes, placenta and umbilical cord. In each tissue, the subunit types in the native isoenzymes were characterized by immunological titration with subunit-specific antibodies and by column chromatography on QAE (quaternary aminoethyl)-Sephadex. Further, the subunits of the partially purified native isoenzymes were resolved by SDS/polyacrylamide-gel electrophoresis, identified by immunoblotting, and quantified by scanning gel densitometry of silver-stained gels and immunoblots. Depending on the type of tissue, one to three subunits were detected. The Mr values of the L, M and C subunits regardless of tissue were 76,700 +/- 1400, 82,500 +/- 1640 and 86,500 +/- 1620. Of the tissues studied, only the muscle PFK isoenzymes exhibited one subunit, which was the M-type subunit. Of the other tissues studied, the PFK isoenzymes contained various amounts of all three subunits. Considering the properties of the native PFK isoenzymes, it is clear that, in human tissues, they are not simply various combinations of two or three homotetrameric isoenzymes, but complex mixtures of homotetramers and heterotetramers. The kinetic/regulatory properties of the various isoenzyme pools were found to be dependent on subunit composition. Images Fig. 1. PMID:2970843

  4. Homologous and unique G protein alpha subunits in the nematode Caenorhabditis elegans.

    PubMed Central

    Lochrie, M A; Mendel, J E; Sternberg, P W; Simon, M I

    1991-01-01

    A cDNA corresponding to a known G protein alpha subunit, the alpha subunit of Go (Go alpha), was isolated and sequenced. The predicted amino acid sequence of C. elegans Go alpha is 80-87% identical to other Go alpha sequences. An mRNA that hybridizes to the C. elegans Go alpha cDNA can be detected on Northern blots. A C. elegans protein that crossreacts with antibovine Go alpha antibody can be detected on immunoblots. A cosmid clone containing the C. elegans Go alpha gene (goa-1) was isolated and mapped to chromosome I. The genomic fragments of three other C. elegans G protein alpha subunit genes (gpa-1, gpa-2, and gpa-3) have been isolated using the polymerase chain reaction. The corresponding cosmid clones were isolated and mapped to disperse locations on chromosome V. The sequences of two of the genes, gpa-1 and gpa-3, were determined. The predicted amino acid sequences of gpa-1 and gpa-3 are only 48% identical to each other. Therefore, they are likely to have distinct functions. In addition they are not homologous enough to G protein alpha subunits in other organisms to be classified. Thus C. elegans has G proteins that are identifiable homologues of mammalian G proteins as well as G proteins that appear to be unique to C. elegans. Study of identifiable G proteins in C. elegans may result in a further understanding of their function in other organisms, whereas study of the novel G proteins may provide an understanding of unique aspects of nematode physiology. Images PMID:1907494

  5. Subunit Arrangement and Function in NMDA Receptors

    SciTech Connect

    Furukawa,H.; Singh, S.; Mancusso, R.; Gouaux, E.

    2005-01-01

    Excitatory neurotransmission mediated by NMDA (N-methyl-D-aspartate) receptors is fundamental to the physiology of the mammalian central nervous system. These receptors are heteromeric ion channels that for activation require binding of glycine and glutamate to the NR1 and NR2 subunits, respectively. NMDA receptor function is characterized by slow channel opening and deactivation, and the resulting influx of cations initiates signal transduction cascades that are crucial to higher functions including learning and memory. Here we report crystal structures of the ligand-binding core of NR2A with glutamate and that of the NR1-NR2A heterodimer with glutamate and glycine. The NR2A-glutamate complex defines the determinants of glutamate and NMDA recognition, and the NR1-NR2A heterodimer suggests a mechanism for ligand-induced ion channel opening. Analysis of the heterodimer interface, together with biochemical and electrophysiological experiments, confirms that the NR1-NR2A heterodimer is the functional unit in tetrameric NMDA receptors and that tyrosine 535 of NR1, located in the subunit interface, modulates the rate of ion channel deactivation.

  6. TSH Receptor Cleavage Into Subunits and Shedding of the A-Subunit; A Molecular and Clinical Perspective.

    PubMed

    Rapoport, Basil; McLachlan, Sandra M

    2016-04-01

    The TSH receptor (TSHR) on the surface of thyrocytes is unique among the glycoprotein hormone receptors in comprising two subunits: an extracellular A-subunit, and a largely transmembrane and cytosolic B-subunit. Unlike its ligand TSH, whose subunits are encoded by two genes, the TSHR is expressed as a single polypeptide that subsequently undergoes intramolecular cleavage into disulfide-linked subunits. Cleavage is associated with removal of a C-peptide region, a mechanism similar in some respects to insulin cleavage into disulfide linked A- and B-subunits with loss of a C-peptide region. The potential pathophysiological importance of TSHR cleavage into A- and B-subunits is that some A-subunits are shed from the cell surface. Considerable experimental evidence supports the concept that A-subunit shedding in genetically susceptible individuals is a factor contributing to the induction and/or affinity maturation of pathogenic thyroid-stimulating autoantibodies, the direct cause of Graves' disease. The noncleaving gonadotropin receptors are not associated with autoantibodies that induce a "Graves' disease of the gonads." We also review herein current information on the location of the cleavage sites, the enzyme(s) responsible for cleavage, the mechanism by which A-subunits are shed, and the effects of cleavage on receptor signaling. PMID:26799472

  7. Sodium channel β subunits: emerging targets in channelopathies

    PubMed Central

    O’Malley, Heather A.; Isom, Lori L.

    2016-01-01

    Voltage-gated sodium channels (VGSCs) are responsible for initiation and propagation of action potentials in excitable cells. VGSCs in mammalian brain are heterotrimeric complexes of α and β subunits. Originally called “auxiliary,” we now know that β subunit proteins are multifunctional signaling molecules that play roles in both excitable and non-excitable cell types, and with or without the pore-forming α subunit present. β subunits function in VGSC and potassium channel modulation, cell adhesion, and gene regulation, with particularly important roles in brain development. Mutations in the genes encoding β subunits are linked to a number of diseases, including epilepsy, sudden death syndromes like SUDEP and SIDS, and cardiac arrhythmia. While VGSC β subunit-specific drugs have not yet been developed, this protein family is an emerging therapeutic target. PMID:25668026

  8. Sodium channel β subunits: emerging targets in channelopathies.

    PubMed

    O'Malley, Heather A; Isom, Lori L

    2015-01-01

    Voltage-gated sodium channels (VGSCs) are responsible for the initiation and propagation of action potentials in excitable cells. VGSCs in mammalian brain are heterotrimeric complexes of α and β subunits. Although β subunits were originally termed auxiliary, we now know that they are multifunctional signaling molecules that play roles in both excitable and nonexcitable cell types and with or without the pore-forming α subunit present. β subunits function in VGSC and potassium channel modulation, cell adhesion, and gene regulation, with particularly important roles in brain development. Mutations in the genes encoding β subunits are linked to a number of diseases, including epilepsy, sudden death syndromes like SUDEP and SIDS, and cardiac arrhythmia. Although VGSC β subunit-specific drugs have not yet been developed, this protein family is an emerging therapeutic target. PMID:25668026

  9. Polymorphism and structure of the gene coding for the alpha 1 subunit of the Artemia franciscana Na/K-ATPase.

    PubMed Central

    García-Sáez, A; Perona, R; Sastre, L

    1997-01-01

    Genomic clones coding for one of the two identified Artemia franciscana Na/K-ATPase alpha subunits, the alpha 1 subunit, have been isolated. Several overlapping clones were obtained, although their restriction maps showed a large heterogeneity. Sequencing of their exons showed that they differ in up to 3.46% of their nucleotides in translated regions and 8.18% in untranslated regions. Southern blot analysis of DNA purified from different lots of A. franciscana cysts and from isolated individuals suggests that the variation is due to the existence of multiple Na/K-ATPase alpha 1 subunit alleles in A. franciscana. The Na/K-ATPase alpha 1 subunit gene is divided into 15 exons. Ten of the 14 introns are located in identical positions in this gene as in the human Na/K-ATPase alpha 3 subunit gene. Analysis of the 5' flanking region of the gene has allowed identification of the transcription-initiation sites. The adjacent upstream region has been shown to have functional promoter activity in cultured mammalian cells, suggesting the evolutionary conservation of some of the promoter regulatory sequences. PMID:9020888

  10. [Molecular cloning of activin betaA subunit mature peptide from peafowl and its application in taxonomy and phylogeny].

    PubMed

    Zou, Fang-Dong; Tong, Xin-Xin; Yue, Bi-Song

    2005-03-01

    The sequences of activin gene betaA subunit mature peptide have been amplified from white peafowl, blue peafowl (pavo cristatus) and green peafowl (pavo muticus) genomic DNA by polymerase chain reaction (PCR) with a pair of degenerate primers. The target fragments were cloned into the vector pMD18-T and sequenced. The length of activin gene betaA subunit mature peptide is 345bp, which encoded a peptide of 115 amino acid residues. Sequence analysis of activin gene betaA subunit mature peptide demonstrated that the identity of nucleotide is 98.0% between blue peaflowl and green peafowl, and the identity of that is 98.8% between blue peaflowl and white peafow. Sequences comparison in NCBI revealed that the sequences of activin gene betaA subunit mature peptides of different species are highly conserved during evolution process. In addition, the restriction enzyme map of activins is high similar between white peafowl and blue peafowl. Phylogenetic tree was constructed with Mega 2 and Clustalxldx software. The result showed that white peafowl has a closer relationship to blue peafowl than to green peafowl. Considered the nucleotide differences of peafowls' activin gene betaA subunit mature peptides, a highly conserved region, we supported that white peafowl was derived from blue peafowl, and it is more possible the hybrid but just the product of color mutation, or maybe as a subspecies of Pavo genus. PMID:15843351

  11. Quantifying the cooperative subunit action in a multimeric membrane receptor

    PubMed Central

    Wongsamitkul, Nisa; Nache, Vasilica; Eick, Thomas; Hummert, Sabine; Schulz, Eckhard; Schmauder, Ralf; Schirmeyer, Jana; Zimmer, Thomas; Benndorf, Klaus

    2016-01-01

    In multimeric membrane receptors the cooperative action of the subunits prevents exact knowledge about the operation and the interaction of the individual subunits. We propose a method that permits quantification of ligand binding to and activation effects of the individual binding sites in a multimeric membrane receptor. The power of this method is demonstrated by gaining detailed insight into the subunit action in olfactory cyclic nucleotide-gated CNGA2 ion channels. PMID:26858151

  12. ADP-glucose pyrophosphorylase large subunit 2 is essential for storage substance accumulation and subunit interactions in rice endosperm.

    PubMed

    Tang, Xiao-Jie; Peng, Cheng; Zhang, Jie; Cai, Yue; You, Xiao-Man; Kong, Fei; Yan, Hai-Gang; Wang, Guo-Xiang; Wang, Liang; Jin, Jie; Chen, Wei-Wei; Chen, Xin-Gang; Ma, Jing; Wang, Peng; Jiang, Ling; Zhang, Wen-Wei; Wan, Jian-Min

    2016-08-01

    ADP-glucose pyrophosphorylase (AGPase) controls a rate-limiting step in the starch biosynthetic pathway in higher plants. Here we isolated a shrunken rice mutant w24. Map-based cloning identified OsAGPL2, a large subunit of the cytosolic AGPase in rice endosperm, as the gene responsible for the w24 mutation. In addition to severe inhibition of starch synthesis and significant accumulation of sugar, the w24 endosperm showed obvious defects in compound granule formation and storage protein synthesis. The defect in OsAGPL2 enhanced the expression levels of the AGPase family. Meanwhile, the elevated activities of starch phosphorylase 1 and sucrose synthase in the w24 endosperm might possibly partly account for the residual starch content in the mutant seeds. Moreover, the expression of OsAGPL2 and its counterpart, OsAGPS2b, was highly coordinated in rice endosperm. Yeast two-hybrid and BiFC assays verified direct interactions between OsAGPL2 and OsAGPS2b as well as OsAGPL1 and OsAGPS1, supporting the model for spatiotemporal complex formation of AGPase isoforms in rice endosperm. Besides, our data provided no evidence for the self-binding of OsAGPS2b, implying that OsAGPS2b might not interact to form higher molecular mass aggregates in the absence of OsAGPL2. Therefore, the molecular mechanism of rice AGPase assembly might differ from that of Arabidopsis. PMID:27297991

  13. Assignment of the gene for the core protein II (UQCRC2) subunit of the mitochondrial cytochrome bc[sub 1] complex to human chromosome 16p12

    SciTech Connect

    Duncan, A.M.V. Kingston General Hospital ); Ozawa, Takayuki; Suzuki, Hiroshi ); Rozen, R. Montreal Children's Hospital )

    1993-11-01

    The mammalian cytochrome be[sub 1] complex (complex III) of the mitochondrial respiratory chain catalyzes electron transfer from ubiquinol to cytochrome c. The complex consists of 10-11 subunits: Core proteins I and II, cytochromes b and c[sub 1], the Rieske iron-sulfur protein, the ubiquinone-binding protein, the hinge protein, and 3-4 subunits of low molecular weight. Cytochrome b is encoded by the mitochondrial genome; the other subunits are encoded by nuclear genes. Both the human cytochrome c[sub 1] and the human ubiquinone-binding protein subunits have been assigned to chromosome 8 by somatic cell hybrid mapping. In this study, the authors used in situ hybridization to map core protein II. In situ hybridization to BrdU-synchronized peripheral blood lymphocytes was performed using the method of Harper and Saunders. Chromosomes were stained with a modified fluorescence, 0.25% Wright's stain procedure. The positions of silver grains directly over or touching well-banded metaphase chromosomes were mapped to an ISCN idiogram. The analysis of the distribution of 200 silver grams following in situ hybridization revealed a significant clustering of grains in the p12 region of chromosome 16. The assignment of the core II subunit to human chromosome 16p12 confirms that it is encoded by the nuclear, rather than the mitochondrial, genome. The identification of a single strong hybridization signal is indicative of one locus with no pseudogenes. 6 refs., 1 fig.

  14. Cryo-EM structure of a tetrameric cyanobacterial photosystem I complex reveals novel subunit interactions.

    PubMed

    Semchonok, Dmitry A; Li, Meng; Bruce, Barry D; Oostergetel, Gert T; Boekema, Egbert J

    2016-09-01

    Photosystem I (PSI) of the thermophilic cyanobacterium Chroococcidiopsis sp. TS-821 (TS-821) forms tetramers Li et al. (2014). Two-dimensional maps obtained by single particle electron microscopy (EM) clearly show that the tetramer lacks four-fold symmetry and is actually composed of a dimer of dimers with C2 symmetry. The resolution of these negative stain 2D maps did not permit the placement of most of the small PSI subunits, except for PsaL. Therefore cryo-EM was used for 3D reconstruction of the PSI tetramer complex. A 3D model at ~11.5Å resolution was obtained and a 2D map within the membrane plane of ~6.1Å. This data was used to build a model that was compared with the high-resolution structure of the PSI of Thermosynechococcus elongatus (T. elongatus) at 2.5Å. This comparison reveals key differences in which subunits are involved in the two different interfaces, interface type 1 within a dimer and interface type 2 between dimers. The type 1 interface in TS-821 is similar to the monomer interface in the trimeric PSI from T. elongatus, with interactions between subunits PsaA, -B, -I, -L and M. In type 2 the interaction is only between PsaA, -B and -L. Unlike the trimeric PSI, the central cavity of the complex is not filled with the PsaL-derived helical bundle, but instead seems filled with lipids. The physiological or evolutionary advantage of the tetramer is unknown. However, the presence of both dimers and tetramers in the thylakoid membrane suggest a dynamic equilibrium that shifts towards the tetramers in high light. PMID:27392600

  15. RICH MAPS

    EPA Science Inventory

    Michael Goodchild recently gave eight reasons why traditional maps are limited as communication devices, and how interactive internet mapping can overcome these limitations. In the past, many authorities in cartography, from Jenks to Bertin, have emphasized the importance of sim...

  16. Kentucky map

    NASA Astrophysics Data System (ADS)

    A wall-sized geological map of Kentucky, the product of 18 years of work, has just been released. Produced by the U.S. Geological Survey (USGS) in cooperation with the Kentucky Geological Survey (KGS) at the University of Kentucky, the map is unique, according to state geologist Donald Haney, because it is the first and only state map ever produced in detailed form from geologic quadrangle maps already available from the KGS.At a scale of 1:250,000, the map shows the surface distribution of various types of rock throughout the state, as well as geologic structure, faults, and surface coal beds. Numerous geologic sections, stratigraphic diagrams, correlation charts, and structure sections accompany the map. Compiled by R. C. McDowell and S. L. Moore of the USGS and by G. J . Grabowski of the KGS, the map was made by photoreducing and generalizing the detailed geologic quadrangle maps.

  17. Map adventures

    USGS Publications Warehouse

    1994-01-01

    Map Adventures, with seven accompanying lessons, is appropriate for grades K-3. Students will learn basic concepts for visualizing objects from different perspectives and how to understand /and use maps.

  18. Compilation of small ribosomal subunit RNA structures.

    PubMed Central

    Neefs, J M; Van de Peer, Y; De Rijk, P; Chapelle, S; De Wachter, R

    1993-01-01

    The database on small ribosomal subunit RNA structure contained 1804 nucleotide sequences on April 23, 1993. This number comprises 365 eukaryotic, 65 archaeal, 1260 bacterial, 30 plastidial, and 84 mitochondrial sequences. These are stored in the form of an alignment in order to facilitate the use of the database as input for comparative studies on higher-order structure and for reconstruction of phylogenetic trees. The elements of the postulated secondary structure for each molecule are indicated by special symbols. The database is available on-line directly from the authors by ftp and can also be obtained from the EMBL nucleotide sequence library by electronic mail, ftp, and on CD ROM disk. PMID:8332525

  19. Dynamic regulation of β1 subunit trafficking controls vascular contractility

    PubMed Central

    Leo, M. Dennis; Bannister, John P.; Narayanan, Damodaran; Nair, Anitha; Grubbs, Jordan E.; Gabrick, Kyle S.; Boop, Frederick A.; Jaggar, Jonathan H.

    2014-01-01

    Ion channels composed of pore-forming and auxiliary subunits control physiological functions in virtually all cell types. A conventional view is that channels assemble with their auxiliary subunits before anterograde plasma membrane trafficking of the protein complex. Whether the multisubunit composition of surface channels is fixed following protein synthesis or flexible and open to acute and, potentially, rapid modulation to control activity and cellular excitability is unclear. Arterial smooth muscle cells (myocytes) express large-conductance Ca2+-activated potassium (BK) channel α and auxiliary β1 subunits that are functionally significant modulators of arterial contractility. Here, we show that native BKα subunits are primarily (∼95%) plasma membrane-localized in human and rat arterial myocytes. In contrast, only a small fraction (∼10%) of total β1 subunits are located at the cell surface. Immunofluorescence resonance energy transfer microscopy demonstrated that intracellular β1 subunits are stored within Rab11A-postive recycling endosomes. Nitric oxide (NO), acting via cGMP-dependent protein kinase, and cAMP-dependent pathways stimulated rapid (≤1 min) anterograde trafficking of β1 subunit-containing recycling endosomes, which increased surface β1 almost threefold. These β1 subunits associated with surface-resident BKα proteins, elevating channel Ca2+ sensitivity and activity. Our data also show that rapid β1 subunit anterograde trafficking is the primary mechanism by which NO activates myocyte BK channels and induces vasodilation. In summary, we show that rapid β1 subunit surface trafficking controls functional BK channel activity in arterial myocytes and vascular contractility. Conceivably, regulated auxiliary subunit trafficking may control ion channel activity in a wide variety of cell types. PMID:24464482

  20. Regulation of the mammalian elongation cycle by subunit rolling: a eukaryotic-specific ribosome rearrangement

    PubMed Central

    Budkevich, Tatyana V.; Giesebrecht, Jan; Behrmann, Elmar; Loerke, Justus; Ramrath, David J.F.; Mielke, Thorsten; Ismer, Jochen; Hildebrand, Peter W.; Tung, Chang-Shung; Nierhaus, Knud H.; Sanbonmatsu, Karissa Y.; Spahn, Christian M.T.

    2014-01-01

    SUMMARY The extent to which bacterial ribosomes and the significantly larger eukaryotic ribosomes share the same mechanisms of ribosomal elongation is unknown. Here, we present sub-nanometer resolution cryo-electron microscopy maps of the mammalian 80S ribosome in the post-translocational state and in complex with the eukaryotic eEF1A•Val-tRNA•GMPPNP ternary complex, revealing significant differences in the elongation mechanism between bacteria and mammals. Surprisingly, and in contrast to bacterial ribosomes, a rotation of the small subunit around its long axis and orthogonal to the well-known intersubunit rotation distinguishes the post-translocational state from the classical pre-translocational state ribosome. We term this motion “subunit rolling”. Correspondingly, a mammalian decoding complex visualized in sub-states before and after codon recognition reveals structural distinctions from the bacterial system. These findings suggest how codon recognition leads to GTPase activation in the mammalian system and demonstrate that in mammalia subunit rolling occurs during tRNA selection. PMID:24995983

  1. Structural aspects of RbfA action during small ribosomal subunit assembly

    PubMed Central

    Datta, Partha P.; Wilson, Daniel N.; Kawazoe, Masahito; Swami, Neil K.; Kaminishi, Tatsuya; Sharma, Manjuli R.; Booth, Timothy M.; Takemoto, Chie; Fucini, Paola; Yokoyama, Shigeyuki; Agrawal, Rajendra K.

    2007-01-01

    Summary Ribosome binding factor A (RbfA) is a bacterial cold-shock response protein, required for an efficient processing of the 5′end of the 16S ribosomal RNA (rRNA) during assembly of the small (30S) ribosomal subunit. Here we present a crystal structure of Thermus thermophilus RbfA and a three-dimensional cryo-electron microscopic (EM) map of the T. thermophilus 30S·RbfA complex. RbfA binds to the 30S subunit in a position overlapping the binding sites of the A- and P-site tRNAs, and RbfA’s functionally important C-terminus extends toward the 5′ end of the 16S rRNA. In the presence of RbfA, a portion of the 16S rRNA encompassing helix 44, which is known to be directly involved in mRNA decoding and tRNA binding, is displaced. These results shed light on the role played by RbfA during maturation of the 30S subunit, and also indicate how RbfA provides cells with a translational advantage under conditions of cold shock. PMID:17996707

  2. Mutations at the Subunit Interface of Yeast Proliferating Cell Nuclear Antigen Reveal a Versatile Regulatory Domain

    PubMed Central

    Halmai, Miklos; Frittmann, Orsolya; Szabo, Zoltan; Daraba, Andreea; Gali, Vamsi K.; Balint, Eva; Unk, Ildiko

    2016-01-01

    Proliferating cell nuclear antigen (PCNA) plays a key role in many cellular processes and due to that it interacts with a plethora of proteins. The main interacting surfaces of Saccharomyces cerevisiae PCNA have been mapped to the interdomain connecting loop and to the carboxy-terminal domain. Here we report that the subunit interface of yeast PCNA also has regulatory roles in the function of several DNA damage response pathways. Using site-directed mutagenesis we engineered mutations at both sides of the interface and investigated the effect of these alleles on DNA damage response. Genetic experiments with strains bearing the mutant alleles revealed that mutagenic translesion synthesis, nucleotide excision repair, and homologous recombination are all regulated through residues at the subunit interface. Moreover, genetic characterization of one of our mutants identifies a new sub-branch of nucleotide excision repair. Based on these results we conclude that residues at the subunit boundary of PCNA are not only important for the formation of the trimer structure of PCNA, but they constitute a regulatory protein domain that mediates different DNA damage response pathways, as well. PMID:27537501

  3. Liposome-Based Adjuvants for Subunit Vaccines: Formulation Strategies for Subunit Antigens and Immunostimulators

    PubMed Central

    Tandrup Schmidt, Signe; Foged, Camilla; Smith Korsholm, Karen; Rades, Thomas; Christensen, Dennis

    2016-01-01

    The development of subunit vaccines has become very attractive in recent years due to their superior safety profiles as compared to traditional vaccines based on live attenuated or whole inactivated pathogens, and there is an unmet medical need for improved vaccines and vaccines against pathogens for which no effective vaccines exist. The subunit vaccine technology exploits pathogen subunits as antigens, e.g., recombinant proteins or synthetic peptides, allowing for highly specific immune responses against the pathogens. However, such antigens are usually not sufficiently immunogenic to induce protective immunity, and they are often combined with adjuvants to ensure robust immune responses. Adjuvants are capable of enhancing and/or modulating immune responses by exposing antigens to antigen-presenting cells (APCs) concomitantly with conferring immune activation signals. Few adjuvant systems have been licensed for use in human vaccines, and they mainly stimulate humoral immunity. Thus, there is an unmet demand for the development of safe and efficient adjuvant systems that can also stimulate cell-mediated immunity (CMI). Adjuvants constitute a heterogeneous group of compounds, which can broadly be classified into delivery systems or immunostimulators. Liposomes are versatile delivery systems for antigens, and they can carefully be customized towards desired immune profiles by combining them with immunostimulators and optimizing their composition, physicochemical properties and antigen-loading mode. Immunostimulators represent highly diverse classes of molecules, e.g., lipids, nucleic acids, proteins and peptides, and they are ligands for pattern-recognition receptors (PRRs), which are differentially expressed on APC subsets. Different formulation strategies might thus be required for incorporation of immunostimulators and antigens, respectively, into liposomes, and the choice of immunostimulator should ideally be based on knowledge regarding the specific PRR

  4. Liposome-Based Adjuvants for Subunit Vaccines: Formulation Strategies for Subunit Antigens and Immunostimulators.

    PubMed

    Tandrup Schmidt, Signe; Foged, Camilla; Korsholm, Karen Smith; Rades, Thomas; Christensen, Dennis

    2016-01-01

    The development of subunit vaccines has become very attractive in recent years due to their superior safety profiles as compared to traditional vaccines based on live attenuated or whole inactivated pathogens, and there is an unmet medical need for improved vaccines and vaccines against pathogens for which no effective vaccines exist. The subunit vaccine technology exploits pathogen subunits as antigens, e.g., recombinant proteins or synthetic peptides, allowing for highly specific immune responses against the pathogens. However, such antigens are usually not sufficiently immunogenic to induce protective immunity, and they are often combined with adjuvants to ensure robust immune responses. Adjuvants are capable of enhancing and/or modulating immune responses by exposing antigens to antigen-presenting cells (APCs) concomitantly with conferring immune activation signals. Few adjuvant systems have been licensed for use in human vaccines, and they mainly stimulate humoral immunity. Thus, there is an unmet demand for the development of safe and efficient adjuvant systems that can also stimulate cell-mediated immunity (CMI). Adjuvants constitute a heterogeneous group of compounds, which can broadly be classified into delivery systems or immunostimulators. Liposomes are versatile delivery systems for antigens, and they can carefully be customized towards desired immune profiles by combining them with immunostimulators and optimizing their composition, physicochemical properties and antigen-loading mode. Immunostimulators represent highly diverse classes of molecules, e.g., lipids, nucleic acids, proteins and peptides, and they are ligands for pattern-recognition receptors (PRRs), which are differentially expressed on APC subsets. Different formulation strategies might thus be required for incorporation of immunostimulators and antigens, respectively, into liposomes, and the choice of immunostimulator should ideally be based on knowledge regarding the specific PRR

  5. The light subunit of system bo,+ is fully functional in the absence of the heavy subunit

    PubMed Central

    Reig, Núria; Chillarón, Josep; Bartoccioni, Paola; Fernández, Esperanza; Bendahan, Annie; Zorzano, Antonio; Kanner, Baruch; Palacín, Manuel; Bertran, Joan

    2002-01-01

    The heteromeric amino acid transporters are composed of a type II glycoprotein and a non-glycosylated polytopic membrane protein. System bo,+ exchanges dibasic for neutral amino acids. It is composed of rBAT and bo,+AT, the latter being the polytopic membrane subunit. Mutations in either of them cause malfunction of the system, leading to cystinuria. bo,+AT-reconstituted systems from HeLa or MDCK cells catalysed transport of arginine that was totally dependent on the presence of one of the bo,+ substrates inside the liposomes. rBAT was essential for the cell surface expression of bo,+AT, but it was not required for reconstituted bo,+AT transport activity. No system bo,+ transport was detected in liposomes derived from cells expressing rBAT alone. The reconstituted bo,+AT showed kinetic asymmetry. Expressing the cystinuria-specific mutant A354T of bo,+AT in HeLa cells together with rBAT resulted in defective arginine uptake in whole cells, which was paralleled by the reconstituted bo,+AT activity. Thus, subunit bo,+AT by itself is sufficient to catalyse transmembrane amino acid exchange. The polytopic subunits may also be the catalytic part in other heteromeric transporters. PMID:12234930

  6. Epitopes from two soybean glycinin subunits antigenic in pigs

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Background: Glycinin is a seed storage protein in soybean (Glycine max) that is allergenic in pigs. Glycinin is a hexamer composed of subunits consisting of a basic and acidic portion joined by disulfide bridges. There are 5 glycinin subunits designated Gy1-Gy5. Results: Twenty seven out of 30 pi...

  7. The Development and Institutionalization of Subunit Power in Organizations.

    ERIC Educational Resources Information Center

    Boeker, Warren

    1989-01-01

    Examines the effects of founding events on the evolution of subunit importance in the semiconductor industry from 1958 to 1985. Distributions of power and subunit importance represent not only influences of current conditions, but also vestiges of earlier events, including the institution's founding. Includes 55 references. (MLH)

  8. Proteopedia Entry: The Large Ribosomal Subunit of "Haloarcula Marismortui"

    ERIC Educational Resources Information Center

    Decatur, Wayne A.

    2010-01-01

    This article presents a "Proteopedia" page that shows the refined version of the structure of the "Haloarcula" large ribosomal subunit as solved by the laboratories of Thomas Steitz and Peter Moore. The landmark structure is of great impact as it is the first atomic-resolution structure of the highly conserved ribosomal subunit which harbors…

  9. Amino Acid Substitutions in the Caenorhabditis elegans RNA Polymerase II Large Subunit AMA-1/RPB-1 that Result in α-Amanitin Resistance and/or Reduced Function.

    PubMed

    Bowman, Elizabeth Anne; Riddle, Donald L; Kelly, William

    2011-11-01

    Mutations in the Caenorhabditis elegans RNA polymerase II AMA-1/RPB-1 subunit that cause α-amanitin resistance and/or developmental defects were isolated previously. We identified 12 of these mutations and mapped them onto the Saccharomyces cerevisiae RPB1 structure to provide insight into AMA-1 regions that are essential for development in a multicellular organism. PMID:22384351

  10. Geranyl diphosphate synthase large subunit, and methods of use

    DOEpatents

    Croteau, Rodney B.; Burke, Charles C.; Wildung, Mark R.

    2001-10-16

    A cDNA encoding geranyl diphosphate synthase large subunit from peppermint has been isolated and sequenced, and the corresponding amino acid sequence has been determined. Replicable recombinant cloning vehicles are provided which code for geranyl diphosphate synthase large subunit). In another aspect, modified host cells are provided that have been transformed, transfected, infected and/or injected with a recombinant cloning vehicle and/or DNA sequence encoding geranyl diphosphate synthase large subunit. In yet another aspect, the present invention provides isolated, recombinant geranyl diphosphate synthase protein comprising an isolated, recombinant geranyl diphosphate synthase large subunit protein and an isolated, recombinant geranyl diphosphate synthase small subunit protein. Thus, systems and methods are provided for the recombinant expression of geranyl diphosphate synthase.

  11. Modulation of Kv4.3 current by accessory subunits.

    PubMed

    Deschênes, Isabelle; Tomaselli, Gordon F

    2002-09-25

    Kv4.3 encodes the pore-forming subunit of the cardiac transient outward potassium current (I(to)). hKv4.3-encoded current does not fully replicate cardiac I(to), suggesting a functionally significant role for accessory subunits. KChIP2 associates with Kv4.3 and modifies hKv4.3-encoded currents but does not replicate native I(to). We examined the effect of several ancillary subunits expressed in the heart on hKv4.3-encoded currents. Remarkably, the ancillary subunits Kvbeta(3), minK, MiRP-1, the Na channel beta(1) and KChIP2 increased the density and modified the gating of hKv4.3 current. hKv4.3 promiscuously assembles with ancillary subunits in vitro, functionally modifying the encoded currents; however, the physiological significance is uncertain. PMID:12297301

  12. Assembly Architecture and DNA Binding of the Bacteriophage P22 Terminase Small Subunit

    PubMed Central

    Němeček, Daniel; Lander, Gabriel C.; Johnson, John E.; Casjens, Sherwood R.; Thomas, George J.

    2008-01-01

    Summary Morphogenesis of bacteriophage P22 involves the packaging of double-stranded DNA into a preassembled procapsid. DNA is translocated by a powerful virally-encoded molecular motor called terminase, which comprises large (gp2, 499 residues) and small (gp3, 162 residues) subunits. While gp2 contains the phosphohydrolase and endonuclease activities of terminase, the function of gp3 may be to regulate specific and nonspecific modes of DNA recognition as well as the enzymatic activities of gp2. Electron microscopy shows that wildtype gp3 self-assembles into a stable and monodisperse nonameric ring. A three-dimensional reconstruction at 18 Å resolution provides the first glimpse of P22 terminase architecture and implies two distinct modes of interaction with DNA – involving a central channel of 20 Å diameter and radial spikes separated by 34 Å. Electromobility shift assays indicate that the gp3 ring binds dsDNA nonspecifically in vitro via electrostatic interactions between the positively charged C-terminus of gp3 (residues 143–152) and phosphates of the DNA backbone. Raman spectra show that nonameric rings formed by subunits truncated at residue 142 retain the subunit fold, despite the loss of DNA-binding activity. Difference density maps between gp3 rings containing full-length and C-terminally truncated subunits are consistent with localization of residues 143–152 along the central channel of the nonameric ring. The results suggest a plausible molecular mechanism for gp3 function in DNA recognition and translocation. PMID:18775728

  13. Nucleotide sequence of the gene for the b subunit of human factor XIII

    SciTech Connect

    Bottenus, R.E.; Ichinose, A.; Davie, E.W. )

    1990-12-01

    Factor XIII (M{sub r} 320 000) is a blood coagulation factor that stabilizes and strengthens the fibrin clot. It circulates in blood as a tetramer composed of two a subunits (M{sub r} 75 000 each) and two b subunits (M{sub r} 80 000 each). The b subunit consists of 641 amino acids and includes 10 tandem repeats of 60 amino acids known as GP-I structures, short consensus repeats (SCR), or sushi domains. In the present study, the human gene for the b subunit has been isolated from three different genomic libraries prepared in {lambda} phage. Fifteen independent phage with inserts coding for the entire gene were isolated and characterized by restriction mapping, Southern blotting, and DNA sequencing. The gene was found to be 28 kilobases in length and consisted of 12 exons (I-XII) separated by 11 intervening sequences. The leader sequence was encoded by exon I, while the carbonyl-terminal region of the protein was encoded by exon XII. Exons II-XI each coded for a single sushi domain, suggesting that the gene evolved through exon shuffling and duplication. The 12 exons in the gene ranged in size from 64 to 222 base pairs, while the introns ranged in size from 87 to 9970 nucleotides and made up 92{percent} of the gene. One nucleotide change was found in the coding region of the gene when its sequence was compared to that of the cDNA. This difference, however, did not result in a change in the amino acid sequence of the protein.

  14. NMR solution structure of the theta subunit of DNA polymerase III from Escherichia coli.

    PubMed Central

    Keniry, M. A.; Berthon, H. A.; Yang, J. Y.; Miles, C. S.; Dixon, N. E.

    2000-01-01

    The catalytic core of Escherichia coli DNA polymerase III contains three tightly associated subunits (alpha, epsilon, and theta). The theta subunit is the smallest, but the least understood of the three. As a first step in a program aimed at understanding its function, the structure of the theta subunit has been determined by triple-resonance multidimensional NMR spectroscopy. Although only a small protein, theta was difficult to assign fully because approximately one-third of the protein is unstructured, and some sections of the remaining structured parts undergo intermediate intramolecular exchange. The secondary structure was deduced from the characteristic nuclear Overhauser effect patterns, the 3J(HN alpha) coupling constants and the consensus chemical shift index. The C-terminal third of the protein, which has many charged and hydrophilic amino acid residues, has no well-defined secondary structure and exists in a highly dynamic state. The N-terminal two-thirds has three helical segments (Gln10-Asp19, Glu38-Glu43, and His47-Glu54), one short extended segment (Pro34-Ala37), and a long loop (Ala20-Glu29), of which part may undergo intermediate conformational exchange. Solution of the three-dimensional structure by NMR techniques revealed that the helices fold in such a way that the surface of theta is bipolar, with one face of the protein containing most of the acidic residues and the other face containing most of the long chain basic residues. Preliminary chemical shift mapping experiments with a domain of the epsilon subunit have identified a loop region (Ala20-Glu29) in theta as the site of association with epsilon. PMID:10794414

  15. Geologic mapping of Europa

    USGS Publications Warehouse

    Greeley, R.; Figueredo, P.H.; Williams, D.A.; Chuang, F.C.; Klemaszewski, J.E.; Kadel, S.D.; Prockter, L.M.; Pappalardo, R.T.; Head, J. W., III; Collins, G.C.; Spaun, N.A.; Sullivan, R.J.; Moore, Johnnie N.; Senske, D.A.; Tufts, B.R.; Johnson, T.V.; Belton, M.J.S.; Tanaka, K.L.

    2000-01-01

    Galileo data enable the major geological units, structures, and surface features to be identified on Europa. These include five primary units (plains, chaos, band, ridge, and crater materials) and their subunits, along with various tectonic structures such as faults. Plains units are the most widespread. Ridged plains material spans a wide range of geological ages, including the oldest recognizable features on Europa, and appears to represent a style of tectonic resurfacing, rather than cryovolcanism. Smooth plains material typically embays other terrains and units, possibly as a type of fluid emplacement, and is among the youngest material units observed. At global scales, plains are typically mapped as undifferentiated plains material, although in some areas differences can be discerned in the near infrared which might be related to differences in ice grain size. Chaos material is composed of plains and other preexisting materials that have been severely disrupted by inferred internal activity; chaos is characterized by blocks of icy material set in a hummocky matrix. Band material is arrayed in linear, curvilinear, wedge-shaped, or cuspate zones with contrasting albedo and surface textures with respect to the surrounding terrain. Bilateral symmetry observed in some bands and the relationships with the surrounding units suggest that band material forms by the lithosphere fracturing, spreading apart, and infilling with material derived from the subsurface. Ridge material is mapped as a unit on local and some regional maps but shown with symbols at global scales. Ridge material includes single ridges, doublet ridges, and ridge complexes. Ridge materials are considered to represent tectonic processes, possibly accompanied by the extrusion or intrusion of subsurface materials, such as diapirs. The tectonic processes might be related to tidal flexing of the icy lithosphere on diurnal or longer timescales. Crater materials include various interior (smooth central

  16. Geologic mapping of Europa

    NASA Astrophysics Data System (ADS)

    Greeley, Ronald; Collins, Geoffrey C.; Spaun, Nicole A.; Sullivan, Robert J.; Moore, Jeffrey M.; Senske, David A.; Tufts, B. Randall; Johnson, Torrence V.; Belton, Michael J. S.; Tanaka, Kenneth L.

    2000-09-01

    Galileo data enable the major geological units, structures, and surface features to be identified on Europa. These include five primary units (plains, chaos, band, ridge, and crater materials) and their subunits, along with various tectonic structures such as faults. Plains units are the most widespread. Ridged plains material spans a wide range of geological ages, including the oldest recognizable features on Europa, and appears to represent a style of tectonic resurfacing, rather than cryovolcanism. Smooth plains material typically embays other terrains and units, possibly as a type of fluid emplacement, and is among the youngest material units observed. At global scales, plains are typically mapped as undifferentiated plains material, although in some areas differences can be discerned in the near infrared which might be related to differences in ice grain size. Chaos material is composed of plains and other preexisting materials that have been severely disrupted by inferred internal activity; chaos is characterized by blocks of icy material set in a hummocky matrix. Band material is arrayed in linear, curvilinear, wedge-shaped, or cuspate zones with contrasting albedo and surface textures with respect to the surrounding terrain. Bilateral symmetry observed in some bands and the relationships with the surrounding units suggest that band material forms by the lithosphere fracturing, spreading apart, and infilling with material derived from the subsurface. Ridge material is mapped as a unit on local and some regional maps but shown with symbols at global scales. Ridge material includes single ridges, doublet ridges, and ridge complexes. Ridge materials are considered to represent tectonic processes, possibly accompanied by the extrusion or intrusion of subsurface materials, such as diapirs. The tectonic processes might be related to tidal flexing of the icy lithosphere on diurnal or longer timescales. Crater materials include various interior (smooth central

  17. Localization of two potassium channel {beta} subunit genes, KCNA1B and KCNA2B

    SciTech Connect

    Schultz, D.; Smith, L.; Thayer, M.

    1996-02-01

    The gating properties and current amplitudes of mammalian voltage-activated Shaker potassium channels are modulated by at least two associated {beta} subunits (Kv{beta}1.1 and Kv{beta}1.2). The human Kv{beta}1.1 gene (KCNA1B) resides on chromosome 3, as indicated by somatic cell hybrid mapping. More precise localization of KCNA1B to 3q26.1 was obtained with fluorescence in situ hybridization (FISH) and was corroborated by PCR screening of the CEPH YAC library. The human Kv{beta}1.2 gene (KCNA2B) resides on chromosome 1, as indicated by somatic cell hybrid mapping, and has been localized by FISH to 1p36.3. 20 refs., 2 figs.

  18. Method for the detection of a polypeptide subunit in the presence of a quaternary protein containing the subunit

    SciTech Connect

    Wands, J.R.; Ozturk, M.; Bellet, D.

    1990-06-12

    This patent describes a method for the determination of a free protein subunit of hCG in a sample containing intact quaternary hCG. It comprises: contacting the sample with a first monoclonal antibody which is bound to a carrier, wherein the first monoclonal antibody binds epitopic determinants bindable only on the free protein subunit; incubating the components for a period of time and under conditions sufficient to form an immune complex between the free protein subunit, the first monoclonal antibody, and the carrier; separating the carrier from the sample; adding to the carrier a detectably labeled second monoclonal antibody, wherein the second monoclonal antibody binds epitopic determinants bindable on both the free protein subunit and the intact quaternary hCG; separating the carrier from the liquid phase; and determining the detectably labeled second monoclonal antibody in the carrier or in the liquid phase, which is a measure of the amount of the free protein subunit in the sample.

  19. Mapping Van

    NASA Technical Reports Server (NTRS)

    1994-01-01

    A NASA Center for the Commercial Development of Space (CCDS) - developed system for satellite mapping has been commercialized for the first time. Global Visions, Inc. maps an area while driving along a road in a sophisticated mapping van equipped with satellite signal receivers, video cameras and computer systems for collecting and storing mapping data. Data is fed into a computerized geographic information system (GIS). The resulting amps can be used for tax assessment purposes, emergency dispatch vehicles and fleet delivery companies as well as other applications.

  20. Stargazin is an AMPA receptor auxiliary subunit.

    PubMed

    Vandenberghe, Wim; Nicoll, Roger A; Bredt, David S

    2005-01-11

    AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid) receptors mediate fast excitatory synaptic transmission in brain and underlie aspects of synaptic plasticity. Numerous AMPA receptor-binding proteins have been implicated in AMPA receptor trafficking and anchoring. However, the relative contributions of these proteins to the composition of native AMPA receptor complexes in brain remain uncertain. Here, we use blue native gel electrophoresis to analyze the composition of native AMPA receptor complexes in cerebellar extracts. We identify two receptor populations: a functional form that contains the transmembrane AMPA receptor-regulatory protein stargazin and an apo-form that lacks stargazin. Limited proteolysis confirms assembly of stargazin with a large proportion of native AMPA receptors. In contrast, other AMPA receptor-interacting proteins, such as synapse-associated protein 97, glutamate receptor-interacting protein 1, protein kinase Calpha binding protein, N-ethylmaleimide-sensitive fusion protein, AP2, and protein 4.1N, do not show significant association with AMPA receptor complexes on native gels. These data identify stargazin as an auxiliary subunit for a neurotransmitter-gated ion channel. PMID:15630087

  1. Stepwise and dynamic assembly of the earliest precursors of small ribosomal subunits in yeast.

    PubMed

    Zhang, Liman; Wu, Chen; Cai, Gaihong; Chen, She; Ye, Keqiong

    2016-03-15

    The eukaryotic ribosomal RNA (rRNA) is associated cotranscriptionally with numerous factors into an enormous 90S preribosomal particle that conducts early processing of small ribosomal subunits. The assembly pathway and structure of the 90S particle is poorly understood. Here, we affinity-purified and analyzed the constituents of yeast 90S particles that were assembled on a series of plasmid-encoded 3'-truncated pre-18S RNAs. We determined the assembly point of 65 proteins and the U3, U14, and snR30 small nucleolar RNAs (snoRNAs), revealing a stepwise and dynamic assembly map. The 5' external transcribed spacer (ETS) alone can nucleate a large complex. When the 18S rRNA is nearly complete, the 90S structure undergoes a dramatic reorganization, releasing U14, snR30, and 14 protein factors that bind earlier. We also identified a reference state of 90S that is fully assembled yet has not undergone 5'ETS processing. The assembly map present here provides a new framework to understand small subunit biogenesis. PMID:26980190

  2. Autocatalytic processing of m-AAA protease subunits in mitochondria.

    PubMed

    Koppen, Mirko; Bonn, Florian; Ehses, Sarah; Langer, Thomas

    2009-10-01

    m-AAA proteases are ATP-dependent proteolytic machines in the inner membrane of mitochondria which are crucial for the maintenance of mitochondrial activities. Conserved nuclear-encoded subunits, termed paraplegin, Afg3l1, and Afg3l2, form various isoenzymes differing in their subunit composition in mammalian mitochondria. Mutations in different m-AAA protease subunits are associated with distinct neuronal disorders in human. However, the biogenesis of m-AAA protease complexes or of individual subunits is only poorly understood. Here, we have examined the processing of nuclear-encoded m-AAA protease subunits upon import into mitochondria and demonstrate autocatalytic processing of Afg3l1 and Afg3l2. The mitochondrial processing peptidase MPP generates an intermediate form of Afg3l2 that is matured autocatalytically. Afg3l1 or Afg3l2 are also required for maturation of newly imported paraplegin subunits after their cleavage by MPP. Our results establish that mammalian m-AAA proteases can act as processing enzymes in vivo and reveal overlapping activities of Afg3l1 and Afg3l2. These findings might be of relevance for the pathogenesis of neurodegenerative disorders associated with mutations in different m-AAA protease subunits. PMID:19656850

  3. RNA polymerase II subunit composition, stoichiometry, and phosphorylation.

    PubMed Central

    Kolodziej, P A; Woychik, N; Liao, S M; Young, R A

    1990-01-01

    RNA polymerase II subunit composition, stoichiometry, and phosphorylation were investigated in Saccharomyces cerevisiae by attaching an epitope coding sequence to a well-characterized RNA polymerase II subunit gene (RPB3) and by immunoprecipitating the product of this gene with its associated polypeptides. The immunopurified enzyme catalyzed alpha-amanitin-sensitive RNA synthesis in vitro. The 10 polypeptides that immunoprecipitated were identical in size and number to those previously described for RNA polymerase II purified by conventional column chromatography. The relative stoichiometry of the subunits was deduced from knowledge of the sequence of the subunits and from the extent of labeling with [35S]methionine. Immunoprecipitation from 32P-labeled cell extracts revealed that three of the subunits, RPB1, RPB2, and RPB6, are phosphorylated in vivo. Phosphorylated and unphosphorylated forms of RPB1 could be distinguished; approximately half of the RNA polymerase II molecules contained a phosphorylated RPB1 subunit. These results more precisely define the subunit composition and phosphorylation of a eucaryotic RNA polymerase II enzyme. Images PMID:2183013

  4. Prokaryotic and eukaryotic RNA polymerases have homologous core subunits.

    PubMed Central

    Sweetser, D; Nonet, M; Young, R A

    1987-01-01

    Eukaryotic RNA polymerases are complex aggregates whose component subunits are functionally ill-defined. The gene that encodes the 140,000-dalton subunit of Saccharomyces cerevisiae RNA polymerase II was isolated and studied in detail to obtain clues to the protein's function. This gene, RPB2, exists in a single copy in the haploid genome. Disruption of the gene is lethal to the yeast cell. RPB2 encodes a protein of 138,750 daltons, which contains sequences implicated in binding purine nucleotides and zinc ions and exhibits striking sequence homology with the beta subunit of Escherichia coli RNA polymerase. These observations suggest that the yeast and the E. coli subunit have similar roles in RNA synthesis, as the beta subunit contains binding sites for nucleotide substrates and a portion of the catalytic site for RNA synthesis. The subunit homologies reported here, and those observed previously with the largest RNA polymerase subunit, indicate that components of the prokaryotic RNA polymerase "core" enzyme have counterparts in eukaryotic RNA polymerases. PMID:3547406

  5. Mutations in GABAA receptor subunits associated with genetic epilepsies.

    PubMed

    Macdonald, Robert L; Kang, Jing-Qiong; Gallagher, Martin J

    2010-06-01

    Mutations in inhibitory GABAA receptor subunit genes (GABRA1, GABRB3, GABRG2 and GABRD) have been associated with genetic epilepsy syndromes including childhood absence epilepsy (CAE), juvenile myoclonic epilepsy (JME), pure febrile seizures (FS), generalized epilepsy with febrile seizures plus (GEFS+), and Dravet syndrome (DS)/severe myoclonic epilepsy in infancy (SMEI). These mutations are found in both translated and untranslated gene regions and have been shown to affect the GABAA receptors by altering receptor function and/or by impairing receptor biogenesis by multiple mechanisms including reducing subunit mRNA transcription or stability, impairing subunit folding, stability, or oligomerization and by inhibiting receptor trafficking. PMID:20308251

  6. Undersea Mapping.

    ERIC Educational Resources Information Center

    DiSpezio, Michael A.

    1991-01-01

    Presented is a cooperative learning activity in which students assume different roles in an effort to produce a relief map of the ocean floor. Materials, procedures, definitions, student roles, and questions are discussed. A reproducible map for the activity is provided. (CW)

  7. Question Mapping

    ERIC Educational Resources Information Center

    Martin, Josh

    2012-01-01

    After accepting the principal position at Farmersville (TX) Junior High, the author decided to increase instructional rigor through question mapping because of the success he saw using this instructional practice at his prior campus. Teachers are the number one influence on student achievement (Marzano, 2003), so question mapping provides a…

  8. Map Adventures.

    ERIC Educational Resources Information Center

    Geological Survey (Dept. of Interior), Reston, VA.

    This curriculum packet about maps, with seven accompanying lessons, is appropriate for students in grades K-3. Students learn basic concepts for visualizing objects from different perspectives and how to understand and use maps. Lessons in the packet center on a story about a little girl, Nikki, who rides in a hot-air balloon that gives her, and…

  9. Atomic-Resolution Structures of the APC/C Subunits Apc4 and the Apc5 N-Terminal Domain

    PubMed Central

    Cronin, Nora B.; Yang, Jing; Zhang, Ziguo; Kulkarni, Kiran; Chang, Leifu; Yamano, Hiroyuki; Barford, David

    2015-01-01

    Many essential biological processes are mediated by complex molecular machines comprising multiple subunits. Knowledge on the architecture of individual subunits and their positions within the overall multimeric complex is key to understanding the molecular mechanisms of macromolecular assemblies. The anaphase-promoting complex/cyclosome (APC/C) is a large multisubunit complex that regulates cell cycle progression by ubiquitinating cell cycle proteins for proteolysis by the proteasome. The holo-complex is composed of 15 different proteins that assemble to generate a complex of 20 subunits. Here, we describe the crystal structures of Apc4 and the N-terminal domain of Apc5 (Apc5N). Apc4 comprises a WD40 domain split by a long α-helical domain, whereas Apc5N has an α-helical fold. In a separate study, we had fitted these atomic models to a 3.6-Å-resolution cryo-electron microscopy map of the APC/C. We describe how, in the context of the APC/C, regions of Apc4 disordered in the crystal assume order through contacts to Apc5, whereas Apc5N shows small conformational changes relative to its crystal structure. We discuss the complementary approaches of high-resolution electron microscopy and protein crystallography to the structure determination of subunits of multimeric complexes. PMID:26343760

  10. The NMDAR subunit NR3A interacts with microtubule-associated protein 1S in the brain

    PubMed Central

    Eriksson, Maria; Samuelsson, Helena; Samuelsson, Eva-Britt; Liu, Leyuan; McKeehan, Wallace L; Benedikz, Eirikur; Sundström, Erik

    2011-01-01

    When screening a brain cDNA library, we found that the N-methyl-d-aspartate receptor subunit NR3A binds to microtubule-associated protein (MAP) 1S/chromosome 19 open reading frame 5 (C19ORF5). The interaction was confirmed in vitro and in vivo, and binding of MAP1S was localized to the membrane-proximal part of the NR3A C-terminus. MAP1S belongs to the same family as MAP1A and MAP1B, and was found to be abundant in both postnatal and adult rat brain. In hippocampal neurons the distribution-pattern of MAP1S resembled that of β-tubulin III, but a fraction of the protein colocalized with synaptic markers synapsin and postsynaptic density protein 95 (PSD95), in β-tubulin III-negative filopodia-like protrusions. There was coexistance between MAP1S and NR3A immunoreactivity in neurite shafts and occasionally in filopodia-like processes. MAP1S potentially links NR3A to the cytoskeleton, and may stabilize NR3A-containing receptors at the synapse and regulate their movement between synaptic and extrasynaptic sites. PMID:17658481

  11. Database on the structure of large ribosomal subunit RNA.

    PubMed Central

    De Rijk, P; Van de Peer, Y; Chapelle, S; De Wachter, R

    1994-01-01

    A database on large ribosomal subunit RNA is made available. It contains 258 sequences. It provides sequence, alignment and secondary structure information in computer-readable formats. Files can be obtained using ftp. PMID:7524023

  12. A process yields large quantities of pure ribosome subunits

    NASA Technical Reports Server (NTRS)

    Friedman, M.; Lu, P.; Rich, A.

    1972-01-01

    Development of process for in-vitro protein synthesis from living cells followed by dissociation of ribosomes into subunits is discussed. Process depends on dialysis or use of chelating agents. Operation of process and advantages over previous methods are outlined.

  13. Primary structure of the ovine pituitary follitropin beta-subunit.

    PubMed Central

    Sairam, M R; Seidah, N G; Chrétien, M

    1981-01-01

    The complete amino acids sequence of the ovine pituitary follitropin beta-subunit was established by studying the tryptic, chymotryptic and thermolytic peptides. The N-terminal sequence of the subunit was confirmed by subjecting the oxidated protein to Edman degradation in an automated sequenator. Automated Edman degradation of the reduced and alkylated (with iodo [14C]acetamide) beta-subunit indicated that most of the molecules used in the sequence studies had lost the N-terminal serine residue. This also confirmed the location of the first five half-cystine residues in the sequence. The proposed structure shows the presence of 111 amino acid residues with the two oligosaccharide moieties linked to asparagine residues located at positions 6 and 23. Heterogeneity occurs at both the termini of the polypeptide chain. Comparison of the sequence of beta-subunit of the ovine hormone with that proposed for human follitropin beta-subunit shows the absence of any deletions in the middle of the peptide chain. Of the 13 replacements, 11 residues can be explained on the basis of a single base change in the codon. The single tryptophan residue of the follitropin occupies an identical position in all the four species that have been studied. The region corresponding to residues 63-105 of the ovine beta-subunit is highly conserved in all the species. PMID:6798969

  14. Transcriptional regulators of Na,K-ATPase subunits

    PubMed Central

    Li, Zhiqin; Langhans, Sigrid A.

    2015-01-01

    The Na,K-ATPase classically serves as an ion pump creating an electrochemical gradient across the plasma membrane that is essential for transepithelial transport, nutrient uptake and membrane potential. In addition, Na,K-ATPase also functions as a receptor, a signal transducer and a cell adhesion molecule. With such diverse roles, it is understandable that the Na,K-ATPase subunits, the catalytic α-subunit, the β-subunit and the FXYD proteins, are controlled extensively during development and to accommodate physiological needs. The spatial and temporal expression of Na,K-ATPase is partially regulated at the transcriptional level. Numerous transcription factors, hormones, growth factors, lipids, and extracellular stimuli modulate the transcription of the Na,K-ATPase subunits. Moreover, epigenetic mechanisms also contribute to the regulation of Na,K-ATPase expression. With the ever growing knowledge about diseases associated with the malfunction of Na,K-ATPase, this review aims at summarizing the best-characterized transcription regulators that modulate Na,K-ATPase subunit levels. As abnormal expression of Na,K-ATPase subunits has been observed in many carcinoma, we will also discuss transcription factors that are associated with epithelial-mesenchymal transition, a crucial step in the progression of many tumors to malignant disease. PMID:26579519

  15. A new look at sodium channel β subunits.

    PubMed

    Namadurai, Sivakumar; Yereddi, Nikitha R; Cusdin, Fiona S; Huang, Christopher L H; Chirgadze, Dimitri Y; Jackson, Antony P

    2015-01-01

    Voltage-gated sodium (Nav) channels are intrinsic plasma membrane proteins that initiate the action potential in electrically excitable cells. They are a major focus of research in neurobiology, structural biology, membrane biology and pharmacology. Mutations in Nav channels are implicated in a wide variety of inherited pathologies, including cardiac conduction diseases, myotonic conditions, epilepsy and chronic pain syndromes. Drugs active against Nav channels are used as local anaesthetics, anti-arrhythmics, analgesics and anti-convulsants. The Nav channels are composed of a pore-forming α subunit and associated β subunits. The β subunits are members of the immunoglobulin (Ig) domain family of cell-adhesion molecules. They modulate multiple aspects of Nav channel behaviour and play critical roles in controlling neuronal excitability. The recently published atomic resolution structures of the human β3 and β4 subunit Ig domains open a new chapter in the study of these molecules. In particular, the discovery that β3 subunits form trimers suggests that Nav channel oligomerization may contribute to the functional properties of some β subunits. PMID:25567098

  16. A new look at sodium channel β subunits

    PubMed Central

    Namadurai, Sivakumar; Yereddi, Nikitha R.; Cusdin, Fiona S.; Huang, Christopher L.-H.; Chirgadze, Dimitri Y.; Jackson, Antony P.

    2015-01-01

    Voltage-gated sodium (Nav) channels are intrinsic plasma membrane proteins that initiate the action potential in electrically excitable cells. They are a major focus of research in neurobiology, structural biology, membrane biology and pharmacology. Mutations in Nav channels are implicated in a wide variety of inherited pathologies, including cardiac conduction diseases, myotonic conditions, epilepsy and chronic pain syndromes. Drugs active against Nav channels are used as local anaesthetics, anti-arrhythmics, analgesics and anti-convulsants. The Nav channels are composed of a pore-forming α subunit and associated β subunits. The β subunits are members of the immunoglobulin (Ig) domain family of cell-adhesion molecules. They modulate multiple aspects of Nav channel behaviour and play critical roles in controlling neuronal excitability. The recently published atomic resolution structures of the human β3 and β4 subunit Ig domains open a new chapter in the study of these molecules. In particular, the discovery that β3 subunits form trimers suggests that Nav channel oligomerization may contribute to the functional properties of some β subunits. PMID:25567098

  17. Identification of the amino acid region involved in the intercellular interaction between the β1 subunits of Na+/K+-ATPase

    PubMed Central

    Tokhtaeva, Elmira; Sachs, George; Sun, Haiying; Dada, Laura A.; Sznajder, Jacob I.; Vagin, Olga

    2012-01-01

    Epithelial junctions depend on intercellular interactions between β1 subunits of the Na+/K+-ATPase molecules of neighboring cells. The interaction between dog and rat subunits is less effective than the interaction between two dog β1 subunits, indicating the importance of species-specific regions for β1–β1 binding. To identify these regions, the species-specific amino acid residues were mapped on a high-resolution structure of the Na+/K+-ATPase β1 subunit to select those exposed towards the β1 subunit of the neighboring cell. These exposed residues were mutated in both dog and rat YFP-linked β1 subunits (YFP–β1) and also in the secreted extracellular domain of the dog β1 subunit. Five rat-like mutations in the amino acid region spanning residues 198–207 of the dog YFP–β1 expressed in Madin–Darby canine kidney (MDCK) cells decreased co-precipitation of the endogenous dog β1 subunit with YFP–β1 to the level observed between dog β1 and rat YFP–β1. In parallel, these mutations impaired the recognition of YFP–β1 by the dog-specific antibody that inhibits cell adhesion between MDCK cells. Accordingly, dog-like mutations in rat YFP–β1 increased both the (YFP–β1)–β1 interaction in MDCK cells and recognition by the antibody. Conversely, rat-like mutations in the secreted extracellular domain of the dog β1 subunit increased its interaction with rat YFP–β1 in vitro. In addition, these mutations resulted in a reduction of intercellular adhesion between rat lung epithelial cells following addition of the secreted extracellular domain of the dog β1 subunit to a cell suspension. Therefore, the amino acid region 198–207 is crucial for both trans-dimerization of the Na+/K+-ATPase β1 subunits and cell–cell adhesion. PMID:22328500

  18. Semantic Mapping.

    ERIC Educational Resources Information Center

    Johnson, Dale D.; And Others

    1986-01-01

    Describes semantic mapping, an effective strategy for vocabulary instruction that involves the categorical structuring of information in graphic form and requires students to relate new words to their own experience and prior knowledge. (HOD)

  19. Mapping Biodiversity.

    ERIC Educational Resources Information Center

    World Wildlife Fund, Washington, DC.

    This document features a lesson plan that examines how maps help scientists protect biodiversity and how plants and animals are adapted to specific ecoregions by comparing biome, ecoregion, and habitat. Samples of instruction and assessment are included. (KHR)

  20. Map Separates

    USGS Publications Warehouse

    U.S. Geological Survey

    2001-01-01

    U.S. Geological Survey (USGS) topographic maps are printed using up to six colors (black, blue, green, red, brown, and purple). To prepare your own maps or artwork based on maps, you can order separate black-and-white film positives or negatives for any color printed on a USGS topographic map, or for one or more of the groups of related features printed in the same color on the map (such as drainage and drainage names from the blue plate.) In this document, examples are shown with appropriate ink color to illustrate the various separates. When purchased, separates are black-and-white film negatives or positives. After you receive a film separate or composite from the USGS, you can crop, enlarge or reduce, and edit to add or remove details to suit your special needs. For example, you can adapt the separates for making regional and local planning maps or for doing many kinds of studies or promotions by using the features you select and then printing them in colors of your choice.

  1. Venus mapping

    NASA Technical Reports Server (NTRS)

    Batson, R. M.; Morgan, H. F.; Sucharski, Robert

    1991-01-01

    Semicontrolled image mosaics of Venus, based on Magellan data, are being compiled at 1:50,000,000, 1:10,000,000, 1:5,000,000, and 1:1,000,000 scales to support the Magellan Radar Investigator (RADIG) team. The mosaics are semicontrolled in the sense that data gaps were not filled and significant cosmetic inconsistencies exist. Contours are based on preliminary radar altimetry data that is subjected to revision and improvement. Final maps to support geologic mapping and other scientific investigations, to be compiled as the dataset becomes complete, will be sponsored by the Planetary Geology and Geophysics Program and/or the Venus Data Analysis Program. All maps, both semicontrolled and final, will be published as I-maps by the United States Geological Survey. All of the mapping is based on existing knowledge of the spacecraft orbit; photogrammetric triangulation, a traditional basis for geodetic control on planets where framing cameras were used, is not feasible with the radar images of Venus, although an eventual shift of coordinate system to a revised spin-axis location is anticipated. This is expected to be small enough that it will affect only large-scale maps.

  2. Conformation of 4.5S RNA in the signal recognition particle and on the 30S ribosomal subunit

    PubMed Central

    GU, SHAN-QING; JÖCKEL, JOHANNES; BEINKER, PHILIPP; WARNECKE, JENS; SEMENKOV, YURI P.; RODNINA, MARINA V.; WINTERMEYER, WOLFGANG

    2005-01-01

    The signal recognition particle (SRP) from Escherichia coli consists of 4.5S RNA and protein Ffh. It is essential for targeting ribosomes that are translating integral membrane proteins to the translocation pore in the plasma membrane. Independently of Ffh, 4.5S RNA also interacts with elongation factor G (EF-G) and the 30S ribosomal subunit. Here we use a cross-linking approach to probe the conformation of 4.5S RNA in SRP and in the complex with the 30S ribosomal subunit and to map the binding site. The UV-activatable cross-linker p-azidophenacyl bromide (AzP) was attached to positions 1, 21, and 54 of wild-type or modified 4.5S RNA. In SRP, cross-links to Ffh were formed from AzP in all three positions in 4.5S RNA, indicating a strongly bent conformation in which the 5′ end (position 1) and the tetraloop region (including position 54) of the molecule are close to one another and to Ffh. In ribosomal complexes of 4.5S RNA, AzP in both positions 1 and 54 formed cross-links to the 30S ribosomal subunit, independently of the presence of Ffh. The major cross-linking target on the ribosome was protein S7; minor cross-links were formed to S2, S18, and S21. There were no cross-links from 4.5S RNA to the 50S subunit, where the primary binding site of SRP is located close to the peptide exit. The functional role of 4.5S RNA binding to the 30S subunit is unclear, as the RNA had no effect on translation or tRNA translocation on the ribosome. PMID:16043501

  3. Single-particle cryoEM analysis at near-atomic resolution from several thousand asymmetric subunits.

    PubMed

    Passos, Dario Oliveira; Lyumkis, Dmitry

    2015-11-01

    A single-particle cryoEM reconstruction of the large ribosomal subunit from Saccharomyces cerevisiae was obtained from a dataset of ∼75,000 particles. The gold-standard and frequency-limited approaches to single-particle refinement were each independently used to determine orientation parameters for the final reconstruction. Both approaches showed similar resolution curves and nominal resolution values for the 60S dataset, estimated at 2.9 Å. The amount of over-fitting present during frequency-limited refinement was quantitatively analyzed using the high-resolution phase-randomization test, and the results showed no apparent over-fitting. The number of asymmetric subunits required to reach specific resolutions was subsequently analyzed by refining subsets of the data in an ab initio manner. With our data collection and processing strategies, sub-nanometer resolution was obtained with ∼200 asymmetric subunits (or, equivalently for the ribosomal subunit, particles). Resolutions of 5.6 Å, 4.5 Å, and 3.8 Å were reached with ∼1000, ∼1600, and ∼5000 asymmetric subunits, respectively. At these resolutions, one would expect to detect alpha-helical pitch, separation of beta-strands, and separation of Cα atoms, respectively. Using this map, together with strategies for ab initio model building and model refinement, we built a region of the ribosomal protein eL6, which was missing in previous models of the yeast ribosome. The relevance for more routine high-resolution structure determination is discussed. PMID:26470814

  4. Proteomic analysis of the mammalian mitochondrial ribosome. Identification of protein components in the 28 S small subunit.

    PubMed

    Suzuki, T; Terasaki, M; Takemoto-Hori, C; Hanada, T; Ueda, T; Wada, A; Watanabe, K

    2001-08-31

    The mammalian mitochondrial ribosome (mitoribosome) has a highly protein-rich composition with a small sedimentation coefficient of 55 S, consisting of 39 S large and 28 S small subunits. In the previous study, we analyzed 39 S large subunit proteins from bovine mitoribosome (Suzuki, T., Terasaki, M., Takemoto-Hori, C., Hanada, T., Ueda, T., Wada, A., and Watanabe, K. (2001) J. Biol. Chem. 276, 21724-21736). The results suggested structural compensation for the rRNA deficit through proteins of increased molecular mass in the mitoribosome. We report here the identification of 28 S small subunit proteins. Each protein was separated by radical-free high-reducing two-dimensional polyacrylamide gel electrophoresis and analyzed by liquid chromatography/mass spectrometry/mass spectrometry using electrospray ionization/ion trap mass spectrometer to identify cDNA sequence by expressed sequence tag data base searches in silico. Twenty one proteins from the small subunit were identified, including 11 new proteins along with their complete cDNA sequences from human and mouse. In addition to these proteins, three new proteins were also identified in the 55 S mitoribosome. We have clearly identified a mitochondrial homologue of S12, which is a key regulatory protein of translation fidelity and a candidate for the autosomal dominant deafness gene, DFNA4. The apoptosis-related protein DAP3 was found to be a component of the small subunit, indicating a new function for the mitoribosome in programmed cell death. In summary, we have mapped a total of 55 proteins from the 55 S mitoribosome on the two-dimensional polyacrylamide gels. PMID:11402041

  5. A bioinformatic and computational study of myosin phosphatase subunit diversity

    PubMed Central

    Dippold, Rachael P.

    2014-01-01

    Variability in myosin phosphatase (MP) subunits may provide specificity in signaling pathways that regulate muscle tone. We utilized public databases and computational algorithms to investigate the phylogenetic diversity of MP regulatory (PPP1R12A-C) and inhibitory (PPP1R14A-D) subunits. The comparison of exonic coding sequences and expression data confirmed or refuted the existence of isoforms and their tissue-specific expression in different model organisms. The comparison of intronic and exonic sequences identified potential expressional regulatory elements. As examples, smooth muscle MP regulatory subunit (PPP1R12A) is highly conserved through evolution. Its alternative exon E24 is present in fish through mammals with two invariant features: 1) a reading frame shift generating a premature termination codon and 2) a hexanucleotide sequence adjacent to the 3′ splice site hypothesized to be a novel suppressor of exon splicing. A characteristic of the striated muscle MP regulatory subunit (PPP1R12B) locus is numerous and phylogenetically variable transcriptional start sites. In fish this locus only codes for the small (M21) subunit, suggesting the primordial function of this gene. Inhibitory subunits show little intragenic variability; their diversity is thought to have arisen by expansion and tissue-specific expression of different gene family members. We demonstrate differences in the regulatory landscape between smooth muscle enriched (PPP1R14A) and more ubiquitously expressed (PPP1R14B) family members and identify deeply conserved intronic sequence and predicted transcriptional cis-regulatory elements. This bioinformatic and computational study has uncovered a number of attributes of MP subunits that supports selection of ideal model organisms and testing of hypotheses regarding their physiological significance and regulated expression. PMID:24898838

  6. Functional diversity of complex I subunits in Candida albicans mitochondria.

    PubMed

    Li, Dongmei; She, Xiaodong; Calderone, Richard

    2016-02-01

    Our interest in the mitochondria of Candida albicans has progressed to the identification of several proteins that are critical to complex I (CI) activity. We speculated that there should be major functional differences at the protein level between mammalian and fungal mitochondria CI. In our pursuit of this idea, we were helped by published data of CI subunit proteins from a broad diversity of species that included two subunit proteins that are not found in mammals. These subunit proteins have been designated as Nuo1p and Nuo2p (NADH-ubiquinone oxidoreductases). Since functional assignments of both C. albicans proteins were unknown, other than having a putative NADH-oxidoreductase activity, we constructed knock-out strains that could be compared to parental cells. The relevance of our research relates to the critical roles of both proteins in cell biology and pathogenesis and their absence in mammals. These features suggest they may be exploited in antifungal drug discovery. Initially, we characterized Goa1p that apparently regulates CI activity but is not a CI subunit protein. We have used the goa1∆ for comparisons to Nuo1p and Nuo2p. We have demonstrated the critical role of these proteins in maintaining CI activities, virulence, and prolonging life span. More recently, transcriptional profiling of the three mutants and an ndh51∆ (protein is a highly conserved CI subunit) has revealed that there are overlapping yet also different functional assignments that suggest subunit specificity. The differences and similarities of each are described below along with our hypotheses to explain these data. Our conclusion and perspective is that the C. albicans CI subunit proteins are highly conserved except for two that define non-mammalian functions. PMID:26373419

  7. Cytochrome c oxidase: Evolution of control via nuclear subunit addition☆

    PubMed Central

    Pierron, Denis; Wildman, Derek E.; Hüttemann, Maik; Markondapatnaikuni, Gopi Chand; Aras, Siddhesh; Grossman, Lawrence I.

    2014-01-01

    According to theory, present eukaryotic cells originated from a beneficial association between two free-living cells. Due to this endosymbiotic event the pre-eukaryotic cell gained access to oxidative phosphorylation (OXPHOS), which produces more than 15 times as much ATP as glycolysis. Because cellular ATP needs fluctuate and OXPHOS both requires and produces entities that can be toxic for eukaryotic cells such as ROS or NADH, we propose that the success of endosymbiosis has largely depended on the regulation of endosymbiont OXPHOS. Several studies have presented cytochrome c oxidase as a key regulator of OXPHOS; for example, COX is the only complex of mammalian OXPHOS with known tissue-specific isoforms of nuclear encoded subunits. We here discuss current knowledge about the origin of nuclear encoded subunits and the appearance of different isozymes promoted by tissue and cellular environments such as hypoxia. We also review evidence for recent selective pressure acting on COX among vertebrates, particularly in primate lineages, and discuss the unique pattern of co-evolution between the nuclear and mitochondrial genomes. Finally, even though the addition of nuclear encoded subunits was a major event in eukaryotic COX evolution, this does not lead to emergence of a more efficient COX, as might be expected from an anthropocentric point of view, for the “higher” organism possessing large brains and muscles. The main function of these subunits appears to be “only” to control the activity of the mitochondrial subunits. We propose that this control function is an as yet underappreciated key point of evolution. Moreover, the importance of regulating energy supply may have caused the addition of subunits encoded by the nucleus in a process comparable to a “domestication scenario” such that the host tends to control more and more tightly the ancestral activity of COX performed by the mtDNA encoded subunits. This article is part of a Special Issue entitled

  8. Cytochrome c oxidase: evolution of control via nuclear subunit addition.

    PubMed

    Pierron, Denis; Wildman, Derek E; Hüttemann, Maik; Markondapatnaikuni, Gopi Chand; Aras, Siddhesh; Grossman, Lawrence I

    2012-04-01

    According to theory, present eukaryotic cells originated from a beneficial association between two free-living cells. Due to this endosymbiotic event the pre-eukaryotic cell gained access to oxidative phosphorylation (OXPHOS), which produces more than 15 times as much ATP as glycolysis. Because cellular ATP needs fluctuate and OXPHOS both requires and produces entities that can be toxic for eukaryotic cells such as ROS or NADH, we propose that the success of endosymbiosis has largely depended on the regulation of endosymbiont OXPHOS. Several studies have presented cytochrome c oxidase as a key regulator of OXPHOS; for example, COX is the only complex of mammalian OXPHOS with known tissue-specific isoforms of nuclear encoded subunits. We here discuss current knowledge about the origin of nuclear encoded subunits and the appearance of different isozymes promoted by tissue and cellular environments such as hypoxia. We also review evidence for recent selective pressure acting on COX among vertebrates, particularly in primate lineages, and discuss the unique pattern of co-evolution between the nuclear and mitochondrial genomes. Finally, even though the addition of nuclear encoded subunits was a major event in eukaryotic COX evolution, this does not lead to emergence of a more efficient COX, as might be expected from an anthropocentric point of view, for the "higher" organism possessing large brains and muscles. The main function of these subunits appears to be "only" to control the activity of the mitochondrial subunits. We propose that this control function is an as yet under appreciated key point of evolution. Moreover, the importance of regulating energy supply may have caused the addition of subunits encoded by the nucleus in a process comparable to a "domestication scenario" such that the host tends to control more and more tightly the ancestral activity of COX performed by the mtDNA encoded subunits. PMID:21802404

  9. Crystal Structure of the 30S Ribosomal Subunit from Thermus Thermophilus. Purification, Crystallization and Structure Determination

    SciTech Connect

    Clemons, William M.; Brodersen, Ditlev E.; McCutcheonn, John P.; May, Joanna L.C.; Carter, Andrew P.; Morgan-Warren, Robert J.; Wimberly, Brian T.; Ramakrishnan, Venki

    2009-10-07

    We describe the crystallization and structure determination of the 30 S ribosomal subunit from Thermus thermophilus. Previous reports of crystals that diffracted to 10 {angstrom} resolution were used as a starting point to improve the quality of the diffraction. Eventually, ideas such as the addition of substrates or factors to eliminate conformational heterogeneity proved less important than attention to detail in yielding crystals that diffracted beyond 3 {angstrom} resolution. Despite improvements in technology and methodology in the last decade, the structure determination of the 30 S subunit presented some very challenging technical problems because of the size of the asymmetric unit, crystal variability and sensitivity to radiation damage. Some steps that were useful for determination of the atomic structure were: the use of anomalous scattering from the LIII edges of osmium and lutetium to obtain the necessary phasing signal; the use of tunable, third-generation synchrotron sources to obtain data of reasonable quality at high resolution; collection of derivative data precisely about a mirror plane to preserve small anomalous differences between Bijvoet mates despite extensive radiation damage and multi-crystal scaling; the pre-screening of crystals to ensure quality, isomorphism and the efficient use of scarce third-generation synchrotron time; pre-incubation of crystals in cobalt hexaammine to ensure isomorphism with other derivatives; and finally, the placement of proteins whose structures had been previously solved in isolation, in conjunction with biochemical data on protein-RNA interactions, to map out the architecture of the 30 S subunit prior to the construction of a detailed atomic-resolution model.

  10. Architecture of the Complex Formed by Large and Small Terminase Subunits from Bacteriophage P22.

    PubMed

    McNulty, Reginald; Lokareddy, Ravi Kumar; Roy, Ankoor; Yang, Yang; Lander, Gabriel C; Heck, Albert J R; Johnson, John E; Cingolani, Gino

    2015-10-01

    Packaging of viral genomes inside empty procapsids is driven by a powerful ATP-hydrolyzing motor, formed in many double-stranded DNA viruses by a complex of a small terminase (S-terminase) subunit and a large terminase (L-terminase) subunit, transiently docked at the portal vertex during genome packaging. Despite recent progress in elucidating the structure of individual terminase subunits and their domains, little is known about the architecture of an assembled terminase complex. Here, we describe a bacterial co-expression system that yields milligram quantities of the S-terminase:L-terminase complex of the Salmonella phage P22. In vivo assembled terminase complex was affinity-purified and stabilized by addition of non-hydrolyzable ATP, which binds specifically to the ATPase domain of L-terminase. Mapping studies revealed that the N-terminus of L-terminase ATPase domain (residues 1-58) contains a minimal S-terminase binding domain sufficient for stoichiometric association with residues 140-162 of S-terminase, the L-terminase binding domain. Hydrodynamic analysis by analytical ultracentrifugation sedimentation velocity and native mass spectrometry revealed that the purified terminase complex consists predominantly of one copy of the nonameric S-terminase bound to two equivalents of L-terminase (1S-terminase:2L-terminase). Direct visualization of this molecular assembly in negative-stained micrographs yielded a three-dimensional asymmetric reconstruction that resembles a "nutcracker" with two L-terminase protomers projecting from the C-termini of an S-terminase ring. This is the first direct visualization of a purified viral terminase complex analyzed in the absence of DNA and procapsid. PMID:26301600

  11. Inhibition of the catalytic subunit of cAMP-dependent protein kinase by dicyclohexylcarbodiimide

    SciTech Connect

    Toner-Webb, J.; Taylor, S.S.

    1987-11-17

    The hydrophobic carbodiimide dicyclohexylcarbodiimide (DCCD) has been shown to inhibit the catalytic (C) subunit of adenosine cyclic 3',5'-phosphate dependent protein kinase in a time-dependent, irreversible manner. The rate of inactivation was first order and showed saturation kinetics with an apparent K/sub i/ of 60 ..mu..M. Magnesium adenosine 5'-triphosphate (MgATP) was capable of protecting against this inhibition, whereas neither a synthetic peptide substrate nor histone afforded protection. Mg alone afforded some protection. When the catalytic subunit was aggregated with the regulatory subunit in the holoenzyme complex, no inhibition was observed. The inhibition was enhanced at low pH, suggesting that a carboxylic acid group was the target for interaction with DCCD. On the basis of the protection studies, it is most likely that this carboxylic acid group is associated with the MgATP binding site, perhaps serving as a ligand for the metal. Efforts to identify the site that was modified by DCCD were made. In no case was radioactivity incorporated into the protein, suggesting that the irreversible inhibition was due to an intramolecular cross-link between a reactive carboxylic acid group and a nearby amino group. Differential peptide mapping identified a single peptide that was consistently lost as a consequence of DCCD inhibition. This peptide (residues 166-189) contained four carboxylic acid residues as well as an internal Lys. Two of these carboxyl groups, Asp-166 and Asp-184, are conserved in all protein kinases, including oncogene transforming proteins and growth factor receptors, and thus are likely to play an essential role.

  12. Cloning DPB3, the gene encoding the third subunit of DNA polymerase II of Saccharomyces cerevisiae.

    PubMed Central

    Araki, H; Hamatake, R K; Morrison, A; Johnson, A L; Johnston, L H; Sugino, A

    1991-01-01

    DNA polymerase II purified from Saccharomyces cerevisiae contains polypeptides with apparent molecular masses of greater than 200, 80, 34, 30 and 29 kDa, the two largest of which (subunits A and B) are encoded by the essential genes POL2 and DPB2. By probing a lambda gt11 expression library of yeast DNA with antiserum against DNA polymerase II, we isolated a single gene, DPB3, that encodes both the 34- and 30-kDa polypeptides (subunit C and C'). The nucleotide sequence of DPB3 contained an open reading frame encoding a 23-kDa protein, significantly smaller than the observed molecular masses, 34- or 30-kDa, which might represent post-translationally modified forms of the DPB3 product. The predicted amino acid sequence contained a possible NTP-binding motif and a glutamate-rich region. NTP-binding motif and a glutamate-rich region. A dpb3 deletion mutant (dpb3 delta) was viable and yielded a DNA polymerase II lacking the 34- and 30-kDa polypeptides. dpb3 delta strains exhibited an increased spontaneous mutation rate, suggesting that the DPB3 product is required to maintain fidelity of chromosomal replication. Since a fifth, 29-kDa polypeptide was present in DNA polymerase II preparations from wild-type cell extracts throughout purification, the subunit composition appears to be A, B, C (or C and C') and D. The 5' nontranscribed region of DPB3 contained the MulI-related sequence ACGCGA, while the 0.9-kb DPB3 transcript accumulated periodically during the cell cycle and peaked at the G1/S boundary. The level of DPB3 transcript thus appears to be under the same cell cycle control as those of POL2, DPB2 and other DNA replication genes. DPB3 was mapped to chromosome II, 30 cM distal to his7. Images PMID:1923754

  13. The first transmembrane domain (TM1) of β2-subunit binds to the transmembrane domain S1 of α-subunit in BK potassium channels

    PubMed Central

    Morera, Francisco J.; Alioua, Abderrahmane; Kundu, Pallob; Salazar, Marcelo; Gonzalez, Carlos; Martinez, Agustin D.; Stefani, Enrico; Toro, Ligia; Latorre, Ramon

    2012-01-01

    The BK channel is one of the most broadly expressed ion channels in mammals. In many tissues, the BK channel pore-forming α-subunit is associated to an auxiliary β-subunit that modulates the voltage- and Ca2+-dependent activation of the channel. Structural components present in β-subunits that are important for the physical association with the α-subunit are yet unknown. Here, we show through co-immunoprecipitation that the intracellular C-terminus, the second transmembrane domain (TM2) and the extracellular loop of the β2-subunit are dispensable for association with the α-subunit pointing transmembrane domain 1 (TM1) as responsible for the interaction. Indeed, the TOXCAT assay for transmembrane protein–protein interactions demonstrated for the first time that TM1 of the β2-subunit physically binds to the transmembrane S1 domain of the α-subunit. PMID:22710124

  14. Molecular characterization of the 28- and 31-kilodalton subunits of the Legionella pneumophila major outer membrane protein.

    PubMed Central

    Hoffman, P S; Seyer, J H; Butler, C A

    1992-01-01

    The major outer membrane protein of Legionella pneumophila exhibits an apparent molecular mass of 100 kDa. Previous studies revealed the oligomer to be composed of 28- and 31-kDa subunits; the latter subunit is covalently bound to peptidoglycan. These proteins exhibit cross-reactivity with polyclonal anti-31-kDa protein serum. In this study, we present evidence to confirm that the 31-kDa subunit is a 28-kDa subunit containing a bound fragment of peptidoglycan. Peptide maps of purified proteins were generated following cyanogen bromide cleavage or proteolysis with staphylococcal V8 protease. A comparison of the banding patterns resulting from sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) revealed a common pattern. Selected peptide fragments were sequenced on a gas phase microsequencer, and the sequence was compared with the sequence obtained for the 28-kDa protein. While the amino terminus of the 31-kDa protein was blocked, peptide fragments generated by cyanogen bromide treatment exhibited a sequence identical to that of the amino terminus of the 28-kDa protein, but beginning at amino acid four (glycine), which is preceded by methionine at the third position. This sequence, (Gly-Thr-Met)-Gly-Pro-Val-Trp-Thr-Pro-Gly-Asn ... , confirms that these proteins have a common amino terminus. An oligonucleotide synthesized from the codons of the common N-terminal amino acid sequence was used to establish by Southern and Northern (RNA) blot analyses that a single gene coded for both proteins. With regard to the putative porin structure, we have identified two major bands at 70 kDa and at approximately 120 kDa by nonreducing SDS-PAGE. The former may represent the typical trimeric motif, while the latter may represent either a double trimer or an aggregate. Analysis of these two forms by two-dimensional SDS-PAGE (first dimensions, nonreducing; second dimensions, reducing) established that both were composed of 31- and 28-kDa subunits cross-linked via

  15. Progress towards development of a cholera subunit vaccine.

    PubMed

    Taylor, Ronald K; Kirn, Thomas J; Bose, Niranjan; Stonehouse, Emily; Tripathi, Shital A; Kovác, Pavol; Wade, William F

    2004-07-01

    Cholera, an enteric disease that can reach pandemic proportions, remains a world-wide problem that is positioned to increase in incidence as changes in global climate or armed conflict spawn the conditions that enhance transmission to humans and, thus, precipitate epidemic cholera. An effective subunit cholera vaccine that can provide protective immunity with one parenteral immunization would be a major advantage over the existing oral vaccines that can require two doses for optimal protection. The existing vaccines are clearly effective in some settings, but are less so in others, especially with respect to specific groups such as young (2-5 years) children. In our efforts to develop a cholera subunit vaccine, we focused on two Vibrio cholerae antigens, LPS (lipopolysaccharide) and TCP (toxin co-regulated pilus), that are known to induce protective antibodies in animal models and, in the case of anti-LPS antibodies, to be associated with clinical protection of V. cholerae exposed or vaccinated individuals. This review discusses the current cholera vaccines and compares the advantages of a cholera subunit vaccine to that of the whole cell vaccines. We discuss the possible subunit antigens and prospective targeted use of a subunit cholera vaccine. PMID:17191897

  16. Parametric mapping

    NASA Astrophysics Data System (ADS)

    Branch, Allan C.

    1998-01-01

    Parametric mapping (PM) lies midway between older and proven artificial landmark based guidance systems and yet to be realized vision based guidance systems. It is a simple yet effective natural landmark recognition system offering freedom from the need for enhancements to the environment. Development of PM systems can be inexpensive and rapid and they are starting to appear in commercial and industrial applications. Together with a description of the structural framework developed to generically describe robot mobility, this paper illustrates clearly the parts of any mobile robot navigation and guidance system and their interrelationships. Among other things, the importance of the richness of the reference map, and not necessarily the sensor map, is introduced, the benefits of dynamic path planners to alleviate the need for separate object avoidance, and the independence of the PM system to the type of sensor input is shown.

  17. Genome-wide promoter binding profiling of protein phosphatase-1 and its major nuclear targeting subunits

    PubMed Central

    Verheyen, Toon; Görnemann, Janina; Verbinnen, Iris; Boens, Shannah; Beullens, Monique; Van Eynde, Aleyde; Bollen, Mathieu

    2015-01-01

    Protein phosphatase-1 (PP1) is a key regulator of transcription and is targeted to promoter regions via associated proteins. However, the chromatin binding sites of PP1 have never been studied in a systematic and genome-wide manner. Methylation-based DamID profiling in HeLa cells has enabled us to map hundreds of promoter binding sites of PP1 and three of its major nuclear interactors, i.e. RepoMan, NIPP1 and PNUTS. Our data reveal that the α, β and γ isoforms of PP1 largely bind to distinct subsets of promoters and can also be differentiated by their promoter binding pattern. PP1β emerged as the major promoter-associated isoform and shows an overlapping binding profile with PNUTS at dozens of active promoters. Surprisingly, most promoter binding sites of PP1 are not shared with RepoMan, NIPP1 or PNUTS, hinting at the existence of additional, largely unidentified chromatin-targeting subunits. We also found that PP1 is not required for the global chromatin targeting of RepoMan, NIPP1 and PNUTS, but alters the promoter binding specificity of NIPP1. Our data disclose an unexpected specificity and complexity in the promoter binding of PP1 isoforms and their chromatin-targeting subunits. PMID:25990731

  18. The Exocyst Subunit Sec6 Interacts with Assembled Exocytic SNARE Complexes.

    PubMed

    Dubuke, Michelle L; Maniatis, Stephanie; Shaffer, Scott A; Munson, Mary

    2015-11-20

    In eukaryotic cells, membrane-bound vesicles carry cargo between intracellular compartments, to and from the cell surface, and into the extracellular environment. Many conserved families of proteins are required for properly localized vesicle fusion, including the multisubunit tethering complexes and the SNARE complexes. These protein complexes work together to promote proper vesicle fusion in intracellular trafficking pathways. However, the mechanism by which the exocyst, the exocytosis-specific multisubunit tethering complex, interacts with the exocytic SNAREs to mediate vesicle targeting and fusion is currently unknown. We have demonstrated previously that the Saccharomyces cerevisiae exocyst subunit Sec6 directly bound the plasma membrane SNARE protein Sec9 in vitro and that Sec6 inhibited the assembly of the binary Sso1-Sec9 SNARE complex. Therefore, we hypothesized that the interaction between Sec6 and Sec9 prevented the assembly of premature SNARE complexes at sites of exocytosis. To map the determinants of this interaction, we used cross-linking and mass spectrometry analyses to identify residues required for binding. Mutation of residues identified by this approach resulted in a growth defect when introduced into yeast. Contrary to our previous hypothesis, we discovered that Sec6 does not change the rate of SNARE assembly but, rather, binds both the binary Sec9-Sso1 and ternary Sec9-Sso1-Snc2 SNARE complexes. Together, these results suggest a new model in which Sec6 promotes SNARE complex assembly, similar to the role proposed for other tether subunit-SNARE interactions. PMID:26446795

  19. Integrative structural analysis of the UTPB complex, an early assembly factor for eukaryotic small ribosomal subunits

    PubMed Central

    Zhang, Cheng; Sun, Qi; Chen, Rongchang; Chen, Xining; Lin, Jinzhong; Ye, Keqiong

    2016-01-01

    Ribosome assembly is an essential and conserved cellular process in eukaryotes that requires numerous assembly factors. The six-subunit UTPB complex is an essential component of the 90S precursor of the small ribosomal subunit. Here, we analyzed the molecular architecture of UTPB using an integrative structural biology approach. We mapped the major interactions that associate each of six UTPB proteins. Crystallographic studies showed that Utp1, Utp21, Utp12 and Utp13 are evolutionarily related and form a dimer of dimers (Utp1–Utp21, Utp12–Utp13) through their homologous helical C-terminal domains. Molecular docking with crosslinking restraints showed that the WD domains of Utp12 and Utp13 are associated, as are the WD domains of Utp1, Utp21 and Utp18. Electron microscopy images of the entire UTPB complex revealed that it predominantly adopts elongated conformations and possesses internal flexibility. We also determined crystal structures of the WD domain of Utp18 and the HAT and deviant HAT domains of Utp6. A structural model of UTPB was derived based on these data. PMID:27330138

  20. RILP interacts with HOPS complex via VPS41 subunit to regulate endocytic trafficking

    PubMed Central

    Lin, Xiaosi; Yang, Ting; Wang, Shicong; Wang, Zhen; Yun, Ye; Sun, Lixiang; Zhou, Yunhe; Xu, Xiaohui; Akazawa, Chihiro; Hong, Wanjin; Wang, Tuanlao

    2014-01-01

    The HOPS complex serves as a tethering complex with GEF activity for Ypt7p in yeast to regulate late endosomal membrane maturation. While the role of HOPS complex is well established in yeast cells, its functional and mechanistic aspects in mammalian cells are less well defined. In this study, we report that RILP, a downstream effector of Rab7, interacts with HOPS complex and recruits HOPS subunits to the late endosomal compartment. Structurally, the amino-terminal portion of RILP interacts with HOPS complex. Unexpectedly, this interaction is independent of Rab7. VPS41 subunit of HOPS complex was defined to be the major partner for interacting with RILP. The carboxyl-terminal region of VPS41 was mapped to be responsible for the interaction. Functionally, either depletion of VPS41 by shRNA or overexpression of VPS41 C-terminal half retarded EGF-induced degradation of EGFR. These results suggest that interaction of RILP with HOPS complex via VPS41 plays a role in endocytic trafficking of EGFR. PMID:25445562

  1. Herpes simplex virus specifies two subunits of ribonucleotide reductase encoded by 3'-coterminal transcripts.

    PubMed Central

    Swain, M A; Galloway, D A

    1986-01-01

    We have previously described a transcription unit located between map coordinates 0.558 and 0.595 on the herpes simplex virus type 2 strain 333 genome which encodes two mRNAs of 5.0 and 1.2 kilobases that share a common 3' terminus, and we have determined the nucleotide sequence of a 38,000-dalton protein specified by the smaller RNA (D. A. Galloway and M. A. Swain, J. Virol. 49:724-730, 1984). The entire nucleotide sequence of the 140,000-dalton protein specified by a 3,432-base-pair open reading frame within the large mRNA is presented, as are transcriptional regulatory sequences upstream of the RNA. The 140,000-dalton protein shows strong homology with the large subunit of well-characterized ribonucleotide reductase enzymes from the mouse and from Escherichia coli and with an Epstein-Barr virus gene. The 38,000-dalton protein has been shown previously to have homology with the small subunit of these enzymes (B.-M. Sjoberg, H. Eklund, J. A. Fuchs, J. Carlson, N. M. Standart, J. V. Ruderman, S. J. Bray, and T. Hunt, FEBS Lett. 183:99-102, 1985). This is the first example of a herpesvirus transcriptional unit that encodes functionally related proteins. PMID:2419588

  2. Position of the CrPV IRES on the 40S subunit and factor dependence of IRES/80S ribosome assembly.

    PubMed

    Pestova, Tatyana V; Lomakin, Ivan B; Hellen, Christopher U T

    2004-09-01

    The cricket paralysis virus intergenic region internal ribosomal entry site (CrPV IGR IRES) can assemble translation initiation complexes by binding to 40S subunits without Met-tRNA(Met)(i) and initiation factors (eIFs) and then by joining directly with 60S subunits, yielding elongation-competent 80S ribosomes. Here, we report that eIF1, eIF1A and eIF3 do not significantly influence IRES/40S subunit binding but strongly inhibit subunit joining and the first elongation cycle. The IRES can avoid their inhibitory effect by its ability to bind directly to 80S ribosomes. The IRES's ability to bind to 40S subunits simultaneously with eIF1 allowed us to use directed hydroxyl radical cleavage to map its position relative to the known position of eIF1. A connecting loop in the IRES's pseudoknot (PK) III domain, part of PK II and the entire domain containing PK I are solvent-exposed and occupy the E site and regions of the P site that are usually occupied by Met-tRNA(Met)(i). PMID:15332113

  3. Real-time tracking of CO migration and binding in the α and β subunits of human hemoglobin via 150-ps time-resolved Laue crystallography

    PubMed Central

    Schotte, Friedrich; Cho, Hyun Sun; Soman, Jayashree; Wulff, Michael; Olson, John S.; Anfinrud, Philip A.

    2014-01-01

    We have developed the method of picosecond Laue crystallography and used this capability to probe ligand dynamics in tetrameric R-state hemoglobin (Hb). Time-resolved, 2 Å-resolution electron density maps of photolyzed HbCO reveal the time-dependent population of CO in the binding (A) and primary docking (B) sites of both α and β subunits from 100 ps to 10 μs. The proximity of the B site in the β subunit is about 0.25 Å closer to its A binding site, and its kBA rebinding rate (~300 μs−1) is six times faster, suggesting distal control of the rebinding dynamics. Geminate rebinding in the β subunit exhibits both prompt and delayed geminate phases. We developed a microscopic model to quantitatively explain the observed kinetics, with three states for the α subunit and four states for the β subunit. This model provides a consistent framework for interpreting rebinding kinetics reported in prior studies of both HbCO and HbO2. PMID:24839343

  4. Dengue vaccine: an update on recombinant subunit strategies.

    PubMed

    Martin, J; Hermida, L

    2016-03-01

    Dengue is an increasing public health problem worldwide, with the four serotypes of the virus infecting over 390 million people annually. There is no specific treatment or antiviral drug for dengue, and prevention is largely limited to controlling the mosquito vectors or disrupting the human-vector contact. Despite the considerable progress made in recent years, an effective vaccine against the virus is not yet available. The development of a dengue vaccine has been hampered by many unique challenges, including the need to ensure the absence of vaccine-induced enhanced severity of disease. Recombinant protein subunit vaccines offer a safer alternative to other vaccine approaches. Several subunit vaccine candidates are presently under development, based on different structural and non-structural proteins of the virus. Novel adjuvants or immunopotentiating strategies are also being tested to improve their immunogenicity. This review summarizes the current status and development trends of subunit dengue vaccines. PMID:26982462

  5. Functional biosynthesis of an allophycocyan beta subunit in Escherichia coli.

    PubMed

    Ge, Baosheng; Sun, Haixiang; Feng, Yang; Yang, Jinying; Qin, Song

    2009-03-01

    Allophycocyanin is a phycobiliprotein with various biological and pharmacological properties. An expression vector was constructed using CpeS as the bilin lyase for the allophycocyanin beta subunit, resulting in overexpression of a fluorescent allophycocyanin beta-subunit in Escherichia coli. A high-density cell culture was developed using a continuous feeding strategy. After 16 h of culture, the dry cell density reached 21.4 g l(-1), the expression of the allophycocyanin beta-subunit was 0.86 g l(-1) broth, and the relative chromoprotein yield was 81.4%. The recombinant protein showed spectral features similar to native allophycocyanin, which provide an efficient methodology for large-scale production of this valuable fluorescent protein. PMID:19269586

  6. Cholera Toxin B: One Subunit with Many Pharmaceutical Applications

    PubMed Central

    Baldauf, Keegan J.; Royal, Joshua M.; Hamorsky, Krystal Teasley; Matoba, Nobuyuki

    2015-01-01

    Cholera, a waterborne acute diarrheal disease caused by Vibrio cholerae, remains prevalent in underdeveloped countries and is a serious health threat to those living in unsanitary conditions. The major virulence factor is cholera toxin (CT), which consists of two subunits: the A subunit (CTA) and the B subunit (CTB). CTB is a 55 kD homopentameric, non-toxic protein binding to the GM1 ganglioside on mammalian cells with high affinity. Currently, recombinantly produced CTB is used as a component of an internationally licensed oral cholera vaccine, as the protein induces potent humoral immunity that can neutralize CT in the gut. Additionally, recent studies have revealed that CTB administration leads to the induction of anti-inflammatory mechanisms in vivo. This review will cover the potential of CTB as an immunomodulatory and anti-inflammatory agent. We will also summarize various recombinant expression systems available for recombinant CTB bioproduction. PMID:25802972

  7. Subunits of the Schizosaccharomyces pombe RNA polymerase II: enzyme purification and structure of the subunit 3 gene.

    PubMed Central

    Azuma, Y; Yamagishi, M; Ishihama, A

    1993-01-01

    To improve our understanding of the structure and function of eukaryotic RNA polymerase II, we purified the enzyme from the fission yeast Schizosaccharomyces pombe. The highly purified RNA polymerase II contained more than eleven polypeptides. The sizes of the largest the second-, and the third-largest polypeptides as measured by SDS-polyacrylamide gel electrophoresis were about 210, 150, and 40 kilodaltons (kDa), respectively, and are similar to those of RPB1, 2, and 3 subunits of Saccharomyces cerevisiae RNA polymerase II. Using the degenerated primers designed after amino acid micro-sequencing of the 40 kDa third-largest polypeptide (subunit 3), we cloned the subunit 3 gene (rpb3) and determined its DNA sequence. Taken together with the sequence of parts of PCR-amplified cDNA, the predicted coding sequence of rpb3, interrupted by two introns, was found to encode a polypeptide of 297 amino acid residues in length with a molecular weight of 34 kDa. The S. pombe subunit 3 contains four structural domains conserved for the alpha-subunit family of RNA polymerase from both eukaryotes and prokaryotes. A putative leucine zipper motif was found to exist in the C-terminal proximal conserved region (domain D). Possible functions of the conserved domains are discussed. Images PMID:8367291

  8. Memphis Maps.

    ERIC Educational Resources Information Center

    Hyland, Stanley; Cox, David; Martin, Cindy

    1998-01-01

    The Memphis Maps program, a collaborative effort of Memphis (Tennessee) educational institutions, public agencies, a bank, and community programs, trains local students in Geographic Information Systems technology and provides the community with valuable demographic and assessment information. The program is described, and factors contributing to…

  9. A remarkably stable TipE gene cluster: evolution of insect Para sodium channel auxiliary subunits

    PubMed Central

    2011-01-01

    Background First identified in fruit flies with temperature-sensitive paralysis phenotypes, the Drosophila melanogaster TipE locus encodes four voltage-gated sodium (NaV) channel auxiliary subunits. This cluster of TipE-like genes on chromosome 3L, and a fifth family member on chromosome 3R, are important for the optional expression and functionality of the Para NaV channel but appear quite distinct from auxiliary subunits in vertebrates. Here, we exploited available arthropod genomic resources to trace the origin of TipE-like genes by mapping their evolutionary histories and examining their genomic architectures. Results We identified a remarkably conserved synteny block of TipE-like orthologues with well-maintained local gene arrangements from 21 insect species. Homologues in the water flea, Daphnia pulex, suggest an ancestral pancrustacean repertoire of four TipE-like genes; a subsequent gene duplication may have generated functional redundancy allowing gene losses in the silk moth and mosquitoes. Intronic nesting of the insect TipE gene cluster probably occurred following the divergence from crustaceans, but in the flour beetle and silk moth genomes the clusters apparently escaped from nesting. Across Pancrustacea, TipE gene family members have experienced intronic nesting, escape from nesting, retrotransposition, translocation, and gene loss events while generally maintaining their local gene neighbourhoods. D. melanogaster TipE-like genes exhibit coordinated spatial and temporal regulation of expression distinct from their host gene but well-correlated with their regulatory target, the Para NaV channel, suggesting that functional constraints may preserve the TipE gene cluster. We identified homology between TipE-like NaV channel regulators and vertebrate Slo-beta auxiliary subunits of big-conductance calcium-activated potassium (BKCa) channels, which suggests that ion channel regulatory partners have evolved distinct lineage-specific characteristics

  10. Micelle-Based Adjuvants for Subunit Vaccine Delivery

    PubMed Central

    Trimaille, Thomas; Verrier, Bernard

    2015-01-01

    In the development of subunit vaccines with purified or recombinant antigens for cancer and infectious diseases, the design of improved and safe adjuvants able to efficiently target the antigen presenting cells, such as dendritic cells, represents a crucial challenge. Nanoparticle-based antigen delivery systems have been identified as an innovative strategy to improve the efficacy of subunit vaccines. Among them, self-assembled micellar nanoparticles from amphiphilic (macro)molecules have recently emerged as promising candidates. In this short review, we report on the recent research findings highlighting the versatility and potential of such systems in vaccine delivery. PMID:26426060

  11. Functional analysis of Drosophila DNA polymerase ε p58 subunit

    PubMed Central

    Sahashi, Ritsuko; Matsuda, Risa; Suyari, Osamu; Kawai, Mieko; Yoshida, Hideki; Cotterill, Sue; Yamaguchi, Masamitsu

    2013-01-01

    DNA polymerase ε (polε) plays a central role in DNA replication in eukaryotic cells, and has been suggested to the main synthetic polymerase on the leading strand. It is a hetero-tetrameric enzyme, comprising a large catalytic subunit (the A subunit ~250 kDa), a B subunit of ~60 kDa in most species (~80 kDa in budding yeast) and two smaller subunits (each ~20 kDa). In Drosophila, two subunits of polε (dpolε) have been identified. One is the 255 kDa catalytic subunit (dpolεp255), and the other is the 58 kDa subunit (dpolεp58). The functions of the B subunit have been mainly studied in budding yeast and mammalian cell culture, few studies have been performed in the context of an intact multicellular organism and therefore its functions in this context remain poorly understood. To address this we examined the in vivo role of dpolεp58 in Drosophila. A homozygous dpolεp58 mutant is pupal lethal, and the imaginal discs are less developed in the third instar larvae. In the eye discs of this mutant S phases, as measured by BrdU incorporation assays, were significantly reduced. In addition staining with an anti-phospho histone H3 (PH3) antibody, (a marker of M phase), was increased in the posterior region of eye discs, where usually cells stop replicating and start differentiation. These results indicate that dpolεp58 is essential for Drosophila development and plays an important role in progression of S phase in mitotic cell cycles. We also observed that the size of nuclei in salivary gland cells were decreased in dpolεp58 mutant, indicating that dpolεp58 also plays a role in endoreplication. Furthermore we detect a putative functional interaction between dpolε and ORC2 in discs suggesting that polε plays a role in the initiation of DNA replication in Drosophila. PMID:24224125

  12. Advancements in the development of subunit influenza vaccines

    PubMed Central

    Zhang, Naru; Zheng, Bo-Jian; Lu, Lu; Zhou, Yusen; Jiang, Shibo; Du, Lanying

    2014-01-01

    The ongoing threat of influenza epidemics and pandemics has emphasized the importance of developing safe and effective vaccines against infections from divergent influenza viruses. In this review, we first introduce the structure and life cycle of influenza A viruses, describing major influenza A virus-caused pandemics. We then compare different types of influenza vaccines and discuss current advancements in the development of subunit influenza vaccines, particularly those based on nucleoprotein (NP), extracellular domain of matrix protein 2 (M2e) and hemagglutinin (HA) proteins. We also illustrate potential strategies for improving the efficacy of subunit influenza vaccines. PMID:25529753

  13. Physical linkage of a GABAA receptor subunit gene to the DXS374 locus in human Xq28.

    PubMed Central

    Bell, M V; Bloomfield, J; McKinley, M; Patterson, M N; Darlison, M G; Barnard, E A; Davies, K E

    1989-01-01

    We report the physical linkage of the gene encoding one of the subunits of the GABAA receptor (GABRA3) to the polymorphic locus DXS374 on the human X chromosome at Xq28. X-linked manic depression and other psychiatric disorders have been mapped to this region, and thus GABRA3 is a potential candidate gene for these disorders. DXS374--and therefore GABRA3--lies distal to the fragile X locus at a recombination fraction of approximately .15. Images Figure 1 Figure 2 PMID:2574000

  14. Aspects of Subunit Interactions in the Chloroplast ATP Synthase (I. Isolation of a Chloroplast Coupling Factor 1-Subunit III Complex from Spinach Thylakoids).

    PubMed Central

    Wetzel, C. M.; McCarty, R. E.

    1993-01-01

    A chloroplast ATP synthase complex (CF1 [chloroplast-coupling factor 1]-CF0 [membrane-spanning portion of chloroplast ATP synthase]) depleted of all CF0 subunits except subunit III (also known as the proteolipid subunit) was purified to study the interaction between CF1 and subunit III. Subunit III has a putative role in proton translocation across the thylakoid membrane during photophosphorylation; therefore, an accurate model of subunit inter-actions involving subunit III will be valuable for elucidating the mechanism and regulation of energy coupling. Purification of the complex from a crude CF1-CF0 preparation from spinach (Spinacia oleracea) thylakoids was accomplished by detergent treatment during anion-exchange chromatography. Subunit III in the complex was positively identified by amino acid analysis and N-terminal sequencing. The association of subunit III with CF1 was verified by linear sucrose gradient centrifugation, immunoprecipitation, and incorporation of the complex into asolectin liposomes. After incorporation into liposomes, CF1 was removed from the CF1-III complex by ethylenediaminetetracetate treatment. The subunit III-proteoliposomes were competent to rebind purified CF1. These results indicate that subunit III directly interacts with CF1 in spinach thylakoids. PMID:12231815

  15. Heterologous expression of plant vacuolar pyrophosphatase in yeast demonstrates sufficiency of the substrate-binding subunit for proton transport

    SciTech Connect

    Kim, E.J.; Zhen, R.G.; Rea, P.A. )

    1994-06-21

    The membrane bounding the vacuole of plant cells contains two electrogenic proton pumps. These are the vacuolar H[sup +]-ATPase (EC 3.6.1.3), an enzyme common to all eukaryotes, and a vacuolar H[sup +]-translocating pyrophosphatase (EC 3.6.1.1), which is ubiquitous in plants but otherwise known in only a few phototrophic bacteria. Although the substrate-binding subunit of the vacuolar H[sup +]-pyrophosphatase has been identified and purified and cDNAs encoding it have been isolated and characterized, the minimal unit competent in pyrophosphate (PPi)-energized H[sup +] translocation is not known. Here the authors address this question and show that heterologous expression of the cDNA (AVP) encoding the substrate-binding subunit of the vacuolar H[sup +]-pyrophosphatase from the vascular plant Arabidopsis thaliana in the yeast Saccharomyces cerevisiae results in the production of vacuolarly localized functional enzyme active in PPi-dependent H[sup +] translocation. Since the heterologously expressed pump is indistinguishable from the native plant enzyme with respect to PPi hydrolysis, H[sub +] translocation, activation by potassium, and selective inhibition by calcium and 1,1-diphosphonates, it is concluded that all of the known catalytic functions of the enzyme map to the one subunit encoded by AVP.

  16. Heterologous expression of plant vacuolar pyrophosphatase in yeast demonstrates sufficiency of the substrate-binding subunit for proton transport.

    PubMed Central

    Kim, E J; Zhen, R G; Rea, P A

    1994-01-01

    The membrane bounding the vacuole of plant cells contains two electrogenic proton pumps. These are the vacuolar H(+)-ATPase (EC 3.6.1.3), an enzyme common to all eukaryotes, and a vacuolar H(+)-translocating pyrophosphatase (EC 3.6.1.1), which is ubiquitous in plants but otherwise known in only a few phototrophic bacteria. Although the substrate-binding subunit of the vacuolar H(+)-pyrophosphatase has been identified and purified and cDNAs encoding it have been isolated and characterized, the minimal unit competent in pyrophosphate (PPi)-energized H+ translocation is not known. Here we address this question and show that heterologous expression of the cDNA (AVP) encoding the substrate-binding subunit of the vacuolar H(+)-pyrophosphatase from the vascular plant Arabidopsis thaliana in the yeast Saccharomyces cerevisiae results in the production of vacuolarly localized functional enzyme active in PPi-dependent H+ translocation. Since the heterologously expressed pump is indistinguishable from the native plant enzyme with respect to PPi hydrolysis, H+ translocation, activation by potassium, and selective inhibition by calcium and 1,1-diphosphonates, it is concluded that all of the known catalytic functions of the enzyme map to the one subunit encoded by AVP. Images PMID:8016125

  17. Mg-chelatase I subunit 1 and Mg-protoporphyrin IX methyltransferase affect the stomatal aperture in Arabidopsis thaliana.

    PubMed

    Tomiyama, Masakazu; Inoue, Shin-Ichiro; Tsuzuki, Tomo; Soda, Midori; Morimoto, Sayuri; Okigaki, Yukiko; Ohishi, Takaya; Mochizuki, Nobuyoshi; Takahashi, Koji; Kinoshita, Toshinori

    2014-07-01

    To elucidate the molecular mechanisms of stomatal opening and closure, we performed a genetic screen using infrared thermography to isolate stomatal aperture mutants. We identified a mutant designated low temperature with open-stomata 1 (lost1), which exhibited reduced leaf temperature, wider stomatal aperture, and a pale green phenotype. Map-based analysis of the LOST1 locus revealed that the lost1 mutant resulted from a missense mutation in the Mg-chelatase I subunit 1 (CHLI1) gene, which encodes a subunit of the Mg-chelatase complex involved in chlorophyll synthesis. Transformation of the wild-type CHLI1 gene into lost1 complemented all lost1 phenotypes. Stomata in lost1 exhibited a partial ABA-insensitive phenotype similar to that of rtl1, a Mg-chelatase H subunit missense mutant. The Mg-protoporphyrin IX methyltransferase (CHLM) gene encodes a subsequent enzyme in the chlorophyll synthesis pathway. We examined stomatal movement in a CHLM knockdown mutant, chlm, and found that it also exhibited an ABA-insensitive phenotype. However, lost1 and chlm seedlings all showed normal expression of ABA-induced genes, such as RAB18 and RD29B, in response to ABA. These results suggest that the chlorophyll synthesis enzymes, Mg-chelatase complex and CHLM, specifically affect ABA signaling in the control of stomatal aperture and have no effect on ABA-induced gene expression. PMID:24840863

  18. GABAB(1) receptor subunit isoforms differentially regulate stress resilience.

    PubMed

    O'Leary, Olivia F; Felice, Daniela; Galimberti, Stefano; Savignac, Hélène M; Bravo, Javier A; Crowley, Tadhg; El Yacoubi, Malika; Vaugeois, Jean-Marie; Gassmann, Martin; Bettler, Bernhard; Dinan, Timothy G; Cryan, John F

    2014-10-21

    Stressful life events increase the susceptibility to developing psychiatric disorders such as depression; however, many individuals are resilient to such negative effects of stress. Determining the neurobiology underlying this resilience is instrumental to the development of novel and more effective treatments for stress-related psychiatric disorders. GABAB receptors are emerging therapeutic targets for the treatment of stress-related disorders such as depression. These receptors are predominantly expressed as heterodimers of a GABAB(2) subunit with either a GABAB(1a) or a GABAB(1b) subunit. Here we show that mice lacking the GABAB(1b) receptor isoform are more resilient to both early-life stress and chronic psychosocial stress in adulthood, whereas mice lacking GABAB(1a) receptors are more susceptible to stress-induced anhedonia and social avoidance compared with wild-type mice. In addition, increased hippocampal expression of the GABAB(1b) receptor subunit is associated with a depression-like phenotype in the helpless H/Rouen genetic mouse model of depression. Stress resilience in GABAB(1b)(-/-) mice is coupled with increased proliferation and survival of newly born cells in the adult ventral hippocampus and increased stress-induced c-Fos activation in the hippocampus following early-life stress. Taken together, the data suggest that GABAB(1) receptor subunit isoforms differentially regulate the deleterious effects of stress and, thus, may be important therapeutic targets for the treatment of depression. PMID:25288769

  19. CMF70 is a subunit of the dynein regulatory complex.

    PubMed

    Kabututu, Zakayi P; Thayer, Michelle; Melehani, Jason H; Hill, Kent L

    2010-10-15

    Flagellar motility drives propulsion of several important pathogens and is essential for human development and physiology. Motility of the eukaryotic flagellum requires coordinate regulation of thousands of dynein motors arrayed along the axoneme, but the proteins underlying dynein regulation are largely unknown. The dynein regulatory complex, DRC, is recognized as a focal point of axonemal dynein regulation, but only a single DRC subunit, trypanin/PF2, is currently known. The component of motile flagella 70 protein, CMF70, is broadly and uniquely conserved among organisms with motile flagella, suggesting a role in axonemal motility. Here we demonstrate that CMF70 is part of the DRC from Trypanosoma brucei. CMF70 is located along the flagellum, co-sediments with trypanin in sucrose gradients and co-immunoprecipitates with trypanin. RNAi knockdown of CMF70 causes motility defects in a wild-type background and suppresses flagellar paralysis in cells with central pair defects, thus meeting the functional definition of a DRC subunit. Trypanin and CMF70 are mutually conserved in at least five of six extant eukaryotic clades, indicating that the DRC was probably present in the last common eukaryotic ancestor. We have identified only the second known subunit of this ubiquitous dynein regulatory system, highlighting the utility of combined genomic and functional analyses for identifying novel subunits of axonemal sub-complexes. PMID:20876659

  20. Transmembrane topography of the nicotinic acetylcholine receptor delta subunit.

    PubMed

    McCrea, P D; Popot, J L; Engelman, D M

    1987-12-01

    Current folding models for the nicotinic acetylcholine receptor (AChR) predict either four or five transmembrane segments per subunit. The N-terminus of each subunit is almost certainly extracellular. We have tested folding models by determining biochemically the cellular location of an intermolecular disulfide bridge thought to lie at the delta subunit C-terminus. Dimers of AChR linked through the delta-delta bridge were prepared from Torpedo marmorata and T.californica electric organ. The disulfide's accessibility to hydrophilic reductants was tested in a reconstituted vesicle system. In right-side-out vesicles (greater than 95% ACh binding sites outwards), the bridge was equally accessible whether or not vesicles had been disrupted by freeze--thawing or by detergents. Control experiments based on the rate of reduction of entrapped diphtheria toxin and measurements of radioactive reductant efflux demonstrated that the vesicles provide an adequate permeability barrier. In reconstituted vesicles containing AChR dimers in scrambled orientations, right-side-out dimers were reduced to monomers three times more rapidly than inside-out dimers, consistent with the measured rate of reductant permeation. These observations indicate that in reconstituted vesicles the delta-delta disulfide bridge lies in the same aqueous space as the ACh binding sites. They are most easily reconciled with folding models that propose an even number of transmembrane crossing per subunit. PMID:3428268

  1. The Essential Anatomical Subunit Approximation Unilateral Cleft Lip Repair.

    PubMed

    Chong, David K; Swanson, Jordan W

    2016-07-01

    The anatomical subunit approximation cleft lip repair advantageously achieves a balanced lip contour, with the line of repair hidden along seams of aesthetic subunits. Dr. David Fisher's original description of the repair reflects the considerable thought that went into the evolution of his design. As his technique has gained acceptance in the intervening 10 years, the authors note several key principles embodied in it that represent a shift in the cleft lip repair paradigm. The authors believe understanding these principles is important to mastery of the anatomical subunit technique, and facilitate its teaching. First, design a plan that adheres to anatomical subunits and perform measurements precisely. Second, identify and adequately release each cleft tissue layer from the lip and nose to enable restoration of balance. Third, drive surgical approximation through inset of the lateral muscle into the superiorly backcut medial orbicularis muscle, followed by skin closure with inferior triangle interposition above the white roll. In this article, the authors present essential components of the technique, and identify several principles that enable its successful execution. PMID:27348690

  2. Bacterial cellulose biosynthesis: diversity of operons, subunits, products and functions

    PubMed Central

    Römling, Ute; Galperin, Michael Y.

    2015-01-01

    Summary Recent studies of bacterial cellulose biosynthesis, including structural characterization of a functional cellulose synthase complex, provided the first mechanistic insight into this fascinating process. In most studied bacteria, just two subunits, BcsA and BcsB, are necessary and sufficient for the formation of the polysaccharide chain in vitro. Other subunits – which differ among various taxa – affect the enzymatic activity and product yield in vivo by modulating expression of biosynthesis apparatus, export of the nascent β-D-glucan polymer to the cell surface, and the organization of cellulose fibers into a higher-order structure. These auxiliary subunits play key roles in determining the quantity and structure of the resulting biofilm, which is particularly important for interactions of bacteria with higher organisms that lead to rhizosphere colonization and modulate virulence of cellulose-producing bacterial pathogens inside and outside of host cells. Here we review the organization of four principal types of cellulose synthase operons found in various bacterial genomes, identify additional bcs genes that encode likely components of the cellulose biosynthesis and secretion machinery, and propose a unified nomenclature for these genes and subunits. We also discuss the role of cellulose as a key component of biofilms formed by a variety of free-living and pathogenic bacteria and, for the latter, in the choice between acute infection and persistence in the host. PMID:26077867

  3. Insecticidal Pilin Subunit from the Insect Pathogen Xenorhabdus nematophila

    PubMed Central

    Khandelwal, Puneet; Choudhury, Devapriya; Birah, Ajanta; Reddy, M. K.; Gupta, Gorakh Prasad; Banerjee, Nirupama

    2004-01-01

    Xenorhabdus nematophila is an insect pathogen and produces protein toxins which kill the larval host. Previously, we characterized an orally toxic, large, outer membrane-associated protein complex from the culture medium of X. nematophila. Here, we describe the cloning, expression, and characterization of a 17-kDa pilin subunit of X. nematophila isolated from that protein complex. The gene was amplified by PCR, cloned, and expressed in Escherichia coli. The recombinant protein was refolded in vitro in the absence of its cognate chaperone by using a urea gradient. The protein oligomerized during in vitro refolding, forming multimers. Point mutations in the conserved N-terminal residues of the pilin protein greatly destabilized its oligomeric organization, demonstrating the importance of the N terminus in refolding and oligomerization of the pilin subunit by donor strand complementation. The recombinant protein was cytotoxic to cultured Helicoverpa armigera larval hemocytes, causing agglutination and subsequent release of the cytoplasmic enzyme lactate dehydrogenase. The agglutination of larval cells by the 17-kDa protein was inhibited by several sugar derivatives. The biological activity of the purified recombinant protein indicated that it has a conformation similar to that of the native protein. The 17-kDa pilin subunit was found to be orally toxic to fourth- or fifth-instar larvae of an important crop pest, H. armigera, causing extensive damage to the midgut epithelial membrane. To our knowledge, this is first report describing an insecticidal pilin subunit of a bacterium. PMID:15375127

  4. Bacterial cellulose biosynthesis: diversity of operons, subunits, products, and functions.

    PubMed

    Römling, Ute; Galperin, Michael Y

    2015-09-01

    Recent studies of bacterial cellulose biosynthesis, including structural characterization of a functional cellulose synthase complex, provided the first mechanistic insight into this fascinating process. In most studied bacteria, just two subunits, BcsA and BcsB, are necessary and sufficient for the formation of the polysaccharide chain in vitro. Other subunits - which differ among various taxa - affect the enzymatic activity and product yield in vivo by modulating (i) the expression of the biosynthesis apparatus, (ii) the export of the nascent β-D-glucan polymer to the cell surface, and (iii) the organization of cellulose fibers into a higher-order structure. These auxiliary subunits play key roles in determining the quantity and structure of resulting biofilms, which is particularly important for the interactions of bacteria with higher organisms - leading to rhizosphere colonization and modulating the virulence of cellulose-producing bacterial pathogens inside and outside of host cells. We review the organization of four principal types of cellulose synthase operon found in various bacterial genomes, identify additional bcs genes that encode components of the cellulose biosynthesis and secretion machinery, and propose a unified nomenclature for these genes and subunits. We also discuss the role of cellulose as a key component of biofilms and in the choice between acute infection and persistence in the host. PMID:26077867

  5. Spectroscopic properties of Carcinus aestuarii hemocyanin and its structural subunits

    NASA Astrophysics Data System (ADS)

    Dolashka-Angelova, Pavlina; Hristova, Rumiyana; Stoeva, Stanka; Voelter, Wolfgang

    1999-12-01

    Hemocyanin (Hc) of Carcinus aestuarii contains three major and one minor electrophoretically separable polypeptide chains which were purified by fast protein liquid chromatography (FPLC) ion exchange chromatography. N-terminal amino acid sequences of four structural subunits (SSs) from C. aestuarii were compared with known N-terminal sequences from other arthropodan hemocyanins. The conformational changes, induced by various treatments, were monitored by far UV, CD and fluorescence spectroscopy. The critical temperatures for the structural subunits, Tc, determined by fluorescence spectroscopy, are in the region of 52-59°C and coincide with the melting temperatures, Tm (49-55°C), determined by CD spectroscopy. The free energy of stabilization in water, Δ GDH 2O , toward guanidinium hydrochloride is about 1.3 times higher for the dodecameric Hc as compared to the isolated subunits and about one time higher for Ca1, comparing with other SSs. The studies reveal that the conformational stability of the native dodecamer towards various denaturants (temperature and guanidinium hydrochloride) indicate that the quaternary structure is stabilized by oligomerization between structural subunits, and the possibility of a structural role of the sugar mojeties cannot be excluded.

  6. The receptor subunits generating NMDA receptor mediated currents in oligodendrocytes

    PubMed Central

    Burzomato, Valeria; Frugier, Guillaume; Pérez-Otaño, Isabel; Kittler, Josef T; Attwell, David

    2010-01-01

    NMDA receptors have been shown to contribute to glutamate-evoked currents in oligodendrocytes. Activation of these receptors damages myelin in ischaemia, in part because they are more weakly blocked by Mg2+ than are most neuronal NMDA receptors. This weak Mg2+ block was suggested to reflect an unusual subunit composition including the NR2C and NR3A subunits. Here we expressed NR1/NR2C and triplet NR1/NR2C/NR3A recombinant receptors in HEK cells and compared their currents with those of NMDA-evoked currents in rat cerebellar oligodendrocytes. NR1/NR2C/3A receptors were less blocked by 2 mm Mg2+ than were NR1/NR2C receptors (the remaining current was 30% and 18%, respectively, of that seen without added Mg2+) and showed less channel noise, suggesting a smaller single channel conductance. NMDA-evoked currents in oligodendrocytes showed a Mg2+ block (to 32%) similar to that observed for NR1/NR2C/NR3A and significantly different from that for NR1/NR2C receptors. Co-immunoprecipitation revealed interactions between NR1, NR2C and NR3A subunits in a purified myelin preparation from rat brain. These data are consistent with NMDA-evoked currents in oligodendrocytes reflecting the activation of receptors containing NR1, NR2C and NR3A subunits. PMID:20660562

  7. Transmembrane topography of the nicotinic acetylcholine receptor delta subunit.

    PubMed Central

    McCrea, P D; Popot, J L; Engelman, D M

    1987-01-01

    Current folding models for the nicotinic acetylcholine receptor (AChR) predict either four or five transmembrane segments per subunit. The N-terminus of each subunit is almost certainly extracellular. We have tested folding models by determining biochemically the cellular location of an intermolecular disulfide bridge thought to lie at the delta subunit C-terminus. Dimers of AChR linked through the delta-delta bridge were prepared from Torpedo marmorata and T.californica electric organ. The disulfide's accessibility to hydrophilic reductants was tested in a reconstituted vesicle system. In right-side-out vesicles (greater than 95% ACh binding sites outwards), the bridge was equally accessible whether or not vesicles had been disrupted by freeze--thawing or by detergents. Control experiments based on the rate of reduction of entrapped diphtheria toxin and measurements of radioactive reductant efflux demonstrated that the vesicles provide an adequate permeability barrier. In reconstituted vesicles containing AChR dimers in scrambled orientations, right-side-out dimers were reduced to monomers three times more rapidly than inside-out dimers, consistent with the measured rate of reductant permeation. These observations indicate that in reconstituted vesicles the delta-delta disulfide bridge lies in the same aqueous space as the ACh binding sites. They are most easily reconciled with folding models that propose an even number of transmembrane crossing per subunit. PMID:3428268

  8. Two orthogonal cleavages separate subunit RNAs in mouse ribosome biogenesis

    PubMed Central

    Wang, Minshi; Anikin, Leonid; Pestov, Dimitri G.

    2014-01-01

    Ribosome biogenesis is a dynamic multistep process, many features of which are still incompletely documented. Here, we show that changes in this pathway can be captured and annotated by means of a graphic set of pre-rRNA ratios, a technique we call Ratio Analysis of Multiple Precursors (RAMP). We find that knocking down a ribosome synthesis factor produces a characteristic RAMP profile that exhibits consistency across a range of depletion levels. This facilitates the inference of affected steps and simplifies comparative analysis. We applied RAMP to examine how endonucleolytic cleavages of the mouse pre-rRNA transcript in the internal transcribed spacer 1 (ITS1) are affected by depletion of factors required for maturation of the small ribosomal subunit (Rcl1, Fcf1/Utp24, Utp23) and the large subunit (Pes1, Nog1). The data suggest that completion of early maturation in a subunit triggers its release from the common pre-rRNA transcript by stimulating cleavage at the proximal site in ITS1. We also find that splitting of pre-rRNA in the 3′ region of ITS1 is prevalent in adult mouse tissues and quiescent cells, as it is in human cells. We propose a model for subunit separation during mammalian ribosome synthesis and discuss its implications for understanding pre-rRNA processing pathways. PMID:25190460

  9. Emergence of ion channel modal gating from independent subunit kinetics.

    PubMed

    Bicknell, Brendan A; Goodhill, Geoffrey J

    2016-09-01

    Many ion channels exhibit a slow stochastic switching between distinct modes of gating activity. This feature of channel behavior has pronounced implications for the dynamics of ionic currents and the signaling pathways that they regulate. A canonical example is the inositol 1,4,5-trisphosphate receptor (IP3R) channel, whose regulation of intracellular Ca(2+) concentration is essential for numerous cellular processes. However, the underlying biophysical mechanisms that give rise to modal gating in this and most other channels remain unknown. Although ion channels are composed of protein subunits, previous mathematical models of modal gating are coarse grained at the level of whole-channel states, limiting further dialogue between theory and experiment. Here we propose an origin for modal gating, by modeling the kinetics of ligand binding and conformational change in the IP3R at the subunit level. We find good agreement with experimental data over a wide range of ligand concentrations, accounting for equilibrium channel properties, transient responses to changing ligand conditions, and modal gating statistics. We show how this can be understood within a simple analytical framework and confirm our results with stochastic simulations. The model assumes that channel subunits are independent, demonstrating that cooperative binding or concerted conformational changes are not required for modal gating. Moreover, the model embodies a generally applicable principle: If a timescale separation exists in the kinetics of individual subunits, then modal gating can arise as an emergent property of channel behavior. PMID:27551100

  10. Abnormal subcellular localization of GABAA receptor subunits in schizophrenia brain.

    PubMed

    Mueller, T M; Remedies, C E; Haroutunian, V; Meador-Woodruff, J H

    2015-01-01

    Inhibitory neurotransmission is primarily mediated by γ-aminobutyric acid (GABA) activating synaptic GABA type A receptors (GABA(A)R). In schizophrenia, presynaptic GABAergic signaling deficits are among the most replicated findings; however, postsynaptic GABAergic deficits are less well characterized. Our lab has previously demonstrated that although there is no difference in total protein expression of the α1-6, β1-3 or γ2 GABA(A)R subunits in the superior temporal gyrus (STG) in schizophrenia, the α1, β1 and β2 GABA(A)R subunits are abnormally N-glycosylated. N-glycosylation is a posttranslational modification that has important functional roles in protein folding, multimer assembly and forward trafficking. To investigate the impact that altered N-glycosylation has on the assembly and trafficking of GABA(A)Rs in schizophrenia, this study used western blot analysis to measure the expression of α1, α2, β1, β2 and γ2 GABA(A)R subunits in subcellular fractions enriched for endoplasmic reticulum (ER) and synapses (SYN) from STG of schizophrenia (N = 16) and comparison (N = 14) subjects and found evidence of abnormal localization of the β1 and β2 GABA(A)R subunits and subunit isoforms in schizophrenia. The β2 subunit is expressed as three isoforms at 52 kDa (β2(52 kDa)), 50 kDa (β2(50 kDa)) and 48 kDa (β2(48 kDa)). In the ER, we found increased total β2 GABA(A)R subunit (β2(ALL)) expression driven by increased β2(50 kDa), a decreased ratio of β(248 kDa):β2(ALL) and an increased ratio of β2(50 kDa):β2(48 kDa). Decreased ratios of β1:β2(ALL) and β1:β2(50 kDa) in both the ER and SYN fractions and an increased ratio of β2(52 kDa):β(248 kDa) at the synapse were also identified in schizophrenia. Taken together, these findings provide evidence that alterations of N-glycosylation may contribute to GABAergic signaling deficits in schizophrenia by disrupting the assembly and trafficking of GABA(A)Rs. PMID:26241350

  11. Abnormal subcellular localization of GABAA receptor subunits in schizophrenia brain

    PubMed Central

    Mueller, T M; Remedies, C E; Haroutunian, V; Meador-Woodruff, J H

    2015-01-01

    Inhibitory neurotransmission is primarily mediated by γ-aminobutyric acid (GABA) activating synaptic GABA type A receptors (GABAAR). In schizophrenia, presynaptic GABAergic signaling deficits are among the most replicated findings; however, postsynaptic GABAergic deficits are less well characterized. Our lab has previously demonstrated that although there is no difference in total protein expression of the α1–6, β1–3 or γ2 GABAAR subunits in the superior temporal gyrus (STG) in schizophrenia, the α1, β1 and β2 GABAAR subunits are abnormally N-glycosylated. N-glycosylation is a posttranslational modification that has important functional roles in protein folding, multimer assembly and forward trafficking. To investigate the impact that altered N-glycosylation has on the assembly and trafficking of GABAARs in schizophrenia, this study used western blot analysis to measure the expression of α1, α2, β1, β2 and γ2 GABAAR subunits in subcellular fractions enriched for endoplasmic reticulum (ER) and synapses (SYN) from STG of schizophrenia (N=16) and comparison (N=14) subjects and found evidence of abnormal localization of the β1 and β2 GABAAR subunits and subunit isoforms in schizophrenia. The β2 subunit is expressed as three isoforms at 52 kDa (β252 kDa), 50 kDa (β250 kDa) and 48 kDa (β248 kDa). In the ER, we found increased total β2 GABAAR subunit (β2ALL) expression driven by increased β250 kDa, a decreased ratio of β248 kDa:β2ALL and an increased ratio of β250 kDa:β248 kDa. Decreased ratios of β1:β2ALL and β1:β250 kDa in both the ER and SYN fractions and an increased ratio of β252 kDa:β248 kDa at the synapse were also identified in schizophrenia. Taken together, these findings provide evidence that alterations of N-glycosylation may contribute to GABAergic signaling deficits in schizophrenia by disrupting the assembly and trafficking of GABAARs. PMID:26241350

  12. Succinate dehydrogenase subunit D and succinate dehydrogenase subunit B mutation analysis in canine phaeochromocytoma and paraganglioma.

    PubMed

    Holt, D E; Henthorn, P; Howell, V M; Robinson, B G; Benn, D E

    2014-07-01

    Phaeochromocytomas (PCs) are tumours of the adrenal medulla chromaffin cells. Paragangliomas (PGLs) arise in sympathetic ganglia (previously called extra-adrenal PCs) or in non-chromaffin parasympathetic ganglia cells that are usually non-secretory. Parenchymal cells from these tumours have a common embryological origin from neural crest ectoderm. Several case series of canine PCs and PGLs have been published and a link between the increased incidence of chemoreceptor neoplasia in brachycephalic dog breeds and chronic hypoxia has been postulated. A similar link to hypoxia in man led to the identification of germline heterozygous mutations in the gene encoding succinate dehydrogenase subunit D (SDHD) and subsequently SDHA, SDHB and SDHC in similar tumours. We investigated canine PCs (n = 6) and PGLs (n = 2) for SDHD and SDHB mutations and in one PGL found a somatic SDHD mutation c.365A>G (p.Lys122Arg) in exon 4, which was not present in normal tissue from this brachycephalic dog. Two PCs were heterozygous for both c.365A>G (p.Lys122Arg) mutation and an exon 3 silent variant c.291G>A. We also identified the heterozygous SDHB exon 2 mutation c.113G>A (p.Arg38Gln) in a PC. These results illustrate that genetic mutations may underlie tumourigenesis in canine PCs and PGLs. The spontaneous nature of these canine diseases and possible association of PGLs with hypoxia in brachycephalic breeds may make them an attractive model for studying the corresponding human tumours. PMID:24813157

  13. Heterotrimeric G protein subunit Gγ13 is critical to olfaction

    PubMed Central

    Li, Feng; Ponissery-Saidu, Samsudeen; Yee, Karen; Wang, Hong; Chen, Meng-Ling; Iguchi, Naoko; Zhang, Genhua; Jiang, Ping; Reisert, Johannes; Huang, Liquan

    2013-01-01

    The activation of G-protein-coupled olfactory receptors on the olfactory sensory neurons (OSNs) triggers a signaling cascade, which is mediated by a heterotrimeric G protein consisting of α, β and γ subunits. Although its α subunit, Gαolf, has been identified and well characterized, the identities of its β and γ subunits and their function in olfactory signal transduction, however, have not been well established yet. We and others have found the expression of Gγ13 in the olfactory epithelium, particularly in the cilia of the OSNs. In this study, we generated a conditional gene knockout mouse line to specifically nullify Gγ13 expression in the olfactory marker protein-expressing OSNs. Immunohistochemical and Western blot results showed that Gγ13 subunit was indeed eliminated in the mutant mice’s olfactory epithelium. Intriguingly, Gαolf, β1 subunits, Ric-8B and CEP290 proteins were also absent in the epithelium whereas the presence of the effector enzyme adenylyl cyclase III remained largely unaltered. Electro-olfactogram studies showed that the mutant animals had greatly reduced responses to a battery of odorants including three presumable pheromones. Behavioral tests indicated that the mutant mice had a remarkably reduced ability to perform an odor-guided search task although their motivation and agility seemed normal. Our results indicate that Gαolf exclusively forms a functional heterotrimeric G protein with Gβ1 and Gγ13 in OSNs, mediating olfactory signal transduction. The identification of the olfactory G protein’s βγ moiety has provided a novel approach to understanding the feedback regulation of olfactory signal transduction pathways as well as the control of subcellular structures of OSNs. PMID:23637188

  14. Antibodies to GABAA receptor α1 and γ2 subunits

    PubMed Central

    Pettingill, Philippa; Kramer, Holger B.; Coebergh, Jan Adriaan; Pettingill, Rosie; Maxwell, Susan; Nibber, Anjan; Malaspina, Andrea; Jacob, Anu; Irani, Sarosh R.; Buckley, Camilla; Beeson, David; Lang, Bethan; Waters, Patrick

    2015-01-01

    Objective: To search for antibodies against neuronal cell surface proteins. Methods: Using immunoprecipitation from neuronal cultures and tandem mass spectrometry, we identified antibodies against the α1 subunit of the γ-aminobutyric acid A receptor (GABAAR) in a patient whose immunoglobulin G (IgG) antibodies bound to hippocampal neurons. We searched 2,548 sera for antibodies binding to GABAAR α, β, and γ subunits on live HEK293 cells and identified the class, subclass, and GABAAR subunit specificities of the positive samples. Results: GABAAR-Abs were identified in 40 of 2,046 (2%) referred sera previously found negative for neuronal antibodies, in 5/502 (1%) previously positive for other neuronal surface antibodies, but not in 92 healthy individuals. The antibodies in 40% bound to either the α1 (9/45, 20%) or the γ2 subunits (9/45, 20%) and were of IgG1 (94%) or IgG3 (6%) subclass. The remaining 60% had lower antibody titers (p = 0.0005), which were mainly immunoglobulin M (IgM) (p = 0.0025), and showed no defined subunit specificity. Incubation of primary hippocampal neurons with GABAAR IgG1 sera reduced surface GABAAR membrane expression. The clinical features of 15 patients (GABAAR α1 n = 6, γ2 n = 5, undefined n = 4) included seizures (47%), memory impairment (47%), hallucinations (33%), or anxiety (20%). Most patients had not been given immunotherapies, but one with new-onset treatment-resistant catatonia made substantial improvement after plasma exchange. Conclusions: The GABAAR α1 and γ2 are new targets for antibodies in autoimmune neurologic disease. The full spectrum of clinical features, treatment responses, correlation with antibody specificity, and in particular the role of the IgM antibodies will need to be assessed in future studies. PMID:25636713

  15. Specific interaction between EF-G and RRF and its implication for GTP-dependent ribosome splitting into subunits

    PubMed Central

    Gao, Ning; Zavialov, Andrey V.; Ehrenberg, Måns; Frank, Joachim

    2008-01-01

    Summary After termination of protein synthesis, the bacterial ribosome is split into its 30S and 50S subunits by the action of ribosome recycling factor (RRF) and elongation factor G (EF-G) in a GTP-hydrolysis dependent manner. Based on a previous cryo-electron microscopy (cryo-EM) study of ribosomal complexes, we have proposed that the binding of EF-G to an RRF containing post-termination ribosome triggers an inter-domain rotation of RRF, which destabilizes two strong intersubunit bridges (B2a and B3) and, ultimately, separates the two subunits. Here, we present a 9 Å (FSC at 0.5 cutoff) cryo-EM map of a 50S EFG GDPNP RRF complex and a quasi-atomic model derived from it, showing the interaction between EF-G and RRF on the 50S subunit in the presence of the non-cleavable GTP analogue GDPNP. The detailed information in this model and a comparative analysis of EF-G structures in various nucleotide- and ribosome-bound states show how rotation of the RRF head domain may be triggered by various domains of EF-G. For validation of our structural model, all known mutations in EF-G and RRF that relate to ribosome recycling have been taken into account. More importantly, our results indicate a substantial conformational change in the Switch I region of EF-G, suggesting that a conformational signal transduction mechanism, similar to that employed in tRNA translocation on the ribosome by EF-G, translates a large-scale movement of EF-G’s domain IV, induced by GTP hydrolysis, into the domain rotation of RRF that eventually splits the ribosome into subunits. PMID:17996252

  16. Pharmacogenomics: mapping monogenic mutations to direct therapy.

    PubMed

    Taylor, Palmer

    2012-07-01

    The molecular mapping of mutations that underlie congenital disorders of monogenic origin can result in both a broader understanding of the molecular basis of the disorder and novel therapeutic insights. Indeed, genotyping patients and then replicating the behavior of the mutant gene products in well-defined biochemical or electrophysiological systems will allow tailoring of therapy to be mutation- and protein sequence-dependent. In this issue of the JCI, Shen and colleagues describe such an approach that identified novel mutations in the α subunit of the nicotinic receptor linked to myasthenia gravis. PMID:22728931

  17. Catalytic Subunit 1 of Protein Phosphatase 2A Is a Subunit of the STRIPAK Complex and Governs Fungal Sexual Development

    PubMed Central

    Beier, Anna; Krisp, Christoph; Wolters, Dirk A.

    2016-01-01

    ABSTRACT The generation of complex three-dimensional structures is a key developmental step for most eukaryotic organisms. The details of the molecular machinery controlling this step remain to be determined. An excellent model system to study this general process is the generation of three-dimensional fruiting bodies in filamentous fungi like Sordaria macrospora. Fruiting body development is controlled by subunits of the highly conserved striatin-interacting phosphatase and kinase (STRIPAK) complex, which has been described in organisms ranging from yeasts to humans. The highly conserved heterotrimeric protein phosphatase PP2A is a subunit of STRIPAK. Here, catalytic subunit 1 of PP2A was functionally characterized. The Δpp2Ac1 strain is sterile, unable to undergo hyphal fusion, and devoid of ascogonial septation. Further, PP2Ac1, together with STRIPAK subunit PRO22, governs vegetative and stress-related growth. We revealed in vitro catalytic activity of wild-type PP2Ac1, and our in vivo analysis showed that inactive PP2Ac1 blocks the complementation of the sterile deletion strain. Tandem affinity purification, followed by mass spectrometry and yeast two-hybrid analysis, verified that PP2Ac1 is a subunit of STRIPAK. Further, these data indicate links between the STRIPAK complex and other developmental signaling pathways, implying the presence of a large interconnected signaling network that controls eukaryotic developmental processes. The insights gained in our study can be transferred to higher eukaryotes and will be important for understanding eukaryotic cellular development in general. PMID:27329756

  18. Subunit composition and chromophore content of R-phycoerythrin from Porphyra haitanensis

    NASA Astrophysics Data System (ADS)

    Gao, Hong-Feng; Ji, Ming-Hou; Cao, Wen-Da

    1996-03-01

    R-phycoerythrin from Porphyra haitanensis exists in two aggregation states with different molecular weights. A more highly aggregated form, RPE I, was chromatographed on Bio-Rex 70 column with urea solution (pH 3.0) as eluent, and the molecular weights of the 3 subunits (α, β, γ) obtained were determined on SDS-PAGE at 18000, 19200 and 30000, respectively. α subunit carried two phycoerythrobilin (PEB); β subunit, three PEB and one phycourobilin (PUB); γ subunit, one PEB and three PUB chromophores. The molar ratio of α, β, and γ subunits of RPE I was 6: 6: 1, and their subunit composition was confired to be (αβ)6γ on account of the molecular weight of RPE I, 232000. A lower aggregated form, RPE II, contained α and β subunits similar to those of RPE I, but its subunit composition was the (αβ) monomer of RPE.

  19. Voltage-gated Na+ channels: Potential for β subunits as therapeutic targets

    PubMed Central

    Brackenbury, William J.; Isom, Lori L.

    2012-01-01

    Background Voltage gated Na+ channels (VGSCs) contain a pore-forming α subunit and one or more β subunits. VGSCs are involved in a wide variety of pathophysiologies, including epilepsy, cardiac arrhythmia, Multiple Sclerosis, periodic paralysis, migraine, neuropathic and inflammatory pain, Huntington’s disease, and cancer. Increasing evidence implicates the β subunits as key players in these disorders. Objective The purpose of this report is to review the recent literature describing the multifunctional roles of VGSC β subunits in the context of their role(s) in disease. Methods An extensive review of the literature on β subunits was performed. Results/conclusion β subunits are multifunctional. As components of VGSC complexes, β subunits mediate signaling processes regulating electrical excitability, adhesion, migration, pathfinding, and transcription. β subunits may prove useful in disease diagnosis and therapy. PMID:18694383

  20. GABAA receptor beta subunit heterogeneity: functional expression of cloned cDNAs.

    PubMed Central

    Ymer, S; Schofield, P R; Draguhn, A; Werner, P; Köhler, M; Seeburg, P H

    1989-01-01

    Cloned cDNAs encoding two new beta subunits of the rat and bovine GABAA receptor have been isolated using a degenerate oligonucleotide probe based on a highly conserved peptide sequence in the second transmembrane domain of GABAA receptor subunits. The beta 2 and beta 3 subunits share approximately 72% sequence identity with the previously characterized beta 1 polypeptide. Northern analysis showed that both beta 2 and beta 3 mRNAs are more abundant in the brain than beta 1 mRNA. All three beta subunit encoding cDNAs were also identified in a library constructed from adrenal medulla RNA. Each beta subunit, when co-expressed in Xenopus oocytes with an alpha subunit, forms functional GABAA receptors. These results, together with the known alpha subunit heterogeneity, suggest that a variety of related but functionally distinct GABAA receptor subtypes are generated by different subunit combinations. Images PMID:2548852

  1. Characterization of the regulatory subunit from brain cyclic AMP-dependent protein kinase II

    SciTech Connect

    Stein, J.C.

    1985-01-01

    Tryptic peptides derived from the regulatory subunits of brain and heart cAMP-dependent protein kinase II were mapped by reverse phase HPLC. At 280 nm, 15 unique peptides were found only in the heart RII digest, while 5 other peptides were obtained only from brain RII. At 210 nm, 13 brain-RII specific and 15 heart-RII specific tryptic peptides were identified and resolved. Two-dimensional mapping analyses revealed that several /sup 37/P-labeled tryptic fragments derived from the autophosphorylation and the photoaffinity labeled cAMP-binding sites of brain RII were separate and distinct from the /sup 32/P-peptides isolated from similarly treated heart RII. The tryptic phosphopeptide containing the autophosphorylation site in brain RII was purified. The sequence and phosphorylation site is: Arg-Ala-Ser(P)-Val-Cys-Ala-Glu-Ala-Tyr-Asn-Pro-Asp-Glu-Glu-Glu-Asp-Asp-Ala-Glu. Astrocytes and neurons exhibit high levels of the brain RII enzyme, while oligodendrocytes contain the heart RII enzyme. Monoclonal antibodies to bovine cerebral cortex RII were made and characterized. The antibodies elucidated a subtle difference between membrane-associated and cytosolic RII from cerebral cortex.

  2. Regulation of expression of a soybean storage protein subunit gene. Progress report

    SciTech Connect

    Thompson, J.F.; Madison, J.T.

    1984-04-23

    We have found that the methionine repression of the ..beta..-subunit gene expression is not due to degradation of the ..beta..-subunit but is due to an effect on synthesis of the ..beta..-subunit. The effect of methionine on the synthesis of the ..beta..-is due to an inhibition of ..beta..-subunit mRNA synthesis. 3 references, 1 figure.

  3. Reverse Genetics of Drosophila RNA Polymerase II: Identification and Characterization of Rpii140, the Genomic Locus for the Second-Largest Subunit

    PubMed Central

    Hamilton, B. J.; Mortin, M. A.; Greenleaf, A. L.

    1993-01-01

    We have used a reverse genetics approach to isolate genes encoding two subunits of Drosophila melanogaster RNA polymerase II. RpII18 encodes the 18-kDa subunit and maps cytogenetically to polytene band region 83A. RpII140 encodes the 140-kDa subunit and maps to polytene band region 88A10:B1,2. Focusing on RpII140, we used in situ hybridization to map this gene to a small subinterval defined by the endpoints of a series of deficiencies impinging on the 88A/B region and showed that it does not represent a previously known genetic locus. Two recently defined complementation groups, A5 and Z6, reside in the same subinterval and thus were candidates for the RpII140 locus. Phenotypes of A5 mutants suggested that they affect RNA polymerase II, in that the lethal phase and the interaction with developmental loci such as Ubx resemble those of mutants in the gene for the largest subunit, RpII215. Indeed, we have achieved complete genetic rescue of representative recessive lethal mutations of A5 with a P-element construct containing a 9.1-kb genomic DNA fragment carrying RpII140. Interestingly, the initial construct also rescued lethal alleles in the neighboring complementation group, Z6, revealing that the 9.1-kb insert carries two genes. Deleting coding region sequences of RpII140, however, yielded a transformation vector that failed to rescue A5 alleles but continued to rescue Z6 alleles. These results strongly support the conclusion that the A5 complementation group is equivalent to the genomic RpII140 locus. PMID:8325487

  4. The structure of cucurbitin: subunit symmetry and organization in situ.

    PubMed

    Colman, P M; Suzuki, E; Van Donkelaar, A

    1980-02-01

    The low-resolution (2 nm) subunit symmetry of cucurbitin, the crystalline seed storage globulin of cucurbits, has been determined by X-ray diffraction. The wet crystals belong to the cubic space group F23 and there are 4 molecules per unit cell. The molecules therefore possess point-group symmetry 23 and contain 12 structural units which at this resolution are indistinguishable. On drying, the crystal lattice dimension shrinks from 13.6 nm to 12.4 nm with no apparent change in symmetry. Diffraction patterns of small crystals spun into a pellet, and sections of dry and wet native seed indicate that in situ the protein is organised in microcrystals of the same unit cell and symmetry. Edestin, the crystalline storage globulin from cannabis, and a crystalline globulin from tobacco seed both have the same crystal lattice as cucurbitin and, very likely, the same subunit symmetry. PMID:7358051

  5. Complete subunit architecture of the proteasome regulatory particle

    PubMed Central

    Lander, Gabriel C.; Estrin, Eric; Matyskiela, Mary E.; Bashore, Charlene; Nogales, Eva; Martin, Andreas

    2011-01-01

    The proteasome is the major ATP-dependent protease in eukaryotic cells, but limited structural information strongly restricts a mechanistic understanding of its activities. The proteasome regulatory particle, consisting of the lid and base subcomplexes, recognizes and processes poly-ubiquitinated substrates. We used electron microscopy and a newly-developed heterologous expression system for the lid to delineate the complete subunit architecture of the regulatory particle. Our studies reveal the spatial arrangement of ubiquitin receptors, deubiquitinating enzymes, and the protein unfolding machinery at subnanometer resolution, outlining the substrate’s path to degradation. Unexpectedly, the ATPase subunits within the base unfoldase are arranged in a spiral staircase, providing insight into potential mechanisms for substrate translocation through the central pore. Large conformational rearrangements of the lid upon holoenzyme formation suggest allosteric regulation of deubiquitination. We provide a structural basis for the ability of the proteasome to degrade a diverse set of substrates and thus regulate vital cellular processes. PMID:22237024

  6. Single subunit type of ferritin from visceral mass of Saccostrea cucullata: cloning, expression and cisplatin-subunit analysis.

    PubMed

    Zhu, Bo; Lin, Qing; Ke, Cai-Huan; Huang, He-Qing

    2011-09-01

    Ferritin, the iron storage protein, plays a key role in iron metabolism. Here, we have cloned an inducible ferritin cDNA with 516 bp within the open reading frame fragment from the visceral mass of Saccostrea cucullata. The subunit sequence of the ferritin was predicted to be a polypeptide of 171 amino acids with a molecular weight (MW) of 19.9182 kDa and an isoelectric point of 5.24. The cDNA sequence of S. cucullata ferritin was constructed into a pET-32a expression system for expressing its relative protein efficiently in the Escherichia coli BL21 strain under isopropyl-β-D-thiogalactoside (IPTG) induction. The recombinant ferritin, which was further purified on a Ni-NTA resin column and digested with enterokinase, was detected as a single subunit of approximately MW 20 kDa using both SDS-PAGE and mass spectrometry. S. cucullata ferritin (ScFer) showed 98% identity with Crassostrea gigas ferritin at the amino acid level. The secondary structure and phosphorylation sites of deduced amino acids were predicted with ExPASy proteomics tools and the NetPhos 2.0 server, respectively, and the subunit space structure of recombinant S. cucullata ferritin (rScFer) was built using the molecular operating environmental software system. The results of both in-gel digestion and identification using MALDI-TOF MS/MS showed that the recombinant protein was ScFer. ICP-MS indicated that rScFer subunit can directly bind to cisplatin[cis-Diaminedichloroplatinum(CDDP)], giving approximately 22.9 CDDP/ferritin subunit for forming a novel complex of CDDP-subunit, which suggests that it constructs a nanometer CDDP core-ferritin for developing a new drug of anti-cancer. The results of both the real-time PCR and Western blotting showed that the expression of ScFer mRNA was up-regulated in the oyster under the stress of Cd(2+). In addition, the expression increment of ScFer mRNA under bacterial challenge indicated that ferritin participated in the immune response of S. cucullata. The

  7. Database on the structure of small ribosomal subunit RNA.

    PubMed Central

    Van de Peer, Y; Caers, A; De Rijk, P; De Wachter, R

    1998-01-01

    About 8600 complete or nearly complete sequences are now available from the Antwerp database on small ribosomal subunit RNA. All these sequences are aligned with one another on the basis of the adopted secondary structure model, which is corroborated by the observation of compensating substitutions in the alignment. Literature references, accession numbers and detailed taxonomic information are also compiled. The database can be consulted via the World Wide Web at URL http://rrna.uia.ac.be/ssu/ PMID:9399829

  8. Database on the structure of large ribosomal subunit RNA.

    PubMed Central

    De Rijk, P; Van de Peer, Y; De Wachter, R

    1996-01-01

    Our database on large ribosomal subunit RNA contained 334 sequences in July, 1995. All sequences in the database are aligned, taking into account secondary structure. The aligned sequences are provided, together with incorporated secondary structure information, in several computer-readable formats. These data can easily be obtained through the World Wide Web. The files in the database are also available via anonymous ftp. PMID:8594610

  9. Database on the structure of large ribosomal subunit RNA.

    PubMed Central

    De Rijk, P; Caers, A; Van de Peer, Y; De Wachter, R

    1998-01-01

    The rRNA WWW Server at URL http://rrna.uia.ac.be/ now provides a database of 496 large subunit ribosomal RNA sequences. All these sequences are aligned, incorporate secondary structure information, and can be obtained in a number of formats. Other information about the sequences, such as literature references, accession numbers and taxonomic information is also available and searchable. If necessary, the data on the server can also be obtained by anonymous ftp. PMID:9399830

  10. Functions of the major tyrosine phosphorylation site of the PDGF receptor beta subunit.

    PubMed Central

    Kazlauskas, A; Durden, D L; Cooper, J A

    1991-01-01

    Two tyrosine phosphorylation sites in the human platelet-derived growth factor receptor (PDGFR) beta subunit have been mapped previously to tyrosine (Y)751, in the kinase insert, and Y857, in the kinase domain. Y857 is the major site of tyrosine phosphorylation in PDGF-stimulated cells. To evaluate the importance of these phosphorylations, we have characterized the wild-type (WT) and mutant human PDGF receptor beta subunits in dog kidney epithelial cells. Replacement of either Y751 or Y857 with phenylalanine (F) reduced PDGF-stimulated DNA synthesis to approximately 50% of the WT level. A mutant receptor with both tyrosines mutated was unable to initiate DNA synthesis, as was a kinase-inactive mutant receptor. Transmodulation of the epidermal growth factor receptor required Y857 but not Y751. We also tested the effects of phosphorylation site mutations on PDGF-stimulated receptor kinase activity. PDGF-induced tyrosine phosphorylation of two cellular proteins, phospholipase C gamma 1 (PLC gamma 1) and the GTPase activating protein of Ras (GAP), was assayed in epithelial cells expressing each of the mutant receptors. Tyrosine phosphorylation of GAP and PLC gamma 1 was reduced markedly by the F857 mutation but not significantly by the F751 mutation. Reduced kinase activity of F857 receptors was also evident in vitro. Immunoprecipitated WT receptors showed a two- to fourfold increase in specific kinase activity if immunoprecipitated from PDGF-stimulated cells. The F751 receptors showed a similar increase in activity, but F857 receptors did not. Our data suggest that phosphorylation of Y857 may be important for stimulation of kinase activity of the receptors and for downstream actions such as epidermal growth factor receptor transmodulation and mitogenesis. Images PMID:1653029

  11. Cryo-EM structure of the small subunit of the mammalian mitochondrial ribosome.

    PubMed

    Kaushal, Prem S; Sharma, Manjuli R; Booth, Timothy M; Haque, Emdadul M; Tung, Chang-Shung; Sanbonmatsu, Karissa Y; Spremulli, Linda L; Agrawal, Rajendra K

    2014-05-20

    The mammalian mitochondrial ribosomes (mitoribosomes) are responsible for synthesizing 13 membrane proteins that form essential components of the complexes involved in oxidative phosphorylation or ATP generation for the eukaryotic cell. The mammalian 55S mitoribosome contains significantly smaller rRNAs and a large mass of mitochondrial ribosomal proteins (MRPs), including large mito-specific amino acid extensions and insertions in MRPs that are homologous to bacterial ribosomal proteins and an additional 35 mito-specific MRPs. Here we present the cryo-EM structure analysis of the small (28S) subunit (SSU) of the 55S mitoribosome. We find that the mito-specific extensions in homologous MRPs generally are involved in inter-MRP contacts and in contacts with mito-specific MRPs, suggesting a stepwise evolution of the current architecture of the mitoribosome. Although most of the mito-specific MRPs and extensions of homologous MRPs are situated on the peripheral regions, they also contribute significantly to the formation of linings of the mRNA and tRNA paths, suggesting a tailor-made structural organization of the mito-SSU for the recruitment of mito-specific mRNAs, most of which do not possess a 5' leader sequence. In addition, docking of previously published coordinates of the large (39S) subunit (LSU) into the cryo-EM map of the 55S mitoribosome reveals that mito-specific MRPs of both the SSU and LSU are involved directly in the formation of six of the 15 intersubunit bridges. PMID:24799711

  12. Cryo-EM structure of the small subunit of the mammalian mitochondrial ribosome

    PubMed Central

    Kaushal, Prem S.; Sharma, Manjuli R.; Booth, Timothy M.; Haque, Emdadul M.; Tung, Chang-Shung; Sanbonmatsu, Karissa Y.; Spremulli, Linda L.; Agrawal, Rajendra K.

    2014-01-01

    The mammalian mitochondrial ribosomes (mitoribosomes) are responsible for synthesizing 13 membrane proteins that form essential components of the complexes involved in oxidative phosphorylation or ATP generation for the eukaryotic cell. The mammalian 55S mitoribosome contains significantly smaller rRNAs and a large mass of mitochondrial ribosomal proteins (MRPs), including large mito-specific amino acid extensions and insertions in MRPs that are homologous to bacterial ribosomal proteins and an additional 35 mito-specific MRPs. Here we present the cryo-EM structure analysis of the small (28S) subunit (SSU) of the 55S mitoribosome. We find that the mito-specific extensions in homologous MRPs generally are involved in inter-MRP contacts and in contacts with mito-specific MRPs, suggesting a stepwise evolution of the current architecture of the mitoribosome. Although most of the mito-specific MRPs and extensions of homologous MRPs are situated on the peripheral regions, they also contribute significantly to the formation of linings of the mRNA and tRNA paths, suggesting a tailor-made structural organization of the mito-SSU for the recruitment of mito-specific mRNAs, most of which do not possess a 5′ leader sequence. In addition, docking of previously published coordinates of the large (39S) subunit (LSU) into the cryo-EM map of the 55S mitoribosome reveals that mito-specific MRPs of both the SSU and LSU are involved directly in the formation of six of the 15 intersubunit bridges. PMID:24799711

  13. Instability of toxin A subunit of AB5 toxins in the bacterial periplasm caused by deficiency of their cognate B subunits

    PubMed Central

    Kim, Sang-Hyun; Ryu, Su Hyang; Lee, Sang-Ho; Lee, Yong-Hoon; Lee, Sang-Rae; Huh, Jae-Won; Kim, Sun-Uk; Kim, Ekyune; Kim, Sunghyun; Jon, Sangyong; Bishop, Russell E.; Chang, Kyu-Tae

    2016-01-01

    Shiga toxin (STx) belongs to the AB5 toxin family and is transiently localized in the periplasm before secretion into the extracellular milieu. While producing outer membrane vesicles (OMVs) containing only A subunit of the toxin (STxA), we created specific STx1B- and STx2B-deficient mutants of E. coli O157:H7. Surprisingly, STxA subunit was absent in the OMVs and periplasm of the STxB-deficient mutants. In parallel, the A subunit of heat-labile toxin (LT) of enterotoxigenic E. coli (ETEC) was absent in the periplasm of the LT-B-deficient mutant, suggesting that instability of toxin A subunit in the absence of the B subunit is a common phenomenon in the AB5 bacterial toxins. Moreover, STx2A was barely detectable in the periplasm of E. coli JM109 when stx2A was overexpressed alone, while it was stably present when stxB was co-expressed. Compared with STx2 holotoxin, purified STx2A was degraded rapidly by periplasmic proteases when assessed for in vitro proteolytic susceptibility, suggesting that the B subunit contributes to stability of the toxin A subunit in the periplasm. We propose a novel role for toxin B subunits of AB5 toxins in protection of the A subunit from proteolysis during holotoxin assembly in the periplasm. PMID:21762677

  14. Purification and subunit heterogeneity of pili of Bordetella bronchiseptica.

    PubMed Central

    Lee, S W; Way, A W; Osen, E G

    1986-01-01

    Pili were isolated and purified from Bordetella bronchiseptica. Electron microscopic observations revealed that pili are ubiquitous in this species. The occurrence of pili and flagella appeared to correlate with growth phase and colonial morphology. Pili were about 3 to 4 nm in diameter and morphologically similar to pili isolated from other gram-negative bacteria. Internal core structure was not evident. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of purified pili showed that up to three different pilus subunit variants could be observed on a single strain, depending on the colonial phase and culture condition. Enzyme immunoassay and immunoblot, however, showed that these subunit variants are serologically related. Mice vaccinated with purified pili were protected against a virulent intraperitoneal challenge of B. bronchiseptica. B. bronchiseptica pili were also found to be similar to Bordetella pertussis pili in morphology and in the molecular size and antigenic structure of pilus subunits. The intact pili of B. bronchiseptica and B. pertussis, however, appeared to have weak serological cross-reactivity. Images PMID:2867974

  15. Ribosomal small subunit domains radiate from a central core.

    PubMed

    Gulen, Burak; Petrov, Anton S; Okafor, C Denise; Vander Wood, Drew; O'Neill, Eric B; Hud, Nicholas V; Williams, Loren Dean

    2016-01-01

    The domain architecture of a large RNA can help explain and/or predict folding, function, biogenesis and evolution. We offer a formal and general definition of an RNA domain and use that definition to experimentally characterize the rRNA of the ribosomal small subunit. Here the rRNA comprising a domain is compact, with a self-contained system of molecular interactions. A given rRNA helix or stem-loop must be allocated uniquely to a single domain. Local changes such as mutations can give domain-wide effects. Helices within a domain have interdependent orientations, stabilities and interactions. With these criteria we identify a core domain (domain A) of small subunit rRNA. Domain A acts as a hub, linking the four peripheral domains and imposing orientational and positional restraints on the other domains. Experimental characterization of isolated domain A, and mutations and truncations of it, by methods including selective 2'OH acylation analyzed by primer extension and circular dichroism spectroscopy are consistent with our architectural model. The results support the utility of the concept of an RNA domain. Domain A, which exhibits structural similarity to tRNA, appears to be an essential core of the small ribosomal subunit. PMID:26876483

  16. RNA degradation paths in a 12-subunit nuclear exosome complex.

    PubMed

    Makino, Debora Lika; Schuch, Benjamin; Stegmann, Elisabeth; Baumgärtner, Marc; Basquin, Claire; Conti, Elena

    2015-08-01

    The eukaryotic exosome is a conserved RNA-degrading complex that functions in RNA surveillance, turnover and processing. How the same machinery can either completely degrade or precisely trim RNA substrates has long remained unexplained. Here we report the crystal structures of a yeast nuclear exosome containing the 9-subunit core, the 3'-5' RNases Rrp44 and Rrp6, and the obligate Rrp6-binding partner Rrp47 in complex with different RNAs. The combined structural and biochemical data of this 12-subunit complex reveal how a single-stranded RNA can reach the Rrp44 or Rrp6 active sites directly or can bind Rrp6 and be threaded via the central channel towards the distal RNase Rrp44. When a bulky RNA is stalled at the entrance of the channel, Rrp6-Rrp47 swings open. The results suggest how the same molecular machine can coordinate processive degradation and partial trimming in an RNA-dependent manner by a concerted swinging mechanism of the two RNase subunits. PMID:26222026

  17. Ribosomal small subunit domains radiate from a central core

    NASA Astrophysics Data System (ADS)

    Gulen, Burak; Petrov, Anton S.; Okafor, C. Denise; Vander Wood, Drew; O'Neill, Eric B.; Hud, Nicholas V.; Williams, Loren Dean

    2016-02-01

    The domain architecture of a large RNA can help explain and/or predict folding, function, biogenesis and evolution. We offer a formal and general definition of an RNA domain and use that definition to experimentally characterize the rRNA of the ribosomal small subunit. Here the rRNA comprising a domain is compact, with a self-contained system of molecular interactions. A given rRNA helix or stem-loop must be allocated uniquely to a single domain. Local changes such as mutations can give domain-wide effects. Helices within a domain have interdependent orientations, stabilities and interactions. With these criteria we identify a core domain (domain A) of small subunit rRNA. Domain A acts as a hub, linking the four peripheral domains and imposing orientational and positional restraints on the other domains. Experimental characterization of isolated domain A, and mutations and truncations of it, by methods including selective 2‧OH acylation analyzed by primer extension and circular dichroism spectroscopy are consistent with our architectural model. The results support the utility of the concept of an RNA domain. Domain A, which exhibits structural similarity to tRNA, appears to be an essential core of the small ribosomal subunit.

  18. Ribosomal small subunit domains radiate from a central core

    PubMed Central

    Gulen, Burak; Petrov, Anton S.; Okafor, C. Denise; Vander Wood, Drew; O’Neill, Eric B.; Hud, Nicholas V.; Williams, Loren Dean

    2016-01-01

    The domain architecture of a large RNA can help explain and/or predict folding, function, biogenesis and evolution. We offer a formal and general definition of an RNA domain and use that definition to experimentally characterize the rRNA of the ribosomal small subunit. Here the rRNA comprising a domain is compact, with a self-contained system of molecular interactions. A given rRNA helix or stem-loop must be allocated uniquely to a single domain. Local changes such as mutations can give domain-wide effects. Helices within a domain have interdependent orientations, stabilities and interactions. With these criteria we identify a core domain (domain A) of small subunit rRNA. Domain A acts as a hub, linking the four peripheral domains and imposing orientational and positional restraints on the other domains. Experimental characterization of isolated domain A, and mutations and truncations of it, by methods including selective 2′OH acylation analyzed by primer extension and circular dichroism spectroscopy are consistent with our architectural model. The results support the utility of the concept of an RNA domain. Domain A, which exhibits structural similarity to tRNA, appears to be an essential core of the small ribosomal subunit. PMID:26876483

  19. Serotonergic modulation of muscle acetylcholine receptors of different subunit composition.

    PubMed Central

    García-Colunga, J; Miledi, R

    1996-01-01

    Modulation of muscle acetylcholine (AcCho) receptors (AcChoRs) by serotonin [5-hydroxytryptamine (5HT)] and other serotonergic compounds was studied in Xenopus laevis oocytes. Various combinations of alpha, beta, gamma, and delta subunit RNAs were injected into oocytes, and membrane currents elicited by AcCho were recorded under voltage clamp. Judging by the amplitudes of AcCho currents generated, the levels of functional receptor expression were: alpha beta gamma delta > alpha beta delta > alpha beta gamma > alpha gamma delta. The alpha beta gamma delta and alpha beta delta AcChoR Subtypes were strongly blocked by 5HT, whereas the alpha beta gamma receptor was blocked only slightly. The order of blocking potency of AcChoRs by 5HT was: alpha beta delta > alpha beta gamma delta > alpha beta gamma. 5HT receptor antagonists, such as methysergide and spiperone, were even more potent blockers of AcChoRs than 5HT but did not show much subunit selectivity. Blockage of alpha beta gamma delta and alpha beta delta receptors by 5HT was voltage-dependent, and the voltage dependence was abolished when the delta subunit was omitted. These findings may need to be taken into consideration when trying to elucidate the mode of action of many clinically important serotonergic compounds. Images Fig. 3 PMID:8633003

  20. Direct Observation of Subunit Exchange along Mature Vimentin Intermediate Filaments

    PubMed Central

    Nöding, Bernd; Herrmann, Harald; Köster, Sarah

    2014-01-01

    Actin filaments, microtubules, and intermediate filaments (IFs) are central elements of the metazoan cytoskeleton. At the molecular level, the assembly mechanism for actin filaments and microtubules is fundamentally different from that of IFs. The former two types of filaments assemble from globular proteins. By contrast, IFs assemble from tetrameric complexes of extended, half-staggered, and antiparallel oriented coiled-coils. These tetramers laterally associate into unit-length filaments; subsequent longitudinal annealing of unit-length filaments yields mature IFs. In vitro, IFs form open structures without a fixed number of tetramers per cross-section along the filament. Therefore, a central question for the structural biology of IFs is whether individual subunits can dissociate from assembled filaments and rebind at other sites. Using the fluorescently labeled IF-protein vimentin for assembly, we directly observe and quantitatively determine subunit exchange events between filaments as well as with soluble vimentin pools. Thereby we demonstrate that the cross-sectional polymorphism of donor and acceptor filaments plays an important role. We propose that in segments of donor filaments with more than the standard 32 molecules per cross-section, subunits are not as tightly bound and are predisposed to be released from the filament. PMID:25517157

  1. Mutant GABA(A) receptor subunits in genetic (idiopathic) epilepsy.

    PubMed

    Hirose, Shinichi

    2014-01-01

    The γ-aminobutyric acid receptor type A (GABAA receptor) is a ligand-gated chloride channel that mediates major inhibitory functions in the central nervous system. GABAA receptors function mainly as pentamers containing α, β, and either γ or δ subunits. A number of antiepileptic drugs have agonistic effects on GABAA receptors. Hence, dysfunctions of GABAA receptors have been postulated to play important roles in the etiology of epilepsy. In fact, mutations or genetic variations of the genes encoding the α1, α6, β2, β3, γ2, or δ subunits (GABRA1, GABRA6, GABRB2, GABRB3, GABRG2, and GABRD, respectively) have been associated with human epilepsy, both with and without febrile seizures. Epilepsy resulting from mutations is commonly one of following, genetic (idiopathic) generalized epilepsy (e.g., juvenile myoclonic epilepsy), childhood absence epilepsy, genetic epilepsy with febrile seizures, or Dravet syndrome. Recently, mutations of GABRA1, GABRB2, and GABRB3 were associated with infantile spasms and Lennox-Gastaut syndrome. These mutations compromise hyperpolarization through GABAA receptors, which is believed to cause seizures. Interestingly, most of the insufficiencies are not caused by receptor gating abnormalities, but by complex mechanisms, including endoplasmic reticulum (ER)-associated degradation, nonsense-mediated mRNA decay, intracellular trafficking defects, and ER stress. Thus, GABAA receptor subunit mutations are now thought to participate in the pathomechanisms of epilepsy, and an improved understanding of these mutations should facilitate our understanding of epilepsy and the development of new therapies. PMID:25194483

  2. Gβ4γ1 as a modulator of M3 muscarinic receptor signalling and novel roles of Gβ1 subunits in the modulation of cellular signalling.

    PubMed

    Khan, Shahriar M; Min, Adam; Gora, Sarah; Houranieh, Geeda M; Campden, Rhiannon; Robitaille, Mélanie; Trieu, Phan; Pétrin, Darlaine; Jacobi, Ashley M; Behlke, Mark A; Angers, Stéphane; Hébert, Terence E

    2015-08-01

    Much is known about the how Gβγ subunits regulate effectors in response to G protein-coupled receptor stimulation. However, there is still a lot we don't know about how specific combinations of Gβ and Gγ are wired into different signalling pathways. Here, using an siRNA screen for different Gβ and Gγ subunits, we examined an endogenous M3 muscarinic receptor signalling pathway in HEK 293 cells. We observed that Gβ(4) subunits were critical for calcium signalling and a downstream surrogate measured as ERK1/2 MAP kinase activity. A number of Gγ subunits could partner with Gβ(4) but the best coupling was seen via Gβ(4)γ(1). Intriguingly, knocking down Gβ(1) actually increased signalling through the M3-mAChR most likely via an increase in Gβ(4) levels. We noted that Gβ(1) occupies the promoter of Gβ(4) and may participate in maturation of its mRNA. This highlights a new role for Gβγ signalling beyond their canonical roles in cellular signalling. PMID:25916507

  3. Cloning and structural analysis of the gene for the regulatory subunit of cAMP-dependent protein kinase in Blastocladiella emersonii.

    PubMed

    Marques, M do V; Gomes, S L

    1992-08-25

    We have isolated and characterized cDNA and genomic DNA clones encoding the regulatory subunit of cAMP-dependent protein kinase in the aquatic fungus Blastocladiella emersonii. Nucleotide sequence analysis has shown that the predicted protein comprises 403 amino acids with a calculated molecular mass of 44,263 Da and an overall 40% identity to mammalian RII subunits, including a serine in the phosphorylation site, which confirms the Blastocladiella protein as a type II regulatory subunit. The B. emersonii R gene presents two introns, one located in the 5'-noncoding region, whereas the other interrupts the coding region, just after the dimerization domain of the protein. The promoter region does not contain recognizable TATA or CCAAT sequences and is very GC rich, a characteristic shared by mammalian cAMP-dependent protein kinase subunit genes previously analyzed. S1 mapping and primer extension experiments revealed multiple transcription initiation sites. Several sequence motifs were identified in the 5'-flanking region which could be responsible for the regulation of this gene. PMID:1512258

  4. cDNA cloning, characterization, and developmental expression of the 20S proteasome alpha5 subunit in the Mediterranean fruit fly Ceratitis capitata.

    PubMed

    Verras, Meletios; Gourzi, Polyxeni; Kalosaka, Katerina; Zacharopoulou, Antigone; Mintzas, Anastassios C

    2008-03-01

    In the present study, we report the cDNA cloning, characterization, and developmental expression of the 20S proteasome alpha5 subunit from the Mediterranean fruit fly Ceratitis capitata (medfly). Using an RT-PCR fragment that corresponds to the amino-terminal region of the Drosophila melanogaster 20S proteasome alpha5 subunit, we isolated a 987-bp cDNA that encodes the complete coding region of the medfly ortholog, which was named CcPSMA5. CcPSMA5 consists of 241 amino acids and has a predicted molecular weight of 26.4 kDa and pI 4.75. Comparison of the CcPSMA5 amino acid sequence with the sequences of all known 20S proteasome alpha5 subunits from different organisms indicated that the medfly 20S proteasome alpha5 subunit has the strongest homology to that of Drosophila. In situ hybridization showed that the CcPSMA5 gene is mapped in the region 44B of chromosome 4. Northern blot hybridization analysis showed that the CcPSMA5 mRNA has a size of approximately 1.2 kb. High levels of the CcPSMA5 mRNA were detected in freshly laid eggs, indicating that they were maternally deposited. The mRNA expression pattern during medfly development suggests that the CcPSMA5 gene is upregulated before mid-embryogenesis and at the onset of metamorphosis. PMID:18163525

  5. Subunit interactions in yeast transcription/repair factor TFIIH. Requirement for Tfb3 subunit in nucleotide excision repair.

    PubMed

    Feaver, W J; Huang, W; Gileadi, O; Myers, L; Gustafsson, C M; Kornberg, R D; Friedberg, E C

    2000-02-25

    A yeast strain harboring a temperature-sensitive allele of TFB3 (tfb3(ts)), the 38-kDa subunit of the RNA polymerase II transcription/nucleotide excision repair factor TFIIH, was found to be sensitive to ultraviolet (UV) radiation and defective for nucleotide excision repair in vitro. Interestingly, tfb3(ts) failed to grow on medium containing caffeine. A comprehensive pairwise two-hybrid analysis between yeast TFIIH subunits identified novel interactions between Rad3 and Tfb3, Tfb4 and Ssl1, as well as Ssl2 and Tfb2. These interactions have facilitated a more complete model of the structure of TFIIH and the nucleotide excision repairosome. PMID:10681587

  6. On the multiple roles of the voltage gated sodium channel β1 subunit in genetic diseases

    PubMed Central

    Baroni, Debora; Moran, Oscar

    2015-01-01

    Voltage-gated sodium channels are intrinsic plasma membrane proteins that initiate the action potential in electrically excitable cells. They are composed of a pore-forming α-subunit and associated β-subunits. The β1-subunit was the first accessory subunit to be cloned. It can be important for controlling cell excitability and modulating multiple aspects of sodium channel physiology. Mutations of β1 are implicated in a wide variety of inherited pathologies, including epilepsy and cardiac conduction diseases. This review summarizes β1-subunit related channelopathies pointing out the current knowledge concerning their genetic background and their underlying molecular mechanisms. PMID:26042039

  7. Hybridization of glutamate aspartate transaminase. Investigation of subunit interaction.

    PubMed

    Boettcher, B; Martinez-Carrion, M

    1975-10-01

    Glutamate aspartate transaminase (EC 2.6.1.1) is a dimeric enzyme with identical subunits with each active site containing pyridoxal 5'-phosphate linked via an internal Shiff's base to a lysine residue. It is not known if these sites interact during catalysis but negative cooperativity has been reported for binding of the coenzyme (Arrio-Dupont, M. (1972), Eur. J. Biochem. 30, 307). Also nonequivalence of its subunits in binding 8-anilinonaphthalene-1-sulfonate (Harris, H.E., and Bayley, P. M. (1975), Biochem. J. 145, 125), in modification of only a single tyrosine with full loss of activity (Christen, P., and Riordan, J.F. (1970), Biochemistry 9, 3025), and following modification with 5,5'-dithiobis(2-nitrobenzoic acid) (Cournil, I., and Arrio-Dupont, M. (1973), Biochemie 55, 103) has been reported. However, steady-state and transient kinetic methods as well as direct titration of the active site chromophore with substrates and substrate analogs have not revealed any cooperative phenomena (Braunstein, A. E. (1973), Enzymes, 3rd Ed. 9, 379). It was therefore decided that a more direct approach should be used to clarify the quistion of subunit interaction during the covalent phase of catalysis. To this end a hybrid method was devised in which a hybrid transaminase was prepared which contained one subunit with a functional active site while the other subunit has the internal Shiff's base reduced with NaBH4. The specific activities and amount of "actively bound" pyridoxal 5'-phosphate are both in a 2:1 ratio for the native and hybrid forms. Comparison of the steady-state kinetic properties of the hybrid and native enzyme forms shows that both forms gave parallel double reciprocal plots which is characteristic of the Ping-Pong Bi-Bi mechanism of transamination. The Km values for the substrates L-aspartic acid and alpha-ketoglutaric acid are nearly identical while the Vmax value for the hybrid is one-half the value of the native transaminase. It therefore appears that

  8. Characterization of the Aalpha and Abeta subunit isoforms of protein phosphatase 2A: differences in expression, subunit interaction, and evolution.

    PubMed Central

    Zhou, Jin; Pham, Huong T; Ruediger, Ralf; Walter, Gernot

    2003-01-01

    Protein phosphatase 2A (PP2A) is very versatile owing to a large number of regulatory subunits and its ability to interact with numerous other proteins. The regulatory A subunit exists as two closely related isoforms designated Aalpha and Abeta. Mutations have been found in both isoforms in a variety of human cancers. Although Aalpha has been intensely studied, little is known about Abeta. We generated Abeta-specific antibodies and determined the cell cycle expression, subcellular distribution, and metabolic stability of Abeta in comparison with Aalpha. Both forms were expressed at constant levels throughout the cell cycle, but Aalpha was expressed at a much higher level than Abeta. Both forms were found predominantly in the cytoplasm, and both had a half-life of approx. 10 h. However, Aalpha and Abeta differed substantially in their expression patterns in normal tissues and in tumour cell lines. Whereas Aalpha was expressed at similarly high levels in all tissues and cell lines, Abeta expression varied greatly. In addition, in vivo studies with epitope-tagged Aalpha and Abeta subunits demonstrated that Abeta is a markedly weaker binder of regulatory B and catalytic C subunits than Aalpha. Construction of phylogenetic trees revealed that the conservation of Aalpha during the evolution of mammals is extraordinarily high in comparison with both Abeta and cytochrome c, suggesting that Aalpha is involved in more protein-protein interactions than Abeta. We also measured the binding of polyoma virus middle tumour antigen and simian virus 40 (SV40) small tumour antigen to Aalpha and Abeta. Whereas both isoforms bound polyoma virus middle tumour antigen equally well, only Aalpha bound SV40 small tumour antigen. PMID:12370081

  9. Two-subunit DNA escort mechanism and inactive subunit bypass in an ultra-fast ring ATPase

    DOE PAGESBeta

    Liu, Ninning; Chistol, Gheorghe; Bustamante, Carlos

    2015-10-09

    SpoIIIE is a homo-hexameric dsDNA translocase responsible for completing chromosome segregation in Bacillus subtilis . Here, we use a single-molecule approach to monitor SpoIIIE translocation when challenged with neutral-backbone DNA and non-hydrolyzable ATP analogs. We show that SpoIIIE makes multiple essential contacts with phosphates on the 5'→3' strand in the direction of translocation. Using DNA constructs with two neutral-backbone segments separated by a single charged base pair, we deduce that SpoIIIE’s step size is 2 bp. Finally, experiments with non-hydrolyzable ATP analogs suggest that SpoIIIE can operate with non-consecutive inactive subunits. We propose a two-subunit escort translocation mechanism thatmore » is strict enough to enable SpoIIIE to track one DNA strand, yet sufficiently compliant to permit the motor to bypass inactive subunits without arrest. We speculate that such a flexible mechanism arose for motors that, like SpoIIIE, constitute functional bottlenecks where the inactivation of even a single motor can be lethal for the cell.« less

  10. Two-subunit DNA escort mechanism and inactive subunit bypass in an ultra-fast ring ATPase

    PubMed Central

    Liu, Ninning; Chistol, Gheorghe; Bustamante, Carlos

    2015-01-01

    SpoIIIE is a homo-hexameric dsDNA translocase responsible for completing chromosome segregation in Bacillus subtilis. Here, we use a single-molecule approach to monitor SpoIIIE translocation when challenged with neutral-backbone DNA and non-hydrolyzable ATP analogs. We show that SpoIIIE makes multiple essential contacts with phosphates on the 5'→3' strand in the direction of translocation. Using DNA constructs with two neutral-backbone segments separated by a single charged base pair, we deduce that SpoIIIE’s step size is 2 bp. Finally, experiments with non-hydrolyzable ATP analogs suggest that SpoIIIE can operate with non-consecutive inactive subunits. We propose a two-subunit escort translocation mechanism that is strict enough to enable SpoIIIE to track one DNA strand, yet sufficiently compliant to permit the motor to bypass inactive subunits without arrest. We speculate that such a flexible mechanism arose for motors that, like SpoIIIE, constitute functional bottlenecks where the inactivation of even a single motor can be lethal for the cell. DOI: http://dx.doi.org/10.7554/eLife.09224.001 PMID:26452092

  11. The alpha subunit of nitrile hydratase is sufficient for catalytic activity and post-translational modification.

    PubMed

    Nelp, Micah T; Astashkin, Andrei V; Breci, Linda A; McCarty, Reid M; Bandarian, Vahe

    2014-06-24

    Nitrile hydratases (NHases) possess a mononuclear iron or cobalt cofactor whose coordination environment includes rare post-translationally oxidized cysteine sulfenic and sulfinic acid ligands. This cofactor is located in the α-subunit at the interfacial active site of the heterodimeric enzyme. Unlike canonical NHases, toyocamycin nitrile hydratase (TNHase) from Streptomyces rimosus is a unique three-subunit member of this family involved in the biosynthesis of pyrrolopyrimidine antibiotics. The subunits of TNHase are homologous to the α- and β-subunits of prototypical NHases. Herein we report the expression, purification, and characterization of the α-subunit of TNHase. The UV-visible, EPR, and mass spectra of the α-subunit TNHase provide evidence that this subunit alone is capable of synthesizing the active site complex with full post-translational modifications. Remarkably, the isolated post-translationally modified α-subunit is also catalytically active with the natural substrate, toyocamycin, as well as the niacin precursor 3-cyanopyridine. Comparisons of the steady state kinetic parameters of the single subunit variant to the heterotrimeric protein clearly show that the additional subunits impart substrate specificity and catalytic efficiency. We conclude that the α-subunit is the minimal sequence needed for nitrile hydration providing a simplified scaffold to study the mechanism and post-translational modification of this important class of catalysts. PMID:24914472

  12. Homodimeric Intrinsic Membrane Proteins. Identification and Modulation of Interactions between Mitochondrial Transporter (Carrier) Subunits

    PubMed Central

    Wohlrab, Hartmut

    2010-01-01

    Transporter (carrier) proteins of the inner mitochondrial membrane link metabolic pathways within the matrix and the cytosol with transport/exchange of metabolites and inorganic ions. Their strict control of these fluxes is required for oxidative phosphorylation. Understanding the ternary complex transport mechanism with which most of these transporters function requires an accounting of the number and interactions of their subunits. The phosphate transporter (PTP, Mir1p) subunit readily forms homodimers with intersubunit affinities changeable by mutations. Cys28, likely at the subunit interface, is a site for mutations yielding transport inhibition or a channel-like transport mode. Such mutations yield a small increase or decrease in affinity between the subunits. The PTP inhibitor N-ethylmaleimide decreases subunit affinity by a small amount. PTP mutations that yield the highest (40%) and the lowest (2%) liposome incorporation efficiencies (LIE) are clustered near Cys28. Such mutant subunits show the lowest and highest subunit affinities respectively. The oxaloacetate transporter (Oac1p) subunit has an almost 2-fold lower affinity than the PTP subunit. The Oac1p, dicarboxylate (Dic1p) and PTP transporter subunits form heterodimers with even lower affinities. These results form a firm basis for detailed studies to establish the effect of subunit affinities on transport mode and activity and for the identification of the mechanism that prevents formation of heterodimers that surely will negatively impact oxidative phosphorylation and ATP levels with serious consequences for the cell. PMID:20171189

  13. Developmental and Regulatory Functions of Na(+) Channel Non-pore-forming β Subunits.

    PubMed

    Winters, J J; Isom, L L

    2016-01-01

    Voltage-gated Na(+) channels (VGSCs) isolated from mammalian neurons are heterotrimeric complexes containing one pore-forming α subunit and two non-pore-forming β subunits. In excitable cells, VGSCs are responsible for the initiation of action potentials. VGSC β subunits are type I topology glycoproteins, containing an extracellular amino-terminal immunoglobulin (Ig) domain with homology to many neural cell adhesion molecules (CAMs), a single transmembrane segment, and an intracellular carboxyl-terminal domain. VGSC β subunits are encoded by a gene family that is distinct from the α subunits. While α subunits are expressed in prokaryotes, β subunit orthologs did not arise until after the emergence of vertebrates. β subunits regulate the cell surface expression, subcellular localization, and gating properties of their associated α subunits. In addition, like many other Ig-CAMs, β subunits are involved in cell migration, neurite outgrowth, and axon pathfinding and may function in these roles in the absence of associated α subunits. In sum, these multifunctional proteins are critical for both channel regulation and central nervous system development. PMID:27586289

  14. Structure of subcomplex Iβ of mammalian respiratory complex I leads to new supernumerary subunit assignments.

    PubMed

    Zhu, Jiapeng; King, Martin S; Yu, Minmin; Klipcan, Liron; Leslie, Andrew G W; Hirst, Judy

    2015-09-29

    Mitochondrial complex I (proton-pumping NADH:ubiquinone oxidoreductase) is an essential respiratory enzyme. Mammalian complex I contains 45 subunits: 14 conserved "core" subunits and 31 "supernumerary" subunits. The structure of Bos taurus complex I, determined to 5-Å resolution by electron cryomicroscopy, described the structure of the mammalian core enzyme and allowed the assignment of 14 supernumerary subunits. Here, we describe the 6.8-Å resolution X-ray crystallography structure of subcomplex Iβ, a large portion of the membrane domain of B. taurus complex I that contains two core subunits and a cohort of supernumerary subunits. By comparing the structures and composition of subcomplex Iβ and complex I, supported by comparisons with Yarrowia lipolytica complex I, we propose assignments for eight further supernumerary subunits in the structure. Our new assignments include two CHCH-domain containing subunits that contain disulfide bridges between CX9C motifs; they are processed by the Mia40 oxidative-folding pathway in the intermembrane space and probably stabilize the membrane domain. We also assign subunit B22, an LYR protein, to the matrix face of the membrane domain. We reveal that subunit B22 anchors an acyl carrier protein (ACP) to the complex, replicating the LYR protein-ACP structural module that was identified previously in the hydrophilic domain. Thus, we significantly extend knowledge of how the mammalian supernumerary subunits are arranged around the core enzyme, and provide insights into their roles in biogenesis and regulation. PMID:26371297

  15. Reconstitution of thermostable ATPase capable of energy coupling from its purified subunits.

    PubMed

    Yoshida, M; Okamoto, H; Sone, N; Hirata, H; Kagawa, Y

    1977-03-01

    Purified dicyclohexylcarbodiimide-sensitive ATPase (TF0-F1) from thermophilic bacterium PS3 is composed of a water soluble part with ATP hydrolytic activity (TF1) and a water insoluble moiety (TF0). All of the five subunits (alpha, beta, gamma, delta, and epsilon) of TF1 were isolated. TF1 was reconstituted from the five subunits, which catalyzed an ATP-32Pi exchange and an ATP-driven enhancement of fluorescence of 1-anilinonaphthalene-8-sulfonate, when adsorbed on proteoliposome inlaid with TF0 (TF3-vesicles). Subunit epsilon and/or delta became firmly bound to TF0-vesicles and there was no preferential sequence in the binding. Both subunits were required for binding of the remaining subunits of TF1 to TF0-vesicles, but they did not modify the high H+ -permeability of TF0-vesicles. The addition of gamma but they did not modify the high H+-permeability of TFO-vesicles. The addition of gamma subunit together with epsilon and delta subunits caused a marked decrease of H+ -permeability of TF0-vesicles, similar to that induced by TF1. We conclude tentatively that the epsilon and delta subunits connect TF0 and the other subunits forming a part of a proton pathway, gamma is a gate of proton flow coupled to ATP hydrolysis (or synthesis), and alpha and beta subunits contain the active site for energy transformation. A possible model of subunit structure of TF1 is proposed. PMID:139610

  16. Generalized epilepsy with febrile seizures plus-associated sodium channel beta1 subunit mutations severely reduce beta subunit-mediated modulation of sodium channel function.

    PubMed

    Xu, R; Thomas, E A; Gazina, E V; Richards, K L; Quick, M; Wallace, R H; Harkin, L A; Heron, S E; Berkovic, S F; Scheffer, I E; Mulley, J C; Petrou, S

    2007-08-10

    Two novel mutations (R85C and R85H) on the extracellular immunoglobulin-like domain of the sodium channel beta1 subunit have been identified in individuals from two families with generalized epilepsy with febrile seizures plus (GEFS+). The functional consequences of these two mutations were determined by co-expression of the human brain NaV1.2 alpha subunit with wild type or mutant beta1 subunits in human embryonic kidney (HEK)-293T cells. Patch clamp studies confirmed the regulatory role of beta1 in that relative to NaV1.2 alone the NaV1.2+beta1 currents had right-shifted voltage dependence of activation, fast and slow inactivation and reduced use dependence. In addition, the NaV1.2+beta1 current entered fast inactivation slightly faster than NaV1.2 channels alone. The beta1(R85C) subunit appears to be a complete loss of function in that none of the modulating effects of the wild type beta1 were observed when it was co-expressed with NaV1.2. Interestingly, the beta1(R85H) subunit also failed to modulate fast kinetics, however, it shifted the voltage dependence of steady state slow inactivation in the same way as the wild type beta1 subunit. Immunohistochemical studies revealed cell surface expression of the wild type beta1 subunit and undetectable levels of cell surface expression for both mutants. The functional studies suggest association of the beta1(R85H) subunit with the alpha subunit where its influence is limited to modulating steady state slow inactivation. In summary, the mutant beta1 subunits essentially fail to modulate alpha subunits which could increase neuronal excitability and underlie GEFS+ pathogenesis. PMID:17629415

  17. Modulation of BK Channel Function by Auxiliary Beta and Gamma Subunits

    PubMed Central

    Li, Q.; Yan, J.

    2016-01-01

    The large-conductance, Ca2+- and voltage-activated K+ (BK) channel is ubiquitously expressed in mammalian tissues and displays diverse biophysical or pharmacological characteristics. This diversity is in part conferred by channel modulation with different regulatory auxiliary subunits. To date, two distinct classes of BK channel auxiliary subunits have been identified: β subunits and γ subunits. Modulation of BK channels by the four auxiliary β (β1–β4) subunits has been well established and intensively investigated over the past two decades. The auxiliary γ subunits, however, were identified only very recently, which adds a new dimension to BK channel regulation and improves our understanding of the physiological functions of BK channels in various tissues and cell types. This chapter will review the current understanding of BK channel modulation by auxiliary β and γ subunits, especially the latest findings. PMID:27238261

  18. Functional diversification of maize RNA polymerase IV and V subtypes via alternative catalytic subunits.

    PubMed

    Haag, Jeremy R; Brower-Toland, Brent; Krieger, Elysia K; Sidorenko, Lyudmila; Nicora, Carrie D; Norbeck, Angela D; Irsigler, Andre; LaRue, Huachun; Brzeski, Jan; McGinnis, Karen; Ivashuta, Sergey; Pasa-Tolic, Ljiljana; Chandler, Vicki L; Pikaard, Craig S

    2014-10-01

    Unlike nuclear multisubunit RNA polymerases I, II, and III, whose subunit compositions are conserved throughout eukaryotes, plant RNA polymerases IV and V are nonessential, Pol II-related enzymes whose subunit compositions are still evolving. Whereas Arabidopsis Pols IV and V differ from Pol II in four or five of their 12 subunits, respectively, and differ from one another in three subunits, proteomic analyses show that maize Pols IV and V differ from Pol II in six subunits but differ from each other only in their largest subunits. Use of alternative catalytic second subunits, which are nonredundant for development and paramutation, yields at least two subtypes of Pol IV and three subtypes of Pol V in maize. Pol IV/Pol V associations with MOP1, RMR1, AGO121, Zm_DRD1/CHR127, SHH2a, and SHH2b extend parallels between paramutation in maize and the RNA-directed DNA methylation pathway in Arabidopsis. PMID:25284785

  19. Individual subunits of bacterial luciferase are molten globules and interact with molecular chaperones.

    PubMed Central

    Flynn, G C; Beckers, C J; Baase, W A; Dahlquist, F W

    1993-01-01

    We have studied the assembly of a large heterodimeric protein, bacterial luciferase, by mixing purified subunits expressed separately in bacteria. The individual subunits alpha and beta contain much (66% and 50%, respectively) of the alpha-helix content of the native heterodimer as measured by circular dichroism, yet the alpha subunit lacks observable tertiary structure as measured by NMR. These results are consistent with the alpha subunit existing in a molten globule or collapsed form prior to assembly. The molecular chaperone GroEL binds reversibly to both subunits prior to assembly. Since these observations were obtained under physiological conditions, we propose that the molten globule exists as a stable form during folding or assembly in the cell. Either the molten globule form of the subunits is an authentic folding intermediate or it is in rapid equilibrium with one. GroEL may function by facilitating assembly through stabilization of these incompletely folded subunits. Images Fig. 4 PMID:7902573

  20. Functional Diversification of Maize RNA Polymerase IV and V subtypes via Alternative Catalytic Subunits

    SciTech Connect

    Haag, Jeremy R.; Brower-Toland, Brent; Krieger, Elysia K.; Sidorenko, Lyudmila; Nicora, Carrie D.; Norbeck, Angela D.; Irsigler, Andre; LaRue, Huachun; Brzeski, Jan; Mcginnis, Karen A.; Ivashuta, Sergey; Pasa-Tolic, Ljiljana; Chandler, Vicki L.; Pikaard, Craig S.

    2014-10-01

    Unlike nuclear multisubunit RNA polymerases I, II, and III, whose subunit compositions are conserved throughout eukaryotes, plant RNA polymerases IV and V are nonessential, Pol II-related enzymes whose subunit compositions are still evolving. Whereas Arabidopsis Pols IV and V differ from Pol II in four or five of their 12 subunits, respectively, and differ from one another in three subunits, proteomic ana- lyses show that maize Pols IV and V differ from Pol II in six subunits but differ from each other only in their largest subunits. Use of alternative catalytic second subunits, which are nonredundant for development and paramutation, yields at least two sub- types of Pol IV and three subtypes of Pol V in maize. Pol IV/Pol V associations with MOP1, RMR1, AGO121, Zm_DRD1/CHR127, SHH2a, and SHH2b extend parallels between paramutation in maize and the RNA-directed DNA methylation pathway in Arabidopsis.

  1. Individual IKs channels at the surface of mammalian cells contain two KCNE1 accessory subunits

    PubMed Central

    Plant, Leigh D.; Xiong, Dazhi; Dai, Hui; Goldstein, Steve A. N.

    2014-01-01

    KCNE1 (E1) β-subunits assemble with KCNQ1 (Q1) voltage-gated K+ channel α-subunits to form IKslow (IKs) channels in the heart and ear. The number of E1 subunits in IKs channels has been an issue of ongoing debate. Here, we use single-molecule spectroscopy to demonstrate that surface IKs channels with human subunits contain two E1 and four Q1 subunits. This stoichiometry does not vary. Thus, IKs channels in cells with elevated levels of E1 carry no more than two E1 subunits. Cells with low levels of E1 produce IKs channels with two E1 subunits and Q1 channels with no E1 subunits—channels with one E1 do not appear to form or are restricted from surface expression. The plethora of models of cardiac function, transgenic animals, and drug screens based on variable E1 stoichiometry do not reflect physiology. PMID:24591645

  2. Human Mind Maps

    ERIC Educational Resources Information Center

    Glass, Tom

    2016-01-01

    When students generate mind maps, or concept maps, the maps are usually on paper, computer screens, or a blackboard. Human Mind Maps require few resources and little preparation. The main requirements are space where students can move around and a little creativity and imagination. Mind maps can be used for a variety of purposes, and Human Mind…

  3. Data on the epitope mapping of soybean A2 and A3 glycinin.

    PubMed

    Saeed, Hanaa; Gagnon, Christine; Cober, Elroy; Gleddie, Steve

    2016-09-01

    The data information provided in this article relate to our research article "Using patient serum to epitope map soybean glycinins reveals common epitopes shared with many legumes and tree nuts" (Saeed et al., 2016) [1]. Here we provide western blot detection of glycinin subunits by soy-sensitive human sera, ELISA screens with overlapping synthetic peptides (epitope mapping), and various database/server epitope searches. PMID:27294180

  4. Carboxymethylation of methionine residues in bovine pituitary luteinizing hormone and its subunits. Effects on the binding activity with receptor sites and interactions between subunits.

    PubMed Central

    Cheng, K W

    1976-01-01

    The reaction of iodoacetic acid with bovine lutropin (luteinizing hormone) at pH 3.0 was specific for methionine residues; it was slow and reached its equilibrium after 12 h at 37 degrees C. The number of modified methionine residues increased proportionately with the amount of the alkylating reagent in the reaction mixture. In the presence of a 20-fold molar excess of iodoacetic acid with respect to methionine, essentially all methionine residues in both subunits of bovine lutropin were carboxymethylated. Studies of various recombinations of modified and native alpha and beta subunits showed that methionine residues in bovine lutropin were not essential for interactions between subunits. Various recombinants were characterized by polyacrylamide-gel electrophoresis and gel filtration of Sephadex G-100. Immunological cross-reactivity by radioimmunoassay of the recombinants of modified alpha and beta subunits was relatively similar to that of the native subunits. However, the biological activity measured by receptor-site binding of the recombinants of alpha and beta chains with a total of three alkylated methionine residues was less than 5% of the activity of native lutropin. It is noteworthy that recombinants of a modified subunit and a native counterpart subunit regenerated 20-30 % of biological activity. These findings suggested that at least 1-2 methionine residues in each subunit are involved in the hormone-receptor interaction for bovine lutropin. Images PLATE 1 PMID:187169

  5. High-affinity ouabain binding by yeast cells expressing Na+, K(+)-ATPase alpha subunits and the gastric H+, K(+)-ATPase beta subunit.

    PubMed Central

    Eakle, K A; Kim, K S; Kabalin, M A; Farley, R A

    1992-01-01

    Recently, a beta subunit for the rat gastric H+,K(+)-ATPase (HK beta), which is structurally similar to the beta subunit of Na+, K(+)-ATPase, has been cloned and characterized. Using heterologous expression in yeast, we have tested the specificity of beta subunit assembly with different isoforms of the alpha subunit of Na+, K(+)-ATPase. Coexpression in yeast cells of the HK beta with both the sheep alpha 1 subunit and the rat alpha 3 subunit isoforms of Na+, K(+)-ATPase (alpha 1 and alpha 3, respectively) leads to the appearance of high-affinity ouabain-binding sites in yeast membranes. These ouabain-binding sites (alpha 1 plus HK beta, alpha 3 plus HK beta) have a high affinity for ouabain (Kd, 5-10 nM) and are expressed at levels similar to those formed with the rat beta 1 subunit of Na+, K(+)-ATPase (beta 1) (alpha 1 plus beta 1 or alpha 3 plus beta 1). Potassium acts as a specific antagonist of ouabain binding by alpha 1 plus HK beta and alpha 3 plus HK beta just like sodium pumps formed with beta 1. Sodium pumps formed with the HK beta, however, show quantitative differences in their affinity for ouabain and in the antagonism of K+ for ouabain binding. These data suggest that the structure of the beta subunit may play a role in sodium pump function. Images PMID:1313569

  6. High-affinity ouabain binding by yeast cells expressing Na+, K(+)-ATPase alpha subunits and the gastric H+, K(+)-ATPase beta subunit.

    PubMed

    Eakle, K A; Kim, K S; Kabalin, M A; Farley, R A

    1992-04-01

    Recently, a beta subunit for the rat gastric H+,K(+)-ATPase (HK beta), which is structurally similar to the beta subunit of Na+, K(+)-ATPase, has been cloned and characterized. Using heterologous expression in yeast, we have tested the specificity of beta subunit assembly with different isoforms of the alpha subunit of Na+, K(+)-ATPase. Coexpression in yeast cells of the HK beta with both the sheep alpha 1 subunit and the rat alpha 3 subunit isoforms of Na+, K(+)-ATPase (alpha 1 and alpha 3, respectively) leads to the appearance of high-affinity ouabain-binding sites in yeast membranes. These ouabain-binding sites (alpha 1 plus HK beta, alpha 3 plus HK beta) have a high affinity for ouabain (Kd, 5-10 nM) and are expressed at levels similar to those formed with the rat beta 1 subunit of Na+, K(+)-ATPase (beta 1) (alpha 1 plus beta 1 or alpha 3 plus beta 1). Potassium acts as a specific antagonist of ouabain binding by alpha 1 plus HK beta and alpha 3 plus HK beta just like sodium pumps formed with beta 1. Sodium pumps formed with the HK beta, however, show quantitative differences in their affinity for ouabain and in the antagonism of K+ for ouabain binding. These data suggest that the structure of the beta subunit may play a role in sodium pump function. PMID:1313569

  7. Concept Mapping

    PubMed Central

    Brennan, Laura K.; Brownson, Ross C.; Kelly, Cheryl; Ivey, Melissa K.; Leviton, Laura C.

    2016-01-01

    Background From 2003 to 2008, 25 cross-sector, multidisciplinary community partnerships funded through the Active Living by Design (ALbD) national program designed, planned, and implemented policy and environmental changes, with complementary programs and promotions. This paper describes the use of concept-mapping methods to gain insights into promising active living intervention strategies based on the collective experience of community representatives implementing ALbD initiatives. Methods Using Concept Systems software, community representatives (n=43) anonymously generated actions and changes in their communities to support active living (183 original statements, 79 condensed statements). Next, respondents (n=26, from 23 partnerships) sorted the 79 statements into self-created categories, or active living intervention approaches. Respondents then rated statements based on their perceptions of the most important strategies for creating community changes (n=25, from 22 partnerships) and increasing community rates of physical activity (n=23, from 20 partnerships). Cluster analysis and multidimensional scaling were used to describe data patterns. Results ALbD community partnerships identified three active living intervention approaches with the greatest perceived importance to create community change and increase population levels of physical activity: changes to the built and natural environment, partnership and collaboration efforts, and land-use and transportation policies. The relative importance of intervention approaches varied according to subgroups of partnerships working with different populations. Conclusions Decision makers, practitioners, and community residents can incorporate what has been learned from the 25 community partnerships to prioritize active living policy, physical project, promotional, and programmatic strategies for work in different populations and settings. PMID:23079266

  8. Maps & minds : mapping through the ages

    USGS Publications Warehouse

    U.S. Geological Survey

    1984-01-01

    Throughout time, maps have expressed our understanding of our world. Human affairs have been influenced strongly by the quality of maps available to us at the major turning points in our history. "Maps & Minds" traces the ebb and flow of a few central ideas in the mainstream of mapping. Our expanding knowledge of our cosmic neighborhood stems largely from a small number of simple but grand ideas, vigorously pursued.

  9. Characterisation of the tryptophan synthase alpha subunit in maize

    PubMed Central

    Kriechbaumer, Verena; Weigang, Linda; Fießelmann, Andreas; Letzel, Thomas; Frey, Monika; Gierl, Alfons; Glawischnig, Erich

    2008-01-01

    Background In bacteria, such as Salmonella typhimurium, tryptophan is synthesized from indole-3-glycerole phosphate (IGP) by a tryptophan synthase αββα heterotetramer. Plants have evolved multiple α (TSA) and β (TSB) homologs, which have probably diverged in biological function and their ability of subunit interaction. There is some evidence for a tryptophan synthase (TS) complex in Arabidopsis. On the other hand maize (Zea mays) expresses the TSA-homologs BX1 and IGL that efficiently cleave IGP, independent of interaction with TSB. Results In order to clarify, how tryptophan is synthesized in maize, two TSA homologs, hitherto uncharacterized ZmTSA and ZmTSAlike, were functionally analyzed. ZmTSA is localized in plastids, the major site of tryptophan biosynthesis in plants. It catalyzes the tryptophan synthase α-reaction (cleavage of IGP), and forms a tryptophan synthase complex with ZmTSB1 in vitro. The catalytic efficiency of the α-reaction is strongly enhanced upon complex formation. A 160 kD tryptophan synthase complex was partially purified from maize leaves and ZmTSA was identified as native α-subunit of this complex by mass spectrometry. ZmTSAlike, for which no in vitro activity was detected, is localized in the cytosol. ZmTSAlike, BX1, and IGL were not detectable in the native tryptophan synthase complex in leaves. Conclusion It was demonstrated in vivo and in vitro that maize forms a tryptophan synthase complex and ZmTSA functions as α-subunit in this complex. PMID:18430213

  10. Covalent dimerization of ribulose bisphosphate carboxylase subunits by UV radiation.

    PubMed

    Ferreira, R M; Franco, E; Teixeira, A R

    1996-08-15

    The effect of UV radiation (UV-A, UV-B and UV-C) on ribulose bisphosphate carboxylase from a variety of plant species was examined. The exposition of plant leaves or the pure enzyme to UV radiation produced a UV-dependent accumulation of a +5 kDa polypeptide (P65). Different approaches were utilized to elucidate the origin and structure of P65: electrophoretic and fluorographic analyses of 35S-labelled ribulose bisphosphate carboxylase exposed to UV radiation and immunological experiments using antibodies specific for P65, for the large and small subunits of ribulose bisphosphate carboxylase and for high-molecular-mass aggregates of the enzyme. These studies revealed that P65 is a dimer, formed by the covalent, non-disulphide linkage of one small subunit with one large subunit of ribulose bisphosphate carboxylase. For short periods of time (< 1 h), the amount of P65 formed increased with the duration of the exposure to the UV radiation and with the energy of the radiation applied. Prolonged exposure to UV radiation (1-6 h) resulted in the formation of high-molecular-mass aggregates of ribulose bisphosphate carboxylase. Formation of P65 was shown to depend on the native state of the protein, was stimulated by inhibitors of enzyme activity, and was inhibited by activators of enzyme activity. A UV-independent accumulation of P65 was also achieved by the in vitro incubation of plant crude extracts. However, the UV-dependent and the UV-independent formation of P65 seemed to occur by distinct molecular mechanisms. The UV-dependent accumulation of P65 was immunologically detected in all species examined, including Lemna minor, Arum italicum, Brassica oleracea, Triticum aestivum, Zea mays, Pisum sativum and Phaseolus vulgaris, suggesting that it may constitute a universal response to UV radiation, common to all photo-synthetic tissues. PMID:8761476

  11. Thermostable Subunit Vaccines for Pulmonary Delivery: How Close Are We?

    PubMed

    Foged, Camilla

    2016-01-01

    In the past century, vaccines have contributed to a significant improvement in global public health by preventing a number of infectious diseases. Despite this, the vaccine field is still facing challenges related to incomplete vaccine coverage and persistent difficult vaccine targets, such as influenza, tuberculosis, and Ebola, for which no good universal vaccines exist. At least two pharmaceutical improvements are expected to help filling this gap: i) The development of thermostable vaccine dosage forms, and ii) the full exploitation of the adjuvant technology for subunit vaccines to potentiate strong immune responses. This review highlights the status and recent advances in formulation and pulmonary delivery of thermostable human subunit vaccines. Such vaccines are very appealing from compliance, distribution and immunological point of view: Being non-invasive, inhalable vaccines are self-administrable, can be distributed independently of functioning freezers and refrigerators, and can be designed to induce mucosal and/or cell-mediated immunity, which is attractive for a number of diseases requiring stimulation of local mucosal immunity for protection. However, the design and delivery of thermostable dry powder-based vaccines represents a technological challenge: It calls for careful formulation and dosage form design, combined with cheap and efficient delivery devices, which must be engineered via a thorough understanding of the physiological barrier and the requirements for induction of mucosal immunity. Here, I review state of the art and perspectives in formulation design and processing methods for powder-based subunit vaccines intended for pulmonary administration, and present dry powder inhaler technologies suitable for translating these vaccines into clinical trials. PMID:26831645

  12. Colocalization of HCN Channel Subunits in Rat Retinal Ganglion Cells

    PubMed Central

    Stradleigh, Tyler W.; Ogata, Genki; Partida, Gloria J.; Oi, Hanako; Greenberg, Kenneth P.; Krempely, Kalen S.; Ishida, Andrew T.

    2011-01-01

    The current-passing pore of mammalian hyperpolarization-activated, cyclic nucleotide-gated ("HCN") channels is formed by subunit isoforms denoted HCN1-4. In various brain areas, antibodies directed against multiple isoforms bind to single neurons and the current ("Ih") passed during hyperpolarizations differs from that of heterologously expressed homomeric channels. By contrast, retinal rod, cone, and bipolar cells appear to use homomeric HCN channels. Here, we assess the generality of this pattern by examining HCN1 and HCN4 immunoreactivity in rat retinal ganglion cells, measuring Ih in dissociated cells, and testing whether HCN1 and HCN4 protein coimmunoprecipitate. Nearly half of the ganglion cells in whole-mounted retinae bound antibodies against both isoforms. Consistent with colocalization and physical association, 8-bromo-cAMP shifted the voltage-sensitivity of Ih less than that of HCN4 channels and more than that of HCN1 channels, and HCN1 coimmunoprecipitated with HCN4 from membrane fraction proteins. Lastly, the immunopositive somata ranged in diameter from the smallest to the largest in rat retina, the dendrites of immunopositive cells arborized at various levels of the inner plexiform layer and over fields of different diameters, and Ih activated with similar kinetics and proportions of fast and slow components in small, medium, and large somata. These results show that different HCN subunits colocalize in single retinal ganglion cells, identify a subunit that can reconcile native Ih properties with the previously reported presence of HCN4 in these cells, and indicate that Ih is biophysically similar in morphologically diverse retinal ganglion cells and differs from Ih in rods, cones, and bipolar cells. PMID:21456027

  13. Mapping: A Course.

    ERIC Educational Resources Information Center

    Whitmore, Paul M.

    1988-01-01

    Reviews the history of cartography. Describes the contributions of Strabo and Ptolemy in early maps. Identifies the work of Gerhard Mercator as the most important advancement in mapping. Discusses present mapping standards from history. (CW)

  14. Subunit specific inhibitors of proteasomes and their potential for immunomodulation

    PubMed Central

    Kisselev, Alexei F; Groettrup, Marcus

    2015-01-01

    Specialized variants of the constitutive 20S proteasome in the immune system like the immunoproteasomes and the thymoproteasome contain active site-bearing subunits which differ in their cleavage priorities and substrate binding pockets. The immunoproteasome plays a crucial role in antigen processing and for the differentiation of pro-inflammatory T helper cells which are involved in the pathogenesis of autoimmunity. Selective inhibitors of the immunoproteasome and constitutive proteasome have recently been generated which interfere with the development and progression of autoimmune diseases. Here we describe these inhibitors and their therapeutic potential as predicted from preclinical models. PMID:25217863

  15. Twisting and subunit rotation in single FOF1-ATP synthase

    PubMed Central

    Sielaff, Hendrik; Börsch, Michael

    2013-01-01

    FOF1-ATP synthases are ubiquitous proton- or ion-powered membrane enzymes providing ATP for all kinds of cellular processes. The mechanochemistry of catalysis is driven by two rotary nanomotors coupled within the enzyme. Their different step sizes have been observed by single-molecule microscopy including videomicroscopy of fluctuating nanobeads attached to single enzymes and single-molecule Förster resonance energy transfer. Here we review recent developments of approaches to monitor the step size of subunit rotation and the transient elastic energy storage mechanism in single FOF1-ATP synthases. PMID:23267178

  16. Database on the structure of small ribosomal subunit RNA.

    PubMed Central

    Van de Peer, Y; Jansen, J; De Rijk, P; De Wachter, R

    1997-01-01

    The Antwerp database on small ribosomal subunit RNA now offers more than 6000 nucleotide sequences (August 1996). All these sequences are stored in the form of an alignment based on the adopted secondary structure model, which is corroborated by the observation of compensating substitutions in the alignment. Besides the primary and secondary structure information, literature references, accession numbers and detailed taxonomic information are also compiled. For ease of use, the complete database is made available to the scientific community via World Wide Web at URL http://rrna.uia.ac.be/ssu/ . PMID:9016516

  17. Database on the structure of small ribosomal subunit RNA.

    PubMed Central

    Van de Peer, Y; Van den Broeck, I; De Rijk, P; De Wachter, R

    1994-01-01

    The database on small ribosomal subunit RNA structure contains (June 1994) 2824 nucleotide sequences. All these sequences are stored in the form of an alignment based on the adopted secondary structure model, which in turn is corroborated by the observation of compensating substitutions in the alignment. The complete database is made available to the scientific community through anonymous ftp on our server in Antwerp. A special effort was made to improve electronic retrieval and a program is supplied that allows to create different file formats. The database can also be obtained from the EMBL nucleotide sequence library. PMID:7524022

  18. Database on the structure of small ribosomal subunit RNA.

    PubMed Central

    Van de Peer, Y; Nicolaï, S; De Rijk, P; De Wachter, R

    1996-01-01

    The Antwerp database on small ribosomal subunit RNA offers over 4300 nucleotide sequences (August 1995). All these sequences are stored in the form of an alignment based on the adopted secondary structure model, which in turn is corroborated by the observation of compensating substitutions in the alignment. Besides the primary and secondary structure information, literature references, accession numbers and detailed taxonomic information are also compiled. The complete database is made available to the scientific community through anonymous ftp and World Wide Web(WWW). PMID:8594609

  19. Database on the structure of large ribosomal subunit RNA.

    PubMed Central

    De Rijk, P; Van de Peer, Y; De Wachter, R

    1997-01-01

    The latest release of the large ribosomal subunit RNA database contains 429 sequences. All these sequences are aligned, and incorporate secondary structure information. The rRNA WWW Server at URL http://rrna.uia.ac.be/ provides researchers with an easily accessible resource to obtain the data in this database in a number of computer-readable formats. A new query interface has been added to the server. If necessary, the data can also be obtained by anonymous ftp from the same site. PMID:9016517

  20. Deciphering the function of the CNGB1b subunit in olfactory CNG channels

    PubMed Central

    Nache, Vasilica; Wongsamitkul, Nisa; Kusch, Jana; Zimmer, Thomas; Schwede, Frank; Benndorf, Klaus

    2016-01-01

    Olfactory cyclic nucleotide-gated (CNG) ion channels are key players in the signal transduction cascade of olfactory sensory neurons. The second messengers cAMP and cGMP directly activate these channels, generating a depolarizing receptor potential. Olfactory CNG channels are composed of two CNGA2 subunits and two modulatory subunits, CNGA4, and CNGB1b. So far the exact role of the modulatory subunits for channel activation is not fully understood. By measuring ligand binding and channel activation simultaneously, we show that in functional heterotetrameric channels not only the CNGA2 subunits and the CNGA4 subunit but also the CNGB1b subunit binds cyclic nucleotides and, moreover, also alone translates this signal to open the pore. In addition, we show that the CNGB1b subunit is the most sensitive subunit in a heterotetrameric channel to cyclic nucleotides and that it accelerates deactivation to a similar extent as does the CNGA4 subunit. In conclusion, the CNGB1b subunit participates in ligand-gated activation of olfactory CNG channels and, particularly, contributes to rapid termination of odorant signal in an olfactory sensory neuron. PMID:27405959

  1. Deciphering the function of the CNGB1b subunit in olfactory CNG channels.

    PubMed

    Nache, Vasilica; Wongsamitkul, Nisa; Kusch, Jana; Zimmer, Thomas; Schwede, Frank; Benndorf, Klaus

    2016-01-01

    Olfactory cyclic nucleotide-gated (CNG) ion channels are key players in the signal transduction cascade of olfactory sensory neurons. The second messengers cAMP and cGMP directly activate these channels, generating a depolarizing receptor potential. Olfactory CNG channels are composed of two CNGA2 subunits and two modulatory subunits, CNGA4, and CNGB1b. So far the exact role of the modulatory subunits for channel activation is not fully understood. By measuring ligand binding and channel activation simultaneously, we show that in functional heterotetrameric channels not only the CNGA2 subunits and the CNGA4 subunit but also the CNGB1b subunit binds cyclic nucleotides and, moreover, also alone translates this signal to open the pore. In addition, we show that the CNGB1b subunit is the most sensitive subunit in a heterotetrameric channel to cyclic nucleotides and that it accelerates deactivation to a similar extent as does the CNGA4 subunit. In conclusion, the CNGB1b subunit participates in ligand-gated activation of olfactory CNG channels and, particularly, contributes to rapid termination of odorant signal in an olfactory sensory neuron. PMID:27405959

  2. Essential 170-kDa subunit for degradation of crystalline cellulose by Clostridium cellulovorans cellulase

    SciTech Connect

    Shoseyov, O.; Doi, R.H. )

    1990-03-01

    The cellulase complex from Clostridium cellulovorans has been purified and its subunit composition determined. The complex exhibits cellulase activity against crystalline cellulose as well as carboxymethylcellulase (CMCase) and cellobiohydrolase activities. Three major subunits are present with molecular masses of 170, 100, and 70 kDa. The 100-kDa subunit is the major CMCase, although at least four other, minor subunits show CMCase activity. The 170-kDa subunit has the highest affinity for cellulose, does not have detectable enzymatic activity, but is necessary for cellulase activity. Immunological studies indicate that the 170-kDa subunit is not required for binding of the catalytic subunits to cellulose and therefore does not function solely as an anchor protein. Thus this core subunit must have multiple functions. The authors propose a working hypothesis that the binding of the 170-kDa subunit converts the crystalline cellulose to a form that is capable of being hydrolyzed in a cooperative fashion by the associated catalytic subunits.

  3. Crystal Structure and Molecular Imaging of the Nav Channel β3 Subunit Indicates a Trimeric Assembly*

    PubMed Central

    Namadurai, Sivakumar; Balasuriya, Dilshan; Rajappa, Rajit; Wiemhöfer, Martin; Stott, Katherine; Klingauf, Jurgen; Edwardson, J. Michael; Chirgadze, Dimitri Y.; Jackson, Antony P.

    2014-01-01

    The vertebrate sodium (Nav) channel is composed of an ion-conducting α subunit and associated β subunits. Here, we report the crystal structure of the human β3 subunit immunoglobulin (Ig) domain, a functionally important component of Nav channels in neurons and cardiomyocytes. Surprisingly, we found that the β3 subunit Ig domain assembles as a trimer in the crystal asymmetric unit. Analytical ultracentrifugation confirmed the presence of Ig domain monomers, dimers, and trimers in free solution, and atomic force microscopy imaging also detected full-length β3 subunit monomers, dimers, and trimers. Mutation of a cysteine residue critical for maintaining the trimer interface destabilized both dimers and trimers. Using fluorescence photoactivated localization microscopy, we detected full-length β3 subunit trimers on the plasma membrane of transfected HEK293 cells. We further show that β3 subunits can bind to more than one site on the Nav 1.5 α subunit and induce the formation of α subunit oligomers, including trimers. Our results suggest a new and unexpected role for the β3 subunits in Nav channel cross-linking and provide new structural insights into some pathological Nav channel mutations. PMID:24567321

  4. Molecular constituents of maxi KCa channels in human coronary smooth muscle: predominant alpha + beta subunit complexes.

    PubMed

    Tanaka, Y; Meera, P; Song, M; Knaus, H G; Toro, L

    1997-08-01

    1. Human large-conductance voltage- and calcium-sensitive K+ (maxi KCa) channels are composed of at least two subunits: the pore-forming subunit, alpha, and a modulatory subunit, beta. Expression of the beta subunit induces dramatic changes in alpha subunit function. It increases the apparent Ca2+ sensitivity and it allows dehydrosoyasaponin I (DHS-I) to upregulate the channel. 2. The functional coupling of maxi KCa channel alpha and beta subunits in freshly dissociated human coronary smooth muscle cells was assessed. To distinguish maxi KCa currents modulated by the beta subunit, we examined (a) their apparent Ca2+ sensitivity, as judged from the voltage necessary to half-activate the channel (V1/2), and (b) their activation by DHS-I. 3. In patches with unitary currents, the majority of channels were half-activated near -85 mV at 18 microM Ca2+, a value similar to that obtained when the human KCa channel alpha (HSLO) and beta (HKV,Ca beta) subunits are co-expressed. A small number of channels half-activated around 0 mV, suggesting the activity of the alpha subunit alone. 4. The properties of macroscopic currents were consistent with the view that most pore-forming alpha subunits were coupled to beta subunits, since the majority of currents had values for V1/2 near to -90 mV, and currents were potentiated by DHS-I. 5. We conclude that in human coronary artery smooth muscle cells, most maxi KCa channels are composed of alpha and beta subunits. The higher Ca2+ sensitivity of maxi KCa channels, resulting from their coupling to beta subunits, suggests an important role of this channel in regulating coronary tone. Their massive activation by micromolar Ca2+ concentrations may lead to a large hyperpolarization causing profound changes in coronary blood flow and cardiac function. PMID:9279807

  5. Molecular constituents of maxi KCa channels in human coronary smooth muscle: predominant alpha + beta subunit complexes.

    PubMed Central

    Tanaka, Y; Meera, P; Song, M; Knaus, H G; Toro, L

    1997-01-01

    1. Human large-conductance voltage- and calcium-sensitive K+ (maxi KCa) channels are composed of at least two subunits: the pore-forming subunit, alpha, and a modulatory subunit, beta. Expression of the beta subunit induces dramatic changes in alpha subunit function. It increases the apparent Ca2+ sensitivity and it allows dehydrosoyasaponin I (DHS-I) to upregulate the channel. 2. The functional coupling of maxi KCa channel alpha and beta subunits in freshly dissociated human coronary smooth muscle cells was assessed. To distinguish maxi KCa currents modulated by the beta subunit, we examined (a) their apparent Ca2+ sensitivity, as judged from the voltage necessary to half-activate the channel (V1/2), and (b) their activation by DHS-I. 3. In patches with unitary currents, the majority of channels were half-activated near -85 mV at 18 microM Ca2+, a value similar to that obtained when the human KCa channel alpha (HSLO) and beta (HKV,Ca beta) subunits are co-expressed. A small number of channels half-activated around 0 mV, suggesting the activity of the alpha subunit alone. 4. The properties of macroscopic currents were consistent with the view that most pore-forming alpha subunits were coupled to beta subunits, since the majority of currents had values for V1/2 near to -90 mV, and currents were potentiated by DHS-I. 5. We conclude that in human coronary artery smooth muscle cells, most maxi KCa channels are composed of alpha and beta subunits. The higher Ca2+ sensitivity of maxi KCa channels, resulting from their coupling to beta subunits, suggests an important role of this channel in regulating coronary tone. Their massive activation by micromolar Ca2+ concentrations may lead to a large hyperpolarization causing profound changes in coronary blood flow and cardiac function. Images Figure 1 PMID:9279807

  6. Meta-analyses of Dehalococcoides mccartyi strain 195 transcriptomic profiles identify a respiration rate-related gene expression transition point and interoperon recruitment of a key oxidoreductase subunit.

    PubMed

    Mansfeldt, Cresten B; Rowe, Annette R; Heavner, Gretchen L W; Zinder, Stephen H; Richardson, Ruth E

    2014-10-01

    A cDNA-microarray was designed and used to monitor the transcriptomic profile of Dehalococcoides mccartyi strain 195 (in a mixed community) respiring various chlorinated organics, including chloroethenes and 2,3-dichlorophenol. The cultures were continuously fed in order to establish steady-state respiration rates and substrate levels. The organization of array data into a clustered heat map revealed two major experimental partitions. This partitioning in the data set was further explored through principal component analysis. The first two principal components separated the experiments into those with slow (1.6±0.6 μM Cl-/h)- and fast (22.9±9.6 μM Cl-/h)-respiring cultures. Additionally, the transcripts with the highest loadings in these principal components were identified, suggesting that those transcripts were responsible for the partitioning of the experiments. By analyzing the transcriptomes (n=53) across experiments, relationships among transcripts were identified, and hypotheses about the relationships between electron transport chain members were proposed. One hypothesis, that the hydrogenases Hup and Hym and the formate dehydrogenase-like oxidoreductase (DET0186-DET0187) form a complex (as displayed by their tight clustering in the heat map analysis), was explored using a nondenaturing protein separation technique combined with proteomic sequencing. Although these proteins did not migrate as a single complex, DET0112 (an FdhB-like protein encoded in the Hup operon) was found to comigrate with DET0187 rather than with the catalytic Hup subunit DET0110. On closer inspection of the genome annotations of all Dehalococcoides strains, the DET0185-to-DET0187 operon was found to lack a key subunit, an FdhB-like protein. Therefore, on the basis of the transcriptomic, genomic, and proteomic evidence, the place of the missing subunit in the DET0185-to-DET0187 operon is likely filled by recruiting a subunit expressed from the Hup operon (DET0112). PMID:25063656

  7. Meta-Analyses of Dehalococcoides mccartyi Strain 195 Transcriptomic Profiles Identify a Respiration Rate-Related Gene Expression Transition Point and Interoperon Recruitment of a Key Oxidoreductase Subunit

    PubMed Central

    Mansfeldt, Cresten B.; Rowe, Annette R.; Heavner, Gretchen L. W.; Zinder, Stephen H.

    2014-01-01

    A cDNA-microarray was designed and used to monitor the transcriptomic profile of Dehalococcoides mccartyi strain 195 (in a mixed community) respiring various chlorinated organics, including chloroethenes and 2,3-dichlorophenol. The cultures were continuously fed in order to establish steady-state respiration rates and substrate levels. The organization of array data into a clustered heat map revealed two major experimental partitions. This partitioning in the data set was further explored through principal component analysis. The first two principal components separated the experiments into those with slow (1.6 ± 0.6 μM Cl−/h)- and fast (22.9 ± 9.6 μM Cl−/h)-respiring cultures. Additionally, the transcripts with the highest loadings in these principal components were identified, suggesting that those transcripts were responsible for the partitioning of the experiments. By analyzing the transcriptomes (n = 53) across experiments, relationships among transcripts were identified, and hypotheses about the relationships between electron transport chain members were proposed. One hypothesis, that the hydrogenases Hup and Hym and the formate dehydrogenase-like oxidoreductase (DET0186-DET0187) form a complex (as displayed by their tight clustering in the heat map analysis), was explored using a nondenaturing protein separation technique combined with proteomic sequencing. Although these proteins did not migrate as a single complex, DET0112 (an FdhB-like protein encoded in the Hup operon) was found to comigrate with DET0187 rather than with the catalytic Hup subunit DET0110. On closer inspection of the genome annotations of all Dehalococcoides strains, the DET0185-to-DET0187 operon was found to lack a key subunit, an FdhB-like protein. Therefore, on the basis of the transcriptomic, genomic, and proteomic evidence, the place of the missing subunit in the DET0185-to-DET0187 operon is likely filled by recruiting a subunit expressed from the Hup operon (DET0112). PMID

  8. Forskolin Regulates L-Type Calcium Channel through Interaction between Actinin 4 and β3 Subunit in Osteoblasts

    PubMed Central

    Guo, Lin; Hei, Hongya; Tian, Lulu; Peng, Wen; Cai, Hui

    2015-01-01

    Voltage-dependent L-type calcium channels that permit cellular calcium influx are essential in calcium-mediated modulation of cellular signaling. Although the regulation of voltage-dependent L-type calcium channels is linked to many factors including cAMP-dependent protein kinase A (PKA) activity and actin cytoskeleton, little is known about the detailed mechanisms underlying the regulation in osteoblasts. Our present study investigated the modulation of L-type calcium channel activities through the effects of forskolin on actin reorganization and on its functional interaction with actin binding protein actinin 4. The results showed that forskolin did not significantly affect the trafficking of pore forming α1c subunit and its interaction with actin binding protein actinin 4, whereas it significantly increased the expression of β3 subunit and its interaction with actinin 4 in osteoblast cells as assessed by co-immunoprecipitation, pull-down assay, and immunostaining. Further mapping showed that the ABD and EF domains of actinin 4 were interaction sites. This interaction is independent of PKA phosphorylation. Knockdown of actinin 4 significantly decreased the activities of L-type calcium channels. Our study revealed a new aspect of the mechanisms by which the forskolin activation of adenylyl cyclase - cAMP cascade regulates the L-type calcium channel in osteoblast cells, besides the PKA mediated phosphorylation of the channel subunits. These data provide insight into the important role of interconnection among adenylyl cyclase, cAMP, PKA, the actin cytoskeleton, and the channel proteins in the regulation of voltage-dependent L-type calcium channels in osteoblast cells. PMID:25902045

  9. The cyanobacterial cytochrome b6f subunit PetP adopts an SH3 fold in solution.

    PubMed

    Veit, Sebastian; Nagadoi, Aritaka; Rögner, Matthias; Rexroth, Sascha; Stoll, Raphael; Ikegami, Takahisa

    2016-06-01

    PetP is a peripheral subunit of the cytochrome b(6)f complex (b(6)f) present in both, cyanobacteria and red algae. It is bound to the cytoplasmic surface of this membrane protein complex where it greatly affects the efficiency of the linear photosynthetic electron flow although it is not directly involved in the electron transfer reactions. Despite the crystal structures of the b(6)f core complex, structural information for the transient regulatory b(6)f subunits is still missing. Here we present the first structure of PetP at atomic resolution as determined by solution NMR. The protein adopts an SH3 fold, which is a common protein motif in eukaryotes but comparatively rare in prokaryotes. The structure of PetP enabled the identification of the potential interaction site for b(6)f binding by conservation mapping. The interaction surface is mainly formed by two large loop regions and one short 310 helix which also exhibit an increased flexibility as indicated by heteronuclear steady-state {(1)H}-(15)N NOE and random coil index parameters. The properties of this potential b(6)f binding site greatly differ from the canonical peptide binding site which is highly conserved in eukaryotic SH3 domains. Interestingly, three other proteins of the photosynthetic electron transport chain share this SH3 fold with PetP: NdhS of the photosynthetic NADH dehydrogenase-like complex (NDH-1), PsaE of the photosystem 1 and subunit α of the ferredoxin-thioredoxin reductase have, similar to PetP, a great impact on the photosynthetic electron transport. Finally, a model is presented to illustrate how SH3 domains modulate the photosynthetic electron transport processes in cyanobacteria. PMID:27033306

  10. Functional characterization of the trans-membrane domain interactions of the Sec61 protein translocation complex beta-subunit

    PubMed Central

    Zhao, Xueqiang; Jäntti, Jussi

    2009-01-01

    Background In eukaryotic cells co- and post-translational protein translocation is mediated by the trimeric Sec61 complex. Currently, the role of the Sec61 complex β-subunit in protein translocation is poorly understood. We have shown previously that in Saccharomyces cerevisiae the trans-membrane domain alone is sufficient for the function of the β-subunit Sbh1p in co-translational protein translocation. In addition, Sbh1p co-purifies not only with the protein translocation channel subunits Sec61p and Sss1p, but also with the reticulon family protein Rtn1p. Results We used random mutagenesis to generate novel Sbh1p mutants in order to functionally map the Sbh1p trans-membrane domain. These mutants were analyzed for their interactions with Sec61p and how they support co-translational protein translocation. The distribution of mutations identifies one side of the Sbh1p trans-membrane domain α-helix that is involved in interactions with Sec61p and that is important for Sbh1p function in protein translocation. At the same time, these mutations do not affect Sbh1p interaction with Rtn1p. Furthermore we show that Sbh1p is found in protein complexes containing not only Rtn1p, but also the two other reticulon-like proteins Rtn2p and Yop1p. Conclusion Our results identify functionally important amino acids in the Sbh1p trans-membrane domain. In addition, our results provide additional support for the involvement of Sec61β in processes unlinked to protein translocation. PMID:19857245

  11. YC-1 BINDING TO THE BETA SUBUNIT OF SOLUBLE GUANYLYL CYCLASE OVERCOMES ALLOSTERIC INHIBITION BY THE ALPHA SUBUNIT

    PubMed Central

    Purohit, Rahul; Fritz, Bradley G.; The, Juliana; Issaian, Aaron; Weichsel, Andrzej; David, Cynthia L.; Campbell, Eric; Hausrath, Andrew C.; Rassouli-Taylor, Leida; Garcin, Elsa D.; Gage, Matthew J.; Montfort, William R.

    2014-01-01

    Soluble guanylate cyclase (sGC) is a heterodimeric heme protein and the primary nitric oxide receptor. NO binding stimulates cyclase activity, leading to regulation of cardiovascular physiology and making sGC an attractive target for drug discovery. YC-1 and related compounds stimulate sGC both independently and synergistically with NO and CO binding; however, where the compounds bind and how they work remains unknown. Using linked-equilibria binding measurements, surface plasmon resonance, and domain truncations in Manduca sexta and bovine sGC, we demonstrate that YC-1 binds near or directly to the heme-containing domain of the beta subunit. In the absence of CO, YC-1 binds with Kd = 9–21 μM, depending on construct. In the presence of CO, these values decrease to 0.6–1.1 μM. Pfizer compound 25 bound ~10-fold weaker than YC-1 in the absence of CO whereas compound BAY 41–2272 bound particularly tightly in the presence of CO (Kd = 30–90 nM). Additionally, we found that CO binding is much weaker to heterodimeric sGC proteins (Kd = 50–100 μM) than to the isolated heme domain (Kd = 0.2 μM for Manduca beta H-NOX/PAS). YC-1 greatly enhanced CO binding to heterodimeric sGC, as expected (Kd = ~1 μM). These data indicate the alpha subunit induces a heme pocket conformation with lower affinity for CO and NO. YC-1 family compounds bind near the heme domain, overcoming the alpha subunit effect and inducing a heme pocket conformation with high affinity. We propose this high-affinity conformation is required for the full-length protein to achieve high catalytic activity. PMID:24328155

  12. The variable subunit associated with protein phosphatase 2A0 defines a novel multimember family of regulatory subunits.

    PubMed Central

    Zolnierowicz, S; Van Hoof, C; Andjelković, N; Cron, P; Stevens, I; Merlevede, W; Goris, J; Hemmings, B A

    1996-01-01

    Two protein phosphatase 2A (PP2A) holoenzymes were isolated from rabbit skeletal muscle containing, in addition to the catalytic and PR65 regulatory subunits, proteins of apparent molecular masses of 61 and 56 kDa respectively. Both holoenzymes displayed low basal phosphorylase phosphatase activity, which could be stimulated by protamine to an extent similar to that of previously characterized PP2A holoenzymes. Protein micro-sequencing of tryptic peptides derived from the 61 kDa protein, termed PR61, yielded 117 residues of amino acid sequence. Molecular cloning by enrichment of specific mRNAs, followed by reverse transcription-PCR and cDNA library screening, revealed that this protein exists in multiple isoforms encoded by at least three genes, one of which gives rise to several splicing variants. Comparisons of these sequences with the available databases identified one more human gene and predicted another based on a rabbit cDNA-derived sequence, thus bringing the number of genes encoding PR61 family members to five. Peptide sequences derived from PR61 corresponded to the deduced amino acid sequences of either alpha or beta isoforms, indicating that the purified PP2A preparation was a mixture of at least two trimers. In contrast, the 56 kDa subunit (termed PR56) seems to correspond to the epsilon isoform of PR61. Several regulatory subunits of PP2A belonging to the PR61 family contain consensus sequences for nuclear localization and might therefore target PP2A to nuclear substrates. PMID:8694763

  13. Phenotypic consequences of deletion of the {gamma}{sub 3}, {alpha}{sub 5}, or {beta}{sub 3} subunit of the type A {gamma}-aminobutyric acid receptor in mice

    SciTech Connect

    Culia, C.T.; Stubbs, L.J.; Montgomery, C.S.; Russell, L.B.; Rinchik, E.M.

    1994-03-29

    Three genes (Gabrg3, Gabra5, and Gabrb3) encoding the {gamma}{sub 3}, {alpha}{sub 5}, and {beta}{sub 3} subunits of the type A {gamma}-aminobutyric acid receptor, respectively, are known to map near the pink-eyed dilution (p) locus in mouse chromosome 7. This region shares homology with a segment of human chromosome 15 that is implicated in Angelman syndrome, an inherited neurobehavioral disorder. By mapping Gabrg3-Gabra5-Gabrb3-telomere. Like Gabrb3, neither the Gabra5 nor Gabrg3 gene is functionally imprinted in adult mouse brain. Mice deleted for all three subunits die at birth with a cleft palate, although there are rare survivors ({approximately} 5%) that do not have a cleft palate but do exhibit a neurological abnormality characterized by tremor, jerky gait, and runtiness. The authors have previously suggested that deficiency of the {beta}{sub 3} subunit may be responsible for the clefting defect. Most notably, however, in this report they describe mice carrying two overlapping, complementing p deletions that fail to express the {gamma}{sub 3} transcript, as well as mice from another line that express neither the {gamma}{sub 3} nor {alpha}{sub 5} transcripts. Surprisingly, mice from both of these lines are phenotypically normal and do not exhibit any of the neurological symptoms characteristic of the rare survivors that are deleted for all three ({gamma}{sub 3}, {alpha}{sub 5}, and {beta}{sub 3}) subunits. These mice therefore provide a whole-organism type A {gamma}-aminobutyric-acid receptor background that is devoid of any receptor subtypes that normally contain the {gamma}{sub 3} and/or {alpha}{sub 5} subunits. The absence of an overt neurological phenotype in mice lacking the {gamma}{sub 3} and/or {alpha}{sub 5} subunits also suggests that mutations in these genes are unlikely to provide useful animal models for Angelman syndrome in humans.

  14. Binding of ATP by pertussis toxin and isolated toxin subunits

    SciTech Connect

    Hausman, S.Z.; Manclark, C.R.; Burns, D.L. )

    1990-07-03

    The binding of ATP to pertussis toxin and its components, the A subunit and B oligomer, was investigated. Whereas, radiolabeled ATP bound to the B oligomer and pertussis toxin, no binding to the A subunit was observed. The binding of ({sup 3}H)ATP to pertussis toxin and the B oligomer was inhibited by nucleotides. The relative effectiveness of the nucleotides was shown to be ATP > GTP > CTP > TTP for pertussis toxin and ATP > GTP > TTP > CTP for the B oligomer. Phosphate ions inhibited the binding of ({sup 3}H)ATP to pertussis toxin in a competitive manner; however, the presence of phosphate ions was essential for binding of ATP to the B oligomer. The toxin substrate, NAD, did not affect the binding of ({sup 3}H)ATP to pertussis toxin, although the glycoprotein fetuin significantly decreased binding. These results suggest that the binding site for ATP is located on the B oligomer and is distinct from the enzymatically active site but may be located near the eukaryotic receptor binding site.

  15. Mechanisms underlying subunit independence in pyramidal neuron dendrites.

    PubMed

    Behabadi, Bardia F; Mel, Bartlett W

    2014-01-01

    Pyramidal neuron (PN) dendrites compartmentalize voltage signals and can generate local spikes, which has led to the proposal that their dendrites act as independent computational subunits within a multilayered processing scheme. However, when a PN is strongly activated, back-propagating action potentials (bAPs) sweeping outward from the soma synchronize dendritic membrane potentials many times per second. How PN dendrites maintain the independence of their voltage-dependent computations, despite these repeated voltage resets, remains unknown. Using a detailed compartmental model of a layer 5 PN, and an improved method for quantifying subunit independence that incorporates a more accurate model of dendritic integration, we first established that the output of each dendrite can be almost perfectly predicted by the intensity and spatial configuration of its own synaptic inputs, and is nearly invariant to the rate of bAP-mediated "cross-talk" from other dendrites over a 100-fold range. Then, through an analysis of conductance, voltage, and current waveforms within the model cell, we identify three biophysical mechanisms that together help make independent dendritic computation possible in a firing neuron, suggesting that a major subtype of neocortical neuron has been optimized for layered, compartmentalized processing under in-vivo-like spiking conditions. PMID:24357611

  16. Thermostable cross-protective subunit vaccine against Brucella species.

    PubMed

    Cherwonogrodzky, John W; Barabé, Nicole D; Grigat, Michelle L; Lee, William E; Poirier, Robert T; Jager, Scott J; Berger, Bradley J

    2014-12-01

    A subunit vaccine candidate was produced from Brucella suis 145 (biovar 4; expressing both the A antigen of Brucella abortus and the M antigen of Brucella melitensis). The preparation consisted mostly of polysaccharide (PS; >90% [wt/wt]; both cell-associated PS and exo-PS were combined) and a small amount of protein (1 to 3%) with no apparent nucleic acids. Vaccinated mice were protected (these had a statistically significant reduction in bacterial colonization compared to that of unvaccinated controls) when challenged with representative strains of three Brucella species most pathogenic for humans, i.e., B. abortus, B. melitensis, and B. suis. As little as 1 ng of the vaccine, without added adjuvant, protected mice against B. suis 145 infection (5 × 10(5) CFU), and a single injection of 1 μg of this subunit vaccine protected mice from B. suis 145 challenge for at least 14 months. A single immunization induced a serum IgG response to Brucella antigens that remained elevated for up to 9 weeks. The use of heat (i.e., boiling-water bath, autoclaving) in the vaccine preparation showed that it was thermostable. This method also ensured safety and security. The vaccine produced was immunogenic and highly protective against multiple strains of Brucella and represents a promising candidate for further evaluation. PMID:25320267

  17. Functional Analysis of AP-2 α and μ2 Subunits

    PubMed Central

    Motley, Alison M.; Berg, Nicola; Taylor, Marcus J.; Sahlender, Daniela A.; Hirst, Jennifer; Owen, David J.

    2006-01-01

    The AP-2 adaptor complex plays a key role in cargo recognition and clathrin-coated vesicle formation at the plasma membrane. To investigate the functions of individual binding sites and domains of the AP-2 complex in vivo, we have stably transfected HeLa cells with wild-type and mutant small interfering RNA–resistant α and μ2 subunits and then used siRNA knockdowns to deplete the endogenous proteins. Mutating the PtdIns(4,5)P2 binding site of α, the phosphorylation site of μ2, or the YXXΦ binding site of μ2 impairs AP-2 function, as assayed by transferrin uptake. In contrast, removing the C-terminal appendage domain of α, or mutating the PtdIns(4,5)P2 binding site of μ2, has no apparent effect. However, adding a C-terminal GFP tag to α renders it completely nonfunctional. These findings demonstrate that there is some functional redundancy in the binding sites of the various AP-2 subunits, because no single mutation totally abolishes function. They also help to explain why GFP-tagged AP-2 never appears to leave the plasma membrane in some live cell imaging studies. Finally, they establish a new model system that can be used both for additional structure-function analyses, and as a way of testing tagged constructs for function in vivo. PMID:17035630

  18. Features of 80S mammalian ribosome and its subunits

    PubMed Central

    Budkevich, Tatyana V.; El'skaya, Anna V.; Nierhaus, Knud H.

    2008-01-01

    It is generally believed that basic features of ribosomal functions are universally valid, but a systematic test still stands out for higher eukaryotic 80S ribosomes. Here we report: (i) differences in tRNA and mRNA binding capabilities of eukaryotic and bacterial ribosomes and their subunits. Eukaryotic 40S subunits bind mRNA exclusively in the presence of cognate tRNA, whereas bacterial 30S do bind mRNA already in the absence of tRNA. 80S ribosomes bind mRNA efficiently in the absence of tRNA. In contrast, bacterial 70S interact with mRNA more productively in the presence rather than in the absence of tRNA. (ii) States of initiation (Pi), pre-translocation (PRE) and post-translocation (POST) of the ribosome were checked and no significant functional differences to the prokaryotic counterpart were observed including the reciprocal linkage between A and E sites. (iii) Eukaryotic ribosomes bind tetracycline with an affinity 15 times lower than that of bacterial ribosomes (Kd 30 μM and 1–2 μM, respectively). The drug does not effect enzymatic A-site occupation of 80S ribosomes in contrast to non-enzymatic tRNA binding to the A-site. Both observations explain the relative resistance of eukaryotic ribosomes to this antibiotic. PMID:18632761

  19. Subunit-specific phenotypes of Salmonella typhimurium HU mutants.

    PubMed Central

    Hillyard, D R; Edlund, M; Hughes, K T; Marsh, M; Higgins, N P

    1990-01-01

    Salmonella hupA and hupB mutants were studied to determine the reasons for the high degree of conservation in HU structure in bacteria. We found one HU-1-specific effect; the F'128 plasmid was 25-fold less stable in hupB compared with hupA or wild-type cells. F' plasmids were 120-fold more unstable in hupA hupB double mutants compared with wild-type cells, and the double mutant also had a significant alteration in plasmid DNA structure. pBR322 DNA isolated from hupA hupB strains was deficient in supercoiling by 10 to 15% compared with wild-type cells, and the topoisomer distribution was significantly more heterogeneous than in wild-type or single-mutant strains. Other systems altered by HU inactivation included flagellar phase variation and phage Mu transposition. However, Mu transposition rates were only about fourfold lower in Salmonella HU double mutants. One reason that Salmonella HU double mutants may be less defective for Mu transposition than E. coli is the synthesis in double mutants of a new, small, basic heat-stable protein, which might partially compensate for the loss of HU. The results indicate that although either HU-1 or HU-2 subunit alone may accommodate the cellular need for general chromosomal organization, the selective pressure to conserve HU-1 and HU-2 structure during evolution could involve specialized roles of the individual subunits. Images PMID:2168381

  20. The Small Ribosomal Subunit RNA Isoforms in Plasmodium Cynomolgi

    PubMed Central

    Corredor, V.; Enea, V.

    1994-01-01

    We report the isolation, characterization and analysis of the small subunit rRNA genes in Plasmodium cynomolgi (Ceylon). As in other Plasmodium species, these genes are present in low copy number, are unlinked and form two types that are distinct in sequence and are expressed stage specifically. The asexually expressed (type A) genes are present in four copies in the Ceylon(-) and in five copies in the Berok(-) strain. Surprisingly, the sexually expressed (type B) gene is present in a single copy. The vast majority of the differences between gene types is confined to the variable regions. The pattern of divergence is different from that observed in Plasmodium berghei or in Plasmodium falciparum. Analysis of the small subunit rRNA sequences of P. cynomolgi, P. berghei and P. falciparum, indicates that the two gene types do not evolve independently but rather interact (through gene conversion or some form of recombination) to such an extent as to erase whatever stage-specific sequence signatures they may have had in the last common ancestor. PMID:8005440

  1. Thermostable Cross-Protective Subunit Vaccine against Brucella Species

    PubMed Central

    Barabé, Nicole D.; Grigat, Michelle L.; Lee, William E.; Poirier, Robert T.; Jager, Scott J.; Berger, Bradley J.

    2014-01-01

    A subunit vaccine candidate was produced from Brucella suis 145 (biovar 4; expressing both the A antigen of Brucella abortus and the M antigen of Brucella melitensis). The preparation consisted mostly of polysaccharide (PS; >90% [wt/wt]; both cell-associated PS and exo-PS were combined) and a small amount of protein (1 to 3%) with no apparent nucleic acids. Vaccinated mice were protected (these had a statistically significant reduction in bacterial colonization compared to that of unvaccinated controls) when challenged with representative strains of three Brucella species most pathogenic for humans, i.e., B. abortus, B. melitensis, and B. suis. As little as 1 ng of the vaccine, without added adjuvant, protected mice against B. suis 145 infection (5 × 105 CFU), and a single injection of 1 μg of this subunit vaccine protected mice from B. suis 145 challenge for at least 14 months. A single immunization induced a serum IgG response to Brucella antigens that remained elevated for up to 9 weeks. The use of heat (i.e., boiling-water bath, autoclaving) in the vaccine preparation showed that it was thermostable. This method also ensured safety and security. The vaccine produced was immunogenic and highly protective against multiple strains of Brucella and represents a promising candidate for further evaluation. PMID:25320267

  2. Evaluation of a Subunit Vaccine to Infectious Hematopoietic Necrosis (IHN) Virus, 1984 FY Annual Report.

    SciTech Connect

    Leong, JoAnn Ching

    1985-07-01

    A prototype subunit vaccine to IHN virus is being developed by recombinant DNA techniques. The techniques involve the isolation and characterization of the glycoprotein gene, which encodes the viral protein responsible for inducing a protective immune response in fish. The viral glycoprotein gene has been cloned and a restriction map of the cloned gene has been prepared. Preliminary DNA sequence analysis of the cloned gene has been initiated so that manipulation of the gene for maximum expression in appropriate plasmid vectors is possible. A recombinant plasmid containing the viral gene inserted in the proper orientation adjacent to a very strong lambda promoter and ribosome binding site has been constructed. Evaluation of this recombinant plasmid for gene expression is being conducted. Immunization trials with purified viral glycoprotein indicate that fish are protected against lethal doses of IHNV after immersion and intraperitoneal methods of immunization. In addition, cross protection immunization trials indicate that Type 2 and Type 1 IHN virus produce glycoproteins that are cross-protective.

  3. Subunit organization in the Dam1 kinetochore complex and its ring around microtubules

    PubMed Central

    Ramey, Vincent H.; Wong, Amanda; Fang, Jie; Howes, Stuart; Barnes, Georjana; Nogales, Eva

    2011-01-01

    All eukaryotic cells must segregate their chromosomes equally between two daughter cells at each division. This process needs to be robust, as errors in the form of loss or gain of genetic material have catastrophic effects on viability. Chromosomes are captured, aligned, and segregated to daughter cells via interaction with spindle microtubules mediated by the kinetochore. In Saccharomyces cerevisiae one microtubule attaches to each kinetochore, requiring extreme processivity from this single connection. The yeast Dam1 complex, an essential component of the outer kinetochore, forms rings around microtubules and in vitro recapitulates much of the functionality of a kinetochore–microtubule attachment. To understand the mechanism of the Dam1 complex at the kinetochore, we must know how it binds to microtubules, how it assembles into rings, and how assembly is regulated. We used electron microscopy to map several subunits within the structure of the Dam1 complex and identify the organization of Dam1 complexes within the ring. Of importance, new data strongly support a more passive role for the microtubule in Dam1 ring formation. Integrating this information with previously published data, we generated a structural model for the Dam1 complex assembly that advances our understanding of its function and will direct future experiments. PMID:21965284

  4. Soybean ribulose bisphosphate carboxylase small subunit: Mechanisms and determinants of RNA turnover. Annual progress report

    SciTech Connect

    Meagher, R.B.

    1993-12-31

    An in vitro degradation system has been developed from petunia and soybean polysomes in order to investigate the mechanisms and determinants controlling RNA turnover in higher plants. This system faithfully degrades soybean ribulose-1,5-bisphosphate carboxylase small subunit (rbcS) mRNA into the same products observed in total RNA preparations. In previous years it was shown that the most stable products represent a nested constellation of fragments, which are shortened from their 3{prime} ends, and have intact 5{prime} ends. Exogenous rbcS RNA tagged with novel 5{prime} sequence 15 or 56 bp long were synthesized in vitro as Sp6 and T7 runoff transcripts, respectively. When added to the system they were degraded faithfully into constellation of products which were 15 or 56 bp longer than the endogenous products, respectively. Detailed kinetics on the appearance of these exogenous products confirmed degradation proceeds in an overall 3{prime} to 5{prime} direction but suggested that there are multiple pathways through which the RNA may be degraded. To further demonstrate a precursor product relationships, in vitro synthesized transcripts truncated at their 3{prime} ends were shown to degrade into the expected smaller fragments previously mapped in the 5{prime} portion of the rbcS RNA.

  5. Cooperative folding kinetics of BBL protein and peripheral subunit-binding domain homologues

    PubMed Central

    Yu, Wookyung; Chung, Kwanghoon; Cheon, Mookyung; Heo, Muyoung; Han, Kyou-Hoon; Ham, Sihyun; Chang, Iksoo

    2008-01-01

    Recent experiments claiming that Naf-BBL protein follows a global downhill folding raised an important controversy as to the folding mechanism of fast-folding proteins. Under the global downhill folding scenario, not only do proteins undergo a gradual folding, but folding events along the continuous folding pathway also could be mapped out from the equilibrium denaturation experiment. Based on the exact calculation using a free energy landscape, relaxation eigenmodes from a master equation, and Monte Carlo simulation of an extended Muñoz–Eaton model that incorporates multiscale-heterogeneous pairwise interactions between amino acids, here we show that the very nature of a two-state cooperative transition such as a bimodal distribution from an exact free energy landscape and biphasic relaxation kinetics manifest in the thermodynamics and folding–unfolding kinetics of BBL and peripheral subunit-binding domain homologues. Our results provide an unequivocal resolution to the fundamental controversy related to the global downhill folding scheme, whose applicability to other proteins should be critically reexamined. PMID:18272497

  6. Oligosaccharyltransferase subunits bind polypeptide substrate to locally enhance N-glycosylation.

    PubMed

    Jamaluddin, M Fairuz B; Bailey, Ulla-Maja; Schulz, Benjamin L

    2014-12-01

    Oligosaccharyltransferase is a multiprotein complex that catalyzes asparagine-linked glycosylation of diverse proteins. Using yeast genetics and glycoproteomics, we found that transient interactions between nascent polypeptide and Ost3p/Ost6p, homologous subunits of oligosaccharyltransferase, were able to modulate glycosylation efficiency in a site-specific manner in vivo. These interactions were driven by hydrophobic and electrostatic complementarity between amino acids in the peptide-binding groove of Ost3p/Ost6p and the sequestered stretch of substrate polypeptide. Based on this dependence, we used in vivo scanning mutagenesis and in vitro biochemistry to map the precise interactions that affect site-specific glycosylation efficiency. We conclude that transient binding of substrate polypeptide by Ost3p/Ost6p increases glycosylation efficiency at asparagines proximal and C-terminal to sequestered sequences. We detail a novel mode of interaction between translocating nascent polypeptide and oligosaccharyltransferase in which binding to Ost3p/Ost6p segregates a short flexible loop of glycosylation-competent polypeptide substrate that is delivered to the oligosaccharyltransferase active site for efficient modification. PMID:25118247

  7. Oligosaccharyltransferase Subunits Bind Polypeptide Substrate to Locally Enhance N-glycosylation*

    PubMed Central

    Jamaluddin, M. Fairuz B.; Bailey, Ulla-Maja; Schulz, Benjamin L.

    2014-01-01

    Oligosaccharyltransferase is a multiprotein complex that catalyzes asparagine-linked glycosylation of diverse proteins. Using yeast genetics and glycoproteomics, we found that transient interactions between nascent polypeptide and Ost3p/Ost6p, homologous subunits of oligosaccharyltransferase, were able to modulate glycosylation efficiency in a site-specific manner in vivo. These interactions were driven by hydrophobic and electrostatic complementarity between amino acids in the peptide-binding groove of Ost3p/Ost6p and the sequestered stretch of substrate polypeptide. Based on this dependence, we used in vivo scanning mutagenesis and in vitro biochemistry to map the precise interactions that affect site-specific glycosylation efficiency. We conclude that transient binding of substrate polypeptide by Ost3p/Ost6p increases glycosylation efficiency at asparagines proximal and C-terminal to sequestered sequences. We detail a novel mode of interaction between translocating nascent polypeptide and oligosaccharyltransferase in which binding to Ost3p/Ost6p segregates a short flexible loop of glycosylation-competent polypeptide substrate that is delivered to the oligosaccharyltransferase active site for efficient modification. PMID:25118247

  8. OsGatB, the Subunit of tRNA-Dependent Amidotransferase, Is Required for Primary Root Development in Rice.

    PubMed

    Qin, Cheng; Cheng, Linming; Zhang, Huanhuan; He, Meiling; Shen, Jingqin; Zhang, Yunhong; Wu, Ping

    2016-01-01

    A short-root rice mutant was isolated from an ethyl methane sulfonate-mutagenized library. From map-based cloning strategy, a point mutation, resulting in an amino acid change from proline to leucine, was identified in the fourth exon of a glutamyl-tRNA (Gln) amidotransferase B subunit family protein (OsGatB, LOC_Os11g34210). This gene is an ortholog of Arabidopsis GatB and yeast PET112. GatB is a subunit of tRNA-dependent amidotransferase (AdT), an essential enzyme involved in Gln-tRNA(Gln) synthesis in mitochondria. Although previous studies have described that cessation in mitochondrial translation is lethal at very early developmental stages in plants, this point mutation resulted in a non-lethal phenotype of smaller root meristem and shorter root cell length. In the root, OsGatB was predominantly expressed in the root tip and played an important role in cell division and elongation there. OsGatB was localized in the mitochondria, and mitochondrial structure and function were all affected in Osgatb root tip cells. PMID:27200067

  9. Troponin T3 regulates nuclear localization of the calcium channel Cavβ1a subunit in skeletal muscle.

    PubMed

    Zhang, Tan; Taylor, Jackson; Jiang, Yang; Pereyra, Andrea S; Messi, Maria Laura; Wang, Zhong-Min; Hereñú, Claudia; Delbono, Osvaldo

    2015-08-15

    The voltage-gated calcium channel (Cav) β1a subunit (Cavβ1a) plays an important role in excitation-contraction coupling (ECC), a process in the myoplasm that leads to muscle-force generation. Recently, we discovered that the Cavβ1a subunit travels to the nucleus of skeletal muscle cells where it helps to regulate gene transcription. To determine how it travels to the nucleus, we performed a yeast two-hybrid screening of the mouse fast skeletal muscle cDNA library and identified an interaction with troponin T3 (TnT3), which we subsequently confirmed by co-immunoprecipitation and co-localization assays in mouse skeletal muscle in vivo and in cultured C2C12 muscle cells. Interacting domains were mapped to the leucine zipper domain in TnT3 COOH-terminus (160-244 aa) and Cavβ1a NH2-terminus (1-99 aa), respectively. The double fluorescence assay in C2C12 cells co-expressing TnT3/DsRed and Cavβ1a/YFP shows that TnT3 facilitates Cavβ1a nuclear recruitment, suggesting that the two proteins play a heretofore unknown role during early muscle differentiation in addition to their classical role in ECC regulation. PMID:25981458

  10. The G protein β subunit controls virulence and multiple growth- and development-related traits in Verticillium dahliae.

    PubMed

    Tzima, Aliki K; Paplomatas, Epaminondas J; Tsitsigiannis, Dimitrios I; Kang, Seogchan

    2012-04-01

    To gain insight into the role of G protein-mediated signaling in virulence and development of the soilborne, wilt causing fungus Verticillium dahliae, the G protein β subunit gene (named as VGB) was disrupted in tomato race 1 strain of V. dahliae. A resulting mutant strain, 70ΔGb15, displayed drastic reduction in virulence, increased microsclerotia formation and conidiation, and decreased ethylene production compared to the corresponding wild type (wt) strain 70wt-r1. Moreover, 70ΔGb15 exhibited an elongated rather than radial growth pattern on agar media. A transformant of 70ΔGb15 (named as 70ΔGbPKAC1) that carries an extra copy of VdPKAC1, a V. dahliae gene encoding the catalytic subunit of the cAMP-dependent protein kinase A, exhibited wt growth pattern and conidiation, was unable to form microsclerotia, produced high amounts of ethylene, and exhibited virulence between that of 70ΔGb15 and 70wt-r1 on tomato plants. Phenotypical changes observed in 70ΔGb15 and 70ΔGbPKAC1 correlated with transcriptional changes in several genes involved in signaling (MAP kinase VMK1) and development (hydrophobin VDH1 and ACC synthase ACS1) of V. dahliae. Results from the present work suggest a linkage between VGB and VdPKAC1 signaling pathways in regulating virulence, hormone production and development in V. dahliae. PMID:22387367

  11. Mutations in the Proteolipid Subunits of the Vacuolar H+-ATPase Provide Resistance to Indolotryptoline Natural Products

    PubMed Central

    2015-01-01

    Indolotryptoline natural products represent a small family of structurally unique chromopyrrolic acid-derived antiproliferative agents. Like many prospective anticancer agents before them, the exploration of their potential clinical utility has been hindered by the limited information known about their mechanism of action. To study the mode of action of two closely related indolotryptolines (BE-54017, cladoniamide A), we selected for drug resistant mutants using a multidrug resistance-suppressed (MDR-sup) Schizosaccharomyces pombe strain. As fission yeast maintains many of the basic cancer-relevant cellular processes present in human cells, it represents an appealing model to use in determining the potential molecular target of antiproliferative natural products through resistant mutant screening. Full genome sequencing of resistant mutants identified mutations in the c and c′ subunits of the proteolipid substructure of the vacuolar H+-ATPase complex (V-ATPase). This collection of resistance-conferring mutations maps to a site that is distant from the nucleotide-binding sites of V-ATPase and distinct from sites found to confer resistance to known V-ATPase inhibitors. Acid vacuole staining, cross-resistance studies, and direct c/c′ subunit mutagenesis all suggest that indolotryptolines are likely a structurally novel class of V-ATPase inhibitors. This work demonstrates the general utility of resistant mutant selection using MDR-sup S. pombe as a rapid and potentially systematic approach for studying the modes of action of cytotoxic natural products. PMID:25319670

  12. YY1 and Sp1 activate transcription of the human NDUFS8 gene encoding the mitochondrial complex I TYKY subunit.

    PubMed

    Lescuyer, Pierre; Martinez, Pascal; Lunardi, Joël

    2002-03-19

    Complex I is the most complicated of the multimeric enzymes that constitute the mitochondrial respiratory chain. It is encoded by both mitochondrial and nuclear genomes. We have previously characterized the human NDUFS8 gene that encodes the TYKY subunit. This essential subunit is thought to participate in the electron transfer and proton pumping activities of complex I. Here, we have analyzed the transcriptional regulation of the NDUFS8 gene. Using primer extension assays, we have identified two transcription start sites. The basal promoter was mapped to a 247 bp sequence upstream from the main transcription start site by reporter gene analysis in HeLa cells and in differentiated or non-differentiated C2C12 cells. Three Sp1 sites and one YY1 site were identified in this minimal promoter. Through gel shift analysis, all sites were shown to bind to their cognate transcription factors. Site-directed mutagenesis revealed that the YY1 site and two upstream adjacent Sp1 sites drive most of the promoter activity. This work represents the first promoter analysis for a complex I gene. Together with previous studies, our results indicate that YY1 and Sp1 control the expression of genes encoding proteins that are involved in almost all steps of the oxidative phosphorylation metabolism. PMID:11955626

  13. OsGatB, the Subunit of tRNA-Dependent Amidotransferase, Is Required for Primary Root Development in Rice

    PubMed Central

    Qin, Cheng; Cheng, Linming; Zhang, Huanhuan; He, Meiling; Shen, Jingqin; Zhang, Yunhong; Wu, Ping

    2016-01-01

    A short-root rice mutant was isolated from an ethyl methane sulfonate-mutagenized library. From map-based cloning strategy, a point mutation, resulting in an amino acid change from proline to leucine, was identified in the fourth exon of a glutamyl-tRNA (Gln) amidotransferase B subunit family protein (OsGatB, LOC_Os11g34210). This gene is an ortholog of Arabidopsis GatB and yeast PET112. GatB is a subunit of tRNA-dependent amidotransferase (AdT), an essential enzyme involved in Gln-tRNAGln synthesis in mitochondria. Although previous studies have described that cessation in mitochondrial translation is lethal at very early developmental stages in plants, this point mutation resulted in a non-lethal phenotype of smaller root meristem and shorter root cell length. In the root, OsGatB was predominantly expressed in the root tip and played an important role in cell division and elongation there. OsGatB was localized in the mitochondria, and mitochondrial structure and function were all affected in Osgatb root tip cells. PMID:27200067

  14. Visualization and molecular analysis of nuclear import of protein kinase CK2 subunits in living cells.

    PubMed

    Martel, V; Filhol, O; Nueda, A; Gerber, D; Benitez, M J; Cochet, C

    2001-11-01

    We have generated fusion proteins between the subunits of CK2 and GFP and characterized their behaviour in living cells. The expressed fusion proteins were functional and interacted with endogenous CK2. Imaging of NIH3T3 cells expressing low level of GFP-CK2alpha or GFP-CK2beta showed that both proteins were mostly nuclear in interphase. Both CK2 subunits contain nuclear localization domains that target them independently to the nucleus. Once in the nucleus, both subunits diffused rapidly in the nucleoplasm. In mitotic cells, CK2 subunits were dispersed throughout the cytoplasm and were not associated to chromatin. Our data are compatible with the idea that each subunit can translocate individually to the nucleus to interact with each other or with important cellular partners. Understanding the molecular mechanisms which regulate the dynamic localization of CK2 subunits will be of central importance. PMID:11827178

  15. Operon structure and cotranslational subunit association direct protein assembly in bacteria.

    PubMed

    Shieh, Yu-Wei; Minguez, Pablo; Bork, Peer; Auburger, Josef J; Guilbride, D Lys; Kramer, Günter; Bukau, Bernd

    2015-11-01

    Assembly of protein complexes is considered a posttranslational process involving random collision of subunits. We show that within the Escherichia coli cytosol, bacterial luciferase subunits LuxA and LuxB assemble into complexes close to the site of subunit synthesis. Assembly efficiency decreases markedly if subunits are synthesized on separate messenger RNAs from genes integrated at distant chromosomal sites. Subunit assembly initiates cotranslationally on nascent LuxB in vivo. The ribosome-associated chaperone trigger factor delays the onset of cotranslational interactions until the LuxB dimer interface is fully exposed. Protein assembly is thus directly coupled to the translation process and involves spatially confined, actively chaperoned cotranslational subunit interactions. Bacterial gene organization into operons therefore reflects a fundamental cotranslational mechanism for spatial and temporal regulation that is vital to effective assembly of protein complexes. PMID:26405228

  16. [Chromatographic and spectroscopic characterization of phycocyanin and its subunits purified from Anabaena variabilis CCC421].

    PubMed

    Chakdar, N; Sakha, S; Pabbi, S

    2014-01-01

    Phycocyanin, a high value pigment was purified from diazotrophic cyanobacteria Anabaena variabilis CCC421 using a strategy involving ammonium sulfate precipitation, dialysis and anion exchange chromatography using DEAE-cellulose column. 36% phycocyanin with a purity of 2.75 was recovered finally after anion exchange chromatography. Purified phycocyanin was found to contain 2 subunits of 17 and 18 kDa which were identified as a-and (3 subunits by SDS-PAGE and MALDI-TOE HPLC method using a C5 column coupled with fluorescence or photodiode-based detection was also developed to separate and detect the A. variabilis CCC421 phycocyanin subunits. The fluorescence method was more sensitive than photodiode one. The purified phycocyanin from A. variabilis CCC421 as well as its subunits was characterized with respect to absorption and IR spectra. Spectral characterization of the subunits revealed that alpha and beta subunits contained one and two phycocyanobilin groups as chromophores, respectively. PMID:25272755

  17. ATP synthases with novel rotor subunits: new insights into structure, function and evolution of ATPases.

    PubMed

    Müller, Volker; Lingl, Astrid; Lewalter, Kim; Fritz, Michael

    2005-12-01

    ATPases with unusual membrane-embedded rotor subunits were found in both F(1)F(0) and A(1)A(0) ATP synthases. The rotor subunit c of A(1)A(0) ATPases is, in most cases, similar to subunit c from F(0). Surprisingly, multiplied c subunits with four, six, or even 26 transmembrane spans have been found in some archaea and these multiplication events were sometimes accompanied by loss of the ion-translocating group. Nevertheless, these enzymes are still active as ATP synthases. A duplicated c subunit with only one ion-translocating group was found along with "normal" F(0) c subunits in the Na(+) F(1)F(0) ATP synthase of the bacterium Acetobacterium woodii. These extraordinary features and exceptional structural and functional variability in the rotor of ATP synthases may have arisen as an adaptation to different cellular needs and the extreme physicochemical conditions in the early history of life. PMID:16691483

  18. The NMDA receptor NR2A subunit regulates proliferation of MKN45 human gastric cancer cells

    SciTech Connect

    Watanabe, Kanako; Kanno, Takeshi; Oshima, Tadayuki; Miwa, Hiroto; Tashiro, Chikara; Nishizaki, Tomoyuki

    2008-03-07

    The present study investigated proliferation of MKN28 and MKN45 human gastric cancer cells regulated by the N-methyl-D-aspartate (NMDA) receptor subunit. The NMDA receptor antagonist DL-2-amino-5-phosphonovaleric acid (AP5) inhibited proliferation of MKN45 cells, but not MKN28 cells. Of the NMDA subunits such as NR1, NR2 (2A, 2B, 2C, and 2D), and NR3 (3A and 3B), all the NMDA subunit mRNAs except for the NR2B subunit mRNA were expressed in both MKN28 and MKN45 cells. MKN45 cells were characterized by higher expression of the NR2A subunit mRNA and lower expression of the NR1 subunit mRNA, but MKN28 otherwise by higher expression of the NR1 subunit mRNA and lower expression of the NR2A subunit mRNA. MKN45 cell proliferation was also inhibited by silencing the NR2A subunit-targeted gene. For MKN45 cells, AP5 or knocking-down the NR2A subunit increased the proportion of cells in the G{sub 1} phase of cell cycling and decreased the proportion in the S/G{sub 2} phase. The results of the present study, thus, suggest that blockage of NMDA receptors including the NR2A subunit suppresses MKN45 cell proliferation due to cell cycle arrest at the G{sub 1} phase; in other words, the NR2A subunit promotes MKN45 cell proliferation by accelerating cell cycling.

  19. Efficient Expression of Functional (α6β2)2β3 AChRs in Xenopus Oocytes from Free Subunits Using Slightly Modified α6 Subunits

    PubMed Central

    Ley, Carson Kai-Kwong; Kuryatov, Alexander; Wang, Jingyi; Lindstrom, Jon Martin

    2014-01-01

    Human (α6β2)(α4β2)β3 nicotinic acetylcholine receptors (AChRs) are essential for addiction to nicotine and a target for drug development for smoking cessation. Expressing this complex AChR is difficult, but has been achieved using subunit concatamers. In order to determine what limits expression of α6* AChRs and to efficiently express α6* AChRs using free subunits, we investigated expression of the simpler (α6β2)2β3 AChR. The concatameric form of this AChR assembles well, but is transported to the cell surface inefficiently. Various chimeras of α6 with the closely related α3 subunit increased expression efficiency with free subunits and produced pharmacologically equivalent functional AChRs. A chimera in which the large cytoplasmic domain of α6 was replaced with that of α3 increased assembly with β2 subunits and transport of AChRs to the oocyte surface. Another chimera replacing the unique methionine 211 of α6 with leucine found at this position in transmembrane domain 1 of α3 and other α subunits increased assembly of mature subunits containing β3 subunits within oocytes. Combining both α3 sequences in an α6 chimera increased expression of functional (α6β2)2β3 AChRs to 12-fold more than with concatamers. This is pragmatically useful, and provides insights on features of α6 subunit structure that limit its expression in transfected cells. PMID:25068303

  20. Neonatal prebiotic (BGOS) supplementation increases the levels of synaptophysin, GluN2A-subunits and BDNF proteins in the adult rat hippocampus.

    PubMed

    Williams, Sarah; Chen, Li; Savignac, Helene M; Tzortzis, George; Anthony, Daniel C; Burnet, Philip W J

    2016-03-01

    Compelling data suggest that perturbations in microbial colonization of the gut in early-life, influences neurodevelopment and adult brain function. If this is the case, then ensuring the growth of beneficial bacteria at an early age will lead to optimal brain development and maturation. We have tested whether feeding neonatal rats daily (from post-natal days 3-21) with a galacto-oligosaccharide prebiotic (Bimuno®, BGOS) or a control solution, alters the levels of hippocampal N-Methyl-D-Aspartate receptor (NMDAR) subunits (GluN1, GluN2A, GluN2B), synaptic proteins (synaptophysin, MAP2, and GAP43) and brain-derived-neurotrophic factor (BDNF), at post-natal days 22 and 56. The administration of BGOS significantly elevated GluN2A subunits, synaptophysin and BDNF in the hippocampus of 22 day old rats. The effect was also observed on day 56 (26 days after the feeding ceased). The levels of all other proteins (GluN1, GluN2B, MAP2, GAP43) remained unaltered. Increased GluN2A, synaptophysin, BDNF, but not MAP2, may suggest that neonatal BGOS feeding alters neurotransmission rather than synaptic architecture. Although the functional consequences of our findings require further investigation, the current study confirms that the manipulation of gut bacteria in early-life, has central effects that persist until at least young adulthood. PMID:26682524

  1. 2.8-Å Cryo-EM Structure of the Large Ribosomal Subunit from the Eukaryotic Parasite Leishmania.

    PubMed

    Shalev-Benami, Moran; Zhang, Yan; Matzov, Donna; Halfon, Yehuda; Zackay, Arie; Rozenberg, Haim; Zimmerman, Ella; Bashan, Anat; Jaffe, Charles L; Yonath, Ada; Skiniotis, Georgios

    2016-07-12

    Leishmania is a single-cell eukaryotic parasite of the Trypanosomatidae family, whose members cause an array of tropical diseases. The often fatal outcome of infections, lack of effective vaccines, limited selection of therapeutic drugs, and emerging resistant strains, underline the need to develop strategies to combat these pathogens. The Trypanosomatid ribosome has recently been highlighted as a promising therapeutic target due to structural features that are distinct from other eukaryotes. Here, we present the 2.8-Å resolution structure of the Leishmania donovani large ribosomal subunit (LSU) derived from a cryo-EM map, further enabling the structural observation of eukaryotic rRNA modifications that play a significant role in ribosome assembly and function. The structure illustrates the unique fragmented nature of leishmanial LSU rRNA and highlights the irregular distribution of rRNA modifications in Leishmania, a characteristic with implications for anti-parasitic drug development. PMID:27373148

  2. Soybean ribulose bisphosphate carboxylase small subunit: Mechanisms and determinants on RNA turnover: Progress report, May 1988--December 1988

    SciTech Connect

    Meagher, R.

    1989-01-01

    The goal of this work is to elucidate the mechanisms and determinants behind the light induced turnover and general chemical instability of Rubisco small subunit (SSU) RNA. Three approaches are being used: (A) to use molecular physiology to help define the phenomena and identify the possible cellular machinery involved in these processes; (B) SSU RNA stability will be assayed in transgenic plants; and (C) in vivo RNA structure is being examined via chemical modification of RNA. The tremendous success we have had with assaying in vivo RNA structure by DMS modification and a need to validate this technique with sufficient controls has resulted in our adding an additional goal to the project: to develop a DMS modification map of 18S rRNA and portions of Rubisco SSU RNA and predict a potential secondary structures.

  3. The testis-specific Cα2 subunit of PKA is kinetically indistinguishable from the common Cα1 subunit of PKA

    PubMed Central

    2011-01-01

    Background The two variants of the α-form of the catalytic (C) subunit of protein kinase A (PKA), designated Cα1 and Cα2, are encoded by the PRKACA gene. Whereas Cα1 is ubiquitous, Cα2 expression is restricted to the sperm cell. Cα1 and Cα2 are encoded with different N-terminal domains. In Cα1 but not Cα2 the N-terminal end introduces three sites for posttranslational modifications which include myristylation at Gly1, Asp-specific deamidation at Asn2 and autophosphorylation at Ser10. Previous reports have implicated specific biological features correlating with these modifications on Cα1. Since Cα2 is not modified in the same way as Cα1 we tested if they have distinct biochemical activities that may be reflected in different biological properties. Results We show that Cα2 interacts with the two major forms of the regulatory subunit (R) of PKA, RI and RII, to form cAMP-sensitive PKAI and PKAII holoenzymes both in vitro and in vivo as is also the case with Cα1. Moreover, using Surface Plasmon Resonance (SPR), we show that the interaction patterns of the physiological inhibitors RI, RII and PKI were comparable for Cα2 and Cα1. This is also the case for their potency to inhibit catalytic activities of Cα2 and Cα1. Conclusion We conclude that the regulatory complexes formed with either Cα1 or Cα2, respectively, are indistinguishable. PMID:21812984

  4. Mapping the Heart

    ERIC Educational Resources Information Center

    Hulse, Grace

    2012-01-01

    In this article, the author describes how her fourth graders made ceramic heart maps. The impetus for this project came from reading "My Map Book" by Sara Fanelli. This book is a collection of quirky, hand-drawn and collaged maps that diagram a child's world. There are maps of her stomach, her day, her family, and her heart, among others. The…

  5. Fundamentals of Physical Mapping

    Technology Transfer Automated Retrieval System (TEKTRAN)

    This book chapter provides an overview of physical mapping in plants and its use for map-based gene cloning. A brief overview of cytogenetics-based physical mapping strategies, and physical mapping approaches currently used and the lessons learnt from the success stories were furnished. The statisti...

  6. Structure of Csm2 elucidates the relationship between small subunits of CRISPR-Cas effector complexes.

    PubMed

    Venclovas, Česlovas

    2016-05-01

    Type I and type III CRISPR-Cas effector complexes share similar architecture and have homologous key subunits. However, the relationship between the so-called small subunits of these complexes remains a contentious issue. Here, it is shown that the recently solved structure of Thermotoga maritima Csm2 represents a dimer with the extensive structure swapping between monomers. Unswapping the structure generates a compact globular monomer which shares similar structure and surface properties with Cmr5, the small subunit of a related Cmr complex. Detailed analysis of available structures of small subunits reveals that they all have a common fold suggesting their common origin. PMID:27091242

  7. Immunoproteasome Assembly: Cooperative Incorporation of Interferon γ (IFN-γ)–inducible Subunits

    PubMed Central

    Griffin, Thomas A.; Nandi, Dipankar; Cruz, Miguel; Fehling, Hans Jörg; Kaer, Luc Van; Monaco, John J.; Colbert, Robert A.

    1998-01-01

    LMP2, LMP7, and MECL are interferon γ–inducible catalytic subunits of vertebrate 20S proteasomes, which can replace constitutive catalytic subunits (delta, X, and Z, respectively) during proteasome biogenesis. We demonstrate that MECL requires LMP2 for efficient incorporation into preproteasomes, and preproteasomes containing LMP2 and MECL require LMP7 for efficient maturation. The latter effect depends on the presequence of LMP7, but not on LMP7 catalytic activity. This cooperative mechanism favors the assembly of homogeneous “immunoproteasomes” containing all three inducible subunits, suggesting that these subunits act in concert to enhance proteasomal generation of major histocompatibility complex class I–binding peptides. PMID:9419215

  8. Thermosensitive TRPV Channel Subunits Coassemble into Heteromeric Channels with Intermediate Conductance and Gating Properties

    PubMed Central

    Cheng, Wei; Yang, Fan; Takanishi, Christina L.; Zheng, Jie

    2007-01-01

    Heat-sensitive transient receptor potential (TRP) channels (TRPV1–4) form the major cellular sensors for detecting temperature increases. Homomeric channels formed by thermosensitive TRPV subunits exhibit distinct temperature thresholds. While these subunits do share significant sequence similarity, whether they can coassemble into heteromeric channels has been controversial. In the present study we investigated the coassembly of TRPV subunits using both spectroscopy-based fluorescence resonance energy transfer (FRET) and single-channel recordings. Fluorescent protein–tagged TRPV subunits were coexpressed in HEK 293 cells; FRET between different subunits was measured as an indication of the formation of heteromeric channels. We observed strong FRET when fluorescence signals were collected selectively from the plasma membrane using a “spectra FRET” approach but much weaker or no FRET from intracellular fluorescence. In addition, no FRET was detected when TRPV subunits were coexpressed with members of the TRPM subfamily or CLC-0 chloride channel subunits. These results indicate that a substantial fraction of TRP channels in the plasma membrane of cotransfected cells were heteromeric. Single-channel recordings confirmed the existence of multiple heteromeric channel forms. Interestingly, heteromeric TRPV channels exhibit intermediate conductance levels and gating kinetic properties. As these subunits coexpress both in sensory neurons and in other tissues, including heart and brain, coassembly between TRPV subunits may contribute to greater functional diversity. PMID:17325193

  9. Bigenomic transcriptional regulation of all thirteen cytochrome c oxidase subunit genes by specificity protein 1

    PubMed Central

    Dhar, Shilpa S.; Johar, Kaid; Wong-Riley, Margaret T. T.

    2013-01-01

    Cytochrome c oxidase (COX) is one of only four known bigenomic proteins, with three mitochondria-encoded subunits and 10 nucleus-encoded ones derived from nine different chromosomes. The mechanism of regulating this multi-subunit, bigenomic enzyme is not fully understood. We hypothesize that specificity protein 1 (Sp1) functionally regulates the 10 nucleus-encoded COX subunit genes directly and the three mitochondrial COX subunit genes indirectly by regulating mitochondrial transcription factors A and B (TFAM, TFB1M and TFB2M) in neurons. By means of in silico analysis, electrophoretic mobility shift and supershift assays, chromatin immunoprecipitation, RNA interference and over-expression experiments, the present study documents that Sp1 is a critical regulator of all 13 COX subunit genes in neurons. This regulation is intimately associated with neuronal activity. Silencing of Sp1 prevented the upregulation of all COX subunits by KCl, and over-expressing Sp1 rescued all COX subunits from being downregulated by tetrodotoxin. Thus, Sp1 and our previously described nuclear respiratory factors 1 and 2 are the three key regulators of all 13 COX subunit genes in neurons. The binding sites for Sp1 on all 10 nucleus-encoded COX subunits, TFAM, TFB1M and TFB2M are highly conserved among mice, rats and humans. PMID:23516108

  10. Characterization of Human RNA Polymerase III Identifies Orthologues for Saccharomyces cerevisiae RNA Polymerase III Subunits

    PubMed Central

    Hu, Ping; Wu, Si; Sun, Yuling; Yuan, Chih-Chi; Kobayashi, Ryuji; Myers, Michael P.; Hernandez, Nouria

    2002-01-01

    Unlike Saccharomyces cerevisiae RNA polymerase III, human RNA polymerase III has not been entirely characterized. Orthologues of the yeast RNA polymerase III subunits C128 and C37 remain unidentified, and for many of the other subunits, the available information is limited to database sequences with various degrees of similarity to the yeast subunits. We have purified an RNA polymerase III complex and identified its components. We found that two RNA polymerase III subunits, referred to as RPC8 and RPC9, displayed sequence similarity to the RNA polymerase II RPB7 and RPB4 subunits, respectively. RPC8 and RPC9 associated with each other, paralleling the association of the RNA polymerase II subunits, and were thus paralogues of RPB7 and RPB4. Furthermore, the complex contained a prominent 80-kDa polypeptide, which we called RPC5 and which corresponded to the human orthologue of the yeast C37 subunit despite limited sequence similarity. RPC5 associated with RPC53, the human orthologue of S. cerevisiae C53, paralleling the association of the S. cerevisiae C37 and C53 subunits, and was required for transcription from the type 2 VAI and type 3 human U6 promoters. Our results provide a characterization of human RNA polymerase III and show that the RPC5 subunit is essential for transcription. PMID:12391170

  11. Cytosolic tail sequences and subunit interactions are critical for synaptic localization of glutamate receptors.

    PubMed

    Chang, Howard Chia-Hao; Rongo, Christopher

    2005-05-01

    AMPA-type glutamate receptors mediate excitatory synaptic transmission in the nervous system. The receptor subunit composition and subcellular localization play an important role in regulating synaptic strength. GLR-1 and GLR-2 are the Caenorhabditis elegans subunits most closely related to the mammalian AMPA-type receptors. These subunits are expressed in overlapping sets of interneurons, and contain type-I PDZ binding motifs in their carboxy-terminal cytosolic tail sequences. We report that GLR-1 and GLR-2 may form a heteromeric complex, the localization of which depends on either GLR-1 or GLR-2 tail sequences. Subunit interactions alone can mediate synaptic localization as endogenous GLR-1, or GLR-2 subunits can rescue the localization defects of subunits lacking tail sequences. Moreover, GLR-2 cytosolic tail sequences are sufficient to confer synaptic localization on a heterologous reporter containing a single-transmembrane domain. The localization of this GLR-2 reporter requires both a PDZ-binding motif in the GLR-2 tail sequence, and sequences outside of this motif. The PDZ protein LIN-10 regulates the localization of the reporter through the sequences outside of the PDZ-binding motif. Our results suggest that multiple synaptic localization signals reside in the cytosolic tail sequence of the receptor subunits, and that channel assembly can rescue the synaptic localization defects of individual mutant subunits as long as there are also wild-type subunits in the receptor complex. PMID:15840655

  12. Enrichment of GABAA Receptor α-Subunits on the Axonal Initial Segment Shows Regional Differences

    PubMed Central

    Gao, Yudong; Heldt, Scott A.

    2016-01-01

    Although it is generally recognized that certain α-subunits of γ-aminobutyric acid type A receptors (GABAARs) form enriched clusters on the axonal initial segment (AIS), the degree to which these clusters vary in different brain areas is not well known. In the current study, we quantified the density, size, and enrichment ratio of fluorescently labeled α1-, α2-, or α3-subunits aggregates co-localized with the AIS-marker ankyrin G and compared them to aggregates in non-AIS locations among different brain areas including hippocampal subfields, basal lateral amygdala (BLA), prefrontal cortex (PFC), and sensory cortex (CTX). We found regional differences in the enrichment of GABAAR α-subunits on the AIS. Significant enrichment was identified in the CA3 of hippocampus for α1-subunits, in the CA1, CA3, and BLA for α2-subunits, and in the BLA for α3-subunits. Using α-subunit knock-out (KO) mice, we found that BLA enrichment of α2- and α3-subunits were physiologically independent of each other, as the enrichment of one subunit was unaffected by the genomic deletion of the other. To further investigate the unique pattern of α-subunit enrichment in the BLA, we examined the association of α2- and α3-subunits with the presynaptic vesicular GABA transporter (vGAT) and the anchoring protein gephyrin (Geph). As expected, both α2- and α3-subunits on the AIS within the BLA received prominent GABAergic innervation from vGAT-positive terminals. Further, we found that the association of α2- and α3-subunits with Geph was weaker in AIS versus non-AIS locations, suggesting that Geph might be playing a lesser role in the enrichment of α2- and α3-subunits on the AIS. Overall, these observations suggest that GABAARs on the AIS differ in subunit composition across brain regions. As with somatodendritic GABAARs, the distinctive expression pattern of AIS-located GABAAR α-subunits in the BLA, and other brain areas, likely contribute to unique forms of GABAergic inhibitory

  13. Phylogenetic mapping of bacterial morphology

    NASA Technical Reports Server (NTRS)

    Siefert, J. L.; Fox, G. E.

    1998-01-01

    The availability of a meaningful molecular phylogeny for bacteria provides a context for examining the historical significance of various developments in bacterial evolution. Herein, the classical morphological descriptions of selected members of the domain Bacteria are mapped upon the genealogical ancestry deduced from comparison of small-subunit rRNA sequences. For the species examined in this study, a distinct pattern emerges which indicates that the coccus shape has arisen and accumulated independently multiple times in separate lineages and typically survived as a persistent end-state morphology. At least two other morphologies persist but have evolved only once. This study demonstrates that although bacterial morphology is not useful in defining bacterial phylogeny, it is remarkably consistent with that phylogeny once it is known. An examination of the experimental evidence available for morphogenesis as well as microbial fossil evidence corroborates these findings. It is proposed that the accumulation of persistent morphologies is a result of the biophysical properties of peptidoglycan and their genetic control, and that an evolved body-plan strategy based on peptidoglycan may have been a fate-sealing step in the evolution of Bacteria. More generally, this study illustrates that significant evolutionary insights can be obtained by examining biological and biochemical data in the context of a reliable phylogenetic structure.

  14. National Atlas maps

    USGS Publications Warehouse

    U.S. Geological Survey

    1991-01-01

    The National Atlas of the United States of America was published by the U.S. Geological Survey in 1970. Its 765 maps and charts are on 335 14- by 19-inch pages. Many of the maps span facing pages. It's worth a quick trip to the library just to leaf through all 335 pages of this book. Rapid scanning of its thematic maps yields rich insights to the geography of issues of continuing national interest. On most maps, the geographic patterns are still valid, though the data are not current. The atlas is out of print, but many of its maps can be purchased separately. Maps that span facing pages in the atlas are printed on one sheet. The maps dated after 1970 are either revisions of original atlas maps, or new maps published in atlas format. The titles of the separate maps are listed here.

  15. A Cooperative Escherichia coli Aspartate Transcarbamoylase without Regulatory Subunits

    SciTech Connect

    Mendes, K.; Kantrowitz, E

    2010-01-01

    Here we report the isolation, kinetic characterization, and X-ray structure determination of a cooperative Escherichia coli aspartate transcarbamoylase (ATCase) without regulatory subunits. The native ATCase holoenzyme consists of six catalytic chains organized as two trimers bridged noncovalently by six regulatory chains organized as three dimers, c{sub 6}r{sub 6}. Dissociation of the native holoenzyme produces catalytically active trimers, c{sub 3}, and nucleotide-binding regulatory dimers, r{sub 2}. By introducing specific disulfide bonds linking the catalytic chains from the upper trimer site specifically to their corresponding chains in the lower trimer prior to dissociation, a new catalytic unit, c{sub 6}, was isolated consisting of two catalytic trimers linked by disulfide bonds. Not only does the c{sub 6} species display enhanced enzymatic activity compared to the wild-type enzyme, but the disulfide bonds also impart homotropic cooperativity, never observed in the wild-type c3. The c{sub 6} ATCase was crystallized in the presence of phosphate and its X-ray structure determined to 2.10 {angstrom} resolution. The structure of c{sub 6} ATCase liganded with phosphate exists in a nearly identical conformation as other R-state structures with similar values calculated for the vertical separation and planar angles. The disulfide bonds linking upper and lower catalytic trimers predispose the active site into a more active conformation by locking the 240s loop into the position characteristic of the high-affinity R state. Furthermore, the elimination of the structural constraints imposed by the regulatory subunits within the holoenzyme provides increased flexibility to the c{sub 6} enzyme, enhancing its activity over the wild-type holoenzyme (c{sub 6}r{sub 6}) and c{sub 3}. The covalent linkage between upper and lower catalytic trimers restores homotropic cooperativity so that a binding event at one or so active sites stimulates binding at the other sites. Reduction

  16. Structure–Function Relationships in Fungal Large-Subunit Catalases

    SciTech Connect

    Diaz, A.; Valdez, V; Rudino-Pinera, E; Horjales, E; Hansberg, W

    2009-01-01

    Neurospora crassa has two large-subunit catalases, CAT-1 and CAT-3. CAT-1 is associated with non-growing cells and accumulates particularly in asexual spores; CAT-3 is associated with growing cells and is induced under different stress conditions. It is our interest to elucidate the structure-function relationships in large-subunit catalases. Here we have determined the CAT-3 crystal structure and compared it with the previously determined CAT-1 structure. Similar to CAT-1, CAT-3 hydrogen peroxide (H{sub 2}O{sub 2}) saturation kinetics exhibited two components, consistent with the existence of two active sites: one saturated in the millimolar range and the other in the molar range. In the CAT-1 structure, we found three interesting features related to its unusual kinetics: (a) a constriction in the channel that conveys H{sub 2}O{sub 2} to the active site; (b) a covalent bond between the tyrosine, which forms the fifth coordination bound to the iron of the heme, and a vicinal cysteine; (c) oxidation of the pyrrole ring III to form a cis-hydroxyl group in C5 and a cis-{gamma}-spirolactone in C6. The site of heme oxidation marks the starts of the central channel that communicates to the central cavity and the shortest way products can exit the active site. CAT-3 has a similar constriction in its major channel, which could function as a gating system regulated by the H{sub 2}O{sub 2} concentration before the gate. CAT-3 functional tyrosine is not covalently bonded, but has instead the electron relay mechanism described for the human catalase to divert electrons from it. Pyrrole ring III in CAT-3 is not oxidized as it is in other large-subunit catalases whose structure has been determined. Different in CAT-3 from these enzymes is an occupied central cavity. Results presented here indicate that CAT-3 and CAT-1 enzymes represent a functional group of catalases with distinctive structural characteristics that determine similar kinetics.

  17. Structure-function relationships in fungal large-subunit catalases.

    PubMed

    Díaz, Adelaida; Valdés, Víctor-Julián; Rudiño-Piñera, Enrique; Horjales, Eduardo; Hansberg, Wilhelm

    2009-02-13

    Neurospora crassa has two large-subunit catalases, CAT-1 and CAT-3. CAT-1 is associated with non-growing cells and accumulates particularly in asexual spores; CAT-3 is associated with growing cells and is induced under different stress conditions. It is our interest to elucidate the structure-function relationships in large-subunit catalases. Here we have determined the CAT-3 crystal structure and compared it with the previously determined CAT-1 structure. Similar to CAT-1, CAT-3 hydrogen peroxide (H(2)O(2)) saturation kinetics exhibited two components, consistent with the existence of two active sites: one saturated in the millimolar range and the other in the molar range. In the CAT-1 structure, we found three interesting features related to its unusual kinetics: (a) a constriction in the channel that conveys H(2)O(2) to the active site; (b) a covalent bond between the tyrosine, which forms the fifth coordination bound to the iron of the heme, and a vicinal cysteine; (c) oxidation of the pyrrole ring III to form a cis-hydroxyl group in C5 and a cis-gamma-spirolactone in C6. The site of heme oxidation marks the starts of the central channel that communicates to the central cavity and the shortest way products can exit the active site. CAT-3 has a similar constriction in its major channel, which could function as a gating system regulated by the H(2)O(2) concentration before the gate. CAT-3 functional tyrosine is not covalently bonded, but has instead the electron relay mechanism described for the human catalase to divert electrons from it. Pyrrole ring III in CAT-3 is not oxidized as it is in other large-subunit catalases whose structure has been determined. Different in CAT-3 from these enzymes is an occupied central cavity. Results presented here indicate that CAT-3 and CAT-1 enzymes represent a functional group of catalases with distinctive structural characteristics that determine similar kinetics. PMID:19109972

  18. Cohesin-Dockerin Interactions of Cellulosomal Subunits of Clostridium cellulovorans

    PubMed Central

    Park, Jae-Seon; Matano, Yutaka; Doi, Roy H.

    2001-01-01

    The cellulosome of Clostridium cellulovorans consists of three major subunits: CbpA, EngE, and ExgS. The C. cellulovorans scaffolding protein (CbpA) contains nine hydrophobic repeated domains (cohesins) for the binding of enzymatic subunits. Cohesin domains are quite homologous, but there are some questions regarding their binding specificity because some of the domains have regions of low-level sequence similarity. Two cohesins which exhibit 60% sequence similarity were investigated for their ability to bind cellulosomal enzymes. Cohesin 1 (Coh1) was found to contain amino acid residues corresponding to amino acids 312 to 453 of CbpA, which contains a total of 1,848 amino acid residues. Coh6 was determined to contain amino acid residues corresponding to residues 1113 to 1254 of CbpA. By genetic construction, these two cohesins were each fused to MalE, producing MalE-Coh1 and MalE-Coh6. The abilities of two fusion proteins to bind to EngE, ExgS, and CbpA were compared. Although MalE-Coh6 could bind EngE and ExgS, little or no binding of the enzymatic subunits was observed with MalE-Coh1. Significantly, the abilities of the two fusion proteins to bind CbpA were similar. The binding of dockerin-containing enzymes to cohesin-containing proteins was suggested as a model for assembly of cellulosomes. In our examination of the role of dockerins, it was also shown that the binding of endoglucanase B (EngB) to CbpA was dependent on the presence of EngB's dockerin. These results suggest that different cohesins may function with differing efficiency and specificity, that cohesins may play some role in the formation of polycellulosomes through Coh-CbpA interactions, and that dockerins play an important role during the interaction of cellulosomal enzymes and cohesins present in CbpA. PMID:11514529

  19. Mutations in the protein kinase A R1α regulatory subunit cause familial cardiac myxomas and Carney complex

    PubMed Central

    Casey, Mairead; Vaughan, Carl J.; He, Jie; Hatcher, Cathy J.; Winter, Jordan M.; Weremowicz, Stanislawa; Montgomery, Kate; Kucherlapati, Raju; Morton, Cynthia C.; Basson, Craig T.

    2000-01-01

    Cardiac myxomas are benign mesenchymal tumors that can present as components of the human autosomal dominant disorder Carney complex. Syndromic cardiac myxomas are associated with spotty pigmentation of the skin and endocrinopathy. Our linkage analysis mapped a Carney complex gene defect to chromosome 17q24. We now demonstrate that the PRKAR1α gene encoding the R1α regulatory subunit of cAMP-dependent protein kinase A (PKA) maps to this chromosome 17q24 locus. Furthermore, we show that PRKAR1α frameshift mutations in three unrelated families result in haploinsufficiency of R1α and cause Carney complex. We did not detect any truncated R1α protein encoded by mutant PRKAR1α. Although cardiac tumorigenesis may require a second somatic mutation, DNA and protein analyses of an atrial myxoma resected from a Carney complex patient with a PRKAR1α deletion revealed that the myxoma cells retain both the wild-type and the mutant PRKAR1α alleles and that wild-type R1α protein is stably expressed. However, in this atrial myxoma, we did observe a reversal of the ratio of R1α to R2β regulatory subunit protein, which may contribute to tumorigenesis. Further investigation will elucidate the cell-specific effects of PRKAR1α haploinsufficiency on PKA activity and the role of PKA in cardiac growth and differentiation. This article may have been published online in advance of the print edition. The date of publication is available from the JCI website, http://www.jci.org. J. Clin. Invest. 106:R31–R38 (2000). PMID:10974026

  20. p53 and TFIIEα share a common binding site on the Tfb1/p62 subunit of TFIIH

    PubMed Central

    Di Lello, Paola; Miller Jenkins, Lisa M.; Mas, Caroline; Langlois, Chantal; Malitskaya, Elena; Fradet-Turcotte, Amélie; Archambault, Jacques; Legault, Pascale; Omichinski, James G.

    2008-01-01

    The general transcription factor IIH is recruited to the transcription preinitiation complex through an interaction between its p62/Tfb1 subunit and the α-subunit of the general transcription factor IIE (TFIIEα). We have determined that the acidic carboxyl terminus of TFIIEα (TFIIEα336–439) directly binds the amino-terminal PH domain of p62/Tfb1 with nanomolar affinity. NMR mapping and mutagenesis studies demonstrate that the TFIIEα binding site on p62/Tfb1 is identical to the binding site for the second transactivation domain of p53 (p53 TAD2). In addition, we demonstrate that TFIIEα336–439 is capable of competing with p53 for a common binding site on p62/Tfb1 and that TFIIEα336–439 and the diphosphorylated form (pS46/pT55) of p53 TAD2 have similar binding constants. NMR structural studies reveal that TFIIEα336–439 contains a small domain (residues 395–433) folded in a novel ββααα topology. NMR mapping studies demonstrate that two unstructured regions (residues 377–393 and residues 433–439) located on either side of the folded domain appear to be required for TFIIEα336–439 binding to p62/Tfb1 and that these two unstructured regions are held close to each other in three-dimensional space by the novel structured domain. We also demonstrate that, like p53, TFIIEα336–439 can activate transcription in vivo. These results point to an important interplay between the general transcription factor TFIIEα and the tumor suppressor protein p53 in regulating transcriptional activation that may be modulated by the phosphorylation status of p53. PMID:18160537

  1. p53 and TFIIEalpha share a common binding site on the Tfb1/p62 subunit of TFIIH.

    PubMed

    Di Lello, Paola; Miller Jenkins, Lisa M; Mas, Caroline; Langlois, Chantal; Malitskaya, Elena; Fradet-Turcotte, Amélie; Archambault, Jacques; Legault, Pascale; Omichinski, James G

    2008-01-01

    The general transcription factor IIH is recruited to the transcription preinitiation complex through an interaction between its p62/Tfb1 subunit and the alpha-subunit of the general transcription factor IIE (TFIIEalpha). We have determined that the acidic carboxyl terminus of TFIIEalpha (TFIIEalpha(336-439)) directly binds the amino-terminal PH domain of p62/Tfb1 with nanomolar affinity. NMR mapping and mutagenesis studies demonstrate that the TFIIEalpha binding site on p62/Tfb1 is identical to the binding site for the second transactivation domain of p53 (p53 TAD2). In addition, we demonstrate that TFIIEalpha(336-439) is capable of competing with p53 for a common binding site on p62/Tfb1 and that TFIIEalpha(336-439) and the diphosphorylated form (pS46/pT55) of p53 TAD2 have similar binding constants. NMR structural studies reveal that TFIIEalpha(336-439) contains a small domain (residues 395-433) folded in a novel betabetaalphaalphaalpha topology. NMR mapping studies demonstrate that two unstructured regions (residues 377-393 and residues 433-439) located on either side of the folded domain appear to be required for TFIIEalpha(336-439) binding to p62/Tfb1 and that these two unstructured regions are held close to each other in three-dimensional space by the novel structured domain. We also demonstrate that, like p53, TFIIEalpha(336-439) can activate transcription in vivo. These results point to an important interplay between the general transcription factor TFIIEalpha and the tumor suppressor protein p53 in regulating transcriptional activation that may be modulated by the phosphorylation status of p53. PMID:18160537

  2. Google Maps: You Are Here

    ERIC Educational Resources Information Center

    Jacobsen, Mikael

    2008-01-01

    Librarians use online mapping services such as Google Maps, MapQuest, Yahoo Maps, and others to check traffic conditions, find local businesses, and provide directions. However, few libraries are using one of Google Maps most outstanding applications, My Maps, for the creation of enhanced and interactive multimedia maps. My Maps is a simple and…

  3. Structure of the large ribosomal subunit from human mitochondria

    PubMed Central

    Bai, Xiao-chen; Sugimoto, Yoichiro; Edwards, Patricia C.; Murshudov, Garib; Scheres, Sjors H. W.; Ramakrishnan, V.

    2014-01-01

    Human mitochondrial ribosomes are highly divergent from all other known ribosomes and are specialized to exclusively translate membrane proteins. They are linked with hereditary mitochondrial diseases, and are often the unintended targets of various clinically useful antibiotics. Using single-particle electron cryo-microscopy we have determined the structure of its large subunit to 3.4 angstrom resolution, revealing 48 proteins, 21 of which are specific to mitochondria. The structure unveils an adaptation of the exit tunnel for hydrophobic nascent peptides, extensive remodeling of the central protuberance including recruitment of mitochondrial tRNAVal to play an integral structural role, and changes in the tRNA binding sites related to the unusual characteristics of mitochondrial tRNAs. PMID:25278503

  4. Interactions between RNase P protein subunits in Archaea

    PubMed Central

    Hall, Thomas A.; Brown, James W.

    2004-01-01

    A yeast two-hybrid system was used to identify protein–protein interactions between the ribonuclease P (RNase P) protein subunits Mth11p, Mth687p, Mth688p and Mth1618p from the archaeon Methanothermobacter thermoautotrophicus. Clear interactions between Mth688p and Mth687p, and between Mth1618p and Mth11p, were confirmed by HIS3 and LacZ reporter expression. Weaker interactions of Mth687p and Mth688p with Mth11p, and Mth11p with itself, are also suggested. These interactions resemble, and confirm, those previously seen among the homologs of these proteins in the more complex yeast RNase P holoenzyme. PMID:15810434

  5. Tropomyosin diffusion over actin subunits facilitates thin filament assembly

    PubMed Central

    Fischer, Stefan; Rynkiewicz, Michael J.; Moore, Jeffrey R.; Lehman, William

    2016-01-01

    Coiled-coil tropomyosin binds to consecutive actin-subunits along actin-containing thin filaments. Tropomyosin molecules then polymerize head-to-tail to form cables that wrap helically around the filaments. Little is known about the assembly process that leads to continuous, gap-free tropomyosin cable formation. We propose that tropomyosin molecules diffuse over the actin-filament surface to connect head-to-tail to partners. This possibility is likely because (1) tropomyosin hovers loosely over the actin-filament, thus binding weakly to F-actin and (2) low energy-barriers provide tropomyosin freedom for 1D axial translation on F-actin. We consider that these unique features of the actin-tropomyosin interaction are the basis of tropomyosin cable formation. PMID:26798831

  6. Immunological characterization of exocyst complex subunits in cell differentiation.

    PubMed

    Wang, Sheng; Hsu, Shu C

    2003-06-01

    We have generated monoclonal antibodies (MAbs) against three proteins sec6, sec15, and exo84. These proteins have been shown to be components of the exocyst complex, a macromolecule required for many biological processes such as kidney epithelial formation and neuronal development. These antibodies can detect the three proteins by enzyme-linked immunoadsorbent assay (ELISA), Western blotting, immunofluorescence microscopy, and immunoprecipitation. Using these antibodies, we found that the three proteins have similar subcellular localization which changes upon cell differentiation. These three proteins also co-immunoprecipitate with each other. These results suggest that at least three exocyst subunits associate with each other in vivo and redistribute in response to cell differentiation. In the future, these antibodies should be useful in the cell biological and functional analysis of the exocyst complex under physiological and pathological conditions. PMID:12954101

  7. Nasal reconstruction based on aesthetic subunits in Orientals.

    PubMed

    Yotsuyanagi, T; Yamashita, K; Urushidate, S; Yokoi, K; Sawada, Y

    2000-07-01

    Reconstruction based on the aesthetic subunit principle has yielded good aesthetic outcomes in patients with moderate to severe nasal defects caused by trauma or tumor resection. However, the topographic subunits previously proposed are often unsuitable for Orientals. Compared with the nose in white patients, the nose in Orientals is low, lacks nasal muscle, and has a flat glabella; the structural features of the underlying cartilage and bone are not distinctly reflected in outward appearance. The authors devised aesthetic subunits suitable for Orientals, and they used these units to reconstruct various parts of the nose. The major difference between these units and those presented previously is the lack of soft triangles and the addition of the glabella as an independent unit. The authors divided the nose into the following five topographic units: the glabella, the nasal dorsum, the nasal tip, and the two alae. The border of the nasal dorsum unit was extended to above the maxillonasal suture. The basic reconstruction techniques use a V-Y advancement flap from the forehead to reconstruct the glabella, an island flap from the forehead to reconstruct the nasal dorsum and nasal tip, a nasolabial flap to reconstruct an ala, and a malar flap to reconstruct the cheek. A combination of flaps was used when the defect involved more than one unit. This concept was used for nasal reconstruction in 24 patients. In one patient undergoing reconstruction of the nasal dorsum and in one undergoing reconstruction of the nasal tip, the texture of the forearm flap did not match well, which resulted in a slightly unsatisfactory aesthetic outcome. In one patient in whom the glabella, nasal dorsum, and part of the cheek were reconstructed simultaneously, a web was formed at the medial ocular angle, and a secondary operation was subsequently performed using Z-plasty. In one patient undergoing reconstruction with a forehead flap, defatting was required to reduce the bulk of the

  8. Map reading tools for map libraries.

    USGS Publications Warehouse

    Greenberg, G.L.

    1982-01-01

    Engineers, navigators and military strategists employ a broad array of mechanical devices to facilitate map use. A larger number of map users such as educators, students, tourists, journalists, historians, politicians, economists and librarians are unaware of the available variety of tools which can be used with maps to increase the speed and efficiency of their application and interpretation. This paper identifies map reading tools such as coordinate readers, protractors, dividers, planimeters, and symbol-templets according to a functional classification. Particularly, arrays of tools are suggested for use in determining position, direction, distance, area and form (perimeter-shape-pattern-relief). -from Author

  9. Effective polymer adjuvants for sustained delivery of protein subunit vaccines.

    PubMed

    Adams, Justin R; Haughney, Shannon L; Mallapragada, Surya K

    2015-03-01

    We have synthesized thermogelling cationic amphiphilic pentablock copolymers that have the potential to act as injectable vaccine carriers and adjuvants that can simultaneously provide sustained delivery and enhance the immunogenicity of released antigen. While these pentablock copolymers have shown efficacy in DNA delivery in past studies, the ability to deliver both DNA and protein for subunit vaccines using the same polymeric carrier can provide greater flexibility and efficacy. We demonstrate the ability of these pentablock copolymers, and the parent triblock Pluronic copolymers to slowly release structurally intact and antigenically stable protein antigens in vitro, create an antigen depot through long-term injection-site persistence and enhance the in vivo immune response to these antigens. We show release of the model protein antigen ovalbumin in vitro from the thermogelling block copolymers with the primary, secondary and tertiary structures of the released protein unchanged compared to the native protein, and its antigenicity preserved upon release. The block copolymers form a gel at physiological temperatures that serves as an antigenic depot and persists in vivo at the site of injection for over 50days. The pentablock copolymers show a significant fivefold enhancement in the immune response compared to soluble protein alone, even 6weeks after the administration, based on measurement of antibody titers. These results demonstrate the potential of these block copolymers hydrogels to persist for several weeks and sustain the release of antigen with minimal effects on protein stability and antigenicity; and their ability to be used simultaneously as a sustained delivery device as well as a subunit vaccine adjuvant platform. PMID:25484331

  10. hERG subunit composition determines differential drug sensitivity

    PubMed Central

    Abi-Gerges, N; Holkham, H; Jones, EMC; Pollard, CE; Valentin, J-P; Robertson, GA

    2011-01-01

    BACKGROUND AND PURPOSE The majority of human ether-a-go-go-related gene (hERG) screens aiming to minimize the risk of drug-induced long QT syndrome have been conducted using heterologous systems expressing the hERG 1a subunit, although both hERG 1a and 1b subunits contribute to the K+ channels producing the repolarizing current IKr. We tested a range of compounds selected for their diversity to determine whether hERG 1a and 1a/1b channels exhibit different sensitivities that may influence safety margins or contribute to a stratified risk analysis. EXPERIMENTAL APPROACH We used the IonWorks™ plate-based electrophysiology device to compare sensitivity of hERG 1a and 1a/1b channels stably expressed in HEK293 cells to 50 compounds previously shown to target hERG channels. Potency was determined as IC50 values (µM) obtained from non-cumulative, eight-point concentration–effect curves of normalized data, fitted to the Hill equation. To minimize possible sources of variability, compound potency was assessed using test plates arranged in alternating columns of cells expressing hERG 1a and 1a/1b. KEY RESULTS Although the potency of most compounds was similar for the two targets, some surprising differences were observed. Fluoxetine (Prozac) was more potent at blocking hERG 1a/1b than 1a channels, yielding a corresponding reduction in the safety margin. In contrast, E-4031 was a more potent blocker of hERG 1a compared with 1a/1b channels, as previously reported, as was dofetilide, another high-affinity blocker. CONCLUSIONS AND IMPLICATIONS The current assays may underestimate the risk of some drugs to cause torsades de pointes arrhythmia, and overestimate the risk of others. PMID:21449979

  11. Proteomic analysis of transducin beta-subunit structural heterogeneity.

    PubMed

    Clack, James W; Juhl, Martha; Rice, Carol A; Li, Junyu; Witzmann, Frank A

    2003-10-01

    Partially purified transducin was resolved using two-dimensional gel electrophoresis (2-DE). Peptide mass fingerprinting of several different spots believed to correspond to the 37 kDa beta-subunit of transducin (T(beta)) was performed. Spots were excised and proteolyzed using modified trypsin. Matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) was performed on the peptide mixture resulting from each spot. As many as six spots with different pI, ranging from 5.2 to 6.1, were observed when separated using 2-DE. MALDI peptide mass fingerprinting determined with high probability that all of the spots were the same gene product, guanine nucleotide-binding protein G(I)/G(S)/G(T) beta-subunit 1 (GNB1; T(beta1)). This suggested that post-translational modification was responsible for the differences in pI. Phosphorylation experiments showed that at least one T(beta1) spot was phosphorylated in vitro with [gamma-(32)P]ATP by an endogenous kinase. Treatment of T(beta) with alkaline phosphatase caused a large change in the spot pattern of T(beta), suggesting that phosphorylated T(beta) is a substrate for alkaline phosphatase. We conclude that T(beta1) constitutes over 99% of the T(beta) expressed in bovine rod outer segments and displays structural heterogeneity that is due to post-translational modification. We also conclude that some, but not all, of the heterogeneity observed is due to phosphorylation of Tb1. PMID:14595696

  12. Differential regulation of thyrotropin subunit apoprotein and carbohydrate biosynthesis by thyroid hormone

    SciTech Connect

    Taylor, T.; Weintraub, B.D.

    1985-04-01

    The regulation of TSH apoprotein and carbohydrate biosynthesis by thyroid hormone was studied by incubating pituitaries from normal and hypothyroid (3 weeks post-thyroidectomy) rats in medium containing (/sup 14/C)alanine and (/sup 3/H) glucosamine. After 6 h, samples were sequentially treated with anti-TSH beta to precipitate TSH and free TSH beta, anti-LH beta to clear the sample of LH and free LH beta, then anti-LH alpha to precipitate free alpha-subunit. Total proteins were acid precipitated. All precipitates were subjected to electrophoresis on sodium dodecyl sulfate-polyacrylamide gels, which were then sliced and assayed by scintillation spectrometry. In hypothyroid pituitaries plus medium, (/sup 14/C)alanine incorporation in combined and free beta-subunits was 26 times normal and considerably greater than the 3.4-fold increase seen in total protein; combined and free alpha-subunits showed no specific increase in apoprotein synthesis. (/sup 3/H)Glucosamine incorporation in combined alpha- and beta-subunits in hypothyroid samples was 13 and 21 times normal, respectively, and was greater than the 1.9-fold increase in total protein; free alpha-subunit showed no specific increase in carbohydrate synthesis. The glucosamine to alanine ratio, reflecting relative glycosylation of newly synthesized molecules, was increased in hypothyroidism for combined alpha-subunits, but not for combined beta-subunits, free alpha-subunits, or total proteins. In summary, short term hypothyroidism selectively stimulated TSH beta apoprotein synthesis and carbohydrate synthesis of combined alpha- and beta-subunits. Hypothyroidism also increased the relative glycosylation of combined alpha-subunit. Thus, thyroid hormone deficiency appears to alter the rate-limiting step in TSH assembly (i.e. beta-subunit synthesis) as well as the carbohydrate structure of TSH, which may play important roles in its biological function.

  13. Valine 904, tyrosine 898, and cysteine 908 in Na,K-ATPase alpha subunits are important for assembly with beta subunits.

    PubMed

    Wang, S G; Farley, R A

    1998-11-01

    A 26-amino acid sequence in an extracellular loop of the Na,K-ATPase alpha subunit between membrane-spanning segments 7 and 8 has been shown to bind to the beta subunit of Na,K-ATPase and to promote alphabeta assembly (Lemas, M. V., Hamrick, M., Takeyasu, K., and Fambrough, D. M. (1994) J. Biol. Chem. 269, 8255-8259) When this 26-amino acid sequence of the rat Na,K-ATPase alpha3 subunit was replaced by the corresponding sequence of the rat gastric H,K-ATPase alpha subunit, the chimeric alpha subunit assembled preferentially with the rat gastric H,K-ATPase beta subunit (Wang, S.-G., Eakle, K. A., Levenson, R., and Farley, R. A. (1997) Am. J. Physiol. 272, C923-C930). In the present study, these 26 amino acids (Asn886-Ala911) of rat Na,K-ATPase alpha3 were replaced by the corresponding amino acids Asn908-Ala933 of rat distal colon H, K-ATPase. Site-directed mutagenesis of the chimeric alpha subunits and Na,K-ATPase alpha3 showed that Val904, Tyr898, and Cys908 in the Na,K-ATPase alpha3 subunit are key residues in alphabeta subunit interactions. The V904Q mutation in Na,K-ATPase alpha3 reduced the Bmax for ouabain binding and the ATPase activity of alpha3beta1 complexes by approximately 95%, and Y898R reduced the Bmax and ATPase activity by approximately 60%. The complementary mutations Q904V and R898Y increased the amount of ouabain bound by yeast membranes expressing the chimera with the colon H,K-ATPase sequence. The amount of ouabain bound by complexes assembled between Na, K-ATPase alpha3 containing the Y898R,C908G mutations and gastric H, K-ATPase beta was less than 10% of wild type Na,K-ATPase alpha3 expressed with the same beta subunit. The R898Y,G908C mutations in the chimeric alpha subunits also increased ouabain binding. PMID:9792642

  14. Roles of the {beta} subunit hinge domain in ATP synthase F{sub 1} sector: Hydrophobic network formed by introduced {beta}Phe174 inhibits subunit rotation

    SciTech Connect

    Nakanishi-Matsui, Mayumi; Kashiwagi, Sachiko; Kojima, Masaki; Nonaka, Takamasa; Futai, Masamitsu

    2010-04-30

    The ATP synthase {beta} subunit hinge domain ({beta}Phe148 {approx} {beta}Gly186, P-loop/{alpha}-helixB/loop/{beta}-sheet4, Escherichia coli residue numbering) dramatically changes in conformation upon nucleotide binding. We previously reported that F{sub 1} with the {beta}Ser174 to Phe mutation in the domain lowered the {gamma} subunit rotation speed, and thus decreased the ATPase activity [M. Nakanishi-Matsui, S. Kashiwagi, T. Ubukata, A. Iwamoto-Kihara, Y. Wada, M. Futai, Rotational catalysis of Escherichia coli ATP synthase F{sub 1} sector. Stochastic fluctuation and a key domain of the {beta} subunit, J. Biol. Chem. 282 (2007) 20698-20704.]. Homology modeling indicates that the amino acid replacement induces a hydrophobic network, in which the {beta}Met159, {beta}Ile163, and {beta}Ala167 residues of the {beta} subunit are involved together with the mutant {beta}Phe174. The network is expected to stabilize the conformation of {beta}{sub DP} (nucleotide-bound form of the {beta} subunit), resulting in increased activation energy for transition to {beta}{sub E} (empty {beta} subunit). The modeling further predicts that replacement of {beta}Met159 with Ala or Ile weakens the hydrophobic network. As expected, these two mutations experimentally suppressed the ATPase activities as well as subunit rotation of {beta}S174F. Furthermore, the rotation rate decreased with the increase of the strength in the hydrophobic network. These results indicate that the smooth conformational change of the {beta} subunit hinge domain is pertinent for the rotational catalysis.

  15. Copolymer semiconductors comprising thiazolothiazole or benzobisthiazole, or benzobisoxazole electron acceptor subunits, and electron donor subunits, and their uses in transistors and solar cells

    DOEpatents

    Jenekhe, Samson A; Subramaniyan, Selvam; Ahmed, Eilaf; Xin, Hao; Kim, Felix Sunjoo

    2014-10-28

    The inventions disclosed, described, and/or claimed herein relate to copolymers comprising copolymers comprising electron accepting A subunits that comprise thiazolothiazole, benzobisthiazole, or benzobisoxazoles rings, and electron donating subunits that comprise certain heterocyclic groups. The copolymers are useful for manufacturing organic electronic devices, including transistors and solar cells. The invention also relates to certain synthetic precursors of the copolymers. Methods for making the copolymers and the derivative electronic devices are also described.

  16. Amino acid sequence of the alpha subunit and computer modelling of the alpha and beta subunits of echicetin from the venom of Echis carinatus (saw-scaled viper).

    PubMed

    Polgár, J; Magnenat, E M; Peitsch, M C; Wells, T N; Saqi, M S; Clemetson, K J

    1997-04-15

    Echicetin, a heterodimeric protein from the venom of Echis carinatus, binds to platelet glycoprotein Ib (GPIb) and so inhibits platelet aggregation or agglutination induced by various platelet agonists acting via GPIb. The amino acid sequence of the beta subunit of echicetin has been reported and found to belong to the recently identified snake venom subclass of the C-type lectin protein family. Echicetin alpha and beta subunits were purified. N-terminal sequence analysis provided direct evidence that the protein purified was echicetin. The paper presents the complete amino acid sequence of the alpha subunit and computer models of the alpha and beta subunits. The sequence of alpha echicetin is highly similar to the alpha and beta chains of various heterodimeric and homodimeric C-type lectins. Neither of the fully reduced and alkylated alpha or beta subunits of echicetin inhibited the platelet agglutination induced by von Willebrand factor-ristocetin or alpha-thrombin. Earlier reports about the inhibitory activity of reduced and alkylated echicetin beta subunit might have been due to partial reduction of the protein. PMID:9163349

  17. Identification of Four Distinct Subunit Types in the Unique 6×6 Hemocyanin of the Centipede Scutigera coleoptrata

    NASA Astrophysics Data System (ADS)

    Gebauer, W.; Markl, J.

    We isolated 6×6 hemocyanin, dissociated it into subunits, and examined it by electron microscopy. The subunits were separated by native polyacrylamide gel electrophoresis (PAGE), sodium dodecyl sulfate PAGE, and crossed immunoelectrophoresis. Single subunits were isolated by gel cutting from native PAGE and identified as hemocyanin by measuring their ultraviolet spectrum. A total of four distinct hemocyanin subunits were identified, and the subunit pattern of the three electrophoresis systems assigned to each other. The relative proportion of subunits a:b:c:d were 2 : 2 :>: 1 as determined by densitometry. Presumably, c and d act as linkers between hexamers.

  18. Mapping the Future of Map Librarianship.

    ERIC Educational Resources Information Center

    Lang, Laura

    1992-01-01

    Discussion of electronic versions of maps focuses on TIGER files (i.e., electronic maps distributed by the U.S. Bureau of the Census) and their manipulation using geographic information system (GIS) technology. Topics addressed include applications of GIS software, projects to improve access to TIGER files, and the role of GIS in libraries. (MES)

  19. Loss of Complex I activity in the Escherichia coli enzyme results from truncating the C-terminus of subunit K, but not from cross-linking it to subunits N or L.

    PubMed

    Zhu, Shaotong; Canales, Alejandra; Bedair, Mai; Vik, Steven B

    2016-06-01

    Complex I is a multi-subunit enzyme of the respiratory chain with seven core subunits in its membrane arm (A, H, J, K, L, M, and N). In the enzyme from Escherichia coli the C-terminal ten amino acids of subunit K lie along the lateral helix of subunit L, and contribute to a junction of subunits K, L and N on the cytoplasmic surface. Using double cysteine mutagenesis, the cross-linking of subunit K (R99C) to either subunit L (K581C) or subunit N (T292C) was attempted. A partial yield of cross-linked product had no effect on the activity of the enzyme, or on proton translocation, suggesting that the C-terminus of subunit K has no dynamic role in function. To further elucidate the role of subunit K genetic deletions were constructed at the C-terminus. Upon the serial deletion of the last 4 residues of the C-terminus of subunit K, various results were obtained. Deletion of one amino acid had little effect on the activity of Complex I, but deletions of 2 or more amino acids led to total loss of enzyme activity and diminished levels of subunits L, M, and N in preparations of membrane vesicles. Together these results suggest that while the C-terminus of subunit K has no dynamic role in energy transduction by Complex I, it is vital for the correct assembly of the enzyme. PMID:26931547

  20. Intracellular dissociation and reassembly of prolyl 4-hydroxylase:the alpha-subunits associated with the immunoglobulin-heavy-chain binding protein (BiP) allowing reassembly with the beta-subunit.

    PubMed Central

    John, D C; Bulleid, N J

    1996-01-01

    Prolyl 4-hydroxylase (P4-H) consists of two distinct polypeptides; the catalytically more important alpha-subunit and the beta-subunit, which is identical to the multifunctional enzyme protein disulphide isomerase. The enzyme appears to be assembled in vivo into an alpha 2 beta 2 tetramer from newly synthesized alpha-subunits associating with an endogenous pool of beta-subunits. Using a cell-free system, we have shown previously that enzyme assembly is redox-dependent and that assembled alpha-subunits are intramolecularly disulphide-bonded [John and Bulleid (1994) Biochemistry 33, 14018-14025]. Here we have studied this assembly process within intact cells by expressing both subunits in COS-1 cells. Newly synthesized alpha-subunits were shown to assemble with the beta-subunit, to form insoluble aggregates, or to remain soluble but not associate with the beta-subunit. Treatment of cells with dithiothreitol (DTT) led to dissociation of P4-H into subunits and on removal of DTT the enzyme reassembled. This reassembly was ATP-dependent, suggesting an interaction with an ATP-dependent chaperone. This was confirmed when immunoglobulin-heavy-chain binding protein (BiP) and alpha-subunits were co-immunoprecipitated with antibodies against the alpha-subunit and BiP, respectively. These results indicate that unassembled alpha-subunits are maintained in an assembly-competent form by interacting with the molecular chaperone BiP. PMID:8760347

  1. Incorporation of high-molecular-weight glutenin subunits into doughs using 2 gram mixograph and extensigraphs

    Technology Transfer Automated Retrieval System (TEKTRAN)

    To study the contributions of high-molecular-weight glutenin subunits (HMW-GS) to the gluten macropolymer and dough properties, wheat HMW-GS (x- and y-types) are synthesized in a bacterial expression system. These subunits are then purified and used to supplement dough mixing and extensigraph experi...

  2. Visualization of Subunit Interactions and Ternary Complexes of Protein Phosphatase 2A in Mammalian Cells

    PubMed Central

    Mo, Shu-Ting; Chiang, Shang-Ju; Lai, Tai-Yu; Cheng, Yu-Ling; Chung, Cheng-En; Kuo, Spencer C. H.; Reece, Kelie M.; Chen, Yung-Cheng; Chang, Nan-Shan; Wadzinski, Brian E.; Chiang, Chi-Wu

    2014-01-01

    Protein phosphatase 2A (PP2A) is a ubiquitous phospho-serine/threonine phosphatase that controls many diverse cellular functions. The predominant form of PP2A is a heterotrimeric holoenzyme consisting of a scaffolding A subunit, a variable regulatory B subunit, and a catalytic C subunit. The C subunit also associates with other interacting partners, such as α4, to form non-canonical PP2A complexes. We report visualization of PP2A complexes in mammalian cells. Bimolecular fluorescence complementation (BiFC) analysis of PP2A subunit interactions demonstrates that the B subunit plays a key role in directing the subcellular localization of PP2A, and confirms that the A subunit functions as a scaffold in recruiting the B and C subunits to form a heterotrimeric holoenzyme. BiFC analysis also reveals that α4 promotes formation of the AC core dimer. Furthermore, we demonstrate visualization of specific ABC holoenzymes in cells by combining BiFC and fluorescence resonance energy transfer (BiFC-FRET). Our studies not only provide direct imaging data to support previous biochemical observations on PP2A complexes, but also offer a promising approach for studying the spatiotemporal distribution of individual PP2A complexes in cells. PMID:25536081

  3. Similar GABAA receptor subunit composition in somatic and axon initial segment synapses of hippocampal pyramidal cells

    PubMed Central

    Kerti-Szigeti, Katalin; Nusser, Zoltan

    2016-01-01

    Hippocampal pyramidal cells (PCs) express many GABAAR subunit types and receive GABAergic inputs from distinct interneurons. Previous experiments revealed input-specific differences in α1 and α2 subunit densities in perisomatic synapses, suggesting distinct IPSC decay kinetics. However, IPSC decays evoked by axo-axonic, parvalbumin- or cholecystokinin-expressing basket cells were found to be similar. Using replica immunogold labeling, here we show that all CA1 PC somatic and AIS synapses contain the α1, α2, β1, β2, β3 and γ2 subunits. In CA3 PCs, 90% of the perisomatic synapses are immunopositive for the α1 subunit and all synapses are positive for the remaining five subunits. Somatic synapses form unimodal distributions based on their immunoreactivity for these subunits. The α2 subunit densities in somatic synapses facing Cav2.1 (i.e. parvalbumin) or Cav2.2 (cholecystokinin) positive presynaptic active zones are comparable. We conclude that perisomatic synapses made by three distinct interneuron types have similar GABAA receptor subunit content. DOI: http://dx.doi.org/10.7554/eLife.18426.001 PMID:27537197

  4. NR2C and NR2D subunits of NMDA receptors in frog and turtle retina.

    PubMed

    Vitanova, Lily Alexandrova

    2012-12-01

    Glutamate NMDA (N-methyl-D-aspartate) receptors are widely distributed in the central nervous system where they are involved in cognitive processes, motor control and many other functions. They are also well studied in the retina, which may be regarded as a biological model of the nervous system. However, little is known about NR2C and NR2D subunits of NMDA receptors, which have some specific features as compared to other subunits. Consequently the aim of the present study was to investigate their distribution in frog (Rana ridibunda) and turtle (Emys orbicularis) retinas which possess mixed and cone types of retina respectively. The experiments were performed using an indirect immunofluorescence method. Four antibodies directed to NR2C and NR2D subunits of NMDA receptor, as well as three antibodies directed to different splice variants of NR1 subunit, which is known to be obligatory for proper functioning of the receptor, were applied. All antibodies caused well expressed labeling in frog and turtle retinas. The NR2C and NR2D subunits were localized in glial Müller cells, while the NR1 subunit had both neuronal and glial localization. Our results show that glial NMDA receptors differ from neuronal ones in their subunit composition. The functional significance of the NMDA receptors and their NR2C and NR2D subunits, in particular for the neuron-glia interactions, is discussed. PMID:22386206

  5. Profiling the expression pattern of GPI transamidase complex subunits in human cancer.

    PubMed

    Nagpal, Jatin K; Dasgupta, Santanu; Jadallah, Sana; Chae, Young K; Ratovitski, Edward A; Toubaji, Antoun; Netto, George J; Eagle, Toby; Nissan, Aviram; Sidransky, David; Trink, Barry

    2008-08-01

    The glycosylphosphatidylinositol transamidase complex (GPIT) consists of five subunits: PIG-U, PIG-T, GPAA1, PIG-S and GPI8, and is important in attaching GPI anchors to target proteins. On the basis of our previous reports incriminating PIG-U as an oncogene in bladder cancer and PIG-T and GPAA1 as oncogenes in breast cancer, we evaluated the expression pattern of the GPIT subunits in 19 different human cancers at both mRNA and protein levels. In general, our results demonstrate a more frequent expression of GPIT subunits in cancers than in normal. Among the 19 anatomic sites compared; breast, ovary and uterus showed consistent evidence of overexpression of specific GPIT subunits. There was also overexpression of PIG-U and GPI8 in lymphoma. In addition, non-small cell lung carcinoma showed significant overexpression of the GPIT subunits as compared to small cell lung carcinoma and normal lung tissue. Also, deregulation of specific GPIT subunits was seen in various other cancers. Forced overexpression of two GPIT subunits; PIG-S and GPI8 alone or in combination induced increased proliferation and invasion of breast cancer cells. Collectively, our study defines a trend involving the deregulated expression and the functional contribution of the GPIT subunits in various cancers with potential implications in diagnosis, prognosis and therapeutic intervention. PMID:18487995

  6. Incorporation of high-molecular-weight glutenin subunits into doughs using 2 gram mixograph and extensigraphs

    Technology Transfer Automated Retrieval System (TEKTRAN)

    To study the contributions of high-molecular-weight glutenin subunits (HMW-GS) to the gluten macropolymer and dough properties, wheat HMW-GS (x- and y-types) are synthesized in a bacterial expression system. These subunits are then purified and used to supplement dough mixing and extensigraph exper...

  7. Similar GABAA receptor subunit composition in somatic and axon initial segment synapses of hippocampal pyramidal cells.

    PubMed

    Kerti-Szigeti, Katalin; Nusser, Zoltan

    2016-01-01

    Hippocampal pyramidal cells (PCs) express many GABAAR subunit types and receive GABAergic inputs from distinct interneurons. Previous experiments revealed input-specific differences in α1 and α2 subunit densities in perisomatic synapses, suggesting distinct IPSC decay kinetics. However, IPSC decays evoked by axo-axonic, parvalbumin- or cholecystokinin-expressing basket cells were found to be similar. Using replica immunogold labeling, here we show that all CA1 PC somatic and AIS synapses contain the α1, α2, β1, β2, β3 and γ2 subunits. In CA3 PCs, 90% of the perisomatic synapses are immunopositive for the α1 subunit and all synapses are positive for the remaining five subunits. Somatic synapses form unimodal distributions based on their immunoreactivity for these subunits. The α2 subunit densities in somatic synapses facing Cav2.1 (i.e. parvalbumin) or Cav2.2 (cholecystokinin) positive presynaptic active zones are comparable. We conclude that perisomatic synapses made by three distinct interneuron types have similar GABAA receptor subunit content. PMID:27537197

  8. Specific Inhibition of Herpes Simplex Virus DNA Polymerase by Helical Peptides Corresponding to the Subunit Interface

    NASA Astrophysics Data System (ADS)

    Digard, Paul; Williams, Kevin P.; Hensley, Preston; Brooks, Ian S.; Dahl, Charles E.; Coen, Donald M.

    1995-02-01

    The herpes simplex virus DNA polymerase consists of two subunits-a catalytic subunit and an accessory subunit, UL42, that increases processivity. Mutations affecting the extreme C terminus of the catalytic subunit specifically disrupt subunit interactions and ablate virus replication, suggesting that new antiviral drugs could be rationally designed to interfere with polymerase heterodimerization. To aid design, we performed circular dichroism (CD) spectroscopy and analytical ultracentrifugation studies, which revealed that a 36-residue peptide corresponding to the C terminus of the catalytic subunit folds into a monomeric structure with partial α-helical character. CD studies of shorter peptides were consistent with a model where two separate regions of α-helix interact to form a hairpin-like structure. The 36-residue peptide and a shorter peptide corresponding to the C-terminal 18 residues blocked UL42-dependent long-chain DNA synthesis at concentrations that had no effect on synthesis by the catalytic subunit alone or by calf thymus DNA polymerase δ and its processivity factor. These peptides, therefore, represent a class of specific inhibitors of herpes simplex virus DNA polymerase that act by blocking accessory-subunit-dependent synthesis. These peptides or their structures may form the basis for the synthesis of clinically effective drugs.

  9. All three subunits of soybean beta-conglycinin are potential food allergens.

    PubMed

    Krishnan, Hari B; Kim, Won-Seok; Jang, Sungchan; Kerley, Monty S

    2009-02-11

    Soybeans are recognized as one of the "big 8" food allergens. IgE antibodies from soybean-sensitive patients recognize more than 15 soybean proteins. Among these proteins only the alpha-subunit of beta-conglycinin, but not the highly homologous alpha'- and beta-subunits, has been shown to be a major allergenic protein. The objective of this study was to examine if the alpha'- and beta-subunits of beta-conglycinin can also serve as potential allergens. Immunoblot analysis using sera collected from soybean-allergic patients revealed the presence of IgE antibodies that recognized several soy proteins including 72, 70, 52, 34, and 21 kDa proteins. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF) analysis of trypsin-digested 72, 70, and 52 kDa proteins indicated that these proteins were the alpha'-, alpha-, and beta-subunits of beta-conglycinin, respectively. Additionally, purified alpha'-, alpha-, and beta-subunits of beta-conglycinin were recognized by IgE antibodies present in the soybean-allergic patients. The IgE reactivity to the beta-subunit of beta-conglycinin was not abolished when this glycoprotein was either deglycosylated using glycosidases or expressed as a recombinant protein in Escherichia coli . The results suggest that in addition to the previously recognized alpha-subunit of beta-conglycinin, the alpha'- and beta-subunits of beta-conglycinin also are potential food allergens. PMID:19138084

  10. Hybrid rotors in F1F(o) ATP synthases: subunit composition, distribution, and physiological significance.

    PubMed

    Brandt, Karsten; Müller, Volker

    2015-09-01

    The c ring of the Na+ F1F(o) ATP synthase from the anaerobic acetogenic bacterium Acetobacterium woodii is encoded by three different genes: atpE1, atpE2 and atpE3. Subunit c1 is similar to typical V-type c subunits and has four transmembrane helices with one ion binding site. Subunit c2 and c3 are identical at the amino acid level and are typical F-type c subunits with one ion binding site in two transmembrane helices. All three constitute a hybrid F(o)V(o) c ring, the first found in nature. To analyze whether other species may have similar hybrid rotors, we searched every genome sequence publicly available as of 23 February 2015 for F1F(o) ATPase operons that have more than one gene encoding the c subunit. This revealed no other species that has three different c subunit encoding genes but twelve species that encode one F(o)- and one V(o)-type c subunit in one operon. Their c subunits have the conserved binding motif for Na+. The organisms are all anaerobic. The advantage of hybrid c rings for the organisms in their environments is discussed. PMID:25838297

  11. Characterization of the Last Subunit of the Arabidopsis COP9 Signalosome

    PubMed Central

    Serino, Giovanna; Su, Hongwen; Peng, Zhaohua; Tsuge, Tomohiko; Wei, Ning; Gu, Hongya; Deng, Xing Wang

    2003-01-01

    The COP9 signalosome (CSN) is an evolutionarily conserved protein complex that resembles the lid subcomplex of proteasomes. Through its ability to regulate specific proteasome-mediated protein degradation events, CSN controls multiple aspects of development. Here, we report the cloning and characterization of AtCSN2, the last uncharacterized CSN subunit from Arabidopsis. We show that the AtCSN2 gene corresponds to the previously identified FUS12 locus and that AtCSN2 copurifies with CSN, confirming that AtCSN2 is an integral component of CSN. AtCSN2 is not only able to interact with the SCFTIR1 subunit AtCUL1, which is partially responsible for the regulatory interaction between CSN and SCFTIR1, but also interacts with AtCUL3, suggesting that CSN is able to regulate the activity of other cullin-based E3 ligases through conserved interactions. Phylogenetic analysis indicated that the duplication and subsequent divergence events that led to the genes that encode CSN and lid subunits occurred before the divergence of unicellular and multicellular eukaryotic organisms and that the CSN subunits were more conserved than the lid subunits during evolution. Comparative analyses of the subunit interaction of CSN revealed a set of conserved subunit contacts and resulted in a model of CSN subunit topology, some aspects of which were substantiated by in vivo cross-link tests. PMID:12615944

  12. Density Equalizing Map Projections

    SciTech Connect

    Close, E. R.; Merrill, D. W.; Holmes, H. H.

    1995-07-01

    A geographic map is mathematically transformed so that the subareas of the map are proportional to a given quantity such as population. In other words, population density is equalized over the entire map. The transformed map can be used as a display tool, or it can be statistically analyzed. For example, cases of disease plotted on the transformed map should be uniformly distributed at random, if disease rates are everywhere equal. Geographic clusters of disease can be readily identified, and their statistical significance determined, on a density equalized map.

  13. Density Equalizing Map Projections

    Energy Science and Technology Software Center (ESTSC)

    1995-07-01

    A geographic map is mathematically transformed so that the subareas of the map are proportional to a given quantity such as population. In other words, population density is equalized over the entire map. The transformed map can be used as a display tool, or it can be statistically analyzed. For example, cases of disease plotted on the transformed map should be uniformly distributed at random, if disease rates are everywhere equal. Geographic clusters of diseasemore » can be readily identified, and their statistical significance determined, on a density equalized map.« less

  14. Mapping of Daedalia Planum Lava Field

    NASA Astrophysics Data System (ADS)

    Giacomini, Lorenza; Carli, Cristian; Massironi, Matteo; Pasquarè, Giorgio; Sgavetti, Maria

    2010-05-01

    Daedalia Planum is one of the Tharsis volcanic plains and is located southwest of the Arsia Mons. MOLA, THEMIS, MOC and OMEGA data have been analysed, providing a multi-scale characterisation of this Martian lava field. According to Mars Global Surveyor's MOLA data, the flanks of Arsia have an average slope <5°, while the surrounding regions, including Daedalia Planum, have slopes <0,5° and commonly <0,1°. Mars Odyssey/THEMIS VIS and IR images show a plain covered by a huge number of lava flows. Older and larger lava flows on the field have a length greater than ~1500 km. Moreover most of the Daedalia flows are associated to wrinkly and ropy surfaces, typical of pahoehoe lavas. On the base of the morphology differences among the flows and through stratigraphic relationships we performed a geological map of the area. MEX/OMEGA spectra were collected in different areas of the lava field. Besides the similar absorption bands OMEGA spectra showed also some differences in reflectance and spectral slope. The spectral map created using the SAM classification reveals that these spectral variations are generally in agreement with the lava flows mapped previously on the base of the flows morphology and stratigraphy. This suggested that such variability is related with different surface textures of the lava flow. Moreover in some cases spectral map highlighted the presence of spectral subunits inside the same stratigraphic unit, due likely to a different mineralogy or rock textures. Therefore spectral analysis revealed useful to improve the geological mapping of the Daedalia Planum region.

  15. Identification of an intra-molecular disulfide bond in the sodium channel β1-subunit.

    PubMed

    Barbieri, Raffaella; Baroni, Debora; Moran, Oscar

    2012-04-01

    The sodium channel β1 subunit is non-covalently associated with the pore-forming α-subunits, and has been proposed to act as a modulator of channel activity, regulator of channel cell surface expression and cell adhesion molecule. Its importance is evident since mutations of the β1 subunit cause neurologic and cardiovascular disorders. The first described β1 subunit mutation is the C121W, that is related to generalized epilepsy with febrile seizures plus (GEFS+), a childhood genetic epilepsy syndrome. This mutation changed a conserved cysteine residue in position 121 into a tryptophan, putatively disrupting a disulfide bridge that should normally maintain the β1 extracellular immunoglobulin-like fold. Using the 2-D-diagonal-SDS-PAGE technique, we demonstrated the existence of this putative disulfide bridge in the Ig-like extracellular domain of the β1 subunit and its disruption in the epileptogenic C121W mutant. PMID:22425777

  16. Protein kinase A catalytic subunit isoform PRKACA; History, function and physiology.

    PubMed

    Turnham, Rigney E; Scott, John D

    2016-02-15

    Our appreciation of the scope and influence of second messenger signaling has its origins in pioneering work on the cAMP-dependent protein kinase. Also called protein kinase A (PKA), this holoenzyme exists as a tetramer comprised of a regulatory (R) subunit dimer and two catalytic (C) subunits. Upon binding of two molecules of the second messenger cAMP to each R subunit, a conformational change in the PKA holoenzyme occurs to release the C subunits. These active kinases phosphorylate downstream targets to propagate cAMP responsive cell signaling events. This article focuses on the discovery, structure, cellular location and physiological effects of the catalytic subunit alpha of protein kinase A (encoded by the gene PRKACA). We also explore the potential role of this essential gene as a molecular mediator of certain disease states. PMID:26687711

  17. A protein kinase from wheat germ that phosphorylates the largest subunit of RNA polymerase II.

    PubMed Central

    Guilfoyle, T J

    1989-01-01

    A protein kinase from wheat germ that phosphorylates the largest subunit of RNA polymerase IIA has been partially purified and characterized. The kinase has a native molecular weight of about 200 kilodaltons. This kinase utilizes Mg2+ and ATP and transfers about 20 phosphates to the heptapeptide repeats Pro-Thr-Ser-Pro-Ser-Tyr-Ser in the carboxyl-terminal domain of the 220-kilodalton subunit of soybean RNA polymerase II. This phosphorylation results in a mobility shift of the 220-kilodalton subunits of a variety of eukaryotic RNA polymerases to polypeptides ranging in size from greater than 220 kilodaltons to 240 kilodaltons on sodium dodecyl sulfate-polyacrylamide gels. The phosphorylation is highly specific to the heptapeptide repeats since a degraded subunit polypeptide of 180 kilodaltons that lacks the heptapeptide repeats is poorly phosphorylated. Synthetic heptapeptide repeat multimers inhibit the phosphorylation of the 220-kilodalton subunit. PMID:2535525

  18. Transcription factor assembly on the nicotinic receptor beta4 subunit gene promoter.

    PubMed

    Scofield, Michael D; Brüschweiler-Li, Lei; Mou, Zhongming; Gardner, Paul D

    2008-04-16

    Nicotinic acetylcholine receptors are involved in a plethora of fundamental biological processes ranging from muscle contraction to formation of memories. The receptors are pentameric proteins whose subunits are encoded by distinct genes. Subunit composition of a mature nicotinic receptor is governed in part by the transcriptional regulation of each subunit gene. Here, using chromatin immunoprecipitation assays, we report the interaction of the transcription factors Sp1, Sp3, c-Jun and Sox10 with the beta4 subunit gene promoter in neuronal-like cell lines and rodent brain tissue. Our results corroborate previous in-vitro data demonstrating that these transcription factors interact with the beta4 promoter. Taken together, these data suggest that Sp1, Sp3, c-Jun and Sox10 regulate expression of the beta4 subunit gene in the mammalian brain. PMID:18382288

  19. Subunit compositions of Arabidopsis RNA polymerases I and III reveal Pol I- and Pol III-specific forms of the AC40 subunit and alternative forms of the C53 subunit

    SciTech Connect

    Ream, Thomas S.; Haag, Jeremy R.; Pontvianne, Frederic; Nicora, Carrie D.; Norbeck, Angela D.; Pasa-Tolic, Ljiljana; Pikaard, Craig S.

    2015-05-02

    Using affinity purification and mass spectrometry, we identified the subunits of Arabidopsis thaliana multisubunit RNA Polymerases I and III (abbreviated as Pol I and Pol III), providing the first description of their physical compositions in plants. AC40 and AC19 subunits are typically common to Pol I (a.k.a. Pol A) and Pol III (a.k.a. Pol C) and are encoded by single genes whose mutation, in humans, is a cause of the craniofacial disorder, Treacher-Collins Syndrome. Surprisingly, A. thaliana, and related species, express two distinct AC40 paralogs, one of which assembles into Pol I and the other of which assembles into Pol III. Changes at eight amino acid positions correlate with this functional divergence of Pol I and Pol III-specific AC40 paralogs. Two genes encode homologs of the yeast C53 subunit, and either variant can assemble into Pol III. By contrast, only one of two potential C17 variants, and one of two potential C31 variants were detected in Pol III. We introduce a new nomenclature system for plant Pol I and Pol III subunits in which the twelve subunits that are structurally and functionally homologous among Pols I through V are assigned equivalent numbers.

  20. Subunit compositions of Arabidopsis RNA polymerases I and III reveal Pol I- and Pol III-specific forms of the AC40 subunit and alternative forms of the C53 subunit

    DOE PAGESBeta

    Ream, Thomas S.; Haag, Jeremy R.; Pontvianne, Frederic; Nicora, Carrie D.; Norbeck, Angela D.; Pasa-Tolic, Ljiljana; Pikaard, Craig S.

    2015-05-02

    Using affinity purification and mass spectrometry, we identified the subunits of Arabidopsis thaliana multisubunit RNA Polymerases I and III (abbreviated as Pol I and Pol III), providing the first description of their physical compositions in plants. AC40 and AC19 subunits are typically common to Pol I (a.k.a. Pol A) and Pol III (a.k.a. Pol C) and are encoded by single genes whose mutation, in humans, is a cause of the craniofacial disorder, Treacher-Collins Syndrome. Surprisingly, A. thaliana, and related species, express two distinct AC40 paralogs, one of which assembles into Pol I and the other of which assembles into Polmore » III. Changes at eight amino acid positions correlate with this functional divergence of Pol I and Pol III-specific AC40 paralogs. Two genes encode homologs of the yeast C53 subunit, and either variant can assemble into Pol III. By contrast, only one of two potential C17 variants, and one of two potential C31 variants were detected in Pol III. We introduce a new nomenclature system for plant Pol I and Pol III subunits in which the twelve subunits that are structurally and functionally homologous among Pols I through V are assigned equivalent numbers.« less

  1. Subunit compositions of Arabidopsis RNA polymerases I and III reveal Pol I- and Pol III-specific forms of the AC40 subunit and alternative forms of the C53 subunit

    PubMed Central

    Ream, Thomas S.; Haag, Jeremy R.; Pontvianne, Frederic; Nicora, Carrie D.; Norbeck, Angela D.; Paša-Tolić, Ljiljana; Pikaard, Craig S.

    2015-01-01

    Using affinity purification and mass spectrometry, we identified the subunits of Arabidopsis thaliana multisubunit RNA polymerases I and III (abbreviated as Pol I and Pol III), the first analysis of their physical compositions in plants. In all eukaryotes examined to date, AC40 and AC19 subunits are common to Pol I (a.k.a. Pol A) and Pol III (a.k.a. Pol C) and are encoded by single genes. Surprisingly, A. thaliana and related species express two distinct AC40 paralogs, one of which assembles into Pol I and the other of which assembles into Pol III. Changes at eight amino acid positions correlate with the functional divergence of Pol I- and Pol III-specific AC40 paralogs. Two genes encode homologs of the yeast C53 subunit and either protein can assemble into Pol III. By contrast, only one of two potential C17 variants, and one of two potential C31 variants were detected in Pol III. We introduce a new nomenclature system for plant Pol I and Pol III subunits in which the 12 subunits that are structurally and functionally homologous among Pols I through V are assigned equivalent numbers. PMID:25813043

  2. 5-HT3 Receptor Brain-Type B-Subunits are Differentially Expressed in Heterologous Systems

    PubMed Central

    2015-01-01

    Genes for five different 5-HT3 receptor subunits have been identified. Most of the subunits have multiple isoforms, but two isoforms of the B subunits, brain-type 1 (Br1) and brain-type 2 (Br2) are of particular interest as they appear to be abundantly expressed in human brain, where 5-HT3B subunit RNA consists of approximately 75% 5-HT3Br2, 24% 5-HT3Br1, and <1% 5-HT3B. Here we use two-electrode voltage-clamp, radioligand binding, fluorescence, whole cell, and single channel patch-clamp studies to characterize the roles of 5-HT3Br1 and 5-HT3Br2 subunits on function and pharmacology in heterologously expressed 5-HT3 receptors. The data show that the 5-HT3Br1 transcriptional variant, when coexpressed with 5-HT3A subunits, alters the EC50, nH, and single channel conductance of the 5-HT3 receptor, but has no effect on the potency of competitive antagonists; thus, 5-HT3ABr1 receptors have the same characteristics as 5-HT3AB receptors. There were some differences in the shapes of 5-HT3AB and 5-HT3ABr1 receptor responses, which were likely due to a greater proportion of homomeric 5-HT3A versus heteromeric 5-HT3ABr1 receptors in the latter, as expression of the 5-HT3Br1 compared to the 5-HT3B subunit is less efficient. Conversely, the 5-HT3Br2 subunit does not appear to form functional channels with the 5-HT3A subunit in either oocytes or HEK293 cells, and the role of this subunit is yet to be determined. PMID:25951416

  3. Topographic antigenic determinants recognized by monoclonal antibodies on human choriogonadotropin beta-subunit

    SciTech Connect

    Bidart, J.M.; Troalen, F.; Salesse, R.; Bousfield, G.R.; Bohuon, C.J.; Bellet, D.H.

    1987-06-25

    We describe a first attempt to study the antibody-combining sites recognized by monoclonal antibodies raised against the beta-subunit of human choriogonadotropin (hCG). Two groups of antibodies were first defined by their ability to recognize only the free beta-subunit or the free and combined subunit. Antibodies FBT-11 and FBT-11-L bind only to hCG beta-subunit but not to hCG, whereas antibodies FBT-10 and D1E8 bind to both the beta-subunit and the hormone. In both cases, the antigenic determinants were localized to the core of the protein (residues 1-112), indicating the weak immunogenicity of the specific carboxyl-terminal extension of hCG-beta. Nine synthetic peptides spanning different regions of hCG-beta and lutropin-beta were assessed for their capacity to inhibit antibody binding. A synthetic peptide inclusive of the NH2-terminal region (residues 1-7) of the hCG beta-subunit was found to inhibit binding to the radiolabeled subunit of a monoclonal antibody specific for free hCG-beta (FBT-11). Further delineation of the antigenic site recognized by this antibody provided evidence for the involvement of fragment 82-92. Moreover, monoclonal antibody FBT-11 inhibited the recombination of hCG-beta to hCG-alpha, indicating that its antigenic determinant might be located nearby or in the hCG-beta portion interacting with the alpha-subunit. Binding of monoclonal antibody FBT-10, corresponding to the second antigenic determinant, was weakly inhibited by fragment 82-105 and did not impair the recombination of the hCG beta-subunit to the hCG alpha-subunit. Its combining site appeared to be located in a region of the intact native choriogonadotropin present at the surface of the hormone-receptor complex.

  4. The action of calcium channel blockers on recombinant L-type calcium channel α1-subunits

    PubMed Central

    Morel, Nicole; Buryi, Vitali; Feron, Olivier; Gomez, Jean-Pierre; Christen, Marie-Odile; Godfraind, Théophile

    1998-01-01

    CHO cells expressing the α1C-a subunit (cardiac isoform) and the α1C-b subunit (vascular isoform) of the voltage-dependent L-type Ca2+ channel were used to investigate whether tissue selectivity of Ca2+ channel blockers could be related to different affinities for α1C isoforms.Inward current evoked by the transfected α1 subunit was recorded by the patch-clamp technique in the whole-cell configuration.Neutral dihydropyridines (nifedipine, nisoldipine, (+)-PN200-110) were more potent inhibitors of α1C-b-subunit than of α1C-a-subunit. This difference was more marked at a holding potential of −100 mV than at −50 mV. SDZ 207-180 (an ionized dihydropyridine) exhibited the same potency on the two isoforms.Pinaverium (ionized non-dihydropyridine derivative) was 2 and 4 fold more potent on α1C-a than on α1C-b subunit at Vh of −100 mV and −50 mV, respectively. Effects of verapamil were identical on the two isoforms at both voltages.[3H]-(+)-PN 200-110 binding experiments showed that neutral dihydropyridines had a higher affinity for the α1C-b than for the α1C-a subunit. SDZ 207-180 had the same affinity for the two isoforms and pinaverium had a higher affinity for the α1C-a subunit than for the α1C-b subunit.These results indicate marked differences among Ca2+ channel blockers in their selectivity for the α1C-a and α1C-b subunits of the Ca2+ channel. PMID:9846638

  5. The action of calcium channel blockers on recombinant L-type calcium channel alpha1-subunits.

    PubMed

    Morel, N; Buryi, V; Feron, O; Gomez, J P; Christen, M O; Godfraind, T

    1998-11-01

    1. CHO cells expressing the alpha(1C-a) subunit (cardiac isoform) and the alpha(1C-b) subunit (vascular isoform) of the voltage-dependent L-type Ca2+ channel were used to investigate whether tissue selectivity of Ca2+ channel blockers could be related to different affinities for alpha1C isoforms. 2. Inward current evoked by the transfected alpha1 subunit was recorded by the patch-clamp technique in the whole-cell configuration. 3. Neutral dihydropyridines (nifedipine, nisoldipine, (+)-PN200-110) were more potent inhibitors of alpha(1C-)b-subunit than of alpha(1C-a)-subunit. This difference was more marked at a holding potential of -100 mV than at -50 mV. SDZ 207-180 (an ionized dihydropyridine) exhibited the same potency on the two isoforms. 4. Pinaverium (ionized non-dihydropyridine derivative) was 2 and 4 fold more potent on alpha(1C-a) than on alpha(1C-b) subunit at Vh of -100 mV and -50 mV, respectively. Effects of verapamil were identical on the two isoforms at both voltages. 5. [3H]-(+)-PN 200-110 binding experiments showed that neutral dihydropyridines had a higher affinity for the alpha(1C-b) than for the alpha(1C-a) subunit. SDZ 207-180 had the same affinity for the two isoforms and pinaverium had a higher affinity for the alpha(1C-a) subunit than for the alpha(1C-b) subunit. 6. These results indicate marked differences among Ca2+ channel blockers in their selectivity for the alpha(1C-a) and alpha(1C-b) subunits of the Ca2+ channel. PMID:9846638

  6. Functional consequences of Kir2.1/Kir2.2 subunit heteromerization.

    PubMed

    Panama, Brian K; McLerie, Meredith; Lopatin, Anatoli N

    2010-10-01

    Kir2 subunits form channels that underlie classical strongly inwardly rectifying potassium currents. While homomeric Kir2 channels display a number of distinct and physiologically important properties, the functional properties of heteromeric Kir2 assemblies, as well as the stoichiometries and the arrangements of Kir2 subunits in native channels, remain largely unknown. Therefore, we have implemented a concatemeric approach, whereby all four cloned Kir2 subunits were linked in tandem, in order to study the effects of Kir2.1 and Kir2.2 heteromerization on properties of the resulting channels. Kir2.2 subunits contributed stronger to single-channel conductance than Kir2.1 subunits, and channels containing two or more Kir2.2 subunits displayed conductances indistinguishable from that of a Kir2.2 homomeric channel. In contrast, single-channel kinetics was a more discriminating property. The open times were significantly shorter in Kir2.2 channels compared with Kir2.1 channels and decreased nearly proportionally to the number of Kir2.2 subunits in the heteromeric channel. Similarly, the sensitivity to block by barium also depended on the proportions of Kir2.1 to Kir2.2 subunits. Overall, the results showed that Kir2.1 and Kir2.2 subunits exert neither a dominant nor an anomalous effect on any of the properties of heteromeric channels. The data highlight opportunities and challenges of using differential properties of Kir2 channels in deciphering the subunit composition of native inwardly rectifying potassium currents. PMID:20676672

  7. Ostreococcus tauri ADP-glucose Pyrophosphorylase Reveals Alternative Paths for the Evolution of Subunit Roles*

    PubMed Central

    Kuhn, Misty L.; Falaschetti, Christine A.; Ballicora, Miguel A.

    2009-01-01

    ADP-glucose pyrophosphorylase controls starch synthesis in plants and is an interesting case to study the evolution and differentiation of roles in heteromeric enzymes. It includes two homologous subunits, small (S) and large (L), that originated from a common photosynthetic eukaryotic ancestor. In present day organisms, these subunits became complementary after loss of certain roles in a process described as subfunctionalization. For instance, the potato tuber enzyme has a noncatalytic L subunit that complements an S subunit with suboptimal allosteric properties. To understand the evolution of catalysis and regulation in this family, we artificially synthesized both subunit genes from the unicellular alga Ostreococcus tauri. This is among the most ancient species in the green lineage that diverged from the ancestor of all green plants and algae. After heterologous gene expression, we purified and characterized the proteins. The O. tauri enzyme was not redox-regulated, suggesting that redox regulation of ADP-glucose pyrophosphorylases appeared later in evolution. The S subunit had a typical low apparent affinity for the activator 3-phosphoglycerate, but it was atypically defective in the catalytic efficiency (Vmax/Km) for the substrate Glc-1-P. The L subunit needed the S subunit for soluble expression. In the presence of a mutated S subunit (to avoid interference), the L subunit had a high apparent affinity for 3-phosphoglycerate and substrates suggesting a leading role in catalysis. Therefore, the subfunctionalization of the O. tauri enzyme was different from previously described cases. To the best of our knowledge, this is the first biochemical description of a system with alternative subfunctionalization paths. PMID:19737928

  8. A neurosteroid potentiation site can be moved among GABAA receptor subunits.

    PubMed

    Bracamontes, John R; Li, Ping; Akk, Gustav; Steinbach, Joe Henry

    2012-11-15

    Endogenous neurosteroids are among the most potent and efficacious potentiators of activation of GABA(A) receptors. It has been proposed that a conserved glutamine residue in the first membrane-spanning region (TM1 region) of the α subunits is required for binding of potentiating neurosteroids. Mutations of this residue can reduce or remove the ability of steroids to potentiate function. However, it is not known whether potentiation requires that a steroid interact with the α subunit, or not. To examine this question we mutated the homologous residue in the β2 and γ2L subunits to glutamine, and found that these mutations could not confer potentiation by allopregnanolone (3α5αP) when expressed in receptors containing ineffective α1 subunits. However, potentiation is restored when the entire TM1 region from the α1 subunit is transferred to the β2 or γ2L subunit. Mutations in the TM1 region that affect potentiation when made in the α1 subunit have similar effects when made in transferred TM1 region. Further, the effects of 3α5αP on single-channel kinetics are similar for wild-type receptors and receptors with moved TM1 regions. These results support the idea that steroids bind in the transmembrane regions of the receptor. The observations are consistent with previous work indicating that neurosteroid potentiation is mediated by an action that affects the receptor as a whole, rather than an individual subunit or pair of subunits, and in addition demonstrate that the mechanism is independent of the nature of the subunit that interacts with steroid. PMID:22988137

  9. Riparian Wetlands: Mapping

    EPA Science Inventory

    Riparian wetlands are critical systems that perform functions and provide services disproportionate to their extent in the landscape. Mapping wetlands allows for better planning, management, and modeling, but riparian wetlands present several challenges to effective mapping due t...

  10. Active Fire Mapping Program

    MedlinePlus

    ... Incidents (Home) New Large Incidents Fire Detection Maps MODIS Satellite Imagery VIIRS Satellite Imagery Fire Detection GIS ... Data Web Services Latest Detected Fire Activity Other MODIS Products Frequently Asked Questions About Active Fire Maps ...

  11. Information-Mapped Chemistry.

    ERIC Educational Resources Information Center

    Olympia, P. L., Jr.

    1979-01-01

    This paper describes the use of information mapping in chemistry and in other related sciences. Information mapping is a way of presenting information without paragraphs and unnecessary transitional phrases. (BB)

  12. Linkage map integration

    SciTech Connect

    Collins, A.; Teague, J.; Morton, N.E.; Keats, B.J.

    1996-08-15

    The algorithms that drive the map+ program for locus-oriented linkage mapping are presented. They depend on the enhanced location database program ldb+ to specify an initial comprehensive map that includes all loci in the summary lod file. Subsequently the map may be edited or order constrained and is automatically improved by estimating the location of each locus conditional on the remainder, beginning with the most discrepant loci. Operating characteristics permit rapid and accurate construction of linkage maps with several hundred loci. The map+ program also performs nondisjunction mapping with tests of nonstandard recombination. We have released map+ on Internet as a source program in the C language together with the location database that now includes the LODSOURCE database. 28 refs., 5 tabs.

  13. Creative Concept Mapping.

    ERIC Educational Resources Information Center

    Brown, David S.

    2002-01-01

    Recommends the use of concept mapping in science teaching and proposes that it be presented as a creative activity. Includes a sample lesson plan of a potato stamp concept mapping activity for astronomy. (DDR)

  14. Using maps in genealogy

    USGS Publications Warehouse

    U.S. Geological Survey

    1994-01-01

    In genealogy, maps are most often used as clues to where public or other records about an ancestor are likely to be found. Searching for maps seldom begins until a newcomer to genealogy has mastered basic genealogical routines

  15. Turning off of GluN2B subunits and turning on of CICR in hippocampal LTD induction after developmental GluN2 subunit switch.

    PubMed

    Yasuda, Hiroki; Mukai, Hideyuki

    2015-11-01

    NMDA receptors (NMDARs) are essential for the induction of synaptic plasticity that mediates activity-dependent refinement of neural circuits during development. GluN2B subunits of NMDARs are abundant at synapses in the immature hippocampus and begin to be replaced by GluN2A subunits with the help of casein kinase 2 activity in the second postnatal week, the critical period for the GluN2 subunit switch (Sanz-Clemente et al. (2000) Neuron 67:984-996). However, the physiological role of GluN2B subunits in the hippocampus during this critical period has not been elucidated. Here, we report that GluN2B subunits mediate the induction of long-term depression (LTD) in the CA1 region of the hippocampus only until this period. Ifenprodil and Ro25-6981, selective inhibitors of NMDARs containing GluN2B subunits, blocked LTD in postnatal Day 11-14 (P11-14) rat hippocampal slices but not in P18-22 hippocampus. Just a few days after P14, synaptic NMDAR currents became narrower than those at P11-14, and calcium influx through NMDARs must be reduced. We found that calcium-induced calcium release (CICR) through ryanodine receptors starts to support the induction of NMDAR-dependent LTD at P18-22. Intracellular application of thapsigargin and ryanodine, inhibitors of Ca2+ -ATP pumps on internal stores and ryanodine receptors, respectively, did not at all affect LTD in the hippocampus at P11-14 but completely blocked LTD in the P18-22 hippocampus. Therefore, calcium influx through NMDAR with GluN2B subunits is sufficient to induce LTD at P11-14, after which CICR compensates for the decrease in calcium influx during LTD induction. PMID:25727316

  16. Cell-free synthesis and assembly of prolyl 4-hydroxylase: the role of the beta-subunit (PDI) in preventing misfolding and aggregation of the alpha-subunit.

    PubMed Central

    John, D C; Grant, M E; Bulleid, N J

    1993-01-01

    Prolyl 4-hydroxylase (P4-H) catalyses a vital post-translational modification in the biosynthesis of collagen. The enzyme consists of two distinct polypeptides forming an alpha 2 beta 2 tetramer (alpha = 64 kDa, beta = 60 kDa), the beta-subunit being identical to the multifunctional enzyme protein disulfide isomerase (PDI). By studying the cell-free synthesis of the rat alpha-subunit of P4-H we have shown that the alpha-subunit can be translocated, glycosylated and the signal peptide cleaved by dog pancreatic microsomal membranes to yield both singly and doubly glycosylated forms. When translations were carried out under conditions which prevent disulfide bond formation, the product synthesized formed aggregates which were associated with the immunoglobulin heavy chain binding protein (BiP). Translations carried out under conditions that promote disulfide bond formation yielded a product that was not associated with BiP but formed a complex with the endogenous beta-subunit (PDI). Complex formation was detected by co-precipitation of the newly synthesized alpha-subunit with antibodies raised against PDI, by sucrose gradient centrifugation and by chemical cross-linking. When microsomal vesicles were depleted of PDI, BiP and other soluble endoplasmic reticulum proteins, no complex formation was observed and the alpha-subunit aggregated even under conditions that promote disulfide bond formation. We have therefore demonstrated that the enzyme P4-H can be assembled at synthesis in a cell-free system and that the solubility of the alpha-subunit is dependent upon its association with PDI. Images PMID:8385607

  17. The hybrid four-CBS-domain KINβγ subunit functions as the canonical γ subunit of the plant energy sensor SnRK1.

    PubMed

    Ramon, Matthew; Ruelens, Philip; Li, Yi; Sheen, Jen; Geuten, Koen; Rolland, Filip

    2013-07-01

    The AMPK/SNF1/SnRK1 protein kinases are a family of ancient and highly conserved eukaryotic energy sensors that function as heterotrimeric complexes. These typically comprise catalytic α subunits and regulatory β and γ subunits, the latter function as the energy-sensing modules of animal AMPK through adenosine nucleotide binding. The ability to monitor accurately and adapt to changing environmental conditions and energy supply is essential for optimal plant growth and survival, but mechanistic insight in the plant SnRK1 function is still limited. In addition to a family of γ-like proteins, plants also encode a hybrid βγ protein that combines the Four-Cystathionine β-synthase (CBS)-domain (FCD) structure in γ subunits with a glycogen-binding domain (GBD), typically found in β subunits. We used integrated functional analyses by ectopic SnRK1 complex reconstitution, yeast mutant complementation, in-depth phylogenetic reconstruction, and a seedling starvation assay to show that only the hybrid KINβγ protein that recruited the GBD around the emergence of the green chloroplast-containing plants, acts as the canonical γ subunit required for heterotrimeric complex formation. Mutagenesis and truncation analysis further show that complex interaction in plant cells and γ subunit function in yeast depend on both a highly conserved FCD and a pre-CBS domain, but not the GBD. In addition to novel insight into canonical AMPK/SNF/SnRK1 γ subunit function, regulation and evolution, we provide a new classification of plant FCD genes as a convenient and reliable tool to predict regulatory partners for the SnRK1 energy sensor and novel FCD gene functions. PMID:23551663

  18. Oil Exploration Mapping

    NASA Technical Reports Server (NTRS)

    1994-01-01

    After concluding an oil exploration agreement with the Republic of Yemen, Chevron International needed detailed geologic and topographic maps of the area. Chevron's remote sensing team used imagery from Landsat and SPOT, combining images into composite views. The project was successfully concluded and resulted in greatly improved base maps and unique topographic maps.

  19. Reading Angles in Maps

    ERIC Educational Resources Information Center

    Izard, Véronique; O'Donnell, Evan; Spelke, Elizabeth S.

    2014-01-01

    Preschool children can navigate by simple geometric maps of the environment, but the nature of the geometric relations they use in map reading remains unclear. Here, children were tested specifically on their sensitivity to angle. Forty-eight children (age 47:15-53:30 months) were presented with fragments of geometric maps, in which angle sections…

  20. Applications of Concept Mapping

    ERIC Educational Resources Information Center

    De Simone, Christina

    2007-01-01

    This article reviews three major uses of the concept-mapping strategies for postsecondary learning: the external representation of concept maps as an external scratch pad to represent major ideas and their organization, the mental construction of concept maps when students are seeking a time-efficient tool, and the electronic construction and…

  1. Mapping Sociological Concepts.

    ERIC Educational Resources Information Center

    Trepagnier, Barbara

    2002-01-01

    Focuses on the use of cognitive mapping within sociology. Describes an assignment where students created a cognitive map that focused on names of theorists and concepts related to them. Discusses sociological imagination in relation to cognitive mapping and the assessment of the assignment. (CMK)

  2. Statistical Mapping by Computer.

    ERIC Educational Resources Information Center

    Utano, Jack J.

    The function of a statistical map is to provide readers with a visual impression of the data so that they may be able to identify any geographic characteristics of the displayed phenomena. The increasingly important role played by the computer in the production of statistical maps is manifested by the varied examples of computer maps in recent…

  3. Using maps in genealogy

    USGS Publications Warehouse

    U.S. Geological Survey

    1999-01-01

    Maps are one of many sources you may need to complete a family tree. In genealogical research, maps can provide clues to where our ancestors may have lived and where to look for written records about them. Beginners should master basic genealogical research techniques before starting to use topographic maps.

  4. Quantitative DNA fiber mapping

    DOEpatents

    Gray, Joe W.; Weier, Heinz-Ulrich G.

    1998-01-01

    The present invention relates generally to the DNA mapping and sequencing technologies. In particular, the present invention provides enhanced methods and compositions for the physical mapping and positional cloning of genomic DNA. The present invention also provides a useful analytical technique to directly map cloned DNA sequences onto individual stretched DNA molecules.

  5. Canine chondrodysplasia caused by a truncating mutation in collagen-binding integrin alpha subunit 10.

    PubMed

    Kyöstilä, Kaisa; Lappalainen, Anu K; Lohi, Hannes

    2013-01-01

    The skeletal dysplasias are disorders of the bone and cartilage tissues. Similarly to humans, several dog breeds have been reported to suffer from different types of genetic skeletal disorders. We have studied the molecular genetic background of an autosomal recessive chondrodysplasia that affects the Norwegian Elkhound and Karelian Bear Dog breeds. The affected dogs suffer from disproportionate short stature dwarfism of varying severity. Through a genome-wide approach, we mapped the chondrodysplasia locus to a 2-Mb region on canine chromosome 17 in nine affected and nine healthy Elkhounds (praw = 7.42×10(-6), pgenome-wide = 0.013). The associated locus contained a promising candidate gene, cartilage specific integrin alpha 10 (ITGA10), and mutation screening of its 30 exons revealed a nonsense mutation in exon 16 (c.2083C>T; p.Arg695*) that segregated fully with the disease in both breeds (p = 2.5×10(-23)). A 24% mutation carrier frequency was indicated in NEs and an 8% frequency in KBDs. The ITGA10 gene product, integrin receptor α10-subunit combines into a collagen-binding α10β1 integrin receptor, which is expressed in cartilage chondrocytes and mediates chondrocyte-matrix interactions during endochondral ossification. As a consequence of the nonsense mutation, the α10-protein was not detected in the affected cartilage tissue. The canine phenotype highlights the importance of the α10β1 integrin in bone growth, and the large animal model could be utilized to further delineate its specific functions. Finally, this study revealed a candidate gene for human chondrodysplasias and enabled the development of a genetic test for breeding purposes to eradicate the disease from the two dog breeds. PMID:24086591

  6. Genomic organization of the murine G protein beta subunit genes and related processed pseudogenes.

    PubMed

    Kitanaka, J; Wang, X B; Kitanaka, N; Hembree, C M; Uhl, G R

    2001-12-01

    The functional significance of heterotrimeric guanine nucleotide binding protein (G protein) for the many physiological processes including the molecular mechanisms of drug addiction have been described. In investigating the changes of mRNA expression after acute psychostimulant administration, we previously identified a cDNA encoding a G protein beta1 subunit (Gbeta1) that was increased up to four-fold in certain brain regions after administration of psychostimulants. The mouse Gbeta1 gene (the mouse genetic symbol, GNB1) was mapped to chromosome 4, but little was known of its genetic features. To characterize the GNB1 gene further, we have cloned and analyzed the genomic structures of the mouse GNBI gene and its homologous sequences. The GNBI gene spans at least 50 kb, and consists of 12 exons and 11 introns. The exon/intron boundaries were determined and found to follow the GT/AG rule. Exons 3-11 encode the Gbeta1 protein, and the exon 2 is an alternative, resulting in putative two splicing variants. Although intron 11 is additional for GNBI compared with GNB2 and GNB3, the intron positions within the protein coding region of GNB1, GNB2 and GNB3 are identical, suggesting that GNB1 should have diverged from the ancestral gene family earlier than the genes for GNB2 and GNB3. We also found the 5'-truncated processed pseudogenes with 71-89% similarities to GNBI mRNA sequence, suggesting that the truncated cDNA copies, which have been reverse-transcribed from a processed mRNA for GNB1, might have been integrated into several new locations in the mouse genome. PMID:11913780

  7. G protein beta gamma subunits stimulate phosphorylation of Shc adapter protein.

    PubMed Central

    Touhara, K; Hawes, B E; van Biesen, T; Lefkowitz, R J

    1995-01-01

    The mechanism of mitogen-activated protein (MAP) kinase activation by pertussis toxin-sensitive Gi-coupled receptors is known to involve the beta gamma subunits of heterotrimeric G proteins (G beta gamma), p21ras activation, and an as-yet-unidentified tyrosine kinase. To investigate the mechanism of G beta gamma-stimulated p21ras activation, G beta gamma-mediated tyrosine phosphorylation was examined by overexpressing G beta gamma or alpha 2-C10 adrenergic receptors (ARs) that couple to Gi in COS-7 cells. Immunoprecipitation of phosphotyrosine-containing proteins revealed a 2- to 3-fold increase in the phosphorylation of two proteins of approximately 50 kDa (designated as p52) in G beta gamma-transfected cells or in alpha 2-C10 AR-transfected cells stimulated with the agonist UK-14304. The latter response was pertussis toxin sensitive. These proteins (p52) were also specifically immunoprecipitated with anti-Shc antibodies and comigrated with two Shc proteins, 46 and 52 kDa. The G beta gamma- or alpha 2-C10 AR-stimulated p52 (Shc) phosphorylation was inhibited by coexpression of the carboxyl terminus of beta-adrenergic receptor kinase (a G beta gamma-binding pleckstrin homology domain peptide) or by the tyrosine kinase inhibitors genistein and herbimycin A, but not by a dominant negative mutant of p21ras. Worthmannin, a specific inhibitor of phosphatidylinositol 3-kinase (PI3K) inhibited phosphorylation of p52 (Shc), implying involvement of PI3K. These results suggest that G beta gamma-stimulated Shc phosphorylation represents an early step in the pathway leading to p21ras activation, similar to the mechanism utilized by growth factor tyrosine kinase receptors. Images Fig. 1 Fig. 3 PMID:7568118

  8. Cholera Toxin B Subunit Shows Transneuronal Tracing after Injection in an Injured Sciatic Nerve

    PubMed Central

    Lai, Bi-Qin; Qiu, Xue-Chen; Zhang, Ke; Zhang, Rong-Yi; Jin, Hui; Li, Ge; Shen, Hui-Yong; Wu, Jin-Lang; Ling, Eng-Ang; Zeng, Yuan-Shan

    2015-01-01

    Cholera toxin B subunit (CTB) has been extensively used in the past for monosynaptic mapping. For decades, it was thought to lack the ability of transneuronal tracing. In order to investigate whether biotin conjugates of CTB (b-CTB) would pass through transneurons in the rat spinal cord, it was injected into the crushed left sciatic nerve. For experimental control, the first order afferent neuronal projections were defined by retrograde transport of fluorogold (FG, a non-transneuronal labeling marker as an experimental control) injected into the crushed right sciatic nerve in the same rat. Neurons containing b-CTB or FG were observed in the dorsal root ganglia (DRG) at the L4-L6 levels ipsilateral to the tracer injection. In the spinal cord, b-CTB labeled neurons were distributed in all laminae ipsilaterally between C7 and S1 segments, but labeling of neurons at the cervical segment was abolished when the T10 segment was transected completely. The interneurons, distributed in the intermediate gray matter and identified as gamma-aminobutyric acid-ergic (GABAergic), were labeled by b-CTB. In contrast, FG labeling was confined to the ventral horn neurons at L4-L6 spinal segments ipsilateral to the injection. b-CTB immunoreactivity remained to be restricted to the soma of neurons and often appeared as irregular patches detected by light and electron microscopy. Detection of monosialoganglioside (GM1) in b-CTB labeled neurons suggests that GM1 ganglioside may specifically enhance the uptake and transneuronal passage of b-CTB, thus supporting the notion that it may be used as a novel transneuronal tracer. PMID:26640949

  9. Proteomic Analysis of Glycine Receptor β Subunit (GlyRβ)-interacting Proteins

    PubMed Central

    del Pino, Isabel; Koch, Dennis; Schemm, Rudolf; Qualmann, Britta; Betz, Heinrich; Paarmann, Ingo

    2014-01-01

    Glycine receptors (GlyRs) mediate inhibitory neurotransmission in spinal cord and brainstem. They are clustered at inhibitory postsynapses via a tight interaction of their β subunits (GlyRβ) with the scaffolding protein gephyrin. In an attempt to isolate additional proteins interacting with GlyRβ, we performed pulldown experiments with rat brain extracts using a glutathione S-transferase fusion protein encompassing amino acids 378–455 of the large intracellular loop of GlyRβ as bait. This identified syndapin I (SdpI) as a novel interaction partner of GlyRβ that coimmunoprecipitates with native GlyRs from brainstem extracts. Both SdpI and SdpII bound efficiently to the intracellular loop of GlyRβ in vitro and colocalized with GlyRβ upon coexpression in COS-7 cells. The SdpI-binding site was mapped to a proline-rich sequence of 22 amino acids within the intracellular loop of GlyRβ. Deletion and point mutation analysis disclosed that SdpI binding to GlyRβ is Src homology 3 domain-dependent. In cultured rat spinal cord neurons, SdpI immunoreactivity was found to partially colocalize with marker proteins of inhibitory and excitatory synapses. When SdpI was acutely knocked down in cultured spinal cord neurons by viral miRNA expression, postsynaptic GlyR clusters were significantly reduced in both size and number. Similar changes in GlyR cluster properties were found in spinal cultures from SdpI-deficient mice. Our results are consistent with a role of SdpI in the trafficking and/or cytoskeletal anchoring of synaptic GlyRs. PMID:24509844

  10. Distinctive interactions of the Arabidopsis homolog of the 30 kD subunit of the cleavage and polyadenylation specificity factor (AtCPSF30) with other polyadenylation factor subunits

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Background: The Arabidopsis ortholog of the 30 kD subunit of the mammalian Cleavage and Polyadenylation Specificity Factor (AtCPSF30) is an RNA-binding endonuclease that is associated with other Arabidopsis CPSF subunits (orthologs of the 160, 100, and 73 kD subunits of CPSF). In order to better u...

  11. Epitopes in α8β1 and other RGD-binding integrins delineate classes of integrin-blocking antibodies and major binding loops in α subunits

    PubMed Central

    Nishimichi, Norihisa; Kawashima, Nagako; Yokosaki, Yasuyuki

    2015-01-01

    Identification of epitopes for integrin-blocking monoclonal antibodies (mAbs) has aided our understanding of structure-function relationship of integrins. We mapped epitopes of chicken anti-integrin-α8-subunit-blocking mAbs by mutational analyses, examining regions that harboured all mapped epitopes recognized by mAbs against other α-subunits in the RGD-binding-integrin subfamily. Six mAbs exhibited blocking function, and these mAbs recognized residues on the same W2:41-loop on the top-face of the β-propeller. Loop-tips sufficiently close to W2:41 (<25 Å) contained within a footprint of the mAbs were mutated, and the loop W3:34 on the bottom face was identified as an additional component of the epitope of one antibody, clone YZ5. Binding sequences on the two loops were conserved in virtually all mammals, and that on W3:34 was also conserved in chickens. These indicate 1) YZ5 binds both top and bottom loops, and the binding to W3:34 is by interactions to conserved residues between immunogen and host species, 2) five other blocking mAbs solely bind to W2:41 and 3) the α8 mAbs would cross-react with most mammals. Comparing with the mAbs against the other α-subunits of RGD-integrins, two classes were delineated; those binding to “W3:34 and an top-loop”, and “solely W2:41”, accounting for 82% of published RGD-integrin-mAbs. PMID:26349930

  12. NMR structure of the amino-terminal domain from the Tfb1 subunit of TFIIH and characterization of its phosphoinositide and VP16 binding sites.

    PubMed

    Di Lello, Paola; Nguyen, Bao D; Jones, Tamara N; Potempa, Krzysztof; Kobor, Michael S; Legault, Pascale; Omichinski, James G

    2005-05-31

    General transcription factor IIH (TFIIH) is recruited to the preinitiation complex (PIC) through direct interactions between its p62 (Tfb1) subunit and the carboxyl-terminal domain of TFIIEalpha. TFIIH has also been shown to interact with a number of transcriptional activator proteins through interactions with the same p62 (Tfb1) subunit. We have determined the NMR solution structure of the amino-terminal domain from the Tfb1 subunit of yeast TFIIH (Tfb1(1-115)). Like the corresponding domain from the human p62 protein, Tfb1(1-115) contains a PH domain fold despite a low level of sequence identity between the two functionally homologous proteins. In addition, we have performed in vitro binding studies that demonstrate that the PH domains of Tfb1 and p62 specifically bind to monophosphorylated inositides [PtdIns(5)P and PtdIns(3)P]. NMR chemical shift mapping demonstrated that the PtdIns(5)P binding site on Tfb1 (p62) is located in the basic pocket formed by beta-strands beta5-beta7 of the PH domain fold. Interestingly, the structural composition of the PtdIns(5)P binding site is different from the composition of the binding sites for phosphoinositides on prototypic PH domains. We have also determined that the PH domains from Tfb1 and p62 are sufficient for binding to the activation domain of VP16. NMR chemical shift mapping demonstrated that the VP16 binding site within the PH domain of Tfb1 (p62) overlaps with the PtdIns(5)P binding site on Tfb1 (p62). These results provide new information about the recognition of phosphoinositides by PH domains, and point to a potential role for phosphoinositides in VP16 regulation. PMID:15909982

  13. Isolation and characterization of temperature-sensitive mutations in RPA190, the gene encoding the largest subunit of RNA polymerase I from Saccharomyces cerevisiae.

    PubMed Central

    Wittekind, M; Dodd, J; Vu, L; Kolb, J M; Buhler, J M; Sentenac, A; Nomura, M

    1988-01-01

    The isolation and characterization of temperature-sensitive mutations in RNA polymerase I from Saccharomyces cerevisiae are described. A plasmid carrying RPA190, the gene encoding the largest subunit of the enzyme, was subjected to in vitro mutagenesis with hydroxylamine. Using a plasmid shuffle screening system, five different plasmids were isolated which conferred a temperature-sensitive phenotype in haploid yeast strains carrying the disrupted chromosomal RPA190 gene. These temperature-sensitive alleles were transferred to the chromosomal RPA190 locus for mapping and physiology experiments. Accumulation of RNA was found to be defective in all mutant strains at the nonpermissive temperature. In addition, analysis of pulse-labeled RNA from two mutant strains at 37 degrees C showed that the transcription of rRNA genes was decreased, while that of 5S RNA was relatively unaffected. RNA polymerase I was partially purified from several of the mutant strains grown at the nonpermissive temperature and was shown to be deficient when assayed in vitro. Fine-structure mapping and sequencing of the mutant alleles demonstrated that all five mutations were unique. The rpa190-1 and rpa190-5 mutations are tightly clustered in region I (S.S. Broyles and B. Moss, Proc. Natl. Acad. Sci. USA 83:3141-3145, 1986), the putative zinc-binding region that is common to all eucaryotic RNA polymerase large subunits. The rpa190-3 mutation is located between regions III and IV, and a strain carrying it behaves as a mutant that is defective in the synthesis of the enzyme. This mutation lies within a previously unidentified segment of highly conserved amino acid sequence homology that is shared among the largest subunits of eucaryotic nuclear RNA polymerases. Another temperature-sensitive mutation, rpa190-2, creates a UGA nonsense codon. Images PMID:3054507

  14. Epitopes in α8β1 and other RGD-binding integrins delineate classes of integrin-blocking antibodies and major binding loops in α subunits.

    PubMed

    Nishimichi, Norihisa; Kawashima, Nagako; Yokosaki, Yasuyuki

    2015-01-01

    Identification of epitopes for integrin-blocking monoclonal antibodies (mAbs) has aided our understanding of structure-function relationship of integrins. We mapped epitopes of chicken anti-integrin-α8-subunit-blocking mAbs by mutational analyses, examining regions that harboured all mapped epitopes recognized by mAbs against other α-subunits in the RGD-binding-integrin subfamily. Six mAbs exhibited blocking function, and these mAbs recognized residues on the same W2:41-loop on the top-face of the β-propeller. Loop-tips sufficiently close to W2:41 (<25 Å) contained within a footprint of the mAbs were mutated, and the loop W3:34 on the bottom face was identified as an additional component of the epitope of one antibody, clone YZ5. Binding sequences on the two loops were conserved in virtually all mammals, and that on W3:34 was also conserved in chickens. These indicate 1) YZ5 binds both top and bottom loops, and the binding to W3:34 is by interactions to conserved residues between immunogen and host species, 2) five other blocking mAbs solely bind to W2:41 and 3) the α8 mAbs would cross-react with most mammals. Comparing with the mAbs against the other α-subunits of RGD-integrins, two classes were delineated; those binding to "W3:34 and an top-loop", and "solely W2:41", accounting for 82% of published RGD-integrin-mAbs. PMID:26349930

  15. The Evolutionarily Conserved Mediator Subunit MDT-15/MED15 Links Protective Innate Immune Responses and Xenobiotic Detoxification

    PubMed Central

    McEwan, Deborah L.; Conery, Annie L.; Ausubel, Frederick M.

    2014-01-01

    Metazoans protect themselves from environmental toxins and virulent pathogens through detoxification and immune responses. We previously identified a small molecule xenobiotic toxin that extends survival of Caenorhabditis elegans infected with human bacterial pathogens by activating the conserved p38 MAP kinase PMK-1 host defense pathway. Here we investigate the cellular mechanisms that couple activation of a detoxification response to innate immunity. From an RNAi screen of 1,420 genes expressed in the C. elegans intestine, we identified the conserved Mediator subunit MDT-15/MED15 and 28 other gene inactivations that abrogate the induction of PMK-1-dependent immune effectors by this small molecule. We demonstrate that MDT-15/MED15 is required for the xenobiotic-induced expression of p38 MAP kinase PMK-1-dependent immune genes and protection from Pseudomonas aeruginosa infection. We also show that MDT-15 controls the induction of detoxification genes and functions to protect the host from bacteria-derived phenazine toxins. These data define a central role for MDT-15/MED15 in the coordination of xenobiotic detoxification and innate immune responses. PMID:24875643

  16. Structure of a herpesvirus nuclear egress complex subunit reveals an interaction groove that is essential for viral replication

    PubMed Central

    Leigh, Kendra E.; Sharma, Mayuri; Mansueto, My Sam; Boeszoermenyi, Andras; Filman, David J.; Hogle, James M.; Wagner, Gerhard; Coen, Donald M.; Arthanari, Haribabu

    2015-01-01

    Herpesviruses require a nuclear egress complex (NEC) for efficient transit of nucleocapsids from the nucleus to the cytoplasm. The NEC orchestrates multiple steps during herpesvirus nuclear egress, including disruption of nuclear lamina and particle budding through the inner nuclear membrane. In the important human pathogen human cytomegalovirus (HCMV), this complex consists of nuclear membrane protein UL50, and nucleoplasmic protein UL53, which is recruited to the nuclear membrane through its interaction with UL50. Here, we present an NMR-determined solution-state structure of the murine CMV homolog of UL50 (M50; residues 1–168) with a strikingly intricate protein fold that is matched by no other known protein folds in its entirety. Using NMR methods, we mapped the interaction of M50 with a highly conserved UL53-derived peptide, corresponding to a segment that is required for heterodimerization. The UL53 peptide binding site mapped onto an M50 surface groove, which harbors a large cavity. Point mutations of UL50 residues corresponding to surface residues in the characterized M50 heterodimerization interface substantially decreased UL50–UL53 binding in vitro, eliminated UL50–UL53 colocalization, prevented disruption of nuclear lamina, and halted productive virus replication in HCMV-infected cells. Our results provide detailed structural information on a key protein–protein interaction involved in nuclear egress and suggest that NEC subunit interactions can be an attractive drug target. PMID:26150520

  17. Immunoproteasome subunit LMP7 Deficiency Improves Obesity and Metabolic Disorders.

    PubMed

    Kimura, Hiroaki; Usui, Fumitake; Karasawa, Tadayoshi; Kawashima, Akira; Shirasuna, Koumei; Inoue, Yoshiyuki; Komada, Takanori; Kobayashi, Motoi; Mizushina, Yoshiko; Kasahara, Tadashi; Suzuki, Koichi; Iwasaki, Yusaku; Yada, Toshihiko; Caturegli, Patrizio; Takahashi, Masafumi

    2015-01-01

    Inflammation plays an important role in the development of obesity and metabolic disorders; however, it has not been fully understood how inflammation occurs and is regulated in their pathogenesis. Low-molecular mass protein-7 (LMP7) is a proteolytic subunit of the immunoproteasome that shapes the repertoire of antigenic peptides on major histocompatibility complex class I molecule. In this study, we investigated the role of LMP7 in the development of obesity and metabolic disorders using LMP7-deficient mice. LMP7 deficiency conveyed resistant to obesity, and improved glucose intolerance and insulin sensitivity in mice fed with high-fat diet (HFD). LMP7 deficiency decreased pancreatic lipase expression, increased fecal lipid contents, and inhibited the increase of plasma triglyceride levels upon oral oil administration or HFD feeding. Using bone marrow-transferred chimeric mice, we found that LMP7 in both bone marrow- and non-bone marrow-derived cells contributes to the development of HFD-induced obesity. LMP7 deficiency decreased inflammatory responses such as macrophage infiltration and chemokine expression while it increased serum adiponection levels. These findings demonstrate a novel role for LMP7 and provide new insights into the mechanisms underlying inflammation in the pathophysiology of obesity and metabolic disorders. PMID:26515636

  18. Subunit association as the stabilizing determinant for archaeal methionine adenosyltransferases.

    PubMed

    Garrido, Francisco; Alfonso, Carlos; Taylor, John C; Markham, George D; Pajares, María A

    2009-07-01

    Archaea contain a class of methionine adenosyltransferases (MATs) that exhibit substantially higher stability than their mesophilic counterparts. Their sequences are highly divergent, but preserve the essential active site motifs of the family. We have investigated the origin of this increased stability using chemical denaturation experiments on Methanococcus jannaschii MAT (Mj-MAT) and mutants containing single tryptophans in place of tyrosine residues. The results from fluorescence, circular dichroism, hydrodynamic, and enzyme activity measurements showed that the higher stability of Mj-MAT derives largely from a tighter association of its subunits in the dimer. Local fluorescence changes, interpreted using secondary structure predictions, further identify the least stable structural elements as the C-terminal ends of beta-strands E2 and E6, and the N-terminus of E3. Dimer dissociation however requires a wider perturbation of the molecule. Additional analysis was initially hindered by the lack of crystal structures for archaeal MATs, a limitation that we overcame by construction of a 3D-homology model of Mj-MAT. This model predicts preservation of the chain topology and three-domain organization typical of this family, locates the least stable structural elements at the flat contact surface between monomers, and shows that alterations in all three domains are required for dimer dissociation. PMID:19348969

  19. NMDA receptor structures reveal subunit arrangement and pore architecture

    PubMed Central

    Lee, Chia-Hsueh; Lü, Wei; Michel, Jennifer Carlisle; Goehring, April; Du, Juan; Song, Xianqiang; Gouaux, Eric

    2014-01-01

    Summary N-methyl-d-aspartate (NMDA) receptors are Hebbian-like coincidence detectors, requiring binding of glycine and glutamate in combination with the relief of voltage-dependent magnesium block to open an ion conductive pore across the membrane bilayer. Despite the importance of the NMDA receptor in the development and function of the brain, a molecular structure of an intact receptor has remained elusive. Here we present x-ray crystal structures of the GluN1/GluN2B NMDA receptor with the allosteric inhibitor, Ro25-6981, partial agonists and the ion channel blocker, MK-801. Receptor subunits are arranged in a 1-2-1-2 fashion, demonstrating extensive interactions between the amino terminal and ligand binding domains. The transmembrane domains harbor a closed-blocked ion channel, a pyramidal central vestibule lined by residues implicated in binding ion channel blockers and magnesium, and a ~2-fold symmetric arrangement of ion channel pore loops. These structures provide new insights into the architecture, allosteric coupling and ion channel function of NMDA receptors. PMID:25008524

  20. Roles of subunit phosphorylation in regulating glutamate receptor function

    PubMed Central

    Wang, John Q.; Guo, Ming-Lei; Jin, Dao-Zhong; Xue, Bing; Fibuch, Eugene E.; Mao, Li-Min

    2014-01-01

    Protein phosphorylation is an important mechanism for regulating ionotropic glutamate receptors (iGluRs). Early studies have established that major iGluR subtypes, including α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptors and N-methyl-D-aspartate (NMDA) receptors, are subject to phosphorylation. Multiple serine, threonine, and tyrosine residues predominantly within the C-terminal regions of AMPA receptor and NMDA receptor subunits have been identified as sensitive phosphorylation sites. These distinct sites undergo either constitutive phosphorylation or activity-dependent phosphorylation induced by changing cellular and synaptic inputs as reversible events. An increasing number of synapse-enriched protein kinases have been found to phosphorylate iGluR. The common kinases include protein kinase A, protein kinase C, Ca2+/calmodulin-dependent protein kinase II, Src/Fyn non-receptor tyrosine kinases, and cyclin dependent kinase-5. Regulated phosphorylation plays a well-documented role in modulating the biochemical, biophysical, and functional properties of the receptor. In the future, identifying the precise mechanisms how phosphorylation regulates iGluR activities and finding the link between iGluR phosphorylation and the pathogenesis of various brain diseases, including psychiatric and neurodegenerative diseases, chronic pain, stroke, Alzheimer’s disease and substance addiction, will be hot topics and could contribute to the development of novel pharmacotherapies, by targeting the defined phosphorylation process, for suppressing iGluR-related disorders. PMID:24291102

  1. Design of a hyperstable 60-subunit protein icosahedron.

    PubMed

    Hsia, Yang; Bale, Jacob B; Gonen, Shane; Shi, Dan; Sheffler, William; Fong, Kimberly K; Nattermann, Una; Xu, Chunfu; Huang, Po-Ssu; Ravichandran, Rashmi; Yi, Sue; Davis, Trisha N; Gonen, Tamir; King, Neil P; Baker, David

    2016-07-01

    The icosahedron is the largest of the Platonic solids, and icosahedral protein structures are widely used in biological systems for packaging and transport. There has been considerable interest in repurposing such structures for applications ranging from targeted delivery to multivalent immunogen presentation. The ability to design proteins that self-assemble into precisely specified, highly ordered icosahedral structures would open the door to a new generation of protein containers with properties custom-tailored to specific applications. Here we describe the computational design of a 25-nanometre icosahedral nanocage that self-assembles from trimeric protein building blocks. The designed protein was produced in Escherichia coli, and found by electron microscopy to assemble into a homogenous population of icosahedral particles nearly identical to the design model. The particles are stable in 6.7 molar guanidine hydrochloride at up to 80 degrees Celsius, and undergo extremely abrupt, but reversible, disassembly between 2 molar and 2.25 molar guanidinium thiocyanate. The icosahedron is robust to genetic fusions: one or two copies of green fluorescent protein (GFP) can be fused to each of the 60 subunits to create highly fluorescent ‘standard candles’ for use in light microscopy, and a designed protein pentamer can be placed in the centre of each of the 20 pentameric faces to modulate the size of the entrance/exit channels of the cage. Such robust and customizable nanocages should have considerable utility in targeted drug delivery, vaccine design and synthetic biology. PMID:27309817

  2. Extensions to the D-Cam sub-unit architecture

    NASA Astrophysics Data System (ADS)

    Ryan, Padraig; Connell, Joseph

    2005-06-01

    Multispectral imaging produces large amounts of data which extend processing, transmission and storage systems to their upper limits. Although there are several interface standards specific to image data acquisition, such as CameraLink, it is Firewire which provides a high-speed data bus, integrated control capability, without loss of flexibility, and which is commonly available as a low cost solution. The class of multispectral imaging requires a different treatment of the processing principals than standard imaging. The same spatial region is captured multiple times using different optical wavelengths. This technique finds application in such diverse areas as coastal monitoring, fruit sorting and automated agriculture. Modifications and additional features to the camera operating and configuration parameters are therefore required which are not generally present with conventional imaging sensors. This paper describes extensions to the IIDC Digital Camera (D-Cam) specification in the development of a Firewire technology platform for transmitting the data structures described and for providing real-time, online control of spectral information acquisition. Additionally, it describes how a set of registers in the sub-unit architecture of the Firewire protocol is augmented to accommodate the demands of a multispectral system. The extensions are specification conformant and do not alter underlining compliance with the base standard. The paper also describes the implementation of the extended D-Cam in the Firewire subsystem of a smart multispectral camera used in commercial applications.

  3. Immunoproteasome subunit LMP7 Deficiency Improves Obesity and Metabolic Disorders

    PubMed Central

    Kimura, Hiroaki; Usui, Fumitake; Karasawa, Tadayoshi; Kawashima, Akira; Shirasuna, Koumei; Inoue, Yoshiyuki; Komada, Takanori; Kobayashi, Motoi; Mizushina, Yoshiko; Kasahara, Tadashi; Suzuki, Koichi; Iwasaki, Yusaku; Yada, Toshihiko; Caturegli, Patrizio; Takahashi, Masafumi

    2015-01-01

    Inflammation plays an important role in the development of obesity and metabolic disorders; however, it has not been fully understood how inflammation occurs and is regulated in their pathogenesis. Low-molecular mass protein-7 (LMP7) is a proteolytic subunit of the immunoproteasome that shapes the repertoire of antigenic peptides on major histocompatibility complex class I molecule. In this study, we investigated the role of LMP7 in the development of obesity and metabolic disorders using LMP7-deficient mice. LMP7 deficiency conveyed resistant to obesity, and improved glucose intolerance and insulin sensitivity in mice fed with high-fat diet (HFD). LMP7 deficiency decreased pancreatic lipase expression, increased fecal lipid contents, and inhibited the increase of plasma triglyceride levels upon oral oil administration or HFD feeding. Using bone marrow-transferred chimeric mice, we found that LMP7 in both bone marrow- and non-bone marrow-derived cells contributes to the development of HFD-induced obesity. LMP7 deficiency decreased inflammatory responses such as macrophage infiltration and chemokine expression while it increased serum adiponection levels. These findings demonstrate a novel role for LMP7 and provide new insights into the mechanisms underlying inflammation in the pathophysiology of obesity and metabolic disorders. PMID:26515636

  4. Design of a hyperstable 60-subunit protein icosahedron

    NASA Astrophysics Data System (ADS)

    Hsia, Yang; Bale, Jacob B.; Gonen, Shane; Shi, Dan; Sheffler, William; Fong, Kimberly K.; Nattermann, Una; Xu, Chunfu; Huang, Po-Ssu; Ravichandran, Rashmi; Yi, Sue; Davis, Trisha N.; Gonen, Tamir; King, Neil P.; Baker, David

    2016-07-01

    The icosahedron is the largest of the Platonic solids, and icosahedral protein structures are widely used in biological systems for packaging and transport. There has been considerable interest in repurposing such structures for applications ranging from targeted delivery to multivalent immunogen presentation. The ability to design proteins that self-assemble into precisely specified, highly ordered icosahedral structures would open the door to a new generation of protein containers with properties custom-tailored to specific applications. Here we describe the computational design of a 25-nanometre icosahedral nanocage that self-assembles from trimeric protein building blocks. The designed protein was produced in Escherichia coli, and found by electron microscopy to assemble into a homogenous population of icosahedral particles nearly identical to the design model. The particles are stable in 6.7 molar guanidine hydrochloride at up to 80 degrees Celsius, and undergo extremely abrupt, but reversible, disassembly between 2 molar and 2.25 molar guanidinium thiocyanate. The icosahedron is robust to genetic fusions: one or two copies of green fluorescent protein (GFP) can be fused to each of the 60 subunits to create highly fluorescent ‘standard candles’ for use in light microscopy, and a designed protein pentamer can be placed in the centre of each of the 20 pentameric faces to modulate the size of the entrance/exit channels of the cage. Such robust and customizable nanocages should have considerable utility in targeted drug delivery, vaccine design and synthetic biology.

  5. Evolution of the primate cytochrome c oxidase subunit II gene.

    PubMed

    Adkins, R M; Honeycutt, R L

    1994-03-01

    We examined the nucleotide and amino acid sequence variation of the cytochrome c oxidase subunit II (COII) gene from 25 primates (4 hominoids, 8 Old World monkeys, 2 New World monkeys, 2 tarsiers, 7 lemuriforms, 2 lorisiforms). Marginal support was found for three phylogenetic conclusions: (1) sister-group relationship between tarsiers and a monkey/ape clade, (2) placement of the aye-aye (Daubentonia) sister to all other strepsirhine primates, and (3) rejection of a sister-group relationship of dwarf lemurs (i.e., Cheirogaleus) with lorisiform primates. Stronger support was found for a sister-group relationship between the ring-tail lemur (Lemur catta) and the gentle lemurs (Hapalemur). In congruence with previous studies on COII, we found that the monkeys and apes have undergone a nearly two-fold increase in the rate of amino acid replacement relative to other primates. Although functionally important amino acids are generally conserved among all primates, the acceleration in amino acid replacements in higher primates is associated with increased variation in the amino terminal end of the protein. Additionally, the replacement of two carboxyl-bearing residues (glutamate and aspartate) at positions 114 and 115 may provide a partial explanation for the poor enzyme kinetics in cross-reactions between the cytochromes c and cytochrome c oxidases of higher primates and other mammals. PMID:8006990

  6. Ral GTPases regulate exocyst assembly through dual subunit interactions.

    PubMed

    Moskalenko, Serge; Tong, Chao; Rosse, Carine; Mirey, Gladys; Formstecher, Etienne; Daviet, Laurent; Camonis, Jacques; White, Michael A

    2003-12-19

    Ral GTPases have been implicated in the regulation of a variety of dynamic cellular processes including proliferation, oncogenic transformation, actin-cytoskeletal dynamics, endocytosis, and exocytosis. Recently the Sec6/8 complex, or exocyst, a multisubunit complex facilitating post-Golgi targeting of distinct subclasses of secretory vesicles, has been identified as a bona fide Ral effector complex. Ral GTPases regulate exocyst-dependent vesicle trafficking and are required for exocyst complex assembly. Sec5, a membrane-associated exocyst subunit, has been identified as a direct target of activated Ral; however, the mechanism by which Ral can modulate exocyst assembly is unknown. Here we report that an additional component of the exocyst, Exo84, is a direct target of activated Ral. We provide evidence that mammalian exocyst components are present as distinct subcomplexes on vesicles and the plasma membrane and that Ral GTPases regulate the assembly interface of a full octameric exocyst complex through interaction with Sec5 and Exo84. PMID:14525976

  7. Subunit Principle of Vulvar Reconstruction: Algorithm and Outcomes

    PubMed Central

    Kang, Gavin Chun-Wui; Tay, Eng Hseon; Por, Yong Chen

    2014-01-01

    Background Vulvar defects result chiefly from oncologic resection of vulvar tumors. Reconstruction of vulvar defects restores form and function for the purpose of coitus, micturition, and defecation. Many surgical options exist for vulvar reconstruction. The purpose of this article is to present our experience with vulvar reconstruction. Methods From 2007 to 2013, 43 women presented to us with vulvar defects for reconstruction. Their mean age at the time of reconstruction was 61.1 years. The most common cause of vulvar defect was from resection of vulvar carcinoma and extramammary Paget's disease of the vulva. Method s of reconstruction ranged from primary closure to skin grafting to the use of pedicled flaps. Results The main complications were that of long term hypertrophic and/or unaesthetic scarring of the donor site in 4 patients. Twenty-two patients (51%) were able to resume sexual intercourse. There were no complications of flap loss, wound dehiscence, and urethral stenosis. Conclusions We present a subunit algorithmic approach to vulvar reconstruction based on defect location within the vulva, dimension of the defect, and patient age and comorbidity. The gracilis and gluteal fold flaps are particularly versatile and aesthetically suited for reconstruction of a variety of vulvar defects. From an aesthetic viewpoint the gluteal fold flap was superior because of the well-concealed donor scar. We advocate the routine use of these 2 flaps for vulvar reconstruction. PMID:25075361

  8. Expansion of transducin subunit gene families in early vertebrate tetraploidizations.

    PubMed

    Lagman, David; Sundström, Görel; Ocampo Daza, Daniel; Abalo, Xesús M; Larhammar, Dan

    2012-10-01

    Hundreds of gene families expanded in the early vertebrate tetraploidizations including many gene families in the phototransduction cascade. We have investigated the evolution of the heterotrimeric G-proteins of photoreceptors, the transducins, in relation to these events using both phylogenetic analyses and synteny comparisons. Three alpha subunit genes were identified in amniotes and the coelacanth, GNAT1-3; two of these were identified in amphibians and teleost fish, GNAT1 and GNAT2. Most tetrapods have four beta genes, GNB1-4, and teleosts have additional duplicates. Finally, three gamma genes were identified in mammals, GNGT1, GNG11 and GNGT2. Of these, GNGT1 and GNGT2 were found in the other vertebrates. In frog and zebrafish additional duplicates of GNGT2 were identified. Our analyses show all three transducin families expanded during the early vertebrate tetraploidizations and the beta and gamma families gained additional copies in the teleost-specific genome duplication. This suggests that the tetraploidizations contributed to visual specialisations. PMID:22814267

  9. Telomerase reverse transcriptase subunit expression is associated with chondrosarcoma malignancy.

    PubMed

    Martin, James A; DeYoung, Barry R; Gitelis, Steven; Weydert, Jamie A; Klingelhutz, Aloysius J; Kurriger, Gail; Buckwalter, Joseph A

    2004-09-01

    Expression of the telomerase reverse transcriptase subunit telomerase reverse transcriptase gene is associated with most human malignancies. Because telomerase reverse transcriptase is rarely expressed in normal tissue, its presence in pathologic specimens is considered a marker of transformed cells. Moreover, high levels of expression have been correlated with poor prognosis in many cancers. Although telomerase activity has been found in chondrosarcomas, its prognostic significance in these malignant cartilage tumors is unknown. Malignancy in cartilage-derived tumors is assessed routinely by histomorphologic grading, but even well differentiated, low-grade lesions can metastasize. This unpredictable behavior greatly complicates the clinical treatment of cartilage tumors, making better prognostic indicators desirable. To address this issue we used immunohistochemistry to compare telomerase reverse transcriptase expression in a collection of 61 tumors consisting of malignant chondrosarcomas of varying grade and benign enchondromas. Associated case histories were reviewed to test the hypothesis that telomerase reverse transcriptase expression levels correlated with subsequent tumor recurrence. We found that the relative abundance of telomerase reverse transcriptase-expressing cells correlated significantly with grade and recurrence. These findings indicate that telomerase reverse transcriptase immunostaining may be a useful adjunct to the conventional three-level grading system. PMID:15346061

  10. Vaults and telomerase share a common subunit, TEP1.

    PubMed

    Kickhoefer, V A; Stephen, A G; Harrington, L; Robinson, M O; Rome, L H

    1999-11-12

    Vaults are large cytoplasmic ribonucleoprotein complexes of undetermined function. Mammalian vaults have two high molecular mass proteins of 193 and 240 kDa. We have identified a partial cDNA encoding the 240-kDa vault protein and determined it is identical to the mammalian telomerase-associated component, TEP1. TEP1 is the mammalian homolog of the Tetrahymena p80 telomerase protein and has been shown to interact specifically with mammalian telomerase RNA and the catalytic protein subunit hTERT. We show that while TEP1 is a component of the vault particle, vaults have no detectable telomerase activity. Using a yeast three-hybrid assay we demonstrate that several of the human vRNAs interact in a sequence-specific manner with TEP1. The presence of 16 WD40 repeats in the carboxyl terminus of the TEP1 protein is a convenient number for this protein to serve a structural or organizing role in the vault, a particle with eight-fold symmetry. The sharing of the TEP1 protein between vaults and telomerase suggests that TEP1 may play a common role in some aspect of ribonucleoprotein structure, function, or assembly. PMID:10551828

  11. Formation of Curvature Subunit of Carbon in Combustion.

    PubMed

    Wu, Xin-Zhou; Yao, Yang-Rong; Chen, Miao-Miao; Tian, Han-Rui; Xiao, Jun; Xu, Yun-Yan; Lin, Min-Song; Abella, Laura; Tian, Cheng-Bo; Gao, Cong-Li; Zhang, Qianyan; Xie, Su-Yuan; Huang, Rong-Bin; Zheng, Lan-Sun

    2016-08-01

    Curvature prevalently exists in the world of carbon materials (e.g., fullerenes, buckyl bowls, carbon nanotubes, and onions), but traditional C2-addition mechanisms fail to elucidate the mechanism responsible for the formation of carbon curvature starting from a pentagonal carbon ring in currently available chemical-physical processes such as combustion. Here, we show a complete series of nascent pentagon-incorporating C5-C18 that are online produced in the flame of acetylene-cyclopentadiene-oxygen and in situ captured by C60 or trapped as polycyclic aromatic hydrocarbons for clarifying the growth of the curved subunit of C20H10. A mechanism regarding C1-substitution and C2-addition has been proposed for understanding the formation of curvature in carbon materials, as exemplified by the typical curved molecule containing a single pentagon completely surrounded by five hexagons. The present mechanism, supported by the intermediates characterized by X-ray crystallography as well as NMR, has been experimentally validated for the rational synthesis of curved molecule in the commercially useful combustion process. PMID:27377559

  12. Characterization of the human rod transducin alpha-subunit gene.

    PubMed Central

    Fong, S L

    1992-01-01

    The human rod transducin alpha subunit (Tr alpha) gene has been cloned. A cDNA clone, HG14, contained a 1.1 kb insertion when compared with the human Tr alpha cDNA published by Van Dop et al. (1). Based on two overlapping clones isolated from a human genomic library, the human Tr alpha gene is 4.9 kb in length and consists of nine exons interrupted by eight introns. Northern blots of human retina total RNA showed that the gene is transcribed by rod photoreceptors into two species of mRNA, 1.3 kb and 2.4 kb in size. Apparently, this is the result of alternative splicing. Two putative transcription initiation sites were determined by primer extension and S1 nuclease protection assays. The putative promoter regions of the human and mouse Tr alpha genes have an identity of 78.1%. As found in the mouse gene (2), no TATA consensus sequence is present in the human gene. Images PMID:1614872

  13. Structure of the Tribolium castaneum Telomerase Catalytic Subunit TERT

    SciTech Connect

    Gillis,A.; Schuller, A.; Skordalakes, E.

    2008-01-01

    A common hallmark of human cancers is the overexpression of telomerase, a ribonucleoprotein complex that is responsible for maintaining the length and integrity of chromosome ends. Telomere length deregulation and telomerase activation is an early, and perhaps necessary, step in cancer cell evolution. Here we present the high-resolution structure of the Tribolium castaneum catalytic subunit of telomerase, TERT. The protein consists of three highly conserved domains, organized into a ring-like structure that shares common features with retroviral reverse transcriptases, viral RNA polymerases and B-family DNA polymerases. Domain organization places motifs implicated in substrate binding and catalysis in the interior of the ring, which can accommodate seven to eight bases of double-stranded nucleic acid. Modelling of an RNA-DNA heteroduplex in the interior of this ring demonstrates a perfect fit between the protein and the nucleic acid substrate, and positions the 3'-end of the DNA primer at the active site of the enzyme, providing evidence for the formation of an active telomerase elongation complex.

  14. Expression, purification, crystallization and preliminary crystallographic analysis of MxiH, a subunit of the Shigella flexneri type III secretion system needle

    SciTech Connect

    Deane, Janet E.; Cordes, Frank S.; Roversi, Pietro; Johnson, Steven; Kenjale, Roma; Picking, William D.; Picking, Wendy L.; Lea, Susan M.; Blocker, Ariel

    2006-03-01

    A monodisperse truncation mutant of MxiH, the subunit of the S. flexneri type III secretion system needle, has been crystallized. SeMet derivatives and a uranyl derivative have undergone preliminary crystallographic analysis. A monodisperse truncation mutant of MxiH, the subunit of the needle from the Shigella flexneri type III secretion system (TTSS), has been overexpressed and purified. Crystals were grown of native and selenomethionine-labelled MxiH{sub CΔ5} and diffraction data were collected to 1.9 Å resolution. The crystals belong to space group C2, with unit-cell parameters a = 183.4, b = 28.1, c = 27.8 Å, β = 96.5°. An anomalous difference Patterson map calculated with the data from the SeMet-labelled crystals revealed a single peak on the Harker section v = 0. Inspection of a uranyl derivative also revealed one peak in the isomorphous difference Patterson map on the Harker section v = 0. Analysis of the self-rotation function indicates the presence of a twofold non-crystallographic symmetry axis approximately along a. The calculated Matthews coefficient is 1.9 Å{sup 3} Da{sup −1} for two molecules per asymmetric unit, corresponding to a solvent content of 33%.

  15. Linkage of TATA-binding protein and proteasome subunit C5 genes in mice and humans reveals synteny conserved between mammals and invertebrates.

    PubMed

    Trachtulec, Z; Hamvas, R M; Forejt, J; Lehrach, H R; Vincek, V; Klein, J

    1997-08-15

    The TATA-binding protein (TBP) is a factor required for the transcription of all classes of eukaryotic genes. Here, we demonstrate that in the mouse the TBP-encoding gene (Tbp) resides next to the proteasomal subunit C5-encoding gene (Psmb1). The genes are located on mouse chromosome 17 in the t complex within the Hybrid sterility 1 (Hst1) region. We demonstrate that the homologous human genes (TBP AND PSMB1) are tightly linked on the long arm of chromosome 6, in a region syntenic with the proximal part of mouse chromosome 17. The mouse Tbp and Psmb1 and the human TBP and PSMB1 genes are transcribed in the opposite orientation. The TATA-binding protein and proteasomal subunit C5 genes are also linked on chromosome III of Caenorhabditis elegans, and together they are linked to other genes whose homologs map to human chromosome 6 and mouse chromosome 17. In the Drosophila genome, the housekeeping TATA-binding protein gene maps close to two other genes with homologs in the mammalian major histocompatibility complex. There thus exists conserved synteny of unrelated genes between mammals and invertebrates. PMID:9286694

  16. Tandem Subunits Effectively Constrain GABAA Receptor Stoichiometry and Recapitulate Receptor Kinetics But Are Insensitive to GABAA Receptor-Associated Protein

    PubMed Central

    Boileau, Andrew J.; Pearce, Robert A.; Czajkowski, Cynthia

    2008-01-01

    GABAergic synapses likely contain multiple GABAA receptor subtypes, making postsynaptic currents difficult to dissect. However, even in heterologous expression systems, analysis of receptors composed of α, β, and γ subunits can be confounded by receptors expressed from α and β subunits alone. To produce recombinant GABAA receptors containing fixed subunit stoichiometry, we coexpressed individual subunits with a “tandem” α1 subunit linked to a β2 subunit. Cotransfection of the γ2 subunit with αβ-tandem subunits in human embryonic kidney 293 cells produced currents that were similar in their macroscopic kinetics, single-channel amplitudes, and pharmacology to overexpression of the γ subunit with nonlinked α1 and β2 subunits. Similarly, expression of α subunits together with αβ-tandem subunits produced receptors having physiological and pharmacological characteristics that closely matched cotransfection of α with β subunits. In this first description of tandem GABAA subunits measured with patch-clamp and rapid agonist application techniques, we conclude that incorporation of αβ-tandem subunits can be used to fix stoichiometry and to establish the intrinsic kinetic properties of α1β2 and α1β2γ2 receptors. We used this method to test whether the accessory protein GABAA receptor-associated protein (GABARAP) alters GABAA receptor properties directly or influences subunit composition. In recombinant receptors with fixed stoichiometry, coexpression of GABARAP-enhanced green fluorescent protein (EGFP) fusion protein had no effect on desensitization, deactivation, or diazepam potentiation of GABA-mediated currents. However, in α1β2γ2S transfections in which stoichiometry was not fixed, GABARAP-EGFP altered desensitization, deactivation, and diazepam potentiation of GABA-mediated currents. The data suggest that GABARAP does not alter receptor kinetics directly but by facilitating surface expression of αβγ receptors. PMID:16339017

  17. Assembly of in Vitro-Synthesized Large Subunits into Ribulose Bisphosphate Carboxylase/Oxygenase Is Sensitive to CI-, Requires ATP, and Does Not Proceed When Large Subunits Are Synthesized at Temperatures [greater than or equal to]32[deg]C.

    PubMed Central

    Hubbs, A. E.; Roy, H.

    1993-01-01

    In higher plants, ribulose bisphosphate carboxylase/oxygenase (Rubisco) consists of eight large "L" subunits, synthesized in chloroplasts, and eight small "S" subunits, synthesized as precursors in the cytosol. Assembly of these into holoenzyme occurs in the chloroplast stroma after import and processing of the S subunits. A chloroplast chaperonin interacts with the L subunits, which dissociate from the chaperonin before they assemble into holoenzyme. Our laboratory has reported L subunit assembly into Rubisco in chloroplast extracts after protein synthesis in leaves, intact chloroplasts, and most recently in membrane-free chloroplast extracts. We report here that the incorporation of in vitro-synthesized L subunits into holoenzyme depends on the conditions of L subunit synthesis. Rubisco assembly did not occur after L subunit synthesis at 160 mM KCI. When L subunit synthesis occurred at approximately 70 mM KCI, assembly depended on the temperature at which L subunit synthesis took place. These phenomena were the result of postsynthetic events taking place during incubation for protein synthesis. We separated these events from protein synthesis by lowering the temperature during protein synthesis. Lower temperatures supported the synthesis of full-length Rubisco L subunits. The assembly of these completed L subunits into Rubisco required intervening incubation with ATP, before addition of S subunits. ATP treatment mobilized L subunits from a complex with the chloroplast chaperonin 60 oligomer. Addition of 130 mM KCI at the beginning of the intervening incubation with ATP blocked the incorporation of L subunits into Rubisco. The inhibitory effect of high KCI was due to CI- and came after association of newly synthesized L subunits with chaperonin 60, but before S subunit addition. It is interesting that L subunits synthesized at [greater than or equal to]32[deg]C failed to assemble into Rubisco under any conditions. These results agree with previous results obtained

  18. Map projections for larger-scale mapping

    NASA Technical Reports Server (NTRS)

    Snyder, J. P.

    1982-01-01

    For the U.S. Geological Survey maps at 1:1,000,000-scale and larger, the most common projections are conformal, such as the Transverse Mercator and Lambert Conformal Conic. Projections for these scales should treat the Earth as an ellipsoid. In addition, the USGS has conceived and designed some new projections, including the Space Oblique Mercator, the first map projection designed to permit low-distortion mapping of the Earth from satellite imagery, continuously following the groundtrack. The USGS has programmed nearly all pertinent projection equations for inverse and forward calculations. These are used to plot maps or to transform coordinates from one projection to another. The projections in current use are described.

  19. The cyclope gene of Drosophila encodes a cytochrome c oxidase subunit VIc homolog.

    PubMed

    Szuplewski, S; Terracol, R

    2001-08-01

    Cytochrome c oxidase is the terminal enzyme of the mitochondrial electron transfer chain. In eukaryotes, the enzyme is composed of 3 mitochondrial DNA-encoded subunits and 7-10 (in mammals) nuclear DNA-encoded subunits. This enzyme has been extensively studied in mammals and yeast but, in Drosophila, very little is known and no mutant has been described so far. Here we report the genetic and molecular characterization of mutations in cyclope (cype) and the cloning of the gene encoding a cytochrome c oxidase subunit VIc homolog. cype is an essential gene whose mutations are lethal and show pleiotropic phenotypes. The 77-amino acid peptide encoded by cype is 46% identical and 59% similar to the human subunit (75 amino acids). The transcripts are expressed maternally and throughout development in localized regions. They are found predominantly in the central nervous system of the embryo; in the central region of imaginal discs; in the germarium, follicular, and nurse cells of the ovary; and in testis. A search in the Genome Annotation Database of Drosophila revealed the absence of subunit VIIb and the presence of 9 putative nuclear cytochrome c oxidase subunits with high identity scores when compared to the 10 human subunits. PMID:11514451

  20. The cyclope gene of Drosophila encodes a cytochrome c oxidase subunit VIc homolog.

    PubMed Central

    Szuplewski, S; Terracol, R

    2001-01-01

    Cytochrome c oxidase is the terminal enzyme of the mitochondrial electron transfer chain. In eukaryotes, the enzyme is composed of 3 mitochondrial DNA-encoded subunits and 7-10 (in mammals) nuclear DNA-encoded subunits. This enzyme has been extensively studied in mammals and yeast but, in Drosophila, very little is known and no mutant has been described so far. Here we report the genetic and molecular characterization of mutations in cyclope (cype) and the cloning of the gene encoding a cytochrome c oxidase subunit VIc homolog. cype is an essential gene whose mutations are lethal and show pleiotropic phenotypes. The 77-amino acid peptide encoded by cype is 46% identical and 59% similar to the human subunit (75 amino acids). The transcripts are expressed maternally and throughout development in localized regions. They are found predominantly in the central nervous system of the embryo; in the central region of imaginal discs; in the germarium, follicular, and nurse cells of the ovary; and in testis. A search in the Genome Annotation Database of Drosophila revealed the absence of subunit VIIb and the presence of 9 putative nuclear cytochrome c oxidase subunits with high identity scores when compared to the 10 human subunits. PMID:11514451

  1. Assembly of eIF3 Mediated by Mutually Dependent Subunit Insertion.

    PubMed

    Smith, M Duane; Arake-Tacca, Luisa; Nitido, Adam; Montabana, Elizabeth; Park, Annsea; Cate, Jamie H

    2016-06-01

    Eukaryotic initiation factor 3 (eIF3), an essential multi-protein complex involved in translation initiation, is composed of 12 tightly associated subunits in humans. While the overall structure of eIF3 is known, the mechanism of its assembly and structural consequences of dysregulation of eIF3 subunit expression seen in many cancers is largely unknown. Here we show that subunits in eIF3 assemble into eIF3 in an interdependent manner. Assembly of eIF3 is governed primarily by formation of a helical bundle, composed of helices extending C-terminally from PCI-MPN domains in eight subunits. We propose that, while the minimal subcomplex of human-like eIF3 functional for translation initiation in cells consists of subunits a, b, c, f, g, i, and m, numerous other eIF3 subcomplexes exist under circumstances of subunit over- or underexpression. Thus, eIF3 subcomplexes formed or "released" due to dysregulated subunit expression may be determining factors contributing to eIF3-related cancers. PMID:27210288

  2. Effects of subunit types of the recombinant GABAA receptor on the response to a neurosteroid.

    PubMed

    Zaman, S H; Shingai, R; Harvey, R J; Darlison, M G; Barnard, E A

    1992-04-10

    When vertebrate brain poly(A)+ RNA is expressed in Xenopus oocytes the response of the GABA receptors formed is found to be inhibited allosterically by a neurosteroid, pregnenolone sulphate (PS). This negative modulation was reproduced after expressing RNAs encoding bovine GABAA receptor subunits in the combinations alpha i + beta 1, or alpha i + beta 1 + gamma 2 (where i = 1, 2 or 3). The characteristics of this inhibition vary significantly with the type of the alpha subunit (alpha 1, alpha 2, or alpha 3) used. When the bovine gamma 2L alternate form of the gamma 2 subunit was replaced by the human gamma 2S subunit, the behaviour was unchanged: the human gamma 2S subunit used is a newly-cloned form, which encodes a polypeptide with two amino acid differences from the human gamma 2 subunit previously described. The results of co-application of PS and 3 alpha-hydroxy-5 alpha-pregnan-ol-20-one, a neurosteroid which is a positive modulator of the GABAA receptor, indicate that these act at different sites on the receptor. PS also increases the desensitisation of the receptor by GABA. This effect, also, is alpha-subunit-type dependent and occurs by an acceleration of the fast phase of desensitisation. PMID:1323476

  3. Kv5, Kv6, Kv8, and Kv9 subunits: No simple silent bystanders

    PubMed Central

    2016-01-01

    Members of the electrically silent voltage-gated K+ (Kv) subfamilies (Kv5, Kv6, Kv8, and Kv9, collectively identified as electrically silent voltage-gated K+ channel [KvS] subunits) do not form functional homotetrameric channels but assemble with Kv2 subunits into heterotetrameric Kv2/KvS channels with unique biophysical properties. Unlike the ubiquitously expressed Kv2 subunits, KvS subunits show a more restricted expression. This raises the possibility that Kv2/KvS heterotetramers have tissue-specific functions, making them potential targets for the development of novel therapeutic strategies. Here, I provide an overview of the expression of KvS subunits in different tissues and discuss their proposed role in various physiological and pathophysiological processes. This overview demonstrates the importance of KvS subunits and Kv2/KvS heterotetramers in vivo and the importance of considering KvS subunits and Kv2/KvS heterotetramers in the development of novel treatments. PMID:26755771

  4. Different patterns of nicotinic acetylcholine receptor subunit transcription in human thymus.

    PubMed

    Bruno, Roxana; Sabater, Lidia; Tolosa, Eva; Sospedra, Mireia; Ferrer-Francesch, Xavier; Coll, Jaume; Foz, Marius; Melms, Arthur; Pujol-Borrell, Ricardo

    2004-04-01

    Clinical observations suggest that the thymus is strongly implicated in the pathogenesis of myasthenia gravis (MG), but questions such as the level and location of nicotinic acetylcholine receptor (AChR) subunit expression that are fundamental to postulate any pathogenic mechanism, remain controversial. We have re-examined this question by combining calibrated RT-PCR and real-time PCR to study nicotinic AChR subunit mRNA expression in a panel of normal and myasthenic thymi. The results suggest that the expression of the different AChR subunits follows three distinct patterns: constitutive for, neonatal for gamma and individually variable for alpha1, beta1 and delta. Experiments using confocal laser microdissection suggest that AChR is mainly expressed in the medullary compartment of the thymus but there is not a clear compartmentalization of subunit expression. The different patterns of subunit expression may influence decisively the level of central tolerance to the subunits and explain the focusing of the T cell response to the alpha and gamma subunits. PMID:15020075

  5. Structural and spectroscopic studies of the native hemocyanin from Maia squinado and its structural subunits

    NASA Astrophysics Data System (ADS)

    Dolashka-Angelova, Pavlina; Hristova, Rumijana; Schuetz, Juergen; Stoeva, Stanka; Schwarz, Heinz; Voelter, Wolfgang

    2000-09-01

    The dodecameric hemocyanin of the crab Maia squinado contains five major electrophoretically separable polypeptide chains (structural subunits) which have been purified by FPLC ion exchange chromatography. The various proteins have been characterized by fluorescence spectroscopy, combined with fluorescence quenching studies, using acrylamide, caesium chloride and potassium iodide as tryptophan quenchers. The results show that the tryptophyl side chains of dodecameric Hc are deeply buried in hydrophobic regions of the hemocyanin aggregates and the quenching efficiency values for the native Hc in comparison with those from the constituent subunits are two to four times less. The conformational stabilities of the native dodecameric aggregate and its isolated structural subunits towards various denaturants (pH, temperature, guanidinium hydrochloride) indicate that the quaternary structure is stabilized by hydrophilic and polar forces, whereby, both, the oxy- and apo-forms of the protein have been considered. The critical temperatures for the structural subunits, Tc, determined by fluorescence spectroscopy, are in the region of 50-60°C, coinciding with the melting temperatures, Tm, determined by CD spectroscopy. The free energy of stabilization in water, Δ GDH 2O , toward guanidinium hydrochloride is about two times higher for the dodecamer as compared to the isolated subunits. These studies reveal that oligomerization between functional subunits has a stabilizing effect on the whole molecule and differences in the primary structures result in different stabilities of the subunits.

  6. Alpha-7 and alpha-4 nicotinic receptor subunit immunoreactivity in genioglossus muscle motoneurons.

    PubMed

    Dehkordi, Ozra; Millis, Richard M; Dennis, Gary C; Coleman, Bernell R; Johnson, Sheree M; Changizi, Loubat; Ovid Trouth, C

    2005-02-15

    In the present study, immunohistochemistry combined with retrograde labeling techniques were used to determine if hypoglossal motoneurons (HMNs), retrogradely labeled after cholera toxin B subunit (CTB) injection to the genioglossus muscle in rats, show immunoreactivity for alpha-7 and alpha-4 subunits of nicotinic acetylcholine receptors (nAChRs). CTB-positive HMNs projecting to the genioglossus muscle were consistently labeled throughout the rostrocaudal extent of the hypoglossal nuclei with the greatest labeling at and caudal to area postrema. Alpha-7 subunit immunoreactivity was found in 39.44+/-5.10% of 870 CTB-labeled motoneurons and the alpha-4 subunit in 51.01+/-3.71% of 983 CTB-positive neurons. Rostrally, the number of genioglossal motoneurons demonstrating immunoreactivity for the alpha-7 subunit was 45.85+/-10.04% compared to 34.96+/-5.11% at and caudal to area postrema (P>0.1). The number of genioglossal motoneurons that showed immunoreactivity for the alpha-4 subunit was 55.03+/-4.83% at and caudal to area postrema compared to 42.98+/-3.90% in rostral areas (P=0.074). These results demonstrate that nAChR immunoreactivity is present in genioglossal motoneurons and suggest a role for alpha-7 and alpha-4 subunits containing nAChRs in the regulation of upper airway patency. PMID:15705531

  7. Tuning the Biological Activity Profile of Antibacterial Polymers via Subunit Substitution Pattern

    PubMed Central

    2015-01-01

    Binary nylon-3 copolymers containing cationic and hydrophobic subunits can mimic the biological properties of host-defense peptides, but relationships between composition and activity are not yet well understood for these materials. Hydrophobic subunits in previously studied examples have been limited mostly to cycloalkane-derived structures, with cyclohexyl proving to be particularly promising. The present study evaluates alternative hydrophobic subunits that are isomeric or nearly isomeric with the cyclohexyl example; each has four sp3 carbons in the side chains. The results show that varying the substitution pattern of the hydrophobic subunit leads to relatively small changes in antibacterial activity but causes significant changes in hemolytic activity. We hypothesize that these differences in biological activity profile arise, at least in part, from variations among the conformational propensities of the hydrophobic subunits. The α,α,β,β-tetramethyl unit is optimal among the subunits we have examined, providing copolymers with potent antibacterial activity and excellent prokaryote vs eukaryote selectivity. Bacteria do not readily develop resistance to the new antibacterial nylon-3 copolymers. These findings suggest that variation in subunit conformational properties could be generally valuable in the development of synthetic polymers for biological applications. PMID:24601599

  8. A Chaperonin Subunit with Unique Structures Is Essential for Folding of a Specific Substrate

    PubMed Central

    Peng, Lianwei; Fukao, Yoichiro; Myouga, Fumiyoshi; Motohashi, Reiko; Shinozaki, Kazuo; Shikanai, Toshiharu

    2011-01-01

    Type I chaperonins are large, double-ring complexes present in bacteria (GroEL), mitochondria (Hsp60), and chloroplasts (Cpn60), which are involved in mediating the folding of newly synthesized, translocated, or stress-denatured proteins. In Escherichia coli, GroEL comprises 14 identical subunits and has been exquisitely optimized to fold its broad range of substrates. However, multiple Cpn60 subunits with different expression profiles have evolved in chloroplasts. Here, we show that, in Arabidopsis thaliana, the minor subunit Cpn60β4 forms a heterooligomeric Cpn60 complex with Cpn60α1 and Cpn60β1–β3 and is specifically required for the folding of NdhH, a subunit of the chloroplast NADH dehydrogenase-like complex (NDH). Other Cpn60β subunits cannot complement the function of Cpn60β4. Furthermore, the unique C-terminus of Cpn60β4 is required for the full activity of the unique Cpn60 complex containing Cpn60β4 for folding of NdhH. Our findings suggest that this unusual kind of subunit enables the Cpn60 complex to assist the folding of some particular substrates, whereas other dominant Cpn60 subunits maintain a housekeeping chaperonin function by facilitating the folding of other obligate substrates. PMID:21483722

  9. Cloning and mapping of murine Nfe2L1

    SciTech Connect

    McKie, J.; Johnstone, K.; Scambler, P.

    1995-02-10

    The murine homologue of the human NFE2L1 basic leucine-zipper gene was isolated from an early embryo library. The deduced amino acid sequence shows 97% identity between the two proteins. Significant sequence similarity is also seen with the p45 subunit of NF-E2 and with the Drosophila CNC protein. Murine Nfe2l1 maps to chromosome 11DE with similar sequences at 7D1-7F1 and 2E4-2G. 14 refs., 2 figs.

  10. Cartographic mapping study

    NASA Technical Reports Server (NTRS)

    Wilson, C.; Dye, R.; Reed, L.

    1982-01-01

    The errors associated with planimetric mapping of the United States using satellite remote sensing techniques are analyzed. Assumptions concerning the state of the art achievable for satellite mapping systems and platforms in the 1995 time frame are made. An analysis of these performance parameters is made using an interactive cartographic satellite computer model, after first validating the model using LANDSAT 1 through 3 performance parameters. An investigation of current large scale (1:24,000) US National mapping techniques is made. Using the results of this investigation, and current national mapping accuracy standards, the 1995 satellite mapping system is evaluated for its ability to meet US mapping standards for planimetric and topographic mapping at scales of 1:24,000 and smaller.

  11. On genetic map functions

    SciTech Connect

    Zhao, Hongyu; Speed, T.P.

    1996-04-01

    Various genetic map functions have been proposed to infer the unobservable genetic distance between two loci from the observable recombination fraction between them. Some map functions were found to fit data better than others. When there are more than three markers, multilocus recombination probabilities cannot be uniquely determined by the defining property of map functions, and different methods have been proposed to permit the use of map functions to analyze multilocus data. If for a given map function, there is a probability model for recombination that can give rise to it, then joint recombination probabilities can be deduced from this model. This provides another way to use map functions in multilocus analysis. In this paper we show that stationary renewal processes give rise to most of the map functions in the literature. Furthermore, we show that the interevent distributions of these renewal processes can all be approximated quite well by gamma distributions. 43 refs., 4 figs.

  12. A novel form of 6-phosphofructokinase. Identification and functional relevance of a third type of subunit in Pichia pastoris.

    PubMed

    Tanneberger, Katrin; Kirchberger, Jürgen; Bär, Jörg; Schellenberger, Wolfgang; Rothemund, Sven; Kamprad, Manja; Otto, Henning; Schöneberg, Torsten; Edelmann, Anke

    2007-08-10

    Classically, 6-phosphofructokinases are homo- and hetero-oligomeric enzymes consisting of alpha subunits and alpha/beta subunits, respectively. Herein, we describe a new form of 6-phosphofructokinase (Pfk) present in several Pichia species, which is composed of three different types of subunit, alpha, beta, and gamma. The sequence of the gamma subunit shows no similarity to classic Pfk subunits or to other known protein sequences. In-depth structural and functional studies revealed that the gamma subunit is a constitutive component of Pfk from Pichia pastoris (PpPfk). Analyses of the purified PpPfk suggest a heterododecameric assembly from the three different subunits. Accordingly, it is the largest and most complex Pfk identified yet. Although, the gamma subunit is not required for enzymatic activity, the gamma subunit-deficient mutant displays a decreased growth on nutrient limitation and reduced cell flocculation when compared with the P. pastoris wild-type strain. Subsequent characterization of purified Pfks from wild-type and gamma subunit-deficient strains revealed that the allosteric regulation of the PpPfk by ATP, fructose 2,6-bisphosphate, and AMP is fine-tuned by the gamma subunit. Therefore, we suggest that the gamma subunit contributes to adaptation of P. pastoris to energy resources. PMID:17522059

  13. Exact Length Distribution of Filamentous Structures Assembled from a Finite Pool of Subunits.

    PubMed

    Harbage, David; Kondev, Jané

    2016-07-01

    Self-assembling filamentous structures made of protein subunits are ubiquitous in cell biology. These structures are often highly dynamic, with subunits in a continuous state of flux, binding to and falling off of filaments. In spite of this constant turnover of their molecular parts, many cellular structures seem to maintain a well-defined size over time, which is often required for their proper functioning. One widely discussed mechanism of size regulation involves the cell maintaining a finite pool of protein subunits available for assembly. This finite pool mechanism can control the length of a single filament by having assembly proceed until the pool of free subunits is depleted to the point when assembly and disassembly are balanced. Still, this leaves open the question of whether the same mechanism can provide size control for multiple filamentous structures that are assembled from a common pool of protein subunits, as is often the case in cells. We address this question by solving the steady-state master equation governing the stochastic assembly and disassembly of multifilament structures made from a shared finite pool of subunits. We find that, while the total number of subunits within a multifilament structure is well-defined, individual filaments within the structure have a wide, power-law distribution of lengths. We also compute the phase diagram for two multifilament structures competing for the same pool of subunits and identify conditions for coexistence when both have a well-defined size. These predictions can be tested in cell experiments in which the size of the subunit pool or the number of filament nucleators is tuned. PMID:27135597

  14. ATP hydrolysis catalyzed by human replication factor C requires participation of multiple subunits.

    PubMed

    Cai, J; Yao, N; Gibbs, E; Finkelstein, J; Phillips, B; O'Donnell, M; Hurwitz, J

    1998-09-29

    Human replication factor C (hRFC) is a five-subunit protein complex (p140, p40, p38, p37, and p36) that acts to catalytically load proliferating cell nuclear antigen onto DNA, where it recruits DNA polymerase delta or epsilon to the primer terminus at the expense of ATP, leading to processive DNA synthesis. We have previously shown that a subcomplex of hRFC consisting of three subunits (p40, p37, and p36) contained DNA-dependent ATPase activity. However, it is not clear which subunit(s) hydrolyzes ATP, as all five subunits include potential ATP binding sites. In this report, we introduced point mutations in the putative ATP-binding sequences of each hRFC subunit and examined the properties of the resulting mutant hRFC complex and the ATPase activity of the hRFC or the p40.p37.p36 complex. A mutation in any one of the ATP binding sites of the p36, p37, p40, or p140 subunits markedly reduced replication activity of the hRFC complex and the ATPase activity of the hRFC or the p40.p37.p36 complex. A mutation in the ATP binding site of the p38 subunit did not alter the replication activity of hRFC. These findings indicate that the replication activity of hRFC is dependent on efficient ATP hydrolysis contributed to by the action of four hRFC subunits. PMID:9751713

  15. Localization of Aggregatibacter actinomycetemcomitans Cytolethal Distending Toxin Subunits during Intoxication of Live Cells

    PubMed Central

    Damek-Poprawa, Monika; Jang, Jae Yeon; Volgina, Alla; Korostoff, Jonathan

    2012-01-01

    The cytolethal distending toxin (Cdt), produced by some clinically important Gram-negative bacterial species, is related to the family of AB-type toxins. Three heterologous proteins (CdtA, CdtB, and CdtC) and a genotoxin mode of action distinguish the Cdt from others in this toxin class. Crystal structures of several species-specific Cdts have provided a basis for predicting subunit interactions and functions. In addition, empirical studies have yielded significant insights into the in vivo interactions of the Cdt subunits. However, there are still critical gaps in information about the intoxication process. In this study, a novel protein tagging technology was used to localize the subunits in Chinese hamster ovary cells (CHO-K1). A tetracysteine motif was engineered in each subunit, and in subunits with mutations in predicted functional domains, to permit detection with the fluorescein arsenical hairpin binding (FlAsH) dye Lumio green. Live-cell imaging, in conjunction with confocal microscopy, was used to capture the locations of the individual subunits in cells intoxicated, under various conditions, with hybrid heterotrimers. Using this approach, we observed the following. (i) The CdtA subunit remains on the cell surface of CHO cells in association with cholesterol-containing and cholesterol-depleted membrane. (ii) The CdtB subunit is exclusively in the cytosol and, after longer exposure times, localizes to the nucleus. (iii) The CdtC subunit is present on the cell surface and, to a greater extent, in the cytosol. These observations suggest that CdtC, but not CdtA, functions as a chaperone for CdtB entry into cells. PMID:22645284

  16. Subunit Conformations and Assembly States of a DNA Translocating Motor: The Terminase of Bacteriophage P22

    PubMed Central

    Němeček, Daniel; Gilcrease, Eddie B.; Kang, Sebyung; Prevelige, Peter E.; Casjens, Sherwood; Thomas, George J.

    2007-01-01

    Bacteriophage P22, a podovirus infecting strains of Salmonella typhimurium, packages a 42 kbp genome using a headful mechanism. DNA translocation is accomplished by the phage terminase, a powerful molecular motor consisting of large and small subunits. Although many of the structural proteins of the P22 virion have been well characterized, little is known about the terminase subunits and their molecular mechanism of DNA translocation. We report here structural and assembly properties of ectopically expressed and highly purified terminase large and small subunits. The large subunit (gp2), which contains the nuclease and ATPase activities of terminase, exists as a stable monomer with an α/β fold. The small subunit (gp3), which recognizes DNA for packaging and may regulate gp2 activity, exhibits a highly α-helical secondary structure and self-associates to form a stable oligomeric ring in solution. For wildtype gp3, the ring contains nine subunits, as demonstrated by hydrodynamic measurements, electron microscopy and native mass spectrometry. We have also characterized a gp3 mutant (Ala 112 → Thr) that forms a ten subunit ring, despite a subunit fold indistinguishable from wildtype. Both the nonameric and decameric gp3 rings exhibit nonspecific DNA binding activity, and gp2 is able to bind strongly to the DNA/gp3 complex but not to DNA alone. We propose a scheme for the roles of P22 terminase large and small subunits in the recruitment and packaging of viral DNA and discuss the model in relation to proposals for terminase-driven DNA translocation in other phages. PMID:17945256

  17. An alternating GluN1-2-1-2 subunit arrangement in mature NMDA receptors.

    PubMed

    Riou, Morgane; Stroebel, David; Edwardson, J Michael; Paoletti, Pierre

    2012-01-01

    NMDA receptors (NMDARs) form glutamate-gated ion channels that play a critical role in CNS physiology and pathology. Together with AMPA and kainate receptors, NMDARs are known to operate as tetrameric complexes with four membrane-embedded subunits associating to form a single central ion-conducting pore. While AMPA and some kainate receptors can function as homomers, NMDARs are obligatory heteromers composed of homologous but distinct subunits, most usually of the GluN1 and GluN2 types. A fundamental structural feature of NMDARs, that of the subunit arrangement around the ion pore, is still controversial. Thus, in a typical NMDAR associating two GluN1 and two GluN2 subunits, there is evidence for both alternating 1/2/1/2 and non-alternating 1/1/2/2 arrangements. Here, using a combination of electrophysiological and cross-linking experiments, we provide evidence that functional GluN1/GluN2A receptors adopt the 1/2/1/2 arrangement in which like subunits are diagonal to one another. Moreover, based on the recent crystal structure of an AMPA receptor, we show that in the agonist-binding and pore regions, the GluN1 subunits occupy a "proximal" position, closer to the central axis of the channel pore than that of GluN2 subunits. Finally, results obtained with reducing agents that differ in their membrane permeability indicate that immature (intracellular) and functional (plasma-membrane inserted) pools of NMDARs can adopt different subunit arrangements, thus stressing the importance of discriminating between the two receptor pools in assembly studies. Elucidating the quaternary arrangement of NMDARs helps to define the interface between the subunits and to understand the mechanism and pharmacology of these key signaling receptors. PMID:22493736

  18. Cooperative subunit interactions mediate fast C-type inactivation of hERG1 K+ channels.

    PubMed

    Wu, Wei; Gardner, Alison; Sanguinetti, Michael C

    2014-10-15

    At depolarized membrane potentials, the conductance of some voltage-gated K(+) channels is reduced by C-type inactivation. This gating process is voltage independent in Kv1 and involves a conformational change in the selectivity filter that is mediated by cooperative subunit interactions. C-type inactivation in hERG1 K(+) channels is voltage-dependent, much faster in onset and greatly attenuates currents at positive potentials. Here we investigate the potential role of subunit interactions in C-type inactivation of hERG1 channels. Point mutations in hERG1 known to eliminate (G628C/S631C), inhibit (S620T or S631A) or enhance (T618A or M645C) C-type inactivation were introduced into subunits that were combined with wild-type subunits to form concatenated tetrameric channels with defined subunit composition and stoichiometry. Channels were heterologously expressed in Xenopus oocytes and the two-microelectrode voltage clamp was used to measure the kinetics and steady-state properties of inactivation of whole cell currents. The effect of S631A or T618A mutations on inactivation was a graded function of the number of mutant subunits within a concatenated tetramer as predicted by a sequential model of cooperative subunit interactions, whereas M645C subunits increased the rate of inactivation of concatemers, as predicted for subunits that act independently of one another. For mutations located within the inactivation gate proper (S620T or G628C/S631C), the presence of a single subunit in a concatenated hERG1 tetramer disrupted gating to the same extent as that observed for mutant homotetramers. Together, our findings indicate that the final step of C-type inactivation of hERG1 channels involves a concerted, all-or-none cooperative interaction between all four subunits, and that probing the mechanisms of channel gating with concatenated heterotypic channels should be interpreted with care, as conclusions regarding the nature of subunit interactions may depend on the

  19. Cooperative subunit interactions mediate fast C-type inactivation of hERG1 K+ channels

    PubMed Central

    Wu, Wei; Gardner, Alison; Sanguinetti, Michael C

    2014-01-01

    At depolarized membrane potentials, the conductance of some voltage-gated K+ channels is reduced by C-type inactivation. This gating process is voltage independent in Kv1 and involves a conformational change in the selectivity filter that is mediated by cooperative subunit interactions. C-type inactivation in hERG1 K+ channels is voltage-dependent, much faster in onset and greatly attenuates currents at positive potentials. Here we investigate the potential role of subunit interactions in C-type inactivation of hERG1 channels. Point mutations in hERG1 known to eliminate (G628C/S631C), inhibit (S620T or S631A) or enhance (T618A or M645C) C-type inactivation were introduced into subunits that were combined with wild-type subunits to form concatenated tetrameric channels with defined subunit composition and stoichiometry. Channels were heterologously expressed in Xenopus oocytes and the two-microelectrode voltage clamp was used to measure the kinetics and steady-state properties of inactivation of whole cell currents. The effect of S631A or T618A mutations on inactivation was a graded function of the number of mutant subunits within a concatenated tetramer as predicted by a sequential model of cooperative subunit interactions, whereas M645C subunits increased the rate of inactivation of concatemers, as predicted for subunits that act independently of one another. For mutations located within the inactivation gate proper (S620T or G628C/S631C), the presence of a single subunit in a concatenated hERG1 tetramer disrupted gating to the same extent as that observed for mutant homotetramers. Together, our findings indicate that the final step of C-type inactivation of hERG1 channels involves a concerted, all-or-none cooperative interaction between all four subunits, and that probing the mechanisms of channel gating with concatenated heterotypic channels should be interpreted with care, as conclusions regarding the nature of subunit interactions may depend on the specific

  20. Molecular cloning of the mouse proteasome subunits MC14 and MECL-1: reciprocally regulated tissue expression of interferon-gamma-modulated proteasome subunits.

    PubMed

    Stohwasser, R; Standera, S; Peters, I; Kloetzel, P M; Groettrup, M

    1997-05-01

    The primary structures of the interferon-gamma-inducible mouse 20S proteasome subunit MECL-1 and its alternate homolog MC14 were determined. Northern analysis of mouse tissues revealed that MECL-1 mRNA predominantly occurred in thymus, lymph nodes, and spleen, whereas small amounts were detected in non-lymphoid tissues such as kidney, muscle, and testis. Unexpectedly, probing RNA blots with MC14 showed that tissues with high MECL-1 expression contained little MC14 and vice versa. A very similar reciprocal tissue expression was subsequently found for the homologous subunit pairs LMP2 and delta as well as LMP7 and MB1. The subunit protein composition of 20S proteasomes purified from liver, thymus, and lung reflected RNA expression. The impact of a regulated reciprocal tissue expression is discussed with respect to thymic selection and the induction of tolerance in potentially autoreactive T cells. PMID:9174609

  1. Solution NMR and calorimetric analysis of Rem2 binding to the Ca2+ channel β4 subunit: a low affinity interaction is required for inhibition of Cav2.1 Ca2+ currents.

    PubMed

    Xu, Xingfu; Zhang, Fangxiong; Zamponi, Gerald W; Horne, William A

    2015-05-01

    Rem, Rad, Kir/Gem (RGK) proteins, including Rem2, mediate profound inhibition of high-voltage activated Ca(2+) channels containing intracellular regulatory β subunits. All RGK proteins bind to voltage-gated Ca(2+) channel β subunit (Cavβ) subunits in vitro, but the necessity of the interaction for current inhibition remains controversial. This study applies NMR and calorimetric techniques to map the binding site for Rem2 on human Cavβ4a and measure its binding affinity. Our experiments revealed 2 binding surfaces on the β4 guanylate kinase domain contributing to a 156 ± 18 µM Kd interaction: a hydrophobic pocket lined by 4 critical residues (L173, N261, H262, and V303), mutation of any of which completely disrupted binding, and a nearby surface containing 3 residues (D206, L209, and D258) that when individually mutated decreased affinity. Voltage-gated Ca(2+) channel α1A subunit (Cav2.1) Ca(2+) currents were completely inhibited by Rem2 when co-expressed with wild-type Cavβ4a, but were unaffected by Rem2 when coexpressed with a Cavβ4a site 1 (L173A/V303A) or site 2 (D258A) mutant. These results provide direct evidence for a low-affinity Rem2/Cavβ4 interaction and show definitively that the interaction is required for Cav2.1 inhibition. PMID:25563298

  2. Prestin forms oligomer with four mechanically independent subunits

    PubMed Central

    Wang, Xiang; Yang, Shiming; Jia, Shuping; He, David Z.Z.

    2010-01-01

    Prestin is the motor protein of cochlear outer hair cells (OHCs) with the unique capability of performing direct, rapid and reciprocal electromechanical conversion. Prestin consists of 744 amino acids with a molecular mass of ~81.4 kDa. The predicted membrane topology and molecular mass of a single prestin molecule appear inadequate to account for the size of intramembrane particles (IMPs) expressed in the OHC membrane. Although recent biochemical evidence suggests that prestin forms homo-oligomers, most likely as a tetramer, the oligomeric structure of prestin in OHCs remains unclear. We obtained the charge density of prestin in the gerbil OHCs by measuring their nonlinear capacitance (NLC). The average charge density (22,608 μm−2) measured was four times the average IMP density (5,686 μm−2) reported in the freeze-fracture study. This suggests that each IMP contains four prestin molecules, based on the general notion that each prestin transfers a single elementary charge. We subsequently compared the voltage dependency and the values of slope factor of NLC and somatic motility simultaneously measured from the same OHCs to determine whether NLC and motility are fully coupled and how prestin subunits function within the tetramer. We showed that the voltage dependency and slope factors of NLC and motility were not statistically different, suggesting that NLC and motility are fully coupled. The fact that the slope factor is the same between NLC and motility suggests that each prestin monomer in the tetramer is in parallel, each interacting independently with cytoplasmic or other partners to facilitate the mechanical response. PMID:20347723

  3. P. berghei Telomerase Subunit TERT is Essential for Parasite Survival

    PubMed Central

    Religa, Agnieszka A.; Ramesar, Jai; Janse, Chris J.; Scherf, Artur; Waters, Andrew P.

    2014-01-01

    Telomeres define the ends of chromosomes protecting eukaryotic cells from chromosome instability and eventual cell death. The complex regulation of telomeres involves various proteins including telomerase, which is a specialized ribonucleoprotein responsible for telomere maintenance. Telomeres of chromosomes of malaria parasites are kept at a constant length during blood stage proliferation. The 7-bp telomere repeat sequence is universal across different Plasmodium species (GGGTTT/CA), though the average telomere length varies. The catalytic subunit of telomerase, telomerase reverse transcriptase (TERT), is present in all sequenced Plasmodium species and is approximately three times larger than other eukaryotic TERTs. The Plasmodium RNA component of TERT has recently been identified in silico. A strategy to delete the gene encoding TERT via double cross-over (DXO) homologous recombination was undertaken to study the telomerase function in P. berghei. Expression of both TERT and the RNA component (TR) in P. berghei blood stages was analysed by Western blotting and Northern analysis. Average telomere length was measured in several Plasmodium species using Telomere Restriction Fragment (TRF) analysis. TERT and TR were detected in blood stages and an average telomere length of ∼950 bp established. Deletion of the tert gene was performed using standard transfection methodologies and we show the presence of tert− mutants in the transfected parasite populations. Cloning of tert- mutants has been attempted multiple times without success. Thorough analysis of the transfected parasite populations and the parasite obtained from extensive parasite cloning from these populations provide evidence for a so called delayed death phenotype as observed in different organisms lacking TERT. The findings indicate that TERT is essential for P. berghei cell survival. The study extends our current knowledge on telomere biology in malaria parasites and validates further investigations

  4. Development of inhibitors of heterotrimeric Gαi subunits

    PubMed Central

    Appleton, Kathryn M.; Bigham, Kevin J.; Lindsey, Christopher C.; Hazard, Starr; Lirjoni, Jonel; Parnham, Stuart; Hennig, Mirko; Peterson, Yuri K.

    2014-01-01

    Heterotrimeric G-proteins are the immediate downstream effectors of G-protein coupled receptors (GPCRs). Endogenous protein guanine nucleotide dissociation inhibitors (GDIs) like AGS3/4 and RGS12/14 function through GPR/Goloco GDI domains. Extensive characterization of GPR domain peptides indicate they function as selective GDIs for Gαi by competing for the GPCR and Gβγ and preventing GDP release. We modified a GPR consensus peptide by testing FGF and TAT leader sequences to make the peptide cell permeable. FGF modification inhibited GDI activity while TAT preserved GDI activity. TAT-GPR suppresses G-protein coupling to the receptor and completely blocked α2-adrenoceptor (α2AR) mediated decreases in cAMP in HEK293 cells at 100 nM. We then sought to discover selective small molecule inhibitors for Gαi. Molecular docking was used to identify potential molecules that bind to and stabilize the Gαi–GDP complex by directly interacting with both Gαi and GDP. Gαi–GTP and Gαq-GDP were used as a computational counter screen and Gαq-GDP was used as a biological counter screen. Thirty-seven molecules were tested using nucleotide exchange. STD NMR assays with compound 0990, a quinazoline derivative, showed direct interaction with Gαi. Several compounds showed Gαi specific inhibition and were able to block α2AR mediated regulation of cAMP. In addition to being a pharmacologic tool, GDI inhibition of Gα subunits has the advantage of circumventing the upstream component of GPCR-related signaling in cases of overstimulation by agonists, mutations, polymorphisms, and expression-related defects often seen in disease. PMID:24818958

  5. Elongated Polyproline Motifs Facilitate Enamel Evolution through Matrix Subunit Compaction

    PubMed Central

    Luan, Xianghong; Dangaria, Smit; Walker, Cameron; Allen, Michael; Kulkarni, Ashok; Gibson, Carolyn; Braatz, Richard; Liao, Xiubei; Diekwisch, Thomas G. H.

    2009-01-01

    Vertebrate body designs rely on hydroxyapatite as the principal mineral component of relatively light-weight, articulated endoskeletons and sophisticated tooth-bearing jaws, facilitating rapid movement and efficient predation. Biological mineralization and skeletal growth are frequently accomplished through proteins containing polyproline repeat elements. Through their well-defined yet mobile and flexible structure polyproline-rich proteins control mineral shape and contribute many other biological functions including Alzheimer's amyloid aggregation and prolamine plant storage. In the present study we have hypothesized that polyproline repeat proteins exert their control over biological events such as mineral growth, plaque aggregation, or viscous adhesion by altering the length of their central repeat domain, resulting in dramatic changes in supramolecular assembly dimensions. In order to test our hypothesis, we have used the vertebrate mineralization protein amelogenin as an exemplar and determined the biological effect of the four-fold increased polyproline tandem repeat length in the amphibian/mammalian transition. To study the effect of polyproline repeat length on matrix assembly, protein structure, and apatite crystal growth, we have measured supramolecular assembly dimensions in various vertebrates using atomic force microscopy, tested the effect of protein assemblies on crystal growth by electron microscopy, generated a transgenic mouse model to examine the effect of an abbreviated polyproline sequence on crystal growth, and determined the structure of polyproline repeat elements using 3D NMR. Our study shows that an increase in PXX/PXQ tandem repeat motif length results (i) in a compaction of protein matrix subunit dimensions, (ii) reduced conformational variability, (iii) an increase in polyproline II helices, and (iv) promotion of apatite crystal length. Together, these findings establish a direct relationship between polyproline tandem repeat fragment

  6. Preclinical and clinical development of a dengue recombinant subunit vaccine.

    PubMed

    Manoff, Susan B; George, Sarah L; Bett, Andrew J; Yelmene, Michele L; Dhanasekaran, Govindarajan; Eggemeyer, Linda; Sausser, Michele L; Dubey, Sheri A; Casimiro, Danilo R; Clements, David E; Martyak, Timothy; Pai, Vidya; Parks, D Elliot; Coller, Beth-Ann G

    2015-12-10

    This review focuses on a dengue virus (DENV) vaccine candidate based on a recombinant subunit approach which targets the DENV envelope glycoprotein (E). Truncated versions of E consisting of the N-terminal portion of E (DEN-80E) have been expressed recombinantly in the Drosophila S2 expression system and shown to have native-like conformation. Preclinical studies demonstrate that formulations containing tetravalent DEN-80E adjuvanted with ISCOMATRIX™ adjuvant induce high titer virus neutralizing antibodies and IFN-γ producing T cells in flavivirus-naïve non-human primates. The preclinical data further suggest that administration of such formulations on a 0, 1, 6 month schedule may result in higher maximum virus neutralizing antibody titers and better durability of those titers compared to administration on a 0, 1, 2 month schedule. In addition, the virus neutralizing antibody titers induced by adjuvanted tetravalent DEN-80E compare favorably to the titers induced by a tetravalent live virus comparator. Furthermore, DEN-80E was demonstrated to be able to boost virus neutralizing antibody titers in macaques that have had a prior DENV exposure. A monovalent version of the vaccine candidate, DEN1-80E, was formulated with Alhydrogel™ and studied in a proof-of-principle Phase I clinical trial by Hawaii Biotech, Inc. (NCT00936429). The clinical trial results demonstrate that both the 10 μg and 50 μg formulations of DEN1-80E with 1.25 mg of elemental aluminum were immunogenic when administered in a 3-injection series (0, 1, 2 months) to healthy, flavivirus-naïve adults. The vaccine formulations induced DENV-1 neutralizing antibodies in the majority of subjects, although the titers in most subjects were modest and waned over time. Both the 10 μg DEN1-80E and the 50 μg DEN1-80E formulations with Alhydrogel™ were generally well tolerated. PMID:26458804

  7. Development of inhibitors of heterotrimeric Gαi subunits.

    PubMed

    Appleton, Kathryn M; Bigham, Kevin J; Lindsey, Christopher C; Hazard, Starr; Lirjoni, Jonel; Parnham, Stuart; Hennig, Mirko; Peterson, Yuri K

    2014-07-01

    Heterotrimeric G-proteins are the immediate downstream effectors of G-protein coupled receptors (GPCRs). Endogenous protein guanine nucleotide dissociation inhibitors (GDIs) like AGS3/4 and RGS12/14 function through GPR/Goloco GDI domains. Extensive characterization of GPR domain peptides indicate they function as selective GDIs for Gαi by competing for the GPCR and Gβγ and preventing GDP release. We modified a GPR consensus peptide by testing FGF and TAT leader sequences to make the peptide cell permeable. FGF modification inhibited GDI activity while TAT preserved GDI activity. TAT-GPR suppresses G-protein coupling to the receptor and completely blocked α2-adrenoceptor (α2AR) mediated decreases in cAMP in HEK293 cells at 100nM. We then sought to discover selective small molecule inhibitors for Gαi. Molecular docking was used to identify potential molecules that bind to and stabilize the Gαi-GDP complex by directly interacting with both Gαi and GDP. Gαi-GTP and Gαq-GDP were used as a computational counter screen and Gαq-GDP was used as a biological counter screen. Thirty-seven molecules were tested using nucleotide exchange. STD NMR assays with compound 0990, a quinazoline derivative, showed direct interaction with Gαi. Several compounds showed Gαi specific inhibition and were able to block α2AR mediated regulation of cAMP. In addition to being a pharmacologic tool, GDI inhibition of Gα subunits has the advantage of circumventing the upstream component of GPCR-related signaling in cases of overstimulation by agonists, mutations, polymorphisms, and expression-related defects often seen in disease. PMID:24818958

  8. Optimized polypeptide for a subunit vaccine against avian reovirus.

    PubMed

    Goldenberg, Dana; Lublin, Avishai; Rosenbluth, Ezra; Heller, E Dan; Pitcovski, Jacob

    2016-06-01

    Avian reovirus (ARV) is a disease-causing agent. The disease is prevented by vaccination with a genotype-specific vaccine while many variants of ARV exist in the field worldwide. Production of new attenuated vaccines is a long-term process and in the case of fast-mutating viruses, an impractical one. In the era of molecular biology, vaccines may be produced by using only the relevant protein for induction of neutralizing antibodies, enabling fast adjustment to the emergence of new genetic strains. Sigma C (SC) protein of ARV is a homotrimer that facilitates host-cell attachment and induce the production and secretion of neutralizing antibodies. The aim of this study was to identify the region of SC that will elicit a protective immune response. Full-length (residues 1-326) and two partial fragments of SC (residues 122-326 and 192-326) were produced in Escherichia coli. The SC fragment of residues 122-326 include the globular head, shaft and hinge domains, while eliminating intra-capsular region. This fragment induces significantly higher levels of anti-ARV antibodies than the shorter fragment or full length SC, which neutralized embryos infection by the virulent strain to a higher extent compared with the antibodies produced in response to the whole virus vaccine. Residues 122-326 fragment is assumed to be folded correctly, exposing linear as well as conformational epitopes that are identical to those of the native protein, while possibly excluding suppressor sequences. The results of this study may serve for the development of a recombinant subunit vaccine for ARV. PMID:27155492

  9. Use of affinity-directed liquid chromatography-mass spectrometry to map the epitopes of a factor VIII inhibitor antibody fraction

    PubMed Central

    Griffiths, Amy E.; Wang, Wensheng; Hagen, Fred K.; Fay, Philip J.

    2011-01-01

    Summary Background Neutralizing factor (F) VIII antibodies develop in ~30% of individuals with hemophilia A and show specificity to multiple sites in the FVIII protein. Methods Reactive epitopes to an immobilized IgG fraction prepared from a high-titer, FVIII inhibitor plasma were determined following immuno-precipitation (IP) of tryptic and chymotryptic peptides derived from digests of the A1 and A2 subunits of FVIIIa and FVIII light chain. Peptides were detected and identified using highly sensitive liquid chromatography-mass spectrometry (LC-MS). Results Coverage maps of the A1 subunit, A2 subunit and light chain represented 79%, 69% and 90%, respectively, of the protein sequences. Dot blots indicated that the inhibitor IgG reacted with epitopes contained within each subunit of FVIIIa. IP coupled with LC-MS identified 19 peptides representing epitopes from all FVIII A and C domains. The majority of peptides (10) were derived from the A2 domain. Three peptides mapped to the C2 domain, while two mapped to the A1 and A3 domains, and single peptides mapped to the a1 segment and C1 domain. Epitopes were typically defined by peptide sequences of <12 residues. Conclusions IP coupled with LC-MS identified extensive antibody reactivity at high resolution over the entire functional FVIII molecule and yielded sequence lengths of less than 15 residues. A number of the peptides identified mapped to known sequences involved in functionally important protein-protein and protein-membrane interactions. PMID:21668738

  10. Biosynthesis of the Torpedo californica Acetylcholine Receptor α Subunit in Yeast

    NASA Astrophysics Data System (ADS)

    Fujita, Norihisa; Nelson, Nathan; Fox, Thomas D.; Claudio, Toni; Lindstrom, Jon; Riezman, Howard; Hess, George P.

    1986-03-01

    Yeast cells were transformed with a plasmid containing complementary DNA encoding the α subunit of the Torpedo californica acetylcholine receptor. These cells synthesized a protein that had the expected molecular weight, antigenic specificity, and ligand-binding properties of the α subunit. The subunit was inserted into the yeast plasma membrane, demonstrating that yeast has the apparatus to express a membrane-bound receptor protein and to insert such a foreign protein into its plasma membrane. The α subunit constituted approximately 1 percent of the total yeast membrane proteins, and its density was about the same in the plasma membrane of yeast and in the receptor-rich electric organ of Electrophorus electricus. In view of the available technology for obtaining large quantities of yeast proteins, it may now be possible to obtain amplified amounts of interesting membrane-bound proteins for physical and biochemical studies.

  11. Is There an Optimal Formulation and Delivery Strategy for Subunit Vaccines?

    PubMed

    Bobbala, Sharan; Hook, Sarah

    2016-09-01

    Modern vaccine design has moved away from attenuated or inactivated whole-pathogen vaccines to more pure and defined subunit vaccines. However subunit antigens have poor bioavailability and stability and lack immunogenicity. To overcome these issues subunit vaccines have to be administered in a suitable delivery system in combination with immune stimulants. Many different delivery systems have been developed and investigated each having different modes of action, for example increasing delivery and/or sustaining delivery of antigen to immune cells. In addition a number of different routes of immunization are possible and these can play a crucial role in determining the fate of an immune response. In this review the different strategies for the delivery of prophylactic and therapeutic subunit vaccines along with the impact of these on the immune responses generated are discussed. PMID:27380191

  12. Localized reconstruction of subunits from electron cryomicroscopy images of macromolecular complexes.

    PubMed

    Ilca, Serban L; Kotecha, Abhay; Sun, Xiaoyu; Poranen, Minna M; Stuart, David I; Huiskonen, Juha T

    2015-01-01

    Electron cryomicroscopy can yield near-atomic resolution structures of highly ordered macromolecular complexes. Often however some subunits bind in a flexible manner, have different symmetry from the rest of the complex, or are present in sub-stoichiometric amounts, limiting the attainable resolution. Here we report a general method for the localized three-dimensional reconstruction of such subunits. After determining the particle orientations, local areas corresponding to the subunits can be extracted and treated as single particles. We demonstrate the method using three examples including a flexible assembly and complexes harbouring subunits with either partial occupancy or mismatched symmetry. Most notably, the method allows accurate fitting of the monomeric RNA-dependent RNA polymerase bound at the threefold axis of symmetry inside a viral capsid, revealing for the first time its exact orientation and interactions with the capsid proteins. Localized reconstruction is expected to provide novel biological insights in a range of challenging biological systems. PMID:26534841

  13. Complex regulation of γ-secretase: from obligatory to modulatory subunits

    PubMed Central

    Gertsik, Natalya; Chiu, Danica; Li, Yue-Ming

    2014-01-01

    γ-Secretase is a four subunit, 19-pass transmembrane enzyme that cleaves amyloid precursor protein (APP), catalyzing the formation of amyloid beta (Aβ) peptides that form amyloid plaques, which contribute to Alzheimer’s disease (AD) pathogenesis. γ-Secretase also cleaves Notch, among many other type I transmembrane substrates. Despite its seemingly promiscuous enzymatic capacity, γ-secretase activity is tightly regulated. This regulation is a function of many cellular entities, including but not limited to the essential γ-secretase subunits, nonessential (modulatory) subunits, and γ-secretase substrates. Regulation is also accomplished by an array of cellular events, such as presenilin (active subunit of γ-secretase) endoproteolysis and hypoxia. In this review we discuss how γ-secretase is regulated with the hope that an advanced understanding of these mechanisms will aid in the development of effective therapeutics for γ-secretase-associated diseases like AD and Notch-addicted cancer. PMID:25610395

  14. Extensive subunit contacts underpin herpesvirus capsid stability and interior-to-exterior allostery.

    PubMed

    Huet, Alexis; Makhov, Alexander M; Huffman, Jamie B; Vos, Matthijn; Homa, Fred L; Conway, James F

    2016-06-01

    The herpesvirus capsid is a complex protein assembly that includes hundreds of copies of four major subunits and lesser numbers of several minor proteins, all of which are essential for infectivity. Cryo-electron microscopy is uniquely suited for studying interactions that govern the assembly and function of such large functional complexes. Here we report two high-quality capsid structures, from human herpes simplex virus type 1 (HSV-1) and the animal pseudorabies virus (PRV), imaged inside intact virions at ~7-Å resolution. From these, we developed a complete model of subunit and domain organization and identified extensive networks of subunit contacts that underpin capsid stability and form a pathway that may signal the completion of DNA packaging from the capsid interior to outer surface, thereby initiating nuclear egress. Differences in the folding and orientation of subunit domains between herpesvirus capsids suggest that common elements have been modified for specific functions. PMID:27111889

  15. Localized reconstruction of subunits from electron cryomicroscopy images of macromolecular complexes

    PubMed Central

    Ilca, Serban L.; Kotecha, Abhay; Sun, Xiaoyu; Poranen, Minna M.; Stuart, David I.; Huiskonen, Juha T.

    2015-01-01

    Electron cryomicroscopy can yield near-atomic resolution structures of highly ordered macromolecular complexes. Often however some subunits bind in a flexible manner, have different symmetry from the rest of the complex, or are present in sub-stoichiometric amounts, limiting the attainable resolution. Here we report a general method for the localized three-dimensional reconstruction of such subunits. After determining the particle orientations, local areas corresponding to the subunits can be extracted and treated as single particles. We demonstrate the method using three examples including a flexible assembly and complexes harbouring subunits with either partial occupancy or mismatched symmetry. Most notably, the method allows accurate fitting of the monomeric RNA-dependent RNA polymerase bound at the threefold axis of symmetry inside a viral capsid, revealing for the first time its exact orientation and interactions with the capsid proteins. Localized reconstruction is expected to provide novel biological insights in a range of challenging biological systems. PMID:26534841

  16. The subunit composition of hinokiresinol synthase controls geometrical selectivity in norlignan formation.

    PubMed

    Suzuki, Shiro; Yamamura, Masaomi; Hattori, Takefumi; Nakatsubo, Tomoyuki; Umezawa, Toshiaki

    2007-12-26

    The selective formation of E- or Z-isomers is an important process in natural product metabolism. We show that the subunit composition of an enzyme can alter the geometrical composition of the enzymatic products. Hinokiresinol synthase, purified from Asparagus officinalis cell cultures, is responsible for the conversion of (7E,7'E)-4-coumaryl 4-coumarate to (Z)-hinokiresinol, the first step in norlignan formation. The protein is most likely a heterodimer composed of two distinct subunits, which share identity with members of the phloem protein 2 gene superfamily. Interestingly, each recombinant subunit of hinokiresinol synthase expressed in Escherichia coli solely converted (7E,7'E)-4-coumaryl 4-coumarate to the unnatural (E)-hinokiresinol, the E-isomer of (Z)-hinokiresinol. By contrast, a mixture of recombinant subunits catalyzed the formation of (Z)-hinokiresinol from the same substrate. PMID:18093914

  17. Nucleotide-Protectable Labeling of Sulfhydryl Groups in Subunit I of the ATPhase from Halobacterium Saccharovorum

    NASA Technical Reports Server (NTRS)

    Sulzner, Michael; Stan-Lotter, Helga; Hochstein, Lawrence I.

    1992-01-01

    A membrane-bound ATPase from the archaebacterium Halobacterium saccharovorum is inhibited by N-ethyl-maleimide in a nucleotide-protectable manner. When the enzyme was incubated with N-[C-14]jethylmaleimide, the bulk of radioactivity was as- sociated with the 87,000-Da subunit (subunit 1). ATP, ADP, or AMP reduced incorporation of the inhibitor. No charge shift of subunit I was detected following labeling with N-ethylmaleimide, indicating an electroneutral reaction. The results are consistent with the selective modification of sulfhydryl groups in subunit I at or near the catalytic site and are further evidence of a resemblance between this archaebacterial ATPase and the vacuolar-type ATPases.

  18. Depressed chemiluminescence response by influenza virus is enhanced after conjugation of viral subunits to muramyl dipeptide.

    PubMed Central

    Masihi, K N; Lange, W; Rohde-Schulz, B; Chedid, L; Jolivet, M

    1985-01-01

    The effect on respiratory burst of murine spleen cells after in vitro exposure to influenza virus, subunits, or subunits conjugated to muramyl dipeptide (MDP) was studied by luminol-dependent chemiluminescence (CL) in response to stimulation by zymosan. CL induced by infectious influenza A virus was depressed but could be elevated to normal levels when MDP was added together with a low, but not with a high, dose of the virus. Profound depression of CL was induced by high doses of influenza A/Brazil, A/Bangkok, and B/Singapore subunits. The same amounts of viral subunits conjugated to MDP restored or even enhanced the CL responses of spleen cells from BALB/c and C57BL/6 mice. Splenic cells from BALB/c mice generated higher levels of CL than did cells from C57BL/6 mice. PMID:4044031

  19. Differential Targeting of Gβγ-Subunit Signaling with Small Molecules

    NASA Astrophysics Data System (ADS)

    Bonacci, Tabetha M.; Mathews, Jennifer L.; Yuan, Chujun; Lehmann, David M.; Malik, Sundeep; Wu, Dianqing; Font, Jose L.; Bidlack, Jean M.; Smrcka, Alan V.

    2006-04-01

    G protein βγ subunits have potential as a target for therapeutic treatment of a number of diseases. We performed virtual docking of a small-molecule library to a site on Gβγ subunits that mediates protein interactions. We hypothesized that differential targeting of this surface could allow for selective modulation of Gβγ subunit functions. Several compounds bound to Gβγ subunits with affinities from 0.1 to 60 μM and selectively modulated functional Gβγ-protein-protein interactions in vitro, chemotactic peptide signaling pathways in HL-60 leukocytes, and opioid receptor-dependent analgesia in vivo. These data demonstrate an approach for modulation of G protein-coupled receptor signaling that may represent an important therapeutic strategy.

  20. Separation of heteromeric potassium channel Kcv towards probing subunit composition-regulated ion permeation and gating

    PubMed Central

    Tan, Qiulin; Shim, Ji Wook; Gu, Li-Qun

    2010-01-01

    The chlorella virus-encoded Kcv can form a homo-tetrameric potassium channel in lipid membranes. This miniature peptide can be synthesized in vitro, and the tetramer purified from the SDS polyacrylamide gel retains the K+ channel functionality. Combining this capability with the mass-tagging method, we propose a simple, straightforward approach that can generically manipulate individual subunits in the tetramer, thereby enabling the detection of contribution from individual subunits to the channel functions. Using this approach, we showed that the structural change from only one subunit in the selectivity filter is sufficient to cause permanent channel inactivation (“all-or-none” mechanism), whereas the mutation near the extracellular entrance additively modifies the ion permeation with the number of mutant subunits in the tetramer (“additive” mechanism). PMID:20303961

  1. Modulation of the Na,K-pump function by beta subunit isoforms

    PubMed Central

    1994-01-01

    To study the role of the Na,K-ATPase beta subunit in the ion transport activity, we have coexpressed the Bufo alpha 1 subunit (alpha 1) with three different isotypes of beta subunits, the Bufo Na,K-ATPase beta 1 (beta 1NaK) or beta 3 (beta 3NaK) subunit or the beta subunit of the rabbit gastric H,K-ATPase (beta HK), by cRNA injection in Xenopus oocyte. We studied the K+ activation kinetics by measuring the Na,K- pump current induced by external K+ under voltage clamp conditions. The endogenous oocyte Na,K-ATPase was selectively inhibited, taking advantage of the large difference in ouabain sensitivity between Xenopus and Bufo Na,K pumps. The K+ half-activation constant (K1/2) was higher in the alpha 1 beta 3NaK than in the alpha 1 beta 1NaK groups in the presence of external Na+, but there was no significant difference in the absence of external Na+. Association of alpha 1 and beta HK subunits produced active Na,K pumps with a much lower apparent affinity for K+ both in the presence and in the absence of external Na+. The voltage dependence of the K1/2 for external K+ was similar with the three beta subunits. Our results indicate that the beta subunit has a significant influence on the ion transport activity of the Na,K pump. The small structural differences between the beta 1NaK and beta 3NaK subunits results in a difference of the apparent affinity for K+ that is measurable only in the presence of external Na+, and thus appears not to be directly related to the K+ binding site. In contrast, association of an alpha 1 subunit with a beta HK subunit results in a Na,K pump in which the K+ binding or translocating mechanisms are altered since the apparent affinity for external K+ is affected even in the absence of external Na+. PMID:8057080

  2. Heat Capacity Mapping Mission

    NASA Technical Reports Server (NTRS)

    Nilsson, C. S.; Andrews, J. C.; Scully-Power, P.; Ball, S.; Speechley, G.; Latham, A. R. (Principal Investigator)

    1980-01-01

    The Tasman Front was delineated by airborne expendable bathythermograph survey; and an Heat Capacity Mapping Mission (HCMM) IR image on the same day shows the same principal features as determined from ground-truth. It is clear that digital enhancement of HCMM images is necessary to map ocean surface temperatures and when done, the Tasman Front and other oceanographic features can be mapped by this method, even through considerable scattered cloud cover.

  3. Map-likelihood phasing

    PubMed Central

    Terwilliger, Thomas C.

    2001-01-01

    The recently developed technique of maximum-likelihood density modification [Terwilliger (2000 ▶), Acta Cryst. D56, 965–972] allows a calculation of phase probabilities based on the likelihood of the electron-density map to be carried out separately from the calculation of any prior phase probabilities. Here, it is shown that phase-probability distributions calculated from the map-likelihood function alone can be highly accurate and that they show minimal bias towards the phases used to initiate the calculation. Map-likelihood phase probabilities depend upon expected characteristics of the electron-density map, such as a defined solvent region and expected electron-density distributions within the solvent region and the region occupied by a macromolecule. In the simplest case, map-likelihood phase-probability distributions are largely based on the flatness of the solvent region. Though map-likelihood phases can be calculated without prior phase information, they are greatly enhanced by high-quality starting phases. This leads to the technique of prime-and-switch phasing for removing model bias. In prime-and-switch phasing, biased phases such as those from a model are used to prime or initiate map-likelihood phasing, then final phases are obtained from map-likelihood phasing alone. Map-likelihood phasing can be applied in cases with solvent content as low as 30%. Potential applications of map-likelihood phasing include unbiased phase calculation from molecular-replacement models, iterative model building, unbiased electron-density maps for cases where 2Fo − Fc or σA-weighted maps would currently be used, structure validation and ab initio phase determination from solvent masks, non-crystallographic symmetry or other knowledge about expected electron density. PMID:11717488

  4. Both alpha and beta subunits of human choriogonadotropin photoaffinity label the hormone receptor.

    PubMed Central

    Ji, I; Ji, T H

    1981-01-01

    It has been shown that a photoactivable derivative of human choriogonadotropin (hCG) labels the lutropin receptor on porcine granulosa cells [Ji, I. & Ji, T. H. (1980) Proc. Natl. Acad. Sci. USA 77, 7167-7170]. In an attempt to identify which of the hCG subunits labeled the receptor, three sets of different hCG derivatives were prepared. In the first set, hCG was coupled to the N-hydroxysuccinimide ester of 4-azidobenzoylglycine and radioiodinated. In the second set, only one of the subunits was radioiodinated, but both subunits were allowed to react with the reagent. In the third set, both the reagent and [125I]iodine were coupled to only one of the subunits. The binding activity of each hormone derivative was comparable to that of 125I-labeled hCG. After binding of these hormone derivatives to the granulosa cell surface, they were photolyzed. After solubilization, autoradiographs of sodium dodecyl sulfate/polyacrylamide gels of each sample revealed a number of labeled bands; the hCG derivatives containing 125I-labeled alpha subunit produced four bands (molecular weights 120,000 +/- 6,000, 96,000 +/- 5,000, 76,000 +/- 4,000, and 73,000 +/- 4,000) and those containing 125I-labeled beta subunit produced three bands (molecular weights 106,000 +/- 6,000, 88,000 +/- 5,000, and 83,000 +/- 4,000). Results were the same when the hormone-receptor complexes were solubilized in 0.5% Triton X-100 and then photolyzed or when the hormone was derivatized with a family of reagents having arms of various lengths. We conclude that both the alpha subunit and the beta subunit of hCG photoaffinity labeled certain membrane polypeptides and that these polypeptides are related to the hormone receptor. Images PMID:6272303

  5. L-type calcium channel β subunit modulates angiotensin II responses in cardiomyocytes.

    PubMed

    Hermosilla, Tamara; Moreno, Cristian; Itfinca, Mircea; Altier, Christophe; Armisén, Ricardo; Stutzin, Andres; Zamponi, Gerald W; Varela, Diego

    2011-01-01

    Angiotensin II regulation of L-type calcium currents in cardiac muscle is controversial and the underlying signaling events are not completely understood. Moreover, the possible role of auxiliary subunit composition of the channels in Angiotensin II modulation of L-type calcium channels has not yet been explored. In this work we study the role of Ca(v)β subunits and the intracellular signaling responsible for L-type calcium current modulation by Angiotensin II. In cardiomyocytes, Angiotensin II exposure induces rapid inhibition of L-type current with a magnitude that is correlated with the rate of current inactivation. Semi-quantitative PCR of cardiomyocytes at different days of culture reveals changes in the Ca(v)β subunits expression pattern that are correlated with the rate of current inactivation and with Angiotensin II effect. Over-expression of individual b subunits in heterologous systems reveals that the magnitude of Angiotensin II inhibition is dependent on the Ca(v)β subunit isoform, with Ca(v)β(1b) containing channels being more strongly regulated. Ca(v)β(2a) containing channels were insensitive to modulation and this effect was partially due to the N-terminal palmitoylation sites of this subunit. Moreover, PLC or diacylglycerol lipase inhibition prevents the Angiotensin II effect on L-type calcium channels, while PKC inhibition with chelerythrine does not, suggesting a role of arachidonic acid in this process. Finally, we show that in intact cardiomyocytes the magnitude of calcium transients on spontaneous beating cells is modulated by Angiotensin II in a Ca(v)β subunit-dependent manner. These data demonstrate that Ca(v)β subunits alter the magnitude of inhibition of L-type current by Angiotensin II. PMID:21525790

  6. Type B Heterotrimeric G Protein γ-Subunit Regulates Auxin and ABA Signaling in Tomato.

    PubMed

    Subramaniam, Gayathery; Trusov, Yuri; Lopez-Encina, Carlos; Hayashi, Satomi; Batley, Jacqueline; Botella, José Ramón

    2016-02-01

    Heterotrimeric G proteins composed of α, β, and γ subunits are central signal transducers mediating the cellular response to multiple stimuli in most eukaryotes. Gγ subunits provide proper cellular localization and functional specificity to the heterotrimer complex. Plant Gγ subunits, divided into three structurally distinct types, are more diverse than their animal counterparts. Type B Gγ subunits, lacking a carboxyl-terminal isoprenylation motif, are found only in flowering plants. We present the functional characterization of type B Gγ subunit (SlGGB1) in tomato (Solanum lycopersicum). We show that SlGGB1 is the most abundant Gγ subunit in tomato and strongly interacts with the Gβ subunit. Importantly, the green fluorescent protein-SlGGB1 fusion protein as well as the carboxyl-terminal yellow fluorescent protein-SlGGB1/amino-terminal yellow fluorescent protein-Gβ heterodimer were localized in the plasma membrane, nucleus, and cytoplasm. RNA interference-mediated silencing of SlGGB1 resulted in smaller seeds, higher number of lateral roots, and pointy fruits. The silenced lines were hypersensitive to exogenous auxin, while levels of endogenous auxins were lower or similar to those of the wild type. SlGGB1-silenced plants also showed strong hyposensitivity to abscisic acid (ABA) during seed germination but not in other related assays. Transcriptome analysis of the transgenic seeds revealed abnormal expression of genes involved in ABA sensing, signaling, and response. We conclude that the type B Gγ subunit SlGGB1 mediates auxin and ABA signaling in tomato. PMID:26668332

  7. Myristoylated. cap alpha. subunits of guanine nucleotide-binding regulatory proteins

    SciTech Connect

    Buss, J.E.; Mumby, S.M.; Casey, P.J.; Gilman, A.G.; Sefton, B.M.

    1987-11-01

    Antisera directed against specific subunits of guanine nucleotide-binding regulatory proteins (G proteins) were used to immunoprecipitate these polypeptides from metabolically labeled cells. This technique detects, in extracts of a human astrocytoma cell line, the ..cap alpha.. subunits of G/sub s/ (stimulatory) (..cap alpha../sub 45/ and ..cap alpha../sub 52/), a 41-kDa subunit of G/sub i/ (inhibitory) (..cap alpha../sub 41/), a 40-kDa protein (..cap alpha../sub 40/), and the 36-kDa ..beta.. subunit. No protein that comigrated with the ..cap alpha.. subunit of G/sup 0/ (unknown function) (..cap alpha../sub 39/) was detected. In cells grown in the presence of (/sup 3/H)myristic acid, ..cap alpha../sub 41/ and ..cap alpha../sub 40/ contained /sup 3/H label, while the ..beta.. subunit did not. Chemical analysis of lipids attached covalently to purified ..cap alpha../sub 41/ and ..cap alpha../sub 39/ from bovine brain also revealed myristic acid. Similar analysis of brain G protein ..beta.. and ..gamma.. subunits and of G/sub t/ (Transducin) subunits (..cap alpha.., ..beta.., and ..gamma..) failed to reveal fatty acids. The fatty acid associated with ..cap alpha../sub 41/ , ..cap alpha../sub 40/, and ..cap alpha../sub 39/ was stable to treatment with base, suggesting that the lipid is linked to the polypeptide via an amide bond. These GTP binding proteins are thus identified as members of a select group of proteins that contains myristic acid covalently attached to the peptide backbone. Myristate may play an important role in stabilizing interactions of G proteins with phospholipid or with membrane-bound proteins.

  8. Pharmacological consequences of the coexpression of BK channel α and auxiliary β subunits

    PubMed Central

    Torres, Yolima P.; Granados, Sara T.; Latorre, Ramón

    2014-01-01

    Coded by a single gene (Slo1, KCM) and activated by depolarizing potentials and by a rise in intracellular Ca2+ concentration, the large conductance voltage- and Ca2+-activated K+ channel (BK) is unique among the superfamily of K+ channels. BK channels are tetramers characterized by a pore-forming α subunit containing seven transmembrane segments (instead of the six found in voltage-dependent K+ channels) and a large C terminus composed of two regulators of K+ conductance domains (RCK domains), where the Ca2+-binding sites reside. BK channels can be associated with accessory β subunits and, although different BK modulatory mechanisms have been described, greater interest has recently been placed on the role that the β subunits may play in the modulation of BK channel gating due to its physiological importance. Four β subunits have currently been identified (i.e., β1, β2, β3, and β4) and despite the fact that they all share the same topology, it has been shown that every β subunit has a specific tissue distribution and that they modify channel kinetics as well as their pharmacological properties and the apparent Ca2+ sensitivity of the α subunit in different ways. Additionally, different studies have shown that natural, endogenous, and synthetic compounds can modulate BK channels through β subunits. Considering the importance of these channels in different pathological conditions, such as hypertension and neurological disorders, this review focuses on the mechanisms by which these compounds modulate the biophysical properties of BK channels through the regulation of β subunits, as well as their potential therapeutic uses for diseases such as those mentioned above. PMID:25346693

  9. Two temporally synthesized charge subunits interact to form the five isoforms of cottonseed (Gossypium hirsutum) catalase.

    PubMed Central

    Ni, W; Trelease, R N; Eising, R

    1990-01-01

    Five charge isoforms of tetrameric catalase were isolated from cotyledons of germinated cotton (Gossypium hirsutum L.) seedlings. Denaturing isoelectric focusing of the individual isoforms in polyacrylamide gels indicated that isoforms A (most anodic) and E (most cathodic) consisted of one subunit of different charge, whereas isoforms B, C and D each consisted of a mixture of these two subunits. Thus the five isoforms apparently were formed through combinations of two subunits in different ratios. Labelling cotyledons in vivo with [35S]methionine at three daily intervals in the dark, and translation in vivo of polyadenylated RNA isolated from cotyledons at the same ages, revealed synthesis of two different subunits. One of the subunits was synthesized in cotyledons at all ages studied (days 1-3), whereas the other subunit was detected only at days 2 and 3. This differential expression of two catalase subunits helped explain previous results from this laboratory showing that the two anodic forms (A and B) found in maturing seeds were supplemented with three cathodic forms (C-E) after the seeds germinated. These subunit data also helped clarify our new findings that proteins of isoforms A, B and C (most active isoforms) accumulated in cotyledons of plants kept in the dark for 3 days, then gradually disappeared during the next several days, whereas isoforms D and E (least active isoforms) remained in the cells. This shift in isoform pattern occurred whether seedlings were kept in the dark or exposed to continuous light after day 3, although exposure to light enhanced this process. These sequential molecular events were responsible for the characteristic developmental changes (rise and fall) in total catalase activity. We believe that the isoform changeover is physiologically related to the changeover in glyoxysome to leaf-type-peroxisome metabolism. Images Fig. 1. Fig. 2. Fig. 3. Fig. 4. Fig. 5. PMID:1695843

  10. Exposing the subunit diversity within protein complexes: a mass spectrometry approach.

    PubMed

    Rozen, Shelly; Tieri, Alessandra; Ridner, Gabriela; Stark, Ann-Kathrin; Schmaler, Tilo; Ben-Nissan, Gili; Dubiel, Wolfgang; Sharon, Michal

    2013-03-01

    Identifying the list of subunits that make up protein complexes constitutes an important step towards understanding their biological functions. However, such knowledge alone does not reveal the full complexity of protein assemblies, as each subunit can take on multiple forms. Proteins can be post-translationally modified or cleaved, multiple products of alternative splicing can exist, and a single subunit may be encoded by more than one gene. Thus, for a complete description of a protein complex, it is necessary to expose the diversity of its subunits. Adding this layer of information is an important step towards understanding the mechanisms that regulate the activity of protein assemblies. Here, we describe a mass spectrometry-based approach that exposes the array of protein variants that comprise protein complexes. Our method relies on denaturing the protein complex, and separating its constituent subunits on a monolithic column prepared in-house. Following the subunit elution from the column, the flow is split into two fractions, using a Triversa NanoMate robot. One fraction is directed straight into an on-line ESI-QToF mass spectrometer for intact protein mass measurements, while the rest of the flow is fractionated into a 96-well plate for subsequent proteomic analysis. The heterogeneity of subunit composition is then exposed by correlating the subunit sequence identity with the accurate mass. Below, we describe in detail the methodological setting of this approach, its application on the endogenous human COP9 signalosome complex, and the significance of the method for structural mass spectrometry analysis of intact protein complexes. PMID:23296018

  11. Crystal Structure of the Oxazolidinone Antibiotic Linezolid Bound to the 50S Ribosomal Subunit

    SciTech Connect

    Ippolito,J.; Kanyo, Z.; Wang, D.; Franceschi, F.; Moore, P.; Steitz, T.; Duffy, E.

    2008-01-01

    The oxazolidinone antibacterials target the 50S subunit of prokaryotic ribosomes. To gain insight into their mechanism of action, the crystal structure of the canonical oxazolidinone, linezolid, has been determined bound to the Haloarcula marismortui 50S subunit. Linezolid binds the 50S A-site, near the catalytic center, which suggests that inhibition involves competition with incoming A-site substrates. These results provide a structural basis for the discovery of improved oxazolidinones active against emerging drug-resistant clinical strains.

  12. Escobar Syndrome Is a Prenatal Myasthenia Caused by Disruption of the Acetylcholine Receptor Fetal γ Subunit

    PubMed Central

    Hoffmann, Katrin; Müller, Juliane S.; Stricker, Sigmar; Megarbane, Andre; Rajab, Anna; Lindner, Tom H.; Cohen, Monika; Chouery, Eliane; Adaimy, Lynn; Ghanem, Ismat; Delague, Valerie; Boltshauser, Eugen; Talim, Beril; Horvath, Rita; Robinson, Peter N.; Lochmüller, Hanns; Hübner, Christoph; Mundlos, Stefan

    2006-01-01

    Escobar syndrome is a form of arthrogryposis multiplex congenita and features joint contractures, pterygia, and respiratory distress. Similar findings occur in newborns exposed to nicotinergic acetylcholine receptor (AChR) antibodies from myasthenic mothers. We performed linkage studies in families with Escobar syndrome and identified eight mutations within the γ-subunit gene (CHRNG) of the AChR. Our functional studies show that γ-subunit mutations prevent the correct localization of the fetal AChR in human embryonic kidney–cell membranes and that the expression pattern in prenatal mice corresponds to the human clinical phenotype. AChRs have five subunits. Two α, one β, and one δ subunit are always present. By switching γ to ɛ subunits in late fetal development, fetal AChRs are gradually replaced by adult AChRs. Fetal and adult AChRs are essential for neuromuscular signal transduction. In addition, the fetal AChRs seem to be the guide for the primary encounter of axon and muscle. Because of this important function in organogenesis, human mutations in the γ subunit were thought to be lethal, as they are in γ-knockout mice. In contrast, many mutations in other subunits have been found to be viable but cause postnatally persisting or beginning myasthenic syndromes. We conclude that Escobar syndrome is an inherited fetal myasthenic disease that also affects neuromuscular organogenesis. Because γ expression is restricted to early development, patients have no myasthenic symptoms later in life. This is the major difference from mutations in the other AChR subunits and the striking parallel to the symptoms found in neonates with arthrogryposis when maternal AChR auto-antibodies crossed the placenta and caused the transient inactivation of the AChR pathway. PMID:16826520

  13. LEGO-NMR spectroscopy: a method to visualize individual subunits in large heteromeric complexes.

    PubMed

    Mund, Markus; Overbeck, Jan H; Ullmann, Janina; Sprangers, Remco

    2013-10-18

    Seeing the big picture: Asymmetric macromolecular complexes that are NMR active in only a subset of their subunits can be prepared, thus decreasing NMR spectral complexity. For the hetero heptameric LSm1-7 and LSm2-8 rings NMR spectra of the individual subunits of the complete complex are obtained, showing a conserved RNA binding site. This LEGO-NMR technique makes large asymmetric complexes accessible to detailed NMR spectroscopic studies. PMID:23946163

  14. Regularity of mappings inverse to Sobolev mappings

    SciTech Connect

    Vodop'yanov, Sergei K

    2012-10-31

    For homeomorphisms {phi}:{Omega}{yields}{Omega}' on Euclidean domains in R{sup n}, n{>=}2, necessary and sufficient conditions ensuring that the inverse mapping belongs to a Sobolev class are investigated. The result obtained is used to describe a new two-index scale of homeomorphisms in some Sobolev class such that their inverses also form a two-index scale of mappings, in another Sobolev class. This scale involves quasiconformal mappings and also homeomorphisms in the Sobolev class W{sup 1}{sub n-1} such that rankD{phi}(x){<=}n-2 almost everywhere on the zero set of the Jacobian det D{phi}(x). Bibliography: 65 titles.

  15. Mapping the Hydrogen Bond Networks in the Catalytic Subunit of Protein Kinase A Using H/D Fractionation Factors.

    PubMed

    Li, Geoffrey C; Srivastava, Atul K; Kim, Jonggul; Taylor, Susan S; Veglia, Gianluigi

    2015-07-01

    Protein kinase A is a prototypical phosphoryl transferase, sharing its catalytic core (PKA-C) with the entire kinase family. PKA-C substrate recognition, active site organization, and product release depend on the enzyme's conformational transitions from the open to the closed state, which regulate its allosteric cooperativity. Here, we used equilibrium nuclear magnetic resonance hydrogen/deuterium (H/D) fractionation factors (φ) to probe the changes in the strength of hydrogen bonds within the kinase upon binding the nucleotide and a pseudosubstrate peptide (PKI5-24). We found that the φ values decrease upon binding both ligands, suggesting that the overall hydrogen bond networks in both the small and large lobes of PKA-C become stronger. However, we observed several important exceptions, with residues displaying higher φ values upon ligand binding. Notably, the changes in φ values are not localized near the ligand binding pockets; rather, they are radiated throughout the entire enzyme. We conclude that, upon ligand and pseudosubstrate binding, the hydrogen bond networks undergo extensive reorganization, revealing that the open-to-closed transitions require global rearrangements of the internal forces that stabilize the enzyme's fold. PMID:26030372

  16. Human cytomegalovirus resistance to deoxyribosylindole nucleosides maps to a transversion mutation in the terminase subunit-encoding gene UL89.

    PubMed

    Gentry, Brian G; Phan, Quang; Hall, Ellie D; Breitenbach, Julie M; Borysko, Katherine Z; Kamil, Jeremy P; Townsend, Leroy B; Drach, John C

    2015-01-01

    Human cytomegalovirus (HCMV) infection can cause severe illnesses, including encephalopathy and mental retardation, in immunocompromised and immunologically immature patients. Current pharmacotherapies for treating systemic HCMV infections include ganciclovir, cidofovir, and foscarnet. However, long-term administration of these agents can result in serious adverse effects (myelosuppression and/or nephrotoxicity) and the development of viral strains with reduced susceptibility to drugs. The deoxyribosylindole (indole) nucleosides demonstrate a 20-fold greater activity in vitro (the drug concentration at which 50% of the number of plaques was reduced with the presence of drug compared to the number in the absence of drug [EC50] = 0.34 μM) than ganciclovir (EC50 = 7.4 μM) without any observed increase in cytotoxicity. Based on structural similarity to the benzimidazole nucleosides, we hypothesize that the indole nucleosides target the HCMV terminase, an enzyme responsible for packaging viral DNA into capsids and cleaving the DNA into genome-length units. To test this hypothesis, an indole nucleoside-resistant HCMV strain was isolated, the open reading frames of the genes that encode the viral terminase were sequenced, and a G766C mutation in exon 1 of UL89 was identified; this mutation resulted in an E256Q change in the amino acid sequence of the corresponding protein. An HCMV wild-type strain, engineered with this mutation to confirm resistance, demonstrated an 18-fold decrease in susceptibility to the indole nucleosides (EC50 = 3.1 ± 0.7 μM) compared to that of wild-type virus (EC50 = 0.17 ± 0.04 μM). Interestingly, this mutation did not confer resistance to the benzimidazole nucleosides (EC50 for wild-type HCMV = 0.25 ± 0.04 μM, EC50 for HCMV pUL89 E256Q = 0.23 ± 0.04 μM). We conclude, therefore, that the G766C mutation that results in the E256Q substitution is unique for indole nucleoside resistance and distinct from previously discovered substitutions that confer both indole and benzimidazole nucleoside resistance (D344E and A355T). PMID:25348532

  17. Making maps with computers

    USGS Publications Warehouse

    Guptill, S.C.; Starr, L.E.

    1988-01-01

    Soon after their introduction in the 1950s, digital computers were used for various phases of the mapping process, especially for trigonometric calculations of survey data and for orientation of aerial photographs on map manuscripts. In addition, computer-controlled plotters were used to draw simple outline maps. The process of collecting data for the plotters was slow and not as precise as those produced by the best manual cartography. Only during the 1980s has it become technologically feasible and cost-effective to assemble and use the data required to automate the mapping process. -from Authors

  18. DENALI IMAGE MAP.

    USGS Publications Warehouse

    Binnie, Douglas R.; Colvocoresses, Alden P.

    1987-01-01

    The Denali National Park and Preserve 1:250,000-scale image map has been prepared and published as part of the US Geological Survey's (USGS) continuing research to improve image mapping techniques. Nine multispectral scanner (MSS) images were geometrically corrected, digitally mosaicked, and enhanced at the National Mapping Division's (NMD) EROS Data Center (EDC). This process involves ground control and digital resampling to the Universal Tranverse Mercator (UTM) projection. This paper specifically discusses the preparation of the digital mosaic and the production peculiarities associated with the Denali National Park and Preserve image map.

  19. Chaotic Polynomial Maps

    NASA Astrophysics Data System (ADS)

    Zhang, Xu

    This paper introduces a class of polynomial maps in Euclidean spaces, investigates the conditions under which there exist Smale horseshoes and uniformly hyperbolic invariant sets, studies the chaotic dynamical behavior and strange attractors, and shows that some maps are chaotic in the sense of Li-Yorke or Devaney. This type of maps includes both the Logistic map and the Hénon map. For some diffeomorphisms with the expansion dimension equal to one or two in three-dimensional spaces, the conditions under which there exist Smale horseshoes and uniformly hyperbolic invariant sets on which the systems are topologically conjugate to the two-sided fullshift on finite alphabet are obtained; for some expanding maps, the chaotic region is analyzed by using the coupled-expansion theory and the Brouwer degree theory. For three types of higher-dimensional polynomial maps with degree two, the conditions under which there are Smale horseshoes and uniformly hyperbolic invariant sets are given, and the topological conjugacy between the maps on the invariant sets and the two-sided fullshift on finite alphabet is obtained. Some interesting maps with chaotic attractors and positive Lyapunov exponents in three-dimensional spaces are found by using computer simulations. In the end, two examples are provided to illustrate the theoretical results.

  20. BOREAS Hardcopy Maps

    NASA Technical Reports Server (NTRS)

    Hall, Forrest G. (Editor); Nelson, Elizabeth; Newcomer, Jeffrey A.

    2000-01-01

    Boreal Ecosystem-Atmospheric Study (BOREAS) hardcopy maps are a collection of approximately 1,000 hardcopy maps representing the physical, climatological, and historical attributes of areas covering primarily the Manitoba and Saskatchewan provinces of Canada. These maps were collected by BOREAS Information System (BORIS) and Canada for Remote Sensing (CCRS) staff to provide basic information about site positions, manmade features, topography, geology, hydrology, land cover types, fire history, climate, and soils of the BOREAS study region. These maps are not available for distribution through the BOREAS project but may be used as an on-site resource. Information is provided within this document for individuals who want to order copies of these maps from the original map source. Note that the maps are not contained on the BOREAS CD-ROM set. An inventory listing file is supplied on the CD-ROM to inform users of the maps that are available. This inventory listing is available from the Earth Observing System Data and Information System (EOSDIS) Oak Ridge National Laboratory (ORNL) Distributed Active Archive Center (DAAC). For hardcopies of the individual maps, contact the sources provided.