Science.gov

Sample records for ribosomal phosphoprotein pfp0

  1. Fine mapping of Plasmodium falciparum ribosomal phosphoprotein PfP0 revealed sequences with highly specific binding activity to human red blood cells.

    PubMed

    Arevalo-Pinzon, Gabriela; Curtidor, Hernando; Reyes, Claudia; Pinto, Martha; Vizcaíno, Carolina; Patarroyo, Manuel A; Patarroyo, Manuel E

    2010-01-01

    The Plasmodium falciparum P0 ribosomal phosphoprotein (PfP0) was identified for the first time by screening a cDNA expression library of P. falciparum parasites with sera from malaria-immune individuals. Due to its localization on the surface of different parasite life-cycle stages (merozoites and gametocytes) and its recognition by invasion-blocking antibodies, PfP0 has been considered a potential malaria-vaccine component. In this study, 16 20-mer-long synthetic peptides spanning the entire PfP0 sequence were evaluated by means of receptor-ligand assays with human red blood cells (RBCs) in order to determine the role played by these peptides in the invasion process. Four RBC high-activity binding peptides (HABPs), located mostly toward the N-terminal region, were identified: HABP 33898 ((1)MAKLSKQQKKQMYIEKLSSL(20)), HABP 33900 ((41)ASVRKSLRGKATILMGKNTRY(60)), HABP 33901 ((61)IRTALKKNLQAVPQIEKLLPY (80)), and HABP 33906 ((161)LIKQGEKVTASSATLLRKFNY(180)). The binding pattern of HABPs 33898 and 33906 to enzyme-treated RBCs suggests receptors of protein nature for these two HABPs, one of which could correspond to a common 58-kDa RBC membrane protein, as indicated by results of cross-linking assays. Both HABPs exhibited high content of alpha-helical features and prevented P. falciparum merozoite invasion to RBCs in vitro by up to 91%. The invasion-blocking ability reported here for these PfP0 HABPs supports their inclusion in immunological studies with the aim of assessing their potential as candidates for a vaccine against P. falciparum malaria.

  2. An abundant nucleolar phosphoprotein is associated with ribosomal DNA in Tetrahymena macronuclei.

    PubMed Central

    McGrath, K E; Smothers, J F; Dadd, C A; Madireddi, M T; Gorovsky, M A; Allis, C D

    1997-01-01

    An abundant 52-kDa phosphoprotein was identified and characterized from macronuclei of the ciliated protozoan Tetrahymena thermophila. Immunoblot analyses combined with light and electron microscopic immunocytochemistry demonstrate that this polypeptide, termed Nopp52, is enriched in the nucleoli of transcriptionally active macronuclei and missing altogether from transcriptionally inert micronuclei. The cDNA sequence encoding Nopp52 predicts a polypeptide whose amino-terminal half consists of multiple acidic/serine-rich regions alternating with basic/proline-rich regions. Multiple serines located in these acidic stretches lie within casein kinase II consensus motifs, and Nopp52 is an excellent substrate for casein kinase II in vitro. The carboxyl-terminal half of Nopp52 contains two RNA recognition motifs and an extreme carboxyl-terminal domain rich in glycine, arginine, and phenylalanine, motifs common in many RNA processing proteins. A similar combination and order of motifs is found in vertebrate nucleolin and yeast NSR1, suggesting that Nopp52 is a member of a family of related nucleolar proteins. NSR1 and nucleolin have been implicated in transcriptional regulation of rDNA and rRNA processing. Consistent with a role in ribosomal gene metabolism, rDNA and Nopp52 colocalize in situ, as well as by cross-linking and immunoprecipitation experiments, demonstrating an association between Nopp52 and rDNA in vivo. Images PMID:9017598

  3. Stable binding of the eukaryotic acidic phosphoproteins to the ribosome is not an absolute requirement for in vivo protein synthesis.

    PubMed

    Remacha, M; Santos, C; Bermejo, B; Naranda, T; Ballesta, J P

    1992-06-15

    The genes encoding the four acidic ribosomal phosphoproteins have been inactivated in Saccharomyces cerevisae by recombination with truncated genes carrying different genetic markers. By crossing single haploid disruptants, strains harboring two simultaneously inactivated acidic protein genes were constructed. None of the six possible double disruptions was lethal, but the simultaneous inactivation of either YP1 alpha and YP1 beta(L44') or YP2 alpha(L44) and YP2 beta(L45) caused an important decrease in the cell growth rate. Ribosomes isolated from these slow-growing strains did not contain acidic proteins, not even the two polypeptides whose genes were still intact, although these proteins were present in the cell extracts and they seem to be able to form high-molecular weight protein complexes. Transformation of a slow-growing double transformant with a plasmid containing one of the disrupted genes restored the presence of the acidic proteins in the ribosomes and normal growth rates. The particles of the slow-growing strains were active in an in vitro amino acid polymerizing system, although their activity could be stimulated by the exogenous addition of the missing proteins. These results indicate that in the absence of either YP1 alpha and YP1 beta(L44') or YP2 alpha (L44) and YP2 beta(L45), the remaining acidic proteins are unable to interact with the ribosome in a stable manner, but that a strong interaction of these ribosomal components with the particle is not an absolute requirement for in vivo and in vitro protein synthesis.

  4. Characterization of P0, a ribosomal phosphoprotein of Plasmodium falciparum. Antibody against amino-terminal domain inhibits parasite growth.

    PubMed

    Goswami, A; Singh, S; Redkar, V D; Sharma, S

    1997-05-02

    A cDNA expression clone of the human malarial parasite Plasmodium falciparum, lambdaPf4, which was reactive only to the immune sera and not to the patient sera, has recently been found to be the P. falciparum homologue of the P0 ribosomal phosphoprotein gene. A Northern analysis of the P0 gene revealed the presence of two transcripts, both present in all the different intraerythrocytic stages of the parasite life cycle. A 138-base pair amino-terminal domain of this gene was expressed as a fusion protein with glutathione S-transferase in Escherichia coli. Polyclonal antibodies raised against this domain immunoprecipitated the expected 38-kDa P0 protein from the 35S-labeled as well as 32P-labeled P. falciparum cultures. Monospecific human immune sera affinity-purified using the expression clone lambdaPf4 also immunoprecipitated the same size protein from [35S]methionine-labeled P. falciparum protein extract. Purified IgG from polyclonal antibodies raised against the amino-terminal domain of P0 protein completely inhibited the growth of P. falciparum in vitro. This inhibition appears to be mainly at the step of erythrocyte invasion by the parasites.

  5. cDNA Cloning and Overexpression of Acidic Ribosomal Phosphoprotein P1 Gene (RPLP1) from the Giant Panda

    PubMed Central

    Du, Yu-Jie; Luo, Xiao-Yan; Hao, Yan-Zhe; Zhang, Tian; Hou, Wan-Ru

    2007-01-01

    RPLP1 is one of acidic ribosomal phosphoproteins encoded by RPLP1 gene, which plays an important role in the elongation step of protein synthesis. The cDNA of RPLP1 was cloned successfully for the first time from the Giant Panda (Ailuropoda melanoleuca) using RT-PCR technology, which was also sequenced, analyzed preliminarily and expressed in E.coli. The cDNA fragment cloned is 449bp in size, containing an open reading frame of 344bp encoding 114 amino acids. Alignment analysis indicated that the nucleotide sequence and the deduced amino acid sequence are highly conserved to other five species studied, including Homo sapiens, Mus musculus, Rattus norvegicus, Bos Taurus and Sus scrofa. The homologies for nucleotide sequences of Giant Panda PPLP1 to that of these species are 92.4%, 89.8%, 89.0%, 91.3% and 87.5%, while the homologies for amino acid sequences are 96.5%, 94.7%, 95.6%, 96.5% and 88.6%. Topology prediction showed there are three Casein kinase II phosphorylation sites and two N-myristoylation sites in the RPLP1 protein of the Giant Panda (Ailuropoda melanoleuca). The RPLP1 gene was overexpressed in E. coli and the result indicated that RPLP1 fusion with the N-terminally His-tagged form gave rise to the accumulation of an expected 18kDa polypeptide, which was in accordance with the predicted protein and could also be used to purify the protein and study its function. PMID:18071584

  6. Effect of acidic ribosomal phosphoprotein mRNA 5'-untranslated region on gene expression and protein accumulation.

    PubMed

    Bermejo, B; Remacha, M; Ortiz-Reyes, B; Santos, C; Ballesta, J P

    1994-02-11

    Constructions were made from genes encoding ribosomal acidic phosphoproteins YP1 beta (L44') and YP2 beta (L45) from Saccharomyces cerevisiae in which different parts of the 5'-untranslated regions were included. The constructs were inserted into centromeric plasmids under the control of the GAL1 promoter and expressed in yeast strains in which the genes coding for each acidic protein family, P1 and P2, had been disrupted. Deletions in the 5' region of the two genes have been found to oppositely affect their expression. Deletion of most of this region strongly stimulates the expression of YP2 beta (L45), increasing the translation efficiency of the mRNA, and generating a 6-fold excess of protein in the cell. A similar deletion in the rpYP1 beta gene represses the expression of the protein, reducing drastically the amount of the mRNA in the cell. The overexpression of rpYP2 beta affects the cell growth by inhibiting protein synthesis at the level of initiation. Reduction of the YP2 beta(L45) overproduction by growing in controlled concentrations of glucose abolishes the inhibitory effect. The excess protein, probably as a high molecular weight complex, apparently interferes with the joining of the 60 S subunit to the initiation complex generating the accumulation of polysome half-mers. In addition, the results indicate the existence of a regulatory mechanism by which each one of the two acidic proteins controls the expression of the other polypeptide. YP1 beta(L44') represses the expression of YP2 beta(L45), while this protein stimulates the expression of YP1 beta(L44').

  7. cDNA cloning and overexpression of acidic ribosomal phosphoprotein P1 gene (RPLP1) from the giant panda.

    PubMed

    Du, Yu-Jie; Luo, Xiao-Yan; Hao, Yan-Zhe; Zhang, Tian; Hou, Wan-Ru

    2007-10-26

    RPLP1 is one of acidic ribosomal phosphoproteins encoded by RPLP1 gene, which plays an important role in the elongation step of protein synthesis. The cDNA of RPLP1 was cloned successfully for the first time from the Giant Panda (Ailuropoda melanoleuca) using RT-PCR technology, which was also sequenced, analyzed preliminarily and expressed in E.coli. The cDNA fragment cloned is 449bp in size, containing an open reading frame of 344bp encoding 114 amino acids. Alignment analysis indicated that the nucleotide sequence and the deduced amino acid sequence are highly conserved to other five species studied, including Homo sapiens, Mus musculus, Rattus norvegicus, Bos Taurus and Sus scrofa. The homologies for nucleotide sequences of Giant Panda PPLP1 to that of these species are 92.4%, 89.8%, 89.0%, 91.3% and 87.5%, while the homologies for amino acid sequences are 96.5%, 94.7%, 95.6%, 96.5% and 88.6%. Topology prediction showed there are three Casein kinase II phosphorylation sites and two N-myristoylation sites in the RPLP1 protein of the Giant Panda (Ailuropoda melanoleuca). The RPLP1 gene was overexpressed in E. coli and the result indicated that RPLP1 fusion with the N-terminally His-tagged form gave rise to the accumulation of an expected 18kDa polypeptide, which was in accordance with the predicted protein and could also be used to purify the protein and study its function.

  8. Plasmodium Riboprotein PfP0 Induces a Deviant Humoral Immune Response in Balb/c Mice

    PubMed Central

    Pathak, Sulabha; Rajeshwari, K.; Garg, Swati; Rajagopal, Sudarsan; Patel, Kalpesh; Das, Bidyut; Pied, Sylviane; Ravindran, Balachandran; Sharma, Shobhona

    2012-01-01

    Passive immunization with antibodies to recombinant Plasmodium falciparum P0 riboprotein (rPfP0, 61–316 amino acids) provides protection against malaria. Carboxy-terminal 16 amino acids of the protein (PfP0C0) are conserved and show 69% identity to human and mouse P0. Antibodies to this domain are found in 10–15% of systemic lupus erythematosus patients. We probed the nature of humoral response to PfP0C0 by repeatedly immunizing mice with rPfP0. We failed to raise stable anti-PfP0C0 hybridomas from any of the 21 mice. The average serum anti-PfP0C0 titer remained low (5.1 ± 1.3 × 104). Pathological changes were observed in the mice after seven boosts. Adsorption with dinitrophenyl hapten revealed that the anti-PfP0C0 response was largely polyreactive. This polyreactivity was distributed across all isotypes. Similar polyreactive responses to PfP0 and PfP0C0 were observed in sera from malaria patients. Our data suggests that PfP0 induces a deviant humoral response, and this may contribute to immune evasion mechanisms of the parasite. PMID:22315513

  9. Ribosomal acidic phosphoproteins P1 and P2 are not required for cell viability but regulate the pattern of protein expression in Saccharomyces cerevisiae.

    PubMed Central

    Remacha, M; Jimenez-Diaz, A; Bermejo, B; Rodriguez-Gabriel, M A; Guarinos, E; Ballesta, J P

    1995-01-01

    Saccharomyces cerevisiae strains with either three inactivated genes (triple disruptants) or four inactivated genes (quadruple disruptants) encoding the four acidic ribosomal phosphoproteins, YP1 alpha, YP1 beta, YP2 alpha, and YP2 beta, present in this species have been obtained. Ribosomes from the triple disruptants and, obviously, those from the quadruple strain do not have bound P proteins. All disrupted strains are viable; however, they show a cold-sensitive phenotype, growing very poorly at 23 degrees C. Cell extracts from the quadruple-disruptant strain are about 30% as active as the control in protein synthesis assays and are stimulated by the addition of free acidic P proteins. Strains lacking acidic proteins do not have a higher suppressor activity than the parental strains, and cell extracts derived from the quadruple disruptant do not show a higher degree of misreading, indicating that the absence of acidic proteins does not affect the accuracy of the ribosomes. However, the patterns of protein expressed in the cells as well as in the cell-free protein system are affected by the absence of P proteins from the particles; a wild-type pattern is restored upon addition of exogenous P proteins to the cell extract. In addition, strains carrying P-protein-deficient ribosomes are unable to sporulate but recover this capacity upon transformation with one of the missing genes. These results indicate that acidic proteins are not an absolute requirement for protein synthesis but regulate the activity of the 60S subunit, affecting the translation of certain mRNAs differently. PMID:7651393

  10. Identification of ribosomal phosphoprotein P0 of Neospora caninum as a potential common vaccine candidate for the control of both neosporosis and toxoplasmosis.

    PubMed

    Zhang, Houshuang; Lee, Eung-goo; Liao, Min; Compaore, Muller K A; Zhang, Guohong; Kawase, Osamu; Fujisaki, Kozo; Sugimoto, Chihiro; Nishikawa, Yoshifumi; Xuan, Xuenan

    2007-06-01

    The characterization of the cross-reactive antigens of two closely related apicomplexan parasites, Neospora caninum and Toxoplasma gondii, is important to elucidate the common mechanisms of parasite-host interactions. In this context, a gene encoding N. caninum ribosomal phosphoprotein P0 (NcP0) was identified by immunoscreening of a N. caninum tachyzoite cDNA expression library with antisera from mice immunized with T. gondii tachyzoites. The NcP0 was encoded by a gene with open reading frame of 936 bp, which encoded a protein of 311 amino acids. The NcP0 gene existed as a single copy in the genome and was interrupted by a 432 bp intron. The NcP0 showed 94.5% amino acid identity to T. gondii P0 (TgP0). Anti-recombinant NcP0 (rNcP0) sera recognized a native parasite protein with a molecular mass of 34 kDa in Western blot analysis. Immunofluorescence analysis showed that the NcP0 was localized to the surface of N. caninum tachyzoites. A purified anti-rNcP0 IgG antibody inhibited the growth of N. caninum and T. gondii in vitro in a concentration-dependent manner. These results indicate that P0 is a cross-reactive antigen between N. caninum and T. gondii and a potential common vaccine candidate to control both parasites.

  11. Thylakoid phosphoproteins

    SciTech Connect

    Coughlan, S.J.; Hind, G.

    1986-01-01

    Thylakoid phosphoproteins were successively fractionated by (1) treatment of /sup 32/P-labeled membranes with 1 M NaBr to remove superficial proteins; (2) extraction with octyl glucoside/cholate; (3) precipitation with ammonium sulfate; (4) size exclusion chromatography on BioGel P300, and (5) sucrose density gradient centrifugation. The detergent extract contained <10% of the original membrane-bound /sup 32/P; it was enriched in cytochrome b/f complex and 64-kDa protein kinase. A 20-kDa protein which copurified with the cytochrome complex and was assumed to be the Rieske protein, was partially phosphorylated. The protein kinase, which phosphorylates itself in vitro, appeared on the sucrose gradient as a phosphoprotein, signalling that it had become labeled in the intact thylakoid. A phosphoprotein of approx.10 kDa which is seen as a product of directly radiolabeling the BioGel P300 extract, was found to differ from the well documented phosphoprotein of this approximate mass that appears in labeled thylakoids. 30 refs., 3 figs., 2 tabs.

  12. The expression of acidic ribosomal phosphoproteins on the surface membrane of different tissues in autoimmune and normal mice which are the target molecules for anti-double-stranded DNA antibodies.

    PubMed Central

    Sun, K H; Liu, W T; Tang, S J; Tsai, C Y; Hsieh, S C; Wu, T H; Han, S H; Yu, C L

    1996-01-01

    Affinity-purified polyclonal anti-double-stranded DNA (anti-dsDNA) antibodies from patients with systemic lupus erythematosus (SLE) exert a cytostatic effect on cultured rat glomerular mesangial cells (MC). The cognate antigens expressed on the surface of MC have been proved to be acidic ribosomal phosphoproteins (P proteins) in our previous study. The mesangial cytostatic effect of anti-dsDNA antibodies is attributed to the cross-reactivity of the antibodies with membrane-expressed P proteins, but not to the effect of minute amounts of anti-ribosomal P proteins antibodies contained in the anti-dsDNA preparations. Immunofluorescence staining of the native cells demonstrated that anti-dsDNA antibodies bound to the surface of rat mesangial cells, rat brain astrocytes (RBA-1) and mouse fibroblasts (3T3). Anti-dsDNA antibodies also exert potent cytostatic effects on these cells in a dose-dependent manner. In addition, the plasma membranes of different cell lines and tissues from normal and autoimmune mice were isolated and probed by anti-dsDNA antibodies in Western blot analysis. We found the actively proliferating cells such as MC, RBA-1 and 3T3 may express both P0 (38,000 MW) and P1 (19,000 MW) on the surface membrane. In addition, the kidney, liver and spleen from either autoimmune MRL-lpr/lpr or BALB/c mice may constantly express P0 protein, but the expression of P1 is inconsistent. In contrast, brain and muscle from either mice failed to express P proteins on their surface. Unexpectedly, a high molecular weight substance (larger than 205,000 MW) with unknown nature appears in the membrane of brain and muscle tissues in both mice. Immunoprecipitation of the surface-biotinylated MC-lysate by anti-dsDNA antibodies further confirmed that P1 (19,000 MW) and P2 (17,000 MW) are really expressed on the cell surface. These results suggest that P proteins expressed on the surface of different tissues become the targets for anti-dsDNA antibodies mediating pleomorphic tissue

  13. Mineral induction by immobilized phosphoproteins

    NASA Technical Reports Server (NTRS)

    Saito, T.; Arsenault, A. L.; Yamauchi, M.; Kuboki, Y.; Crenshaw, M. A.

    1997-01-01

    Dentin phosphoproteins are thought to have a primary role in the deposition of mineral on the collagen of dentin. In this study we determined the type of binding between collagen and phosphoproteins necessary for mineral formation onto collagen fibrils and whether the phosphate esters are required. Bovine dentin phosphophoryn or phosvitin from egg yolk were immobilized on reconstituted skin type I collagen fibrils by adsorption or by covalent cross-linking. In some samples the ester phosphate was removed from the covalently cross-linked phosphoproteins by treatment with acid phosphatase. All samples were incubated at 37 degrees C in metastable solutions that do not spontaneously precipitate. Reconstituted collagen fibrils alone did not induce mineral formation. The phosphoproteins adsorbed to the collagen fibrils desorbed when the mineralization medium was added, and mineral was not induced. The mineral induced by the cross-linked phosphoproteins was apatite, and the crystals were confined to the surface of the collagen fibrils. With decreasing medium saturation the time required for mineral induction increased. The interfacial tensions calculated for apatite formation by either phosphoprotein cross-linked to collagen were about the same as that for phosphatidic acid liposomes and hydroxyapatite. This similarity in values indicates that the nucleation potential of these highly phosphorylated surfaces is about the same. It is concluded that phosphoproteins must be irreversibly bound to collagen fibrils for the mineralization of the collagen network in solutions that do not spontaneously precipitate. The phosphate esters of phosphoproteins are required for mineral induction, and the carboxylate groups are not sufficient.

  14. Mineral induction by immobilized phosphoproteins

    NASA Technical Reports Server (NTRS)

    Saito, T.; Arsenault, A. L.; Yamauchi, M.; Kuboki, Y.; Crenshaw, M. A.

    1997-01-01

    Dentin phosphoproteins are thought to have a primary role in the deposition of mineral on the collagen of dentin. In this study we determined the type of binding between collagen and phosphoproteins necessary for mineral formation onto collagen fibrils and whether the phosphate esters are required. Bovine dentin phosphophoryn or phosvitin from egg yolk were immobilized on reconstituted skin type I collagen fibrils by adsorption or by covalent cross-linking. In some samples the ester phosphate was removed from the covalently cross-linked phosphoproteins by treatment with acid phosphatase. All samples were incubated at 37 degrees C in metastable solutions that do not spontaneously precipitate. Reconstituted collagen fibrils alone did not induce mineral formation. The phosphoproteins adsorbed to the collagen fibrils desorbed when the mineralization medium was added, and mineral was not induced. The mineral induced by the cross-linked phosphoproteins was apatite, and the crystals were confined to the surface of the collagen fibrils. With decreasing medium saturation the time required for mineral induction increased. The interfacial tensions calculated for apatite formation by either phosphoprotein cross-linked to collagen were about the same as that for phosphatidic acid liposomes and hydroxyapatite. This similarity in values indicates that the nucleation potential of these highly phosphorylated surfaces is about the same. It is concluded that phosphoproteins must be irreversibly bound to collagen fibrils for the mineralization of the collagen network in solutions that do not spontaneously precipitate. The phosphate esters of phosphoproteins are required for mineral induction, and the carboxylate groups are not sufficient.

  15. Differential Phosphoprotein Profiling of Tamoxifen Response

    DTIC Science & Technology

    2009-08-01

    need for global phosphoproteome analysis. I have developed a method for comparison of global phosphoprotein profiles involving stable isotope...growth and survival, highlighting the need for global phosphoproteome analysis. Although many biomarkers for breast cancer prognosis and therapy...goal of this project is obtain global phosphoprotein profiles of tamoxifen response and to compare responses in tamoxifen sensitive and resistant cell

  16. Differential Phosphoprotein Profiling of Tamoxifen Response

    DTIC Science & Technology

    2008-08-01

    cataloging global phosphorylation events in response to tamoxifen treatment in tamoxifen sensitive and resistant cells we will provide better... global phosphoprotein profiles. Our methodology involves stable isotope labeling 2, a phosphoprotein affinity step, 1-D SDS-PAGE and LC-MS/MS 3. I...with Xpress ratio less than 0.6 and about 28 proteins with ratio larger than 1.66. Manual analysis is underway to confirm the protein abundance

  17. Oligomerization of Mumps Virus Phosphoprotein

    PubMed Central

    Pickar, Adrian; Elson, Andrew; Yang, Yang; Xu, Pei; Luo, Ming

    2015-01-01

    ABSTRACT The mumps virus (MuV) genome encodes a phosphoprotein (P) that is important for viral RNA synthesis. P forms the viral RNA-dependent RNA polymerase with the large protein (L). P also interacts with the viral nucleoprotein (NP) and self-associates to form a homotetramer. The P protein consists of three domains, the N-terminal domain (PN), the oligomerization domain (PO), and the C-terminal domain (PC). While PN is known to relax the NP-bound RNA genome, the roles of PO and PC are not clear. In this study, we investigated the roles of PO and PC in viral RNA synthesis using mutational analysis and a minigenome system. We found that PN and PC functions can be trans-complemented. However, this complementation requires PO, indicating that PO is essential for P function. Using this trans-complementation system, we found that P forms parallel dimers (PN to PN and PC to PC). Furthermore, we found that residues R231, K238, K253, and K260 in PO are critical for P's functions. We identified PC to be the domain that interacts with L. These results provide structure-function insights into the role of MuV P. IMPORTANCE MuV, a paramyxovirus, is an important human pathogen. The P protein of MuV is critical for viral RNA synthesis. In this work, we established a novel minigenome system that allows the domains of P to be complemented in trans. Using this system, we confirmed that MuV P forms parallel dimers. An understanding of viral RNA synthesis will allow the design of better vaccines and the development of antivirals. PMID:26311887

  18. The Role of Acidic Phosphoproteins in Biomineralization

    PubMed Central

    Alvares, Keith

    2014-01-01

    Biomineralization is the process by which living organisms deposit mineral in the extracellular matrix. In nature, almost 50% of biominerals are calcium-bearing minerals. In addition to calcium, we also find biominerals formed from silica and magnetite. Calcium containing biominerals could be either calcium phosphate as in apatite found in vertebrates or calcium carbonate as in calcite and aragonite found in many invertebrates. Since all biomineralization is matrix mediated, an understanding of the nature of the proteins involved is essential in elucidating its mechanism. This review will discuss some of the proteins involved in the process of biomineralization involving calcium. Two proteins, dentin matrix protein 1 and dentin phosphoprotein (Phosphophoryn) will serve as models for the vertebrate system, and two others - P16 and phosphodontin will serve as models for the invertebrate system. PMID:24437603

  19. The role of acidic phosphoproteins in biomineralization.

    PubMed

    Alvares, Keith

    2014-01-01

    Biomineralization is the process by which living organisms deposit mineral in the extracellular matrix. In nature, almost 50% of biominerals are calcium-bearing minerals. In addition to calcium, we find biominerals formed from silica and magnetite. Calcium-containing biominerals could be either calcium phosphate as in apatite found in vertebrates or calcium carbonate as in calcite and aragonite found in many invertebrates. Since all biomineralization is matrix mediated, an understanding of the nature of the proteins involved is essential in elucidating its mechanism. This review will discuss some of the proteins involved in the process of biomineralization involving calcium. Two proteins, dentin matrix protein 1 and dentin phosphoprotein (Phosphophoryn) will serve as models for the vertebrate system, and two others - P16 and phosphodontin will serve as models for the invertebrate system.

  20. Revealing phosphoproteins playing role in tobacco pollen activated in vitro.

    PubMed

    Fíla, Jan; Matros, Andrea; Radau, Sonja; Zahedi, René Peiman; Capková, Věra; Mock, Hans-Peter; Honys, David

    2012-11-01

    The transition between the quiescent mature and the metabolically active germinating pollen grain most probably involves changes in protein phosphorylation status, since phosphorylation has been implicated in the regulation of many cellular processes. Given that, only a minor proportion of cellular proteins are phosphorylated at any one time, and that phosphorylated and nonphosphorylated forms of many proteins can co-exist within a cell, the identification of phosphoproteins requires some prior enrichment from a crude protein extract. Here, we have used metal oxide/hydroxide affinity chromatography (MOAC) based on an aluminum hydroxide matrix for this purpose, and have generated a population of phosphoprotein candidates from both mature and in vitro activated tobacco pollen grains. Both electrophoretic and nonelectrophoretic methods, allied to MS, were applied to these extracts to identify a set of 139 phosphoprotein candidates. In vitro phosphorylation was also used to validate the spectrum of phosphoprotein candidates obtained by the MOAC phosphoprotein enrichment. Since only one phosphorylation site was detected by the above approach, titanium dioxide phosphopeptide enrichment of trypsinized mature pollen crude extract was performed as well. It resulted in a detection of additional 51 phosphorylation sites giving a total of 52 identified phosphosites in this set of 139 phosphoprotein candidates.

  1. Molecular Evolution of Phosphoprotein Phosphatases in Drosophila

    PubMed Central

    Miskei, Márton; Ádám, Csaba; Kovács, László; Karányi, Zsolt; Dombrádi, Viktor

    2011-01-01

    Phosphoprotein phosphatases (PPP), these ancient and important regulatory enzymes are present in all eukaryotic organisms. Based on the genome sequences of 12 Drosophila species we traced the evolution of the PPP catalytic subunits and noted a substantial expansion of the gene family. We concluded that the 18–22 PPP genes of Drosophilidae were generated from a core set of 8 indispensable phosphatases that are present in most of the insects. Retropositons followed by tandem gene duplications extended the phosphatase repertoire, and sporadic gene losses contributed to the species specific variations in the PPP complement. During the course of these studies we identified 5, up till now uncharacterized phosphatase retrogenes: PpY+, PpD5+, PpD6+, Pp4+, and Pp6+ which are found only in some ancient Drosophila. We demonstrated that all of these new PPP genes exhibit a distinct male specific expression. In addition to the changes in gene numbers, the intron-exon structure and the chromosomal localization of several PPP genes was also altered during evolution. The G−C content of the coding regions decreased when a gene moved into the heterochromatic region of chromosome Y. Thus the PPP enzymes exemplify the various types of dynamic rearrangements that accompany the molecular evolution of a gene family in Drosophilidae. PMID:21789237

  2. Leishmanial phosphatase hydrolyzes phosphoproteins and inositol phosphates

    SciTech Connect

    Saha, A.K.; Das, S.; Glew, R.H.

    1986-05-01

    An extensively purified preparation of the predominant, tartrate-resistant acid phosphatase (ACP) from the external surface of Leishmania donovani promastigotes form catalyzes the dephosphorylation of several phosphoproteins; these include: pyruvate kinase, phosphorylase kinase and histones. However, the protein phosphatase activity of ACP is very low compared with that of other protein phosphates known to be involved in regulating various metabolic pathways. /sup 32/P-labelled inositoltriphosphate (IP3), a well-established second messenger derived from phosphatidylinositol-4,5-diphosphate (PIP2), was a substrate for the leishmanial acid phosphatase; incubation of the IP3 preparation with 13.2 milliunits (1 unit equals 1 ..mu..mol 4-methylumbelliferyl phosphate (MUP) cleaved per min at pH 5.5) of ACP at pH 5.5 for 4 hr resulted in hydrolysis of 75% of the radiolabelled substrate resulting in a mixture of inositoldiphosphate and inositolmonophosphate. In addition PIP2 was hydrolyzed rapidly by ACP at pH 5.5 (V/sub max/, 71 units/mg protein; k/sub m/, 4.16 ..mu..M). In contrast, to MUP which is hydrolzyed most rapidly at pH 5.5, PIP2 hydrolysis was optimal at pH 6.8. These observations raise the possibility that ACP could play a role in the host-phagocyte interaction by degrading the precursor of the second messenger, PIP2 or the second messenger itself, IP3.

  3. Analysis of flagellar phosphoproteins from Chlamydomonas reinhardtii.

    PubMed

    Boesger, Jens; Wagner, Volker; Weisheit, Wolfram; Mittag, Maria

    2009-07-01

    Cilia and flagella are cell organelles that are highly conserved throughout evolution. For many years, the green biflagellate alga Chlamydomonas reinhardtii has served as a model for examination of the structure and function of its flagella, which are similar to certain mammalian cilia. Proteome analysis revealed the presence of several kinases and protein phosphatases in these organelles. Reversible protein phosphorylation can control ciliary beating, motility, signaling, length, and assembly. Despite the importance of this posttranslational modification, the identities of many ciliary phosphoproteins and knowledge about their in vivo phosphorylation sites are still missing. Here we used immobilized metal affinity chromatography to enrich phosphopeptides from purified flagella and analyzed them by mass spectrometry. One hundred forty-one phosphorylated peptides were identified, belonging to 32 flagellar proteins. Thereby, 126 in vivo phosphorylation sites were determined. The flagellar phosphoproteome includes different structural and motor proteins, kinases, proteins with protein interaction domains, and many proteins whose functions are still unknown. In several cases, a dynamic phosphorylation pattern and clustering of phosphorylation sites were found, indicating a complex physiological status and specific control by reversible protein phosphorylation in the flagellum.

  4. Post-translational processing of chicken bone phosphoproteins. Identification of bone (phospho)protein kinase.

    PubMed Central

    Mikuni-Takagaki, Y; Glimcher, M J

    1990-01-01

    We have detected a protein kinase which phosphorylates bone phosphoproteins (BPPs) in the detergent extract of the membranous fractions in the periosteal bone strips of 12-day-embryonic-chick tibia. This enzyme, tentatively named BPP kinase, has a catalytic subunit of Mr approximately 39,000, utilizes GTP as well as ATP as a phospho-group donor, is inhibited by 2,3-bisphosphoglycerate and heparin, and is therefore similar to casein kinase II. The enzyme can phosphorylate dephosphorylated proteins such as casein, phosvitin and chicken BPPs, but the last-named are preferred substrates. The in vitro-phosphorylation-assay products of this enzyme in the extract were indistinguishable on an SDS/polyacrylamide gel from the major [32P]phosphoproteins metabolically labelled in the embryonic-chick bone tissue. The regulatory mechanisms of the phosphorylation process of BPPs by BPP kinase as well as the potential role of this enzyme in mineralization are discussed. Images Fig. 1. Fig. 4. PMID:2363697

  5. Phosphoproteins and protein kinases of the Golgi apparatus membrane

    SciTech Connect

    Capasso, J.M.; Abeijon, C.; Hirschberg, C.B.

    1985-11-25

    Incubation of a highly purified fraction derived from rat liver Golgi apparatus with (gamma-TSP)ATP results in phosphorylation of several endogenous phosphoproteins. One phosphoprotein with an apparent Mr of 48,300 is radiolabeled to an apparent extent at least 5-fold higher than any other phosphoprotein as part of either the Golgi apparatus or highly purified rat liver fractions derived from the rough endoplasmic reticulum, mitochondria, plasma membrane, coated vesicles, cytosol, and total homogenate. Approximately 70% of the 48.3-kDa phosphoprotein appears to be a specific extrinsic Golgi membrane protein with the phosphorylated amino acid being threonine. The protein kinase which phosphorylates the 48.3-kDa protein is an intrinsic Golgi membrane protein and is dependent on MgS , independent of CaS , calmodulin, and cAMP, and is inhibited by N-ethylmaleimide. Preliminary evidence suggests that there are also intrinsic membrane protein kinases in the Golgi apparatus which are dependent on CaS and cAMP. The physiological role of the above phosphoproteins and protein kinases is not known.

  6. Rapid, Multiplexed Phosphoprotein Profiling Using Silicon Photonic Sensor Arrays

    PubMed Central

    2015-01-01

    Extracellular signaling is commonly mediated through post-translational protein modifications that propagate messages from membrane-bound receptors to ultimately regulate gene expression. Signaling cascades are ubiquitously intertwined, and a full understanding of function can only be gleaned by observing dynamics across multiple key signaling nodes. Importantly, targets within signaling cascades often represent opportunities for therapeutic development or can serve as diagnostic biomarkers. Protein phosphorylation is a particularly important post-translational modification that controls many essential cellular signaling pathways. Not surprisingly, aberrant phosphorylation is found in many human diseases, including cancer, and phosphoprotein-based biomarker signatures hold unrealized promise for disease monitoring. Moreover, phosphoprotein analysis has wide-ranging applications across fundamental chemical biology, as many drug discovery efforts seek to target nodes within kinase signaling pathways. For both fundamental and translational applications, the analysis of phosphoprotein biomarker targets is limited by a reliance on labor-intensive and/or technically challenging methods, particularly when considering the simultaneous monitoring of multiplexed panels of phosphoprotein biomarkers. We have developed a technology based upon arrays of silicon photonic microring resonator sensors that fills this void, facilitating the rapid and automated analysis of multiple phosphoprotein levels from both cell lines and primary human tumor samples requiring only minimal sample preparation. PMID:26539563

  7. Localization of Phosphoproteins within the Barnacle Adhesive Interface.

    PubMed

    Dickinson, Gary H; Yang, Xu; Wu, Fanghui; Orihuela, Beatriz; Rittschof, Dan; Beniash, Elia

    2016-06-01

    Barnacles permanently adhere to nearly any inert substrate using proteinaceous glue. The glue consists of at least ten major proteins, some of which have been isolated and sequenced. Questions still remain about the chemical mechanisms involved in adhesion and the potential of the glue to serve as a platform for mineralization of the calcified base plate. We tested the hypothesis that barnacle glue contains phosphoproteins, which have the potential to play a role in both adhesion and mineralization. Using a combination of phosphoprotein-specific gel staining and Western blotting with anti-phosphoserine antibody, we identified multiple phosphorylated proteins in uncured glue secretions from the barnacle Amphibalanus amphitrite The protein composition of the glue and the quantity and abundance of phosphoproteins varied distinctly among individual barnacles, possibly due to cyclical changes in the glue secretion over time. We assessed the location of the phosphoproteins within the barnacle glue layer using decalcified barnacle base plates and residual glue deposited by reattached barnacles. Phosphoproteins were found throughout the organic matrix of the base plate and within the residual glue. Staining within the residual glue appeared most intensely in regions where capillary glue ducts, which are involved in cyclical release of glue, had been laid down. Lastly, mineralization studies of glue proteins separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) indicated that proteins identified as phosphorylated possibly induce mineralization of calcium carbonate (CaCO3). These results contribute to our understanding of the protein composition of barnacle glue, and provide new insights into the potential roles of phosphoproteins in underwater bioadhesives.

  8. RIBOSOME-MEMBRANE INTERACTION

    PubMed Central

    Adelman, M. R.; Sabatini, David D.; Blobel, Günter

    1973-01-01

    In a medium of high ionic strength, rat liver rough microsomes can be nondestructively disassembled into ribosomes and stripped membranes if nascent polypeptides are discharged from the bound ribosomes by reaction with puromycin. At 750 mM KCl, 5 mM MgCl2, 50 mM Tris·HCl, pH 7 5, up to 85% of all bound ribosomes are released from the membranes after incubation at room temperature with 1 mM puromycin. The ribosomes are released as subunits which are active in peptide synthesis if programmed with polyuridylic acid. The ribosome-denuded, or stripped, rough microsomes (RM) can be recovered as intact, essentially unaltered membranous vesicles Judging from the incorporation of [3H]puromycin into hot acid-insoluble material and from the release of [3H]leucine-labeled nascent polypeptide chains from bound ribosomes, puromycin coupling occurs almost as well at low (25–100 mM) as at high (500–1000 mM) KCl concentrations. Since puromycin-dependent ribosome release only occurs at high ionic strength, it appears that ribosomes are bound to membranes via two types of interactions: a direct one between the membrane and the large ribosomal subunit (labile at high KCl concentration) and an indirect one in which the nascent chain anchors the ribosome to the membrane (puromycin labile). The nascent chains of ribosomes specifically released by puromycin remain tightly associated with the stripped membranes. Some membrane-bound ribosomes (up to 40%) can be nondestructively released in high ionic strength media without puromycin; these appear to consist of a mixture of inactive ribosomes and ribosomes containing relatively short nascent chains. A fraction (∼15%) of the bound ribosomes can only be released from membranes by exposure of RM to ionic conditions which cause extensive unfolding of ribosomal subunits, the nature and significance of these ribosomes is not clear. PMID:4682341

  9. Driving ribosome assembly.

    PubMed

    Kressler, Dieter; Hurt, Ed; Bassler, Jochen

    2010-06-01

    Ribosome biogenesis is a fundamental process that provides cells with the molecular factories for cellular protein production. Accordingly, its misregulation lies at the heart of several hereditary diseases (e.g., Diamond-Blackfan anemia). The process of ribosome assembly comprises the processing and folding of the pre-rRNA and its concomitant assembly with the ribosomal proteins. Eukaryotic ribosome biogenesis relies on a large number (>200) of non-ribosomal factors, which confer directionality and accuracy to this process. Many of these non-ribosomal factors fall into different families of energy-consuming enzymes, notably including ATP-dependent RNA helicases, AAA-ATPases, GTPases, and kinases. Ribosome biogenesis is highly conserved within eukaryotic organisms; however, due to the combination of powerful genetic and biochemical methods, it is best studied in the yeast Saccharomyces cerevisiae. This review summarizes our current knowledge on eukaryotic ribosome assembly, with particular focus on the molecular role of the involved energy-consuming enzymes.

  10. Isolation of Mitochondrial Ribosomes.

    PubMed

    Carroll, Adam J

    2017-01-01

    Translation of mitochondrial encoded mRNAs by mitochondrial ribosomes is thought to play a major role in regulating the expression of mitochondrial proteins. However, the structure and function of plant mitochondrial ribosomes remains poorly understood. To study mitochondrial ribosomes, it is necessary to separate them from plastidic and cytosolic ribosomes that are generally present at much higher concentrations. Here, a straight forward protocol for the preparation of fractions highly enriched in mitochondrial ribosomes from plant cells is described. The method begins with purification of mitochondria followed by mitochondrial lysis and ultracentrifugation of released ribosomes through sucrose cushions and gradients. Dark-grown Arabidopsis cells were used in this example because of the ease with which good yields of pure mitochondria can be obtained from them. However, the steps for isolation of ribosomes from mitochondria could be applied to mitochondria obtained from other sources. Proteomic analyses of resulting fractions have confirmed strong enrichment of mitochondrial ribosomal proteins.

  11. The Ribosome Filter Redux

    PubMed Central

    Mauro, Vincent P.; Edelman, Gerald M.

    2010-01-01

    The ribosome filter hypothesis postulates that ribosomes are not simply translation machines but also function as regulatory elements that differentially affect or filter the translation of particular mRNAs. On the basis of new information, we take the opportunity here to review the ribosome filter hypothesis, suggest specific mechanisms of action, and discuss recent examples from the literature that support it. PMID:17890902

  12. Identification of phosphoproteins in Arabidopsis thaliana leaves using polyethylene glycol fractionation, immobilized metal-ion affinity chromatography, two-dimensional gel electrophoresis and mass spectrometry.

    PubMed

    Aryal, Uma K; Krochko, Joan E; Ross, Andrew R S

    2012-01-01

    Reversible protein phosphorylation is a key regulatory mechanism in cells. Identification and characterization of phosphoproteins requires specialized enrichment methods, due to the relatively low abundance of these proteins, and is further complicated in plants by the high abundance of Rubisco in green tissues. We present a novel method for plant phosphoproteome analysis that depletes Rubisco using polyethylene glycol fractionation and utilizes immobilized metal-ion affinity chromatography to enrich phosphoproteins. Subsequent protein separation by one- and two-dimensional gel electrophoresis is further improved by extracting the PEG-fractionated protein samples with SDS/phenol and methanol/chloroform to remove interfering compounds. Using this approach, we identified 132 phosphorylated proteins in a partial Arabidopsis leaf extract. These proteins are involved in a range of biological processes, including CO(2) fixation, protein assembly and folding, stress response, redox regulation, and cellular metabolism. Both large and small subunits of Rubisco were phosphorylated at multiple sites, and depletion of Rubisco enhanced detection of less abundant phosphoproteins, including those associated with state transitions between photosystems I and II. The discovery of a phosphorylated form of AtGRP7, a self-regulating RNA-binding protein that affects floral transition, as well as several previously uncharacterized ribosomal proteins confirm the utility of this approach for phosphoproteome analysis and its potential to increase our understanding of growth and development in plants.

  13. Enrichment and Analysis of Intact Phosphoproteins in Arabidopsis Seedlings.

    PubMed

    Aryal, Uma K; Ross, Andrew R S; Krochko, Joan E

    2015-01-01

    Protein phosphorylation regulates diverse cellular functions and plays a key role in the early development of plants. To complement and expand upon previous investigations of protein phosphorylation in Arabidopsis seedlings we used an alternative approach that combines protein extraction under non-denaturing conditions with immobilized metal-ion affinity chromatography (IMAC) enrichment of intact phosphoproteins in Rubisco-depleted extracts, followed by identification using two-dimensional gel electrophoresis (2-DE) and liquid chromatography-tandem mass spectrometry (LC-MS/MS). In-gel trypsin digestion and analysis of selected gel spots identified 144 phosphorylated peptides and residues, of which only 18 phosphopeptides and 8 phosphosites were found in the PhosPhAt 4.0 and P3DB Arabidopsis thaliana phosphorylation site databases. More than half of the 82 identified phosphoproteins were involved in carbohydrate metabolism, photosynthesis/respiration or oxidative stress response mechanisms. Enrichment of intact phosphoproteins prior to 2-DE and LC-MS/MS appears to enhance detection of phosphorylated threonine and tyrosine residues compared with methods that utilize peptide-level enrichment, suggesting that the two approaches are somewhat complementary in terms of phosphorylation site coverage. Comparing results for young seedlings with those obtained previously for mature Arabidopsis leaves identified five proteins that are differentially phosphorylated in these tissues, demonstrating the potential of this technique for investigating the dynamics of protein phosphorylation during plant development.

  14. Structure of an Enzyme-Derived Phosphoprotein Recognition Domain

    PubMed Central

    Johnston, Christopher A.; Doe, Chris Q.; Prehoda, Kenneth E.

    2012-01-01

    Membrane Associated Guanylate Kinases (MAGUKs) contain a protein interaction domain (GKdom) derived from the enzyme Guanylate Kinase (GKenz). Here we show that GKdom from the MAGUK Discs large (Dlg) is a phosphoprotein recognition domain, specifically recognizing the phosphorylated form of the mitotic spindle orientation protein Partner of Inscuteable (Pins). We determined the structure of the Dlg-Pins complex to understand the dramatic transition from nucleotide kinase to phosphoprotein recognition domain. The structure reveals that the region of the GKdom that once served as the GMP binding domain (GBD) has been co-opted for protein interaction. Pins makes significantly more contact with the GBD than does GMP, but primarily with residues that are conserved between enzyme and domain revealing the versatility of the GBD as a platform for nucleotide and protein interactions. Mutational analysis reveals that the GBD is also used to bind the GK ligand MAP1a, suggesting that this is a common mode of MAGUK complex assembly. The GKenz undergoes a dramatic closing reaction upon GMP binding but the protein-bound GKdom remains in the ‘open’ conformation indicating that the dramatic conformational change has been lost in the conversion from nucleotide kinase to phosphoprotein recognition domain. PMID:22545154

  15. The Modular Adaptive Ribosome.

    PubMed

    Yadav, Anupama; Radhakrishnan, Aparna; Panda, Anshuman; Singh, Amartya; Sinha, Himanshu; Bhanot, Gyan

    2016-01-01

    The ribosome is an ancient machine, performing the same function across organisms. Although functionally unitary, recent experiments suggest specialized roles for some ribosomal proteins. Our central thesis is that ribosomal proteins function in a modular fashion to decode genetic information in a context dependent manner. We show through large data analyses that although many ribosomal proteins are essential with consistent effect on growth in different conditions in yeast and similar expression across cell and tissue types in mice and humans, some ribosomal proteins are used in an environment specific manner. The latter set of variable ribosomal proteins further function in a coordinated manner forming modules, which are adapted to different environmental cues in different organisms. We show that these environment specific modules of ribosomal proteins in yeast have differential genetic interactions with other pathways and their 5'UTRs show differential signatures of selection in yeast strains, presumably to facilitate adaptation. Similarly, we show that in higher metazoans such as mice and humans, different modules of ribosomal proteins are expressed in different cell types and tissues. A clear example is nervous tissue that uses a ribosomal protein module distinct from the rest of the tissues in both mice and humans. Our results suggest a novel stratification of ribosomal proteins that could have played a role in adaptation, presumably to optimize translation for adaptation to diverse ecological niches and tissue microenvironments.

  16. The Modular Adaptive Ribosome

    PubMed Central

    Yadav, Anupama; Radhakrishnan, Aparna; Panda, Anshuman; Singh, Amartya; Sinha, Himanshu; Bhanot, Gyan

    2016-01-01

    The ribosome is an ancient machine, performing the same function across organisms. Although functionally unitary, recent experiments suggest specialized roles for some ribosomal proteins. Our central thesis is that ribosomal proteins function in a modular fashion to decode genetic information in a context dependent manner. We show through large data analyses that although many ribosomal proteins are essential with consistent effect on growth in different conditions in yeast and similar expression across cell and tissue types in mice and humans, some ribosomal proteins are used in an environment specific manner. The latter set of variable ribosomal proteins further function in a coordinated manner forming modules, which are adapted to different environmental cues in different organisms. We show that these environment specific modules of ribosomal proteins in yeast have differential genetic interactions with other pathways and their 5’UTRs show differential signatures of selection in yeast strains, presumably to facilitate adaptation. Similarly, we show that in higher metazoans such as mice and humans, different modules of ribosomal proteins are expressed in different cell types and tissues. A clear example is nervous tissue that uses a ribosomal protein module distinct from the rest of the tissues in both mice and humans. Our results suggest a novel stratification of ribosomal proteins that could have played a role in adaptation, presumably to optimize translation for adaptation to diverse ecological niches and tissue microenvironments. PMID:27812193

  17. How Ribosomes Translate Cancer.

    PubMed

    Sulima, Sergey O; Hofman, Isabel J F; De Keersmaecker, Kim; Dinman, Jonathan D

    2017-09-18

    A wealth of novel findings, including congenital ribosomal mutations in ribosomopathies and somatic ribosomal mutations in various cancers, have significantly increased our understanding of the relevance of ribosomes in oncogenesis. Here, we explore the growing list of mechanisms by which the ribosome is involved in carcinogenesis-from the hijacking of ribosomes by oncogenic factors and dysregulated translational control, to the effects of mutations in ribosomal components on cellular metabolism. Of clinical importance, the recent success of RNA polymerase inhibitors highlights the dependence on "onco-ribosomes" as an Achilles' heel of cancer cells and a promising target for further therapeutic intervention.Significance: The recent discovery of somatic mutations in ribosomal proteins in several cancers has strengthened the link between ribosome defects and cancer progression, while also raising the question of which cellular mechanisms such defects exploit. Here, we discuss the emerging molecular mechanisms by which ribosomes support oncogenesis, and how this understanding is driving the design of novel therapeutic strategies. Cancer Discov; 7(10); 1-19. ©2017 AACR. ©2017 American Association for Cancer Research.

  18. Combined effect of tissue stabilization and protein extraction methods on phosphoprotein analysis.

    PubMed

    Kofanova, Olga A; Fack, Fred; Niclou, Simone P; Betsou, Fay

    2013-06-01

    Preanalytical conditions applied during sample collection and processing can affect the detection or quantification of unstable phosphoprotein biomarkers. We evaluated the consequences of tissue stabilization and protein extraction methods on phosphoprotein analysis. The effects of stabilization techniques (heat stabilization, snap-freezing) and time on the levels of phosphoproteins, including phospho-Akt, p-ERK 1/2, p-IkBα, p-JNK, and p38 MAPK, were evaluated using a BioPlex phosphoprotein assay. Additionally, two different protein extraction protocols, using different extraction buffers (8 M urea buffer, or Bio-Rad buffer without urea) were tested. For snap-frozen samples, protein extraction yields were comparable with the two buffer systems. For heat-stabilized samples, total protein yields were significantly lower following extraction in non-urea buffer. However, the concentrations of specific phosphoproteins were significantly higher in heat-stabilized samples than in the corresponding snap-frozen samples, indicating that this tissue processing method better preserved phosphoproteins. Significant differences were found between the measured phosphoprotein levels in heat-stabilized and snap-frozen tissue, suggesting that alterations occur very rapidly after tissue excision. Our results suggest that heat stabilization can be used as a tissue processing method for subsequent phosphoprotein analyses, but also suggest that the BioPlex phosphoprotein assay could be used as a possible quality control method to assess tissue sample integrity.

  19. The ribosomal database project.

    PubMed

    Larsen, N; Olsen, G J; Maidak, B L; McCaughey, M J; Overbeek, R; Macke, T J; Marsh, T L; Woese, C R

    1993-07-01

    The Ribosomal Database Project (RDP) is a curated database that offers ribosome data along with related programs and services. The offerings include phylogenetically ordered alignments of ribosomal RNA (rRNA) sequences, derived phylogenetic trees, rRNA secondary structure diagrams and various software packages for handling, analyzing and displaying alignments and trees. The data are available via ftp and electronic mail. Certain analytic services are also provided by the electronic mail server.

  20. The ribosomal database project.

    PubMed Central

    Larsen, N; Olsen, G J; Maidak, B L; McCaughey, M J; Overbeek, R; Macke, T J; Marsh, T L; Woese, C R

    1993-01-01

    The Ribosomal Database Project (RDP) is a curated database that offers ribosome data along with related programs and services. The offerings include phylogenetically ordered alignments of ribosomal RNA (rRNA) sequences, derived phylogenetic trees, rRNA secondary structure diagrams and various software packages for handling, analyzing and displaying alignments and trees. The data are available via ftp and electronic mail. Certain analytic services are also provided by the electronic mail server. PMID:8332524

  1. The Ribosomal Database Project.

    PubMed

    Maidak, B L; Larsen, N; McCaughey, M J; Overbeek, R; Olsen, G J; Fogel, K; Blandy, J; Woese, C R

    1994-09-01

    The Ribosomal Database Project (RDP) is a curated database that offers ribosome-related data, analysis services, and associated computer programs. The offerings include phylogenetically ordered alignments of ribosomal RNA (rRNA) sequences, derived phylogenetic trees, rRNA secondary structure diagrams, and various software for handling, analyzing and displaying alignments and trees. The data are available via anonymous ftp (rdp.life.uiuc.edu), electronic mail (server/rdp.life.uiuc.edu) and gopher (rdpgopher.life.uiuc.edu). The electronic mail server also provides ribosomal probe checking, approximate phylogenetic placement of user-submitted sequences, screening for chimeric nature of newly sequenced rRNAs, and automated alignment.

  2. Phosphoprotein phosphatase of bovine spleen cell nuclei: physicochemical properties

    SciTech Connect

    Rezyapkin, V.I.; Leonova, L.E.; Komkova, A.I.

    1986-01-10

    The physicochemical properties of phosphoprotein phosphatase (EC 1.3.1.16) from bovine spleen cell nuclei were studied. The enzyme possesses broad substrate specificity and catalyzes the dephosphorylation of phosphocasein, ATP, ADP, and p-nitrophenyl phosphate (pNPP). K/sub m/ for ATP, ADP, and pNPP are equal to 0.44, 0.43, and 1.25 mM, respectively. M/sub r/ of the enzyme, according to the data of gel filtraction of Sephadex G-75 and electrophoresis in polyacrylamide gel of various concentrations is approx. 33,000. In electrophoresis in the presence of SDS, two protein bands with M/sub r/ 12,000 and 18,000 are detected. In the enzyme molecule, acid amino acid residues predominate; two free SH groups and two disulfide bridges are detected. Phosphoprotein phosphatase is a glycoprotein, containing approx. 22% carbonhydrates. The protein possesses a supplementary absorption maximum at 560 nm. Ammonium molybdate is a competitive inhibitor with K/sub i/ 0.37 ..mu..M, while sodium fluoride is a noncompetitive inhibitor with K/sub i/ 1.3 mM. Incubation in the presence of 2 mM phenylmethylsulfonyl fluoride for 25 h leads to a loss of approx. 46% of the enzymatic activity. Ammonium molybdate, sodium fluoride, and PMSF are reversible inhibitors. Modifications of the SH groups, NH/sub 2/ groups, and histidine leads to a decrease in the enzymatic activity. Incubation of phosphoprotein phosphatase with (..gamma..-/sup 32/P)ATP leads to the incorporation of 0.33 mole /sup 33/P per mole of the enzyme. The mechanism of hydrolysis of the phosphodiester bond, catalyzed by the enzyme, is discussed.

  3. Involvement of the Rabies Virus Phosphoprotein Gene in Neuroinvasiveness

    PubMed Central

    Yamaoka, Satoko; Ito, Naoto; Ohka, Seii; Kaneda, Shohei; Nakamura, Hiroko; Agari, Takahiro; Masatani, Tatsunori; Nakagawa, Keisuke; Okada, Kazuma; Okadera, Kota; Mitake, Hiromichi; Fujii, Teruo

    2013-01-01

    Rabies virus (RABV), which is transmitted via a bite wound caused by a rabid animal, infects peripheral nerves and then spreads to the central nervous system (CNS) before causing severe neurological symptoms and death in the infected individual. Despite the importance of this ability of the virus to spread from a peripheral site to the CNS (neuroinvasiveness) in the pathogenesis of rabies, little is known about the mechanism underlying the neuroinvasiveness of RABV. In this study, to obtain insights into the mechanism, we conducted comparative analysis of two fixed RABV strains, Nishigahara and the derivative strain Ni-CE, which cause lethal and asymptomatic infections, respectively, in mice after intramuscular inoculation. Examination of a series of chimeric viruses harboring the respective genes from Nishigahara in the genetic background of Ni-CE revealed that the Nishigahara phosphoprotein (P) gene plays a major role in the neuroinvasiveness by mediating infection of peripheral nerves. The results obtained from both in vivo and in vitro experiments strongly suggested that the Nishigahara P gene, but not the Ni-CE P gene, is important for stable viral replication in muscle cells. Further investigation based on the previous finding that RABV phosphoprotein counteracts the host interferon (IFN) system demonstrated that the Nishigahara P gene, but not the Ni-CE P gene, functions to suppress expression of the beta interferon (IFN-β) gene (Ifn-β) and IFN-stimulated genes in muscle cells. In conclusion, we provide the first data strongly suggesting that RABV phosphoprotein assists viral replication in muscle cells by counteracting the host IFN system and, consequently, enhances infection of peripheral nerves. PMID:24027304

  4. The relationship between the MMP system, adrenoceptors and phosphoprotein phosphatases

    PubMed Central

    Rietz, A; Spiers, JP

    2012-01-01

    The MMPs and their inhibitors [tissue inhibitor of MMPs (TIMPs) ] form the mainstay of extracellular matrix homeostasis. They are expressed in response to numerous stimuli including cytokines and GPCR activation. This review highlights the importance of adrenoceptors and phosphoprotein phosphatases (PPP) in regulating MMPs in the cardiovascular system, which may help explain some of the beneficial effects of targeting the adrenoceptor system in tissue remodelling and will establish emerging crosstalk between these three systems. Although α- and β-adrenoceptor activation increases MMP but decreases TIMP expression, MMPs are implicated in the growth stimulatory effects of adrenoceptor activation through transactivation of epidermal growth factor receptor. Furthermore, they have recently been found to catalyse the proteolysis of β-adrenoceptors and modulate vascular tone. While the mechanisms underpinning these effects are not well defined, reversible protein phosphorylation by kinases and phosphatases may be key. In particular, PPP (Ser/Thr phosphatases) are not only critical in resensitization and internalization of adrenoceptors but also modulate MMP expression. The interrelationship is complex as isoprenaline (ISO) inhibits okadaic acid [phosphoprotein phosphatase type 1/phosphoprotein phosphatase type 2A (PP2A) inhibitor]-mediated MMP expression. While this may be simply due to its ability to transiently increase PP2A activity, there is evidence for MMP-9 that ISO prevents okadaic acid-mediated expression of MMP-9 through a β-arrestin, NF-κB-dependent pathway, which is abolished by knock-down of PP2A. It is essential that crosstalk between MMPs, adrenoceptors and PPP are investigated further as it will provide important insight into how adrenoceptors modulate cardiovascular remodelling, and may identify new targets for pharmacological manipulation of the MMP system. PMID:22364165

  5. The effect of complexing phosphoproteins to decalcified collagen on in vitro calcification.

    PubMed

    Endo, A; Glimcher, M J

    1989-01-01

    Decalcified samples of chicken bone containing phosphoproteins of varying concentrations were used to assess the effect of phosphoproteins and of protein-bound Ser(P) and Thr(P) in the in vitro nucleation of a Ca-P solid phase from metastable solutions of Ca and P. Phosphoproteins of bone as well as the phosphoproteins from egg yolk (phosvitin) were used. Increasing concentrations of phosphoprotein [as measured by the amount of protein bound Ser(P) and Thr(P)] in the decalcified bone particles significantly reduced the time required for nucleation to occur after exposure to metastable solutions of Ca and P (decreased operational lag times). Treatment with wheat germ acid phosphatase markedly reduced the concentration of Ser(P) and Thr(P) in the decalcified bone samples and in the decalcified bone collagen samples complexed with phosphoproteins (almost to zero). The loss of the organic phosphate groups significantly increased the operational lag time, but did not abolish nucleation of apatite crystals by the bone collagen fibrils essentially devoid of Ser(P) and Thr(P). Bone phosphoproteins were not specific; substitution of phosvitin for bone phosphoproteins as complexes with bone collagen also proved to be effective facilitators of nucleation, which was interesting since both types of phosphoproteins have certain common chemical and structural characteristics. Noncollagenous components other than phosphoproteins were present in the decalcified bone samples. However, the marked dependence of the lag time on the Ser(P) and Thr(P) concentrations and the very marked diminution in the efficacy of the nucleation phenomenon as a result of treatment with wheat germ acid phosphatase, clearly suggests that the organic phosphate residues of the phosphoproteins play a direct and significant role in the process of in vitro nucleation of a solid phase of Ca and P (apatite) by bone collagen, and by implication, possibly in in vivo mineralization as well.

  6. The Ribosomal Database Project

    PubMed Central

    Olsen, Gary J.; Overbeek, Ross; Larsen, Niels; Marsh, Terry L.; McCaughey, Michael J.; Maciukenas, Michael A.; Kuan, Wen-Min; Macke, Thomas J.; Xing, Yuqing; Woese, Carl R.

    1992-01-01

    The Ribosomal Database Project (RDP) compiles ribosomal sequences and related data, and redistributes them in aligned and phylogenetically ordered form to its user community. It also offers various software packages for handling, analyzing and displaying sequences. In addition, the RDP offers (or will offer) certain analytic services. At present the project is in an intermediate stage of development. PMID:1598241

  7. The Ribosomal Database Project

    NASA Technical Reports Server (NTRS)

    Olsen, G. J.; Overbeek, R.; Larsen, N.; Marsh, T. L.; McCaughey, M. J.; Maciukenas, M. A.; Kuan, W. M.; Macke, T. J.; Xing, Y.; Woese, C. R.

    1992-01-01

    The Ribosomal Database Project (RDP) complies ribosomal sequences and related data, and redistributes them in aligned and phylogenetically ordered form to its user community. It also offers various software packages for handling, analyzing and displaying sequences. In addition, the RDP offers (or will offer) certain analytic services. At present the project is in an intermediate stage of development.

  8. The Ribosomal Database Project.

    PubMed

    Olsen, G J; Overbeek, R; Larsen, N; Marsh, T L; McCaughey, M J; Maciukenas, M A; Kuan, W M; Macke, T J; Xing, Y; Woese, C R

    1992-05-11

    The Ribosomal Database Project (RDP) complies ribosomal sequences and related data, and redistributes them in aligned and phylogenetically ordered form to its user community. It also offers various software packages for handling, analyzing and displaying sequences. In addition, the RDP offers (or will offer) certain analytic services. At present the project is in an intermediate stage of development.

  9. Queuine mediated inhibition in phosphorylation of tyrosine phosphoproteins in cancer.

    PubMed

    Pathak, Chandramani; Jaiswal, Yogesh K; Vinayak, Manjula

    2008-09-01

    Protein phosphorylation or dephosphorylation is the most important regulatory switch of signal transduction contributing to control of cell proliferation. The reversibility of phosphorylation and dephosphorylation is due to the activities of kinases and phosphatase, which determine protein phosphorylation level of cell under different physiological and pathological conditions. Receptor tyrosine kinase (RTK) mediated cellular signaling is precisely coordinated and tightly controlled in normal cells which ensures regulated mitosis. Deregulation of RTK signaling resulting in aberrant activation in RTKs leads to malignant transformation. Queuine is one of the modified base of tRNA which participates in down regulation of tyrosine kinase activity. The guanine analogue queuine is a nutrient factor to eukaryotes and occurs as free base or modified nucleoside queuosine into the first anticodon position of specific tRNAs. The tRNAs are often queuine deficient in cancer and fast proliferating tissues. The present study is aimed to investigate queuine mediated inhibition in phosphorylation of tyrosine phosphorylated proteins in lymphoma bearing mouse. The result shows high level of cytosolic and membrane associated tyrosine phosphoprotein in DLAT cancerous mouse liver compared to normal. Queuine treatments down regulate the level of tyrosine phosphoproteins, which suggests that queuine is involved in regulation of mitotic signaling pathways.

  10. Adsorption and interactions of dentine phosphoprotein with hydroxyapatite and collagen.

    PubMed

    Milan, Anna M; Sugars, Rachael V; Embery, Graham; Waddington, Rachel J

    2006-06-01

    Dentine phosphoprotein (DPP) has been proposed to both promote and inhibit mineral deposition during dentinogenesis. The present study aimed to investigate the molecular interactions of DPP and dephosphorylated DPP (DPP-p) with hydroxyapatite (HAP). Bovine DPP was purified and dephosphorylated by alkaline phosphatase to obtain DPP-p. DPP and DPP-p adsorption to HAP was determined along with their ability, when free in solution or bound to collagen, to influence HAP-induced crystal growth. Absorption isotherms suggested that lower DPP concentrations (1.5-6.25 microg ml(-1)) demonstrated a reduced affinity for HAP compared with higher protein concentrations (12.5-50.0 microg ml(-1)). Dephosphorylated DPP had a much reduced affinity for HAP compared with DPP. Dentine phosphoprotein inhibited seeded HAP crystal growth, in a dose-dependent manner, whilst removal of the phosphate groups reduced this inhibition. When bound to collagen fibrils, DPP significantly promoted the rate of HAP crystal growth over 0-8 min. Conversely, DPP-p and collagen significantly decreased the rate of crystal growth over 0-18 min. These results indicate a major role for the phosphate groups present on DPP in HAP crystal growth. In addition, concentration-dependent conformational changes to DPP, and the interaction with other matrix components, such as collagen, are important in predicting its dual role in the mineralization of dentine.

  11. Functional Specialization of Ribosomes?

    PubMed Central

    Gilbert, Wendy V.

    2011-01-01

    Ribosomes are highly conserved macromolecular machines responsible for protein synthesis in all living organisms. Work published in the past year shows that changes to the ribosome core can affect the mechanism of translation initiation that is favored in the cell, potentially leading to specific changes in the relative efficiencies with which different proteins are made. Here I examine recent data from expression and proteomic studies suggesting that cells make slightly different ribosomes under different growth conditions and discuss genetic evidence that such differences are functional. In particular, I will argue that eukaryotic cells likely produce ribosomes that lack one or more ‘core’ ribosomal proteins (RPs) under some conditions, and that ‘core’ RPs contribute differentially to translation of distinct subpopulations of mRNAs. PMID:21242088

  12. Assembly of bacterial ribosomes.

    PubMed

    Shajani, Zahra; Sykes, Michael T; Williamson, James R

    2011-01-01

    The assembly of ribosomes from a discrete set of components is a key aspect of the highly coordinated process of ribosome biogenesis. In this review, we present a brief history of the early work on ribosome assembly in Escherichia coli, including a description of in vivo and in vitro intermediates. The assembly process is believed to progress through an alternating series of RNA conformational changes and protein-binding events; we explore the effects of ribosomal proteins in driving these events. Ribosome assembly in vivo proceeds much faster than in vitro, and we outline the contributions of several of the assembly cofactors involved, including Era, RbfA, RimJ, RimM, RimP, and RsgA, which associate with the 30S subunit, and CsdA, DbpA, Der, and SrmB, which associate with the 50S subunit.

  13. Powering through ribosome assembly

    PubMed Central

    Strunk, Bethany S.; Karbstein, Katrin

    2009-01-01

    Ribosome assembly is required for cell growth in all organisms. Classic in vitro work in bacteria has led to a detailed understanding of the biophysical, thermodynamic, and structural basis for the ordered and correct assembly of ribosomal proteins on ribosomal RNA. Furthermore, it has enabled reconstitution of active subunits from ribosomal RNA and proteins in vitro. Nevertheless, recent work has shown that eukaryotic ribosome assembly requires a large macromolecular machinery in vivo. Many of these assembly factors such as ATPases, GTPases, and kinases hydrolyze nucleotide triphosphates. Because these enzymes are likely regulatory proteins, much work to date has focused on understanding their role in the assembly process. Here, we review these factors, as well as other sources of energy, and their roles in the ribosome assembly process. In addition, we propose roles of energy-releasing enzymes in the assembly process, to explain why energy is used for a process that occurs largely spontaneously in bacteria. Finally, we use literature data to suggest testable models for how these enzymes could be used as targets for regulation of ribosome assembly. PMID:19850913

  14. 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

  15. Protein profiling and phosphoprotein analysis by isoelectric focusing.

    PubMed

    Maccarrone, Giuseppina; Filiou, Michaela D

    2015-01-01

    Protein profiling enables the qualitative characterization of a proteome of interest. Phosphorylation is a post-translational modification with regulatory functions in a plethora of cell processes. We present an experimental workflow for simultaneous analysis of the proteome and phosphoproteome with no additional enrichment for phosphoproteins/phosphopeptides. Our approach is based on isoelectric focusing (IEF) which allows the separation of peptide mixtures on an immobilized pH gradient (IPG) according to their isoelectric point. Due to the negative charge of the phosphogroup, most of the phosphopeptides migrate toward acidic pH values. Peptides and phosphopeptides are then identified by mass spectrometry (MS) and phosphopeptide spectra are manually checked for the assignment of phosphorylation sites. Here, we apply this methodology to investigate synaptosome extracts from whole mouse brain. IEF-based peptide separation is an efficient method for peptide and phosphopeptide identification.

  16. Hyperphosphatemia, Phosphoprotein Phosphatases, and Microparticle Release in Vascular Endothelial Cells

    PubMed Central

    Abbasian, Nima; Burton, James O.; Herbert, Karl E.; Tregunna, Barbara-Emily; Brown, Jeremy R.; Ghaderi-Najafabadi, Maryam; Brunskill, Nigel J.; Goodall, Alison H.

    2015-01-01

    Hyperphosphatemia in patients with advanced CKD is thought to be an important contributor to cardiovascular risk, in part because of endothelial cell (EC) dysfunction induced by inorganic phosphate (Pi). Such patients also have an elevated circulating concentration of procoagulant endothelial microparticles (MPs), leading to a prothrombotic state, which may contribute to acute occlusive events. We hypothesized that hyperphosphatemia leads to MP formation from ECs through an elevation of intracellular Pi concentration, which directly inhibits phosphoprotein phosphatases, triggering a global increase in phosphorylation and cytoskeletal changes. In cultured human ECs (EAhy926), incubation with elevated extracellular Pi (2.5 mM) led to a rise in intracellular Pi concentration within 90 minutes. This was mediated by PiT1/slc20a1 Pi transporters and led to global accumulation of tyrosine- and serine/threonine-phosphorylated proteins, a marked increase in cellular Tropomyosin-3, plasma membrane blebbing, and release of 0.1- to 1-μm-diameter MPs. The effect of Pi was independent of oxidative stress or apoptosis. Similarly, global inhibition of phosphoprotein phosphatases with orthovanadate or fluoride yielded a global protein phosphorylation response and rapid release of MPs. The Pi-induced MPs expressed VE-cadherin and superficial phosphatidylserine, and in a thrombin generation assay, they displayed significantly more procoagulant activity than particles derived from cells incubated in medium with a physiologic level of Pi (1 mM). These data show a mechanism of Pi-induced cellular stress and signaling, which may be widely applicable in mammalian cells, and in ECs, it provides a novel pathologic link between hyperphosphatemia, generation of MPs, and thrombotic risk. PMID:25745026

  17. An integrated workflow for characterizing intact phosphoproteins from complex mixtures

    PubMed Central

    Wu, Si; Yang, Feng; Zhao, Rui; Tolić, Nikola; Robinson, Errol W.; Camp, David; Smith, Richard D.; Paša-Tolić, Ljiljana

    2014-01-01

    The phosphorylation of any site on a given protein can affect its activity, degradation rate, ability to dock with other proteins or bind divalent cations, and/or its localization. These effects can operate within the same protein; in fact, multisite phosphorylation is a key mechanism for achieving signal integration in cells. Hence, knowing the overall phosphorylation signature of a protein is essential for understanding the "state" of a cell. However, current technologies to monitor the phosphorylation status of proteins are inefficient at determining the relative stoichiometries of phosphorylation at multiple sites. Here we report a new capability for comprehensive liquid chromatography mass spectrometry (LC/MS) analysis of intact phosphoproteins. The technology platform built upon integrated bottom-up and top-down approach that is facilitated by intact protein reversed-phase (RP)LC concurrently coupled with Fourier transform ion cyclotron resonance (FTICR) MS and fraction collection. As the use of conventional RPLC systems for phosphopeptide identification has proven challenging due to the formation of metal ion complexes at various metal surfaces during LC/MS and ESI-MS analysis, we have developed a “metal-free” RPLC-ESI-MS platform for phosphoprotein characterization. This platform demonstrated a significant sensitivity enhancement for phosphorylated casein proteins enriched from a standard protein mixture and revealed the presence of over 20 casein isoforms arising from genetic variants with varying numbers of phosphorylation sites. The integrated workflow was also applied to an enriched yeast phosphoproteome to evaluate the feasibility of this strategy for characterizing complex biological systems, and revealed ~16% of the detected yeast proteins to have multiple phosphorylation isoforms. Intact protein LC/MS platform for characterization of combinatorial posttranslational modifications (PTMs), with special emphasis on multisite phosphorylation, holds

  18. Paradigms of ribosome synthesis: Lessons learned from ribosomal proteins

    PubMed Central

    Gamalinda, Michael; Woolford, John L

    2015-01-01

    The proteome in all cells is manufactured via the intricate process of translation by multimolecular factories called ribosomes. Nevertheless, these ribonucleoprotein particles, the largest of their kind, also have an elaborate assembly line of their own. Groundbreaking discoveries that bacterial ribosomal subunits can be self-assembled in vitro jumpstarted studies on how ribosomes are constructed. Until recently, ribosome assembly has been investigated almost entirely in vitro with bacterial small subunits under equilibrium conditions. In light of high-resolution ribosome structures and a more sophisticated toolkit, the past decade has been defined by a burst of kinetic studies in vitro and, importantly, also a shift to examining ribosome maturation in living cells, especially in eukaryotes. In this review, we summarize the principles governing ribosome assembly that emerged from studies focusing on ribosomal proteins and their interactions with rRNA. Understanding these paradigms has taken center stage, given the linkage between anomalous ribosome biogenesis and proliferative disorders. PMID:26779413

  19. Ribosome dynamics during decoding.

    PubMed

    Rodnina, Marina V; Fischer, Niels; Maracci, Cristina; Stark, Holger

    2017-03-19

    Elongation factors Tu (EF-Tu) and SelB are translational GTPases that deliver aminoacyl-tRNAs (aa-tRNAs) to the ribosome. In each canonical round of translation elongation, aa-tRNAs, assisted by EF-Tu, decode mRNA codons and insert the respective amino acid into the growing peptide chain. Stop codons usually lead to translation termination; however, in special cases UGA codons are recoded to selenocysteine (Sec) with the help of SelB. Recruitment of EF-Tu and SelB together with their respective aa-tRNAs to the ribosome is a multistep process. In this review, we summarize recent progress in understanding the role of ribosome dynamics in aa-tRNA selection. We describe the path to correct codon recognition by canonical elongator aa-tRNA and Sec-tRNA(Sec) and discuss the local and global rearrangements of the ribosome in response to correct and incorrect aa-tRNAs. We present the mechanisms of GTPase activation and GTP hydrolysis of EF-Tu and SelB and summarize what is known about the accommodation of aa-tRNA on the ribosome after its release from the elongation factor. We show how ribosome dynamics ensures high selectivity for the cognate aa-tRNA and suggest that conformational fluctuations, induced fit and kinetic discrimination play major roles in maintaining the speed and fidelity of translation.This article is part of the themed issue 'Perspectives on the ribosome'.

  20. Principles and examples of gel-based approaches for phosphoprotein analysis.

    PubMed

    Steinberger, Birgit; Mayrhofer, Corina

    2015-01-01

    Methods for analyzing the phosphorylation status of proteins are essential to investigate in detail key cellular processes, including signal transduction and cell metabolism. The transience of this post-translational modification and the generally low abundance of phosphoproteins require specific enrichment and/or detection steps prior to analysis. Here, we describe three gel-based approaches for the analysis of differentially expressed phosphoproteins. These approaches comprise (1) the sequential fluorescence staining of two-dimensional (2-D) gels using Pro-Q(®) Diamond and SYPRO(®) Ruby dyes to visualize and quantify phosphoproteins in total cellular lysates as well as (2) affinity enrichment of phosphoproteins in conjunction with sequential fluorescence staining of the 2-D gels and (3) affinity enrichment of proteins prior to pre-electrophoretic fluorescence labeling and 2-D gel electrophoresis.

  1. Purification of 70S ribosomes.

    PubMed

    Rivera, Maria C; Maguire, Bruce; Lake, James A

    2015-03-02

    Here we describe the further purification of prokaryotic ribosomal particles obtained after the centrifugation of a crude cell lysate through a sucrose cushion. In this final purification step, a fraction containing ribosomes, ribosomal subunits, and polysomes is centrifuged through a 7%-30% (w/w) linear sucrose gradient to isolate tight couple 70S ribosomes, as well as dissociated 30S and 50S subunits. The tight couples fraction, or translationally active ribosome fraction, is composed of intact vacant ribosomes that can be used in cell-free translation systems.

  2. Ribosome maturation in E. coli.

    PubMed

    Silengo, L; Altruda, F; Dotto, G P; Lacquaniti, F; Perlo, C; Turco, E; Mangiarotti, G

    1977-01-01

    In vivo and in vitro experiments have shown that processing of ribosomal RNA is a late event in ribosome biogenesis. The precursor form of RNA is probably necessary to speed up the assembly of ribomal proteins. Newly formed ribosomal particles which have already entered polyribosomes differ from mature ribosomes not only in their RNA content but also in their susceptibility to unfolding in low Mg concentration and to RNase attack. Final maturation of new ribosomes is probably dependent on their functioning in protein synthesis. Thus only those ribosomes which have proven to be functional may be converted into stable cellular structures.

  3. Large scale identification and quantitative profiling of phosphoproteins expressed during seed filling in oilseed rape.

    PubMed

    Agrawal, Ganesh Kumar; Thelen, Jay J

    2006-11-01

    Seed filling is a dynamic, temporally regulated phase of seed development that determines the composition of storage reserves in mature seeds. Although the metabolic pathways responsible for storage reserve synthesis such as carbohydrates, oils, and proteins are known, little is known about their regulation. Protein phosphorylation is a ubiquitous form of regulation that influences many aspects of dynamic cellular behavior in plant biology. Here a systematic study has been conducted on five sequential stages (2, 3, 4, 5, and 6 weeks after flowering) of seed development in oilseed rape (Brassica napus L. Reston) to survey the presence and dynamics of phosphoproteins. High resolution two-dimensional gel electrophoresis in combination with a phosphoprotein-specific Pro-Q Diamond phosphoprotein fluorescence stain revealed approximately 300 phosphoprotein spots. Of these, quantitative expression profiles for 234 high quality spots were established, and hierarchical cluster analyses revealed the occurrence of six principal expression trends during seed filling. The identity of 103 spots was determined using LC-MS/MS. The identified spots represented 70 non-redundant phosphoproteins belonging to 10 major functional categories including energy, metabolism, protein destination, and signal transduction. Furthermore phosphorylation within 16 non-redundant phosphoproteins was verified by mapping the phosphorylation sites by LC-MS/MS. Although one of these sites was postulated previously, the remaining sites have not yet been reported in plants. Phosphoprotein data were assembled into a web database. Together this study provides evidence for the presence of a large number of functionally diverse phosphoproteins, including global regulatory factors like 14-3-3 proteins, within developing B. napus seed.

  4. Ribosome dynamics during decoding

    PubMed Central

    Maracci, Cristina; Stark, Holger

    2017-01-01

    Elongation factors Tu (EF-Tu) and SelB are translational GTPases that deliver aminoacyl-tRNAs (aa-tRNAs) to the ribosome. In each canonical round of translation elongation, aa-tRNAs, assisted by EF-Tu, decode mRNA codons and insert the respective amino acid into the growing peptide chain. Stop codons usually lead to translation termination; however, in special cases UGA codons are recoded to selenocysteine (Sec) with the help of SelB. Recruitment of EF-Tu and SelB together with their respective aa-tRNAs to the ribosome is a multistep process. In this review, we summarize recent progress in understanding the role of ribosome dynamics in aa-tRNA selection. We describe the path to correct codon recognition by canonical elongator aa-tRNA and Sec-tRNASec and discuss the local and global rearrangements of the ribosome in response to correct and incorrect aa-tRNAs. We present the mechanisms of GTPase activation and GTP hydrolysis of EF-Tu and SelB and summarize what is known about the accommodation of aa-tRNA on the ribosome after its release from the elongation factor. We show how ribosome dynamics ensures high selectivity for the cognate aa-tRNA and suggest that conformational fluctuations, induced fit and kinetic discrimination play major roles in maintaining the speed and fidelity of translation. This article is part of the themed issue ‘Perspectives on the ribosome’. PMID:28138068

  5. Context Specificity in Causal Signaling Networks Revealed by Phosphoprotein Profiling.

    PubMed

    Hill, Steven M; Nesser, Nicole K; Johnson-Camacho, Katie; Jeffress, Mara; Johnson, Aimee; Boniface, Chris; Spencer, Simon E F; Lu, Yiling; Heiser, Laura M; Lawrence, Yancey; Pande, Nupur T; Korkola, James E; Gray, Joe W; Mills, Gordon B; Mukherjee, Sach; Spellman, Paul T

    2017-01-25

    Signaling networks downstream of receptor tyrosine kinases are among the most extensively studied biological networks, but new approaches are needed to elucidate causal relationships between network components and understand how such relationships are influenced by biological context and disease. Here, we investigate the context specificity of signaling networks within a causal conceptual framework using reverse-phase protein array time-course assays and network analysis approaches. We focus on a well-defined set of signaling proteins profiled under inhibition with five kinase inhibitors in 32 contexts: four breast cancer cell lines (MCF7, UACC812, BT20, and BT549) under eight stimulus conditions. The data, spanning multiple pathways and comprising ∼70,000 phosphoprotein and ∼260,000 protein measurements, provide a wealth of testable, context-specific hypotheses, several of which we experimentally validate. Furthermore, the data provide a unique resource for computational methods development, permitting empirical assessment of causal network learning in a complex, mammalian setting. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

  6. Positive Expression of Human Cytomegalovirus Phosphoprotein 65 in Atherosclerosis

    PubMed Central

    Wang, Zhe; Cai, Jun; Zhang, Mingming; Wang, Xiaojing; Chi, Hongjie; Feng, Haijun

    2016-01-01

    Previous studies showed that human cytomegalovirus (HCMV) is associated with atherosclerosis. However, local vascular atherosclerosis related HCMV infection and protein expression remain unclear. This study aimed to assess the relationship between HCMV infection and atherosclerosis. Formalin-fixed, paraffin-embedded peripheral artery specimens were obtained from 15 patients with atherosclerosis undergoing vascular surgery from 2008 to 2010 at Zhongnan Hospital, Wuhan University. Pathological analyses were carried out after hematoxylin and eosin (H&E) and Masson trichrome staining. In situ hybridization and immunohistochemistry with two different monoclonal antibodies were employed to detect HCMV nucleic acids and proteins, respectively. H&E and Masson trichrome staining showed homogeneous extracellular matrix in femoral artery, while smooth muscle fibers were interlaced with collagen fibers; in carotid artery, inflammatory cell infiltration, foam cell vascular change, cholesterol crystals, and layered collagen fibers were observed. In situ hybridization showed no expression of HCMV nucleic acids in all 15 cases. Immunohistochemical staining for protein immediate-early protein (IE1 72) was negative in all cases, while phosphoprotein 65 (pp65) expression was detected in 14 cases. A high rate of positive pp65 signals was found in patients with atherosclerosis, suggesting that local HCMV infection may be associated with the pathogenesis of atherosclerosis. Further studies on this relationship are warranted. PMID:27990427

  7. Transgenic Expression of Dentin Phosphoprotein Inhibits Skeletal Development

    PubMed Central

    Zhang, H.; Liu, P.; Wang, S.; Liu, C.; Jani, P.; Lu, Y.; Qin, C.

    2016-01-01

    Dentin sialophosphoprotein (DSPP) is proteolytically processed into an NH2-terminal fragment called dentin sialoprotein (DSP) and a COOH-terminal fragment known as dentin phosphoprotein (DPP). These two fragments are believed to perform distinct roles in formation of bone and dentin. To investigate the functions of DPP in skeletal development, we generated transgenic mice to overexpress hemagglutinin (HA)-tagged DPP under the control of a 3.6 kb type I collagen (Col1a1) promoter (designated as Col1a1-HA-DPP). The Col1a1-HA-DPP transgenic mice were significantly smaller by weight, had smaller skeletons and shorter long bones than their wild type littermates, as demonstrated by X-ray radiography. They displayed reduced trabecular bone formation and narrower zones of proliferative and hypertrophic chondrocytes in the growth plates of the long bones. Histological analyses showed that the transgenic mice had reduced cell proliferation in the proliferating zone, but lacked obvious defects in the chondrocyte differentiation. In addition, the transgenic mice with a high level of transgene expression developed spontaneous long bone fractures. In conclusion, overexpressing DPP inhibited skeletal development, suggesting that the balanced actions between the NH2- and COOH-terminal fragments of DSPP may be required for normal skeletal development. PMID:26972716

  8. Epitope mapping of Canine distemper virus phosphoprotein by monoclonal antibodies.

    PubMed

    Sugai, Akihiro; Kooriyama, Takanori; Sato, Hiroki; Yoneda, Misako; Kai, Chieko

    2009-12-01

    The gene for phosphoprotein (P) of CDV encodes three different proteins, P, V, and C. The P protein is involved in viral gene transcription and replication. In the present study, we produced MAbs against a unique domain of the CDV-P protein, from aa 232 to 507, and determined their antigenic sites. By immunizing BALB/c mice with the recombinant P protein-specific fragment, we obtained six MAbs. Competitive binding inhibition assays revealed that they recognized two distinct regions of the P protein. Western blot analysis and immunofluorescence assays using deletion mutants of the unique C-terminus of the CDV-P protein revealed that all MAbs recognized a central short region (aa 233-303) of the CDV-P protein. In addition, linear and conformational epitopes have been determined, and at least four antigenic sites exist in the P protein central region. Furthermore, four of the MAbs were found to react with the P protein of recent Japanese field isolates but not with that of the older CDV strains, including a vaccine strain. Thus, these MAbs could be clinically useful for quick diagnosis during the CDV outbreaks.

  9. Analysis of Flagellar Phosphoproteins from Chlamydomonas reinhardtii▿ †

    PubMed Central

    Boesger, Jens; Wagner, Volker; Weisheit, Wolfram; Mittag, Maria

    2009-01-01

    Cilia and flagella are cell organelles that are highly conserved throughout evolution. For many years, the green biflagellate alga Chlamydomonas reinhardtii has served as a model for examination of the structure and function of its flagella, which are similar to certain mammalian cilia. Proteome analysis revealed the presence of several kinases and protein phosphatases in these organelles. Reversible protein phosphorylation can control ciliary beating, motility, signaling, length, and assembly. Despite the importance of this posttranslational modification, the identities of many ciliary phosphoproteins and knowledge about their in vivo phosphorylation sites are still missing. Here we used immobilized metal affinity chromatography to enrich phosphopeptides from purified flagella and analyzed them by mass spectrometry. One hundred forty-one phosphorylated peptides were identified, belonging to 32 flagellar proteins. Thereby, 126 in vivo phosphorylation sites were determined. The flagellar phosphoproteome includes different structural and motor proteins, kinases, proteins with protein interaction domains, and many proteins whose functions are still unknown. In several cases, a dynamic phosphorylation pattern and clustering of phosphorylation sites were found, indicating a complex physiological status and specific control by reversible protein phosphorylation in the flagellum. PMID:19429781

  10. TSC1 Sets the Rate of Ribosome Export and Protein Synthesis through Nucleophosmin Translation

    PubMed Central

    Pelletier, Corey L.; Maggi, Leonard B.; Brady, Suzanne N.; Scheidenhelm, Danielle K.; Gutmann, David H.; Weber, Jason D.

    2008-01-01

    Nucleophosmin (B23) is a nucleolar phosphoprotein that has been implicated in numerous cellular processes. In particular, nucleophosmin interacts with nucleolar components of newly synthesized ribosomes to promote ribosome nuclear export. Nucleophosmin is a classic mitogen-induced protein, with changes in its expression correlating with growth factor stimulation. In this study, we examined the underlying mechanism of nucleophosmin induction and showed that hyperproliferative signals emanating from oncogenic H-RasV12 cause tremendous increases in nucleophosmin protein expression. Nucleophosmin protein accumulation was dependent on mammalian target of rapamycin (mTOR) activation, as rapamycin completely prevented nucleophosmin induction. Consistent with this finding, genetic ablation of Tsc1, a major upstream inhibitor of mTOR, resulted in nucleophosmin protein induction through increased translation of existing nucleophosmin mRNAs. Increases in nucleophosmin protein accumulation were suppressed by reintroduction of TSC1. Induction of nucleophosmin through Tsc1 loss resulted in a greater pool of actively translating ribosomes in the cytoplasm, higher overall rates of protein synthesis, and increased cell proliferation, all of which were dependent on efficient nucleophosmin nuclear export. Nucleophosmin protein accumulation in the absence of Tsc1 promoted the nuclear export of maturing ribosome subunits, providing a mechanistic link between TSC1/mTOR signaling, nucleophosmin-mediated nuclear export of ribosome subunits, protein synthesis levels, and cell growth. PMID:17308101

  11. EXPANDING THE RIBOSOMAL UNIVERSE

    PubMed Central

    Dinman, Jonathan D.; Kinzy, Terri Goss

    2009-01-01

    SUMMARY In this issue of Structure, Frank and colleagues (Taylor et al., 2009) present the most complete model of a eukaryotic ribosome to date. This achievement represents a critical milestone along the path to structurally defining the unique aspects of the eukaryotic protein synthetic machinery. PMID:20004156

  12. Ribosomal Antibiotics: Contemporary Challenges

    PubMed Central

    Auerbach-Nevo, Tamar; Baram, David; Bashan, Anat; Belousoff, Matthew; Breiner, Elinor; Davidovich, Chen; Cimicata, Giuseppe; Eyal, Zohar; Halfon, Yehuda; Krupkin, Miri; Matzov, Donna; Metz, Markus; Rufayda, Mruwat; Peretz, Moshe; Pick, Ophir; Pyetan, Erez; Rozenberg, Haim; Shalev-Benami, Moran; Wekselman, Itai; Zarivach, Raz; Zimmerman, Ella; Assis, Nofar; Bloch, Joel; Israeli, Hadar; Kalaora, Rinat; Lim, Lisha; Sade-Falk, Ofir; Shapira, Tal; Taha-Salaime, Leena; Tang, Hua; Yonath, Ada

    2016-01-01

    Most ribosomal antibiotics obstruct distinct ribosomal functions. In selected cases, in addition to paralyzing vital ribosomal tasks, some ribosomal antibiotics are involved in cellular regulation. Owing to the global rapid increase in the appearance of multi-drug resistance in pathogenic bacterial strains, and to the extremely slow progress in developing new antibiotics worldwide, it seems that, in addition to the traditional attempts at improving current antibiotics and the intensive screening for additional natural compounds, this field should undergo substantial conceptual revision. Here, we highlight several contemporary issues, including challenging the common preference of broad-range antibiotics; the marginal attention to alterations in the microbiome population resulting from antibiotics usage, and the insufficient awareness of ecological and environmental aspects of antibiotics usage. We also highlight recent advances in the identification of species-specific structural motifs that may be exploited for the design and the creation of novel, environmental friendly, degradable, antibiotic types, with a better distinction between pathogens and useful bacterial species in the microbiome. Thus, these studies are leading towards the design of “pathogen-specific antibiotics,” in contrast to the current preference of broad range antibiotics, partially because it requires significant efforts in speeding up the discovery of the unique species motifs as well as the clinical pathogen identification. PMID:27367739

  13. Expanding the ribosomal universe.

    PubMed

    Dinman, Jonathan D; Kinzy, Terri Goss

    2009-12-09

    In this issue of Structure, Taylor et al. (2009) present the most complete model of an eukaryotic ribosome to date. This achievement represents a critical milestone along the path to structurally defining the unique aspects of the eukaryotic protein synthetic machinery.

  14. Ribosome-inactivating proteins

    PubMed Central

    Walsh, Matthew J; Dodd, Jennifer E; Hautbergue, Guillaume M

    2013-01-01

    Ribosome-inactivating proteins (RIPs) were first isolated over a century ago and have been shown to be catalytic toxins that irreversibly inactivate protein synthesis. Elucidation of atomic structures and molecular mechanism has revealed these proteins to be a diverse group subdivided into two classes. RIPs have been shown to exhibit RNA N-glycosidase activity and depurinate the 28S rRNA of the eukaryotic 60S ribosomal subunit. In this review, we compare archetypal RIP family members with other potent toxins that abolish protein synthesis: the fungal ribotoxins which directly cleave the 28S rRNA and the newly discovered Burkholderia lethal factor 1 (BLF1). BLF1 presents additional challenges to the current classification system since, like the ribotoxins, it does not possess RNA N-glycosidase activity but does irreversibly inactivate ribosomes. We further discuss whether the RIP classification should be broadened to include toxins achieving irreversible ribosome inactivation with similar turnovers to RIPs, but through different enzymatic mechanisms. PMID:24071927

  15. Constructing ribosomes along the Danube

    PubMed Central

    Warner, Jonathan R.

    2010-01-01

    The EMBO Conference on Ribosome Synthesis held last summer explored the latest breakthroughs in ribosome assembly and how it affects disease. Both of these topics have recently seen important advances that enlighten how almost 200 proteins cooperate to produce a ribosome and how the cell responds to a malfunction in this process. PMID:20010797

  16. Estimating the total number of phosphoproteins and phosphorylation sites in eukaryotic proteomes

    PubMed Central

    Vlastaridis, Panayotis; Kyriakidou, Pelagia; Chaliotis, Anargyros; Van de Peer, Yves; Oliver, Stephen G.

    2017-01-01

    Abstract Background: Phosphorylation is the most frequent post-translational modification made to proteins and may regulate protein activity as either a molecular digital switch or a rheostat. Despite the cornucopia of high-throughput (HTP) phosphoproteomic data in the last decade, it remains unclear how many proteins are phosphorylated and how many phosphorylation sites (p-sites) can exist in total within a eukaryotic proteome. We present the first reliable estimates of the total number of phosphoproteins and p-sites for four eukaryotes (human, mouse, Arabidopsis, and yeast). Results: In all, 187 HTP phosphoproteomic datasets were filtered, compiled, and studied along with two low-throughput (LTP) compendia. Estimates of the number of phosphoproteins and p-sites were inferred by two methods: Capture-Recapture, and fitting the saturation curve of cumulative redundant vs. cumulative non-redundant phosphoproteins/p-sites. Estimates were also adjusted for different levels of noise within the individual datasets and other confounding factors. We estimate that in total, 13 000, 11 000, and 3000 phosphoproteins and 230 000, 156 000, and 40 000 p-sites exist in human, mouse, and yeast, respectively, whereas estimates for Arabidopsis were not as reliable. Conclusions: Most of the phosphoproteins have been discovered for human, mouse, and yeast, while the dataset for Arabidopsis is still far from complete. The datasets for p-sites are not as close to saturation as those for phosphoproteins. Integration of the LTP data suggests that current HTP phosphoproteomics appears to be capable of capturing 70 % to 95 % of total phosphoproteins, but only 40 % to 60 % of total p-sites. PMID:28327990

  17. Proteomic Analysis of Phosphoproteins in the Rice Nucleus During the Early Stage of Seed Germination.

    PubMed

    Li, Ming; Yin, Xiaojian; Sakata, Katsumi; Yang, Pingfang; Komatsu, Setsuko

    2015-07-02

    The early stage of seed germination is the first step in the plant life cycle without visible morphological change. To investigate the mechanism controlling the early stage of rice seed germination, we performed gel-and label-free nuclear phosphoproteomics. A total of 3467 phosphopeptides belonging to 102 nuclear phosphoproteins from rice embryos were identified. Protein-synthesis-related proteins were mainly phosphorylated. During the first 24 h following imbibition, 115 nuclear phosphoproteins were identified, and significant changes in the phosphorylation level over time were observed in 29 phosphoproteins. Cluster analysis indicated that nucleotide-binding proteins and zinc finger CCCH- and BED-type proteins increased in abundance during the first 12 h of imbibition and then decreased. The in silico protein-protein interactions for 29 nuclear phosphoproteins indicated that the Sas10/Utp3 protein, which functions in snoRNA binding and gene silencing, was the center of the phosphoprotein network in nuclei. The germination rate of seeds was significantly slowed with phosphatase inhibitor treatment. The mRNA expression of the zinc finger CCCH-type protein did not change, and the zinc finger BED-type protein was upregulated in rice embryos during the early stage of germination with phosphatase inhibitor treatment. These results suggest that the phosphorylation and dephosphorylation of nuclear proteins are involved in rice seed germination. Furthermore, transcription factors such as zinc finger CCCH- and BED-type proteins might play a key role through nuclear phosphoproteins, and Sas10/Utp3 protein might interact with nuclear phosphoproteins in rice embryos to mediate the early stage of seed germination.

  18. Isolation of ribosomes and polysomes.

    PubMed

    Rivera, Maria C; Maguire, Bruce; Lake, James A

    2015-03-02

    Here we describe a preparative differential centrifugation protocol for the isolation of ribosomes from a crude cell homogenate. The subcellular fraction obtained is enriched in ribosome monomers and polysomes. The protocol has been optimized for the homogenization and collection of the ribosomal fraction from prokaryotic cells, mammalian and plant tissues, reticulocytes, and chloroplasts. The quality of the ribosomal preparation is enhanced by the removal of the remaining cellular components and adsorbed proteins by pelleting through a sucrose cushion with a high concentration of monovalent salts, NH4Cl or KCl. The different components of the ribosomal fraction isolated using this protocol can be further purified by sucrose gradient centrifugation.

  19. The generation of phosphoserine stretches in phosphoproteins: mechanism and significance.

    PubMed

    Cesaro, Luca; Pinna, Lorenzo A

    2015-10-01

    In the infancy of studies on protein phosphorylation the occurrence of clusters of three or more consecutive phosphoseryl residues in secreted and in cellular phosphoproteins was reported. Later however, while the reversible phosphorylation of Ser, Thr and Tyr residues was recognized to be the most frequent and general mechanism of cell regulation and signal transduction, the phenomenon of multi-phosphorylation of adjacent residues was entirely neglected. Nowadays, in the post-genomic era, the availability of large phosphoproteomics database makes possible a comprehensive re-visitation of this intriguing aspect of protein phosphorylation, aimed at shedding light on both its mechanistic occurrence and its functional meaning. Here we describe an analysis of the human phosphoproteome disclosing the existence of more than 800 rows of 3 to >10 consecutive phosphoamino acids, composed almost exclusively of phosphoserine, while clustered phosphothreonines and phosphotyrosines are almost absent. A scrutiny of these phosphorylated rows supports the conclusion that they are generated through the major contribution of a few hierarchical protein kinases, with special reference to CK2. Also well documented is the combined intervention of CK1 and GSK3, the former acting as priming and primed, the latter as primed kinase. The by far largest proportion of proteins containing (pS)n clusters display a nuclear localization where they play a prominent role in the regulation of transcription. Consistently the molecular function of the by far largest majority of these proteins is the ability to bind other macromolecules and/or nucleotides and metal ions. A "String" analysis performed under stringent conditions reveals that >80% of them are connected to each other by physical and/or functional links, and that this network of interactions mostly take place at the nuclear level.

  20. Structural insights into ribosome translocation

    PubMed Central

    Ling, Clarence

    2016-01-01

    During protein synthesis, tRNA and mRNA are translocated from the A to P to E sites of the ribosome thus enabling the ribosome to translate one codon of mRNA after the other. Ribosome translocation along mRNA is induced by the universally conserved ribosome GTPase, elongation factor G (EF‐G) in bacteria and elongation factor 2 (EF‐2) in eukaryotes. Recent structural and single‐molecule studies revealed that tRNA and mRNA translocation within the ribosome is accompanied by cyclic forward and reverse rotations between the large and small ribosomal subunits parallel to the plane of the intersubunit interface. In addition, during ribosome translocation, the ‘head’ domain of small ribosomal subunit undergoes forward‐ and back‐swiveling motions relative to the rest of the small ribosomal subunit around the axis that is orthogonal to the axis of intersubunit rotation. tRNA/mRNA translocation is also coupled to the docking of domain IV of EF‐G into the A site of the small ribosomal subunit that converts the thermally driven motions of the ribosome and tRNA into the forward translocation of tRNA/mRNA inside the ribosome. Despite recent and enormous progress made in the understanding of the molecular mechanism of ribosome translocation, the sequence of structural rearrangements of the ribosome, EF‐G and tRNA during translocation is still not fully established and awaits further investigation. WIREs RNA 2016, 7:620–636. doi: 10.1002/wrna.1354 For further resources related to this article, please visit the WIREs website. PMID:27117863

  1. Highly efficient precipitation of phosphoproteins using trivalent europium, terbium, and erbium ions.

    PubMed

    Güzel, Yüksel; Rainer, Matthias; Mirza, Munazza Raza; Bonn, Günther K

    2012-05-01

    This study describes a highly efficient method for the selective precipitation of phosphoproteins by trivalent europium, terbium, and erbium metal ions. These metal cations belong to the group of lanthanides and are known to be hard acceptors with an overwhelming preference for oxygen-containing anions such as phosphates to which they form very tight ionic bonds. The method could be successfully applied to specifically precipitate phosphoproteins from complex samples including milk and egg white by forming solid metal-protein complexes. Owing to the low solubility product of the investigated lanthanide salts, the produced metal-protein complexes showed high stability. The protein pellets were extensively washed to remove nonphosphorylated proteins and contaminants. For the analysis of proteins the pellets were first dissolved in 30 % formic acid and subjected to matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) MS. For peptide mass-fingerprint analysis the precipitated phosphoproteins were enzymatically digested using microwave-assisted digestion. The method was found to be highly specific for the isolation and purification of phosphoproteins. Protein quantification was performed by colorimetric detection of total precipitated phosphoproteins and revealed more than 95 % protein recovery for each lanthanide salt.

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

    PubMed Central

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

    2013-01-01

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

  3. Ribosomal Database Project II

    DOE Data Explorer

    The Ribosomal Database Project (RDP) provides ribosome related data and services to the scientific community, including online data analysis and aligned and annotated Bacterial small-subunit 16S rRNA sequences. As of March 2008, RDP Release 10 is available and currently (August 2009) contains 1,074,075 aligned 16S rRNA sequences. Data that can be downloaded include zipped GenBank and FASTA alignment files, a histogram (in Excel) of the number of RDP sequences spanning each base position, data in the Functional Gene Pipeline Repository, and various user submitted data. The RDP-II website also provides numerous analysis tools.[From the RDP-II home page at http://rdp.cme.msu.edu/index.jsp

  4. Mitochondrial ribosomes in cancer.

    PubMed

    Kim, Hyun-Jung; Maiti, Priyanka; Barrientos, Antoni

    2017-04-23

    Mitochondria play fundamental roles in the regulation of life and death of eukaryotic cells. They mediate aerobic energy conversion through the oxidative phosphorylation (OXPHOS) system, and harbor and control the intrinsic pathway of apoptosis. As a descendant of a bacterial endosymbiont, mitochondria retain a vestige of their original genome (mtDNA), and its corresponding full gene expression machinery. Proteins encoded in the mtDNA, all components of the multimeric OXPHOS enzymes, are synthesized in specialized mitochondrial ribosomes (mitoribosomes). Mitoribosomes are therefore essential in the regulation of cellular respiration. Additionally, an increasing body of literature has been reporting an alternative role for several mitochondrial ribosomal proteins as apoptosis-inducing factors. No surprisingly, the expression of genes encoding for mitoribosomal proteins, mitoribosome assembly factors and mitochondrial translation factors is modified in numerous cancers, a trait that has been linked to tumorigenesis and metastasis. In this article, we will review the current knowledge regarding the dual function of mitoribosome components in protein synthesis and apoptosis and their association with cancer susceptibility and development. We will also highlight recent developments in targeting mitochondrial ribosomes for the treatment of cancer. Copyright © 2017 Elsevier Ltd. All rights reserved.

  5. Improved staining of phosphoproteins with high sensitivity in polyacrylamide gels using Stains-All.

    PubMed

    Cong, Wei-Tao; Ye, Wei-Jian; Chen, Mao; Zhao, Ting; Zhu, Zhong-Xin; Niu, Chao; Ruan, Dan-Dan; Ni, Mao-Wei; Zhou, Xuan; Jin, Li-Tai

    2013-12-01

    An improved Stains-All (ISA) staining method for phosphoproteins in SDS-PAGE was described. Down to 0.5-1 ng phosphoproteins (α-casein, β-casein, or phosvitin) can be successfully selectively detected by ISA stain, which is approximately 120-fold higher than that of original Stains-All stain, but is similar to that of commonly used Pro-Q Diamond stain. Furthermore, unlike the original Stains-All protocol that was time consuming and light unstable, ISA stain could be completed within 60 min without resorting to protect the gels from light during the whole staining procedure. According to the results, it is concluded that ISA stain is a rapid, sensitive, specific, and economic staining method for a broad application to the research of phosphoproteins.

  6. Molecular evolution of dentin phosphoprotein among toothed and toothless animals

    PubMed Central

    2009-01-01

    Background Dentin sialophosphoprotein (DSPP) is the largest member of the SIBLING family and is the most abundant noncollagenous protein in dentin. DSPP is also expressed in non-mineralized tissues including metabolically active ductal epithelia and some cancers. Its function, however, is poorly defined. The carboxy-terminal fragment, dentin phosphoprotein (DPP) is encoded predominantly by a large repetitive domain that requires separate cloning/sequencing reactions and is, therefore, often incomplete in genomic databases. Comparison of DPP sequences from at least one member of each major branch in the mammalian evolutionary tree (including some "toothless" mammals) as well as one reptile and bird may help delineate its possible functions in both dentin and ductal epithelia. Results The BMP1-cleavage and translation-termination domains were sufficiently conserved to permit amplification/cloning/sequencing of most species' DPP. While the integrin-binding domain, RGD, was present in about half of species, only vestigial remnants of this tripeptide were identified in the others. The number of tandem repeats of the nominal SerSerAsp phosphorylation motif in toothed mammals (including baleen whale and platypus which lack teeth as adults), ranged from ~75 (elephant) to >230 (human). These repeats were not perfect, however, and patterns of intervening sequences highlight the rapidity of changes among even closely related species. Two toothless anteater species have evolved different sets of nonsense mutations shortly after their BMP1 motifs suggesting that while cleavage may be important for DSPP processing in other tissues, the DPP domain itself may be required only in dentin. The lizard DSPP had an intact BMP1 site, a remnant RGD motif, as well as a distinctly different Ser/Asp-rich domain compared to mammals. Conclusions The DPP domain of DSPP was found to change dramatically within mammals and was lost in two truly toothless animals. The defining aspect of DPP, the

  7. Ribosome recycling induces optimal translation rate at low ribosomal availability.

    PubMed

    Marshall, E; Stansfield, I; Romano, M C

    2014-09-06

    During eukaryotic cellular protein synthesis, ribosomal translation is made more efficient through interaction between the two ends of the messenger RNA (mRNA). Ribosomes reaching the 3' end of the mRNA can thus recycle and begin translation again on the same mRNA, the so-called 'closed-loop' model. Using a driven diffusion lattice model of translation, we study the effects of ribosome recycling on the dynamics of ribosome flow and density on the mRNA. We show that ribosome recycling induces a substantial increase in ribosome current. Furthermore, for sufficiently large values of the recycling rate, the lattice does not transition directly from low to high ribosome density, as seen in lattice models without recycling. Instead, a maximal current phase becomes accessible for much lower values of the initiation rate, and multiple phase transitions occur over a wide region of the phase plane. Crucially, we show that in the presence of ribosome recycling, mRNAs can exhibit a peak in protein production at low values of the initiation rate, beyond which translation rate decreases. This has important implications for translation of certain mRNAs, suggesting that there is an optimal concentration of ribosomes at which protein synthesis is maximal, and beyond which translational efficiency is impaired.

  8. Prognostic significance of peroxiredoxin 1 and ezrin-radixin-moesin-binding phosphoprotein 50 in cholangiocarcinoma.

    PubMed

    Yonglitthipagon, Ponlapat; Pairojkul, Chawalit; Chamgramol, Yaovalux; Loukas, Alex; Mulvenna, Jason; Bethony, Jeffrey; Bhudhisawasdi, Vajarabhongsa; Sripa, Banchob

    2012-10-01

    We performed a comparative proteomic analysis of protein expression profiles in 4 cholangiocarcinoma cell lines: K100, M156, M213, and M139. The H69 biliary cell line was used as a control. Peroxiredoxin 1 and ezrin-radixin-moesin-binding phosphoprotein 50 were selected for further validation by immunohistochemistry using a cholangiocarcinoma tissue microarray (n = 301) to assess their prognostic value in this cancer. Both peroxiredoxin 1 and ezrin-radixin-moesin-binding phosphoprotein 50 were overexpressed in cholangiocarcinoma tissues compared with normal liver tissues. Of the 301 cholangiocarcinoma cases, overexpression of peroxiredoxin 1 in 103 (34.3%) was associated with an age-related effect in young patients (P = .011) and the absence of cholangiocarcinoma in lymphatic vessels and perineural tissues (P = .004 and P = .037, respectively). Expression of radixin-moesin-binding phosphoprotein 50 correlated with histopathologic type, with 180 (59.8%) of moderately or poorly differentiated tumors (P = .039) being higher, and was associated with the presence of cholangiocarcinoma in lymphatic and vascular vessels (P < .001 and P < .001, respectively). The high expression of radixin-moesin-binding phosphoprotein 50 and the low expression of peroxiredoxin 1 correlated with reduced survival by univariate analysis (P = .017 and P = .048, respectively). Moreover, the impact of peroxiredoxin 1 and radixin-moesin-binding phosphoprotein 50 expression on patient survival was an independent predictor in multivariate analyses (P = .004 and P = .025, respectively). Therefore, altered expression of peroxiredoxin 1 and radixin-moesin-binding phosphoprotein 50 may be used as prognostic markers in cholangiocarcinoma.

  9. Mitochondrial ribosomes in a trypanosome.

    PubMed

    Tittawella, Ivor; Yasmin, Lubna; Baranov, Vladimir

    2003-08-01

    The nature, and even the existence, of trypanosome mitochondrial ribosomes has been the subject of some debate. We investigated this further in the insect trypanosome, Crithidia fasciculata. In sucrose gradients of parasite lysates, mitochondrial ribosomal RNA co-sediments at approximately 35S with nascent peptides synthesized in the presence of the cytosolic translational inhibitor, cycloheximide. Co-sedimenting peptides in this peak are much reduced when the parasites are treated with the bacterial translational inhibitor, chloramphenicol. In CsCl gradients this peak resolves at a buoyant density of 1.42 g/cm(3), a value typical for mito-ribosomes. Electron microscopy of peak material shows particles smaller than cytosolic ribosomes, but with characteristic ribosomal shapes. We propose that these particles represent the parasite's mitochondrial ribosomes.

  10. PAPE (Prefractionation-Assisted Phosphoprotein Enrichment): A Novel Approach for Phosphoproteomic Analysis of Green Tissues from Plants

    PubMed Central

    Lassowskat, Ines; Naumann, Kai; Lee, Justin; Scheel, Dierk

    2013-01-01

    Phosphorylation is an important post-translational protein modification with regulatory roles in diverse cellular signaling pathways. Despite recent advances in mass spectrometry, the detection of phosphoproteins involved in signaling is still challenging, as protein phosphorylation is typically transient and/or occurs at low levels. In green plant tissues, the presence of highly abundant proteins, such as the subunits of the RuBisCO complex, further complicates phosphoprotein analysis. Here, we describe a simple, but powerful, method, which we named prefractionation-assisted phosphoprotein enrichment (PAPE), to increase the yield of phosphoproteins from Arabidopsis thaliana leaf material. The first step, a prefractionation via ammonium sulfate precipitation, not only depleted RuBisCO almost completely, but, serendipitously, also served as an efficient phosphoprotein enrichment step. When coupled with a subsequent metal oxide affinity chromatography (MOAC) step, the phosphoprotein content was highly enriched. The reproducibility and efficiency of phosphoprotein enrichment was verified by phospho-specific staining and, further, by mass spectrometry, where it could be shown that the final PAPE fraction contained a significant number of known and additionally novel (potential) phosphoproteins. Hence, this facile two-step procedure is a good prerequisite to probe the phosphoproteome and gain deeper insight into plant phosphorylation-based signaling events. PMID:28250405

  11. BALANCED PRODUCTION OF RIBOSOMAL PROTEINS

    PubMed Central

    Perry, Robert P.

    2017-01-01

    Eukaryotic ribosomes contain one molecule each of 79 different proteins. The genes encoding these proteins are usually at widely scattered loci and have distinctive promoters with certain common features. This minireview discusses the means by which cells manage to balance the production of ribosomal proteins so as to end up with equimolar quantities in the ribosome. Regulation at all levels of gene expression, from transcription to protein turnover, is considered. PMID:17689889

  12. Isolation of ribosomes by chromatography.

    PubMed

    Maguire, Bruce A

    2015-04-01

    Mixed-mode chromatography on cysteine-SulfoLink resin efficiently separates ribosomes from cell lysates and is particularly effective at rapidly removing endogenous proteases and nucleases, resulting in ribosomes of improved purity, integrity, and activity. Binding occurs partly by anion exchange of the RNA of the ribosomes, so that cells must be lysed in a buffer of moderate ionic strength (conductivity no more than 20 mS for chromatography of bacterial ribosomes) without any highly charged additives (e.g., heparin, which is used to inhibit RNases in yeast). A robust protocol for Escherichia coli is given here as an example.

  13. Ribonuclease selection for ribosome profiling

    PubMed Central

    Gerashchenko, Maxim V.; Gladyshev, Vadim N.

    2017-01-01

    Ribosome profiling has emerged as a powerful method to assess global gene translation, but methodological and analytical challenges often lead to inconsistencies across labs and model organisms. A critical issue in ribosome profiling is nuclease treatment of ribosome–mRNA complexes, as it is important to ensure both stability of ribosomal particles and complete conversion of polysomes to monosomes. We performed comparative ribosome profiling in yeast and mice with various ribonucleases including I, A, S7 and T1, characterized their cutting preferences, trinucleotide periodicity patterns and coverage similarities across coding sequences, and showed that they yield comparable estimations of gene expression when ribosome integrity is not compromised. However, ribosome coverage patterns of individual transcripts had little in common between the ribonucleases. We further examined their potency at converting polysomes to monosomes across other commonly used model organisms, including bacteria, nematodes and fruit flies. In some cases, ribonuclease treatment completely degraded ribosome populations. Ribonuclease T1 was the only enzyme that preserved ribosomal integrity while thoroughly converting polysomes to monosomes in all examined species. This study provides a guide for ribonuclease selection in ribosome profiling experiments across most common model systems. PMID:27638886

  14. Molecular inventory control in ribosome biosynthesis.

    PubMed

    Warner, J R; Johnson, S P

    1986-11-01

    The eukaryotic cell coordinates the accumulation of each ribosomal protein with every other ribosomal protein, with ribosomal RNA and with the needs of the cell. To do so it regulates the transcription, processing, translation and lifetime of the mRNA for ribosomal proteins. When all else fails, it rapidly degrades any excess ribosomal protein which is synthesized.

  15. Ribosomal vaccines. I. Immunogenicity of ribosomal fractions isolated from Salmonella typhimurium and Yersinia pestis.

    PubMed

    Johnson, W

    1972-06-01

    The immunogenicity of ribosomes and ribosomal subfractions isolated from Yersina pestis and Salmonella typhimurium has been studied. Ribosomes and ribosomal protein isolated from S. typhimurium protected mice against lethal challenge. Ribosomal ribonucleic acid isolated by phenol extraction failed to induce any significant level of protection in mice. None of the ribosomes or ribosomal subfractions isolated from Y. pestis were effective in inducing immunity to lethal challenge. These results suggest that the immunogen of the ribosomal vaccine is protein.

  16. Mechanistic insights into phosphoprotein-binding FHA domains.

    PubMed

    Liang, Xiangyang; Van Doren, Steven R

    2008-08-01

    of FHA domains by a bioinformatics approach. The phosphopeptide-dependent dynamics of an FHA domain, SH2 domain, and PTB domain suggest a common theme: rigid, preformed binding surfaces support van der Waals contacts that provide favorable binding enthalpy. Despite the lack of pronounced conformational changes in FHA domains linked to binding events, more subtle adjustments may be possible. In the one FHA domain tested, phosphothreonine peptide binding is accompanied by increased flexibility just outside the binding site and increased rigidity across the beta-sandwich. The folding of the same FHA domain progresses through near-native intermediates that stabilize the recognition loops in the center of the phosphoprotein-binding surface; this may promote rigidity in the interface and affinity for targets phosphorylated on threonine.

  17. Ribosomes in a Stacked Array

    PubMed Central

    Yamashita, Yui; Kadokura, Yoshitomo; Sotta, Naoyuki; Fujiwara, Toru; Takigawa, Ichigaku; Satake, Akiko; Onouchi, Hitoshi; Naito, Satoshi

    2014-01-01

    Expression of CGS1, which codes for an enzyme of methionine biosynthesis, is feedback-regulated by mRNA degradation in response to S-adenosyl-l-methionine (AdoMet). In vitro studies revealed that AdoMet induces translation arrest at Ser-94, upon which several ribosomes stack behind the arrested one, and mRNA degradation occurs at multiple sites that presumably correspond to individual ribosomes in a stacked array. Despite the significant contribution of stacked ribosomes to inducing mRNA degradation, little is known about the ribosomes in the stacked array. Here, we assigned the peptidyl-tRNA species of the stacked second and third ribosomes to their respective codons and showed that they are arranged at nine-codon intervals behind the Ser-94 codon, indicating tight stacking. Puromycin reacts with peptidyl-tRNA in the P-site, releasing the nascent peptide as peptidyl-puromycin. This reaction is used to monitor the activity of the peptidyltransferase center (PTC) in arrested ribosomes. Puromycin reaction of peptidyl-tRNA on the AdoMet-arrested ribosome, which is stalled at the pre-translocation step, was slow. This limited reactivity can be attributed to the peptidyl-tRNA occupying the A-site at this step rather than to suppression of PTC activity. In contrast, puromycin reactions of peptidyl-tRNA with the stacked second and third ribosomes were slow but were not as slow as pre-translocation step ribosomes. We propose that the anticodon end of peptidyl-tRNA resides in the A-site of the stacked ribosomes and that the stacked ribosomes are stalled at an early step of translocation, possibly at the P/E hybrid state. PMID:24652291

  18. Molecular signatures of ribosomal evolution.

    PubMed

    Roberts, Elijah; Sethi, Anurag; Montoya, Jonathan; Woese, Carl R; Luthey-Schulten, Zaida

    2008-09-16

    Ribosomal signatures, idiosyncrasies in the ribosomal RNA (rRNA) and/or proteins, are characteristic of the individual domains of life. As such, insight into the early evolution of the domains can be gained from a comparative analysis of their respective signatures in the translational apparatus. In this work, we identify signatures in both the sequence and structure of the rRNA and analyze their contributions to the universal phylogenetic tree using both sequence- and structure-based methods. Domain-specific ribosomal proteins can be considered signatures in their own right. Although it is commonly assumed that they developed after the universal ribosomal proteins, we present evidence that at least one may have been present before the divergence of the organismal lineages. We find correlations between the rRNA signatures and signatures in the ribosomal proteins showing that the rRNA signatures coevolved with both domain-specific and universal ribosomal proteins. Finally, we show that the genomic organization of the universal ribosomal components contains these signatures as well. From these studies, we propose the ribosomal signatures are remnants of an evolutionary-phase transition that occurred as the cell lineages began to coalesce and so should be reflected in corresponding signatures throughout the fabric of the cell and its genome.

  19. Molecular signatures of ribosomal evolution

    PubMed Central

    Roberts, Elijah; Sethi, Anurag; Montoya, Jonathan; Woese, Carl R.; Luthey-Schulten, Zaida

    2008-01-01

    Ribosomal signatures, idiosyncrasies in the ribosomal RNA (rRNA) and/or proteins, are characteristic of the individual domains of life. As such, insight into the early evolution of the domains can be gained from a comparative analysis of their respective signatures in the translational apparatus. In this work, we identify signatures in both the sequence and structure of the rRNA and analyze their contributions to the universal phylogenetic tree using both sequence- and structure-based methods. Domain-specific ribosomal proteins can be considered signatures in their own right. Although it is commonly assumed that they developed after the universal ribosomal proteins, we present evidence that at least one may have been present before the divergence of the organismal lineages. We find correlations between the rRNA signatures and signatures in the ribosomal proteins showing that the rRNA signatures coevolved with both domain-specific and universal ribosomal proteins. Finally, we show that the genomic organization of the universal ribosomal components contains these signatures as well. From these studies, we propose the ribosomal signatures are remnants of an evolutionary-phase transition that occurred as the cell lineages began to coalesce and so should be reflected in corresponding signatures throughout the fabric of the cell and its genome. PMID:18768810

  20. A portrait of tissue phosphoprotein stability in the clinical tissue procurement process.

    PubMed

    Espina, Virginia; Edmiston, Kirsten H; Heiby, Michael; Pierobon, Mariaelena; Sciro, Manuela; Merritt, Barbara; Banks, Stacey; Deng, Jianghong; VanMeter, Amy J; Geho, David H; Pastore, Lucia; Sennesh, Joel; Petricoin, Emanuel F; Liotta, Lance A

    2008-10-01

    Little is known about the preanalytical fluctuations of phosphoproteins during tissue procurement for molecular profiling. This information is crucial to establish guidelines for the reliable measurement of these analytes. To develop phosphoprotein profiles of tissue subjected to the trauma of excision, we measured the fidelity of 53 signal pathway phosphoproteins over time in tissue specimens procured in a community clinical practice. This information provides strategies for potential surrogate markers of stability and the design of phosphoprotein preservative/fixation solutions. Eleven different specimen collection time course experiments revealed augmentation (+/-20% from the time 0 sample) of signal pathway phosphoprotein levels as well as decreases over time independent of tissue type, post-translational modification, and protein subcellular location (tissues included breast, colon, lung, ovary, and uterus (endometrium/myometrium) and metastatic melanoma). Comparison across tissue specimens showed an >20% decrease of protein kinase B (AKT) Ser-473 (p < 0.002) and myristoylated alanine-rich C-kinase substrate protein Ser-152/156 (p < 0.0001) within the first 90-min postexcision. Proteins in apoptotic (cleaved caspase-3 Asp-175 (p < 0.001)), proliferation/survival/hypoxia (IRS-1 Ser-612 (p < 0.0003), AMP-activated protein kinase beta Ser-108 (p < 0.005), ERK Thr-202/Tyr-204 (p < 0.003), and GSK3alphabeta Ser-21/9 (p < 0.01)), and transcription factor pathways (STAT1 Tyr-701 (p < 0.005) and cAMP response element-binding protein Ser-133 (p < 0.01)) showed >20% increases within 90-min postprocurement. Endothelial nitric-oxide synthase Ser-1177 did not change over the time period evaluated with breast or leiomyoma tissue. Treatment with phosphatase or kinase inhibitors alone revealed that tissue kinase pathways are active ex vivo. Combinations of kinase and phosphatase inhibitors appeared to stabilize proteins that exhibited increases in the presence of phosphatase

  1. Ribosomal Peptide Natural Products: Bridging the Ribosomal and Nonribosomal Worlds

    PubMed Central

    McIntosh, John A.; Donia, Mohamed S.; Schmidt, Eric W.

    2010-01-01

    Ribosomally synthesized bacterial natural products rival the nonribosomal peptides in their structural and functional diversity. The last decade has seen substantial progress in the identification and characterization of biosynthetic pathways leading to ribosomal peptide natural products with new and unusual structural motifs. In some of these cases, the motifs are similar to those found in nonribosomal peptides, and many are constructed by convergent or even paralogous enzymes. Here, we summarize the major structural and biosynthetic categories of ribosomally synthesized bacterial natural products and, where applicable, compare them to their homologs from nonribosomal biosynthesis. PMID:19642421

  2. Supernumerary proteins of mitochondrial ribosomes.

    PubMed

    Rackham, Oliver; Filipovska, Aleksandra

    2014-04-01

    Messenger RNAs encoded by mitochondrial genomes are translated on mitochondrial ribosomes that have unique structure and protein composition compared to prokaryotic and cytoplasmic ribosomes. Mitochondrial ribosomes are a patchwork of core proteins that share homology with prokaryotic ribosomal proteins and new, supernumerary proteins that can be unique to different organisms. In mammals, there are specific supernumerary ribosomal proteins that are not present in other eukaryotes. Here we discuss the roles of supernumerary proteins in the regulation of mitochondrial gene expression and compare them among different eukaryotic systems. Furthermore, we consider if differences in the structure and organization of mitochondrial genomes may have contributed to the acquisition of mitochondrial ribosomal proteins with new functions. The distinct and diverse compositions of mitochondrial ribosomes illustrate the high evolutionary divergence found between mitochondrial genetic systems. Elucidating the role of the organism-specific supernumerary proteins may provide a window into the regulation of mitochondrial gene expression through evolution in response to distinct evolutionary paths taken by mitochondria in different organisms. This article is part of a Special Issue entitled Frontiers of Mitochondrial Research. © 2013.

  3. Transcriptome-wide measurement of ribosomal occupancy by ribosome profiling.

    PubMed

    Aeschimann, Florian; Xiong, Jieyi; Arnold, Andreas; Dieterich, Christoph; Grosshans, Helge

    2015-09-01

    Gene expression profiling provides a tool to analyze the internal states of cells or organisms, and their responses to perturbations. While global measurements of mRNA levels have thus been widely used for many years, it is only through the recent development of the ribosome profiling technique that an analogous examination of global mRNA translation programs has become possible. Ribosome profiling reveals which RNAs are being translated to what extent and where the translated open reading frames are located. In addition, different modes of translation regulation can be distinguished and characterized. Here, we present an optimized, step-by-step protocol for ribosome profiling. Although established in Caenorhabditis elegans, our protocol and optimization approaches should be equally usable for other model organisms or cell culture with little adaptation. Next to providing a protocol, we compare two different methods for isolation of single ribosomes and two different library preparations, and describe strategies to optimize the RNase digest and to reduce ribosomal RNA contamination in the libraries. Moreover, we discuss bioinformatic strategies to evaluate the quality of the data and explain how the data can be analyzed for different applications. In sum, this article seeks to facilitate the understanding, execution, and optimization of ribosome profiling experiments. Copyright © 2015 Elsevier Inc. All rights reserved.

  4. Eukaryotic ribosome assembly, transport and quality control.

    PubMed

    Peña, Cohue; Hurt, Ed; Panse, Vikram Govind

    2017-09-07

    Eukaryotic ribosome synthesis is a complex, energy-consuming process that takes place across the nucleolus, nucleoplasm and cytoplasm and requires more than 200 conserved assembly factors. Here, we discuss mechanisms by which the ribosome assembly and nucleocytoplasmic transport machineries collaborate to produce functional ribosomes. We also highlight recent cryo-EM studies that provided unprecedented snapshots of ribosomes during assembly and quality control.

  5. The RDP (Ribosomal Database Project).

    PubMed

    Maidak, B L; Olsen, G J; Larsen, N; Overbeek, R; McCaughey, M J; Woese, C R

    1997-01-01

    The Ribosomal Database Project (RDP) is a curated database that offers ribosome-related data, analysis services and associated computer programs. The offerings include phylogenetically ordered alignments of ribosomal RNA (rRNA) sequences, derived phylogenetic trees, rRNA secondary structure diagrams, and various software for handling, analyzing and displaying alignments and trees. The data are available via anonymous FTP (rdp.life.uiuc.edu), electronic mail (server@rdp.life.uiuc.edu), gopher (rdpgopher.life.uiuc.edu) and WWW (http://rdpwww.life.uiuc.edu/ ). The electronic mail and WWW servers provide ribosomal probe checking, approximate phylogenetic placement of user-submitted sequences, screening for possible chimeric rRNA sequences, automated alignment, and a suggested placement of an unknown sequence on an existing phylogenetic tree.

  6. The Ribosomal Database Project (RDP).

    PubMed

    Maidak, B L; Olsen, G J; Larsen, N; Overbeek, R; McCaughey, M J; Woese, C R

    1996-01-01

    The Ribosomal Database Project (RDP) is a curated database that offers ribosome-related data, analysis services and associated computer programs. The offerings include phylogenetically ordered alignments of ribosomal RNA (rRNA) sequences, derived phylogenetic trees, rRNA secondary structure diagrams and various software for handling, analyzing and displaying alignments and trees. The data are available via anonymous ftp (rdp.life.uiuc.edu), electronic mail (server@rdp.life.uiuc.edu), gopher (rdpgopher.life.uiuc.edu) and World Wide Web (WWW)(http://rdpwww.life.uiuc.edu/). The electronic mail and WWW servers provide ribosomal probe checking, screening for possible chimeric rRNA sequences, automated alignment and approximate phylogenetic placement of user-submitted sequences on an existing phylogenetic tree.

  7. The RDP (Ribosomal Database Project).

    PubMed Central

    Maidak, B L; Olsen, G J; Larsen, N; Overbeek, R; McCaughey, M J; Woese, C R

    1997-01-01

    The Ribosomal Database Project (RDP) is a curated database that offers ribosome-related data, analysis services and associated computer programs. The offerings include phylogenetically ordered alignments of ribosomal RNA (rRNA) sequences, derived phylogenetic trees, rRNA secondary structure diagrams, and various software for handling, analyzing and displaying alignments and trees. The data are available via anonymous FTP (rdp.life.uiuc.edu), electronic mail (server@rdp.life.uiuc.edu), gopher (rdpgopher.life.uiuc.edu) and WWW (http://rdpwww.life.uiuc.edu/ ). The electronic mail and WWW servers provide ribosomal probe checking, approximate phylogenetic placement of user-submitted sequences, screening for possible chimeric rRNA sequences, automated alignment, and a suggested placement of an unknown sequence on an existing phylogenetic tree. PMID:9016515

  8. Expression of truncated phosphoproteins of Nipah virus and Hendra virus in Escherichia coli for the differentiation of henipavirus infections.

    PubMed

    Chen, Ji-Ming; Yaiw, Koon Chu; Yu, Meng; Wang, Lin-Fa; Wang, Qing-Hua; Crameri, Gary; Wang, Zhi-Liang

    2007-06-01

    The genus Henipavirus in the family Paramyxoviridae compromises two newly identified dangerous pathogens, Nipah virus and Hendra virus. Phosphoprotein of the two viruses is one of the major immunodominant antigens and the most divergent protein in the viral genomes. We have now expressed two pairs of truncated phosphoproteins of the two viruses in Escherichia coli in a soluble form using a vector tailored from pET32a. The truncated recombinant phosphoproteins were purified with His-Tag affinity chromatography and their antigenicity was determined by western blotting and ELISA. The longer pair of truncated recombinant phosphoproteins, covering amino acid residues 4-550, was more antigenic than the shorter one and of potential utility in the serological differentiation of henipavirus infections.

  9. RNA folding and ribosome assembly.

    PubMed

    Woodson, Sarah A

    2008-12-01

    Ribosome synthesis is a tightly regulated process that is crucial for cell survival. Chemical footprinting, mass spectrometry, and cryo-electron microscopy are revealing how these complex cellular machines are assembled. Rapid folding of the rRNA provides a platform for protein-induced assembly of the bacterial 30S ribosome. Multiple assembly pathways increase the flexibility of the assembly process, while accessory factors and modification enzymes chaperone the late stages of assembly and control the quality of the mature subunits.

  10. Identification of the 64 kilodalton chloroplast stromal phosphoprotein as phosphoglucomutase. [Pisum sativum

    SciTech Connect

    Salvucci, M.E.; Drake, R.R.; Broadbent, K.P.; Haley, B.E. ); Hanson, K.R.; McHale, N.A. )

    1990-05-01

    Phosphorylation of the 64 kilodalton stromal phosphoprotein by incubation of pea (Pisum sativum) chloroplast extracts with ({gamma}-{sup 32}P)ATP decreased in the presence of Glc-6-P and Glc-1,6-P{sub 2}, but was stimulated by glucose. Two-dimensional gel electrophoresis following incubation of intact chloroplasts and stromal extracts with ({gamma}-{sup 32}P)ATP, or incubation of stromal extracts and partially purified phosphoglucomutase (EC 2.7.5.1) with ({sup 32}P)Glc-1-P showed that the identical 64 kilodalton polypeptide was labeled. A 62 kilodalton polypeptide was phosphorylated by incubation of tobacco (Nicotiana sylvestris) stromal extracts with either ({gamma}-{sup 32}P)ATP or ({sup 32}P)Glc-1-P. In contrast, an analogous polypeptide was not phosphorylated in extracts from a tobacco mutant deficient in plastid phosphoglucomutase activity. The results indicate that the 64 (or 62) kilodalton chloroplast stromal phosphoprotein is phosphoglucomutase.

  11. Virion basic phosphoprotein from human cytomegalovirus contains O-linked N-acetylglucosamine.

    PubMed Central

    Benko, D M; Haltiwanger, R S; Hart, G W; Gibson, W

    1988-01-01

    A 149-kDa virion protein of human strains of cytomegalovirus is the principal acceptor for galactose added in vitro by bovine milk galactosyltransferase. Peptide comparisons with other biochemical characteristics of the galactosylated protein identified it as the virus-encoded basic phosphoprotein. This protein is an abundant constituent of the virion and is located in the tegument region, between the capsid and the envelope, rather than in the envelope layer with the recognized virion glycoproteins. The galactosylated carbohydrate was resistant to a commercial preparation of endoglycosidase F but was sensitive to removal by alkali-induced beta-elimination, indicating an O-linkage to the protein. Chromatographic and electrophoretic determinations identified the beta-eliminated material as the alditol of Gal beta 1-4GlcNAc, establishing that the human cytomegalovirus virion basic phosphoprotein contains single O-linked residues of N-acetylglucosamine. Images PMID:2833746

  12. Targeting ricin to the ribosome.

    PubMed

    May, Kerrie L; Yan, Qing; Tumer, Nilgun E

    2013-07-01

    The plant toxin ricin is highly toxic for mammalian cells and is of concern for bioterrorism. Ricin belongs to a family of functionally related toxins, collectively referred to as ribosome inactivating proteins (RIPs), which disable ribosomes and halt protein synthesis. Currently there are no specific antidotes against ricin or related RIPs. The catalytic subunit of ricin is an N-glycosidase that depurinates a universally conserved adenine residue within the sarcin/ricin loop (SRL) of the 28S rRNA. This depurination activity inhibits translation and its biochemistry has been intensively studied. Yet, recent developments paint a more complex picture of toxicity, with ribosomal proteins and cellular signaling pathways contributing to the potency of ricin. In particular, several studies have now established the importance of the ribosomal stalk structure in facilitating the depurination activity and ribosome specificity of ricin and other RIPs. This review highlights recent developments defining toxin-ribosome interactions and examines the significance of these interactions for toxicity and therapeutic intervention.

  13. Combined use of nuclear phosphoprotein c-Myc and cellular phosphoprotein p53 for hepatocellular carcinoma detection in high-risk chronic hepatitis C patients.

    PubMed

    Attallah, A M; El-Far, M; Abdelrazek, M A; Omran, M M; Attallah, A A; Elkhouly, A A; Elkenawy, H M; Farid, K

    2017-10-01

    Hepatocellular carcinoma (HCC) is a multistage process resulting from various genetic changes. We aimed to determine nuclear phosphoprotein c-Myc and cellular phosphoprotein p53 expression and to evaluate their importance in HCC diagnosis. One hundred and twenty chronic hepatitis C (CHC) patients (60 non-HCC CHC patients and 60 HCC patients who had a single small (<5 cm) tumour) were recruited. The gene products of c-Myc and p53 were identified in liver tissues and serum samples using immunostaining, western blot and ELISA. Immunohistochemical detection of c-Myc and p53 with monospecific antibodies revealed intense and diffuse cytoplasmic staining patterns. Accumulated mutant proteins, released from tumour cells into the extracellular serum, were detected at 62 KDa, for c-Myc, and 53 KDa, for p53, using western blotting. In contrast to alpha feto-protein, there was a significant increase (p < 0.0001) in the positivity rate of c-Myc (86.7% vs. 6.7%) and p53 (78.3% vs. 8.3%) in the malignant vs. non-malignant patients. The parallel combination of c-Myc and p53 reach the absolute sensitivity (100%), for more accurate and reliable HCC detection (specificity was 87%). c-Myc and p53 are potential HCC diagnostic biomarkers, and convenient combinations of them could improve diagnostic accuracy of HCC.

  14. Ribosome dynamics and the evolutionary history of ribosomes

    NASA Astrophysics Data System (ADS)

    Fox, George E.; Paci, Maxim; Tran, Quyen; Petrov, Anton S.; Williams, Loren D.

    2015-09-01

    The ribosome is a dynamic nanomachine responsible for coded protein synthesis. Its major subsystems were essentially in place at the time of the last universal common ancestor (LUCA). Ribosome evolutionary history thus potentially provides a window into the pre- LUCA world. This history begins with the origins of the peptidyl transferase center where the actual peptide is synthesized and then continues over an extended timeframe as additional functional centers including the GTPase center are added. The large ribosomal RNAs (rRNAs) have grown over time by an accretion process and a model exists that proposes a relative age of each accreted element. We have compared atomic resolution ribosome structures before and after EF-G bound GTP hydrolysis and thereby identified the location of 23 pivot points in the large rRNAs that facilitate ribosome dynamics. Pivots in small subunit helices h28 and h44 appear to be especially central to the process and according to the accretion model significantly older than the other helices containing pivots. Overall, the results suggest that ribosomal dynamics occurred in two phases. In the first phase, an inherently mobile h28/h44 combination provided the flexibility needed to create a dynamic ribosome that was essentially a Brownian machine. This addition likely made coded peptide synthesis possible by facilitating movement of a primitive mRNA. During the second phase, addition of pivoting elements and the creation of a factor binding site allowed the regulation of the inherent motion created by h28/h44. All of these events likely occurred before LUCA.

  15. Atrazine Affects Phosphoprotein and Protein Expression in MCF-10A Human Breast Epithelial Cells

    PubMed Central

    Huang, Peixin; Yang, John; Song, Qisheng; Sheehan, David

    2014-01-01

    Atrazine, a member of the 2-chloro-s-triazine family of herbicides, is the most widely used pesticide in the world and often detected in agriculture watersheds. Although it was generally considered as an endocrine disruptor, posing a potential threat to human health, the molecular mechanisms of atrazine effects remain unclear. Using two-dimensional gel electrophoresis, we identified a panel of differentially expressed phosphoproteins and total proteins in human breast epithelial MCF-10A cells after being exposed to environmentally relevant concentrations of atrazine. Atrazine treatments for 6 h resulted in differential expression of 4 phosphoproteins and 8 total-proteins as compared to the control cells (>1.5-fold, p < 0.05). MALDI-TOF MS/MS analysis revealed that the differentially expressed proteins belong to various cellular compartments (nucleus, cytosol, membrane) and varied in function, including those regulating the stress response such as peroxiredoxin I, HSP70 and HSP27; structural proteins such as tropomyosin and profilin 1; and oncogenesis proteins such as ANP32A. Six of the 12 identified proteins were verified by quantitative PCR for their transcript levels. The most up-regulated phosphoprotein by atrazine treatment, ANP32A, was further analyzed for its expression, distribution and cellular localization using Western blot and immunocytochemical approaches. The results revealed that ANP32 expression after atrazine treatment increased dose and time dependently and was primarily located in the nucleus. This study may provide new evidence on the potential toxicity of atrazine in human cells. PMID:25275270

  16. Highly selective recovery of phosphopeptides using trypsin-assisted digestion of precipitated lanthanide-phosphoprotein complexes.

    PubMed

    Güzel, Yüksel; Rainer, Matthias; Mirza, Munazza R; Messner, Christoph B; Bonn, Günther K

    2013-05-21

    The basic idea of this study was to recover phosphopeptides after trypsin-assisted digestion of precipitated phosphoproteins using trivalent lanthanide ions. In the first step, phosphoproteins were extracted from the protein solution by precipitation with La(3+) and Ce(3+) ions, forming stable pellets. Additionally, the precipitated lanthanide-phosphoprotein complexes were suspended and directly digested on-pellet using trypsin. Non-phosphorylated peptides were released into the supernatants by enzymatic cleavage and phosphopeptides remained bound on the precipitated pellet. Further washing steps improved the removal of non-phosphorylated peptides. For the recovery of phosphopeptides the precipitated pellets were dissolved in 3.7% hydrochloric acid. The performance of this method was evaluated by several experiments using MALDI-TOF MS measurements and delivered the highest selectivity for phosphopeptides. This can be explained by the overwhelming preference of lanthanides for binding to oxygen-containing anions such as phosphates. The developed enrichment method was evaluated with several types of biological samples, including fresh milk and egg white. The uniqueness and the main advantages of the presented approach are the enrichment on the protein-level and the recovery of phosphopeptides on the peptide-level. This allows much easier handling, as the number of molecules on the peptide level is unavoidably higher, by complicating every enrichment strategy.

  17. Inhibition of hydroxyapatite growth by casein, a potential salivary phosphoprotein homologue.

    PubMed

    Romero, Maria J R H; Nakashima, Syozi; Nikaido, Toru; Ichinose, Shizuko; Sadr, Alireza; Tagami, Junji

    2015-08-01

    Salivary phosphoproteins are essential in tooth mineral regulation but are often overlooked in vitro. This study aimed to evaluate the effect of casein, as a salivary phosphoprotein homologue, on the deposition and growth of hydroxyapatite (HA) on tooth surfaces. Hydroxyapatite growth was quantified using seeded crystal systems. Artificial saliva (AS) containing HA powder and 0, 10, 20, 50, or 100 μg ml(-1) of casein, or 100 μg ml(-1) of dephosphorylated casein (Dcasein), was incubated for 0-8 h at 37°C, pH 7.2. Calcium concentrations were measured using atomic absorption spectroscopy (AAS). Surface precipitation of HA on bovine enamel and dentine blocks, incubated in similar conditions for 7 d, was examined using field emission scanning electron microscopy (FE-SEM) and transmission electron microscopy (TEM) with selected area electron diffraction (SAED). Casein adsorption was assessed using modified Lowry assays and zeta-potential measurements. The AAS results revealed a concentration-dependent inhibition of calcium consumption. Hydroxyapatite precipitation occurred when no casein was present, whereas precipitation of HA was apparently completely inhibited in casein-containing groups. Adsorption data demonstrated increasingly negative zeta-potential with increased casein concentration and an affinity constant similar to proline-rich proteins with Langmuir modelling. Casein inhibited the deposition and growth of HA primarily through the binding of esterized phosphate to HA active sites, indicating its potential as a mineral-regulating salivary phosphoprotein homologue in vitro.

  18. Acidic-phosphoprotein phosphatase activity of rat ventral prostate nuclei: apparent lack of effect of androgens.

    PubMed

    Wilson, M J; Ahmed, K; Fischbach, T J

    1978-08-03

    A protein phosphatase activity has been demonstrated in nuclei of rat ventral prostate utilizing 32P-labelled phosvitin as a model acidic phosphoprotein substrate. This phosphoprotein phosphatase has a pH optimum of 6.7, is unaffected by the sulphydryl protecting agent 2-mercaptoethanol, and requires a divalent cation for maximal activity. Of the various divalent cations tested, Mg2+ is the most effective in reactivating the EDTA-inhibited enzyme. The phosphatase is inhibited by sodium flouride, sodium oxalate, N-ethylmaleimide, ATP and ADP but is relatively insensitive to ammonium molybdate. Increased ionic strength of the reaction medium also causes a reduction in the enzyme activity, e.g., by 48% at 200 mM sodium chloride. The activity of the acidic phosphoprotein phosphatase did not change significantly at 48 h or 96 h post-orchiectomy when expressed per unit of nuclear protein. However, it is reduced by approx. 30% at these times after castration if based on DNA content. The decline in activity per nucleus reflects the decrease in the realtive nuclear protein content observed at 48 h or 96 h post-orchiectomy. This suggests that the decline in the phosphorylation of prostatic nuclear acidic proteins which occurs upon androgen withdrawal is not due to increased nuclear phosphatase activity.

  19. Atrazine affects phosphoprotein and protein expression in MCF-10A human breast epithelial cells.

    PubMed

    Huang, Peixin; Yang, John; Song, Qisheng

    2014-10-01

    Atrazine, a member of the 2-chloro-s-triazine family of herbicides, is the most widely used pesticide in the world and often detected in agriculture watersheds. Although it was generally considered as an endocrine disruptor, posing a potential threat to human health, the molecular mechanisms of atrazine effects remain unclear. Using two-dimensional gel electrophoresis, we identified a panel of differentially expressed phosphoproteins and total proteins in human breast epithelial MCF-10A cells after being exposed to environmentally relevant concentrations of atrazine. Atrazine treatments for 6 h resulted in differential expression of 4 phosphoproteins and 8 total-proteins as compared to the control cells (>1.5-fold, p<0.05). MALDI-TOF MS/MS analysis revealed that the differentially expressed proteins belong to various cellular compartments (nucleus, cytosol, membrane) and varied in function, including those regulating the stress response such as peroxiredoxin I, HSP70 and HSP27; structural proteins such as tropomyosin and profilin 1; and oncogenesis proteins such as ANP32A. Six of the 12 identified proteins were verified by quantitative PCR for their transcript levels. The most up-regulated phosphoprotein by atrazine treatment, ANP32A, was further analyzed for its expression, distribution and cellular localization using Western blot and immunocytochemical approaches. The results revealed that ANP32 expression after atrazine treatment increased dose and time dependently and was primarily located in the nucleus. This study may provide new evidence on the potential toxicity of atrazine in human cells.

  20. Sustained mitogen-activated protein kinase activation reprograms defense metabolism and phosphoprotein profile in Arabidopsis thaliana.

    PubMed

    Lassowskat, Ines; Böttcher, Christoph; Eschen-Lippold, Lennart; Scheel, Dierk; Lee, Justin

    2014-01-01

    Mitogen-activated protein kinases (MAPKs) target a variety of protein substrates to regulate cellular signaling processes in eukaryotes. In plants, the number of identified MAPK substrates that control plant defense responses is still limited. Here, we generated transgenic Arabidopsis thaliana plants with an inducible system to simulate in vivo activation of two stress-activated MAPKs, MPK3, and MPK6. Metabolome analysis revealed that this artificial MPK3/6 activation (without any exposure to pathogens or other stresses) is sufficient to drive the production of major defense-related metabolites, including various camalexin, indole glucosinolate and agmatine derivatives. An accompanying (phospho)proteome analysis led to detection of hundreds of potential phosphoproteins downstream of MPK3/6 activation. Besides known MAPK substrates, many candidates on this list possess typical MAPK-targeted phosphosites and in many cases, the corresponding phosphopeptides were detected by mass spectrometry. Notably, several of these putative phosphoproteins have been reported to be associated with the biosynthesis of antimicrobial defense substances (e.g., WRKY transcription factors and proteins encoded by the genes from the "PEN" pathway required for penetration resistance to filamentous pathogens). Thus, this work provides an inventory of candidate phosphoproteins, including putative direct MAPK substrates, for future analysis of MAPK-mediated defense control. (Proteomics data are available with the identifier PXD001252 via ProteomeXchange, http://proteomecentral.proteomexchange.org).

  1. [Ribosomal RNA Evolution

    NASA Technical Reports Server (NTRS)

    1997-01-01

    It is generally believed that an RNA World existed at an early stage in the history of life. During this early period, RNA molecules are seen to be potentially involved in both catalysis and the storage of genetic information. Translation presents several interrelated themes of inquiry for exobiology. First, it is essential, for understanding the very origin of life, how peptides and eventually proteins might have come to be made on the early Earth in a template directed manner. Second, it is necessary to understand how a machinery of similar complexity to that found in the ribosomes of modern organisms came to exist by the time of the last common ancestor (as detected by 16S rRNA sequence studies). Third, the ribosomal RNAs themselves likely had a very early origin and studies of their history may be very informative about the nature of the RNA World. Moreover, studies of these RNAs will contribute to a better understanding of the potential roles of RNA in early evolution.During the past year we have ave conducted a comparative study of four completely sequenced bacterial genoames. We have focused initially on conservation of gene order. The second component of the project continues to build on the model system for studying the validity of variant 5S rRNA sequences in the vicinity of the modern Vibrio proteolyticus 5S rRNA that we established earlier. This system has made it possible to conduct a detailed and extensive analysis of a local portion of the sequence space. These core methods have been used to construct numerous mutants during the last several years. Although it has been a secondary focus, this work has continued over the last year such that we now have in excess of 125 V. proteolyticus derived constructs which have been made and characterized. We have also continued high resolution NMR work on RNA oligomers originally initiated by G. Kenneth Smith who was funded by a NASA Graduate Student Researcher's Fellowship Award until May of 1996. Mr. Smith

  2. Neuron-Like Networks Between Ribosomal Proteins Within the Ribosome

    NASA Astrophysics Data System (ADS)

    Poirot, Olivier; Timsit, Youri

    2016-05-01

    From brain to the World Wide Web, information-processing networks share common scale invariant properties. Here, we reveal the existence of neural-like networks at a molecular scale within the ribosome. We show that with their extensions, ribosomal proteins form complex assortative interaction networks through which they communicate through tiny interfaces. The analysis of the crystal structures of 50S eubacterial particles reveals that most of these interfaces involve key phylogenetically conserved residues. The systematic observation of interactions between basic and aromatic amino acids at the interfaces and along the extension provides new structural insights that may contribute to decipher the molecular mechanisms of signal transmission within or between the ribosomal proteins. Similar to neurons interacting through “molecular synapses”, ribosomal proteins form a network that suggest an analogy with a simple molecular brain in which the “sensory-proteins” innervate the functional ribosomal sites, while the “inter-proteins” interconnect them into circuits suitable to process the information flow that circulates during protein synthesis. It is likely that these circuits have evolved to coordinate both the complex macromolecular motions and the binding of the multiple factors during translation. This opens new perspectives on nanoscale information transfer and processing.

  3. Preparation and proteomic analysis of chloroplast ribosomes.

    PubMed

    Yamaguchi, Kenichi

    2011-01-01

    Proteomics of chloroplast ribosomes in spinach and Chlamydomonas revealed unique protein composition and structures of plastid ribosomes. These studies have suggested the presence of some ribosomal proteins unique to plastid ribosomes which may be involved in plastid-unique translation regulation. Considering the strong background of genetic analysis and molecular biology in Arabidopsis, the in-depth proteomic characterization of Arabidopsis plastid ribosomes would facilitate further understanding of plastid translation in higher plants. Here, I describe simple and rapid methods for the preparation of plastid ribosomes from Chlamydomonas and Arabidopsis using sucrose gradients. I also describe purity criteria and methods for yield estimation of the purified plastid ribosomes and subunits, methods for the preparation of plastid ribosomal proteins, as well as the identification of some Arabidopsis plastid ribosomal proteins by matrix-assisted laser desorption/ionization mass spectrometry.

  4. Chloroplast ribosomes and protein synthesis.

    PubMed Central

    Harris, E H; Boynton, J E; Gillham, N W

    1994-01-01

    Consistent with their postulated origin from endosymbiotic cyanobacteria, chloroplasts of plants and algae have ribosomes whose component RNAs and proteins are strikingly similar to those of eubacteria. Comparison of the secondary structures of 16S rRNAs of chloroplasts and bacteria has been particularly useful in identifying highly conserved regions likely to have essential functions. Comparative analysis of ribosomal protein sequences may likewise prove valuable in determining their roles in protein synthesis. This review is concerned primarily with the RNAs and proteins that constitute the chloroplast ribosome, the genes that encode these components, and their expression. It begins with an overview of chloroplast genome structure in land plants and algae and then presents a brief comparison of chloroplast and prokaryotic protein-synthesizing systems and a more detailed analysis of chloroplast rRNAs and ribosomal proteins. A description of the synthesis and assembly of chloroplast ribosomes follows. The review concludes with discussion of whether chloroplast protein synthesis is essential for cell survival. PMID:7854253

  5. Simulating activity of the bacterial ribosome.

    PubMed

    Trylska, Joanna

    2009-11-01

    Computational modeling studies that investigate activity of the bacterial ribosome were reviewed. Computational approaches became possible with the availability of three-dimensional atomic resolution structures of the ribosomal subunits. However, due to the enormous size of the system, theoretical efforts to study the ribosome are few and challenging. For example, to extend the simulation timescales to biologically relevant ones, often, reduced models that require tedious parameterizations need to be applied. To that end, modeling of the ribosome focused on its internal dynamics, electrostatic properties, inhibition by antibiotics, polypeptide folding in the ribosome tunnel and assembly mechanisms driving the formation of the small ribosomal subunit.

  6. Comprehensive Molecular Structure of the Eukaryotic Ribosome

    PubMed Central

    Taylor, Derek J.; Devkota, Batsal; Huang, Andrew D.; Topf, Maya; Narayanan, Eswar; Sali, Andrej; Harvey, Stephen C.; Frank, Joachim

    2009-01-01

    Despite the emergence of a large number of X-ray crystallographic models of the bacterial 70S ribosome over the past decade, an accurate atomic model of the eukaryotic 80S ribosome is still not available. Eukaryotic ribosomes possess more ribosomal proteins and ribosomal RNA than bacterial ribosomes, which are implicated in extra-ribosomal functions in the eukaryotic cells. By combining cryo-EM with RNA and protein homology modeling, we obtained an atomic model of the yeast 80S ribosome complete with all ribosomal RNA expansion segments and all ribosomal proteins for which a structural homolog can be identified. Mutation or deletion of 80S ribosomal proteins can abrogate maturation of the ribosome, leading to several human diseases. We have localized one such protein unique to eukaryotes, rpS19e, whose mutations are associated with Diamond-Blackfan anemia in humans. Additionally, we characterize crucial and novel interactions between the dynamic stalk base of the ribosome with eukaryotic elongation factor 2. PMID:20004163

  7. Challenges in describing ribosome dynamics

    NASA Astrophysics Data System (ADS)

    Nguyen, Kien; Whitford, Paul Charles

    2017-04-01

    For decades, protein folding and functional dynamics have been described in terms of diffusive motion across an underlying energy landscape. With continued advances in structural biology and high-performance computing, the field is positioned to extend these approaches to large biomolecular assemblies. Through the application of energy landscape techniques to the ribosome, one may work towards establishing a comprehensive description of the dynamics, which will bridge theoretical concepts and experimental observations. In this perspective, we discuss a few of the challenges that will need to be addressed as we extend the application of landscape principles to the ribosome.

  8. Chromatographic purification of highly active yeast ribosomes.

    PubMed

    Meskauskas, Arturas; Leshin, Jonathan A; Dinman, Jonathan D

    2011-10-24

    Eukaryotic ribosomes are much more labile as compared to their eubacterial and archael counterparts, thus posing a significant challenge to researchers. Particularly troublesome is the fact that lysis of cells releases a large number of proteases and nucleases which can degrade ribosomes. Thus, it is important to separate ribosomes from these enzymes as quickly as possible. Unfortunately, conventional differential ultracentrifugation methods leaves ribosomes exposed to these enzymes for unacceptably long periods of time, impacting their structural integrity and functionality. To address this problem, we utilize a chromatographic method using a cysteine charged Sulfolink resin. This simple and rapid application significantly reduces co-purifying proteolytic and nucleolytic activities, producing high yields of intact, highly biochemically active yeast ribosomes. We suggest that this method should also be applicable to mammalian ribosomes. The simplicity of the method, and the enhanced purity and activity of chromatographically purified ribosome represents a significant technical advancement for the study of eukaryotic ribosomes.

  9. Significant and unique changes in phosphorylation levels of four phosphoproteins in two apple rootstock genotypes under drought stress.

    PubMed

    Ren, Jing; Mao, Juan; Zuo, Cunwu; Calderón-Urrea, Alejandro; Dawuda, Mohammed Mujitaba; Zhao, Xin; Li, Xinwen; Chen, Baihong

    2017-07-14

    Drought stress is a major problem around the world and there is still little molecular mechanism about how fruit crops deal with moderate drought stress. Here, the physiological and phosphoproteomic responses of drought-sensitive genotype (M26) and drought-tolerant genotype (MBB) under moderate drought stress were investigated. Our results of the physiology analysis indicated that the MBB genotype could produce more osmosis-regulating substances. Furthermore, phosphoproteins from leaves of both genotypes under moderate drought stress were analyzed using the isobaric tags for relative and absolute quantification technology. A total of 595 unique phosphopeptides, 682 phosphorylated sites, and 446 phosphoproteins were quantitatively analyzed in the two genotypes. Five and thirty-five phosphoproteins with the phosphorylation levels significantly changed (PLSC) were identified in M26 and MBB, respectively. Among these, four PLSC phosphoproteins were common to both genotypes, perhaps indicating a partial overlap of the mechanisms to moderate drought stress. Gene ontology analyses revealed that the PLSC phosphoproteins represent a unique combination of metabolism, transcription, translation, and protein processing, suggesting that the response in apple to moderate drought stress encompasses a new and unique homeostasis of major cellular processes. The basic trend was an increase in protein and organic molecules abundance related to drought. These increases were higher in MBB than in M26. Our study is the first to address the phosphoproteome of apple rootstocks in response to moderate drought stress, and provide insights into the molecular regulation mechanisms of apple rootstock under moderate drought stress.

  10. Complete sequence analysis of cDNA clones encoding rat whey phosphoprotein: homology to a protease inhibitor.

    PubMed

    Dandekar, A M; Robinson, E A; Appella, E; Qasba, P K

    1982-07-01

    Lactoprotein clones have been isolated from a rat mammary gland recombinant library of cDNA plasmids. Clones p-Wp 52 and p-Wp 47 were shown by hybrid selection, in vitro translation, and immunoprecipitation to represent a cloned DNA sequence encoding rat whey phosphoprotein. We report here the nucleotide sequence of the cDNA insert of p-Wp 52 and shows that it encodes the complete whey phosphoprotein sequence. The encoded sequence shows a high content of half-cystine, glutamic acid, aspartic acid, and serine but an absence of tyrosine. The half-cystines appear in unique arrangements and are repeated in two domains of the protein. The second domain has striking similarities with the second domain of the red sea turtle protease inhibitor. Clone p-Wp 52 has allowed the study of expression of whey phosphoprotein mRNA during functional differentiation of rat mammary gland and in mammary tumors. The whey phosphoprotein mRNA is detected during midpregnancy and lactation in the rat mammary gland but is barely detected in mammary tumors in which other milk protein mRNAs are expressed. The whey phosphoprotein gene in these tumors is hypermethylated, correlating with the reduced expression of this gene.

  11. Characterization of the interactions between the nucleoprotein and the phosphoprotein of Henipavirus.

    PubMed

    Habchi, Johnny; Blangy, Stéphanie; Mamelli, Laurent; Jensen, Malene Ringkjøbing; Blackledge, Martin; Darbon, Hervé; Oglesbee, Michael; Shu, Yaoling; Longhi, Sonia

    2011-04-15

    The Henipavirus genome is encapsidated by the nucleoprotein (N) within a helical nucleocapsid that recruits the polymerase complex via the phosphoprotein (P). In a previous study, we reported that in henipaviruses, the N-terminal domain of the phosphoprotein and the C-terminal domain of the nucleoprotein (N(TAIL)) are both intrinsically disordered. Here we show that Henipavirus N(TAIL) domains are also disordered in the context of full-length nucleoproteins. We also report the cloning, purification, and characterization of the C-terminal X domains (P(XD)) of Henipavirus phosphoproteins. Using isothermal titration calorimetry, we show that N(TAIL) and P(XD) form a 1:1 stoichiometric complex that is stable under NaCl concentrations as high as 1 M and has a K(D) in the μM range. Using far-UV circular dichroism and nuclear magnetic resonance, we show that P(XD) triggers an increase in the α-helical content of N(TAIL). Using fluorescence spectroscopy, we show that P(XD) has no impact on the chemical environment of a Trp residue introduced at position 527 of the Henipavirus N(TAIL) domain, thus arguing for the lack of stable contacts between the C termini of N(TAIL) and P(XD). Finally, we present a tentative structural model of the N(TAIL)-P(XD) interaction in which a short, order-prone region of N(TAIL) (α-MoRE; amino acids 473-493) adopts an α-helical conformation and is embedded between helices α2 and α3 of P(XD), leading to a relatively small interface dominated by hydrophobic contacts. The present results provide the first detailed experimental characterization of the N-P interaction in henipaviruses and designate the N(TAIL)-P(XD) interaction as a valuable target for rational antiviral approaches.

  12. Identification of Crosstalk between Phosphoprotein Signaling Pathways in RAW 264.7 Macrophage Cells

    PubMed Central

    Gupta, Shakti; Maurya, Mano Ram; Subramaniam, Shankar

    2010-01-01

    Signaling pathways mediate the effect of external stimuli on gene expression in cells. The signaling proteins in these pathways interact with each other and their phosphorylation levels often serve as indicators for the activity of signaling pathways. Several signaling pathways have been identified in mammalian cells but the crosstalk between them is not well understood. Alliance for Cellular Signaling (AfCS) has measured time-course data in RAW 264.7 macrophage cells on important phosphoproteins, such as the mitogen-activated protein kinases (MAPKs) and signal transducer and activator of transcription (STATs), in single- and double-ligand stimulation experiments for 22 ligands. In the present work, we have used a data-driven approach to analyze the AfCS data to decipher the interactions and crosstalk between signaling pathways in stimulated macrophage cells. We have used dynamic mapping to develop a predictive model using a partial least squares approach. Significant interactions were selected through statistical hypothesis testing and were used to reconstruct the phosphoprotein signaling network. The proposed data-driven approach is able to identify most of the known signaling interactions such as protein kinase B (Akt) → glycogen synthase kinase 3α/β (GSKα/β) etc., and predicts potential novel interactions such as P38 → RSK and GSK → ezrin/radixin/moesin. We have also shown that the model has good predictive power for extrapolation. Our novel approach captures the temporal causality and directionality in intracellular signaling pathways. Further, case specific analysis of the phosphoproteins in the network has led us to propose hypothesis about inhibition (phosphorylation) of GSKα/β via P38. PMID:20126526

  13. AMPLIFICATION OF RIBOSOMAL RNA SEQUENCES

    EPA Science Inventory

    This book chapter offers an overview of the use of ribosomal RNA sequences. A history of the technology traces the evolution of techniques to measure bacterial phylogenetic relationships and recent advances in obtaining rRNA sequence information. The manual also describes procedu...

  14. AMPLIFICATION OF RIBOSOMAL RNA SEQUENCES

    EPA Science Inventory

    This book chapter offers an overview of the use of ribosomal RNA sequences. A history of the technology traces the evolution of techniques to measure bacterial phylogenetic relationships and recent advances in obtaining rRNA sequence information. The manual also describes procedu...

  15. All Ribosomes Are Created Equal. Really?

    PubMed

    Preiss, Thomas

    2016-02-01

    Ribosomes are generally thought of as molecular machines with a constitutive rather than regulatory role during protein synthesis. A study by Slavov et al.[1] now shows that ribosomes of distinct composition and functionality exist within eukaryotic cells, giving credence to the concept of 'specialized' ribosomes.

  16. Reconstitution of functional eukaryotic ribosomes from Dictyostelium discoideum ribosomal proteins and RNA.

    PubMed

    Mangiarotti, G; Chiaberge, S

    1997-08-08

    40 and 60 S ribosomal subunits have been reconstituted in vitro from purified ribosomal RNA and ribosomal proteins of Dictyostelium discoideum. The functionality of the reconstituted ribosomes was demonstrated in in vitro mRNA-directed protein synthesis. The reassembly proceeded well with immature precursors of ribosomal RNA but poorly if at all with mature cytoplasmic RNA species. Reassembly also required a preparation of small nuclear RNA(s), acting as morphopoietic factor(s).

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

    PubMed Central

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

    1983-01-01

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

  18. A simple, rapid and low-cost staining method for gel-electrophoresis separated phosphoproteins via the fluorescent purpurin dye.

    PubMed

    Cong, Weitao; Shen, Jiayi; Xuan, Yuanhu; Zhu, Xinliang; Ni, Maowei; Zhu, Zhongxin; Hong, Guoying; Lu, Xianghong; Jin, Litai

    2014-12-07

    A novel fluorescence detection method for phosphoproteins in 1-D and 2-D SDS-PAGE by using purpurin is developed in this study. Phosphoproteins as low as 4-8 ng could be specifically detected by purpurin within 60 min, and the detection limit is similar to or better than that of Pro-Q Diamond staining. Only 2 steps (staining and destaining) are needed for purpurin staining without requiring excessive fixing and washing steps, and for single use, $0.8 is enough for purpurin staining. By comprehensively comparing with Pro-Q Diamond staining, it is concluded that purpurin staining is a simple, rapid and low-cost staining method for a broad application to the research of phosphoproteins.

  19. Phosphoproteomes of Strongylocentrotus purpuratus shell and tooth matrix: identification of a major acidic sea urchin tooth phosphoprotein, phosphodontin

    PubMed Central

    2010-01-01

    Background Sea urchin is a major model organism for developmental biology and biomineralization research. However, identification of proteins involved in larval skeleton formation and mineralization processes in the embryo and adult, and the molecular characterization of such proteins, has just gained momentum with the sequencing of the Strongylocentrotus purpuratus genome and the introduction of high-throughput proteomics into the field. Results The present report contains the determination of test (shell) and tooth organic matrix phosphoproteomes. Altogether 34 phosphoproteins were identified in the biomineral organic matrices. Most phosphoproteins were specific for one compartment, only two were identified in both matrices. The sea urchin phosphoproteomes contained several obvious orthologs of mammalian proteins, such as a Src family tyrosine kinase, protein kinase C-delta 1, Dickkopf-1 and other signal transduction components, or nucleobindin. In most cases phosphorylation sites were conserved between sea urchin and mammalian proteins. However, the majority of phosphoproteins had no mammalian counterpart. The most interesting of the sea urchin-specific phosphoproteins, from the perspective of biomineralization research, was an abundant highly phosphorylated and very acidic tooth matrix protein composed of 35 very similar short sequence repeats, a predicted N-terminal secretion signal sequence, and an Asp-rich C-terminal motif, contained in [Glean3:18919]. Conclusions The 64 phosphorylation sites determined represent the most comprehensive list of experimentally identified sea urchin protein phosphorylation sites at present and are an important addition to the recently analyzed Strongylocentrotus purpuratus shell and tooth proteomes. The identified phosphoproteins included a major, highly phosphorylated protein, [Glean3:18919], for which we suggest the name phosphodontin. Although not sequence-related to such highly phosphorylated acidic mammalian dental

  20. GTPases involved in bacterial ribosome maturation.

    PubMed

    Goto, Simon; Muto, Akira; Himeno, Hyouta

    2013-05-01

    The ribosome is an RNA- and protein-based macromolecule having multiple functional domains to facilitate protein synthesis, and it is synthesized through multiple steps including transcription, stepwise cleavages of the primary transcript, modifications of ribosomal proteins and RNAs and assemblies of ribosomal proteins with rRNAs. This process requires dozens of trans-acting factors including GTP- and ATP-binding proteins to overcome several energy-consuming steps. Despite accumulation of genetic, biochemical and structural data, the entire process of bacterial ribosome synthesis remains elusive. Here, we review GTPases involved in bacterial ribosome maturation.

  1. Robust production of recombinant phosphoproteins using cell-free protein synthesis

    PubMed Central

    Oza, Javin P.; Aerni, Hans R.; Pirman, Natasha L.; Barber, Karl W.; ter Haar, Charlotte M.; Rogulina, Svetlana; Amrofell, Matthew B.; Isaacs, Farren J.; Rinehart, Jesse; Jewett, Michael C.

    2015-01-01

    Understanding the functional and structural consequences of site-specific protein phosphorylation has remained limited by our inability to produce phosphoproteins at high yields. Here we address this limitation by developing a cell-free protein synthesis (CFPS) platform that employs crude extracts from a genomically recoded strain of Escherichia coli for site-specific, co-translational incorporation of phosphoserine into proteins. We apply this system to the robust production of up to milligram quantities of human MEK1 kinase. Then, we recapitulate a physiological signalling cascade in vitro to evaluate the contributions of site-specific phosphorylation of mono- and doubly phosphorylated forms on MEK1 activity. We discover that only one phosphorylation event is necessary and sufficient for MEK1 activity. Our work sets the stage for using CFPS as a rapid high-throughput technology platform for direct expression of programmable phosphoproteins containing multiple phosphorylated residues. This work will facilitate study of phosphorylation-dependent structure–function relationships, kinase signalling networks and kinase inhibitor drugs. PMID:26350765

  2. Use of intein-mediated phosphoprotein arrays to study substrate specificity of protein phosphatases.

    PubMed

    Kochinyan, Samvel; Sun, Luo; Ghosh, Inca; Barshevsky, Tanya; Xu, Jie; Xu, Ming-Qun

    2007-01-01

    Synthetic peptides incorporating various chemical moieties, for example, phosphate groups, are convenient tools for investigating protein modification enzymes, such as protein phosphatases (PPs). However, short peptides are sometimes poor substrates, and their binding to commonly used matrices is unpredictable and variable. In general, protein substrates for PPs are superior for enzymatic assays, binding to various matrices, and Western blot analysis. The preparation and characterization of phosphoproteins, however can be difficult and technically demanding. In this study, the intein-mediated protein ligation (IPL) technique was used to readily generate phosphorylated protein substrates by ligating a synthetic phosphopeptide to an intein-generated carrier protein (CP) possessing a carboxyl-terminal thioester with a one-to-one stoichiometry. The ligated phosphoprotein (LPP) substrate was treated with a PP and subsequently subjected to array or Western blot analysis with a phospho-specific antibody. This approach is highly effective in producing arrays of protein substrates containing phosphorylated amino acid residues and has been applied for screening of PPs with specificity toward phosphorylated tyrosine, serine, or threonine residues, resulting in an approximately 240-fold increase in sensitivity in dot blot analysis compared with the use of synthetic peptides. The IPL technique overcomes the disadvantages of current methods and is a versatile system for the facile production of protein substrates containing well-defined structural motifs for the study of protein modification enzymes.

  3. Phosphoproteins with Stability Against All Urinary Phosphatases as Potential Biomarkers in Urine.

    PubMed

    Zhao, Mindi; Liu, Kehui; Gao, Youhe

    2015-01-01

    Urine, by accumulating all kinds of changes, was proposed to be a better source for biomarker discovery. As one of the most common post-translational modifications, phosphorylation plays a vital role in many biological activities. However, the urine phosphoproteome has been largely neglected due to the low abundance of phosphoproteins and the presence of various phosphatases in urine. The low level of background phosphorylation in urine is actually advantageous, as urinary phosphopeptides/proteins that are stable to the phosphatases present in urine have the potential to serve as valuable disease biomarkers. Using a TiO2 enrichment strategy, this study aimed to create a comprehensive proteomic profile of human urinary phosphoproteins and to characterize the changes in the urine phosphoproteome after incubation of urine with renal carcinoma cell lysates. In total, 106 urine phosphorylation sites corresponding to 64 proteins, including 80 previously unidentified human urine protein phosphorylation sites, were identified by mass spectrometry. Fifteen phosphopeptides, together averaging 47% of the total phosphopeptides, were found in samples from three individuals. Cellular proteins are potential source of biomarker in urine phosphorylated proteins. Addition of renal carcinoma cellular proteins to urine did not significantly change the phosphorylation level of urine proteins. But there were still a few phosphopeptides from cell lysates survived urinary phosphatases; such phosphopeptides represent potential biomarkers in urine.

  4. Proteomic profiling of the phosphoproteins in the rat thalamus, hippocampus and frontal lobe after propofol anesthesia

    PubMed Central

    2014-01-01

    Background Propofol is a safe and effective intravenous anesthetic that is widely used for the induction and maintenance of anesthesia during surgery. However, the mechanism by which propofol exerts its anesthetic effect remains unknown. The rapid onset of phosphorylation modifications coincides with that of propofol anesthesia. Methods Propofol-anesthetized rat models were built and phosphorylated proteins in the thalamus, hippocampus and frontal lobe were enriched the to analyze the changes in these phosphoproteins after propofol anesthesia. Results Sixteen of these phosphoprotein spots were successfully identified using MALDI-TOF MS and a subsequent comparative sequence search in the Mascot database. Of these proteins, keratin 18 and the tubulin 2c chain are cytoskeletal proteins; keratin 18 and gelsolin are relevant to alcohol drowsiness. Based on Western blot analysis, we also confirmed that the phosphorylation of these proteins is directly induced by propofol, indicating that propofol anesthesia may be relevant to cytoskeletal proteins and alcohol drowsiness. Conclusions These identified propofol-induced phosphorylations of proteins provide meaningful contributions for further studying the anesthetic mechanism of propofol. PMID:24410762

  5. Selection and characterization of single-chain recombinant antibodies against phosphoprotein of Newcastle disease virus.

    PubMed

    Li, Benqiang; Ye, Jiaxin; Lin, Yuan; Wang, Man; Jia, Rui; Zhu, Jianguo

    2014-09-01

    Phosphoprotein (P), involved in virus RNA replication and transcription, had become a new target for the research on treating Newcastle disease virus (NDV). Here we described the cloning and expression of phosphoprotein from NDV, and then screened the anti-P antibodies from the chicken single chain fragment variable (scFv) library, which were generated from chickens immunized with the ND vaccines. As a first step, the recombinant expression vector pET28a-P was successfully constructed. In a following step, two anti-P positive scFv clones from the scFv library were selected by indirect enzyme-linked immunosorbent assay (ELISA) method. The sequence analysis of two positive clones showed that there were more variation in complementary determine region (CDR) of VH and VL, and the CDR3 in VH exhibited a significant change in amino acid number and type. In another experiment, the purified scFv antibodies used in the assay was shown to be specific for NDV-P by western blot. The results indicated that the strategy we used in this experiment proved to be convenient way for screening scFv antibody, which paved a new way for the immunization diagnosis and the exploration of integrated control of NDV.

  6. Characterization of hibernating ribosomes in mammalian cells

    PubMed Central

    Majumder, Mithu; Mullins, Michael R; Yuan, Celvie L; Papadopoulou, Barbara; Merrick, William C; Komar, Anton A

    2011-01-01

    Protein synthesis across kingdoms involves the assembly of 70S (prokaryotes) or 80S (eukaryotes) ribosomes on the mRNAs to be translated. 70S ribosomes are protected from degradation in bacteria during stationary growth or stress conditions by forming dimers that migrate in polysome profiles as 100S complexes. Formation of ribosome dimers in Escherichia coli is mediated by proteins, namely the ribosome modulation factor (RMF), which is induced in the stationary phase of cell growth. It is reported here a similar ribosomal complex of 110S in eukaryotic cells, which forms during nutrient starvation. The dynamic nature of the 110S ribosomal complex (mammalian equivalent of the bacterial 100S) was supported by the rapid conversion into polysomes upon nutrient-refeeding via a mechanism sensitive to inhibitors of translation initiation. Several experiments were used to show that the 110S complex is a dimer of nontranslating ribosomes. Cryo-electron microscopy visualization of the 110S complex revealed that two 80S ribosomes are connected by a flexible, albeit localized, interaction. We conclude that, similarly to bacteria, rat cells contain stress-induced ribosomal dimers. The identification of ribosomal dimers in rat cells will bring new insights in our thinking of the ribosome structure and its function during the cellular response to stress conditions. PMID:21768774

  7. Characterizing inactive ribosomes in translational profiling

    PubMed Central

    Liu, Botao; Qian, Shu-Bing

    2016-01-01

    ABSTRACT The broad impact of translational regulation has emerged explosively in the last few years in part due to the technological advance in genome-wide interrogation of gene expression. During mRNA translation, the majority of actively translating ribosomes exist as polysomes in cells with multiple ribosomes loaded on a single transcript. The importance of the monosome, however, has been less appreciated in translational profiling analysis. Here we report that the monosome fraction isolated by sucrose sedimentation contains a large quantity of inactive ribosomes that do not engage on mRNAs to direct translation. We found that the elongation factor eEF2, but not eEF1A, stably resides in these non-translating ribosomes. This unique feature permits direct evaluation of ribosome status under various stress conditions and in the presence of translation inhibitors. Ribosome profiling reveals that the monosome has a similar but not identical pattern of ribosome footprints compared to the polysome. We show that the association of free ribosomal subunits minimally contributes to ribosome occupancy outside of the coding region. Our results not only offer a quantitative method to monitor ribosome availability, but also uncover additional layers of ribosome status needed to be considered in translational profiling analysis. PMID:27335722

  8. Characterization of hibernating ribosomes in mammalian cells.

    PubMed

    Krokowski, Dawid; Gaccioli, Francesca; Majumder, Mithu; Mullins, Michael R; Yuan, Celvie L; Papadopoulou, Barbara; Merrick, William C; Komar, Anton A; Taylor, Derek; Hatzoglou, Maria

    2011-08-15

    Protein synthesis across kingdoms involves the assembly of 70S (prokaryotes) or 80S (eukaryotes) ribosomes on the mRNAs to be translated. 70S ribosomes are protected from degradation in bacteria during stationary growth or stress conditions by forming dimers that migrate in polysome profiles as 100S complexes. Formation of ribosome dimers in Escherichia coli is mediated by proteins, namely the ribosome modulation factor (RMF), which is induced in the stationary phase of cell growth. It is reported here a similar ribosomal complex of 110S in eukaryotic cells, which forms during nutrient starvation. The dynamic nature of the 110S ribosomal complex (mammalian equivalent of the bacterial 100S) was supported by the rapid conversion into polysomes upon nutrient-refeeding via a mechanism sensitive to inhibitors of translation initiation. Several experiments were used to show that the 110S complex is a dimer of nontranslating ribosomes. Cryo-electron microscopy visualization of the 110S complex revealed that two 80S ribosomes are connected by a flexible, albeit localized, interaction. We conclude that, similarly to bacteria, rat cells contain stress-induced ribosomal dimers. The identification of ribosomal dimers in rat cells will bring new insights in our thinking of the ribosome structure and its function during the cellular response to stress conditions.

  9. Structure of Ribosomal Silencing Factor Bound to Mycobacterium tuberculosis Ribosome.

    PubMed

    Li, Xiaojun; Sun, Qingan; Jiang, Cai; Yang, Kailu; Hung, Li-Wei; Zhang, Junjie; Sacchettini, James C

    2015-10-06

    The ribosomal silencing factor RsfS slows cell growth by inhibiting protein synthesis during periods of diminished nutrient availability. The crystal structure of Mycobacterium tuberculosis (Mtb) RsfS, together with the cryo-electron microscopy (EM) structure of the large subunit 50S of Mtb ribosome, reveals how inhibition of protein synthesis by RsfS occurs. RsfS binds to the 50S at L14, which, when occupied, blocks the association of the small subunit 30S. Although Mtb RsfS is a dimer in solution, only a single subunit binds to 50S. The overlap between the dimer interface and the L14 binding interface confirms that the RsfS dimer must first dissociate to a monomer in order to bind to L14. RsfS interacts primarily through electrostatic and hydrogen bonding to L14. The EM structure shows extended rRNA density that it is not found in the Escherichia coli ribosome, the most striking of these being the extended RNA helix of H54a.

  10. Crystal structure of the eukaryotic ribosome.

    PubMed

    Ben-Shem, Adam; Jenner, Lasse; Yusupova, Gulnara; Yusupov, Marat

    2010-11-26

    Crystal structures of prokaryotic ribosomes have described in detail the universally conserved core of the translation mechanism. However, many facets of the translation process in eukaryotes are not shared with prokaryotes. The crystal structure of the yeast 80S ribosome determined at 4.15 angstrom resolution reveals the higher complexity of eukaryotic ribosomes, which are 40% larger than their bacterial counterparts. Our model shows how eukaryote-specific elements considerably expand the network of interactions within the ribosome and provides insights into eukaryote-specific features of protein synthesis. Our crystals capture the ribosome in the ratcheted state, which is essential for translocation of mRNA and transfer RNA (tRNA), and in which the small ribosomal subunit has rotated with respect to the large subunit. We describe the conformational changes in both ribosomal subunits that are involved in ratcheting and their implications in coordination between the two associated subunits and in mRNA and tRNA translocation.

  11. [About the ribosomal biogenesis in human].

    PubMed

    Tafforeau, Lionel

    2015-01-01

    Ribosomes are cellular ribonucleoprotein particles required for a fundamental mechanism, translation of the genetic information into proteins. Ribosome biogenesis is a highly complex pathway involving many maturation steps: ribosomal RNA (rRNA) synthesis, rRNA processing, pre-rRNA modifications, its assembly with ribosomal proteins in the nuceolus, export of the subunit precursors to the nucleoplasm and the cytoplasm. Ribosome biogenesis has mainly being investigated in yeast during these last 25 years. However, recent works have shown that, despite many similarities between yeast and human ribosome structure and biogenesis, human pre-rRNA processing is far more complex than in yeast. In order to better understand diseases related to a malfunction in ribosome synthesis, the ribosomopathies, research should be conducted directly in human cells and animal models. © 2015 médecine/sciences – Inserm.

  12. 5S rRNA and ribosome.

    PubMed

    Gongadze, G M

    2011-12-01

    5S rRNA is an integral component of the ribosome of all living organisms. It is known that the ribosome without 5S rRNA is functionally inactive. However, the question about the specific role of this RNA in functioning of the translation apparatus is still open. This review presents a brief history of the discovery of 5S rRNA and studies of its origin and localization in the ribosome. The previously expressed hypotheses about the role of this RNA in the functioning of the ribosome are discussed considering the unique location of 5S rRNA in the ribosome and its intermolecular contacts. Based on analysis of the current data on ribosome structure and its functional complexes, the role of 5S rRNA as an intermediary between ribosome functional domains is discussed.

  13. Ribosomal targets for antibiotic drug discovery

    DOEpatents

    Blanchard, Scott C.; Feldman, Michael Brian; Wang, Leyi; Doudna Cate, James H.; Pulk, Arto; Altman, Roger B.; Wasserman, Michael R

    2016-09-13

    The present invention relates to methods to identify molecules that binds in the neomycin binding pocket of a bacterial ribosome using structures of an intact bacterial ribosome that reveal how the ribosome binds tRNA in two functionally distinct states, determined by x-ray crystallography. One state positions tRNA in the peptidyl-tRNA binding site. The second, a fully rotated state, is stabilized by ribosome recycling factor (RRF) and binds tRNA in a highly bent conformation in a hybrid peptidyl/exit (P/E) site. Additionally, the invention relates to various assays, including single-molecule assay for ribosome recycling, and methods to identify compounds that interfere with ribosomal function by detecting newly identified intermediate FRET states using known and novel FRET pairs on the ribosome. The invention also provides vectors and compositions with an N-terminally tagged S13 protein.

  14. Eukaryotic ribosome biogenesis at a glance.

    PubMed

    Thomson, Emma; Ferreira-Cerca, Sébastien; Hurt, Ed

    2013-11-01

    Ribosomes play a pivotal role in the molecular life of every cell. Moreover, synthesis of ribosomes is one of the most energetically demanding of all cellular processes. In eukaryotic cells, ribosome biogenesis requires the coordinated activity of all three RNA polymerases and the orchestrated work of many (>200) transiently associated ribosome assembly factors. The biogenesis of ribosomes is a tightly regulated activity and it is inextricably linked to other fundamental cellular processes, including growth and cell division. Furthermore, recent studies have demonstrated that defects in ribosome biogenesis are associated with several hereditary diseases. In this Cell Science at a Glance article and the accompanying poster, we summarise the current knowledge on eukaryotic ribosome biogenesis, with an emphasis on the yeast model system.

  15. Interrelationships between yeast ribosomal protein assembly events and transient ribosome biogenesis factors interactions in early pre-ribosomes.

    PubMed

    Jakob, Steffen; Ohmayer, Uli; Neueder, Andreas; Hierlmeier, Thomas; Perez-Fernandez, Jorge; Hochmuth, Eduard; Deutzmann, Rainer; Griesenbeck, Joachim; Tschochner, Herbert; Milkereit, Philipp

    2012-01-01

    Early steps of eukaryotic ribosome biogenesis require a large set of ribosome biogenesis factors which transiently interact with nascent rRNA precursors (pre-rRNA). Most likely, concomitant with that initial contacts between ribosomal proteins (r-proteins) and ribosome precursors (pre-ribosomes) are established which are converted into robust interactions between pre-rRNA and r-proteins during the course of ribosome maturation. Here we analysed the interrelationship between r-protein assembly events and the transient interactions of ribosome biogenesis factors with early pre-ribosomal intermediates termed 90S pre-ribosomes or small ribosomal subunit (SSU) processome in yeast cells. We observed that components of the SSU processome UTP-A and UTP-B sub-modules were recruited to early pre-ribosomes independently of all tested r-proteins. On the other hand, groups of SSU processome components were identified whose association with early pre-ribosomes was affected by specific r-protein assembly events in the head-platform interface of the SSU. One of these components, Noc4p, appeared to be itself required for robust incorporation of r-proteins into the SSU head domain. Altogether, the data reveal an emerging network of specific interrelationships between local r-protein assembly events and the functional interactions of SSU processome components with early pre-ribosomes. They point towards some of these components being transient primary pre-rRNA in vivo binders and towards a role for others in coordinating the assembly of major SSU domains.

  16. RIBOSOME PRECURSOR PARTICLES IN NUCLEOLI

    PubMed Central

    Liau, Ming C.; Perry, Robert P.

    1969-01-01

    Ribonucleoprotein (RNP) particles containing the precursors of ribosomal RNA were extracted from L cell nucleoli and analyzed under conditions comparable to those used in the characterization of cytoplasmic ribosomes. Using nucleoli from cells suitably labeled with 3H-uridine, we detected three basic RNP components, sedimenting at approximately 62S, 78S, and 110S in sucrose gradients containing magnesium. A fourth particle, sedimenting at about 95S, appears to be a dimer of the 62S and 78S components. When centrifuged in gradients containing EDTA, the 62S, 78S, and 110S particles sediment at about 55S, 65S, and 80S, respectively. RNA was extracted from RNP particles which were prepared by two cycles of zonal centrifugation. The 62S particles yielded 32S RNA and a detectable amount of 28S RNA, the 78S structures, 32S RNA and possibly some 36S RNA, and the 110S particles, a mixture of 45S, 36S, and 32S RNA's. When cells were pulsed briefly and further incubated in the presence of actinomycin D, there was a gradual shift of radioactivity from heavier to lighter particles. This observation is consistent with the scheme of maturation: 110S → 78S → 62S. The principal buoyant densities in cesium chloride of the 110S, 78S, and 62S particles are 1.465, 1.490, and 1.545, respectively. These densities are all significantly lower than 1.570, which is characteristic of the mature large subunit of cytoplasmic ribosomes, suggesting that the precursor particles have a relatively higher ratio of protein to RNA, and that ribosome maturation involves, in addition to decrease in the size of the RNA molecules, a progressive decrease in the proportion of associated protein. PMID:5815062

  17. Ribosome engineering to promote new crystal forms

    SciTech Connect

    Selmer, Maria; Gao, Yong-Gui; Weixlbaumer, Albert; Ramakrishnan, V.

    2012-05-01

    Truncation of ribosomal protein L9 in T. thermophilus allows the generation of new crystal forms and the crystallization of ribosome–GTPase complexes. Crystallographic studies of the ribosome have provided molecular details of protein synthesis. However, the crystallization of functional complexes of ribosomes with GTPase translation factors proved to be elusive for a decade after the first ribosome structures were determined. Analysis of the packing in different 70S ribosome crystal forms revealed that regardless of the species or space group, a contact between ribosomal protein L9 from the large subunit and 16S rRNA in the shoulder of a neighbouring small subunit in the crystal lattice competes with the binding of GTPase elongation factors to this region of 16S rRNA. To prevent the formation of this preferred crystal contact, a mutant strain of Thermus thermophilus, HB8-MRCMSAW1, in which the ribosomal protein L9 gene has been truncated was constructed by homologous recombination. Mutant 70S ribosomes were used to crystallize and solve the structure of the ribosome with EF-G, GDP and fusidic acid in a previously unobserved crystal form. Subsequent work has shown the usefulness of this strain for crystallization of the ribosome with other GTPase factors.

  18. Isolation of a calcium-binding phosphoprotein from the oocytes and hemolymph of the blood-sucking insect Rhodnius prolixus.

    PubMed

    Silva-Neto, M A; Atella, G C; Fialho, E; Paes, M C; Zingali, R B; Petretski, J H; Alves, E W; Masuda, H

    1996-11-22

    A novel calcium-binding phosphoprotein was isolated from the oocytes of the blood-sucking bug Rhodnius prolixus. This protein exhibits an apparent molecular mass of 18 kDa on gel filtration, but migrates as an 8-kDa band on N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine/SDS-polyacrylamide gels. It has a high content of serine (24% of the total number of residues), and phosphoserine is the sole amino acid phosphorylated in vivo. A similar protein was partially purified from the hemolymph. It resembles the oocyte form of the protein in its NH2-terminal sequence and its ability to be taken up by growing ovaries. 45Ca binding to the oocyte phosphoprotein was determined after SDS-polyacrylamide gel electrophoresis followed by blotting on nitrocellulose membranes. Titration of Ca2+-binding sites shows a high capacity (approximately 50 mol/mol of protein), but a low affinity (K0.5 congruent with 10(-3) M). Based on these characteristics, we have named this protein Rhodnius calcium-binding phosphoprotein. It resembles phosvitin, a phosphoprotein present in the oocytes of nonmammalian vertebrates.

  19. Identification of nuclear phosphoproteins as novel tobacco markers in mouse lung tissue following short-term exposure to tobacco smoke

    PubMed Central

    Niimori-Kita, Kanako; Ogino, Kiyoshi; Mikami, Sayaka; Kudoh, Shinji; Koizumi, Daikai; Kudoh, Noritaka; Nakamura, Fumiko; Misumi, Masahiro; Shimomura, Tadasuke; Hasegawa, Koki; Usui, Fumihiko; Nagahara, Noriyuki; Ito, Takaaki

    2014-01-01

    Smoking is a risk factor for lung diseases, including chronic obstructive pulmonary disease and lung cancer. However, the molecular mechanisms mediating the progression of these diseases remain unclear. Therefore, we sought to identify signaling pathways activated by tobacco-smoke exposure, by analyzing nuclear phosphoprotein expression using phosphoproteomic analysis of lung tissue from mice exposed to tobacco smoke. Sixteen mice were exposed to tobacco smoke for 1 or 7 days, and the expression of phosphorylated peptides was analyzed by mass spectrometry. A total of 253 phosphoproteins were identified, including FACT complex subunit SPT16 in the 1-day exposure group, keratin type 1 cytoskeletal 18 (K18), and adipocyte fatty acid-binding protein, in the 7-day exposure group, and peroxiredoxin-1 (OSF3) and spectrin β chain brain 1 (SPTBN1), in both groups. Semi-quantitative analysis of the identified phosphoproteins revealed that 33 proteins were significantly differentially expressed between the control and exposed groups. The identified phosphoproteins were classified according to their biological functions. We found that the identified proteins were related to inflammation, regeneration, repair, proliferation, differentiation, morphogenesis, and response to stress and nicotine. In conclusion, we identified proteins, including OSF3 and SPTBN1, as candidate tobacco smoke-exposure markers; our results provide insights into the mechanisms of tobacco smoke-induced diseases. PMID:25349779

  20. Ca2+ uptake, Ca2+-ATPase activity, phosphoprotein formation and phosphate turnover in a microsomal fraction of smooth muscle.

    PubMed

    Raeymaekers, L; Hasselbach, W

    1981-05-15

    Vesicles capable of phosphate-stimulated calcium uptake were isolated from the microsomal fraction of the smooth muscle of the pig stomach according to a previously described procedure which consists in increasing the density of the vesicles by loading them with calcium phosphate and isolating them by centrifugation [Raeymaekers, L., Agostini, B., and Hasselbach, W. (1981) Histochemistry, 70, 139--150]. These vesicles, which contain calcium phosphate deposits, are able to accumulate an additional amount of calcium. This calcium uptake is accompanied by calcium-stimulated ATPase activity and by the formation of an acid-stable phosphoprotein. The acid-denatured phosphoprotein is dephosphorylated by hydroxylamine, which indicates that an acylphosphate is formed. This phosphoprotein probably represents a phosphorylated transport intermediate similar to that seen with the Ca2+-ATPase of sarcoplasmic reticulum of skeletal muscle. As with the Ca2+-ATPase of sarcoplasmic reticulum vesicles, this vesicular fraction catalyses an exchange between inorganic phosphate and the gamma-phosphate of ATP (ATP-Pi exchange) which is dependent on the presence of intravesicular calcium, and an exchange of phosphate between ATP and ADP (ATP-ADP exchange). The results further indicate that the turnover rate of the calcium pump, calculated from the ratio of calcium-stimulated ATPase activity to the steady-state level of phosphoprotein, is similar to that of Ca2+-ATPase of sarcoplasmic reticulum of skeletal muscle.

  1. Synthesis of ribosomes in Saccharomyces cerevisiae.

    PubMed Central

    Warner, J R

    1989-01-01

    The assembly of a eucaryotic ribosome requires the synthesis of four ribosomal ribonucleic acid (RNA) molecules and more than 75 ribosomal proteins. It utilizes all three RNA polymerases; it requires the cooperation of the nucleus and the cytoplasm, the processing of RNA, and the specific interaction of RNA and protein molecules. It is carried out efficiently and is exquisitely sensitive to the needs of the cell. Our current understanding of this process in the genetically tractable yeast Saccharomyces cerevisiae is reviewed. The ribosomal RNA genes are arranged in a tandem array of 100 to 200 copies. This tandem array has led to unique ways of carrying out a number of functions. Replication is asymmetric and does not initiate from every autonomously replicating sequence. Recombination is suppressed. Transcription of the major ribosomal RNA appears to involve coupling between adjacent transcription units, which are separated by the 5S RNA transcription unit. Genes for many ribosomal proteins have been cloned and sequenced. Few are linked; most are duplicated; most have an intron. There is extensive homology between yeast ribosomal proteins and those of other species. Most, but not all, of the ribosomal protein genes have one or two sites that are essential for their transcription and that bind a common transcription factor. This factor binds also to many other places in the genome, including the telomeres. There is coordinated transcription of the ribosomal protein genes under a variety of conditions. However, the cell seems to possess no mechanism for regulating the transcription of individual ribosomal protein genes in response either to a deficiency or an excess of a particular ribosomal protein. A deficiency causes slow growth. Any excess ribosomal protein is degraded very rapidly, with a half-life of 1 to 5 min. Unlike most types of cells, yeast cells appear not to regulate the translation of ribosomal proteins. However, in the case of ribosomal protein L32

  2. The Ribosome Modulates Nascent Protein Folding

    PubMed Central

    Kaiser, Christian M.; Goldman, Daniel H.; Chodera, John D.; Tinoco, Ignacio; Bustamante, Carlos

    2014-01-01

    Proteins are synthesized by the ribosome and generally must fold to become functionally active. Although it is commonly assumed that the ribosome affects the folding process, this idea has been extremely difficult to demonstrate. We have developed an experimental system to investigate the folding of single ribosome-bound stalled nascent polypeptides with optical tweezers. In T4 lysozyme, synthesized in a reconstituted in vitro translation system, the ribosome slows the formation of stable tertiary interactions and the attainment of the native state relative to the free protein. Incomplete T4 lysozyme polypeptides misfold and aggregate when free in solution, but they remain folding-competent near the ribosomal surface. Altogether, our results suggest that the ribosome not only decodes the genetic information and synthesizes polypeptides, but also promotes efficient de novo attainment of the native state. PMID:22194581

  3. BECN1-dependent CASP2 incomplete autophagy induction by binding to rabies virus phosphoprotein

    PubMed Central

    Liu, Juan; Wang, Hailong; Gu, Jinyan; Deng, Tingjuan; Yuan, Zhuangchuan; Hu, Boli; Xu, Yunbin; Yan, Yan; Zan, Jie; Liao, Min; DiCaprio, Erin; Li, Jianrong; Su, Shuo; Zhou, Jiyong

    2017-01-01

    ABSTRACT Autophagy is an essential component of host immunity and used by viruses for survival. However, the autophagy signaling pathways involved in virus replication are poorly documented. Here, we observed that rabies virus (RABV) infection triggered intracellular autophagosome accumulation and results in incomplete autophagy by inhibiting autophagy flux. Subsequently, we found that RABV infection induced the reduction of CASP2/caspase 2 and the activation of AMP-activated protein kinase (AMPK)-AKT-MTOR (mechanistic target of rapamycin) and AMPK-MAPK (mitogen-activated protein kinase) pathways. Further investigation revealed that BECN1/Beclin 1 binding to viral phosphoprotein (P) induced an incomplete autophagy via activating the pathways CASP2-AMPK-AKT-MTOR and CASP2-AMPK-MAPK by decreasing CASP2. Taken together, our data first reveals a crosstalk of BECN1 and CASP2-dependent autophagy pathways by RABV infection. PMID:28129024

  4. Structure of the C-Terminal Domain of Lettuce Necrotic Yellows Virus Phosphoprotein

    PubMed Central

    Martinez, Nicolas; Ribeiro, Euripedes A.; Leyrat, Cédric; Tarbouriech, Nicolas; Ruigrok, Rob W. H.

    2013-01-01

    Lettuce necrotic yellows virus (LNYV) is a prototype of the plant-adapted cytorhabdoviruses. Through a meta-prediction of disorder, we localized a folded C-terminal domain in the amino acid sequence of its phosphoprotein. This domain consists of an autonomous folding unit that is monomeric in solution. Its structure, solved by X-ray crystallography, reveals a lollipop-shaped structure comprising five helices. The structure is different from that of the corresponding domains of other Rhabdoviridae, Filoviridae, and Paramyxovirinae; only the overall topology of the polypeptide chain seems to be conserved, suggesting that this domain evolved under weak selective pressure and varied in size by the acquisition or loss of functional modules. PMID:23785215

  5. Cytorhabdovirus phosphoprotein shows RNA silencing suppressor activity in plants, but not in insect cells.

    PubMed

    Mann, Krin S; Johnson, Karyn N; Dietzgen, Ralf G

    2015-02-01

    RNA silencing in plants and insects provides an antiviral defense and as a countermeasure most viruses encode RNA silencing suppressors (RSS). For the family Rhabdoviridae, no detailed functional RSS studies have been reported in plant hosts and insect vectors. In agroinfiltrated Nicotiana benthamiana leaves we show for the first time for a cytorhabdovirus, lettuce necrotic yellows virus (LNYV), that one of the nucleocapsid core proteins, phosphoprotein (P) has relatively weak local RSS activity and delays systemic silencing of a GFP reporter. Analysis of GFP small RNAs indicated that the P protein did not prevent siRNA accumulation. To explore RSS activity in insects, we used a Flock House virus replicon system in Drosophila S2 cells. In contrast to the plant host, LNYV P protein did not exhibit RSS activity in the insect cells. Taken together our results suggest that P protein may target plant-specific components of RNA silencing post siRNA biogenesis.

  6. Recombinant phosphoprotein based single serum dilution ELISA for rapid serological detection of Newcastle disease virus.

    PubMed

    Das, Moushumee; Kumar, Sachin

    2015-12-01

    Newcastle disease virus (NDV) is the causative agent of a highly contagious disease in avian species. All strains of NDV belong to avian paramyxovirus serotype-1. The disease is endemic in different parts of the world and vaccination is the only way to protect birds from NDV infection. The virus non-structural phosphoprotein (P) is the second most abundant protein and a major modulator of viral replication. Although P protein shows lesser evolutionary divergence among NDV isolates, it is known to be highly divergent among different avian paramyxovirus serotypes. In the present study, a recombinant P protein based single serum dilution ELISA was developed which showed better sensitivity, specificity and accuracy as compared to conventional methods for NDV detection. The recombinant P protein based ELISA could be an alternative to existing diagnostics against NDV infection in chickens.

  7. Ribosome-associated protein quality control

    PubMed Central

    Brandman, Onn; Hegde, Ramanujan S

    2016-01-01

    Protein synthesis by the ribosome can fail for numerous reasons including faulty mRNA, insufficient availability of charged tRNAs and genetic errors. All organisms have evolved mechanisms to recognize stalled ribosomes and initiate pathways for recycling, quality control and stress signaling. Here we review the discovery and molecular dissection of the eukaryotic ribosome-associated quality-control pathway for degradation of nascent polypeptides arising from interrupted translation. PMID:26733220

  8. Ribosomes are optimized for autocatalytic production

    NASA Astrophysics Data System (ADS)

    Reuveni, Shlomi; Ehrenberg, Måns; Paulsson, Johan

    2017-07-01

    Many fine-scale features of ribosomes have been explained in terms of function, revealing a molecular machine that is optimized for error-correction, speed and control. Here we demonstrate mathematically that many less well understood, larger-scale features of ribosomes—such as why a few ribosomal RNA molecules dominate the mass and why the ribosomal protein content is divided into 55-80 small, similarly sized segments—speed up their autocatalytic production.

  9. Secreted Phosphoprotein 1 and Sex-Specific Differences in Silica-Induced Pulmonary Fibrosis in Mice

    PubMed Central

    Latoche, Joseph D.; Ufelle, Alexander Chukwuma; Fazzi, Fabrizio; Ganguly, Koustav; Leikauf, George D.; Fattman, Cheryl L.

    2016-01-01

    Background: Fibrotic lung diseases occur predominantly in males, and reports describe better survival in affected females. Male mice are more sensitive to silica-induced lung fibrosis than silica-treated female mice. Secreted phosphoprotein 1 (SPP1, also known as osteopontin) increases in pulmonary fibrosis, and Spp1 transcription may be regulated by estrogen or estrogen receptor–related receptors. Objective: We determined whether differences in silica-induced SPP1 levels contribute to sex differences in lung fibrosis. Methods: Male and female mice were treated with 0.2 g/kg intratracheal silica, and lung injury was assessed 1, 3, or 14 days post-exposure. Gene-targeted (Spp1–/–) mice, control Spp1+/+ (C57BL/6J) mice, ovariectomized (OVX) female mice, and estrogen-treated male mice were treated with silica, and lung injury was assessed. Results: Silica-induced SPP1 in lung tissue, bronchoalveolar lavage, and serum increased more in male than in female mice. Following silica treatment, bronchoalveolar lavage cell infiltrates decreased in female Spp1–/– mice compared with female Spp1+/+ mice, and lung hydroxyproline decreased in male Spp1–/– mice compared with male Spp1+/+ mice. OVX female mice had increased lung SPP1 expression in response to silica compared with silica-treated sham female mice. Silica-induced lung collagen and hydroxyproline (markers of fibrosis), and SPP1 levels decreased in estrogen-treated males compared with untreated males. Conclusion: These findings suggest that sex-specific differences in SPP1 levels contribute to the differential sensitivity of male and female mice to the development of silica-induced fibrosis. Citation: Latoche JD, Ufelle AC, Fazzi F, Ganguly K, Leikauf GD, Fattman CL. 2016. Secreted phosphoprotein 1 and sex-specific differences in silica-induced pulmonary fibrosis in mice. Environ Health Perspect 124:1199–1207; http://dx.doi.org/10.1289/ehp.1510335 PMID:26955063

  10. Regulation and subcellular localization of the microtubule-destabilizing stathmin family phosphoproteins in cortical neurons.

    PubMed

    Gavet, Olivier; El Messari, Saïd; Ozon, Sylvie; Sobel, André

    2002-06-01

    Stathmin is a ubiquitous cytosolic phosphoprotein, preferentially expressed in the nervous system, and the generic element of a protein family that includes the neural-specific proteins SCG10, SCLIP, and RB3 and its splice variants, RB3' and RB3". All phosphoproteins of the family share with stathmin its tubulin binding and microtubule (MT)-destabilizing activities. To understand better the specific roles of these proteins in neuronal cells, we performed a comparative study of their expression, regulation, and intracellular distribution in embryonic cortical neurons in culture. We found that stathmin is highly expressed ( approximately 0.25% of total proteins) and uniformly present in the various neuronal compartments (cell body, dendrites, axon, growth cones). It appeared mainly unphosphorylated or weakly phosphorylated on one site, and antisera to specific phosphorylated sites (serines 16, 25, or 38) did not reveal a differential regulation of its phosphorylation among neuronal cell compartments. However, they revealed a subpopulation of cells in which stathmin was highly phosphorylated on serine 16, possibly by CaM kinase II also active in a similar subpopulation. The other proteins of the stathmin family are expressed about 100-fold less than stathmin in partially distinct neuronal populations, RB3 being detected in only about 20% of neurons in culture. In contrast to stathmin, they are each mostly concentrated at the Golgi apparatus and are also present along dendrites and axons, including growth cones. Altogether, our results suggest that the different members of the stathmin family have complementary, at least partially distinct functions in neuronal cell regulation, in particular in relation to MT dynamics. Copyright 2002 Wiley-Liss, Inc.

  11. Spatiotemporal phosphoprotein distribution and associated cytokine response of a traumatic injury.

    PubMed

    Han, Alice A; Currie, Holly N; Loos, Matthew S; Vrana, Julie A; Fabyanic, Emily B; Prediger, Maren S; Boyd, Jonathan W

    2016-03-01

    Molecular mechanisms of wound healing have been extensively characterized, providing a better understanding of the processes involved in wound repair and offering advances in treatment methods. Both spatial and temporal investigations of injury biomarkers have helped to pinpoint significant time points and locations during the recovery process, which may be vital in managing the injury and making the appropriate diagnosis. This study addresses spatial and temporal differences of phosphoproteins found in skeletal muscle tissue following a traumatic femur fracture, which were further compared to co-localized cytokine responses. In particular, several proteins (Akt, ERK, c-Jun, CREB, JNK, MEK1, and p38) and post-translational phosphorylations (p-Akt, p-c-Jun, p-CREB, p-ERK1/2, p-MEK1, p-p38, p-GSK3α/β, p-HSP27, p-p70S6K, and p-STAT3) associated with inflammation, new tissue formation, and remodeling were found to exhibit significant spatial and temporal differences in response to the traumatic injury. Quadratic discriminant analysis of all measured responses, including cytokine concentrations from previously published findings, was used to classify temporal and spatial observations at high predictive rates, further confirming that distinct spatiotemporal distributions for total protein, phosphorylation signaling, and cytokine (IL-1α, IL-1ß, IL2, IL6, TNF-α, and MIP-1α) responses exist. Finally, phosphoprotein measurements were found to be significantly correlated to cytokine concentrations, suggesting coordinated intracellular and extracellular activity during crucial periods of repair. This study represents a first attempt to monitor and assess integrated changes in extracellular and intracellular signaling in response to a traumatic injury in muscle tissues, which may provide a framework for future research to improve both our understanding of wounds and their treatment options.

  12. Ribosome biogenesis in the yeast Saccharomyces cerevisiae.

    PubMed

    Woolford, John L; Baserga, Susan J

    2013-11-01

    Ribosomes are highly conserved ribonucleoprotein nanomachines that translate information in the genome to create the proteome in all cells. In yeast these complex particles contain four RNAs (>5400 nucleotides) and 79 different proteins. During the past 25 years, studies in yeast have led the way to understanding how these molecules are assembled into ribosomes in vivo. Assembly begins with transcription of ribosomal RNA in the nucleolus, where the RNA then undergoes complex pathways of folding, coupled with nucleotide modification, removal of spacer sequences, and binding to ribosomal proteins. More than 200 assembly factors and 76 small nucleolar RNAs transiently associate with assembling ribosomes, to enable their accurate and efficient construction. Following export of preribosomes from the nucleus to the cytoplasm, they undergo final stages of maturation before entering the pool of functioning ribosomes. Elaborate mechanisms exist to monitor the formation of correct structural and functional neighborhoods within ribosomes and to destroy preribosomes that fail to assemble properly. Studies of yeast ribosome biogenesis provide useful models for ribosomopathies, diseases in humans that result from failure to properly assemble ribosomes.

  13. Assembly of the 30S ribosomal subunit.

    PubMed

    Williamson, James R

    2005-11-01

    The assembly of ribosomes requires a significant fraction of the energy expenditure for rapidly growing bacteria. The ribosome is composed of three large RNA molecules and over 50 small proteins that must be rapidly and efficiently assembled into the molecular machine responsible for protein synthesis. For over 30 years, the 30S ribosome has been a key model system for understanding the process of ribosome biogenesis through in vitro assembly experiments. We have recently developed an isotope pulse-chase experiment using quantitative mass spectrometry that permits assembly kinetics to be measured in real time. Kinetic studies have revealed an assembly energy landscape that ensures efficient assembly by a flexible and robust pathway.

  14. Role of GTPases in bacterial ribosome assembly.

    PubMed

    Britton, Robert A

    2009-01-01

    The assembly of the ribosome, a complex molecular machine composed of RNA and protein, is a poorly understood process. Recent work has demonstrated that GTPases are likely to play key roles in the assembly of ribosomes in bacteria and eukaryotes. This review highlights several bacterial ribosome assembly GTPases (RA-GTPases) and discusses possible functions for these proteins in the biogenesis of individual ribosomal subunits and subunit joining. RA-GTPases appear to link various aspects of the cell cycle and metabolism with translation. How these RA-GTPases may coordinate these connections are discussed.

  15. Ribosome Biogenesis in the Yeast Saccharomyces cerevisiae

    PubMed Central

    Woolford, John L.; Baserga, Susan J.

    2013-01-01

    Ribosomes are highly conserved ribonucleoprotein nanomachines that translate information in the genome to create the proteome in all cells. In yeast these complex particles contain four RNAs (>5400 nucleotides) and 79 different proteins. During the past 25 years, studies in yeast have led the way to understanding how these molecules are assembled into ribosomes in vivo. Assembly begins with transcription of ribosomal RNA in the nucleolus, where the RNA then undergoes complex pathways of folding, coupled with nucleotide modification, removal of spacer sequences, and binding to ribosomal proteins. More than 200 assembly factors and 76 small nucleolar RNAs transiently associate with assembling ribosomes, to enable their accurate and efficient construction. Following export of preribosomes from the nucleus to the cytoplasm, they undergo final stages of maturation before entering the pool of functioning ribosomes. Elaborate mechanisms exist to monitor the formation of correct structural and functional neighborhoods within ribosomes and to destroy preribosomes that fail to assemble properly. Studies of yeast ribosome biogenesis provide useful models for ribosomopathies, diseases in humans that result from failure to properly assemble ribosomes. PMID:24190922

  16. Insights into the Mechanism of Ribosomal Incorporation of Mammalian L13a Protein during Ribosome Biogenesis

    PubMed Central

    Das, Priyanka; Basu, Abhijit; Biswas, Aditi; Poddar, Darshana; Andrews, Joel; Barik, Sailen; Komar, Anton A.

    2013-01-01

    In contrast to prokaryotes, the precise mechanism of incorporation of ribosomal proteins into ribosomes in eukaryotes is not well understood. For the majority of eukaryotic ribosomal proteins, residues critical for rRNA binding, a key step in the hierarchical assembly of ribosomes, have not been well defined. In this study, we used the mammalian ribosomal protein L13a as a model to investigate the mechanism(s) underlying eukaryotic ribosomal protein incorporation into ribosomes. This work identified the arginine residue at position 68 of L13a as being essential for L13a binding to rRNA and incorporation into ribosomes. We also demonstrated that incorporation of L13a takes place during maturation of the 90S preribosome in the nucleolus, but that translocation of L13a into the nucleolus is not sufficient for its incorporation into ribosomes. Incorporation of L13a into the 90S preribosome was required for rRNA methylation within the 90S complex. However, mutations abolishing ribosomal incorporation of L13a did not affect its ability to be phosphorylated or its extraribosomal function in GAIT element-mediated translational silencing. These results provide new insights into the mechanism of ribosomal incorporation of L13a and will be useful in guiding future studies aimed at fully deciphering mammalian ribosome biogenesis. PMID:23689135

  17. Ribosomal protein methyltransferases in the yeast Saccharomyces cerevisiae: Roles in ribosome biogenesis and translation.

    PubMed

    Al-Hadid, Qais; White, Jonelle; Clarke, Steven

    2016-02-12

    A significant percentage of the methyltransferasome in Saccharomyces cerevisiae and higher eukaryotes is devoted to methylation of the translational machinery. Methylation of the RNA components of the translational machinery has been studied extensively and is important for structure stability, ribosome biogenesis, and translational fidelity. However, the functional effects of ribosomal protein methylation by their cognate methyltransferases are still largely unknown. Previous work has shown that the ribosomal protein Rpl3 methyltransferase, histidine protein methyltransferase 1 (Hpm1), is important for ribosome biogenesis and translation elongation fidelity. In this study, yeast strains deficient in each of the ten ribosomal protein methyltransferases in S. cerevisiae were examined for potential defects in ribosome biogenesis and translation. Like Hpm1-deficient cells, loss of four of the nine other ribosomal protein methyltransferases resulted in defects in ribosomal subunit synthesis. All of the mutant strains exhibited resistance to the ribosome inhibitors anisomycin and/or cycloheximide in plate assays, but not in liquid culture. Translational fidelity assays measuring stop codon readthrough, amino acid misincorporation, and programmed -1 ribosomal frameshifting, revealed that eight of the ten enzymes are important for translation elongation fidelity and the remaining two are necessary for translation termination efficiency. Altogether, these results demonstrate that ribosomal protein methyltransferases in S. cerevisiae play important roles in ribosome biogenesis and translation.

  18. The other lives of ribosomal proteins

    PubMed Central

    2010-01-01

    Despite the fact that ribosomal proteins are the constituents of an organelle that is present in every cell, they show a surprising level of regulation, and several of them have also been shown to have other extra-ribosomal functions, such in replication, transcription, splicing or even ageing. This review provides a comprehensive summary of these important aspects. PMID:20650820

  19. In vitro integration of ribosomal RNA synthesis, ribosome assembly, and translation

    PubMed Central

    Jewett, Michael C; Fritz, Brian R; Timmerman, Laura E; Church, George M

    2013-01-01

    Purely in vitro ribosome synthesis could provide a critical step towards unraveling the systems biology of ribosome biogenesis, constructing minimal cells from defined components, and engineering ribosomes with new functions. Here, as an initial step towards this goal, we report a method for constructing Escherichia coli ribosomes in crude S150 E. coli extracts. While conventional methods for E. coli ribosome reconstitution are non-physiological, our approach attempts to mimic chemical conditions in the cytoplasm, thus permitting several biological processes to occur simultaneously. Specifically, our integrated synthesis, assembly, and translation (iSAT) technology enables one-step co-activation of rRNA transcription, assembly of transcribed rRNA with native ribosomal proteins into functional ribosomes, and synthesis of active protein by these ribosomes in the same compartment. We show that iSAT makes possible the in vitro construction of modified ribosomes by introducing a 23S rRNA mutation that mediates resistance against clindamycin. We anticipate that iSAT will aid studies of ribosome assembly and open new avenues for making ribosomes with altered properties. PMID:23799452

  20. In vitro integration of ribosomal RNA synthesis, ribosome assembly, and translation.

    PubMed

    Jewett, Michael C; Fritz, Brian R; Timmerman, Laura E; Church, George M

    2013-06-25

    Purely in vitro ribosome synthesis could provide a critical step towards unraveling the systems biology of ribosome biogenesis, constructing minimal cells from defined components, and engineering ribosomes with new functions. Here, as an initial step towards this goal, we report a method for constructing Escherichia coli ribosomes in crude S150 E. coli extracts. While conventional methods for E. coli ribosome reconstitution are non-physiological, our approach attempts to mimic chemical conditions in the cytoplasm, thus permitting several biological processes to occur simultaneously. Specifically, our integrated synthesis, assembly, and translation (iSAT) technology enables one-step co-activation of rRNA transcription, assembly of transcribed rRNA with native ribosomal proteins into functional ribosomes, and synthesis of active protein by these ribosomes in the same compartment. We show that iSAT makes possible the in vitro construction of modified ribosomes by introducing a 23S rRNA mutation that mediates resistance against clindamycin. We anticipate that iSAT will aid studies of ribosome assembly and open new avenues for making ribosomes with altered properties.

  1. Analysis of ribosome biogenesis factor-modules in yeast cells depleted from pre-ribosomes

    PubMed Central

    Merl, Juliane; Jakob, Steffen; Ridinger, Katrin; Hierlmeier, Thomas; Deutzmann, Rainer; Milkereit, Philipp; Tschochner, Herbert

    2010-01-01

    Formation of eukaryotic ribosomes requires more than 150 biogenesis factors which transiently interact with the nascent ribosomal subunits. Previously, many pre-ribosomal intermediates could be distinguished by their protein composition and rRNA precursor (pre-rRNA) content. We purified complexes of ribosome biogenesis factors from yeast cells in which de novo synthesis of rRNA precursors was down-regulated by genetic means. We compared the protein composition of these largely pre-rRNA free assemblies with the one of analogous pre-ribosomal preparations by semi-quantitative mass spectrometry. The experimental setup minimizes the possibility that the analysed pre-rRNA free protein modules were derived from (partially) disrupted pre-ribosomal particles and provides thereby strong evidence for their pre-ribosome independent existence. In support of the validity of this approach (i) the predicted composition of the analysed protein modules was in agreement with previously described rRNA-free complexes and (ii) in most of the cases we could identify new candidate members of reported protein modules. An unexpected outcome of these analyses was that free large ribosomal subunits are associated with a specific set of ribosome biogenesis factors in cells where neo-production of nascent ribosomes was blocked. The data presented strengthen the idea that assembly of eukaryotic pre-ribosomal particles can result from transient association of distinct building blocks. PMID:20100801

  2. Complementary roles of initiation factor 1 and ribosome recycling factor in 70S ribosome splitting

    PubMed Central

    Pavlov, Michael Y; Antoun, Ayman; Lovmar, Martin; Ehrenberg, Måns

    2008-01-01

    We demonstrate that ribosomes containing a messenger RNA (mRNA) with a strong Shine–Dalgarno sequence are rapidly split into subunits by initiation factors 1 (IF1) and 3 (IF3), but slowly split by ribosome recycling factor (RRF) and elongation factor G (EF-G). Post-termination-like (PTL) ribosomes containing mRNA and a P-site-bound deacylated transfer RNA (tRNA) are split very rapidly by RRF and EF-G, but extremely slowly by IF1 and IF3. Vacant ribosomes are split by RRF/EF-G much more slowly than PTL ribosomes and by IF1/IF3 much more slowly than mRNA-containing ribosomes. These observations reveal complementary splitting of different ribosomal complexes by IF1/IF3 and RRF/EF-G, and suggest the existence of two major pathways for ribosome splitting into subunits in the living cell. We show that the identity of the deacylated tRNA in the PTL ribosome strongly affects the rate by which it is split by RRF/EF-G and that IF3 is involved in the mechanism of ribosome splitting by IF1/IF3 but not by RRF/EF-G. With support from our experimental data, we discuss the principally different mechanisms of ribosome splitting by IF1/IF3 and by RRF/EF-G. PMID:18497739

  3. Non-ribosomal factors in ribosome subunit assembly are emerging targets for new antibacterial drugs.

    PubMed

    Comartin, David J; Brown, Eric D

    2006-10-01

    It is becoming increasingly clear that bacterial ribosome assembly is catalyzed by a variety of non-ribosomal factors. Newly characterized factors in bacterial ribosome biogenesis are broadly conserved and often indispensable proteins that can be classified either as chaperones facilitating assembly, or enzymes with ribosomal RNA- and ribosomal protein-modifying functions. Accumulating evidence indicates that the proteins Era, Obg, YjeQ, YlqF and RimM are chaperones which may be crucial to bacterial ribosome assembly, and therefore represent novel targets for modern antibacterial drug discovery. Ongoing work aimed at understanding ribosome biogenesis is expected to continue to yield additional factors crucial to this process, and provide new targets with drug discovery potential.

  4. Ribosome defects in disorders of erythropoiesis.

    PubMed

    Narla, Anupama; Hurst, Slater N; Ebert, Benjamin L

    2011-02-01

    Over the past decade, genetic lesions that cause ribosome dysfunction have been identified in both congenital and acquired human disorders. These discoveries have established a new category of disorders, known as ribosomopathies, in which the primary pathophysiology is related to impaired ribosome function. The protoptypical disorders are Diamond-Blackfan anemia, a congenital bone marrow failure syndrome, and the 5q- syndrome, a subtype of myelodysplastic syndrome. In both of these disorders, impaired ribosome function causes a severe macrocytic anemia. In this review, we will discuss the evidence that defects in ribosomal biogenesis cause the hematologic phenotype of Diamond-Blackfan anemia and the 5q- syndrome. We will also explore the potential mechanisms by which a ribosomal defect, which would be expected to have widespread consequences, may lead to specific defects in erythropoiesis.

  5. Import of ribosomal proteins into yeast mitochondria.

    PubMed

    Woellhaf, Michael W; Hansen, Katja G; Garth, Christoph; Herrmann, Johannes M

    2014-12-01

    Mitochondrial ribosomes of baker's yeast contain at least 78 protein subunits. All but one of these proteins are nuclear-encoded, synthesized on cytosolic ribosomes, and imported into the matrix for biogenesis. The import of matrix proteins typically relies on N-terminal mitochondrial targeting sequences that form positively charged amphipathic helices. Interestingly, the N-terminal regions of many ribosomal proteins do not closely match the characteristics of matrix targeting sequences, suggesting that the import processes of these proteins might deviate to some extent from the general import route. So far, the biogenesis of only two ribosomal proteins, Mrpl32 and Mrp10, was studied experimentally and indeed showed surprising differences to the import of other preproteins. In this review article we summarize the current knowledge on the transport of proteins into the mitochondrial matrix, and thereby specifically focus on proteins of the mitochondrial ribosome.

  6. Biophysical studies of bacterial ribosome assembly.

    PubMed

    Williamson, James R

    2008-06-01

    The assembly of the bacterial ribosome involves the association of over 50 proteins to 3 large RNA molecules, and it represents a major metabolic activity for rapidly growing bacteria. The availability of atomic structures of the ribosome and the application of biochemical and biophysical methods have led to rapid progress in understanding the mechanistic details of ribosome assembly. The basic steps required to assemble a ribosome are outlined, and the contributions of mass spectrometry, computational methods, and RNA-folding studies in understanding these steps are detailed. This complex process takes place with both sequential and parallel processing that is coordinated to ensure efficient and complete assembly of ribosomes to meet the demands of cell growth.

  7. Differential Stoichiometry among Core Ribosomal Proteins

    PubMed Central

    Slavov, Nikolai; Semrau, Stefan; Airoldi, Edoardo; Budnik, Bogdan; van Oudenaarden, Alexander

    2015-01-01

    Summary Understanding the regulation and structure of ribosomes is essential to understanding protein synthesis and its dysregulation in disease. While ribosomes are believed to have a fixed stoichiometry among their core ribosomal proteins (RPs), some experiments suggest a more variable composition. Testing such variability requires direct and precise quantification of RPs. We used mass spectrometry to directly quantify RPs across monosomes and polysomes of mouse embryonic stem cells (ESC) and budding yeast. Our data show that the stoichiometry among core RPs in wild-type yeast cells and ESC depends both on the growth conditions and on the number of ribosomes bound per mRNA. Furthermore, we find that the fitness of cells with a deleted RP-gene is inversely proportional to the enrichment of the corresponding RP in polysomes. Together, our findings support the existence of ribosomes with distinct protein composition and physiological function. PMID:26565899

  8. Interaction of Chloramphenicol Tripeptide Analogs with Ribosomes.

    PubMed

    Tereshchenkov, A G; Shishkina, A V; Tashlitsky, V N; Korshunova, G A; Bogdanov, A A; Sumbatyan, N V

    2016-04-01

    Chloramphenicol amine peptide derivatives containing tripeptide fragments of regulatory "stop peptides" - MRL, IRA, IWP - were synthesized. The ability of the compounds to form ribosomal complexes was studied by displacement of the fluorescent erythromycin analog from its complex with E. coli ribosomes. It was found that peptide chloramphenicol analogs are able to bind to bacterial ribosomes. The dissociation constants were 4.3-10 µM, which is 100-fold lower than the corresponding values for chloramphenicol amine-ribosome complex. Interaction of the chloramphenicol peptide analogs with ribosomes was simulated by molecular docking, and the most probable contacts of "stop peptide" motifs with the elements of nascent peptide exit tunnel were identified.

  9. How should we think about the ribosome?

    PubMed

    Moore, Peter B

    2012-01-01

    In a few years we are likely to have structures for the ribosome in all the conformations it assumes during protein synthesis. The golden age of ribosome structure determination is thus drawing to a close, and as it does the focus in the field will shift from structure determination to understanding why the ribosome's structure changes the way it does as it performs its function. Thus in the future, kinetic and thermodynamic experiments will become increasingly important, and as they do, the field will have to start thinking about the dynamics of the ribosome far more carefully than it has in the past. The reasoning that underlies these assertions will be explained, and a more general issue explored, namely what can be said today about the modus operandi of the ribosome. What kind of a device is it?

  10. Protein synthesis by ribosomes with tethered subunits.

    PubMed

    Orelle, Cédric; Carlson, Erik D; Szal, Teresa; Florin, Tanja; Jewett, Michael C; Mankin, Alexander S

    2015-08-06

    The ribosome is a ribonucleoprotein machine responsible for protein synthesis. In all kingdoms of life it is composed of two subunits, each built on its own ribosomal RNA (rRNA) scaffold. The independent but coordinated functions of the subunits, including their ability to associate at initiation, rotate during elongation, and dissociate after protein release, are an established model of protein synthesis. Furthermore, the bipartite nature of the ribosome is presumed to be essential for biogenesis, since dedicated assembly factors keep immature ribosomal subunits apart and prevent them from translation initiation. Free exchange of the subunits limits the development of specialized orthogonal genetic systems that could be evolved for novel functions without interfering with native translation. Here we show that ribosomes with tethered and thus inseparable subunits (termed Ribo-T) are capable of successfully carrying out protein synthesis. By engineering a hybrid rRNA composed of both small and large subunit rRNA sequences, we produced a functional ribosome in which the subunits are covalently linked into a single entity by short RNA linkers. Notably, Ribo-T was not only functional in vitro, but was also able to support the growth of Escherichia coli cells even in the absence of wild-type ribosomes. We used Ribo-T to create the first fully orthogonal ribosome-messenger RNA system, and demonstrate its evolvability by selecting otherwise dominantly lethal rRNA mutations in the peptidyl transferase centre that facilitate the translation of a problematic protein sequence. Ribo-T can be used for exploring poorly understood functions of the ribosome, enabling orthogonal genetic systems, and engineering ribosomes with new functions.

  11. Phosphoprotein affinity purification identifies proteins involved in S-adenosyl-L-methionine-induced enhancement of antibiotic production in Streptomyces coelicolor.

    PubMed

    Meng, Lingzhu; Yang, Seung Hwan; Palaniyandi, Sasikumar Arunachalam; Lee, Sung-Kwon; Lee, In-Ae; Kim, Tae-Jong; Suh, Joo-Won

    2011-01-01

    Streptomycetes are the major natural source of clinical antibiotics. The enhanced secondary metabolite production of many streptomycetes by S-adenosylmethionine (SAM) in previous studies suggested the existence of a common SAM regulatory effect. We screened nine proteins using the phosphoprotein purification column from Streptomyces coelicolor. Among them, genes (SCO5477, SCO5113, SCO4647, SCO4885 and SCO1793) for five proteins were disrupted by insertion mutation. The undecylprodigiosin and actinorhodin productions were changed in all mutations. The SAM-induced enhancement of actinorhodin production was abolished by all mutations except SCO4885 mutation, which reduced the production of actinorhodin and undecylprodigiosin with SAM treatment. This study demonstrates that phosphoprotein affinity purification can be used as a screening method to identify the proteins involved SAM signaling.

  12. A Druggable Pocket at the Nucleocapsid/Phosphoprotein Interaction Site of Human Respiratory Syncytial Virus

    PubMed Central

    Ouizougun-Oubari, Mohamed; Pereira, Nelson; Tarus, Bogdan; Galloux, Marie; Lassoued, Safa; Fix, Jenna; Tortorici, M. Alejandra; Hoos, Sylviane; Baron, Bruno; England, Patrick; Desmaële, Didier; Couvreur, Patrick; Bontems, François; Rey, Félix A.; Eléouët, Jean-François; Slama-Schwok, Anny

    2015-01-01

    ABSTRACT Presently, respiratory syncytial virus (RSV), the main cause of severe respiratory infections in infants, cannot be treated efficiently with antivirals. However, its RNA-dependent polymerase complex offers potential targets for RSV-specific drugs. This includes the recognition of its template, the ribonucleoprotein complex (RNP), consisting of genomic RNA encapsidated by the RSV nucleoprotein, N. This recognition proceeds via interaction between the phosphoprotein P, which is the main polymerase cofactor, and N. The determinant role of the C terminus of P, and more particularly of the last residue, F241, in RNP binding and viral RNA synthesis has been assessed previously. Here, we provide detailed structural insight into this crucial interaction for RSV polymerase activity. We solved the crystallographic structures of complexes between the N-terminal domain of N (N-NTD) and C-terminal peptides of P and characterized binding by biophysical approaches. Our results provide a rationale for the pivotal role of F241, which inserts into a well-defined N-NTD pocket. This primary binding site is completed by transient contacts with upstream P residues outside the pocket. Based on the structural information of the N-NTD:P complex, we identified inhibitors of this interaction, selected by in silico screening of small compounds, that efficiently bind to N and compete with P in vitro. One of the compounds displayed inhibitory activity on RSV replication, thereby strengthening the relevance of N-NTD for structure-based design of RSV-specific antivirals. IMPORTANCE Respiratory syncytial virus (RSV) is a widespread pathogen that is a leading cause of acute lower respiratory infections in infants worldwide. RSV cannot be treated efficiently with antivirals, and no vaccine is presently available, with the development of pediatric vaccines being particularly challenging. Therefore, there is a need for new therapeutic strategies that specifically target RSV. The interaction

  13. Borna disease virus phosphoprotein modulates epigenetic signaling in neurons to control viral replication.

    PubMed

    Bonnaud, Emilie M; Szelechowski, Marion; Bétourné, Alexandre; Foret, Charlotte; Thouard, Anne; Gonzalez-Dunia, Daniel; Malnou, Cécile E

    2015-06-01

    Understanding the modalities of interaction of neurotropic viruses with their target cells represents a major challenge that may improve our knowledge of many human neurological disorders for which viral origin is suspected. Borna disease virus (BDV) represents an ideal model to analyze the molecular mechanisms of viral persistence in neurons and its consequences for neuronal homeostasis. It is now established that BDV ensures its long-term maintenance in infected cells through a stable interaction of viral components with the host cell chromatin, in particular, with core histones. This has led to our hypothesis that such an interaction may trigger epigenetic changes in the host cell. Here, we focused on histone acetylation, which plays key roles in epigenetic regulation of gene expression, notably for neurons. We performed a comparative analysis of histone acetylation patterns of neurons infected or not infected by BDV, which revealed that infection decreases histone acetylation on selected lysine residues. We showed that the BDV phosphoprotein (P) is responsible for these perturbations, even when it is expressed alone independently of the viral context, and that this action depends on its phosphorylation by protein kinase C. We also demonstrated that BDV P inhibits cellular histone acetyltransferase activities. Finally, by pharmacologically manipulating cellular acetylation levels, we observed that inhibiting cellular acetyl transferases reduces viral replication in cell culture. Our findings reveal that manipulation of cellular epigenetics by BDV could be a means to modulate viral replication and thus illustrate a fascinating example of virus-host cell interaction. Persistent DNA viruses often subvert the mechanisms that regulate cellular chromatin dynamics, thereby benefitting from the resulting epigenetic changes to create a favorable milieu for their latent and persistent states. Here, we reasoned that Borna disease virus (BDV), the only RNA virus known to

  14. Phosphoprotein Isotope-Coded Solid-Phase Tag Approach for Enrichment and Quantitative Analysis of Phosphopeptides from Complex Mixtures

    SciTech Connect

    Qian, Weijun ); Goshe, Michael B.; Camp, David G. ); Yu, Li-Rong ); Tang, Keqi ); Smith, Richard D. )

    2003-10-15

    Many cellular processes are regulated by reversible protein phosphorylation and the ability to identify and quantify phosphoproteins from proteomes is essential for gaining a better understanding of these dynamic cellular processes. However, a sensitive, efficient and global method capable of addressing the phosphoproteome has yet to be developed. Here we describe an improved stable-isotope labeling method using a Phosphoprotein Isotope-coded Solid-phase Tag (PhIST) for isolating and measuring the relative abundance of phosphorylated peptides from complex peptide mixtures resulting from the enzymatic digestion of extracted proteins. The PhIST approach is an extension of the previously reported Phosphoprotein Isotope-coded Affinity Tag (PhIAT)approach developed by our laboratory1-2, where the O-phosphate moiety on phosphoseryl or phosphothreonyl residues were derivatized by hydroxide ion-medated B-elimination followed by the addition of 1,2-ethanedithiol (EDT). Instead of using the biotin affinity tag, peptides containing the EDT moiety were captured and labeled in one step using isotope-coded solid-phase reagents containing either light (12C6, 14N) or heavy (13C6, 15N) stable isotopes. The captured peptides labeled with the isotope-coded tags were released from the solid-phase support by UV photocleavage and analyzed by capillary LC-MS/MS. The efficiency and sensitivity of the PhIST labeling approach for identification of phosphopeptides from mixtures was demonstrated using casein phosphoproteins. Its utility for proteomic applications is demonstrated by the labeling of soluble proteins from human breast cancer cell line.

  15. Characterization of phosphoproteins and protein kinase activity of virions, noninfectious enveloped particles, and dense bodies of human cytomegalovirus.

    PubMed Central

    Roby, C; Gibson, W

    1986-01-01

    Phosphorylation of the proteins of human cytomegalovirus (CMV) virions, noninfectious enveloped particles (NIEPs), and dense bodies was investigated. Analyses of particles phosphorylated in vivo showed the following. Virions contain three predominant phosphoproteins (i.e., basic phosphoprotein and upper and lower matrix proteins) and at least nine minor phosphorylated species. NIEPs contain all of these and one additional major species, the assembly protein. Dense bodies contain only one (i.e., lower matrix) of the predominant and four of the minor virion phosphoproteins. Two-dimensional (charge-size) separations in denaturing polyacrylamide gels showed that the relative net charges of the predominant phosphorylated species ranged from the basic phosphoprotein to the more neutral upper matrix protein. In vitro assays showed that purified virions of human CMV have an associated protein kinase activity. The activity was detected only after disrupting the envelope; it had a pH optimum of approximately 9 to 9.5 and required a divalent cation, preferring magnesium to manganese. In vitro, this activity catalyzed phosphorylation of the virion proteins observed to be phosphorylated in vivo. Peptide comparisons indicated that the sites phosphorylated in vitro are a subset of those phosphorylated in vivo, underscoring the probable biological relevance of the kinase activity. Casein, phosvitin, and to a minor extent lysine-rich histones served as exogenous phosphate acceptors. Arginine-rich and lysine-rich histones and protamine sulfate, as well as the polyamines spermine and spermidine, stimulated incorporation of phosphate into the endogenous viral proteins. Virions of all human and simian CMV strains tested showed this activity. Analyses of other virus particles, including three intracellular capsid forms (i.e., A, B, and C capsids), NIEPs, and dense bodies, indicated that the active enzyme was not present in the capsid. Rate-velocity sedimentation of disrupted virions

  16. Growth-associated phosphoprotein expression is increased in the supragranular regions of the dentate gyrus following pilocarpine-induced seizures in rats.

    PubMed

    Naffah-Mazzacoratti, M G; Funke, M G; Sanabria, E R; Cavalheiro, E A

    1999-01-01

    Neuroplasticity has been investigated considering the neuronal growth-associated phosphoprotein as a marker of neuronal adaptive capabilities. In the present work, studying the hippocampal reorganization observed in the epilepsy model induced by pilocarpine, we carried out quantitative western blotting associated with immunohistochemistry to determine the distribution of growth-associated phosphoprotein in the hippocampus of rats in acute, silent and chronic periods of this epilepsy model. The fibers and punctate elements from the inner molecular layer of the dentate gyrus were strongly immunostained in animals killed 5 h after status epilepticus, compared with the same region in control animals. Rats presenting partial seizures showed no alterations in the immunostaining pattern compared with saline-treated animals. The hippocampal dentate gyrus of animals during the seizure-free period and presenting spontaneous recurrent seizures was also characterized by strong growth-associated phosphoprotein immunostaining of fibers and punctate elements in the inner molecular layer, contrasting with the control group. As determined by western blotting analysis, growth-associated phosphoprotein levels increased following status epilepticus and remained elevated at the later time-points, both during the silent period and during the period of chronic recurring seizures. Pilocarpine-treated animals, which did not develop status epilepticus, showed no change in growth-associated phosphoprotein levels, indicating that status epilepticus is important to induce growth-associated phosphoprotein overexpression. The measurement of this overexpression could represent one of the early signals of hippocampal reorganization due to status epilepticus-induced damage.

  17. Functional analysis of Saccharomyces cerevisiae ribosomal protein Rpl3p in ribosome synthesis

    PubMed Central

    Rosado, Iván V.; Kressler, Dieter; de la Cruz, Jesús

    2007-01-01

    Ribosome synthesis in eukaryotes requires a multitude of trans-acting factors. These factors act at many steps as the pre-ribosomal particles travel from the nucleolus to the cytoplasm. In contrast to the well-studied trans-acting factors, little is known about the contribution of the ribosomal proteins to ribosome biogenesis. Herein, we have analysed the role of ribosomal protein Rpl3p in 60S ribosomal subunit biogenesis. In vivo depletion of Rpl3p results in a deficit in 60S ribosomal subunits and the appearance of half-mer polysomes. This phenotype is likely due to the instability of early and intermediate pre-ribosomal particles, as evidenced by the low steady-state levels of 27SA3, 27SBS and 7SL/S precursors. Furthermore, depletion of Rpl3p impairs the nucleocytoplasmic export of pre-60S ribosomal particles. Interestingly, flow cytometry analysis indicates that Rpl3p-depleted cells arrest in the G1 phase. Altogether, we suggest that upon depletion of Rpl3p, early assembly of 60S ribosomal subunits is aborted and subsequent steps during their maturation and export prevented. PMID:17569673

  18. Comparative Phosphoproteomic Analysis under High-Nitrogen Fertilizer Reveals Central Phosphoproteins Promoting Wheat Grain Starch and Protein Synthesis

    PubMed Central

    Zhen, Shoumin; Deng, Xiong; Zhang, Ming; Zhu, Gengrui; Lv, Dongwen; Wang, Yaping; Zhu, Dong; Yan, Yueming

    2017-01-01

    Nitrogen (N) is a macronutrient important for plant growth and development. It also strongly influences starch and protein synthesis, closely related to grain yield and quality. We performed the first comparative phosphoproteomic analysis of developing wheat grains in response to high-N fertilizer. Physiological and biochemical analyses showed that application of high-N fertilizer resulted in significant increases in leaf length and area, chlorophyll content, the activity of key enzymes in leaves such as nitrate reductase (NR), and in grains such as sucrose phosphate synthase (SPS), sucrose synthase (SuSy), and ADP glucose pyrophosphorylase (AGPase). This enhanced enzyme activity led to significant improvements in starch content, grain yield, and ultimately, bread making quality. Comparative phosphoproteomic analysis of developing grains under the application of high-N fertilizer performed 15 and 25 days post-anthesis identified 2470 phosphosites among 1372 phosphoproteins, of which 411 unique proteins displayed significant changes in phosphorylation level (>2-fold or <0.5-fold). These phosphoproteins are involved mainly in signaling transduction, starch synthesis, energy metabolism. Pro-Q diamond staining and Western blotting confirmed our phosphoproteomic results. We propose a putative pathway to elucidate the important roles of the central phosphoproteins regulating grain starch and protein synthesis. Our results provide new insights into the molecular mechanisms of protein phosphorylation modifications involved in grain development, yield and quality formation. PMID:28194157

  19. DNA topoisomerase II alpha is the major chromosome protein recognized by the mitotic phosphoprotein antibody MPM-2.

    PubMed Central

    Taagepera, S; Rao, P N; Drake, F H; Gorbsky, G J

    1993-01-01

    We have determined that the major mitotic phosphoprotein in chromosomes recognized by the antiphosphoprotein antibody MPM-2 is the 170-kDa isoform of topoisomerase II (topo II), the isoform predominant in proliferating cells. As a prerequisite to making this discovery, it was necessary to develop protocols to protect chromosomal proteins from dephosphorylation during cell extraction and chromosome isolation procedures. Immunofluorescence analysis of the large chromosomes prepared from Indian Muntjac cells revealed colocalization of MPM-2 and anti-topo II antibodies to the chromosomal centromeres and to the axial regions of the chromosomal arms. For biochemical fractionation studies, large quantities of chromosomes from the P388D1 mouse lymphocyte cell line were isolated and treated to remove DNA and histone proteins. Immunoblot and immunoprecipitation experiments with this chromosome scaffold fraction identified the major MPM-2-reactive phosphoprotein to be DNA topo II. Using a panel of anti-peptide antibodies specific to the isoforms of topo II, we determined that the major phosphoprotein recognized by MPM-2 is the 170-kDa isoform of topo II, topo II alpha. The 180-kDa isoform, topo II beta, present in the isolated chromosomes in much smaller quantities, is also recognized by MPM-2. The mitotic phosphorylation of the topo II proteins may be critical for proper chromosome condensation and segregation. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 PMID:7690961

  20. The transforming proteins of PRCII virus and Rous sarcoma virus form a complex with the same two cellular phosphoproteins.

    PubMed Central

    Adkins, B; Hunter, T; Sefton, B M

    1982-01-01

    P105 and P110, the presumptive transforming proteins of PRCII avian sarcoma virus, have been found to be present in transformed chicken cells in two forms: as monomers and as part of a complex which contains both a 50,000-dalton and a 90,000-dalton cellular phosphoprotein. The 90,000-dalton cellular protein was found to be identical to one of the proteins in chicken cells whose synthesis is induced by stress. The 50,000-dalton protein was found to contain phosphotyrosine when isolated from the complex and therefore may be a substrate for the tyrosine protein kinase activity which is associated with P105 and P110. These same two cellular phosphoproteins have previously been shown to be present in a complex with pp60src, the tyrosine protein kinase which is the transforming protein of Rous sarcoma virus. However, not all avian sarcoma virus transforming proteins with associated tyrosine protein kinase activities form a complex efficiently with these cellular proteins. Little if any of P90, the putative transforming protein of Yamaguchi 73 virus, was found in a complex with the 50,000-dalton and 90,000-dalton cellular phosphoproteins. Images PMID:6180178

  1. The PAXgene® Tissue System Preserves Phosphoproteins in Human Tissue Specimens and Enables Comprehensive Protein Biomarker Research

    PubMed Central

    Gündisch, Sibylle; Schott, Christina; Wolff, Claudia; Tran, Kai; Beese, Christian; Viertler, Christian; Zatloukal, Kurt; Becker, Karl-Friedrich

    2013-01-01

    Precise quantitation of protein biomarkers in clinical tissue specimens is a prerequisite for accurate and effective diagnosis, prognosis, and personalized medicine. Although progress is being made, protein analysis from formalin-fixed and paraffin-embedded tissues is still challenging. In previous reports, we showed that the novel formalin-free tissue preservation technology, the PAXgene Tissue System, allows the extraction of intact and immunoreactive proteins from PAXgene-fixed and paraffin-embedded (PFPE) tissues. In the current study, we focused on the analysis of phosphoproteins and the applicability of two-dimensional gel electrophoresis (2D-PAGE) and enzyme-linked immunosorbent assay (ELISA) to the analysis of a variety of malignant and non-malignant human tissues. Using western blot analysis, we found that phosphoproteins are quantitatively preserved in PFPE tissues, and signal intensities are comparable to that in paired, frozen tissues. Furthermore, proteins extracted from PFPE samples are suitable for 2D-PAGE and can be quantified by ELISA specific for denatured proteins. In summary, the PAXgene Tissue System reliably preserves phosphoproteins in human tissue samples, even after prolonged fixation or stabilization times, and is compatible with methods for protein analysis such as 2D-PAGE and ELISA. We conclude that the PAXgene Tissue System has the potential to serve as a versatile tissue fixative for modern pathology. PMID:23555997

  2. Ribosomopathies: human disorders of ribosome dysfunction

    PubMed Central

    Narla, Anupama

    2010-01-01

    Ribosomopathies compose a collection of disorders in which genetic abnormalities cause impaired ribosome biogenesis and function, resulting in specific clinical phenotypes. Congenital mutations in RPS19 and other genes encoding ribosomal proteins cause Diamond-Blackfan anemia, a disorder characterized by hypoplastic, macrocytic anemia. Mutations in other genes required for normal ribosome biogenesis have been implicated in other rare congenital syndromes, Schwachman-Diamond syndrome, dyskeratosis congenita, cartilage hair hypoplasia, and Treacher Collins syndrome. In addition, the 5q− syndrome, a subtype of myelodysplastic syndrome, is caused by a somatically acquired deletion of chromosome 5q, which leads to haploinsufficiency of the ribosomal protein RPS14 and an erythroid phenotype highly similar to Diamond-Blackfan anemia. Acquired abnormalities in ribosome function have been implicated more broadly in human malignancies. The p53 pathway provides a surveillance mechanism for protein translation as well as genome integrity and is activated by defects in ribosome biogenesis; this pathway appears to be a critical mediator of many of the clinical features of ribosomopathies. Elucidation of the mechanisms whereby selective abnormalities in ribosome biogenesis cause specific clinical syndromes will hopefully lead to novel therapeutic strategies for these diseases. PMID:20194897

  3. Ribosomopathies: human disorders of ribosome dysfunction.

    PubMed

    Narla, Anupama; Ebert, Benjamin L

    2010-04-22

    Ribosomopathies compose a collection of disorders in which genetic abnormalities cause impaired ribosome biogenesis and function, resulting in specific clinical phenotypes. Congenital mutations in RPS19 and other genes encoding ribosomal proteins cause Diamond-Blackfan anemia, a disorder characterized by hypoplastic, macrocytic anemia. Mutations in other genes required for normal ribosome biogenesis have been implicated in other rare congenital syndromes, Schwachman-Diamond syndrome, dyskeratosis congenita, cartilage hair hypoplasia, and Treacher Collins syndrome. In addition, the 5q- syndrome, a subtype of myelodysplastic syndrome, is caused by a somatically acquired deletion of chromosome 5q, which leads to haploinsufficiency of the ribosomal protein RPS14 and an erythroid phenotype highly similar to Diamond-Blackfan anemia. Acquired abnormalities in ribosome function have been implicated more broadly in human malignancies. The p53 pathway provides a surveillance mechanism for protein translation as well as genome integrity and is activated by defects in ribosome biogenesis; this pathway appears to be a critical mediator of many of the clinical features of ribosomopathies. Elucidation of the mechanisms whereby selective abnormalities in ribosome biogenesis cause specific clinical syndromes will hopefully lead to novel therapeutic strategies for these diseases.

  4. Quantitative determination of ribosome nascent chain stability

    PubMed Central

    Samelson, Avi J.; Jensen, Madeleine K.; Soto, Randy A.; Cate, Jamie H. D.; Marqusee, Susan

    2016-01-01

    Accurate protein folding is essential for proper cellular and organismal function. In the cell, protein folding is carefully regulated; changes in folding homeostasis (proteostasis) can disrupt many cellular processes and have been implicated in various neurodegenerative diseases and other pathologies. For many proteins, the initial folding process begins during translation while the protein is still tethered to the ribosome; however, most biophysical studies of a protein’s energy landscape are carried out in isolation under idealized, dilute conditions and may not accurately report on the energy landscape in vivo. Thus, the energy landscape of ribosome nascent chains and the effect of the tethered ribosome on nascent chain folding remain unclear. Here we have developed a general assay for quantitatively measuring the folding stability of ribosome nascent chains, and find that the ribosome exerts a destabilizing effect on the polypeptide chain. This destabilization decreases as a function of the distance away from the peptidyl transferase center. Thus, the ribosome may add an additional layer of robustness to the protein-folding process by avoiding the formation of stable partially folded states before the protein has completely emerged from the ribosome. PMID:27821780

  5. Comparative anatomy of a regulatory ribosomal protein.

    PubMed

    Worbs, Michael; Wahl, Markus C; Lindahl, Lasse; Zengel, Janice M

    2002-08-01

    Ribosomal protein L4 is a crucial folding mediator and an important architectural component of the large ribosomal subunit. Furthermore, Escherichia coli L4 produced in excess of its rRNA binding sites downregulates the transcription and translation of its own S10 operon, encoding 11 ribosomal proteins. Genetic experiments and the crystal structure of Thermotoga maritima L4 had implicated separable regions on L4 in ribosome association and expression control while RNA competition experiments and the regulatory capacity of heterologous L4 had suggested an overlap of the protein sequences involved in the two functions. We report herein that contrary to other foreign bacterial L4 proteins, L4 from T. maritima only weakly controlled expression of the S10 operon in E. coli. Also, wildtype T. maritima L4 was more weakly associated with E. coli ribosomes than with the E. coli analog. Rational mutageneses were performed to try to increase the regulatory competence of T. maritima L4. The ribosome incorporation of the mutant proteins was also investigated. Two different deletions removing T. maritima-specific sequences had little effects on regulation although one did improve ribosome association. Interestingly, a set of multiple mutations, which rendered the region around helices alpha4 and alpha5 in T. maritima L4 more E. coli-like, had no influence on the incorporation of the protein into the large ribosomal subunit but considerably improved its regulatory potential. Therefore, the area around helices alpha4 and alpha5, which is critical for the initial folding steps of the large subunit, is also a central element of autogenous control, presumably by contacting the S10 mRNA leader. Ribosome association is compounded at later stages of assembly by additional rRNA contacts through L4 areas which do not participate in regulation. Similarly, sequences outside the alpha4/alpha5 region aid expression control.

  6. Origin and evolution of the ribosome.

    PubMed

    Fox, George E

    2010-09-01

    The modern ribosome was largely formed at the time of the last common ancestor, LUCA. Hence its earliest origins likely lie in the RNA world. Central to its development were RNAs that spawned the modern tRNAs and a symmetrical region deep within the large ribosomal RNA, (rRNA), where the peptidyl transferase reaction occurs. To understand pre-LUCA developments, it is argued that events that are coupled in time are especially useful if one can infer a likely order in which they occurred. Using such timing events, the relative age of various proteins and individual regions within the large rRNA are inferred. An examination of the properties of modern ribosomes strongly suggests that the initial peptides made by the primitive ribosomes were likely enriched for l-amino acids, but did not completely exclude d-amino acids. This has implications for the nature of peptides made by the first ribosomes. From the perspective of ribosome origins, the immediate question regarding coding is when did it arise rather than how did the assignments evolve. The modern ribosome is very dynamic with tRNAs moving in and out and the mRNA moving relative to the ribosome. These movements may have become possible as a result of the addition of a template to hold the tRNAs. That template would subsequently become the mRNA, thereby allowing the evolution of the code and making an RNA genome useful. Finally, a highly speculative timeline of major events in ribosome history is presented and possible future directions discussed.

  7. Mumps Virus Nucleoprotein Enhances Phosphorylation of the Phosphoprotein by Polo-Like Kinase 1.

    PubMed

    Pickar, Adrian; Zengel, James; Xu, Pei; Li, Zhuo; He, Biao

    2015-11-25

    The viral RNA-dependent RNA polymerases (vRdRps) of nonsegmented, negative-sense viruses (NNSVs) consist of the enzymatic large protein (L) and the phosphoprotein (P). P is heavily phosphorylated, and its phosphorylation plays a critical role in viral RNA synthesis. Since NNSVs do not encode kinases, P is phosphorylated by host kinases. In this study, we investigate the roles that viral proteins play in the phosphorylation of mumps virus (MuV) P. We found that nucleoprotein (NP) enhances the phosphorylation of P. We have identified the serine/threonine kinase Polo-like kinase 1 (PLK1) as a host kinase that phosphorylates P and have found that phosphorylation of P by PLK1 is enhanced by NP. The PLK1 binding site in MuV P was mapped to residues 146 to 148 within the S(pS/T)P motif, and the phosphorylation site was identified as residues S292 and S294. It has previously been shown that P acts as a chaperone for NP, which encapsidates viral genomic RNA to form the NP-RNA complex, the functional template for viral RNA synthesis. Thus, it is assumed that phosphorylation of P may regulate NP's ability to form the NP-RNA complex, thereby regulating viral RNA synthesis. Our work demonstrates that MuV NP affects phosphorylation of P, suggesting that NP can regulate viral RNA synthesis by regulating phosphorylation of P. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

  8. Mumps Virus Nucleoprotein Enhances Phosphorylation of the Phosphoprotein by Polo-Like Kinase 1

    PubMed Central

    Pickar, Adrian; Zengel, James; Xu, Pei; Li, Zhuo

    2015-01-01

    ABSTRACT The viral RNA-dependent RNA polymerases (vRdRps) of nonsegmented, negative-sense viruses (NNSVs) consist of the enzymatic large protein (L) and the phosphoprotein (P). P is heavily phosphorylated, and its phosphorylation plays a critical role in viral RNA synthesis. Since NNSVs do not encode kinases, P is phosphorylated by host kinases. In this study, we investigate the roles that viral proteins play in the phosphorylation of mumps virus (MuV) P. We found that nucleoprotein (NP) enhances the phosphorylation of P. We have identified the serine/threonine kinase Polo-like kinase 1 (PLK1) as a host kinase that phosphorylates P and have found that phosphorylation of P by PLK1 is enhanced by NP. The PLK1 binding site in MuV P was mapped to residues 146 to 148 within the S(pS/T)P motif, and the phosphorylation site was identified as residues S292 and S294. IMPORTANCE It has previously been shown that P acts as a chaperone for NP, which encapsidates viral genomic RNA to form the NP-RNA complex, the functional template for viral RNA synthesis. Thus, it is assumed that phosphorylation of P may regulate NP's ability to form the NP-RNA complex, thereby regulating viral RNA synthesis. Our work demonstrates that MuV NP affects phosphorylation of P, suggesting that NP can regulate viral RNA synthesis by regulating phosphorylation of P. PMID:26608325

  9. Knockdown of Golgi phosphoprotein 2 inhibits hepatocellular carcinoma cell proliferation and motility

    PubMed Central

    Liu, Yiming; Zhang, Xiaodi; Sun, Ting; Jiang, Junchang; Li, Ying; Chen, Mingliang; Wei, Zhen; Jiang, Weiqin; Zhou, Linfu

    2016-01-01

    Golgi phosphoprotein 2 (GP73) is highly expressed in hepatocellular carcinoma (HCC) cells, where it serves as a biomarker and indicator of disease progression. We used MTS assays, anchorage-independent cell colony formation assays and a xenograft tumor model to show that GP73-specific siRNAs inhibit HCC proliferation in HepG2, SMMC-7721, and Huh7 cell lines and in vivo. Following GP73 silencing, levels of p-Rb, a factor related to metastasis, were reduced, but cell cycle progression was unaffected. Our results suggest that GP73 silencing may not directly suppress proliferation, but may instead inhibit cell motility. Results from proliferation assays suggest GP73 reduces expression of epithelial mesenchymal transition (EMT)-related factors and promotes cell motility, while transwell migration and invasion assays indicated a possible role in metastasis. Immunofluorescence co-localization microscopy and immunoblotting showed that GP73 decreases expression of N-cadherin and E-cadherin, two key factors in EMT, which may in turn decrease intracellular adhesive forces and promote cell motility. This study confirmed that GP73 expression leads to increased expression of EMT-related proteins and that GP73 silencing reduces HCC cell migration in vitro. These findings suggest that GP73 silencing through siRNA delivery may provide a novel low-toxicity therapy for the inhibition of tumor proliferation and metastasis. PMID:26870893

  10. Stress-induced phosphoprotein-1 maintains the stability of JAK2 in cancer cells

    PubMed Central

    Jung, Shih-Ming; Tsai, Chi-Neu; Lin, Chiao-Yun; Chen, Shun-Hua; Sue, Shih-Che; Wang, Tzu-Hao; Wang, Hsin-Shih; Lai, Chyong-Huey

    2016-01-01

    Overexpression of stress-induced phosphoprotein 1 (STIP1) − a co-chaperone of heat shock protein (HSP) 70/HSP90 – and activation of the JAK2-STAT3 pathway occur in several tumors. Combined treatment with a HSP90 inhibitor and a JAK2 inhibitor exert synergistic anti-cancer effects. Here, we show that STIP1 stabilizes JAK2 protein in ovarian and endometrial cancer cells. Knock-down of endogenous STIP1 decreased JAK2 and phospho-STAT3 protein levels. The N-terminal fragment of STIP1 interacts with the N-terminus of JAK2, whereas the C-terminal DP2 domain of STIP1 mediates the interaction with HSP90 and STAT3. A peptide fragment in the DP2 domain of STIP1 (peptide 520) disrupted the interaction between STIP1 and HSP90 and induced cell death through JAK2 suppression. In an animal model, treatment with peptide 520 inhibited tumor growth. In summary, STIP1 modulates the function of the HSP90-JAK2-STAT3 complex. Peptide 520 may have therapeutic potential in the treatment of JAK2-overexpressing tumors. PMID:27409672

  11. Short interfering RNAs targeting a vampire-bat related rabies virus phosphoprotein mRNA.

    PubMed

    Ono, Ekaterina Alexandrovna Durymanova; Taniwaki, Sueli Akemi; Brandão, Paulo

    The aim of this study was to assess the in vitro and in vivo effects of short-interfering RNAs (siRNAs) against rabies virus phosphoprotein (P) mRNA in a post-infection treatment for rabies as an extension of a previous report (Braz J Microbiol. 2013 Nov 15;44(3):879-82). To this end, rabies virus strain RABV-4005 (related to the Desmodus rotundus vampire bat) were used to inoculate BHK-21 cells and mice, and the transfection with each of the siRNAs was made with Lipofectamine-2000™. In vitro results showed that siRNA 360 was able to inhibit the replication of strain RABV-4005 with a 1log decrease in virus titter and 5.16-fold reduction in P mRNA, 24h post-inoculation when compared to non-treated cells. In vivo, siRNA 360 was able to induce partial protection, but with no significant difference when compared to non-treated mice. These results indicate that, despite the need for improvement for in vivo applications, P mRNA might be a target for an RNAi-based treatment for rabies. Copyright © 2017 Sociedade Brasileira de Microbiologia. Published by Elsevier Editora Ltda. All rights reserved.

  12. Nicotinamide attenuates the decrease of astrocytic phosphoprotein PEA-15 in focal cerebral ischemic injury.

    PubMed

    Koh, Phil-Ok

    2012-03-01

    Nicotinamide exerts neuroprotective effects against focal cerebral ischemic injury. Phosphoprotein enriched in astrocytes 15 (PEA-15) is prominently expressed in astrocytes that exert broad anti-apoptotic functions. This study investigated whether nicotinamide modulates PEA-15 and levels of two phosphorylated PEA-15 (Serine 104 and 116) in an animal model of middle cerebral artery occlusion (MCAO)-induced injury. Adult male rats were treated with vehicle or nicotinamide (500 mg/kg) 2 hr after the onset of MCAO and cerebral cortices were collected at 24 hr after MCAO. In a proteomic approach, MCAO induced decreases of PEA-15 levels, while nicotinamide treatment attenuated the injury-induced decrease in PEA-15. The results of Western blot analysis suggest that nicotinamide prevented injury-induced reduction in phospho-PEA-15 (Serine 104) and phospho-PEA-15 (Serine 116) levels. The phosphorylation of PEA-15 exerts anti-apoptotic functions, and reduction of PEA-15 phosphorylation leads to apoptotic cell death. These results suggest that nicotinamide exerts a neuroprotective effect by attenuating the injury-induced decreases of PEA-15 and phospho-PEA-15 (Ser 104 and Ser 116) proteins.

  13. The measles virus phosphoprotein interacts with the linker domain of STAT1

    SciTech Connect

    Devaux, Patricia Priniski, Lauren; Cattaneo, Roberto

    2013-09-15

    The measles virus (MV) phosphoprotein (P) and V proteins block the interferon (IFN) response by impeding phosphorylation of the signal transducer and activator of transcription 1 (STAT1) by the Janus kinase 1 (JAK1). We characterized how STAT1 mutants interact with P and JAK1 phosphorylation. Certain mutants of the linker, the Src-homology 2 domain (SH2), or the transactivation domain had reduced or abolished phosphorylation through JAK1 after IFN treatment. Other mutants, mainly localized in the linker, failed to interact with P as documented by the lack of interference with nuclear translocation. Thus the functional footprint of P on STAT1 localizes mainly to the linker domain; there is also some overlap with the STAT1 phosphorylation functional footprint on the SH2 domain. Based on these observations, we discuss how the MV-P might operate to inhibit the JAK/STAT pathway. - Highlights: • Residue in the linker and SH2 domains of STAT1 are important for MV-P interaction. • Residue in the linker and SH2 domains of STAT1 are important for STAT1 phosphorylation. • Residues interferring with both functions have similar location on STAT1. • The viral P and V proteins may operate in concert to inhibit the JAK/STAT pathway.

  14. In Vivo Analysis of the Major Exocytosis-sensitive Phosphoprotein in Tetrahymena

    PubMed Central

    Chilcoat, N. Doane; Turkewitz, Aaron P.

    1997-01-01

    Phosphoglucomutase (PGM) is a ubiquitous highly conserved enzyme involved in carbohydrate metabolism. A number of recently discovered PGM-like proteins in a variety of organisms have been proposed to function in processes other than metabolism. In addition, sequence analysis suggests that several of these may lack PGM enzymatic activity. The best studied PGM-like protein is parafusin, a major phosphoprotein in the ciliate Paramecium tetraurelia that undergoes rapid and massive dephosphorylation when cells undergo synchronous exocytosis of their dense-core secretory granules. Indirect genetic and biochemical evidence also supports a role in regulated exocytotic membrane fusion. To examine this matter directly, we have identified and cloned the parafusin homologue in Tetrahymena thermophila, a ciliate in which protein function can be studied in vivo. The unique T. thermophila gene, called PGM1, encodes a protein that is closely related to parafusin by sequence and by characteristic post-translational modifications. Comparison of deduced protein sequences, taking advantage of the known atomic structure of rabbit muscle PGM, suggests that both ciliate enzymes and all other PGM-like proteins have PGM activity. We evaluated the activity and function of PGM1 through gene disruption. Surprisingly, ΔPGM1 cells displayed no detectable defect in exocytosis, but showed a dramatic decrease in PGM activity. Both our results, and reinterpretation of previous data, suggest that any potential role for PGM-like proteins in regulated exocytosis is unlikely to precede membrane fusion. PMID:9382866

  15. Frameshift mutations in dentin phosphoprotein and dependence of dentin disease phenotype on mutation location.

    PubMed

    Nieminen, Pekka; Papagiannoulis-Lascarides, Lisa; Waltimo-Siren, Janna; Ollila, Päivi; Karjalainen, Sara; Arte, Sirpa; Veerkamp, Jaap; Tallon Walton, Victoria; Chimenos Küstner, Eduard; Siltanen, Tarja; Holappa, Heidi; Lukinmaa, Pirjo-Liisa; Alaluusua, Satu

    2011-04-01

    We describe results from a mutational analysis of the region of the dentin sialophosphoprotein (DSPP) gene encoding dentin phosphoprotein (DPP) in 12 families with dominantly inherited dentin diseases. In eight families (five mutations in the N-terminal third of DPP), the clinical and radiologic features were uniform and compatible with dentin dysplasia type II (DD-II) with major clinical signs in the deciduous dentition. In the other families (four mutations in the more C-terminal part), the permanent teeth also were affected, and the diseases could be classified as variants of dentinogenesis imperfecta. Attrition was not prominent, but periapical infections were common. Discoloring with varying intensity was evident, and pulps and root canals were obliterated in the permanent dentition. All mutations caused a frameshift that replaced the Ser-Ser-Asx repeat by a code for a hydrophobic downstream sequence of approximately original length. We conclude that frameshift mutations in DSPP explain a significant part of dentin diseases. Furthermore, we propose that the location of the mutation is reflected in the phenotypic features as a gradient from DD-II to more severe disease that does not conform to the classic definitions of DI-II. Copyright © 2011 American Society for Bone and Mineral Research.

  16. The phosphoprotein gene of a dolphin morbillivirus isolate exhibits genomic variation at the editing site.

    PubMed

    Bolt, G; Alexandersen, S; Blixenkrone-Møller, M

    1995-12-01

    The nucleotide sequence of the phosphoprotein (P) gene of a dolphin morbillivirus (DMV) isolate was determined. Like those of other morbilliviruses the DMV P gene encoded P and C proteins in overlapping open reading frames and V protein by editing the P gene transcript. Among P mRNA based clones the editing site variants GGGC, GGGG, GAGC and GGGGGGC predicting a P protein, and the variants GGGGC and GGGGGG predicting a V protein, were found. Surprisingly, the three variants GGGC, GGGG and GAGC were also found among clones generated from genomic RNA of the DMV isolate. Thus, more than one viral genome type appeared to be present in cells infected with the DMV isolate. By a similar analysis of the virus genomes in the tissue from which the DMV isolate was obtained, only the GGGC type was found, indicating that the GGGG and GAGC types arose during adaptation of the virus to growth in cell cultures. No editing site variants likely to have arisen by editing the GAGC type were encountered, and it remains ot be determined whether mRNA encoding V protein can be transcribed from genomes with this editing site. Using antisera raised against the common N terminus and unique C termini of the predicted P and V proteins, the in vivo expression of these proteins was demonstrated.

  17. Ser/Thr-phosphoprotein phosphatases in chondrogenesis: neglected components of a two-player game.

    PubMed

    Matta, Csaba; Mobasheri, Ali; Gergely, Pál; Zákány, Róza

    2014-10-01

    Protein phosphorylation plays a determining role in the regulation of chondrogenesis in vitro. While signalling pathways governed by protein kinases including PKA, PKC, and mitogen-activated protein kinases (MAPK) have been mapped in great details, published data relating to the specific role of phosphoprotein phosphatases (PPs) in differentiating chondroprogenitor cells or in mature chondrocytes is relatively sparse. This review discusses the known functions of Ser/Thr-specific PPs in the molecular signalling pathways of chondrogenesis. PPs are clearly equally important as protein kinases to counterbalance the effect of reversible protein phosphorylation. Of the main Ser/Thr PPs, some of the functions of PP1, PP2A and PP2B have been characterised in the context of chondrogenesis. While PP1 and PP2A appear to negatively regulate chondrogenic differentiation and maintenance of chondrocyte phenotype, calcineurin is an important stimulatory mediator during chondrogenesis but becomes inhibitory in mature chondrocytes. Furthermore, PPs are implicated to be mediators during the pathogenesis of osteoarthritis that makes them potential therapeutic targets to be exploited in the close future. Among the many yet unexplored targets of PPs, modulation of plasma membrane ion channel function and participation in mechanotransduction pathways are emerging novel aspects of signalling during chondrogenesis that should be further elucidated. Besides the regulation of cellular ion homeostasis, other potentially significant novel roles for PPs during the regulation of in vitro chondrogenesis are discussed. Copyright © 2014 Elsevier Inc. All rights reserved.

  18. IgM antigen receptor complex contains phosphoprotein products of B29 and mb-1 genes.

    PubMed Central

    Campbell, K S; Hager, E J; Friedrich, R J; Cambier, J C

    1991-01-01

    Membrane immunoglobulin M (mIgM) and mIgD are major B-lymphocyte antigen receptors, which function by internalizing antigens for processing and presentation to T cells and by transducing essential signals for proliferation and differentiation. Although ligation of mIgM or mIgD results in rapid activation of a phospholipase C and a tyrosine kinase(s), these receptors have cytoplasmic tails of only three amino acid residues (Lys-Val-Lys), which seem ill suited for direct physical coupling with cytoplasmic signal transduction structures. In this report, we identify the alpha, beta, and gamma components of the mIgM-associated phosphoprotein complex, which may play a role in signal transduction. Proteolytic peptide mapping demonstrated that the IgM-alpha chain differs from Ig-beta and Ig-gamma. The chains were purified, and amino-terminal sequencing revealed identity with two previously cloned B-cell-specific genes. One component, IgM-alpha, is a product of the mb-1 gene, and the two additional components, Ig-beta and Ig-gamma, are products of the B29 gene. Immunoblotting analysis using rabbit antibodies prepared against predicted peptide sequences of each gene product confirmed the identification of these mIgM-associated proteins. The deduced sequence indicates that these receptor subunits lack inherent protein kinase domains but include common tyrosine-containing sequence motifs, which are likely sites of induced tyrosine phosphorylation. Images PMID:2023945

  19. Sulphation of secreted phosphoprotein I (SPPI, osteopontin) is associated with mineralized tissue formation

    SciTech Connect

    Nagata, T.; Todescan, R.; Goldberg, H.A.; Zhang, Q.; Sodek, J. )

    1989-11-30

    Secreted phosphoprotein I (SPPI) is a prominent structural protein in mineralized connective tissues. Rat bone cells in culture produce several forms of SPPI that differ in post-translational modifications such as phosphorylation and sulphation. To determine the significance of protein sulphation in bone formation, the synthesis of SPPI was studied in vitro using rat bone marrow cells (RBMC) which form bone-like tissue when grown in the presence of dexamethasone (Dex) and beta-glycerophosphate (beta-GP). In the presence of 10(-7) M Dex SPPI expression was stimulated 4-5-fold. Radiolabelling multilayered RBMCs for 48 h with (35S)-methionine, Na2(35SO4), or Na3(32PO4) revealed that two major phosphorylated forms of SPPI were secreted into the culture medium: a highly phosphorylated form migrating at 44 kDa on 15% SDS-PAGE and a less phosphorylated 55 kDa form. In the mineralized tissue formed in the presence of Dex and beta-GP, both forms of SPPI, in addition to proteoglycans and a 67 kDa protein, incorporated significant amounts of (35SO4). Sulphation of SPPI was not observed in the absence of mineral formation, indicating that the sulphation of SPPI is closely associated with mineralization and that it can be used as a sensitive and specific marker for the osteoblastic phenotype.

  20. Borna Disease Virus Phosphoprotein Impairs the Developmental Program Controlling Neurogenesis and Reduces Human GABAergic Neurogenesis

    PubMed Central

    Scordel, Chloé; Szelechowski, Marion; Poulet, Aurélie; Richardson, Jennifer; Benchoua, Alexandra; Gonzalez-Dunia, Daniel; Eloit, Marc; Coulpier, Muriel

    2015-01-01

    It is well established that persistent viral infection may impair cellular function of specialized cells without overt damage. This concept, when applied to neurotropic viruses, may help to understand certain neurologic and neuropsychiatric diseases. Borna disease virus (BDV) is an excellent example of a persistent virus that targets the brain, impairs neural functions without cell lysis, and ultimately results in neurobehavioral disturbances. Recently, we have shown that BDV infects human neural progenitor cells (hNPCs) and impairs neurogenesis, revealing a new mechanism by which BDV may interfere with brain function. Here, we sought to identify the viral proteins and molecular pathways that are involved. Using lentiviral vectors for expression of the bdv-p and bdv-x viral genes, we demonstrate that the phosphoprotein P, but not the X protein, diminishes human neurogenesis and, more particularly, GABAergic neurogenesis. We further reveal a decrease in pro-neuronal factors known to be involved in neuronal differentiation (ApoE, Noggin, TH and Scg10/Stathmin2), demonstrating that cellular dysfunction is associated with impairment of specific components of the molecular program that controls neurogenesis. Our findings thus provide the first evidence that a viral protein impairs GABAergic human neurogenesis, a process that is dysregulated in several neuropsychiatric disorders. They improve our understanding of the mechanisms by which a persistent virus may interfere with brain development and function in the adult. PMID:25923687

  1. Signaling in striatal neurons: the phosphoproteins of reward, addiction, and dyskinesia.

    PubMed

    Girault, Jean-Antoine

    2012-01-01

    The striatum is a deep region of the forebrain involved in action selection, control of movement, and motivation. It receives a convergent excitatory glutamate input from the cerebral cortex and the thalamus, controlled by dopamine (DA) released in response to unexpected rewards and other salient stimuli. Striatal function and its dysfunction in drug addiction or Parkinson's disease depend on the interplay between these neurotransmitters. Signaling cascades in striatal medium-sized spiny neurons (MSNs) involve multiple kinases, phosphatases, and phosphoproteins, some of which are highly enriched in these neurons. They control the properties of ion channels and the plasticity of MSNs, in part through their effects on gene transcription. This chapter summarizes signaling in MSNs and focuses on the regulation of multiple protein phosphatases through DA and glutamate receptors and the role of ERK. It is hypothesized that these pathways are particularly adapted to the specific computing properties of MSNs and the function of the basal ganglia circuits in which they participate. Copyright © 2012 Elsevier Inc. All rights reserved.

  2. Retinophilin is a light-regulated phosphoprotein required to suppress photoreceptor dark noise in Drosophila.

    PubMed

    Mecklenburg, Kirk L; Takemori, Nobuaki; Komori, Naoka; Chu, Brian; Hardie, Roger C; Matsumoto, Hiroyuki; O'Tousa, Joseph E

    2010-01-27

    Photoreceptor cells achieve high sensitivity, reliably detecting single photons, while limiting the spontaneous activation events responsible for dark noise. We used proteomic, genetic, and electrophysiological approaches to characterize Retinophilin (RTP) (CG10233) in Drosophila photoreceptors and establish its involvement in dark-noise suppression. RTP possesses membrane occupation and recognition nexus (MORN) motifs, a structure shared with mammalian junctophilins and other membrane-associated proteins found within excitable cells. We show the MORN repeats, and both the N- and C-terminal domains, are required for RTP localization in the microvillar light-gathering organelle, the rhabdomere. RTP exists in multiple phosphorylated isoforms under dark conditions and is dephosphorylated by light exposure. An RTP deletion mutant exhibits a high rate of spontaneous membrane depolarization events in dark conditions but retains the normal kinetics of the light response. Photoreceptors lacking neither inactivation nor afterpotential C (NINAC) myosin III, a motor protein/kinase, also display a similar dark-noise phenotype as the RTP deletion. We show that NINAC mutants are depleted for RTP. These results suggest the increase in dark noise in NINAC mutants is attributable to lack of RTP and, furthermore, defines a novel role for NINAC in the rhabdomere. We propose that RTP is a light-regulated phosphoprotein that organizes rhabdomeric components to suppress random activation of the phototransduction cascade and thus increases the signaling fidelity of dark-adapted photoreceptors.

  3. Dentin sialoprotein and dentin phosphoprotein have distinct roles in dentin mineralization

    PubMed Central

    Suzuki, Shigeki; Sreenath, Taduru; Haruyama, Naoto; Honeycutt, Cherlita; Terse, Anita; Cho, Andrew; Kohler, Thomas; Müller, Ralph; Goldberg, Michel; Kulkarni, Ashok B.

    2009-01-01

    Dentin sialophosphoprotein (DSPP), a major non-collagenous matrix protein of odontoblasts, is proteolytically cleaved into dentin sialoprotein (DSP) and dentin phosphoprotein (DPP). Our previous studies revealed that DSPP null mice display a phenotype similar to human autosomal dominant dentinogenesis imperfecta, in which teeth have widened predentin and irregular dentin mineralization resulting in sporadic unmineralized areas in dentin and frequent pulp exposure. Earlier in vitro studies suggested that DPP, but not DSP, plays a significant role in initiation and maturation of dentin mineralization. However, the precise in vivo roles of DSP and DPP are far from clear. Here we report the generation of DPPcKO mice, in which only DSP is expressed in a DSPP null background, resulting in a conditional DPP knockout. DPPcKO teeth show a partial rescue of the DSPP null phenotype with the restored predentin width, an absence of irregular unmineralized areas in dentin, and less frequent pulp exposure. Micro-computed tomography (micro-CT) analysis of DPPcKO molars further confirmed this partial rescue with a significant recovery in the dentin volume, but not in the dentin mineral density. These results indicate distinct roles of DSP and DPP in dentin mineralization, with DSP regulating initiation of dentin mineralization, and DPP being involved in the maturation of mineralized dentin. PMID:19348940

  4. Structural studies on the authentic mumps virus nucleocapsid showing uncoiling by the phosphoprotein

    PubMed Central

    Cox, Robert; Pickar, Adrian; Qiu, Shihong; Tsao, Jun; Rodenburg, Cynthia; Dokland, Terje; Elson, Andrew; He, Biao; Luo, Ming

    2014-01-01

    Mumps virus (MuV) is a highly contagious pathogen, and despite extensive vaccination campaigns, outbreaks continue to occur worldwide. The virus has a negative-sense, single-stranded RNA genome that is encapsidated by the nucleocapsid protein (N) to form the nucleocapsid (NC). NC serves as the template for both transcription and replication. In this paper we solved an 18-Å–resolution structure of the authentic MuV NC using cryo-electron microscopy. We also observed the effects of phosphoprotein (P) binding on the MuV NC structure. The N-terminal domain of P (PNTD) has been shown to bind NC and appeared to induce uncoiling of the helical NC. Additionally, we solved a 25-Å–resolution structure of the authentic MuV NC bound with the C-terminal domain of P (PCTD). The location of the encapsidated viral genomic RNA was defined by modeling crystal structures of homologous negative strand RNA virus Ns in NC. Both the N-terminal and C-terminal domains of MuV P bind NC to participate in access to the genomic RNA by the viral RNA-dependent-RNA polymerase. These results provide critical insights on the structure-function of the MuV NC and the structural alterations that occur through its interactions with P. PMID:25288750

  5. A new system for naming ribosomal proteins.

    PubMed

    Ban, Nenad; Beckmann, Roland; Cate, Jamie H D; Dinman, Jonathan D; Dragon, François; Ellis, Steven R; Lafontaine, Denis L J; Lindahl, Lasse; Liljas, Anders; Lipton, Jeffrey M; McAlear, Michael A; Moore, Peter B; Noller, Harry F; Ortega, Joaquin; Panse, Vikram Govind; Ramakrishnan, V; Spahn, Christian M T; Steitz, Thomas A; Tchorzewski, Marek; Tollervey, David; Warren, Alan J; Williamson, James R; Wilson, Daniel; Yonath, Ada; Yusupov, Marat

    2014-02-01

    A system for naming ribosomal proteins is described that the authors intend to use in the future. They urge others to adopt it. The objective is to eliminate the confusion caused by the assignment of identical names to ribosomal proteins from different species that are unrelated in structure and function. In the system proposed here, homologous ribosomal proteins are assigned the same name, regardless of species. It is designed so that new names are similar enough to old names to be easily recognized, but are written in a format that unambiguously identifies them as 'new system' names.

  6. Isolation of Bacterial Ribosomes with Monolith Chromatography

    PubMed Central

    Trauner, Andrej; Bennett, Mark H.; Williams, Huw D.

    2011-01-01

    We report the development of a rapid chromatographic method for the isolation of bacterial ribosomes from crude cell lysates in less than ten minutes. Our separation is based on the use of strong anion exchange monolithic columns. Using a simple stepwise elution program we were able to purify ribosomes whose composition is comparable to those isolated by sucrose gradient ultracentrifugation, as confirmed by quantitative proteomic analysis (iTRAQ). The speed and simplicity of this approach could accelerate the study of many different aspects of ribosomal biology. PMID:21326610

  7. Effects of anti-C23 (nucleolin) antibody on transcription of ribosomal DNA in Chironomus salivary gland cells

    SciTech Connect

    Egyhazi, E.; Pigon, A. ); Chang, Jinhong; Ghaffari, S.H.; Dreesen, T.D.; Wellman, S.E.; Case, S.T.; Olson, M.O.J. )

    1988-10-01

    Protein C23 (also called nucleolin or 100-kDa nucleolar protein) is a major nucleolar phosphoprotein involved in ribosome biogenesis. To determine the effects of protein C23 on preribosomal RNA (pre-rRNA) synthesis anti-C23 antiserum was microinjected into nuclei of Chironomus tentans salivary glands. Transcription was measured by incubation of the glands with {sup 32}P-labeled RNA precursors followed by microdissection of nucleoli, RNA extraction, and electrophoretic analyses. Injection of the anti-C23 antibody caused a 2- to 3.5-fold stimulation of {sup 32}P incorporation into 38 S pre-rRNA. No stimulation was observed in salivary glands injected with preimmune serum or antiserum preabsorbed with protein C23. The stimulatory effect was selective for pre-rRNA as indicated by the lack of stimulation of {sup 32}P incorporation into extranucleolar RNA. Injection of the antiserum produced little or no effect on pre-RNA processing as measured by the relative amounts of {sup 32}P-labeled intermediate cleavage products of pre-rRNA in stimulated versus control glands. These results suggest that protein C23 not only is involved in ribosome assembly but also plays a role in regulating the transcription of the preribosomal RNA.

  8. Hold on to your friends: Dedicated chaperones of ribosomal proteins: Dedicated chaperones mediate the safe transfer of ribosomal proteins to their site of pre-ribosome incorporation.

    PubMed

    Pillet, Benjamin; Mitterer, Valentin; Kressler, Dieter; Pertschy, Brigitte

    2017-01-01

    Eukaryotic ribosomes are assembled from their components, the ribosomal RNAs and ribosomal proteins, in a tremendously complex, multi-step process, which primarily takes place in the nuclear compartment. Therefore, most ribosomal proteins have to travel from the cytoplasm to their incorporation site on pre-ribosomes within the nucleus. However, due to their particular characteristics, such as a highly basic amino acid composition and the presence of unstructured extensions, ribosomal proteins are especially prone to aggregation and degradation in their unassembled state, hence specific mechanisms must operate to ensure their safe delivery. Recent studies have uncovered a group of proteins, termed dedicated chaperones, specialized in accompanying and guarding individual ribosomal proteins. In this essay, we review how these dedicated chaperones utilize different folds to interact with their ribosomal protein clients and how they ensure their soluble expression and interconnect their intracellular transport with their efficient assembly into pre-ribosomes.

  9. Regulation of ribosomal protein synthesis in an Escherichia coli mutant missing ribosomal protein L1.

    PubMed Central

    Jinks-Robertson, S; Nomura, M

    1981-01-01

    In an Escherichia coli B strain missing ribosomal protein L1, the synthesis rate of L11 is 50% greater than that of other ribosomal proteins. This finding is in agreement with the previous conclusion that L1 regulates synthesis of itself and L11 and indicates that this regulation is important for maintaining the balanced synthesis of ribosomal proteins under physiological conditions. PMID:7009590

  10. Illuminating parasite protein production by ribosome profiling

    PubMed Central

    Parsons, Marilyn; Myler, Peter J.

    2016-01-01

    While technologies for global enumeration of transcript abundance are well-developed, those that assess protein abundance require tailoring to penetrate to low abundance proteins. Ribosome profiling circumvents this challenge by measuring global protein production via sequencing small mRNA fragments protected by the assembled ribosome. This powerful approach is now being applied to protozoan parasites, including trypanosomes and Plasmodium. It has been used to identify new protein coding sequences (CDSs) and clarify the boundaries of previously annotated CDSs in Trypanosoma brucei. Ribosome profiling has demonstrated that translation efficiencies vary widely between genes and, for trypanosomes at least, for the same gene across stages. The ribosomal proteins are themselves subjected to translational control, suggesting a means of reinforcing global translational regulation. PMID:27061497

  11. The Circadian Clock Coordinates Ribosome Biogenesis

    PubMed Central

    Symul, Laura; Martin, Eva; Atger, Florian; Naef, Felix; Gachon, Frédéric

    2013-01-01

    Biological rhythms play a fundamental role in the physiology and behavior of most living organisms. Rhythmic circadian expression of clock-controlled genes is orchestrated by a molecular clock that relies on interconnected negative feedback loops of transcription regulators. Here we show that the circadian clock exerts its function also through the regulation of mRNA translation. Namely, the circadian clock influences the temporal translation of a subset of mRNAs involved in ribosome biogenesis by controlling the transcription of translation initiation factors as well as the clock-dependent rhythmic activation of signaling pathways involved in their regulation. Moreover, the circadian oscillator directly regulates the transcription of ribosomal protein mRNAs and ribosomal RNAs. Thus the circadian clock exerts a major role in coordinating transcription and translation steps underlying ribosome biogenesis. PMID:23300384

  12. Eukaryotic Ribosome Assembly and Nuclear Export.

    PubMed

    Nerurkar, Purnima; Altvater, Martin; Gerhardy, Stefan; Schütz, Sabina; Fischer, Ute; Weirich, Christine; Panse, Vikram Govind

    2015-01-01

    Accurate translation of the genetic code into functional polypeptides is key to cellular growth and proliferation. This essential process is carried out by the ribosome, a ribonucleoprotein complex of remarkable size and intricacy. Although the structure of the mature ribosome has provided insight into the mechanism of translation, our knowledge regarding the assembly, quality control, and intracellular targeting of this molecular machine is still emerging. Assembly of the eukaryotic ribosome begins in the nucleolus and requires more than 350 conserved assembly factors, which transiently associate with the preribosome at specific maturation stages. After accomplishing their tasks, early-acting assembly factors are released, preparing preribosomes for nuclear export. Export competent preribosomal subunits are transported through nuclear pore complexes into the cytoplasm, where they undergo final maturation steps, which are closely connected to quality control, before engaging in translation. In this chapter, we focus on the final events that commit correctly assembled ribosomal subunits for translation.

  13. Quantitative studies of ribosome conformational dynamics.

    PubMed

    Fraser, Christopher S; Doudna, Jennifer A

    2007-05-01

    The ribosome is a dynamic machine that undergoes many conformational rearrangements during the initiation of protein synthesis. Significant differences exist between the process of protein synthesis initiation in eubacteria and eukaryotes. In particular, the initiation of eukaryotic protein synthesis requires roughly an order of magnitude more initiation factors to promote efficient mRNA recruitment and ribosomal recognition of the start codon than are needed for eubacterial initiation. The mechanisms by which these initiation factors promote ribosome conformational changes during stages of initiation have been studied using cross-linking, footprinting, site-directed probing, cryo-electron microscopy, X-ray crystallography, fluorescence spectroscopy and single-molecule techniques. Here, we review how the results of these different approaches have begun to converge to yield a detailed molecular understanding of the dynamic motions that the eukaryotic ribosome cycles through during the initiation of protein synthesis.

  14. Potential extra-ribosomal functions of ribosomal proteins in Saccharomyces cerevisiae.

    PubMed

    Lu, Hui; Zhu, Yi-Fei; Xiong, Juan; Wang, Rong; Jia, Zhengping

    2015-08-01

    Ribosomal proteins (RPs), are essential components of the ribosomes, the molecular machines that turn mRNA blueprints into proteins, as they serve to stabilize the structure of the rRNA, thus improving protein biosynthesis. In addition, growing evidence suggests that RPs can function in other cellular roles. In the present review, we summarize several potential extra-ribosomal functions of RPs in ribosomal biogenesis, transcription activity, translation process, DNA repair, replicative life span, adhesive growth, and morphological transformation in Saccharomyces cerevisiae. However, the future in-depth studies are needed to identify these novel secondary functions of RPs in S. cerevisiae.

  15. Ribosome Inactivating Proteins from Rosaceae.

    PubMed

    Shang, Chenjing; Rougé, Pierre; Van Damme, Els J M

    2016-08-22

    Ribosome-inactivating proteins (RIPs) are widespread among higher plants of different taxonomic orders. In this study, we report on the RIP sequences found in the genome/transcriptome of several important Rosaceae species, including many economically important edible fruits such as apple, pear, peach, apricot, and strawberry. All RIP domains from Rosaceae share high sequence similarity with conserved residues in the catalytic site and the carbohydrate binding sites. The genomes of Malus domestica and Pyrus communis contain both type 1 and type 2 RIP sequences, whereas for Prunus mume, Prunus persica, Pyrus bretschneideri, and Pyrus communis a complex set of type 1 RIP sequences was retrieved. Heterologous expression and purification of the type 1 as well as the type 2 RIP from apple allowed to characterize the biological activity of the proteins. Both RIPs from Malus domestica can inhibit protein synthesis. Furthermore, molecular modelling suggests that RIPs from Rosaceae possess three-dimensional structures that are highly similar to the model proteins and can bind to RIP substrates. Screening of the recombinant type 2 RIP from apple on a glycan array revealed that this type 2 RIP interacts with terminal sialic acid residues. Our data suggest that the RIPs from Rosaceae are biologically active proteins.

  16. Role of GTPases in ribosome assembly.

    PubMed

    Karbstein, Katrin

    2007-09-01

    GTPases are a universally conserved class of regulatory proteins involved in such diverse cellular functions as signal transduction, translation, cytoskeleton formation, and intracellular transport. GTPases are also required for ribosome assembly in eukaryotes and bacteria, where they present themselves as possible regulatory molecules. Strikingly, in bacteria they represent the largest class of essential assembly factors. A review of their common structural, biochemical and genetic interactions is presented and integrated with models for their function in ribosome assembly. 2007 Wiley Periodicals, Inc

  17. Atomic mutagenesis at the ribosomal decoding site.

    PubMed

    Schrode, Pius; Huter, Paul; Clementi, Nina; Erlacher, Matthias

    2017-01-02

    Ribosomal decoding is an essential process in every living cell. During protein synthesis the 30S ribosomal subunit needs to accomplish binding and accurate decoding of mRNAs. From mutational studies and high-resolution crystal structures nucleotides G530, A1492 and A1493 of the 16S rRNA came into focus as important elements for the decoding process. Recent crystallographic data challenged the so far accepted model for the decoding mechanism. To biochemically investigate decoding in greater detail we applied an in vitro reconstitution approach to modulate single chemical groups at A1492 and A1493. The modified ribosomes were subsequently tested for their ability to efficiently decode the mRNA. Unexpectedly, the ribosome was rather tolerant toward modifications of single groups either at the base or at the sugar moiety in terms of translation activity. Concerning translation fidelity, the elimination of single chemical groups involved in a hydrogen bonding network between the tRNA, mRNA and rRNA did not change the accuracy of the ribosome. These results indicate that the contribution of those chemical groups and the formed hydrogen bonds are not crucial for ribosomal decoding.

  18. Atomic mutagenesis at the ribosomal decoding site

    PubMed Central

    Schrode, Pius; Huter, Paul; Clementi, Nina; Erlacher, Matthias

    2017-01-01

    ABSTRACT Ribosomal decoding is an essential process in every living cell. During protein synthesis the 30S ribosomal subunit needs to accomplish binding and accurate decoding of mRNAs. From mutational studies and high-resolution crystal structures nucleotides G530, A1492 and A1493 of the 16S rRNA came into focus as important elements for the decoding process. Recent crystallographic data challenged the so far accepted model for the decoding mechanism. To biochemically investigate decoding in greater detail we applied an in vitro reconstitution approach to modulate single chemical groups at A1492 and A1493. The modified ribosomes were subsequently tested for their ability to efficiently decode the mRNA. Unexpectedly, the ribosome was rather tolerant toward modifications of single groups either at the base or at the sugar moiety in terms of translation activity. Concerning translation fidelity, the elimination of single chemical groups involved in a hydrogen bonding network between the tRNA, mRNA and rRNA did not change the accuracy of the ribosome. These results indicate that the contribution of those chemical groups and the formed hydrogen bonds are not crucial for ribosomal decoding. PMID:27841727

  19. Spontaneous Intersubunit Rotation in Single Ribosomes

    PubMed Central

    Cornish, Peter V.; Ermolenko, Dmitri N.; Noller, Harry F.; Ha, Taekjip

    2008-01-01

    Summary During the elongation cycle, tRNA and mRNA undergo coupled translocation through the ribosome catalyzed by elongation factor EF-G. Cryo-EM reconstructions of certain EF-G-containing complexes led to the proposal that the mechanism of translocation involves rotational movement between the two ribosomal subunits. Here, using single-molecule FRET we observe that pre-translocation ribosomes undergo spontaneous intersubunit rotational movement in the absence of EF-G, fluctuating between two conformations corresponding to the classical and hybrid states of the translocational cycle. In contrast, post-translocation ribosomes are fixed predominantly in the classical, non-rotated state. Movement of the acceptor stem of deacylated tRNA into the 50S E site and EF-G binding to the ribosome both contribute to stabilization of the rotated, hybrid state. Furthermore, the acylation state of P-site tRNA has a dramatic effect on the frequency of intersubunit rotation. Our results provide direct evidence that the intersubunit rotation that underlies ribosomal translocation is thermally driven. PMID:18538656

  20. Ribonuclease Sensitivity of Escherichia coli Ribosomes

    PubMed Central

    Santer, Melvin; Smith, Josephine R.

    1966-01-01

    Santer, Melvin (Haverford College, Haverford, Pa.), and Josephine R. Smith. Ribonuclease sensitivity of Escherichia coli ribosomes. J. Bacteriol. 92:1099–1110. 1966.—The ribonucleic acid (RNA) contained in 70S ribosomes and in 50S and 30S subunits was hydrolyzed by pancreatic ribonuclease. A 7% amount of the RNA was removed from the 70S particle; at 10−4m magnesium concentration, a maximum of 24 and 30% of the RNA in the 50S and the 30S fractions, respectively, was removed by ribonuclease. At the two lower magnesium ion concentrations, 50S ribosomes did not lose any protein, whereas 30S ribosomes lost protein as a result of ribonuclease treatment. A number of proteins were removed from the 30S particles by ribonuclease, and these proteins were antigenically related to proteins present in 50S ribosomes. The differential effect of ribonuclease on 50S and 30S ribosomes suggested that they have structural dissimilarities. Images PMID:5332866

  1. A recent intermezzo at the Ribosome Club.

    PubMed

    Pavlov, Michael Y; Liljas, Anders; Ehrenberg, Måns

    2017-03-19

    Two sets of ribosome structures have recently led to two different interpretations of what limits the accuracy of codon translation by transfer RNAs. In this review, inspired by this intermezzo at the Ribosome Club, we briefly discuss accuracy amplification by energy driven proofreading and its implementation in genetic code translation. We further discuss general ways by which the monitoring bases of 16S rRNA may enhance the ultimate accuracy (d-values) and how the codon translation accuracy is reduced by the actions of Mg(2+) ions and the presence of error inducing aminoglycoside antibiotics. We demonstrate that complete freezing-in of cognate-like tautomeric states of ribosome-bound nucleotide bases in transfer RNA or messenger RNA is not compatible with recent experiments on initial codon selection by transfer RNA in ternary complex with elongation factor Tu and GTP. From these considerations, we suggest that the sets of 30S subunit structures from the Ramakrishnan group and 70S structures from the Yusupov/Yusupova group may, after all, reflect two sides of the same coin and how the structurally based intermezzo at the Ribosome Club may be resolved simply by taking the dynamic aspects of ribosome function into account.This article is part of the themed issue 'Perspectives on the ribosome'.

  2. Mapping the interaction of SmpB with ribosomes by footprinting of ribosomal RNA

    PubMed Central

    Ivanova, Natalia; Pavlov, Michael Y.; Bouakaz, Elli; Ehrenberg, Måns; Schiavone, Lovisa Holmberg

    2005-01-01

    In trans-translation transfer messenger RNA (tmRNA) and small protein B (SmpB) rescue ribosomes stalled on truncated or in other ways problematic mRNAs. SmpB promotes the binding of tmRNA to the ribosome but there is uncertainty about the number of participating SmpB molecules as well as their ribosomal location. Here, the interaction of SmpB with ribosomal subunits and ribosomes was studied by isolation of SmpB containing complexes followed by chemical modification of ribosomal RNA with dimethyl sulfate, kethoxal and hydroxyl radicals. The results show that SmpB binds 30S and 50S subunits with 1:1 molar ratios and the 70S ribosome with 2:1 molar ratio. SmpB-footprints are similar on subunits and the ribosome. In the 30S subunit, SmpB footprints nucleotides that are in the vicinity of the P-site facing the E-site, and in the 50S subunit SmpB footprints nucleotides that are located below the L7/L12 stalk in the 3D structure of the ribosome. Based on these results, we suggest a mechanism where two molecules of SmpB interact with tmRNA and the ribosome during trans-translation. The first SmpB molecule binds near the factor-binding site on the 50S subunit helping tmRNA accommodation on the ribosome, whereas the second SmpB molecule may functionally substitute for a missing anticodon stem–loop in tmRNA during later steps of trans-translation. PMID:15972795

  3. Phosphorylation of paramyxovirus phosphoprotein and its role in viral gene expression

    PubMed Central

    Fuentes, Sandra M; Sun, Dengyun; Schmitt, Anthony P; He, Biao

    2010-01-01

    Paramyxoviruses include many important human and animal pathogens such as measles virus, mumps virus, human parainfluenza viruses, and respiratory syncytial virus, as well as emerging viruses such as Nipah virus and Hendra virus. The paramyxovirus RNA-dependent RNA polymerase consists of the phosphoprotein (P) and the large protein. Both of these proteins are essential for viral RNA synthesis. The P protein is phosphorylated at multiple sites, probably by more than one host kinase. While it is thought that the phosphorylation of P is important for its role in viral RNA synthesis, the precise role of P protein phosphorylation remains an enigma. For instance, it was demonstrated that the putative CKII phosphorylation sites of the P protein of respiratory syncytial virus play a role in viral RNA synthesis using a minigenome replicon system; however, mutating these putative CKII phosphorylation sites within a viral genome had no effect on viral RNA synthesis, leading to the hypothesis that P protein phosphorylation, at least by CKII, does not play a role in viral RNA synthesis. Recently, it has been reported that the phosphorylation state of the P protein of parainfluenza virus 5, a prototypical paramyxovirus, correlates with the ability of P protein to synthesize viral RNA, indicating that P protein phosphorylation does in fact play a role in viral RNA synthesis. Furthermore, host kinases PLK1, as well as AKT1 have been found to play critical roles in paramyxovirus RNA synthesis through regulation of P protein phosphorylation status. Beyond furthering our understanding of paramyxovirus RNA replication, these recent discoveries may also result in a new paradigm in treating infections caused by these viruses, as host kinases that regulate paramyxovirus replication are investigated as potential targets of therapeutic intervention. PMID:20020826

  4. Vasodilator-Stimulated Phosphoprotein Deficiency Potentiates PAR-1-induced Increase in Endothelial Permeability in Mouse Lungs

    PubMed Central

    Profirovic, Jasmina; Han, Jingyan; Andreeva, Alexandra V.; Neamu, Radu F.; Pavlovic, Sasha; Vogel, Stephen M.; Walter, Ulrich; Voyno-Yasenetskaya, Tatyana A.

    2010-01-01

    Vasodilator-stimulated phosphoprotein (VASP) is implicated in the protection of the endothelial barrier in vitro and in vivo. VASP function in thrombin signaling in the endothelial cells (ECs) is not known. For the first time we studied the effects of VASP deficiency on EC permeability and pulmonary vascular permeability in response to thrombin receptor stimulation. We provided the evidence that VASP deficiency potentiates the increase in endothelial permeability induced by activation of thrombin receptor in cultured human umbilical vein endothelial cells (HUVECs) and isolated mouse lungs. Using transendothelial resistance measurement, we showed that siRNA-mediated VASP downregulation in HUVECs leads to a potentiation of thrombin- and protease-activated receptor 1 (PAR-1) agonist-induced increase in endothelial permeability. Compared to control cells, VASP-deficient HUVECs had delayed endothelial junctional reassembly and abrogated VE-cadherin cytoskeletal anchoring in the recovery phase after thrombin stimulation, as demonstrated by immunofluorescence studies and cell fractionation analysis, respectively. Measurement of the capillary filtration coefficient in isolated mouse lungs demonstrated that VASP−/− mice have increased microvascular permeability in response to infusion with PAR-1 agonist compared to wild type mice. Lack of VASP led to decreased Rac1 activation both in VASP-deficient HUVECs after thrombin stimulation and VASP−/− mouse lungs after PAR-1 agonist infusion, indicating that VASP effects on thrombin signaling may correlated with changes in Rac1 activity. This study demonstrates that VASP may play critical and complex role in the regulation of thrombin-dependent disruption of the endothelial barrier function. PMID:20945373

  5. Structural disorder within Henipavirus nucleoprotein and phosphoprotein: from predictions to experimental assessment.

    PubMed

    Habchi, Johnny; Mamelli, Laurent; Darbon, Hervé; Longhi, Sonia

    2010-07-21

    Henipaviruses are newly emerged viruses within the Paramyxoviridae family. Their negative-strand RNA genome is packaged by the nucleoprotein (N) within alpha-helical nucleocapsid that recruits the polymerase complex made of the L protein and the phosphoprotein (P). To date structural data on Henipaviruses are scarce, and their N and P proteins have never been characterized so far. Using both computational and experimental approaches we herein show that Henipaviruses N and P proteins possess large intrinsically disordered regions. By combining several disorder prediction methods, we show that the N-terminal domain of P (PNT) and the C-terminal domain of N (NTAIL) are both mostly disordered, although they contain short order-prone segments. We then report the cloning, the bacterial expression, purification and characterization of Henipavirus PNT and NTAIL domains. By combining gel filtration, dynamic light scattering, circular dichroism and nuclear magnetic resonance, we show that both NTAIL and PNT belong to the premolten globule sub-family within the class of intrinsically disordered proteins. This study is the first reported experimental characterization of Henipavirus P and N proteins. The evidence that their respective N-terminal and C-terminal domains are highly disordered under native conditions is expected to be invaluable for future structural studies by helping to delineate N and P protein domains amenable to crystallization. In addition, following previous hints establishing a relationship between structural disorder and protein interactivity, the present results suggest that Henipavirus PNT and NTAIL domains could be involved in manifold protein-protein interactions.

  6. Structural Disorder within Henipavirus Nucleoprotein and Phosphoprotein: From Predictions to Experimental Assessment

    PubMed Central

    Darbon, Hervé; Longhi, Sonia

    2010-01-01

    Henipaviruses are newly emerged viruses within the Paramyxoviridae family. Their negative-strand RNA genome is packaged by the nucleoprotein (N) within α-helical nucleocapsid that recruits the polymerase complex made of the L protein and the phosphoprotein (P). To date structural data on Henipaviruses are scarce, and their N and P proteins have never been characterized so far. Using both computational and experimental approaches we herein show that Henipaviruses N and P proteins possess large intrinsically disordered regions. By combining several disorder prediction methods, we show that the N-terminal domain of P (PNT) and the C-terminal domain of N (NTAIL) are both mostly disordered, although they contain short order-prone segments. We then report the cloning, the bacterial expression, purification and characterization of Henipavirus PNT and NTAIL domains. By combining gel filtration, dynamic light scattering, circular dichroism and nuclear magnetic resonance, we show that both NTAIL and PNT belong to the premolten globule sub-family within the class of intrinsically disordered proteins. This study is the first reported experimental characterization of Henipavirus P and N proteins. The evidence that their respective N-terminal and C-terminal domains are highly disordered under native conditions is expected to be invaluable for future structural studies by helping to delineate N and P protein domains amenable to crystallization. In addition, following previous hints establishing a relationship between structural disorder and protein interactivity, the present results suggest that Henipavirus PNT and NTAIL domains could be involved in manifold protein-protein interactions. PMID:20657787

  7. Borna Disease Virus Phosphoprotein Represses p53-Mediated Transcriptional Activity by Interference with HMGB1

    PubMed Central

    Zhang, Guoqi; Kobayashi, Takeshi; Kamitani, Wataru; Komoto, Satoshi; Yamashita, Makiko; Baba, Satoko; Yanai, Hideyuki; Ikuta, Kazuyoshi; Tomonaga, Keizo

    2003-01-01

    Borna disease virus (BDV) is a noncytolytic, neurotropic RNA virus that has a broad host range in warm-blooded animals, probably including humans. Recently, it was demonstrated that a 24-kDa phosphoprotein (P) of BDV directly binds to a multifunctional protein, amphoterin-HMGB1, and inhibits its function in cultured neural cells (W. Kamitani, Y. Shoya, T. Kobayashi, M. Watanabe, B. J. Lee, G. Zhang, K. Tomonaga, and K. Ikuta, J. Virol. 75:8742-8751, 2001). This observation suggested that expression of BDV P may cause deleterious effects in cellular functions by interference with HMGB1. In this study, we further investigated the significance of the binding between P and HMGB1. We demonstrated that P directly binds to the A-box domain on HMGB1, which is also responsible for interaction with a tumor suppression factor, p53. Recent works have demonstrated that binding between HMGB1 and p53 enhances p53-mediated transcriptional activity. Thus, we examined whether BDV P affects the transcriptional activity of p53 by interference with HMGB1. Mammalian two-hybrid analysis revealed that p53 and P competitively interfere with the binding of each protein to HMGB1 in a p53-deficient cell line, NCI-H1299. In addition, P was able to significantly decrease p53-mediated transcriptional activation of the cyclin G promoter. Furthermore, we showed that activation of p21waf1 expression was repressed in cyclosporine-treated BDV-infected cells, as well as p53-transduced NCI-H1299 cells. These results suggested that BDV P may be a unique inhibitor of p53 activity via binding to HMGB1. PMID:14581561

  8. 14-3-3 phosphoprotein interaction networks – does isoform diversity present functional interaction specification?

    PubMed Central

    Paul, Anna-Lisa; Denison, Fiona C.; Schultz, Eric R.; Zupanska, Agata K.; Ferl, Robert J.

    2012-01-01

    The 14-3-3 proteins have emerged as major phosphoprotein interaction proteins and thereby constitute a key node in the Arabidopsis Interactome Map, a node through which a large number of important signals pass. Throughout their history of discovery and description, the 14-3-3s have been described as protein families and there has been some evidence that the different 14-3-3 family members within any organism might carry isoform-specific functions. However, there has also been evidence for redundancy of 14-3-3 function, suggesting that the perceived 14-3-3 diversity may be the accumulation of neutral mutations over evolutionary time and as some 14-3-3 genes develop tissue or organ-specific expression. This situation has led to a currently unresolved question – does 14-3-3 isoform sequence diversity indicate functional diversity at the biochemical or cellular level? We discuss here some of the key observations on both sides of the resulting debate, and present a set of contrastable observations to address the theory functional diversity does exist among 14-3-3 isoforms. The resulting model suggests strongly that there are indeed functional specificities in the 14-3-3s of Arabidopsis. The model further suggests that 14-3-3 diversity and specificity should enter into the discussion of 14-3-3 roles in signal transduction and be directly approached in 14-3-3 experimentation. It is hoped that future studies involving 14-3-3s will continue to address specificity in experimental design and analysis. PMID:22934100

  9. Rabies virus phosphoprotein interacts with mitochondrial Complex I and induces mitochondrial dysfunction and oxidative stress.

    PubMed

    Kammouni, Wafa; Wood, Heidi; Saleh, Ali; Appolinario, Camila M; Fernyhough, Paul; Jackson, Alan C

    2015-08-01

    Our previous studies in an experimental model of rabies showed neuronal process degeneration in association with severe clinical disease. Cultured adult rodent dorsal root ganglion neurons infected with challenge virus standard (CVS)-11 strain of rabies virus (RABV) showed axonal swellings and reduced axonal growth with evidence of oxidative stress. We have shown that CVS infection alters a variety of mitochondrial parameters and increases reactive oxygen species (ROS) production and mitochondrial Complex I activity vs. mock infection. We have hypothesized that a RABV protein targets mitochondria and triggers dysfunction. Mitochondrial extracts of mouse neuroblastoma cells were analyzed with a proteomics approach. We have identified peptides belonging to the RABV nucleocapsid protein (N), phosphoprotein (P), and glycoprotein (G), and our data indicate that the extract was most highly enriched with P. P was also detected by immunoblotting in RABV-infected purified mitochondrial extracts and also in Complex I immunoprecipitates from the extracts but not in mock-infected extracts. A plasmid expressing P in cells increased Complex I activity and increased ROS generation, whereas expression of other RABV proteins did not. We have analyzed recombinant plasmids encoding various P gene segments. Expression of a peptide from amino acid 139-172 increased Complex I activity and ROS generation similar to expression of the entire P protein, whereas peptides that did not contain this region did not increase Complex I activity or induce ROS generation. These results indicate that a region of the RABV P interacts with Complex I in mitochondria causing mitochondrial dysfunction, increased generation of ROS, and oxidative stress.

  10. Secreted Phosphoprotein 1 Is a Determinant of Lung Function Development in Mice

    PubMed Central

    Martin, Timothy M.; Concel, Vincent J.; Upadhyay, Swapna; Bein, Kiflai; Brant, Kelly A.; George, Leema; Mitra, Ankita; Thimraj, Tania A.; Fabisiak, James P.; Vuga, Louis J.; Fattman, Cheryl; Kaminski, Naftali; Schulz, Holger; Leikauf, George D.

    2014-01-01

    Secreted phosphoprotein 1 (Spp1) is located within quantitative trait loci associated with lung function that was previously identified by contrasting C3H/HeJ and JF1/Msf mouse strains that have extremely divergent lung function. JF1/Msf mice with diminished lung function had reduced lung SPP1 transcript and protein during the peak stage of alveologenesis (postnatal day [P]14–P28) as compared with C3H/HeJ mice. In addition to a previously identified genetic variant that altered runt-related transcription factor 2 (RUNX2) binding in the Spp1 promoter, we identified another promoter variant in a putative RUNX2 binding site that increased the DNA protein binding. SPP1 induced dose-dependent mouse lung epithelial-15 cell proliferation. Spp1(−/−) mice have decreased specific total lung capacity/body weight, higher specific compliance, and increased mean airspace chord length (Lm) compared with Spp1(+/+) mice. Microarray analysis revealed enriched gene ontogeny categories, with numerous genes associated with lung development and/or respiratory disease. Insulin-like growth factor 1, Hedgehog-interacting protein, wingless-related mouse mammary tumor virus integration site 5A, and NOTCH1 transcripts decreased in the lung of P14 Spp1(−/−) mice as determined by quantitative RT-PCR analysis. SPP1 promotes pneumocyte growth, and mice lacking SPP1 have smaller, more compliant lungs with enlarged airspace (i.e., increased Lm). Microarray analysis suggests a dysregulation of key lung developmental transcripts in gene-targeted Spp1(−/−) mice, particularly during the peak phase of alveologenesis. In addition to its known roles in lung disease, this study supports SPP1 as a determinant of lung development in mice. PMID:24816281

  11. Phosphoprotein enriched in astrocytes (PEA)-15: A potential therapeutic target in multiple disease states

    PubMed Central

    Greig, Fiona H.; Nixon, Graeme F.

    2014-01-01

    Phosphoprotein enriched in astrocytes-15 (PEA-15) is a cytoplasmic protein that sits at an important junction in intracellular signalling and can regulate diverse cellular processes, such as proliferation and apoptosis, dependent upon stimulation. Regulation of these processes occurs by virtue of the unique interaction of PEA-15 with other signalling proteins. PEA-15 acts as a cytoplasmic tether for the mitogen-activated protein kinases, extracellular signal-regulated kinase 1/2 (ERK1/2) preventing nuclear localisation. In order to release ERK1/2, PEA-15 requires to be phosphorylated via several potential pathways. PEA-15 (and its phosphorylation state) therefore regulates many ERK1/2-dependent processes, including proliferation, via regulating ERK1/2 nuclear translocation. In addition, PEA-15 contains a death effector domain (DED) which allows interaction with other DED-containing proteins. PEA-15 can bind the DED-containing apoptotic adaptor molecule, Fas-associated death domain protein (FADD) which is also dependent on the phosphorylation status of PEA-15. PEA-15 binding of FADD can inhibit apoptosis as bound FADD cannot participate in the assembly of apoptotic signalling complexes. Through these protein–protein interactions, PEA-15-regulated cellular effects have now been investigated in a number of disease-related studies. Changes in PEA-15 expression and regulation have been observed in diabetes mellitus, cancer, neurological disorders and the cardiovascular system. These changes have been suggested to contribute to the pathology related to each of these disease states. As such, new therapeutic targets based around PEA-15 and its associated interactions are now being uncovered and could provide novel avenues for treatment strategies in multiple diseases. PMID:24657708

  12. Specific dephosphorylation of phosphoproteins by protein-serine and -tyrosine kinases.

    PubMed

    Kole, H K; Abdel-Ghany, M; Racker, E

    1988-08-01

    Five protein kinases are shown to serve as specific phosphatases in the absence of ADP. Although the rates of hydrolysis are very slow compared to the forward phosphorylation rates under optimal conditions, they are of the same order as the reverse reaction in the presence of ADP. Because cells contain approximately equal to 3 mM ATP, neither the reverse reaction nor the phosphatase is likely to play a physiological role. beta-casein B phosphorylated by the catalytic subunit of cAMP-dependent protein kinase (protein kinase A) is specifically dephosphorylated by protein kinase A but not by polypeptide-dependent protein kinase (protein kinase P). beta-casein B phosphorylated by protein kinase P is specifically dephosphorylated by protein kinase P but not by protein kinase A. Histone H1 phosphorylated by protein kinase C is dephosphorylated by the same enzyme in the absence of ADP. In all cases tested addition of ADP and F1-ATPase accelerates moderately the rate of dephosphorylation. Native H+-ATPase from yeast plasma membranes is isolated mainly in the phosphorylated form. It is dephosphorylated and rephosphorylated by protein kinase P but not by protein kinase A. Protein-tyrosine kinase of the epidermal growth factor receptor phosphorylates the random synthetic polypeptide poly(Glu80Tyr20). The phosphorylated polymer is specifically dephosphorylated in the absence of ADP by epidermal growth factor receptor preparations but not by insulin receptor preparations. The same polymer phosphorylated by insulin receptor is dephosphorylated by insulin receptor but not by epidermal growth factor receptor preparations. By using a cycle of dephosphorylation-rephosphorylation, it is possible to identify proteins that are phosphorylated by these protein kinases in vivo. Should this method be applicable to additional protein kinases, it should be possible to estimate the quantitative contribution of each protein kinase to a single phosphoprotein.

  13. Specific dephosphorylation of phosphoproteins by protein-serine and -tyrosine kinases.

    PubMed Central

    Kole, H K; Abdel-Ghany, M; Racker, E

    1988-01-01

    Five protein kinases are shown to serve as specific phosphatases in the absence of ADP. Although the rates of hydrolysis are very slow compared to the forward phosphorylation rates under optimal conditions, they are of the same order as the reverse reaction in the presence of ADP. Because cells contain approximately equal to 3 mM ATP, neither the reverse reaction nor the phosphatase is likely to play a physiological role. beta-casein B phosphorylated by the catalytic subunit of cAMP-dependent protein kinase (protein kinase A) is specifically dephosphorylated by protein kinase A but not by polypeptide-dependent protein kinase (protein kinase P). beta-casein B phosphorylated by protein kinase P is specifically dephosphorylated by protein kinase P but not by protein kinase A. Histone H1 phosphorylated by protein kinase C is dephosphorylated by the same enzyme in the absence of ADP. In all cases tested addition of ADP and F1-ATPase accelerates moderately the rate of dephosphorylation. Native H+-ATPase from yeast plasma membranes is isolated mainly in the phosphorylated form. It is dephosphorylated and rephosphorylated by protein kinase P but not by protein kinase A. Protein-tyrosine kinase of the epidermal growth factor receptor phosphorylates the random synthetic polypeptide poly(Glu80Tyr20). The phosphorylated polymer is specifically dephosphorylated in the absence of ADP by epidermal growth factor receptor preparations but not by insulin receptor preparations. The same polymer phosphorylated by insulin receptor is dephosphorylated by insulin receptor but not by epidermal growth factor receptor preparations. By using a cycle of dephosphorylation-rephosphorylation, it is possible to identify proteins that are phosphorylated by these protein kinases in vivo. Should this method be applicable to additional protein kinases, it should be possible to estimate the quantitative contribution of each protein kinase to a single phosphoprotein. Images PMID:2901092

  14. Characterization of the major phosphoprotein and its kinase on the surface of the rat adipocyte

    SciTech Connect

    Kang, E.S.; Chiang, T.M.

    1986-12-01

    Intact rat fat cell exposed to 12.5 ..mu..M (..gamma..-32P)ATP incorporate label into specific proteins within minutes. By solubilizing the reaction mixture with SDS which bypasses the subcellular fractionation steps, the labeled proteins can be identified in autoradiographs of SDS-PAGE gels. The most prominently labeled protein has an M/sub r/ of 42,000. Localization of this component to the cell surface can be made on the basis of inhibition of phosphorylation by addition of a protein derived from the rat brain with protein kinase inhibitory property, susceptibility of the phosphorylated protein to the tryptic digestion, inhibition of phosphorylation of this protein after brief exposure to melittin. To rule out the possibility that the cell surface protein might be a mitochondrial contaminant from broken cells, /sup 32/Pi-labeled and (..gamma..-/sup 32/P)ATP-labeled cells were chromatographed on a rabbit anti-pyruvate dehydrogenase antibody-Sepharose 4B column. A single labeled peak was detected upon elution of the bound fraction only in the /sup 32/pi-labeled sample, and not in the (..gamma..-/sup 32/P)ATP-labeled sample. Subcellular fractionation studies of intact cells labeled depending on whether a continuous sucrose gradient or a discontinuous sucrose gradient was used. Finally, comparison of the autoradiographs of two-dimensional (2D) gels show different isoelectric points for 42,000 M/sub r/ components in (..gamma..-/sup 32/P)ATP- and /sup 32/Pi-labeled cells. These and other experiments support the likelihood that phosphoproteins of 42,000 M/sub r/ are present at two sites in the intact rat fat cell, the cell surface and at an intracellular site, most likely the mitochondria.

  15. Vasodilator-Stimulated Phosphoprotein Activity Is Required for Coxiella burnetii Growth in Human Macrophages

    PubMed Central

    Colonne, Punsiri M.; Winchell, Caylin G.; Graham, Joseph G.; Onyilagha, Frances I.; MacDonald, Laura J.; Doeppler, Heike R.; Storz, Peter; Kurten, Richard C.; Beare, Paul A.; Voth, Daniel E.

    2016-01-01

    Coxiella burnetii is an intracellular bacterial pathogen that causes human Q fever, an acute flu-like illness that can progress to chronic endocarditis and liver and bone infections. Humans are typically infected by aerosol-mediated transmission, and C. burnetii initially targets alveolar macrophages wherein the pathogen replicates in a phagolysosome-like niche known as the parasitophorous vacuole (PV). C. burnetii manipulates host cAMP-dependent protein kinase (PKA) signaling to promote PV formation, cell survival, and bacterial replication. In this study, we identified the actin regulatory protein vasodilator-stimulated phosphoprotein (VASP) as a PKA substrate that is increasingly phosphorylated at S157 and S239 during C. burnetii infection. Avirulent and virulent C. burnetii triggered increased levels of phosphorylated VASP in macrophage-like THP-1 cells and primary human alveolar macrophages, and this event required the Cα subunit of PKA. VASP phosphorylation also required bacterial protein synthesis and secretion of effector proteins via a type IV secretion system, indicating the pathogen actively triggers prolonged VASP phosphorylation. Optimal PV formation and intracellular bacterial replication required VASP activity, as siRNA-mediated depletion of VASP reduced PV size and bacterial growth. Interestingly, ectopic expression of a phospho-mimetic VASP (S239E) mutant protein prevented optimal PV formation, whereas VASP (S157E) mutant expression had no effect. VASP (S239E) expression also prevented trafficking of bead-containing phagosomes to the PV, indicating proper VASP activity is critical for heterotypic fusion events that control PV expansion in macrophages. Finally, expression of dominant negative VASP (S157A) in C. burnetii-infected cells impaired PV formation, confirming importance of the protein for proper infection. This study provides the first evidence of VASP manipulation by an intravacuolar bacterial pathogen via activation of PKA in human

  16. Mineralization induction effects of osteopontin, bone sialoprotein, and dentin phosphoprotein on a biomimetic collagen substrate.

    PubMed

    Zurick, Kevin M; Qin, Chunlin; Bernards, Matthew T

    2013-06-01

    Native bone tissue is composed of a matrix of collagen, noncollagenous proteins, and calcium phosphate minerals, which are primarily hydroxyapatite. The SIBLING (small integrin-binding ligand, N-linked glycoprotein) family of proteins is the primary noncollagenous protein group found in mineralized tissues. In this work, the mineralization induction capabilities of three of the SIBLING members, bone sialoprotein (BSP), osteopontin (OPN), and the calcium-binding subdomain of dentin sialophosphoprotein, dentin phosphoprotein (DPP), are directly compared on a biomimetic collagen substrate. A self-assembled, loosely aligned collagen fibril substrate was prepared, and then (125) I-radiolabeled adsorption isotherms were developed for BSP, OPN, and DPP. The results showed that BSP exhibited the highest binding capacity for collagen at lower concentrations, followed by DPP and OPN. However, at the highest concentrations, all three proteins had similar adsorption levels. The adsorption isotherms were then used to identify conditions that resulted in identical amounts of adsorbed protein. These substrates were prepared and placed in simulated body fluid for 5, 10, and 24 h at 37°C. The resulting mineral morphology was assessed by atomic force microscopy, and the composition was determined using photochemical assays. Mineralization was seen in the presence of all the proteins. However, DPP was seen to be the only protein that formed individual mineral nodules similar to those seen in developing bone. This suggests that DPP plays a significant role in the biomineralization process and that the incorporation of DPP into tissue engineering constructs may facilitate the induction of biomimetic mineral formation.

  17. Phosphoprotein secretome of tumor cells as a source of candidates for breast cancer biomarkers in plasma.

    PubMed

    Zawadzka, Anna M; Schilling, Birgit; Cusack, Michael P; Sahu, Alexandria K; Drake, Penelope; Fisher, Susan J; Benz, Christopher C; Gibson, Bradford W

    2014-04-01

    Breast cancer is a heterogeneous disease whose molecular diversity is not well reflected in clinical and pathological markers used for prognosis and treatment selection. As tumor cells secrete proteins into the extracellular environment, some of these proteins reach circulation and could become suitable biomarkers for improving diagnosis or monitoring response to treatment. As many signaling pathways and interaction networks are altered in cancerous tissues by protein phosphorylation, changes in the secretory phosphoproteome of cancer tissues could reflect both disease progression and subtype. To test this hypothesis, we compared the phosphopeptide-enriched fractions obtained from proteins secreted into conditioned media (CM) derived from five luminal and five basal type breast cancer cell lines using label-free quantitative mass spectrometry. Altogether over 5000 phosphosites derived from 1756 phosphoproteins were identified, several of which have the potential to qualify as phosphopeptide plasma biomarker candidates for the more aggressive basal and also the luminal-type breast cancers. The analysis of phosphopeptides from breast cancer patient plasma and controls allowed us to construct a discovery list of phosphosites under rigorous collection conditions, and second to qualify discovery candidates generated from the CM studies. Indeed, a set of basal-specific phosphorylation CM site candidates derived from IBP3, CD44, OPN, FSTL3, LAMB1, and STC2, and luminal-specific candidates derived from CYTC and IBP5 were selected and, based on their presence in plasma, quantified across all cell line CM samples using Skyline MS1 intensity data. Together, this approach allowed us to assemble a set of novel cancer subtype specific phosphopeptide candidates for subsequent biomarker verification and clinical validation.

  18. Secreted phosphoprotein 24 kD inhibits nerve root inflammation induced by bone morphogenetic protein-2.

    PubMed

    Tian, Haijun; Li, Chen-Shuang; Scott, Trevor P; Montgomery, Scott R; Phan, Kevin; Lao, Lifeng; Zhang, Wei; Li, Yawei; Hayashi, Tetsuo; Takahashi, Shinji; Alobaidaan, Raed; Ruangchainikom, Monchai; Zhao, Ke-Wei; Brochmann, Elsa J; Murray, Samuel S; Wang, Jeffrey C; Daubs, Michael D

    2015-02-01

    Bone morphogenetic protein-2 (BMP-2) has been used to successfully promote spine fusion, but side-effects including nerve inflammation have been observed. To investigate the direct neurotoxic effects of BMP-2 and test the hypotheses that the use of BMP binding proteins, such as secreted phosphoprotein 24 kD (Spp24), can reduce or eliminate these effects. In vitro experiments and in vivo analysis in a rodent model. In vitro, dorsal root ganglion cells were cultured in the presence of BMP-2 with and without Spp24 and calcitonin gene-related peptide and Substance P, markers of neuroinflammation, were measured by immunohistochemistry. In vivo, rats underwent a left-sided laminotomy at L5 to expose the S1 nerve root and were randomized into four different groups according to the intervention at the laminotomy site: collagen sponge only (no BMP-2 or Spp24), BMP-2 in a collagen sponge only, BMP-2 in a collagen sponge+an empty collagen sponge to act as a barrier, and BMP-2 in a collagen sponge+Spp24 in a collagen sponge to act as a barrier. Functional evaluation was done using the Basso, Beattie, and Bresnahan scale and immunohistochemical analyses were performed using calcitonin gene-related peptide and Substance P staining. The neuroinflammatory effects of BMP-2 in vitro were ameliorated by the addition of Spp24. Similarly, in vivo, Spp24 reduced the expression of markers on neuroinflammation in animals treated with BMP-2 and also improved the function after BMP-2 administration. These results confirm that BMP binding proteins have great potential as adjuvant therapies to limit BMP-2 related side-effects in spine surgery. Copyright © 2015 Elsevier Inc. All rights reserved.

  19. Neuronal stathmins: a family of phosphoproteins cooperating for neuronal development, plasticity and regeneration.

    PubMed

    Chauvin, Stéphanie; Sobel, André

    2015-03-01

    Nervous system development, plasticity and regeneration require numerous, coordinated and finely tuned subcellular mechanisms. Phosphoproteins of the stathmin family, originally identified as intracellular signal relay proteins, are mostly or exclusively expressed in the nervous system with a high level of expression during brain development. Vertebrate stathmins 1-4 all possess a C-terminal "stathmin-like domain" that binds or releases tubulin in a phosphorylation dependent way, and hence participates in the control of microtubule dynamics, an essential process for neuronal differentiation. Contrary to stathmin 1, stathmins 2-4 possess an N-terminal extension whose reversible palmitoylation specifically targets them to the Golgi and intracellular membranes. Regulation of stathmins 2-4 palmitoylation is therefore an important regulatory mechanism that controls their shuttling to various neuronal compartments where they can then act locally. Expression of stathmins is upregulated during neuronal differentiation and plasticity, and altered in numerous neurodegenerative diseases. Experimental perturbation of stathmins expression in Drosophila or in neurons in culture revealed their importance in neuronal growth and differentiation, each stathmin fulfilling at least partially distinct and likely complementary roles. On the other hand, knock-out of stathmins in mice, with the exception of stathmin 2, resulted in mostly mild or no detected phenotype, revealing likely compensations among stathmins. Altogether, through their combinatorial expression and regulation by phosphorylation and by palmitoylation, and through their interactions with tubulin and other neuronal protein targets, the various stathmins appear as essential regulators of neuronal differentiation at the various stages during development and plasticity of the nervous system.

  20. Distribution of dwell times of a ribosome: effects of infidelity, kinetic proofreading and ribosome crowding

    NASA Astrophysics Data System (ADS)

    Sharma, Ajeet K.; Chowdhury, Debashish

    2011-04-01

    Ribosome is a molecular machine that polymerizes a protein where the sequence of the amino acid residues, the monomers of the protein, is dictated by the sequence of codons (triplets of nucleotides) on a messenger RNA (mRNA) that serves as the template. The ribosome is a molecular motor that utilizes the template mRNA strand also as the track. Thus, in each step the ribosome moves forward by one codon and, simultaneously, elongates the protein by one amino acid. We present a theoretical model that captures most of the main steps in the mechanochemical cycle of a ribosome. The stochastic movement of the ribosome consists of an alternating sequence of pause and translocation; the sum of the durations of a pause and the following translocation is the time of dwell of the ribosome at the corresponding codon. We derive the analytical expression for the distribution of the dwell times of a ribosome in our model. Wherever experimental data are available, our theoretical predictions are consistent with those results. We suggest appropriate experiments to test the new predictions of our model, particularly the effects of the quality control mechanism of the ribosome and that of their crowding on the mRNA track.

  1. Distribution of dwell times of a ribosome: effects of infidelity, kinetic proofreading and ribosome crowding.

    PubMed

    Sharma, Ajeet K; Chowdhury, Debashish

    2011-04-01

    Ribosome is a molecular machine that polymerizes a protein where the sequence of the amino acid residues, the monomers of the protein, is dictated by the sequence of codons (triplets of nucleotides) on a messenger RNA (mRNA) that serves as the template. The ribosome is a molecular motor that utilizes the template mRNA strand also as the track. Thus, in each step the ribosome moves forward by one codon and, simultaneously, elongates the protein by one amino acid. We present a theoretical model that captures most of the main steps in the mechanochemical cycle of a ribosome. The stochastic movement of the ribosome consists of an alternating sequence of pause and translocation; the sum of the durations of a pause and the following translocation is the time of dwell of the ribosome at the corresponding codon. We derive the analytical expression for the distribution of the dwell times of a ribosome in our model. Wherever experimental data are available, our theoretical predictions are consistent with those results. We suggest appropriate experiments to test the new predictions of our model, particularly the effects of the quality control mechanism of the ribosome and that of their crowding on the mRNA track.

  2. Stepwise splitting of ribosomal proteins from yeast ribosomes by LiCl.

    PubMed

    Piir, Kerli; Tamm, Tiina; Kisly, Ivan; Tammsalu, Triin; Remme, Jaanus

    2014-01-01

    Structural studies have revealed that the core of the ribosome structure is conserved among ribosomes of all kingdoms. Kingdom-specific ribosomal proteins (r-proteins) are located in peripheral parts of the ribosome. In this work, the interactions between rRNA and r-proteins of eukaryote Saccharomyces cerevisiae ribosome were investigated applying LiCl induced splitting and quantitative mass spectrometry. R-proteins were divided into four groups according to their binding properties to the rRNA. Most yeast r-proteins are removed from rRNA by 0.5-1 M LiCl. Eukaryote-specific r-proteins are among the first to dissociate. The majority of the strong binders are known to be required for the early ribosome assembly events. As compared to the bacterial ribosome, yeast r-proteins are dissociated from rRNA at lower ionic strength. Our results demonstrate that the nature of protein-RNA interactions in the ribosome is not conserved between different kingdoms.

  3. Antibiotic-induced ribosomal assembly defects result from changes in the synthesis of ribosomal proteins.

    PubMed

    Siibak, Triinu; Peil, Lauri; Dönhöfer, Alexandra; Tats, Age; Remm, Maido; Wilson, Daniel N; Tenson, Tanel; Remme, Jaanus

    2011-04-01

    Inhibitors of protein synthesis cause defects in the assembly of ribosomal subunits. In response to treatment with the antibiotics erythromycin or chloramphenicol, precursors of both large and small ribosomal subunits accumulate. We have used a pulse-labelling approach to demonstrate that the accumulating subribosomal particles maturate into functional 70S ribosomes. The protein content of the precursor particles is heterogeneous and does not correspond with known assembly intermediates. Mass spectrometry indicates that production of ribosomal proteins in the presence of the antibiotics correlates with the amounts of the individual ribosomal proteins within the precursor particles. Thus, treatment of cells with chloramphenicol or erythromycin leads to an unbalanced synthesis of ribosomal proteins, providing the explanation for formation of assembly-defective particles. The operons for ribosomal proteins show a characteristic pattern of antibiotic inhibition where synthesis of the first proteins is inhibited weakly but gradually increases for the subsequent proteins in the operon. This phenomenon most likely reflects translational coupling and allows us to identify other putative coupled non-ribosomal operons in the Escherichia coli chromosome. © 2011 Blackwell Publishing Ltd.

  4. Saccharomyces cerevisiae ribosomal protein L26 is not essential for ribosome assembly and function.

    PubMed

    Babiano, Reyes; Gamalinda, Michael; Woolford, John L; de la Cruz, Jesús

    2012-08-01

    Ribosomal proteins play important roles in ribosome biogenesis and function. Here, we study the evolutionarily conserved L26 in Saccharomyces cerevisiae, which assembles into pre-60S ribosomal particles in the nucle(ol)us. Yeast L26 is one of the many ribosomal proteins encoded by two functional genes. We have disrupted both genes; surprisingly, the growth of the resulting rpl26 null mutant is apparently identical to that of the isogenic wild-type strain. The absence of L26 minimally alters 60S ribosomal subunit biogenesis. Polysome analysis revealed the appearance of half-mers. Analysis of pre-rRNA processing indicated that L26 is mainly required to optimize 27S pre-rRNA maturation, without which the release of pre-60S particles from the nucle(ol)us is partially impaired. Ribosomes lacking L26 exhibit differential reactivity to dimethylsulfate in domain I of 25S/5.8S rRNAs but apparently are able to support translation in vivo with wild-type accuracy. The bacterial homologue of yeast L26, L24, is a primary rRNA binding protein required for 50S ribosomal subunit assembly in vitro and in vivo. Our results underscore potential differences between prokaryotic and eukaryotic ribosome assembly. We discuss the reasons why yeast L26 plays such an apparently nonessential role in the cell.

  5. Comparative analysis of salt-responsive phosphoproteins in maize leaves using Ti(4+)--IMAC enrichment and ESI-Q-TOF MS.

    PubMed

    Hu, Yufeng; Guo, Shuangxi; Li, Xuexian; Ren, Xueqin

    2013-02-01

    Salinity is one of the most common abiotic stresses encountered by plants. Reversible protein phosphorylation is involved in plant defense processes against salinity stress. Here, we performed global phosphopeptide mapping through enrichment by our synthesized PVA-phosphate-Ti(4+) IMAC coupled with subsequent identification by ESI-Q-TOF MS. A total of 104 peptide sequences containing 139 phosphorylation sites were determined from 70 phosphoproteins of the control leaves. In contrast, 124 phosphopeptides containing 143 phosphorylated sites from 92 phosphoproteins were identified in salt-stressed maize leaves. Compared with the control, 47 proteins were phosphorylated, 25 were dephosphorylated, and 45 overlapped. Among the 72 differential phosphoproteins, 35 were known salt stress response proteins and the rest had not been reported in the literature. To dissect the differential phosphorylation, gene ontology annotations were retrieved for the differential phosphoproteins. The results revealed that cell signaling pathway members such as calmodulin and 14-3-3 proteins were regulated in response to 24-h salt stress. Multiple putative salt-responsive phosphoproteins seem to be involved in the regulation of photosynthesis-related processes. These results may help to understand the salt-inducible phosphorylation processes of maize leaves.

  6. The structure of ribosome-lankacidin complex reveals ribosomal sites for synergistic antibiotics

    SciTech Connect

    Auerbach, Tamar; Mermershtain, Inbal; Davidovich, Chen; Bashan, Anat; Belousoff, Matthew; Wekselman, Itai; Zimmerman, Ella; Xiong, Liqun; Klepacki, Dorota; Arakawa, Kenji; Kinashi, Haruyasu; Mankin, Alexander S.; Yonath, Ada

    2010-04-26

    Crystallographic analysis revealed that the 17-member polyketide antibiotic lankacidin produced by Streptomyces rochei binds at the peptidyl transferase center of the eubacterial large ribosomal subunit. Biochemical and functional studies verified this finding and showed interference with peptide bond formation. Chemical probing indicated that the macrolide lankamycin, a second antibiotic produced by the same species, binds at a neighboring site, at the ribosome exit tunnel. These two antibiotics can bind to the ribosome simultaneously and display synergy in inhibiting bacterial growth. The binding site of lankacidin and lankamycin partially overlap with the binding site of another pair of synergistic antibiotics, the streptogramins. Thus, at least two pairs of structurally dissimilar compounds have been selected in the course of evolution to act synergistically by targeting neighboring sites in the ribosome. These results underscore the importance of the corresponding ribosomal sites for development of clinically relevant synergistic antibiotics and demonstrate the utility of structural analysis for providing new directions for drug discovery.

  7. Dealing with stable structures at ribosome binding sites: bacterial translation and ribosome standby.

    PubMed

    Unoson, Cecilia; Wagner, E Gerhart H

    2007-11-01

    Bacterial ribosomes have great difficulties to initiate translation on stable structures within mRNAs. Translational coupling and induced structure changes are strategies to open up inhibitory RNA structures encompassing ribosome binding sites (RBS). There are, however, mRNAs in which stable structures are not unfolded, but that are nevertheless efficiently initiated at high rates. de Smit and van Duin(1) proposed a "ribosome standby" model to theoretically solve this paradox: the 30S ribosome binds nonspecifically to an accessible site on the mRNA (standby site), waiting for a transient opening of a stable RBS hairpin. Upon unfolding, the 30S subunit relocates to form a productive initiation complex. Recent reports have provided experimental support for this model. This review will describe and compare two different flavors of standby sites, their properties, and their likely implications. We also discuss the possibility that ribosome standby may be a more general strategy to obtain high translation rates.

  8. The structure of ribosome-lankacidin complex reveals ribosomal sites for synergistic antibiotics

    PubMed Central

    Auerbach, Tamar; Mermershtain, Inbal; Davidovich, Chen; Bashan, Anat; Belousoff, Matthew; Wekselman, Itai; Zimmerman, Ella; Xiong, Liqun; Klepacki, Dorota; Arakawa, Kenji; Kinashi, Haruyasu; Mankin, Alexander S.; Yonath, Ada

    2010-01-01

    Crystallographic analysis revealed that the 17-member polyketide antibiotic lankacidin produced by Streptomyces rochei binds at the peptidyl transferase center of the eubacterial large ribosomal subunit. Biochemical and functional studies verified this finding and showed interference with peptide bond formation. Chemical probing indicated that the macrolide lankamycin, a second antibiotic produced by the same species, binds at a neighboring site, at the ribosome exit tunnel. These two antibiotics can bind to the ribosome simultaneously and display synergy in inhibiting bacterial growth. The binding site of lankacidin and lankamycin partially overlap with the binding site of another pair of synergistic antibiotics, the streptogramins. Thus, at least two pairs of structurally dissimilar compounds have been selected in the course of evolution to act synergistically by targeting neighboring sites in the ribosome. These results underscore the importance of the corresponding ribosomal sites for development of clinically relevant synergistic antibiotics and demonstrate the utility of structural analysis for providing new directions for drug discovery. PMID:20080686

  9. HflX is a ribosome-splitting factor rescuing stalled ribosomes under stress conditions.

    PubMed

    Zhang, Yanqing; Mandava, Chandra Sekhar; Cao, Wei; Li, Xiaojing; Zhang, Dejiu; Li, Ningning; Zhang, Yixiao; Zhang, Xiaoxiao; Qin, Yan; Mi, Kaixia; Lei, Jianlin; Sanyal, Suparna; Gao, Ning

    2015-11-01

    Adverse cellular conditions often lead to nonproductive translational stalling and arrest of ribosomes on mRNAs. Here, we used fast kinetics and cryo-EM to characterize Escherichia coli HflX, a GTPase with unknown function. Our data reveal that HflX is a heat shock-induced ribosome-splitting factor capable of dissociating vacant as well as mRNA-associated ribosomes with deacylated tRNA in the peptidyl site. Structural data demonstrate that the N-terminal effector domain of HflX binds to the peptidyl transferase center in a strikingly similar manner as that of the class I release factors and induces dramatic conformational changes in central intersubunit bridges, thus promoting subunit dissociation. Accordingly, loss of HflX results in an increase in stalled ribosomes upon heat shock. These results suggest a primary role of HflX in rescuing translationally arrested ribosomes under stress conditions.

  10. Multimer staining of cytomegalovirus phosphoprotein 65-specific T cells for diagnosis and therapeutic purposes: a comparative study.

    PubMed

    Yao, Junxia; Bechter, Clemens; Wiesneth, Markus; Härter, Georg; Götz, Marlies; Germeroth, Lothar; Guillaume, Philippe; Hasan, Ferishte; von Harsdorf, Stephanie; Mertens, Thomas; Michel, Detlef; Döhner, Hartmut; Bunjes, Donald; Schmitt, Michael; Schmitt, Anita

    2008-05-15

    Cytomegalovirus (CMV) disease represents a serious complication after allogeneic peripheral blood stem cell (PBSC) transplantation. If possible, stem cell donors for transplantation are selected on the basis of their CMV serostatus. However, the cytomegalovirus-specific immune status can be further characterized by measuring CMV phosphoprotein 65-specific CD8(+) T cell frequencies using tetramers, pentamers, and streptamers. We therefore investigated the specificity and sensitivity of all 3 methods and compared the results to patient serostatus. Twenty-three samples from CMV-seropositive healthy volunteers and 15 samples from CMV-seropositive patients before and after allogeneic PBSC transplantation were stained with tetramers, pentamers, or streptamers and analyzed by flow cytometry. Similar frequencies of CD8(+) and multimer(+) T cells could be measured by all 3 multimer technologies. The lowest background signals (< or =0.02%) were obtained using tetramer technology. Frequencies of 0.19%-2.48% of CMV phosphoprotein 65 495-503-specific CD8(+) T cells were detected in healthy volunteers. Antigen-specific T cells were detected in only 11 (48%) of 23 seropositive healthy volunteers. CMV antigenemia before day 100 after allogeneic PBSC transplantation occurred in 2 of 3 patients without any specific T cells. These findings demonstrate the power of multimer staining and a certain limitation of serologic testing to define appropriate donors for transplantation. Therefore, whenever possible, CMV-seropositive donors of transplants to seropositive recipients should be screened for their CD8(+) T cell frequency. All 3 multimer technologies can be used, yielding similar results. The streptamer technology additionally offers the advantage of selecting CMV phosphoprotein 65-specific CD8(+) T cells at the good manufacturing practice level for adoptive T cell transfer.

  11. Purification of a 53kD pI 4. 8 cytosolic phosphoprotein from HL60

    SciTech Connect

    Biser, P.S.; Spearman, T.N.; Bruzzone, M.; Durham, J.P.

    1987-05-01

    In order to study the potential role of a 53kD pI 4.8 phosphoprotein in the differentiation of HL60 using monoclonal antibodies, a partial purification has been carried out. Cytosol from cells differentiated with 1 M retinoic acid was applied to a DEAE-cellulose column and eluted with a linear NaCl gradient. Fractions were screened by in vitro phosphorylation of aliquots using /sup 32/P ATP and highly purified protein kinase C, SDS-PAGE, and autoradiography. Fraction which showed autoradiographic bands of the correct molecular weight were further analyzed using 2-D electrophoresis involving isolectric focusing over a pH range of 4-6 followed by SDS-PAGE on a 10% slab gel. Autoradiograms of these gels showed the 53 kD pI 4.8 phosphoprotein to elute with a peak at 0.24 NaCl. This 53 kD pI 4.8 protein was identified as the 53kD pI 4.8 phosphoprotein whose synthesis and phosphorylation is induced by retinoic acid by DEAE chromatography of cytosol from cells labelled in vivo with /sup 32/PO/sub 4//sup -2/ followed by 2-D electrophoresis. Fractions containing the 53 kD pI 4.8 protein were concentrated and applied to a chromatofocusing column which was eluted with a gradient from pH 6 to 4. Analysis of fractions via in vitro phosphorylation and SDS PAGE showed the 53 kD pI 4.8 protein eluting with a peak at pH 4.8 as a silver-stained band well separated from contaminating proteins. Experiments are currently in progress to produce monoclonal antibodies to the 53 kD pI 4.8 protein using the partially purified antigen.

  12. High-resolution structure of the Escherichia coli ribosome

    DOE PAGES

    Noeske, Jonas; Wasserman, Michael R.; Terry, Daniel S.; ...

    2015-03-16

    Protein synthesis by the ribosome is highly dependent on the ionic conditions in the cellular environment, but the roles of ribosome solvation remain poorly understood. Moreover, the function of modifications to ribosomal RNA and ribosomal proteins are unclear. Here we present the structure of the Escherichia coli 70S ribosome to 2.4 Å resolution. The structure reveals details of the ribosomal subunit interface that are conserved in all domains of life, and suggest how solvation contributes to ribosome integrity and function. The structure also suggests how the conformation of ribosomal protein uS12 likely impacts its contribution to messenger RNA decoding. Inmore » conclusion, this structure helps to explain the phylogenetic conservation of key elements of the ribosome, including posttranscriptional and posttranslational modifications and should serve as a basis for future antibiotic development.« less

  13. [Study of the surface of Escherichia coli ribosomes and ribosomal particles by the tritium bombardment method].

    PubMed

    Iusupov, M M; Spirin, A S

    1986-11-01

    A new technique of atomic tritium bombardment has been used to study the surface topography of Escherichia coli ribosomes and ribosomal subunits. The technique provides for the labeling of proteins exposed on the surface of ribosomal particles, the extent of protein labeling being proportional to the degree of exposure. The following proteins were considerably tritiated in the 70S ribosomes: S1, S4, S7, S9 and/or S11, S12 and/or L20, S13, S18, S20, S21, L1, L5, L6, L7/L12, L10, L11, L16, L17, L24, L26 and L27. A conclusion is drawn that these proteins are exposed on the ribosome surface to an essentially greater extent than the others. Dissociation of 70S ribosomes into the ribosomal subunits by decreasing Mg2+ concentration does not lead to the exposure of additional ribosomal proteins. This implies that there are no proteins on the contacting surfaces of the subunits. However, if a mixture of subunits has been subjected to centrifugation in a low Mg2+ concentration at high concentrations of a monovalent cation, proteins S3, S5, S7, S14, S18 and L16 are more exposed on the surface of the isolated 30S and 50S subunits than in the subunit mixture or in the 70S ribosomes. The exposure of additional proteins is explained by distortion of the native quaternary structure of ribosomal subunits as a result of the separation procedure. Reassociation of isolated subunits at high Mg2+ concentration results in shielding of proteins S3, S5, S7 and S18 and can be explained by reconstitution of the intact 30S subunit structure.

  14. Determination of alkali-labile phosphoprotein phosphorus from fish plasma using the Tb(3+)-tiron complex as a fluorescence probe.

    PubMed

    Lv, Xue-Fei; Zhao, Yi-Bing; Zhou, Qun-Fang; Jiang, Gui-Bin; Song, Mao-Yong

    2007-01-01

    A sensitive method based on the fluorescence quenching effect of the Tb(3+)-Tiron complex is proposed for the determination of alkali-labile phosphoprotein phosphorus (ALP) released from fish plasma. The detection limit was 5.4 ng/ml (S/N = 2), and the relative standard deviation of the quenching effect (6 replicates) was 4.6%. The results obtained by the proposed method were in good agreement with those obtained by the colorimetric assay. The advantages of the present method are its relatively simple detection procedure, the lack of toxic organic solvents, and high sensitivity.

  15. One-Step Preservation of Phosphoproteins and Tissue Morphology at Room Temperature for Diagnostic and Research Specimens

    PubMed Central

    Mueller, Claudius; Edmiston, Kirsten H.; Carpenter, Calvin; Gaffney, Eoin; Ryan, Ciara; Ward, Ronan; White, Susan; Memeo, Lorenzo; Colarossi, Cristina; Petricoin, Emanuel F.; Liotta, Lance A.; Espina, Virginia

    2011-01-01

    Background There is an urgent need to measure phosphorylated cell signaling proteins in cancer tissue for the individualization of molecular targeted kinase inhibitor therapy. However, phosphoproteins fluctuate rapidly following tissue procurement. Snap-freezing preserves phosphoproteins, but is unavailable in most clinics and compromises diagnostic morphology. Formalin fixation preserves tissue histomorphology, but penetrates tissue slowly, and is unsuitable for stabilizing phosphoproteins. We originated and evaluated a novel one-step biomarker and histology preservative (BHP) chemistry that stabilizes signaling protein phosphorylation and retains formalin-like tissue histomorphology with equivalent immunohistochemistry in a single paraffin block. Results Total protein yield extracted from BHP-fixed, routine paraffin-embedded mouse liver was 100% compared to snap-frozen tissue. The abundance of 14 phosphorylated proteins was found to be stable over extended fixation times in BHP fixed paraffin embedded human colon mucosa. Compared to matched snap-frozen tissue, 8 phosphoproteins were equally preserved in mouse liver, while AMPKβ1 Ser108 was slightly elevated after BHP fixation. More than 25 tissues from mouse, cat and human specimens were evaluated for preservation of histomorphology. Selected tissues were evaluated in a multi-site, independent pathology review. Tissue fixed with BHP showed equivalent preservation of cytoplasmic and membrane cytomorphology, with significantly better nuclear chromatin preservation by BHP compared to formalin. Immunohistochemical staining of 13 non-phosphorylated proteins, including estrogen receptor alpha, progesterone receptor, Ki-67 and Her2, was equal to or stronger in BHP compared to formalin. BHP demonstrated significantly improved immunohistochemical detection of phosphorylated proteins ERK Thr202/Tyr204, GSK3-α/β Ser21/Ser9, p38-MAPK Thr180/Tyr182, eIF4G Ser1108 and Acetyl-CoA Carboxylase Ser79. Conclusion In a single

  16. A recent intermezzo at the Ribosome Club

    PubMed Central

    Pavlov, Michael Y.; Liljas, Anders

    2017-01-01

    Two sets of ribosome structures have recently led to two different interpretations of what limits the accuracy of codon translation by transfer RNAs. In this review, inspired by this intermezzo at the Ribosome Club, we briefly discuss accuracy amplification by energy driven proofreading and its implementation in genetic code translation. We further discuss general ways by which the monitoring bases of 16S rRNA may enhance the ultimate accuracy (d-values) and how the codon translation accuracy is reduced by the actions of Mg2+ ions and the presence of error inducing aminoglycoside antibiotics. We demonstrate that complete freezing-in of cognate-like tautomeric states of ribosome-bound nucleotide bases in transfer RNA or messenger RNA is not compatible with recent experiments on initial codon selection by transfer RNA in ternary complex with elongation factor Tu and GTP. From these considerations, we suggest that the sets of 30S subunit structures from the Ramakrishnan group and 70S structures from the Yusupov/Yusupova group may, after all, reflect two sides of the same coin and how the structurally based intermezzo at the Ribosome Club may be resolved simply by taking the dynamic aspects of ribosome function into account. This article is part of the themed issue ‘Perspectives on the ribosome’. PMID:28138071

  17. Ribosomes in the squid giant axon.

    PubMed

    Bleher, R; Martin, R

    2001-01-01

    Ribosome clusters, referred to as endoaxoplasmic plaques, were documented and quantitatively analyzed in the squid giant axon at the light and electron microscopic levels. The methods included nonspecific high affinity fluorescence staining of RNA by YOYO-1, specific immunofluorescence labeling of ribosomal RNA, electron energy loss spectroscopic mapping of ribosomal phosphorus, and conventional transmission electron microscopy. The endoaxoplasmic plaques were sharply defined, oval in shape, and less than 2 microm in diameter. While they were very numerous in the postsynaptic axonal area of the giant synapse, the frequency of occurrence was much lower in the peripheral giant axon, with a density of about 1 plaque/1000 microm3. Their distribution was random within axoplasm, with no preferential localization near the membrane. The several thousand ribosomes in a plaque usually were not membrane bound, but vesicular structures were observed in or near plaques; plaques were often surrounded by mitochondria. We conclude that ribosomes, a requisite machinery for protein synthesis, are present in the squid giant axon in discrete configurations.

  18. Mitochondrial ribosomal proteins (MRPs) of yeast.

    PubMed Central

    Graack, H R; Wittmann-Liebold, B

    1998-01-01

    Mitochondrial ribosomal proteins (MRPs) are the counterparts in that organelle of the cytoplasmic ribosomal proteins in the host. Although the MRPs fulfil similar functions in protein biosynthesis, they are distinct in number, features and primary structures from the latter. Most progress in the eludication of the properties of individual MRPs, and in the characterization of the corresponding genes, has been made in baker's yeast (Saccharomyces cerevisiae). To date, 50 different MRPs have been determined, although biochemical data and mutational analysis propose a total number which is substantially higher. Surprisingly, only a minority of the MRPs that have been characterized show significant sequence similarities to known ribosomal proteins from other sources, thus limiting the deduction of their functions by simple comparison of amino acid sequences. Further, individual MRPs have been characterized functionally by mutational studies, and the regulation of expression of MRP genes has been described. The interaction of the mitochondrial ribosomes with transcription factors specific for individual mitochondrial mRNAs, and the communication between mitochondria and the nucleus for the co-ordinated expression of ribosomal constituents, are other aspects of current MRP research. Although the mitochondrial translational system is still far from being described completely, the yeast MRP system serves as a model for other organisms, including that of humans. PMID:9445368

  19. Functional Importance of Mobile Ribosomal Proteins.

    PubMed

    Chang, Kai-Chun; Wen, Jin-Der; Yang, Lee-Wei

    2015-01-01

    Although the dynamic motions and peptidyl transferase activity seem to be embedded in the rRNAs, the ribosome contains more than 50 ribosomal proteins (r-proteins), whose functions remain largely elusive. Also, the precise forms of some of these r-proteins, as being part of the ribosome, are not structurally solved due to their high flexibility, which hinders the efforts in their functional elucidation. Owing to recent advances in cryo-electron microscopy, single-molecule techniques, and theoretical modeling, much has been learned about the dynamics of these r-proteins. Surprisingly, allosteric regulations have been found in between spatially separated components as distant as those in the opposite sides of the ribosome. Here, we focus on the functional roles and intricate regulations of the mobile L1 and L12 stalks and L9 and S1 proteins. Conformational flexibility also enables versatile functions for r-proteins beyond translation. The arrangement of r-proteins may be under evolutionary pressure that fine-tunes mass distributions for optimal structural dynamics and catalytic activity of the ribosome.

  20. Gateway role for rRNA precursors in ribosome assembly.

    PubMed

    Gutgsell, Nancy S; Jain, Chaitanya

    2012-12-01

    In Escherichia coli, rRNAs are initially transcribed with precursor sequences, which are subsequently removed through processing reactions. To investigate the role of precursor sequences, we analyzed ribosome assembly in strains containing mutations in the processing RNases. We observed that defects in 23S rRNA processing resulted in an accumulation of ribosomal subunits and caused a significant delay in ribosome assembly. These observations suggest that precursor residues in 23S rRNA control ribosome assembly and could be serving a regulatory role to couple ribosome assembly to rRNA processing. The possible mechanisms through which rRNA processing and ribosome assembly could be linked are discussed.

  1. Large-scale isolation of mitochondrial ribosomes from mammalian tissues.

    PubMed

    Spremulli, Linda L

    2007-01-01

    The preparation of mammalian mitochondrial ribosomes in sufficient quantities for biochemical studies is best done beginning with whole tissue rather than from cells in culture. This issue arises because of the low abundance of these ribosomes in cells, making their isolation a challenge. Crude mitochondrial preparations are made by differential centrifugation and are treated with digitonin to remove the outer membrane. This treatment also effectively removes most contamination by cytoplasmic ribosomes. Purification of mammalian mitochondrial ribosomes requires treatment with detergents to release the ribosomes from their association with the membrane. Sucrose density gradient centrifugation is used to separate the ribosomes from other large oligomeric complexes from this organelle.

  2. Structural basis for selectivity and toxicity of ribosomal antibiotics

    PubMed Central

    Böttger, Erik C.; Springer, Burkhard; Prammananan, Therdsak; Kidan, Yishak; Sander, Peter

    2001-01-01

    Ribosomal antibiotics must discriminate between bacterial and eukaryotic ribosomes to various extents. Despite major differences in bacterial and eukaryotic ribosome structure, a single nucleotide or amino acid determines the selectivity of drugs affecting protein synthesis. Analysis of resistance mutations in bacteria allows the prediction of whether cytoplasmic or mitochondrial ribosomes in eukaryotic cells will be sensitive to the drug. This has important implications for drug specificity and toxicity. Together with recent data on the structure of ribosomal subunits these data provide the basis for development of new ribosomal antibiotics by rationale drug design. PMID:11306553

  3. One core, two shells: bacterial and eukaryotic ribosomes.

    PubMed

    Melnikov, Sergey; Ben-Shem, Adam; Garreau de Loubresse, Nicolas; Jenner, Lasse; Yusupova, Gulnara; Yusupov, Marat

    2012-06-05

    Ribosomes are universally conserved enzymes that carry out protein biosynthesis. Bacterial and eukaryotic ribosomes, which share an evolutionarily conserved core, are thought to have evolved from a common ancestor by addition of proteins and RNA that bestow different functionalities to ribosomes from different domains of life. Recently, structures of the eukaryotic ribosome, determined by X-ray crystallography, have allowed us to compare these structures to previously determined structures of bacterial ribosomes. Here we describe selected bacteria- or eukaryote-specific structural features of the ribosome and discuss the functional implications of some of them.

  4. Modified ribosome profiling reveals high abundance of ribosome protected mRNA fragments derived from 3′ untranslated regions

    PubMed Central

    Miettinen, Teemu P.; Björklund, Mikael

    2015-01-01

    Ribosome profiling identifies ribosome positions on translated mRNAs. A prominent feature of published datasets is the near complete absence of ribosomes in 3′ untranslated regions (3′UTR) although substantial ribosome density can be observed on non-coding RNAs. Here we perform ribosome profiling in cultured Drosophila and human cells and show that different features of translation are revealed depending on the nuclease and the digestion conditions used. Most importantly, we observe high abundance of ribosome protected fragments in 3′UTRs of thousands of genes without manipulation of translation termination. Affinity purification of ribosomes indicates that the 3′UTR reads originate from ribosome protected fragments. Association of ribosomes with the 3′UTR may be due to ribosome migration through the stop codon or 3′UTR mRNA binding to ribosomes on the coding sequence. This association depends primarily on the relative length of the 3′UTR and may be related to translational regulation or ribosome recycling, for which the efficiency is known to inversely correlate with 3′UTR length. Together our results indicate that ribosome profiling is highly dependent on digestion conditions and that ribosomes commonly associate with the 3′UTR, which may have a role in translational regulation. PMID:25550424

  5. Phosphoprotein F1: purification and characterization of a brain kinase C substrate related to plasticity.

    PubMed

    Chan, S Y; Murakami, K; Routtenberg, A

    1986-12-01

    To study the role of protein kinase C (PKC) and its substrates in neuronal function, we have investigated the in vitro endogenous phosphorylation of the neuronal phosphoprotein F1 after induction of synaptic plasticity by long-term potentiation (LTP). The protein F1 phosphorylation was found to increase 5 min (Routtenberg et al., 1985), 1 hr (Lovinger et al., 1986) and 3 d (Lovinger et al., 1985) after LTP. The characteristics of this protein bear close similarities to a number of proteins characterized in various neuronal systems, such as B50 (brain specific, synaptosome-enriched protein), pp46 (a growth cone protein), and GAP 43 (nerve growth and regeneration-associated protein). A positive identification of the purified protein F1 with these proteins would link protein F1 to the developmental growth of axons, nerve regeneration, and polyphosphoinositide metabolism, as well as adult plasticity. We have therefore purified and partially characterized native protein F1 so that a meaningful comparison among the properties of these proteins can be made. Using synaptosomal plasma membrane (P2') as starting material, subsequent purification involved pH extraction, 40-80% ammonium sulfate precipitation, hydroxylapatite, and phenyl-Sepharose column chromatography. This procedure achieved greater than 800-fold purification and about 45% yield relative to P2'. Purified protein F1 (Mr = 47,000, pI = 4.5) was found to be a hydrophilic molecule and was phosphorylated by 1000-fold purified PKC in the presence of phosphatidylserine (PS) and Ca2+. The Ka of PS activation is about 15 micrograms/ml (approximately 20 microM), and that of Ca2+ is about 25 microM. Diolein and DiC:8 (a synthetic diacylglycerol) lowered the requirement of Ca2+ for maximal stimulation from 100 to 5 microM. Ca2+-calmodulin kinases type I and II did not phosphorylate protein F1. The phosphoamino acid analysis showed that 97% of the total incorporated 32P-phosphate was on the serine residue. Phosphopeptide

  6. Vasodilator-stimulated phosphoprotein-phosphorylation assay in patients on clopidogrel: does standardisation matter?

    PubMed

    Freynhofer, Matthias K; Bruno, Veronika; Willheim, Martin; Hübl, Wolfgang; Wojta, Johann; Huber, Kurt

    2012-03-01

    The vasodilator-stimulated phosphoprotein-phosphorylation (VASP-P) flow-cytometric assay is mainly used in clinical trials to measure thienopyridine effects. However, there are remarkable differences in the reported optimal cut-offs, ranging from 48-61% platelet reactivity index (PRI). We therefore investigated whether a lack of standardisation might explain the differences in the cut-offs. We measured VASP-P in 62 individuals. PRI was calculated using the mean, geometric mean and median fluorescence intensities (FI). Stability of the blood-samples (time-to-assay, 0-2 days) and stability of the processed samples (0-120 minutes) within the recommended time-span were tested. Time-to-assay significantly influenced the PRI (p<0.001): the PRI from mean FI after two days was lower compared to values on day 1 (52 ± 22.9 vs. 57.7 ± 24.1, p<0.001). The PRI from the geometric mean FI after two days was lower compared to day 0 as well as day 1 (51.3 ± 23 vs. 58.2 ± 24.2 and vs. 59.1 ± 23.7, both p<0.001). The PRI from median FI was stable over time (day 0: 59.1 ± 25%, day 1: 59.7 ± 24.1% and day 2: 56.4 ± 23.9%, all p=ns). Furthermore, the lag time of the processed samples significantly altered the PRI (all p<0.001) with a maximum difference for PRI based on geometric mean FI after 90 minutes compared to baseline (Δ=3.92%PRI, p<0.001). The differences in the reported cut-offs might be explained by a lack of standardisation. More precise standardisation is inevitable, as the PRI significantly depends on the method of calculation, the time-to-assay as well as on the lag time after processing. Tolerably stable results were obtained for the PRI from the median FI.

  7. Structural snapshots of actively translating human ribosomes.

    PubMed

    Behrmann, Elmar; Loerke, Justus; Budkevich, Tatyana V; Yamamoto, Kaori; Schmidt, Andrea; Penczek, Pawel A; Vos, Matthijn R; Bürger, Jörg; Mielke, Thorsten; Scheerer, Patrick; Spahn, Christian M T

    2015-05-07

    Macromolecular machines, such as the ribosome, undergo large-scale conformational changes during their functional cycles. Although their mode of action is often compared to that of mechanical machines, a crucial difference is that, at the molecular dimension, thermodynamic effects dominate functional cycles, with proteins fluctuating stochastically between functional states defined by energetic minima on an energy landscape. Here, we have used cryo-electron microscopy to image ex-vivo-derived human polysomes as a source of actively translating ribosomes. Multiparticle refinement and 3D variability analysis allowed us to visualize a variety of native translation intermediates. Significantly populated states include not only elongation cycle intermediates in pre- and post-translocational states, but also eEF1A-containing decoding and termination/recycling complexes. Focusing on the post-translocational state, we extended this assessment to the single-residue level, uncovering striking details of ribosome-ligand interactions and identifying both static and functionally important dynamic elements.

  8. Genome Mining for Ribosomally Synthesized Natural Products

    PubMed Central

    Velásquez, Juan E.; van der Donk, Wilfred

    2011-01-01

    In recent years, the number of known peptide natural products that are synthesized via the ribosomal pathway has rapidly grown. Taking advantage of sequence homology among genes encoding precursor peptides or biosynthetic proteins, in silico mining of genomes combined with molecular biology approaches has guided the discovery of a large number of new ribosomal natural products, including lantipeptides, cyanobactins, linear thiazole/oxazole-containing peptides, microviridins, lasso peptides, amatoxins, cyclotides, and conopeptides. In this review, we describe the strategies used for the identification of these ribosomally-synthesized and posttranslationally modified peptides (RiPPs) and the structures of newly identified compounds. The increasing number of chemical entities and their remarkable structural and functional diversity may lead to novel pharmaceutical applications. PMID:21095156

  9. Functions of Ribosomal Proteins in Assembly of Eukaryotic Ribosomes In Vivo

    PubMed Central

    2016-01-01

    The proteome of cells is synthesized by ribosomes, complex ribonucleoproteins that in eukaryotes contain 79–80 proteins and four ribosomal RNAs (rRNAs) more than 5,400 nucleotides long. How these molecules assemble together and how their assembly is regulated in concert with the growth and proliferation of cells remain important unanswered questions. Here, we review recently emerging principles to understand how eukaryotic ribosomal proteins drive ribosome assembly in vivo. Most ribosomal proteins assemble with rRNA cotranscriptionally; their association with nascent particles is strengthened as assembly proceeds. Each subunit is assembled hierarchically by sequential stabilization of their subdomains. The active sites of both subunits are constructed last, perhaps to prevent premature engagement of immature ribosomes with active subunits. Late-assembly intermediates undergo quality-control checks for proper function. Mutations in ribosomal proteins that affect mostly late steps lead to ribosomopathies, diseases that include a spectrum of cell type–specific disorders that often transition from hypoproliferative to hyperproliferative growth. PMID:25706898

  10. Sequestration of Ribosome during Protein Aggregate Formation: Contribution of ribosomal RNA

    PubMed Central

    Pathak, Bani K.; Mondal, Surojit; Banerjee, Senjuti; Ghosh, Amar Nath; Barat, Chandana

    2017-01-01

    An understanding of the mechanisms underlying protein aggregation and cytotoxicity of the protein aggregates is crucial in the prevention of several diseases in humans. Ribosome, the cellular protein synthesis machine is capable of acting as a protein folding modulator. The peptidyltransferase center residing in the domain V of large ribosomal subunit 23S rRNA is the centre for the protein folding ability of the ribosome and is also the cellular target of several antiprion compounds. Our in vitro studies unexpectedly reveal that the partial unfolding or aggregation of lysozyme under reducing conditions in presence of the ribosome can induce aggregation of ribosomal components. Electrostatic interactions complemented by specific rRNA-protein interaction drive the ribosome-protein aggregation process. Under similar conditions the rRNA, especially the large subunit rRNA and in vitro transcribed RNA corresponding to domain V of 23S rRNA (bDV RNA) stimulates lysozyme aggregation leading to RNA-protein aggregate formation. Protein aggregation during the refolding of non-disulfide containing protein BCAII at high concentrations also induces ribosome aggregation. BCAII aggregation was also stimulated in presence of the large subunit rRNA. Our observations imply that the specific sequestration of the translation machine by aggregating proteins might contribute to their cytotoxicity. PMID:28169307

  11. Functions of ribosomal proteins in assembly of eukaryotic ribosomes in vivo.

    PubMed

    de la Cruz, Jesús; Karbstein, Katrin; Woolford, John L

    2015-01-01

    The proteome of cells is synthesized by ribosomes, complex ribonucleoproteins that in eukaryotes contain 79-80 proteins and four ribosomal RNAs (rRNAs) more than 5,400 nucleotides long. How these molecules assemble together and how their assembly is regulated in concert with the growth and proliferation of cells remain important unanswered questions. Here, we review recently emerging principles to understand how eukaryotic ribosomal proteins drive ribosome assembly in vivo. Most ribosomal proteins assemble with rRNA cotranscriptionally; their association with nascent particles is strengthened as assembly proceeds. Each subunit is assembled hierarchically by sequential stabilization of their subdomains. The active sites of both subunits are constructed last, perhaps to prevent premature engagement of immature ribosomes with active subunits. Late-assembly intermediates undergo quality-control checks for proper function. Mutations in ribosomal proteins that affect mostly late steps lead to ribosomopathies, diseases that include a spectrum of cell type-specific disorders that often transition from hypoproliferative to hyperproliferative growth.

  12. Studies on structural stability of thermophilic Sulfolobus acidocaldarius ribosomes.

    PubMed

    Yangala, Kalavathi; Suryanarayana, Tangirala

    2007-02-01

    Structural stability of thermophilic archaeon Sulfolobus acidocaldarius ribosomes, with respect their susceptibility to pancreatic RNase A and stability to temperature (deltaTm), on treatment with various stabilizing (polyamines) and destabilizing (sulfhydryl and intercalating) agents were studied and compared with mesophilic E. coli ribosomes, to understand the structural differences between thermophilic and mesophilic ribosomes. Thermophilic archaeal ribosomes and their subunits were 10-times less susceptible to pancreatic RNase A, compared to mesophilic ribosomes, showing the presence of strong and compact structural organization in them. Thermophilic ribosomes treated with destabilizing agents, such as sulfhydryl reagents [5,5'-Dithio-bis-(2-nitrobenzoic acid), N-ethylmaleimide and p-hydroxymercurybenzoate) and intercalating agents (ethidium bromide, EtBr) showed higher stability to RNase A, compared to similarly treated mesophilic ribosomes, indicating the unavailability of thiol-reactive groups and the presence of strong solvent inaccessible inner core. Higher stability of thermophilic ribosomes compared to mesophilic ribosomes to unfolding agents like urea further supported the presence of strong inner core particle. Thermophilic ribosomes treated with intercalating agents, such as EtBr were less susceptible to RNase A, though they bound to more reagent, showing the rigidity or resilience of their macromolecular structure to alterations caused by destabilizing agents. Overall, these results indicated that factors such as presence of strong solvent inaccessible inner core and rigidity of ribosome macromolecular structure contributed stability of thermophilic ribosomes to RNase A and other destabilizing agents, when compared to mesophilic ribosomes.

  13. Proteomic characterization of archaeal ribosomes reveals the presence of novel archaeal-specific ribosomal proteins.

    PubMed

    Márquez, Viter; Fröhlich, Thomas; Armache, Jean-Paul; Sohmen, Daniel; Dönhöfer, Alexandra; Mikolajka, Aleksandra; Berninghausen, Otto; Thomm, Michael; Beckmann, Roland; Arnold, Georg J; Wilson, Daniel N

    2011-02-04

    Protein synthesis occurs in macromolecular particles called ribosomes. All ribosomes are composed of RNA and proteins. While the protein composition of bacterial and eukaryotic ribosomes has been well-characterized, a systematic analysis of archaeal ribosomes has been lacking. Here we report the first comprehensive two-dimensional PAGE and mass spectrometry analysis of archaeal ribosomes isolated from the thermophilic Pyrobaculum aerophilum and the thermoacidophilic Sulfolobus acidocaldarius Crenarchaeota. Our analysis identified all 66 ribosomal proteins (r-proteins) of the P. aerophilum small and large subunits, as well as all but two (62 of 64; 97%) r-proteins of the S. acidocaldarius small and large subunits that are predicted genomically. Some r-proteins were identified with one or two lysine methylations and N-terminal acetylations. In addition, we identify three hypothetical proteins that appear to be bona fide r-proteins of the S. acidocaldarius large subunit. Dissociation of r-proteins from the S. acidocaldarius large subunit indicates that the novel r-proteins establish tighter interactions with the large subunit than some integral r-proteins. Furthermore, cryo electron microscopy reconstructions of the S. acidocaldarius and P. aerophilum 50S subunits allow for a tentative localization of the binding site of the novel r-proteins. This study illustrates not only the potential diversity of the archaeal ribosomes but also the necessity to experimentally analyze the archaeal ribosomes to ascertain their protein composition. The discovery of novel archaeal r-proteins and factors may be the first step to understanding how archaeal ribosomes cope with extreme environmental conditions.

  14. Low molecular mass phosphoproteins from the frog rod outer segments form a complex with 48 kDa protein.

    PubMed

    Krapivinsky, G B; Malenyov, A L; Zaikina, I V; Fesenko, E E

    1992-09-01

    Upon separation of cAMP-dependent low molecular mass phosphoproteins [Components I and II; Polans et al. (1979) J. gen. Physiol. 74, 595-613] from the frog rod outer segments by gel-chromatography, isoelectric focusing, non-denaturating electrophoresis and ion-exchange chromatography, they behave like subunits of the oligomeric complex. Apparent molecular mass of the complex determined by gel-chromatography is 52-57 kDa and by non-denaturating gradient electrophoresis is 62-66 kDa. The isoelectric point of the complex is 5.5. The elution profile of Components I and II upon gel-chromatography and ion-exchange chromatography coincides with that of major rod outer segment 48 kDa protein. The isoelectric point for them also coincides with the isoelectric point of 48 kDa protein. The amount of low molecular mass phosphoproteins is sealed rods is equal to one molecule per 60 rhodopsin molecules and coincides with that of a 48 kDa protein. It is suggested that in solution Components I and II form an oligomeric complex with 48 kDa protein.

  15. A peptide targeted against phosphoprotein and leader RNA interaction inhibits growth of Chandipura virus -- an emerging rhabdovirus.

    PubMed

    Roy, Arunava; Chakraborty, Prasenjit; Polley, Smarajit; Chattopadhyay, Dhrubajyoti; Roy, Siddhartha

    2013-11-01

    The fatal illness caused by Chandipura virus (CHPV), an emerging pathogen, presently lacks any therapeutic option. Previous research suggested that interaction between the virally encoded phosphoprotein (P) and the positive sense leader RNA (le-RNA) may play an important role in the viral lifecycle. In this report, we have identified a β-sheet/loop motif in the C-terminal domain of the CHPV P protein as essential for this interaction. A synthetic peptide encompassing this motif and spanning a continuous stretch of 36 amino acids (Pep208-243) was found to bind the le-RNA in vitro and inhibit CHPV growth in infected cells. Furthermore, a stretch of three amino acid residues at position 217-219 was identified as essential for this interaction, both in vitro and in infected cells. siRNA knockdown-rescue experiments demonstrated that these three amino acid residues are crucial for the leader RNA binding function of P protein in the CHPV life cycle. Mutations of these three amino acid residues render the peptide completely ineffective against CHPV. Effect of inhibition of phosphoprotein-leader RNA interaction on viral replication was assayed. Peptide Pep208-243 tagged with a cell penetrating peptide was found to inhibit CHPV replication as ascertained by real time RT-PCR. The specific inhibition of viral growth observed using this peptide suggests a new possibility for designing of anti-viral agents against Mononegavirale group of human viruses.

  16. A rapid and simple 8-quinolinol-based fluorescent stain of phosphoproteins in polyacrylamide gel after electrophoresis.

    PubMed

    Wang, Xu; Hwang, Sun-Young; Cong, Wei-Tao; Jin, Li-Tai; Choi, Jung-Kap

    2015-10-01

    In order to obtain an easy and rapid protocol to visualize phosphoproteins in SDS-PAGE, a fluorescent detection method named 8-Quinolinol (8-Q) stain is described. 8-Q can form ternary complexes in the gel matrix contributed by the affinity of aluminum ion (Al(3+) ) to the phosphate groups on the proteins and the metal chelating property of 8-Quinolinol, exhibiting strong fluorescence in ultraviolet light. It can visualize as little as 4∼8 ng of α-casein and β-casein, 16∼32 ng of ovalbumin and κ-casein which is more sensitive than Stains-All but less sensitive than Pro-Q Diamond. The protocol of 8-Q requires only 70 min in 0.75 mm mini-size or 1.0 mm large-size gels with five changes of solutions without destaining step; Pro-Q takes at least 250 min with 11 changes of solutions. In addition, the new method was confirmed by the study of dephosphorylation and LC-MS/MS, respectively. The approach to visualize phosphoprotein utilizing 8-Q could be an alternative to simplify the analytical operations for phosphoproteomics research.

  17. Convergent evolution led ribosome inactivating proteins to interact with ribosomal stalk.

    PubMed

    Lapadula, Walter J; Sanchez-Puerta, M Virginia; Ayub, Maximiliano Juri

    2012-03-01

    Ribosome-inactivating proteins (RIPs) inhibit protein synthesis by depurinating an adenine on the sarcin-ricin loop (SRL) of the large subunit ribosomal RNA. Several RIPs interact with the C-terminal end of ribosomal stalk P proteins, and this interaction is required for their full activity. In contrast, the activity of Pokeweed Antiviral Protein is not affected by blocking this stalk component. Here, we provide evidence from phylogenetic analyses and sequence alignments suggesting that the interaction with the C-terminal end of P proteins evolved independently in different RIPs by convergent evolution.

  18. Dom34 Rescues Ribosomes in 3´ Untranslated Regions

    PubMed Central

    Guydosh, Nicholas R.; Green, Rachel

    2014-01-01

    SUMMARY Ribosomes that stall before completing peptide synthesis must be recycled and returned to the cytoplasmic pool. The protein Dom34 and cofactors Hbs1 and Rli1 can dissociate stalled ribosomes in vitro, but the identity of targets in the cell is unknown. Here we extend ribosome profiling methodology to reveal a high-resolution molecular characterization of Dom34 function in vivo. Dom34 removes stalled ribosomes from truncated mRNAs, but, in contrast, does not generally dissociate ribosomes on coding sequences known to trigger stalling, such as polyproline. We also show that Dom34 targets arrested ribosomes near the ends of 3´ UTRs. These ribosomes appear to gain access to the 3 UTR via a mechanism that does not require decoding of the mRNA. These results suggest that ribosomes frequently enter downstream noncoding regions and that Dom34 carries out the important task of rescuing them. PMID:24581494

  19. Dom34 rescues ribosomes in 3' untranslated regions.

    PubMed

    Guydosh, Nicholas R; Green, Rachel

    2014-02-27

    Ribosomes that stall before completing peptide synthesis must be recycled and returned to the cytoplasmic pool. The protein Dom34 and cofactors Hbs1 and Rli1 can dissociate stalled ribosomes in vitro, but the identity of targets in the cell is unknown. Here, we extend ribosome profiling methodology to reveal a high-resolution molecular characterization of Dom34 function in vivo. Dom34 removes stalled ribosomes from truncated mRNAs, but, in contrast, does not generally dissociate ribosomes on coding sequences known to trigger stalling, such as polyproline. We also show that Dom34 targets arrested ribosomes near the ends of 3' UTRs. These ribosomes appear to gain access to the 3' UTR via a mechanism that does not require decoding of the mRNA. These results suggest that ribosomes frequently enter downstream noncoding regions and that Dom34 carries out the important task of rescuing them.

  20. Sprouty-Related Ena/Vasodilator-Stimulated Phosphoprotein Homology 1-Domain-Containing Protein-2 Critically Regulates Influenza A Virus-Induced Pneumonia.

    PubMed

    Ito, Toshihiro; Itakura, Junya; Takahashi, Sakuma; Sato, Miwa; Mino, Megumi; Fushimi, Soichiro; Yamada, Masao; Morishima, Tuneo; Kunkel, Steven L; Matsukawa, Akihiro

    2016-07-01

    Influenza A virus causes acute respiratory infections that induce annual epidemics and occasional pandemics. Although a number of studies indicated that the virus-induced intracellular signaling events are important in combating influenza virus infection, the mechanism how specific molecule plays a critical role among various intracellular signaling events remains unknown. Raf/MEK/extracellular signal-regulated kinase cascade is one of the key signaling pathways during influenza virus infection, and the Sprouty-related Ena/vasodilator-stimulated phosphoprotein homology 1-domain-containing protein has recently been identified as a negative regulator of Raf-dependent extracellular signal-regulated kinase activation. Here, we examined the role of Raf/MEK/extracellular signal-regulated kinase cascade through sprouty-related Ena/vasodilator-stimulated phosphoprotein homology 1-domain-containing protein in influenza A viral infection because the expression of sprouty-related Ena/vasodilator-stimulated phosphoprotein homology 1-domain-containing protein was significantly enhanced in human influenza viral-induced pneumonia autopsy samples. Prospective animal trial. Research laboratory. Wild-type and sprouty-related Ena/vasodilator-stimulated phosphoprotein homology 1-domain-containing protein-2 knockout mice inoculated with influenza A. Wild-type or sprouty-related Ena/vasodilator-stimulated phosphoprotein homology 1-domain-containing protein-2 knockout mice were infected by intranasal inoculation of influenza A (A/PR/8). An equal volume of phosphate-buffered saline was inoculated intranasally into mock-infected mice. Influenza A infection of sprouty-related Ena/vasodilator-stimulated phosphoprotein homology 1-domain-containing protein-2 knockout mice led to higher mortality with greater viral load, excessive inflammation, and enhanced cytokine production than wild-type mice. Administration of MEK inhibitor, U0126, improved mortality and reduced both viral load and

  1. Peptide Bond Formation Mechanism Catalyzed by Ribosome.

    PubMed

    Świderek, Katarzyna; Marti, Sergio; Tuñón, Iñaki; Moliner, Vicent; Bertran, Juan

    2015-09-23

    In this paper we present a study of the peptide bond formation reaction catalyzed by ribosome. Different mechanistic proposals have been explored by means of Free Energy Perturbation methods within hybrid QM/MM potentials, where the chemical system has been described by the M06-2X functional and the environment by means of the AMBER force field. According to our results, the most favorable mechanism in the ribosome would proceed through an eight-membered ring transition state, involving a proton shuttle mechanism through the hydroxyl group of the sugar and a water molecule. This transition state is similar to that described for the reaction in solution (J. Am. Chem. Soc. 2013, 135, 8708-8719), but the reaction mechanisms are noticeably different. Our simulations reproduce the experimentally determined catalytic effect of ribosome that can be explained by the different behavior of the two environments. While the solvent reorganizes during the chemical process involving an entropic penalty, the ribosome is preorganized in the formation of the Michaelis complex and does not suffer important changes along the reaction, dampening the charge redistribution of the chemical system.

  2. Peptide Bond Formation Mechanism Catalyzed by Ribosome

    PubMed Central

    Świderek, Katarzyna; Marti, Sergio; Tuñón, Iñaki; Moliner, Vicent; Bertran, Juan

    2015-01-01

    In this paper we present a study of the peptide bond formation reaction catalyzed by ribosome. Different mechanistic proposals have been explored by means of Free Energy Perturbation methods within hybrid QM/MM potentials, where the chemical system has been described by the M06-2X functional and the environment by means of the AMBER force field. According to our results, the most favourable mechanism in the ribosome would proceed through an eight-membered ring transition state, involving a proton shuttle mechanism through the hydroxyl group of the sugar and a water molecule. This transition state is similar to that described for the reaction in solution (J. Am. Chem. Soc. 2013, 135, 8708–8719) but the reaction mechanisms are noticeable different. Our simulations reproduce the experimentally determined catalytic effect of ribosome that can be explained by the different behaviour of the two environments. While the solvent reorganizes during the chemical process involving an entropic penalty, the ribosome is preorganized in the formation of the Michaelis complex and does not suffer important changes along the reaction, dampening the charge redistribution of the chemical system. PMID:26325003

  3. Diamond-Blackfan anemia, ribosome and erythropoiesis

    PubMed Central

    Costa, L. Da; Moniz, H.; Simansour, M.; Tchernia, G.; Mohandas, N.; Leblanc, T.

    2010-01-01

    Diamond-Blackfan anemia is a rare inherited bone marrow failure syndrome (5 to 7 cases/million live births) characterized by an are generative, usually macrocytic anemia with an absence or less than 5% of erythroid precursors (erythroblastopenia) in an otherwise normal bone marrow. The platelet and the white cell counts are usually normal but neutropenia, thrombopenia or thrombocytosis have been noted at diagnosis. In 40 to 50% of DBA patients, congenital abnormalities mostly in the cephalic area and in thumbs and upper limbs have been described. Recent analysis did show a phenotype/genotype correlation. Congenital erythroblastopenia of DBA is the first human disease identified to result from defects in ribosomal biogenesis. The first ribosomal gene involved in DBA, ribosomal protein (RP) gene S19 (RPS19 gene), was identified in 1999. Subsequently, mutations in 12 other RP genes out of a total of 78 RP genes have been identified in DBA. All RP gene mutations described to date are heterozygous and dominant inheritance has been documented in 40 to 45% of affected individuals. As RP mutations are yet to be identified in approximately 50% of DBA cases, it is likely that other yet to be identified genes involved in ribosomal biogenesis or other pathways may be responsible for DBA phenotype. PMID:20655265

  4. Ribosome. Mechanical force releases nascent chain-mediated ribosome arrest in vitro and in vivo.

    PubMed

    Goldman, Daniel H; Kaiser, Christian M; Milin, Anthony; Righini, Maurizio; Tinoco, Ignacio; Bustamante, Carlos

    2015-04-24

    Protein synthesis rates can affect gene expression and the folding and activity of the translation product. Interactions between the nascent polypeptide and the ribosome exit tunnel represent one mode of regulating synthesis rates. The SecM protein arrests its own translation, and release of arrest at the translocon has been proposed to occur by mechanical force. Using optical tweezers, we demonstrate that arrest of SecM-stalled ribosomes can indeed be rescued by force alone and that the force needed to release stalling can be generated in vivo by a nascent chain folding near the ribosome tunnel exit. We formulate a kinetic model describing how a protein can regulate its own synthesis by the force generated during folding, tuning ribosome activity to structure acquisition by a nascent polypeptide. Copyright © 2015, American Association for the Advancement of Science.

  5. Reductive alkylation of ribosomes as a probe to the topography of ribosomal proteins*

    PubMed Central

    Moore, Graham; Crichton, Robert R.

    1974-01-01

    Escherichia coli ribosomes were treated with a number of different aldehydes of various sizes in the presence of NaBH4. After incorporation of either 3H or 14C, the ribosomal proteins were separated by two-dimensional polyacrylamide-gel electrophoresis and the extent of alkylation of the lysine residues in each protein was measured. The same pattern of alkylation was observed with the four reagents used, namely formaldehyde, acetone, benzaldehyde and 3,4,5-trimethoxybenzaldehyde. Every protein in 30S and 50S subunits was modified, although there was considerable variation in the degree of alkylation of individual proteins. A topographical classification of ribosomal proteins is presented, based on the degree of exposure of lysine residues. The data indicate that every protein of the ribosome has at least one lysine residue exposed at or near the surface of the ribonucleo-protein complex. PMID:4462744

  6. Dynamic relationships between ribosomal conformational and RNA positional changes during ribosomal translocation.

    PubMed

    Xie, Ping

    2016-12-01

    Ribosomal translocation catalyzed by EF-G hydrolyzing GTP entails multiple conformational changes of ribosome and positional changes of tRNAs and mRNA in the ribosome. However, the detailed dynamic relations among these changes and EF-G sampling are not clear. Here, based on our proposed pathway of ribosomal translocation, we study theoretically the dynamic relations among these changes exhibited in the single molecule data and those exhibited in the ensemble kinetic data. It is shown that the timing of these changes in the single molecule data and that in the ensemble kinetic data show very different. The theoretical results are in agreement with both the available ensemble kinetic experimental data and the single molecule experimental data.

  7. Crystal structure of eukaryotic ribosome and its complexes with inhibitors.

    PubMed

    Yusupova, Gulnara; Yusupov, Marat

    2017-03-19

    A high-resolution structure of the eukaryotic ribosome has been determined and has led to increased interest in studying protein biosynthesis and regulation of biosynthesis in cells. The functional complexes of the ribosome crystals obtained from bacteria and yeast have permitted researchers to identify the precise residue positions in different states of ribosome function. This knowledge, together with electron microscopy studies, enhances our understanding of how basic ribosome processes, including mRNA decoding, peptide bond formation, mRNA, and tRNA translocation and cotranslational transport of the nascent peptide, are regulated. In this review, we discuss the crystal structure of the entire 80S ribosome from yeast, which reveals its eukaryotic-specific features, and application of X-ray crystallography of the 80S ribosome for investigation of the binding mode for distinct compounds known to inhibit or modulate the protein-translation function of the ribosome. We also refer to a challenging aspect of the structural study of ribosomes, from higher eukaryotes, where the structures of major distinctive features of higher eukaryote ribosome-the high-eukaryote-specific long ribosomal RNA segments (about 1MDa)-remain unresolved. Presently, the structures of the major part of these high-eukaryotic expansion ribosomal RNA segments still remain unresolved.This article is part of the themed issue 'Perspectives on the ribosome'.

  8. Acidic Ribosomal Proteins from the Extreme ’Halobacterium cutirubrum’,

    DTIC Science & Technology

    the extreme halophilic bacterium, Halobacterium cutirubrum. The identification of the protein moieties involved in these and other interactions in...the halophile ribosome requires a rapid and reproducible screening method for the separation, enumeration and identification of these acidic...polypeptides in the complex ribosomal protein mixtures. In this paper the authors present the results of analyses of the halophile ribosomal proteins using a

  9. On the pathway of ribosomal translocation.

    PubMed

    Xie, Ping

    2016-11-01

    The translocation of tRNAs coupled with mRNA in the ribosome is a critical process in the elongation cycle of protein synthesis. The translocation entails large-scale conformational changes of the ribosome and involves several intermediate states with tRNAs in different positions with respect to 30S and 50S ribosomal subunits. However, the detailed role of the intermediate states is unknown and the detailed mechanism and pathway of translocation is unclear. Here based on previous structural, biochemical and single-molecule data we present a translocation pathway by incorporating several intermediate states. With the pathway, we study theoretically (i) the kinetics of 30S head rotation associated with translocation catalyzed by wild-type EF-G, (ii) the dynamics of fluctuations between different tRNA states during translocation interfered with EF-G mutants and translocation-specific antibiotics, (iii) the kinetics of tRNA movement in 50S subunit and mRNA movement in 30S subunit in the presence of wild-type EF-G, EF-G mutants and translocation-specific antibiotics, (iv) the dynamics of EF-G sampling to the ribosome during translocation, etc., providing consistent and quantitative explanations of various available biochemical and single-molecule experimental data published in the literature. Moreover, we study the kinetics of 30S head rotation in the presence of EF-G mutants, providing predicted results. These have significant implications for the molecular mechanism and pathway of ribosomal translocation. Copyright © 2016 Elsevier B.V. All rights reserved.

  10. A RanGTP-independent mechanism allows ribosomal protein nuclear import for ribosome assembly

    PubMed Central

    Schütz, Sabina; Fischer, Ute; Altvater, Martin; Nerurkar, Purnima; Peña, Cohue; Gerber, Michaela; Chang, Yiming; Caesar, Stefanie; Schubert, Olga T; Schlenstedt, Gabriel; Panse, Vikram G

    2014-01-01

    Within a single generation time a growing yeast cell imports ∼14 million ribosomal proteins (r-proteins) into the nucleus for ribosome production. After import, it is unclear how these intrinsically unstable and aggregation-prone proteins are targeted to the ribosome assembly site in the nucleolus. Here, we report the discovery of a conserved nuclear carrier Tsr2 that coordinates transfer of the r-protein eS26 to the earliest assembling pre-ribosome, the 90S. In vitro studies revealed that Tsr2 efficiently dissociates importin:eS26 complexes via an atypical RanGTP-independent mechanism that terminates the import process. Subsequently, Tsr2 binds the released eS26, shields it from proteolysis, and ensures its safe delivery to the 90S pre-ribosome. We anticipate similar carriers—termed here escortins—to securely connect the nuclear import machinery with pathways that deposit r-proteins onto developing pre-ribosomal particles. DOI: http://dx.doi.org/10.7554/eLife.03473.001 PMID:25144938

  11. A RanGTP-independent mechanism allows ribosomal protein nuclear import for ribosome assembly.

    PubMed

    Schütz, Sabina; Fischer, Ute; Altvater, Martin; Nerurkar, Purnima; Peña, Cohue; Gerber, Michaela; Chang, Yiming; Caesar, Stefanie; Schubert, Olga T; Schlenstedt, Gabriel; Panse, Vikram G

    2014-08-21

    Within a single generation time a growing yeast cell imports ∼14 million ribosomal proteins (r-proteins) into the nucleus for ribosome production. After import, it is unclear how these intrinsically unstable and aggregation-prone proteins are targeted to the ribosome assembly site in the nucleolus. Here, we report the discovery of a conserved nuclear carrier Tsr2 that coordinates transfer of the r-protein eS26 to the earliest assembling pre-ribosome, the 90S. In vitro studies revealed that Tsr2 efficiently dissociates importin:eS26 complexes via an atypical RanGTP-independent mechanism that terminates the import process. Subsequently, Tsr2 binds the released eS26, shields it from proteolysis, and ensures its safe delivery to the 90S pre-ribosome. We anticipate similar carriers-termed here escortins-to securely connect the nuclear import machinery with pathways that deposit r-proteins onto developing pre-ribosomal particles. Copyright © 2014, Schütz et al.

  12. Ribosomal Protein S14 Unties the MDM2-p53 Loop Upon Ribosomal Stress

    PubMed Central

    Zhou, Xiang; Hao, Qian; Liao, Jun-ming; Zhang, Qi; Lu, Hua

    2013-01-01

    The MDM2-p53 feedback loop is crucially important for restricting p53 level and activity during normal cell growth and proliferation, and is thus subjected to dynamic regulation in order for cells to activate p53 upon various stress signals. Several ribosomal proteins, such as RPL11, RPL5, RPL23, RPL26, or RPS7, have been shown to play a role in regulation of this feedback loop in response to ribosomal stress. Here, we identify another ribosomal protein S14, which is highly associated with 5q-syndrome, as a novel activator of p53 by inhibiting MDM2 activity. We found that RPS14, but not RPS19, binds to the central acidic domain of MDM2, like RPL5 and RPL23, and inhibits its E3 ubiquitin ligase activity toward p53. This RPS14-MDM2 binding was induced upon ribosomal stress caused by actinomycin D or mycophenolic acid. Overexpression of RPS14, but not RPS19, elevated p53 level and activity, leading to G1 or G2 arrest. Conversely, knockdown of RPS14 alleviated p53 induction by these two reagents. Interestingly, knockdown of either RPS14 or RPS19 caused a ribosomal stress that led to p53 activation, which was impaired by further knocking down the level of RPL11 or RPL5. Together, our results demonstrate that RPS14 and RPS19 play distinct roles in regulating the MDM2-p53 feedback loop in response to ribosomal stress. PMID:22391559

  13. Arabidopsis protein arginine methyltransferase 3 is required for ribosome biogenesis by affecting precursor ribosomal RNA processing

    PubMed Central

    Hang, Runlai; Liu, Chunyan; Ahmad, Ayaz; Zhang, Yong; Lu, Falong; Cao, Xiaofeng

    2014-01-01

    Ribosome biogenesis is a fundamental and tightly regulated cellular process, including synthesis, processing, and assembly of rRNAs with ribosomal proteins. Protein arginine methyltransferases (PRMTs) have been implicated in many important biological processes, such as ribosome biogenesis. Two alternative precursor rRNA (pre-rRNA) processing pathways coexist in yeast and mammals; however, how PRMT affects ribosome biogenesis remains largely unknown. Here we show that Arabidopsis PRMT3 (AtPRMT3) is required for ribosome biogenesis by affecting pre-rRNA processing. Disruption of AtPRMT3 results in pleiotropic developmental defects, imbalanced polyribosome profiles, and aberrant pre-rRNA processing. We further identify an alternative pre-rRNA processing pathway in Arabidopsis and demonstrate that AtPRMT3 is required for the balance of these two pathways to promote normal growth and development. Our work uncovers a previously unidentified function of PRMT in posttranscriptional regulation of rRNA, revealing an extra layer of complexity in the regulation of ribosome biogenesis. PMID:25352672

  14. Development of pre-implantation porcine embryos cultured within alginate hydrogel systems either supplemented with secreted phosphoprotein 1 or conjugated with arg-gly-asp peptide

    USDA-ARS?s Scientific Manuscript database

    Although deficiencies in porcine embryo elongation play a significant role on early embryonic mortality and establishment of within–litter developmental variation, the exact mechanisms of elongation are poorly understood. Secreted phosphoprotein 1 (SPP1) is increased within the uterine milieu during...

  15. Ribosomal suppressors and antisuppressors in Podospora anserina: resistance to cycloheximide.

    PubMed Central

    Coppin-Raynal, E

    1977-01-01

    Informational suppressors and antisuppressors have been previously isolated in Podospora anserina, and a range of exclusively genetic arguments have led to the assumption that they correspond to ribosomal mutations. An in vivo and in vitro comparison of the effect of the ribosomal inhibitor cycloheximide on wildtype and mutant strains described in this paper confirms the ribosomal hypothesis for at least some mutants. Indeed, the four mutants in the AS3 gene were cycloheximide resistant, and their ribosomes were found to be resistant when analyzed by polyuridyl-directed polyphenylalanine systhesis. On the other hand, ribosomes from two su 1 mutants were hypersensitive to the drug. PMID:893344

  16. Identification and characterization of a Dictyostelium discoideum ribosomal protein gene.

    PubMed Central

    Szymkowski, D E; Deering, R A

    1990-01-01

    We have identified a developmentally repressed large-subunit ribosomal protein gene of Dictyostelium discoideum based on sequence similarity to other ribosomal proteins. Protein rpl7 is homologous to large subunit ribosomal proteins from the rat and possibly to Mycoplasma capricolum and Escherichia coli, but is not similar to three sequenced ribosomal proteins in Dictyostelium. The rpl7 gene is present at one copy per genome, as are six other cloned Dictyostelium ribosomal proteins. Restriction fragment length polymorphisms exist for ribosomal protein genes rpl7, rp1024, and rp110 in strain HU182; most Dictyostelium ribosomal protein genes examined are linked no closer than 30-100 kb to each other in the genome. Dictyostelium ribosomal proteins are known to be developmentally regulated, and levels of rpl7 transcript gradually decrease during the 24-hour development cycle. This drop correlates with that of rp1024, indicating these and other ribosomal protein genes may be coordinately regulated. To determine the cellular location of the protein, we raised antibodies to an rpl7-derived branched synthetic peptide. These antibodies cross-reacted with one protein of the expected size in a ribosomal protein fraction of Dictyostelium, indicating that the product of gene rpl7 is localized in the ribosome. Images PMID:1975664

  17. M phase phosphoprotein 1 is a human plus-end-directed kinesin-related protein required for cytokinesis

    PubMed Central

    Abaza, Aouatef; Soleilhac, Jean-Marc; Westendorf, Joanne; Piel, Matthieu; Crevel, Isabelle; Roux, Aurelien; Pirollet, Fabienne

    2003-01-01

    Summary The human M-Phase Phosphoprotein 1 (MPP1), previously identified through a screening of a subset of proteins specifically phosphorylated at the G2/M transition (1), is characterized as a plus-end-directed kinesin-related protein. Recombinant MPP1 exhibits in vitro microtubule-binding and microtubule-bundling properties as well as microtubules-stimulated ATPase activity. In gliding experiments using polarity-marked microtubules, MPP1 is a slow molecular motor that moves towards the microtubule plus-end at a 0.07 μm/s speed. In cycling cells, MPP1 localizes mainly to the nuclei in interphase. During mitosis, MPP1 is diffuse throughout the cytoplasm in metaphase and subsequently localizes to the midzone to further concentrate on the midbody. MPP1 suppression by RNA interference induces failure of cell division late in cytokinesis. We conclude that MPP1 is a new mitotic molecular motor required for completion of cytokinesis. PMID:12740395

  18. Structural Disorder within Paramyxoviral Nucleoproteins and Phosphoproteins in Their Free and Bound Forms: From Predictions to Experimental Assessment

    PubMed Central

    Habchi, Johnny; Longhi, Sonia

    2015-01-01

    We herein review available computational and experimental data pointing to the abundance of structural disorder within the nucleoprotein (N) and phosphoprotein (P) from three paramyxoviruses, namely the measles (MeV), Nipah (NiV) and Hendra (HeV) viruses. We provide a detailed molecular description of the mechanisms governing the disorder-to-order transition that the intrinsically disordered C-terminal domain (NTAIL) of their N proteins undergoes upon binding to the C-terminal X domain (PXD) of the homologous P proteins. We also show that NTAIL–PXD complexes are “fuzzy”, i.e., they possess a significant residual disorder, and discuss the possible functional significance of this fuzziness. Finally, we emphasize the relevance of N–P interactions involving intrinsically disordered proteins as promising targets for new antiviral approaches, and end up summarizing the general functional advantages of disorder for viruses. PMID:26184170

  19. Cloning and characterization of DNA complementary to the canine distemper virus mRNA encoding matrix, phosphoprotein, and nucleocapsid protein

    SciTech Connect

    Rozenblatt, S.; Eizenberg, O.; Englund, G.; Bellini, W.J.

    1985-02-01

    Double-stranded cDNA synthesized from total polyadenylate-containing mRNA, extracted from monkey kidney cells infected with canine distemper virus (CDV), has been cloned into the PstI site of Escherichia coli plasmid pBR322. Clones containing canine distemper virus DNA were identified by hybridization to a canine distemper virus-specific, /sup 32/P-labeled cDNA. Four specific clones containing different classes of sequences have been identified. The cloned plasmids contain inserts of 800 (clone 44-80), 960 (clone 74-16), 1700 (clone 364), and 950 (clone 40-9) base pairs. The sizes of the mRNA species complementary to these inserts are 1500, 1850, 1850 and 2500 nucleotides, respectively, as determined by the Northern technique. Three of the cloned DNA fragments were further identified as the reverse transcripts of the mRNA coding for the matrix, phosphoprotein, and nucleocapsid protein of CDV.

  20. How order and disorder within paramyxoviral nucleoproteins and phosphoproteins orchestrate the molecular interplay of transcription and replication.

    PubMed

    Longhi, Sonia; Bloyet, Louis-Marie; Gianni, Stefano; Gerlier, Denis

    2017-09-01

    In this review, we summarize computational and experimental data gathered so far showing that structural disorder is abundant within paramyxoviral nucleoproteins (N) and phosphoproteins (P). In particular, we focus on measles, Nipah, and Hendra viruses and highlight both commonalities and differences with respect to the closely related Sendai virus. The molecular mechanisms that control the disorder-to-order transition undergone by the intrinsically disordered C-terminal domain (NTAIL) of their N proteins upon binding to the C-terminal X domain (XD) of the homologous P proteins are described in detail. By having a significant residual disorder, NTAIL-XD complexes are illustrative examples of "fuzziness", whose possible functional significance is discussed. Finally, the relevance of N-P interactions as promising targets for innovative antiviral approaches is underscored, and the functional advantages of structural disorder for paramyxoviruses are pinpointed.

  1. Inflammation-associated repression of vasodilator-stimulated phosphoprotein (VASP) reduces alveolar-capillary barrier function during acute lung injury

    PubMed Central

    Henes, Janek; Schmit, Marthe A.; Morote-Garcia, Julio C.; Mirakaj, Valbona; Köhler, David; Glover, Louise; Eldh, Therese; Walter, Ulrich; Karhausen, Jörn; Colgan, Sean P.; Rosenberger, Peter

    2009-01-01

    Acute lung injury (ALI) is an inflammatory disorder associated with reduced alveolar-capillary barrier function, increased pulmonary vascular permeability, and infiltration of leukocytes into the alveolar space. Pulmonary function might be compromised, its most severe form being the acute respiratory distress syndrome. A protein central to physiological barrier properties is vasodilator-stimulated phosphoprotein (VASP). Given the fact that VASP expression is reduced during periods of cellular hypoxia, we investigated the role of VASP during ALI. Initial studies revealed reduced VASP expressional levels through cytokines in vitro. Studies in the putative human VASP promoter identified NF-κB as a key regulator of VASP transcription. This VASP repression results in increased paracellular permeability and migration of neutrophils in vitro. In a model of LPS-induced ALI, VASP−/− mice demonstrated increased pulmonary damage compared with wild-type animals. These findings were confirmed in a second model of ventilator-induced lung injury. Studies employing bone marrow chimeric animals identified tissue-specific repression of VASP as the underlying cause of decreased barrier properties of the alveolar-capillary barrier during ALI. Taken together these studies identify tissue-specific VASP as a central protein in the control of the alveolar-capillary barrier properties during ALI.—Henes, J., Schmit, M. A., Morote-Garcia, J. C., Mirakaj, V., Köhler, D., Glover, L., Eldh, T., Walter, U., Karhausen, J., Colgan, S. P., Rosenberger, P. Inflammation-associated repression of vasodilator-stimulated phosphoprotein (VASP) reduces alveolar-capillary barrier function during acute lung injury. PMID:19690214

  2. Determination of the non-ionic detergent insolubility and phosphoprotein associations of glycosylphosphatidylinositol-anchored proteins expressed on T cells.

    PubMed Central

    Solomon, K R; Mallory, M A; Finberg, R W

    1998-01-01

    Glycosylphosphatidylinositol (GPI)-anchored proteins are poorly solublized in non-ionic detergents such as Triton X-100 and Nonidet P40, but are easily solublized by detergents with high critical micelle concentrations such as octylglucoside. This solubility profile has been suggested to be due to the localization of GPI-anchored proteins to lipid microdomains rich in cholesterol and sphingolipids. Additionally, GPI-anchored proteins expressed on haemopoietic cells have been shown to associate with src-family tyrosine kinases and heterotrimeric G proteins. Despite these observations, the non-ionic detergent insolubility of GPI-anchored proteins on haemopoietic cells has not been quantified nor has a relationship between the non-ionic detergent insolubility of these proteins and their association with signal-transduction molecules been identified. Here we show that GPI-anchored proteins found on T-cell tumours and activated T cells, although significantly more insoluble then transmembrane proteins, are not uniform in their detergent insolubility. Whereas CD59 was between 4% and 13% soluble, CD48 was between 13% and 25% soluble, CD55 was between 20% and 30% soluble, and CD109 was between 34% and 75% soluble. The ability of these GPI-anchored proteins to associate with phosphoproteins was correlated with their detergent insolubility: the more detergent-insoluble that a GPI-anchored protein was, the greater the level of phosphoprotein associations. These experiments reveal a relationship between non-ionic detergent insolubility and association with signal-transduction molecules and suggest a cause-and-effect relationship between these two properties. In total, these experiments support the hypothesis that the association of GPI-anchored proteins with signalling molecules is due to their sorting to lipid microdomains. PMID:9716490

  3. Intracellular receptor for somatostatin in gastric mucosal cells: decomposition and reconstitution of somatostatin-stimulated phosphoprotein phosphatases.

    PubMed

    Reyl, F J; Lewin, M J

    1982-02-01

    Using 32P-labeled histone as exogenous substrate, we showed a potent stimulatory effect of somatostatin on cytosolic phosphoprotein phosphatases (PPPases; phosphoprotein phosphohydrolase, EC 3.1.3.16) in rat gastric mucosal cells. Partial purification of cytosolic fraction in DEAE-Sephadex ion-exchange chromatography and further gel filtration on Sephadex C-75 and Sephadex G-100 separated somatostatin-dependent PPPases into three distinct molecular species. One corresponding to Mr 130,000 was devoid of any PPPase activity but specifically bound [Tyr1]somatostatin 125I-labeled on the Tyr ([125I-Tyr1]somatostatin) with an apparent equilibrium dissociation constant of 3 x 10(-10) M. The two other molecular species corresponded to Mrs 64,000 and 13,000. They produced catalytic dephosphorylation of 32P-labeled histone, but they were not sensitive to somatostatin and did not show any specific binding to radiolabeled hormone. Mixing of the larger with either of the two smaller molecular species resulted in concentration -dependent inhibition of PPPase activity. However this inhibition was reversed by increased concentrations of somatostatin, with the concentration for half-maximal reactivation on being close to 0.1 nM. Furthermore somatostatin stimulation in reconstituted materials developed according to a rapid time course (t1/2, less than 5 sec), consistent with that observed for binding of [125I-Tyr1]somatostatin. These results strongly argue for the presence of an intracellular somatostatin receptor in gastric mucosal cells and characterize this receptor as a PPPase regulatory subunit. Thus, substrate dephosphorylation could be the primary event triggering physiological effects of somatostatin in stomach and perhaps other organs of the digestive tract [Reyl, F. & Lewin, M. J.l M. (1981) Biochim. Biophys. Acta 675, 297-300].

  4. Heat Shock Protein 70 Regulates Degradation of the Mumps Virus Phosphoprotein via the Ubiquitin-Proteasome Pathway

    PubMed Central

    Kubota, Toru; Kita, Shunsuke; Nakatsu, Yuichiro; Aoki, Natsuko; Mori, Yoshio; Maenaka, Katsumi; Takeda, Makoto; Kidokoro, Minoru

    2014-01-01

    ABSTRACT Mumps virus (MuV) infection induces formation of cytoplasmic inclusion bodies (IBs). Growing evidence indicates that IBs are the sites where RNA viruses synthesize their viral RNA. However, in the case of MuV infection, little is known about the viral and cellular compositions and biological functions of the IBs. In this study, pulldown purification and N-terminal amino acid sequencing revealed that stress-inducible heat shock protein 70 (Hsp72) was a binding partner of MuV phosphoprotein (P protein), which was an essential component of the IB formation. Immunofluorescence and immunoblotting analyses revealed that Hsp72 was colocalized with the P protein in the IBs, and its expression was increased during MuV infection. Knockdown of Hsp72 using small interfering RNAs (siRNAs) had little, if any, effect on viral propagation in cultured cells. Knockdown of Hsp72 caused accumulation of ubiquitinated P protein and delayed P protein degradation. These results show that Hsp72 is recruited to IBs and regulates the degradation of MuV P protein through the ubiquitin-proteasome pathway. IMPORTANCE Formation of cytoplasmic inclusion bodies (IBs) is a common characteristic feature in mononegavirus infections. IBs are considered to be the sites of viral RNA replication and transcription. However, there have been few studies focused on host factors recruited to the IBs and their biological functions. Here, we identified stress-inducible heat shock protein 70 (Hsp72) as the first cellular partner of mumps virus (MuV) phosphoprotein (P protein), which is an essential component of the IBs and is involved in viral RNA replication/transcription. We found that the Hsp72 mobilized to the IBs promoted degradation of the MuV P protein through the ubiquitin-proteasome pathway. Our data provide new insight into the role played by IBs in mononegavirus infection. PMID:25552722

  5. Determination of the non-ionic detergent insolubility and phosphoprotein associations of glycosylphosphatidylinositol-anchored proteins expressed on T cells.

    PubMed

    Solomon, K R; Mallory, M A; Finberg, R W

    1998-09-01

    Glycosylphosphatidylinositol (GPI)-anchored proteins are poorly solublized in non-ionic detergents such as Triton X-100 and Nonidet P40, but are easily solublized by detergents with high critical micelle concentrations such as octylglucoside. This solubility profile has been suggested to be due to the localization of GPI-anchored proteins to lipid microdomains rich in cholesterol and sphingolipids. Additionally, GPI-anchored proteins expressed on haemopoietic cells have been shown to associate with src-family tyrosine kinases and heterotrimeric G proteins. Despite these observations, the non-ionic detergent insolubility of GPI-anchored proteins on haemopoietic cells has not been quantified nor has a relationship between the non-ionic detergent insolubility of these proteins and their association with signal-transduction molecules been identified. Here we show that GPI-anchored proteins found on T-cell tumours and activated T cells, although significantly more insoluble then transmembrane proteins, are not uniform in their detergent insolubility. Whereas CD59 was between 4% and 13% soluble, CD48 was between 13% and 25% soluble, CD55 was between 20% and 30% soluble, and CD109 was between 34% and 75% soluble. The ability of these GPI-anchored proteins to associate with phosphoproteins was correlated with their detergent insolubility: the more detergent-insoluble that a GPI-anchored protein was, the greater the level of phosphoprotein associations. These experiments reveal a relationship between non-ionic detergent insolubility and association with signal-transduction molecules and suggest a cause-and-effect relationship between these two properties. In total, these experiments support the hypothesis that the association of GPI-anchored proteins with signalling molecules is due to their sorting to lipid microdomains.

  6. Immobilization of a phosphonated analog of matrix phosphoproteins within cross-linked collagen as a templating mechanism for biomimetic mineralization

    PubMed Central

    Gu, Li-sha; Kim, Young Kyung; Liu, Yan; Takahashi, Kei; Arun, Senthil; Wimmer, Courtney E.; Osorio, Raquel; Ling, Jun-qi; Looney, Stephen W.; Pashley, David H.; Tay, Franklin R.

    2010-01-01

    Immobilization of phosphoproteins on a collagen matrix is important for induction of intrafibrillar apatite mineralization. Unlike phosphate esters, polyphosphonic acid has no reactive sites for covalent binding to collagen amine groups. Binding of polyvinylphosphonic acid (PVPA), a biomimetic templating analog of matrix phosphoproteins, to collagen was found to be electrostatic in nature. Thus, an alternative retention mechanism was designed for immobilization of PVPA to collagen by cross-linking the latter with carbodiimide (EDC). This mechanism is based on the principle of size exclusion entrapment of PVPA molecules within the internal water compartments of collagen. By cross-linking collagen with EDC, a zero-length cross-linking agent, the sieving property of collagen is increased, enabling the PVPA to be immobilized within the collagen. Absence of covalent cross-linking between PVPA and collagen was confirmed by FT-IR spectroscopy. Based on these results, a concentration range for immobilized PVPA to template intrafibrillar apatite deposition was established and validated using a single-layer reconstituted type I collagen mineralization model. In the presence of a polyacrylic acid-containing mineralization medium, optimal intrafibrillar mineralization of the EDC-cross-linked collagen was achieved using 500 and 1,000 μg/mL PVPA. The mineralized fibrils exhibited a hierarchical order of intrafibrillar mineral infiltration, as manifested by the appearance of electron-dense periodicity within unstained fibrils. Understanding the basic processes in intrafibrillar mineralization of reconstituted collagen creates opportunities for the design of tissue engineering materials for hard tissue repair and regeneration. PMID:20688200

  7. RPG: the Ribosomal Protein Gene database

    PubMed Central

    Nakao, Akihiro; Yoshihama, Maki; Kenmochi, Naoya

    2004-01-01

    RPG (http://ribosome.miyazaki-med.ac.jp/) is a new database that provides detailed information about ribosomal protein (RP) genes. It contains data from humans and other organisms, including Drosophila melanogaster, Caenorhabditis elegans, Saccharo myces cerevisiae, Methanococcus jannaschii and Escherichia coli. Users can search the database by gene name and organism. Each record includes sequences (genomic, cDNA and amino acid sequences), intron/exon structures, genomic locations and information about orthologs. In addition, users can view and compare the gene structures of the above organisms and make multiple amino acid sequence alignments. RPG also provides information on small nucleolar RNAs (snoRNAs) that are encoded in the introns of RP genes. PMID:14681386

  8. RPG: the Ribosomal Protein Gene database.

    PubMed

    Nakao, Akihiro; Yoshihama, Maki; Kenmochi, Naoya

    2004-01-01

    RPG (http://ribosome.miyazaki-med.ac.jp/) is a new database that provides detailed information about ribosomal protein (RP) genes. It contains data from humans and other organisms, including Drosophila melanogaster, Caenorhabditis elegans, Saccharo myces cerevisiae, Methanococcus jannaschii and Escherichia coli. Users can search the database by gene name and organism. Each record includes sequences (genomic, cDNA and amino acid sequences), intron/exon structures, genomic locations and information about orthologs. In addition, users can view and compare the gene structures of the above organisms and make multiple amino acid sequence alignments. RPG also provides information on small nucleolar RNAs (snoRNAs) that are encoded in the introns of RP genes.

  9. Tertiary interactions within the ribosomal exit tunnel.

    PubMed

    Kosolapov, Andrey; Deutsch, Carol

    2009-04-01

    Although tertiary folding of whole protein domains is prohibited by the cramped dimensions of the ribosomal tunnel, dynamic tertiary interactions may permit folding of small elementary units within the tunnel. To probe this possibility, we used a beta-hairpin and an alpha-helical hairpin from the cytosolic N terminus of a voltage-gated potassium channel and determined a probability of folding for each at defined locations inside and outside the tunnel. Minimalist tertiary structures can form near the exit port of the tunnel, a region that provides an entropic window for initial exploration of local peptide conformations. Tertiary subdomains of the nascent peptide fold sequentially, but not independently, during translation. These studies offer an approach for diagnosing the molecular basis for folding defects that lead to protein malfunction and provide insight into the role of the ribosome during early potassium channel biogenesis.

  10. Cryo-EM structure of the spinach chloroplast ribosome reveals the location of plastid-specific ribosomal proteins and extensions.

    PubMed

    Graf, Michael; Arenz, Stefan; Huter, Paul; Dönhöfer, Alexandra; Nováček, Jiří; Wilson, Daniel N

    2016-12-15

    Ribosomes are the protein synthesizing machines of the cell. Recent advances in cryo-EM have led to the determination of structures from a variety of species, including bacterial 70S and eukaryotic 80S ribosomes as well as mitoribosomes from eukaryotic mitochondria, however, to date high resolution structures of plastid 70S ribosomes have been lacking. Here we present a cryo-EM structure of the spinach chloroplast 70S ribosome, with an average resolution of 5.4 Å for the small 30S subunit and 3.6 Å for the large 50S ribosomal subunit. The structure reveals the location of the plastid-specific ribosomal proteins (RPs) PSRP1, PSRP4, PSRP5 and PSRP6 as well as the numerous plastid-specific extensions of the RPs. We discover many features by which the plastid-specific extensions stabilize the ribosome via establishing additional interactions with surrounding ribosomal RNA and RPs. Moreover, we identify a large conglomerate of plastid-specific protein mass adjacent to the tunnel exit site that could facilitate interaction of the chloroplast ribosome with the thylakoid membrane and the protein-targeting machinery. Comparing the Escherichia coli 70S ribosome with that of the spinach chloroplast ribosome provides detailed insight into the co-evolution of RP and rRNA.

  11. Disassembly of yeast 80S ribosomes into subunits is a concerted action of ribosome-assisted folding of denatured protein.

    PubMed

    Chakraborty, Biprashekhar; Bhakta, Sayan; Sengupta, Jayati

    2016-01-22

    It has been shown by several groups that ribosome can assist folding of denatured protein in vitro and the process is conserved across the species. Domain V of large ribosomal rRNA which occupies the intersubunit side of the large subunit was identified as the key player responsible for chaperoning the folding process. Thus, it is conceivable that denatured protein needs to access the intersubunit space of the ribosome in order to get folded. In this study, we have investigated the mechanism of release of the protein from the eukaryotic ribosome following reactivation. We have observed significant splitting of yeast 80S ribosome when incubated with the denatured BCAII protein. Energy-free disassembly mechanism functions in low Mg(+2) ion concentration for prokaryotic ribosomes. Eukaryotic ribosomes do not show significant splitting even at low Mg(+2) ion concentration. In this respect, denatured protein-induced disassembly of eukaryotic ribosome without the involvement of any external energy source is intriguing. For prokaryotic ribosomes, it was reported that the denatured protein induces ribosome splitting into subunits in order to access domain V-rRNA. In contrast, our results suggest an alternative mechanism for eukaryotic ribosomal rRNA-mediated protein folding and subsequent separation of the subunits by which release of the activated-protein occurs.

  12. Release of nonstop ribosomes is essential.

    PubMed

    Feaga, Heather A; Viollier, Patrick H; Keiler, Kenneth C

    2014-11-11

    Bacterial ribosomes frequently translate to the 3' end of an mRNA without terminating at a stop codon. Almost all bacteria use the transfer-messenger RNA (tmRNA)-based trans-translation pathway to release these "nonstop" ribosomes and maintain protein synthesis capacity. trans-translation is essential in some species, but in others, such as Caulobacter crescentus, trans-translation can be inactivated. To determine why trans-translation is dispensable in C. crescentus, a Tn-seq screen was used to identify genes that specifically alter growth in cells lacking ssrA, the gene encoding tmRNA. One of these genes, CC1214, was essential in ΔssrA cells. Purified CC1214 protein could release nonstop ribosomes in vitro. CC1214 is a homolog of the Escherichia coli ArfB protein, and using the CC1214 sequence, ArfB homologs were identified in the majority of bacterial phyla. Most species in which ssrA has been deleted contain an ArfB homolog, suggesting that release of nonstop ribosomes may be essential in most or all bacteria. Genes that are conserved across large phylogenetic distances are expected to confer a selective advantage. The genes required for trans-translation, ssrA and smpB, have been found in >99% of sequenced bacterial genomes, suggesting that they are broadly important. However, these genes can be deleted in some species without loss of viability. The identification and characterization of C. crescentus ArfB reveals why trans-translation is not essential in C. crescentus and suggests that many other bacteria are likely to use ArfB to survive when trans-translation is compromised. Copyright © 2014 Feaga et al.

  13. Mitochondrial ribosome assembly in health and disease

    PubMed Central

    De Silva, Dasmanthie; Tu, Ya-Ting; Amunts, Alexey; Fontanesi, Flavia; Barrientos, Antoni

    2015-01-01

    The ribosome is a structurally and functionally conserved macromolecular machine universally responsible for catalyzing protein synthesis. Within eukaryotic cells, mitochondria contain their own ribosomes (mitoribosomes), which synthesize a handful of proteins, all essential for the biogenesis of the oxidative phosphorylation system. High-resolution cryo-EM structures of the yeast, porcine and human mitoribosomal subunits and of the entire human mitoribosome have uncovered a wealth of new information to illustrate their evolutionary divergence from their bacterial ancestors and their adaptation to synthesis of highly hydrophobic membrane proteins. With such structural data becoming available, one of the most important remaining questions is that of the mitoribosome assembly pathway and factors involved. The regulation of mitoribosome biogenesis is paramount to mitochondrial respiration, and thus to cell viability, growth and differentiation. Moreover, mutations affecting the rRNA and protein components produce severe human mitochondrial disorders. Despite its biological and biomedical significance, knowledge on mitoribosome biogenesis and its deviations from the much-studied bacterial ribosome assembly processes is scarce, especially the order of rRNA processing and assembly events and the regulatory factors required to achieve fully functional particles. This article focuses on summarizing the current available information on mitoribosome assembly pathway, factors that form the mitoribosome assembly machinery, and the effect of defective mitoribosome assembly on human health. PMID:26030272

  14. Mitochondrial ribosome assembly in health and disease.

    PubMed

    De Silva, Dasmanthie; Tu, Ya-Ting; Amunts, Alexey; Fontanesi, Flavia; Barrientos, Antoni

    2015-01-01

    The ribosome is a structurally and functionally conserved macromolecular machine universally responsible for catalyzing protein synthesis. Within eukaryotic cells, mitochondria contain their own ribosomes (mitoribosomes), which synthesize a handful of proteins, all essential for the biogenesis of the oxidative phosphorylation system. High-resolution cryo-EM structures of the yeast, porcine and human mitoribosomal subunits and of the entire human mitoribosome have uncovered a wealth of new information to illustrate their evolutionary divergence from their bacterial ancestors and their adaptation to synthesis of highly hydrophobic membrane proteins. With such structural data becoming available, one of the most important remaining questions is that of the mitoribosome assembly pathway and factors involved. The regulation of mitoribosome biogenesis is paramount to mitochondrial respiration, and thus to cell viability, growth and differentiation. Moreover, mutations affecting the rRNA and protein components produce severe human mitochondrial disorders. Despite its biological and biomedical significance, knowledge on mitoribosome biogenesis and its deviations from the much-studied bacterial ribosome assembly processes is scarce, especially the order of rRNA processing and assembly events and the regulatory factors required to achieve fully functional particles. This article focuses on summarizing the current available information on mitoribosome assembly pathway, factors that form the mitoribosome assembly machinery, and the effect of defective mitoribosome assembly on human health.

  15. The ribosome challenge to the RNA world.

    PubMed

    Bowman, Jessica C; Hud, Nicholas V; Williams, Loren Dean

    2015-04-01

    An RNA World that predated the modern world of polypeptide and polynucleotide is one of the most widely accepted models in origin of life research. In this model, the translation system shepherded the RNA World into the extant biology of DNA, RNA, and protein. Here, we examine the RNA World Hypothesis in the context of increasingly detailed information available about the origins, evolution, functions, and mechanisms of the translation system. We conclude that the translation system presents critical challenges to RNA World Hypotheses. Firstly, a timeline of the RNA World is problematic when the ribosome is incorporated. The mechanism of peptidyl transfer of the ribosome appears distinct from evolved enzymes, signaling origins in a chemical rather than biological milieu. Secondly, we have no evidence that the basic biochemical toolset of life is subject to substantive change by Darwinian evolution, as required for the transition from the RNA world to extant biology. Thirdly, we do not see specific evidence for biological takeover of ribozyme function by protein enzymes. Finally, we can find no basis for preservation of the ribosome as ribozyme or the universality of translation, if it were the case that other information transducing ribozymes, such as ribozyme polymerases, were replaced by protein analogs and erased from the phylogenetic record. We suggest that an updated model of the RNA World should address the current state of knowledge of the translation system.

  16. Quantitative profiling of initiating ribosomes in vivo.

    PubMed

    Gao, Xiangwei; Wan, Ji; Liu, Botao; Ma, Ming; Shen, Ben; Qian, Shu-Bing

    2015-02-01

    Cells have evolved exquisite mechanisms to fine-tune the rate of protein synthesis in response to stress. Systemic mapping of start-codon positions and precise measurement of the corresponding initiation rate would transform our understanding of translational control. Here we present quantitative translation initiation sequencing (QTI-seq), with which the initiating ribosomes can be profiled in real time at single-nucleotide resolution. Resultant initiation maps not only delineated variations of start-codon selection but also highlighted a dynamic range of initiation rates in response to nutrient starvation. The integrated data set provided unique insights into principles of alternative translation and mechanisms controlling different aspects of translation initiation. With RiboTag mice, QTI-seq permitted tissue-specific profiling of initiating ribosomes in vivo. Liver cell-specific ribosome profiling uncovered a robust translational reprogramming of the proteasome system in fasted mice. Our findings illuminated the prevalence and dynamic nature of translational regulation pivotal to physiological adaptation in vivo.

  17. Prefabrication of a ribosomal protein subcomplex essential for eukaryotic ribosome formation

    PubMed Central

    Peña, Cohue; Schütz, Sabina; Fischer, Ute; Chang, Yiming; Panse, Vikram G

    2016-01-01

    Spatial clustering of ribosomal proteins (r-proteins) through tertiary interactions is a striking structural feature of the eukaryotic ribosome. However, the functional importance of these intricate inter-connections, and how they are established is currently unclear. Here, we reveal that a conserved ATPase, Fap7, organizes interactions between neighboring r-proteins uS11 and eS26 prior to their delivery to the earliest ribosome precursor, the 90S. In vitro, uS11 only when bound to Fap7 becomes competent to recruit eS26 through tertiary contacts found between these r-proteins on the mature ribosome. Subsequently, Fap7 ATPase activity unloads the uS11:eS26 subcomplex onto its rRNA binding site, and therefore ensures stoichiometric integration of these r-proteins into the 90S. Fap7-depletion in vivo renders uS11 susceptible to proteolysis, and precludes eS26 incorporation into the 90S. Thus, prefabrication of a native-like r-protein subcomplex drives efficient and accurate construction of the eukaryotic ribosome. DOI: http://dx.doi.org/10.7554/eLife.21755.001 PMID:27929371

  18. Functional dichotomy of ribosomal proteins during the synthesis of mammalian 40S ribosomal subunits.

    PubMed

    O'Donohue, Marie-Françoise; Choesmel, Valérie; Faubladier, Marlène; Fichant, Gwennaële; Gleizes, Pierre-Emmanuel

    2010-09-06

    Our knowledge of the functions of metazoan ribosomal proteins in ribosome synthesis remains fragmentary. Using siRNAs, we show that knockdown of 31 of the 32 ribosomal proteins of the human 40S subunit (ribosomal protein of the small subunit [RPS]) strongly affects pre-ribosomal RNA (rRNA) processing, which often correlates with nucleolar chromatin disorganization. 16 RPSs are strictly required for initiating processing of the sequences flanking the 18S rRNA in the pre-rRNA except at the metazoan-specific early cleavage site. The remaining 16 proteins are necessary for progression of the nuclear and cytoplasmic maturation steps and for nuclear export. Distribution of these two subsets of RPSs in the 40S subunit structure argues for a tight dependence of pre-rRNA processing initiation on the folding of both the body and the head of the forming subunit. Interestingly, the functional dichotomy of RPS proteins reported in this study is correlated with the mutation frequency of RPS genes in Diamond-Blackfan anemia.

  19. Functional Interaction between Ribosomal Protein L6 and RbgA during Ribosome Assembly

    PubMed Central

    Davis, Joseph H.; Williamson, James R.; Britton, Robert A.

    2014-01-01

    RbgA is an essential GTPase that participates in the assembly of the large ribosomal subunit in Bacillus subtilis and its homologs are implicated in mitochondrial and eukaryotic large subunit assembly. How RbgA functions in this process is still poorly understood. To gain insight into the function of RbgA we isolated suppressor mutations that partially restored the growth of an RbgA mutation (RbgA-F6A) that caused a severe growth defect. Analysis of these suppressors identified mutations in rplF, encoding ribosomal protein L6. The suppressor strains all accumulated a novel ribosome intermediate that migrates at 44S in sucrose gradients. All of the mutations cluster in a region of L6 that is in close contact with helix 97 of the 23S rRNA. In vitro maturation assays indicate that the L6 substitutions allow the defective RbgA-F6A protein to function more effectively in ribosome maturation. Our results suggest that RbgA functions to properly position L6 on the ribosome, prior to the incorporation of L16 and other late assembly proteins. PMID:25330043

  20. Ribosome biogenesis; the KsgA protein throws a methyl-mediated switch in ribosome assembly.

    PubMed

    Mangat, Chand S; Brown, Eric D

    2008-12-01

    Many trans-acting factors that aid in ribosome biogenesis have been identified in higher organisms but relatively few such factors are known in prokaryotes. In bacteria, the list of such factors includes ATP-energized helicases and chaperones as well as an emerging cadre of switch GTPases. The KsgA protein is a universally conserved methyltransferase that dimethylates both A1518 and A1519 of the 16S rRNA of the small ribosomal subunit. Methylation has long been thought to be solely for fine-tuning of protein translation. In this issue of Molecular Microbiology, Connolly et al. present data suggesting KsgA might function in the assembly of the small subunit of the ribosome. Indeed, the work indicates that KsgA might have a checkpoint role in ribosome biogenesis where methylation by this protein marks the completion of its assembly role. These findings open our thinking to new candidate assembly factors and provide a new direction for understanding ribosome assembly.

  1. Functional interaction between ribosomal protein L6 and RbgA during ribosome assembly.

    PubMed

    Gulati, Megha; Jain, Nikhil; Davis, Joseph H; Williamson, James R; Britton, Robert A

    2014-10-01

    RbgA is an essential GTPase that participates in the assembly of the large ribosomal subunit in Bacillus subtilis and its homologs are implicated in mitochondrial and eukaryotic large subunit assembly. How RbgA functions in this process is still poorly understood. To gain insight into the function of RbgA we isolated suppressor mutations that partially restored the growth of an RbgA mutation (RbgA-F6A) that caused a severe growth defect. Analysis of these suppressors identified mutations in rplF, encoding ribosomal protein L6. The suppressor strains all accumulated a novel ribosome intermediate that migrates at 44S in sucrose gradients. All of the mutations cluster in a region of L6 that is in close contact with helix 97 of the 23S rRNA. In vitro maturation assays indicate that the L6 substitutions allow the defective RbgA-F6A protein to function more effectively in ribosome maturation. Our results suggest that RbgA functions to properly position L6 on the ribosome, prior to the incorporation of L16 and other late assembly proteins.

  2. Polar bears, antibiotics, and the evolving ribosome (Nobel Lecture).

    PubMed

    Yonath, Ada

    2010-06-14

    High-resolution structures of ribosomes, the cellular machines that translate the genetic code into proteins, revealed the decoding mechanism, detected the mRNA path, identified the sites of the tRNA molecules in the ribosome, elucidated the position and the nature of the nascent proteins exit tunnel, illuminated the interactions of the ribosome with non-ribosomal factors, such as the initiation, release and recycling factors, and provided valuable information on ribosomal antibiotics, their binding sites, modes of action, principles of selectivity and the mechanisms leading to their resistance. Notably, these structures proved that the ribosome is a ribozyme whose active site, namely where the peptide bonds are being formed, is situated within a universal symmetrical region that is embedded in the otherwise asymmetric ribosome structure. As this symmetrical region is highly conserved and provides the machinery required for peptide bond formation and for ribosome polymerase activity, it may be the remnant of the proto-ribosome, a dimeric prebiotic machine that formed peptide bonds and non-coded polypeptide chains. Structures of complexes of ribosomes with antibiotics targeting them revealed the principles allowing for their clinical use, identified resistance mechanisms and showed the structural bases for discriminating pathogenic bacteria from hosts, hence providing valuable structural information for antibiotics improvement and for the design of novel compounds that can serve as antibiotics.

  3. Fluorescence-based monitoring of ribosome assembly landscapes.

    PubMed

    Nikolay, Rainer; Schloemer, Renate; Mueller, Silke; Deuerling, Elke

    2015-02-25

    Ribosomes and functional complexes of them have been analyzed at the atomic level. Far less is known about the dynamic assembly and degradation events that define the half-life of ribosomes and guarantee their quality control. We developed a system that allows visualization of intact ribosomal subunits and assembly intermediates (i.e. assembly landscapes) by convenient fluorescence-based analysis. To this end, we labeled the early assembly ribosomal proteins L1 and S15 with the fluorescent proteins mAzami green and mCherry, respectively, using chromosomal gene insertion. The reporter strain harbors fluorescently labeled ribosomal subunits that operate wild type-like, as shown by biochemical and growth assays. Using genetic and chemical perturbations by depleting genes encoding the ribosomal proteins L3 and S17, respectively, or using ribosome-targeting antibiotics, we provoked ribosomal subunit assembly defects. These defects were readily identified by fluorometric analysis after sucrose density centrifugation in unprecedented resolution. This strategy is useful to monitor and characterize subunit specific assembly defects caused by ribosome-targeting drugs that are currently used and to characterize new molecules that affect ribosome assembly and thereby constitute new classes of antibacterial agents.

  4. An overview of pre-ribosomal RNA processing in eukaryotes

    PubMed Central

    Henras, Anthony K; Plisson-Chastang, Célia; O'Donohue, Marie-Françoise; Chakraborty, Anirban; Gleizes, Pierre-Emmanuel

    2015-01-01

    Ribosomal RNAs are the most abundant and universal noncoding RNAs in living organisms. In eukaryotes, three of the four ribosomal RNAs forming the 40S and 60S subunits are borne by a long polycistronic pre-ribosomal RNA. A complex sequence of processing steps is required to gradually release the mature RNAs from this precursor, concomitant with the assembly of the 79 ribosomal proteins. A large set of trans-acting factors chaperone this process, including small nucleolar ribonucleoparticles. While yeast has been the gold standard for studying the molecular basis of this process, recent technical advances have allowed to further define the mechanisms of ribosome biogenesis in animals and plants. This renewed interest for a long-lasting question has been fueled by the association of several genetic diseases with mutations in genes encoding both ribosomal proteins and ribosome biogenesis factors, and by the perspective of new anticancer treatments targeting the mechanisms of ribosome synthesis. A consensus scheme of pre-ribosomal RNA maturation is emerging from studies in various kinds of eukaryotic organisms. However, major differences between mammalian and yeast pre-ribosomal RNA processing have recently come to light. WIREs RNA 2015, 6:225–242. doi: 10.1002/wrna.1269 PMID:25346433

  5. Structure of a mitochondrial ribosome with minimal RNA

    PubMed Central

    Sharma, Manjuli R.; Booth, Timothy M.; Simpson, Larry; Maslov, Dmitri A.; Agrawal, Rajendra K.

    2009-01-01

    The Leishmania tarentolae mitochondrial ribosome (Lmr) is a minimal ribosomal RNA (rRNA)-containing ribosome. We have obtained a cryo-EM map of the Lmr. The map reveals several features that have not been seen in previously-determined structures of eubacterial or eukaryotic (cytoplasmic or organellar) ribosomes to our knowledge. Comparisons of the Lmr map with X-ray crystallographic and cryo-EM maps of the eubacterial ribosomes and a cryo-EM map of the mammalian mitochondrial ribosome show that (i) the overall structure of the Lmr is considerably more porous, (ii) the topology of the intersubunit space is significantly different, with fewer intersubunit bridges, but more tunnels, and (iii) several of the functionally-important rRNA regions, including the α-sarcin-ricin loop, have different relative positions within the structure. Furthermore, the major portions of the mRNA channel, the tRNA passage, and the nascent polypeptide exit tunnel contain Lmr-specific proteins, suggesting that the mechanisms for mRNA recruitment, tRNA interaction, and exiting of the nascent polypeptide in Lmr must differ markedly from the mechanisms deduced for ribosomes in other organisms. Our study identifies certain structural features that are characteristic solely of mitochondrial ribosomes and other features that are characteristic of both mitochondrial and chloroplast ribosomes (i.e., organellar ribosomes). PMID:19497863

  6. Ribosomal History Reveals Origins of Modern Protein Synthesis

    PubMed Central

    Harish, Ajith; Caetano-Anollés, Gustavo

    2012-01-01

    The origin and evolution of the ribosome is central to our understanding of the cellular world. Most hypotheses posit that the ribosome originated in the peptidyl transferase center of the large ribosomal subunit. However, these proposals do not link protein synthesis to RNA recognition and do not use a phylogenetic comparative framework to study ribosomal evolution. Here we infer evolution of the structural components of the ribosome. Phylogenetic methods widely used in morphometrics are applied directly to RNA structures of thousands of molecules and to a census of protein structures in hundreds of genomes. We find that components of the small subunit involved in ribosomal processivity evolved earlier than the catalytic peptidyl transferase center responsible for protein synthesis. Remarkably, subunit RNA and proteins coevolved, starting with interactions between the oldest proteins (S12 and S17) and the oldest substructure (the ribosomal ratchet) in the small subunit and ending with the rise of a modern multi-subunit ribosome. Ancestral ribonucleoprotein components show similarities to in vitro evolved RNA replicase ribozymes and protein structures in extant replication machinery. Our study therefore provides important clues about the chicken-or-egg dilemma associated with the central dogma of molecular biology by showing that ribosomal history is driven by the gradual structural accretion of protein and RNA structures. Most importantly, results suggest that functionally important and conserved regions of the ribosome were recruited and could be relics of an ancient ribonucleoprotein world. PMID:22427882

  7. Ribosomal history reveals origins of modern protein synthesis.

    PubMed

    Harish, Ajith; Caetano-Anollés, Gustavo

    2012-01-01

    The origin and evolution of the ribosome is central to our understanding of the cellular world. Most hypotheses posit that the ribosome originated in the peptidyl transferase center of the large ribosomal subunit. However, these proposals do not link protein synthesis to RNA recognition and do not use a phylogenetic comparative framework to study ribosomal evolution. Here we infer evolution of the structural components of the ribosome. Phylogenetic methods widely used in morphometrics are applied directly to RNA structures of thousands of molecules and to a census of protein structures in hundreds of genomes. We find that components of the small subunit involved in ribosomal processivity evolved earlier than the catalytic peptidyl transferase center responsible for protein synthesis. Remarkably, subunit RNA and proteins coevolved, starting with interactions between the oldest proteins (S12 and S17) and the oldest substructure (the ribosomal ratchet) in the small subunit and ending with the rise of a modern multi-subunit ribosome. Ancestral ribonucleoprotein components show similarities to in vitro evolved RNA replicase ribozymes and protein structures in extant replication machinery. Our study therefore provides important clues about the chicken-or-egg dilemma associated with the central dogma of molecular biology by showing that ribosomal history is driven by the gradual structural accretion of protein and RNA structures. Most importantly, results suggest that functionally important and conserved regions of the ribosome were recruited and could be relics of an ancient ribonucleoprotein world.

  8. The Ribosomal Protein Rpl22 Controls Ribosome Composition by Directly Repressing Expression of Its Own Paralog, Rpl22l1

    PubMed Central

    O'Leary, Monique N.; Schreiber, Katherine H.; Zhang, Yong; Duc, Anne-Cécile E.; Rao, Shuyun; Hale, J. Scott; Academia, Emmeline C.; Shah, Shreya R.; Morton, John F.; Holstein, Carly A.; Martin, Dan B.; Kaeberlein, Matt; Ladiges, Warren C.; Fink, Pamela J.; MacKay, Vivian L.; Wiest, David L.; Kennedy, Brian K.

    2013-01-01

    Most yeast ribosomal protein genes are duplicated and their characterization has led to hypotheses regarding the existence of specialized ribosomes with different subunit composition or specifically-tailored functions. In yeast, ribosomal protein genes are generally duplicated and evidence has emerged that paralogs might have specific roles. Unlike yeast, most mammalian ribosomal proteins are thought to be encoded by a single gene copy, raising the possibility that heterogenous populations of ribosomes are unique to yeast. Here, we examine the roles of the mammalian Rpl22, finding that Rpl22−/− mice have only subtle phenotypes with no significant translation defects. We find that in the Rpl22−/− mouse there is a compensatory increase in Rpl22-like1 (Rpl22l1) expression and incorporation into ribosomes. Consistent with the hypothesis that either ribosomal protein can support translation, knockdown of Rpl22l1 impairs growth of cells lacking Rpl22. Mechanistically, Rpl22 regulates Rpl22l1 directly by binding to an internal hairpin structure and repressing its expression. We propose that ribosome specificity may exist in mammals, providing evidence that one ribosomal protein can influence composition of the ribosome by regulating its own paralog. PMID:23990801

  9. The Structure and Function of the Eukaryotic Ribosome

    PubMed Central

    Wilson, Daniel N.; Doudna Cate, Jamie H.

    2012-01-01

    Structures of the bacterial ribosome have provided a framework for understanding universal mechanisms of protein synthesis. However, the eukaryotic ribosome is much larger than it is in bacteria, and its activity is fundamentally different in many key ways. Recent cryo-electron microscopy reconstructions and X-ray crystal structures of eukaryotic ribosomes and ribosomal subunits now provide an unprecedented opportunity to explore mechanisms of eukaryotic translation and its regulation in atomic detail. This review describes the X-ray crystal structures of the Tetrahymena thermophila 40S and 60S subunits and the Saccharomyces cerevisiae 80S ribosome, as well as cryo-electron microscopy reconstructions of translating yeast and plant 80S ribosomes. Mechanistic questions about translation in eukaryotes that will require additional structural insights to be resolved are also presented. PMID:22550233

  10. Reducing Ribosome Biosynthesis Promotes Translation during Low Mg(2+) Stress.

    PubMed

    Pontes, Mauricio H; Yeom, Jinki; Groisman, Eduardo A

    2016-11-03

    The synthesis of ribosomes is regulated by both amino acid abundance and the availability of ATP, which regenerates guanosine triphosphate (GTP), powers ribosomes, and promotes transcription of rRNA genes. We now report that bacteria supersede both of these controls when experiencing low cytosolic magnesium (Mg(2+)), a divalent cation essential for ribosome stabilization and for neutralization of ATP's negative charge. We uncover a regulatory circuit that responds to low cytosolic Mg(2+) by promoting expression of proteins that import Mg(2+) and lower ATP amounts. This response reduces the levels of ATP and ribosomes, making Mg(2+) ions available for translation. Mutants defective in Mg(2+) uptake and unable to reduce ATP levels accumulate non-functional ribosomal components and undergo translational arrest. Our findings establish a paradigm whereby cells reduce the amounts of translating ribosomes to carry out protein synthesis. Copyright © 2016. Published by Elsevier Inc.

  11. Active yeast ribosome preparation using monolithic anion exchange chromatography.

    PubMed

    Munoz, Antonio M; Yourik, Paul; Rajagopal, Vaishnavi; Nanda, Jagpreet S; Lorsch, Jon R; Walker, Sarah E

    2017-02-01

    In vitro studies of translation provide critical mechanistic details, yet purification of large amounts of highly active eukaryotic ribosomes remains a challenge for biochemists and structural biologists. Here, we present an optimized method for preparation of highly active yeast ribosomes that could easily be adapted for purification of ribosomes from other species. The use of a nitrogen mill for cell lysis coupled with chromatographic purification of the ribosomes results in 10-fold-increased yield and less variability compared with the traditional approach, which relies on sedimentation through sucrose cushions. We demonstrate that these ribosomes are equivalent to those made using the traditional method in a host of in vitro assays, and that utilization of this new method will consistently produce high yields of active yeast ribosomes.

  12. Macrolide antibiotics allosterically predispose the ribosome for translation arrest

    PubMed Central

    Sothiselvam, Shanmugapriya; Liu, Bo; Han, Wei; Ramu, Haripriya; Klepacki, Dorota; Atkinson, Gemma Catherine; Brauer, Age; Remm, Maido; Tenson, Tanel; Schulten, Klaus; Vázquez-Laslop, Nora; Mankin, Alexander S.

    2014-01-01

    Translation arrest directed by nascent peptides and small cofactors controls expression of important bacterial and eukaryotic genes, including antibiotic resistance genes, activated by binding of macrolide drugs to the ribosome. Previous studies suggested that specific interactions between the nascent peptide and the antibiotic in the ribosomal exit tunnel play a central role in triggering ribosome stalling. However, here we show that macrolides arrest translation of the truncated ErmDL regulatory peptide when the nascent chain is only three amino acids and therefore is too short to be juxtaposed with the antibiotic. Biochemical probing and molecular dynamics simulations of erythromycin-bound ribosomes showed that the antibiotic in the tunnel allosterically alters the properties of the catalytic center, thereby predisposing the ribosome for halting translation of specific sequences. Our findings offer a new view on the role of small cofactors in the mechanism of translation arrest and reveal an allosteric link between the tunnel and the catalytic center of the ribosome. PMID:24961372

  13. Active yeast ribosome preparation using monolithic anion exchange chromatography

    PubMed Central

    Munoz, Antonio M.; Yourik, Paul; Rajagopal, Vaishnavi; Lorsch, Jon R.

    2017-01-01

    ABSTRACT In vitro studies of translation provide critical mechanistic details, yet purification of large amounts of highly active eukaryotic ribosomes remains a challenge for biochemists and structural biologists. Here, we present an optimized method for preparation of highly active yeast ribosomes that could easily be adapted for purification of ribosomes from other species. The use of a nitrogen mill for cell lysis coupled with chromatographic purification of the ribosomes results in 10-fold-increased yield and less variability compared with the traditional approach, which relies on sedimentation through sucrose cushions. We demonstrate that these ribosomes are equivalent to those made using the traditional method in a host of in vitro assays, and that utilization of this new method will consistently produce high yields of active yeast ribosomes. PMID:27981882

  14. Placeholder factors in ribosome biogenesis: please, pave my way

    PubMed Central

    Espinar-Marchena, Francisco J.; Babiano, Reyes; Cruz, Jesús

    2017-01-01

    The synthesis of cytoplasmic eukaryotic ribosomes is an extraordinarily energy-demanding cellular activity that occurs progressively from the nucleolus to the cytoplasm. In the nucleolus, precursor rRNAs associate with a myriad of trans-acting factors and some ribosomal proteins to form pre-ribosomal particles. These factors include snoRNPs, nucleases, ATPases, GTPases, RNA helicases, and a vast list of proteins with no predicted enzymatic activity. Their coordinate activity orchestrates in a spatiotemporal manner the modification and processing of precursor rRNAs, the rearrangement reactions required for the formation of productive RNA folding intermediates, the ordered assembly of the ribosomal proteins, and the export of pre-ribosomal particles to the cytoplasm; thus, providing speed, directionality and accuracy to the overall process of formation of translation-competent ribosomes. Here, we review a particular class of trans-acting factors known as "placeholders". Placeholder factors temporarily bind selected ribosomal sites until these have achieved a structural context that is appropriate for exchanging the placeholder with another site-specific binding factor. By this strategy, placeholders sterically prevent premature recruitment of subsequently binding factors, premature formation of structures, avoid possible folding traps, and act as molecular clocks that supervise the correct progression of pre-ribosomal particles into functional ribosomal subunits. We summarize the current understanding of those factors that delay the assembly of distinct ribosomal proteins or subsequently bind key sites in pre-ribosomal particles. We also discuss recurrent examples of RNA-protein and protein-protein mimicry between rRNAs and/or factors, which have clear functional implications for the ribosome biogenesis pathway. PMID:28685141

  15. Commandeering the Ribosome: Lessons Learned from Dicistroviruses about Translation

    PubMed Central

    Kerr, Craig H.

    2016-01-01

    To replicate, all viruses depend entirely on the enslavement of host cell ribosomes for their own advantage. To this end, viruses have evolved a multitude of translational strategies to usurp the ribosome. RNA-based structures known as internal ribosome entry sites (IRESs) are among the most notable mechanisms employed by viruses to seize host ribosomes. In this article, we spotlight the intergenic region IRES from the Dicistroviridae family of viruses and its importance as a model for IRES-dependent translation and in understanding fundamental properties of translation. PMID:27053555

  16. 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

  17. Ribosome hibernation factor promotes Staphylococcal survival and differentially represses translation

    PubMed Central

    Basu, Arnab; Yap, Mee-Ngan F.

    2016-01-01

    In opportunistic Gram-positive Staphylococcus aureus, a small protein called hibernation-promoting factor (HPFSa) is sufficient to dimerize 2.5-MDa 70S ribosomes into a translationally inactive 100S complex. Although the 100S dimer is observed in only the stationary phase in Gram-negative gammaproteobacteria, it is ubiquitous throughout all growth phases in S. aureus. The biological significance of the 100S ribosome is poorly understood. Here, we reveal an important role of HPFSa in preserving ribosome integrity and poising cells for translational restart, a process that has significant clinical implications for relapsed staphylococcal infections. We found that the hpf null strain is severely impaired in long-term viability concomitant with a dramatic loss of intact ribosomes. Genome-wide ribosome profiling shows that eliminating HPFSa drastically increased ribosome occupancy at the 5′ end of specific mRNAs under nutrient-limited conditions, suggesting that HPFSa may suppress translation initiation. The protective function of HPFSa on ribosomes resides at the N-terminal conserved basic residues and the extended C-terminal segment, which are critical for dimerization and ribosome binding, respectively. These data provide significant insight into the functional consequences of 100S ribosome loss for protein synthesis and stress adaptation. PMID:27001516

  18. Can Structures Lead to Better Drugs? Lessons from Ribosome Research

    NASA Astrophysics Data System (ADS)

    Yonath, Ada

    Ribosome research has undergone astonishing progress in recent years. Crystal structures have shed light on the functional properties of the translation machinery and revealed how the ribosome's striking architecture is ingeniously designed as the framework for its unique capabilities: precise decoding, substrate mediated peptide-bond formation and efficient poly-merase activity. New findings include the two concerted elements of tRNA translocation: sideways shift and a ribosomal-navigated rotatory motion; the dynamics of the nascent chain exit tunnel and the shelter formed by the ribosome-bound trigger-factor, which acts as a chaperone to prevent nascent chain aggregation and misfolding.

  19. Alterations in the ribosomal machinery in cancer and hematologic disorders

    PubMed Central

    2012-01-01

    Ribosomes are essential components of the protein translation machinery and are composed of more than 80 unique large and small ribosomal proteins. Recent studies show that in addition to their roles in protein translation, ribosomal proteins are also involved in extra-ribosomal functions of DNA repair, apoptosis and cellular homeostasis. Consequently, alterations in the synthesis or functioning of ribosomal proteins can lead to various hematologic disorders. These include congenital anemias such as Diamond Blackfan anemia and Shwachman Diamond syndrome; both of which are associated with mutations in various ribosomal genes. Acquired uniallelic deletion of RPS14 gene has also been shown to lead to the 5q syndrome, a distinct subset of MDS associated with macrocytic anemia. Recent evidence shows that specific ribosomal proteins are overexpressed in liver, colon, prostate and other tumors. Ribosomal protein overexpression can promote tumorigenesis by interactions with the p53 tumor suppressor pathway and also by direct effects on various oncogenes. These data point to a broad role of ribosome protein alterations in hematologic and oncologic diseases. PMID:22709827

  20. Quantitative proteomic analysis of ribosome assembly and turnover in vivo.

    PubMed

    Sykes, Michael T; Shajani, Zahra; Sperling, Edit; Beck, Andrea H; Williamson, James R

    2010-10-29

    Although high-resolution structures of the ribosome have been solved in a series of functional states, relatively little is known about how the ribosome assembles, particularly in vivo. Here, a general method is presented for studying the dynamics of ribosome assembly and ribosomal assembly intermediates. Since significant quantities of assembly intermediates are not present under normal growth conditions, the antibiotic neomycin is used to perturb wild-type Escherichia coli. Treatment of E. coli with the antibiotic neomycin results in the accumulation of a continuum of assembly intermediates for both the 30S and 50S subunits. The protein composition and the protein stoichiometry of these intermediates were determined by quantitative mass spectrometry using purified unlabeled and (15)N-labeled wild-type ribosomes as external standards. The intermediates throughout the continuum are heterogeneous and are largely depleted of late-binding proteins. Pulse-labeling with (15)N-labeled medium time-stamps the ribosomal proteins based on their time of synthesis. The assembly intermediates contain both newly synthesized proteins and proteins that originated in previously synthesized intact subunits. This observation requires either a significant amount of ribosome degradation or the exchange or reuse of ribosomal proteins. These specific methods can be applied to any system where ribosomal assembly intermediates accumulate, including strains with deletions or mutations of assembly factors. This general approach can be applied to study the dynamics of assembly and turnover of other macromolecular complexes that can be isolated from cells. Copyright © 2010 Elsevier Ltd. All rights reserved.

  1. Effects of detergents on ribosomal precursor subunits of Bacillus megaterium.

    PubMed

    Body, A; Brownstein, B H

    1978-01-01

    Cell extracts prepared by osmotic lysis of protoplasts were analyzed by sucrose gradient sedimentation. In the absence of detergents, ribosomal precursor particles were found in a gradient fraction which sedimented faster than mature 50S subunits and in two other fractions coincident with mature 50S and 30S ribosomal subunits. Phospholipid, an indicator of membrane, was shown to be associated with only the fastest-sedimenting ribosomal precursor particle fraction. After the extracts were treated with detergents, all phospholipid was found at the top of the gradients. Brij 58, Triton X-100, and Nonidet P-40 did not cause a change in the sedimentation values of precursors; however, the detergents deoxycholate or LOC (Amway Corp.) disrupted the fastest-sedimenting precursor and converted the ribosomal precursor subunits which sedimented at the 50S and 30S positions to five different classes of more slowly sedimenting particles. Earlier reports on the in vivo assembly of ribosomal subunits have shown that several stages of ribosomal precursor subunits exist, and, in the presence of the detergents deoxycholate and LOC, which had been used to prepare cell extracts, the precursors sedimented more slowly. Our data are consistent with the hypothesis that those detergents selectively modify the structure of ribosomal precursors and lend further support to the hypothesis that the in vivo ribosomal precursor subunits have 50S and 30S sedimentation values. In addition, these data support the idea that the ribosomal precursor particles found in the fast-sedimenting fraction may constitute a unique precursor fraction.

  2. Effects of Detergents on Ribosomal Precursor Subunits of Bacillus megaterium

    PubMed Central

    Body, Barbara A.; Brownstein, Bernard H.

    1978-01-01

    Cell extracts prepared by osmotic lysis of protoplasts were analyzed by sucrose gradient sedimentation. In the absence of detergents, ribosomal precursor particles were found in a gradient fraction which sedimented faster than mature 50S subunits and in two other fractions coincident with mature 50S and 30S ribosomal subunits. Phospholipid, an indicator of membrane, was shown to be associated with only the fastest-sedimenting ribosomal precursor particle fraction. After the extracts were treated with detergents, all phospholipid was found at the top of the gradients. Brij 58, Triton X-100, and Nonidet P-40 did not cause a change in the sedimentation values of precursors; however, the detergents deoxycholate or LOC (Amway Corp.) disrupted the fastest-sedimenting precursor and converted the ribosomal precursor subunits which sedimented at the 50S and 30S positions to five different classes of more slowly sedimenting particles. Earlier reports on the in vivo assembly of ribosomal subunits have shown that several stages of ribosomal precursor subunits exist, and, in the presence of the detergents deoxycholate and LOC, which had been used to prepare cell extracts, the precursors sedimented more slowly. Our data are consistent with the hypothesis that those detergents selectively modify the structure of ribosomal precursors and lend further support to the hypothesis that the in vivo ribosomal precursor subunits have 50S and 30S sedimentation values. In addition, these data support the idea that the ribosomal precursor particles found in the fast-sedimenting fraction may constitute a unique precursor fraction. PMID:412833

  3. Ribosome Footprint Profiling of Translation throughout the Genome

    PubMed Central

    Ingolia, Nicholas T.

    2016-01-01

    Ribosome profiling has emerged as a technique for measuring translation comprehensively and quantitatively by deep sequencing of ribosome-protected mRNA fragments. By identifying the precise positions of ribosomes, footprinting experiments have unveiled key insights into the composition and regulation of the expressed proteome, including delineating potentially functional micropeptides, revealing pervasive translation on cytosolic RNAs, and identifying differences in elongation rates driven by codon usage or other factors. This Primer looks at important experimental and analytical concerns for executing ribosome profiling experiments and surveys recent examples where the approach was developed to explore protein biogenesis and homeostasis. PMID:27015305

  4. Ribosome recycling: An essential process of protein synthesis.

    PubMed

    Kiel, Michael C; Kaji, Hideko; Kaji, Akira

    2007-01-01

    A preponderance of textbooks outlines cellular protein synthesis (translation) in three basic steps: initiation, elongation, and termination. However, researchers in the field of translation accept that a vital fourth step exists; this fourth step is called ribosome recycling. Ribosome recycling occurs after the nascent polypeptide has been released during the termination step. Despite the release of the polypeptide, ribosomes remain bound to the mRNA and tRNA. It is only during the fourth step of translation that ribosomes are ultimately released from the mRNA, split into subunits, and are free to bind new mRNA, thus the term "ribosome recycling." This step is essential to the viability of cells. In bacteria, it is catalyzed by two proteins, elongation factor G and ribosome recycling factor, a near perfect structural mimic of tRNA. Eukaryotic organelles such as mitochondria and chloroplasts possess ribosome recycling factor and elongation factor G homologues, but the nature of ribosome recycling in eukaryotic cytoplasm is still under investigation. In this review, the discovery of ribosome recycling and the basic mechanisms involved are discussed so that textbook writers and teachers can include this vital step, which is just as important as the three conventional steps, in sections dealing with protein synthesis.

  5. On the Ribosomal Density that Maximizes Protein Translation Rate

    PubMed Central

    Zarai, Yoram; Margaliot, Michael; Tuller, Tamir

    2016-01-01

    During mRNA translation, several ribosomes attach to the same mRNA molecule simultaneously translating it into a protein. This pipelining increases the protein translation rate. A natural and important question is what ribosomal density maximizes the protein translation rate. Using mathematical models of ribosome flow along both a linear and a circular mRNA molecules we prove that typically the steady-state protein translation rate is maximized when the ribosomal density is one half of the maximal possible density. We discuss the implications of our results to endogenous genes under natural cellular conditions and also to synthetic biology. PMID:27861564

  6. Effect of Ribosomal Wash Factors on Inhibition by Chloramphenicol

    PubMed Central

    Cameron, Helen J.; Rogers, Samuel J.; Julian, Gordon R.

    1972-01-01

    In vitro protein-synthesizing systems from Escherichia coli can be categorized as either chloramphenicol-sensitive or chloramphenicol-insensitive. The chloramphenicol-sensitive systems used in this study required the presence of factors removed from ribosomes with 1.0 m NH4Cl when chromatographically purified ribosomes were used for amino acid incorporation. These ribosomal wash factors inhibited but did not eliminate amino acid incorporation in chloramphenicol-insensitive systems. For both systems, addition of increasing amounts of the ribosomal wash factors increased the sensitivity to chloramphenicol inhibition. PMID:4597705

  7. Investigation of Ribosomes Using Molecular Dynamics Simulation Methods.

    PubMed

    Makarov, G I; Makarova, T M; Sumbatyan, N V; Bogdanov, A A

    2016-12-01

    The ribosome as a complex molecular machine undergoes significant conformational changes while synthesizing a protein molecule. Molecular dynamics simulations have been used as complementary approaches to X-ray crystallography and cryoelectron microscopy, as well as biochemical methods, to answer many questions that modern structural methods leave unsolved. In this review, we demonstrate that all-atom modeling of ribosome molecular dynamics is particularly useful in describing the process of tRNA translocation, atomic details of behavior of nascent peptides, antibiotics, and other small molecules in the ribosomal tunnel, and the putative mechanism of allosteric signal transmission to functional sites of the ribosome.

  8. Complete kinetic mechanism for recycling of the bacterial ribosome.

    PubMed

    Borg, Anneli; Pavlov, Michael; Ehrenberg, Måns

    2016-01-01

    How EF-G and RRF act together to split a post-termination ribosomal complex into its subunits has remained obscure. Here, using stopped-flow experiments with Rayleigh light scattering detection and quench-flow experiments with radio-detection of GTP hydrolysis, we have clarified the kinetic mechanism of ribosome recycling and obtained precise estimates of its kinetic parameters. Ribosome splitting requires that EF-G binds to an already RRF-containing ribosome. EF-G binding to RRF-free ribosomes induces futile rounds of GTP hydrolysis and inhibits ribosome splitting, implying that while RRF is purely an activator of recycling, EF-G acts as both activator and competitive inhibitor of RRF in recycling of the post-termination ribosome. The ribosome splitting rate and the number of GTPs consumed per splitting event depend strongly on the free concentrations of EF-G and RRF. The maximal recycling rate, here estimated as 25 sec(-1), is approached at very high concentrations of EF-G and RRF with RRF in high excess over EF-G. The present in vitro results, suggesting an in vivo ribosome recycling rate of ∼5 sec(-1), are discussed in the perspective of rapidly growing bacterial cells.

  9. Complete kinetic mechanism for recycling of the bacterial ribosome

    PubMed Central

    Borg, Anneli; Pavlov, Michael

    2016-01-01

    How EF-G and RRF act together to split a post-termination ribosomal complex into its subunits has remained obscure. Here, using stopped-flow experiments with Rayleigh light scattering detection and quench-flow experiments with radio-detection of GTP hydrolysis, we have clarified the kinetic mechanism of ribosome recycling and obtained precise estimates of its kinetic parameters. Ribosome splitting requires that EF-G binds to an already RRF-containing ribosome. EF-G binding to RRF-free ribosomes induces futile rounds of GTP hydrolysis and inhibits ribosome splitting, implying that while RRF is purely an activator of recycling, EF-G acts as both activator and competitive inhibitor of RRF in recycling of the post-termination ribosome. The ribosome splitting rate and the number of GTPs consumed per splitting event depend strongly on the free concentrations of EF-G and RRF. The maximal recycling rate, here estimated as 25 sec−1, is approached at very high concentrations of EF-G and RRF with RRF in high excess over EF-G. The present in vitro results, suggesting an in vivo ribosome recycling rate of ∼5 sec−1, are discussed in the perspective of rapidly growing bacterial cells. PMID:26527791

  10. Homoiterons and expansion in ribosomal RNAs

    PubMed Central

    Parker, Michael S.; Sallee, Floyd R.; Park, Edwards A.; Parker, Steven L.

    2015-01-01

    Ribosomal RNAs in both prokaryotes and eukaryotes feature numerous repeats of three or more nucleotides with the same nucleobase (homoiterons). In prokaryotes these repeats are much more frequent in thermophile compared to mesophile or psychrophile species, and have similar frequency in both large RNAs. These features point to use of prokaryotic homoiterons in stabilization of both ribosomal subunits. The two large RNAs of eukaryotic cytoplasmic ribosomes have expanded to a different degree across the evolutionary ladder. The big RNA of the larger subunit (60S LSU) evolved expansion segments of up to 2400 nucleotides, and the smaller subunit (40S SSU) RNA acquired expansion segments of not more than 700 nucleotides. In the examined eukaryotes abundance of rRNA homoiterons generally follows size and nucleotide bias of the expansion segments, and increases with GC content and especially with phylogenetic rank. Both the nucleotide bias and frequency of homoiterons are much larger in metazoan and angiosperm LSU compared to the respective SSU RNAs. This is especially pronounced in the tetrapod vertebrates and seems to culminate in the hominid mammals. The stability of secondary structure in polyribonucleotides would significantly connect to GC content, and should also relate to G and C homoiteron content. RNA modeling points to considerable presence of homoiteron-rich double-stranded segments especially in vertebrate LSU RNAs, and homoiterons with four or more nucleotides in the vertebrate and angiosperm LSU RNAs are largely confined to the expansion segments. These features could mainly relate to protein export function and attachment of LSU to endoplasmic reticulum and other subcellular networks. PMID:26636029

  11. Homoiterons and expansion in ribosomal RNAs.

    PubMed

    Parker, Michael S; Sallee, Floyd R; Park, Edwards A; Parker, Steven L

    2015-01-01

    Ribosomal RNAs in both prokaryotes and eukaryotes feature numerous repeats of three or more nucleotides with the same nucleobase (homoiterons). In prokaryotes these repeats are much more frequent in thermophile compared to mesophile or psychrophile species, and have similar frequency in both large RNAs. These features point to use of prokaryotic homoiterons in stabilization of both ribosomal subunits. The two large RNAs of eukaryotic cytoplasmic ribosomes have expanded to a different degree across the evolutionary ladder. The big RNA of the larger subunit (60S LSU) evolved expansion segments of up to 2400 nucleotides, and the smaller subunit (40S SSU) RNA acquired expansion segments of not more than 700 nucleotides. In the examined eukaryotes abundance of rRNA homoiterons generally follows size and nucleotide bias of the expansion segments, and increases with GC content and especially with phylogenetic rank. Both the nucleotide bias and frequency of homoiterons are much larger in metazoan and angiosperm LSU compared to the respective SSU RNAs. This is especially pronounced in the tetrapod vertebrates and seems to culminate in the hominid mammals. The stability of secondary structure in polyribonucleotides would significantly connect to GC content, and should also relate to G and C homoiteron content. RNA modeling points to considerable presence of homoiteron-rich double-stranded segments especially in vertebrate LSU RNAs, and homoiterons with four or more nucleotides in the vertebrate and angiosperm LSU RNAs are largely confined to the expansion segments. These features could mainly relate to protein export function and attachment of LSU to endoplasmic reticulum and other subcellular networks.

  12. Ribosomal RNA: a key to phylogeny

    NASA Technical Reports Server (NTRS)

    Olsen, G. J.; Woese, C. R.

    1993-01-01

    As molecular phylogeny increasingly shapes our understanding of organismal relationships, no molecule has been applied to more questions than have ribosomal RNAs. We review this role of the rRNAs and some of the insights that have been gained from them. We also offer some of the practical considerations in extracting the phylogenetic information from the sequences. Finally, we stress the importance of comparing results from multiple molecules, both as a method for testing the overall reliability of the organismal phylogeny and as a method for more broadly exploring the history of the genome.

  13. Ribosomal RNA: a key to phylogeny.

    PubMed

    Olsen, G J; Woese, C R

    1993-01-01

    As molecular phylogeny increasingly shapes our understanding of organismal relationships, no molecule has been applied to more questions than have ribosomal RNAs. We review this role of the rRNAs and some of the insights that have been gained from them. We also offer some of the practical considerations in extracting the phylogenetic information from the sequences. Finally, we stress the importance of comparing results from multiple molecules, both as a method for testing the overall reliability of the organismal phylogeny and as a method for more broadly exploring the history of the genome.

  14. The Epigenetic Pathways to Ribosomal DNA Silencing

    PubMed Central

    Srivastava, Rakesh; Srivastava, Rashmi

    2016-01-01

    SUMMARY Heterochromatin is the transcriptionally repressed portion of eukaryotic chromatin that maintains a condensed appearance throughout the cell cycle. At sites of ribosomal DNA (rDNA) heterochromatin, epigenetic states contribute to gene silencing and genome stability, which are required for proper chromosome segregation and a normal life span. Here, we focus on recent advances in the epigenetic regulation of rDNA silencing in Saccharomyces cerevisiae and in mammals, including regulation by several histone modifications and several protein components associated with the inner nuclear membrane within the nucleolus. Finally, we discuss the perturbations of rDNA epigenetic pathways in regulating cellular aging and in causing various types of diseases. PMID:27250769

  15. [Fragment reaction catalyzed by E. coli ribosomes].

    PubMed

    Kotusov, V V; Kukhanova, M K; Sal'nikova, N E; Nikolaeva, L V; Kraevskiĭ, A A

    1977-01-01

    It has been shown that 50S subunits of E. coli MRE-600 ribosomes catalyze the reaction of N-(formyl)-methionyl ester of adenosine 5'-phosphate acting as peptide donor, with Phe-tRNA or CACCA-Phe serving as a peptide acceptor. The reaction is stimulated by cytidine 5'phosphate and inhibited by lincomycin, puromycin and chloramphenicol. The obtained results show that the structure of the donor site of peptidyltransferase is completely assembled on the 50S subunit and 30S subunit is not required for its formation.

  16. A Molecular Titration System Coordinates Ribosomal Protein Gene Transcription with Ribosomal RNA Synthesis.

    PubMed

    Albert, Benjamin; Knight, Britta; Merwin, Jason; Martin, Victoria; Ottoz, Diana; Gloor, Yvonne; Bruzzone, Maria Jessica; Rudner, Adam; Shore, David

    2016-11-17

    Cell growth potential is determined by the rate of ribosome biogenesis, a complex process that requires massive and coordinated transcriptional output. In the yeast Saccharomyces cerevisiae, ribosome biogenesis is highly regulated at the transcriptional level. Although evidence for a system that coordinates ribosomal RNA (rRNA) and ribosomal protein gene (RPG) transcription has been described, the molecular mechanisms remain poorly understood. Here we show that an interaction between the RPG transcriptional activator Ifh1 and the rRNA processing factor Utp22 serves to coordinate RPG transcription with that of rRNA. We demonstrate that Ifh1 is rapidly released from RPG promoters by a Utp22-independent mechanism following growth inhibition, but that its long-term dissociation requires Utp22. We present evidence that RNA polymerase I activity inhibits the ability of Utp22 to titrate Ifh1 from RPG promoters and propose that a dynamic Ifh1-Utp22 interaction fine-tunes RPG expression to coordinate RPG and rRNA transcription. Copyright © 2016 Elsevier Inc. All rights reserved.

  17. Ribosome-messenger recognition: mRNA target sites for ribosomal protein S1.

    PubMed Central

    Boni, I V; Isaeva, D M; Musychenko, M L; Tzareva, N V

    1991-01-01

    Ribosomal protein S1 is known to play an important role in translational initiation, being directly involved in recognition and binding of mRNAs by 30S ribosomal particles. Using a specially developed procedure based on efficient crosslinking of S1 to mRNA induced by UV irradiation, we have identified S1 binding sites on several phage RNAs in preinitiation complexes. Targets for S1 on Q beta and fr RNAs are localized upstream from the coat protein gene and contain oligo(U)-sequences. In the case of Q beta RNA, this S1 binding site overlaps the S-site for Q beta replicase and the site for S1 binding within a binary complex. It is reasonable that similar U-rich sequences represent S1 binding sites on bacterial mRNAs. To test this idea we have used E. coli ssb mRNA prepared in vitro with the T7 promoter/RNA polymerase system. By the methods of toeprinting, enzymatic footprinting, and UV crosslinking we have shown that binding of the ssb mRNA to 30S ribosomes is S1-dependent. The oligo(U)-sequence preceding the SD domain was found to be the target for S1. We propose that S1 binding sites, represented by pyrimidine-rich sequences upstream from the SD region, serve as determinants involved in recognition of mRNA by the ribosome. Images PMID:2011495

  18. Resonance assignment of the ribosome binding domain of E. coli ribosomal protein S1.

    PubMed

    Giraud, Pierre; Créchet, Jean-Bernard; Uzan, Marc; Bontems, François; Sizun, Christina

    2015-04-01

    Ribosomal protein S1 is an essential actor for protein synthesis in Escherichia coli. It is involved in mRNA recruitment by the 30S ribosomal subunit and recognition of the correct start codon during translation initiation. E. coli S1 is a modular protein that contains six repeats of an S1 motif, which have distinct functions despite structural homology. Whereas the three central repeats have been shown to be involved in mRNA recognition, the two first repeats that constitute the N-terminal domain of S1 are responsible for binding to the 30S subunit. Here we report the almost complete (1)H, (13)C and (15)N resonance assignment of two fragments of the 30S binding region of S1. The first fragment comprises only the first repeat. The second corresponds to the entire ribosome binding domain. Since S1 is absent from all high resolution X-ray structures of prokaryotic ribosomes, these data provide a first step towards atomic level structural characterization of this domain by NMR. Chemical shift analysis of the first repeat provides evidence for structural divergence from the canonical OB-fold of an S1 motif. In contrast the second domain displays the expected topology for an S1 motif, which rationalizes the functional specialization of the two subdomains.

  19. Transition State Analogues Rescue Ribosomes from Saporin-L1 Ribosome Inactivating Protein†

    PubMed Central

    Sturm, Matthew B.; Tyler, Peter C.; Evans, Gary B.; Schramm, Vern L.

    2009-01-01

    Ribosome-inactivating proteins (RIPs) catalyze the hydrolytic depurination of one or more adenosine residues from eukaryotic ribosomes. Depurination of the ribosomal sarcin-ricin tetraloop (GAGA) causes inhibition of protein synthesis and cellular death. We characterized the catalytic properties of saporin-L1 from Saponaria officinalis (soapwort) leaves and demonstrate robust activity against defined nucleic acid substrates and mammalian ribosomes. Transition state analogue mimics of small oligonucleotide substrates of saporin-L1 are powerful, slow-onset inhibitors when adenosine is replaced with the transition state mimic 9-deazaadenine-9-methylene-N-hydroxypyrrolidine (DADMeA). Linear, cyclic and stem-loop oligonucleotide inhibitors containing DADMeA and based on the GAGA sarcin-ricin tetraloop gave slow-onset tight-binding inhibition constants (Ki*) of 2.3 to 8.7 nM at physiological conditions and bind up to 40,000-fold tighter than RNA substrates. Saporin-L1 inhibition of rabbit reticulocyte translation was protected by these inhibitors. Transition state analogues of saporin-L1 have potential in cancer therapy that employs saporin-L1 linked immunotoxins. PMID:19764816

  20. Ultraviolet light-induced crosslinking of two major phosphoproteins and poly(A)+RNA from free polyribosomes; changes in phosphorylation by inhibitors of transcription and translation

    SciTech Connect

    Schweiger, A.; Kostka, G.; Weiss, E.

    1986-04-14

    Polyribosomes were isolated without the use of detergents, irradiated with ultraviolet light and labelled in the presence of (gamma-/sup 32/P) adenosine 5'-triphosphate. Poly(A)+RNA-protein structures separated by chromatography on oligo (dT)-cellulose contained up to 1o crosslinked proteins as shown by SDS-polyacrylamide gel electrophoresis. These included a 71 kDa poly(A)-bound species and two major phosphoproteins of 66 and 13o kDa. Pretreatment of rats with inhibitors of transcription and translation caused different and significant alterations in the labelling of the two phosphoproteins, suggesting that phosphorylation of proteins closely associated with mRNA may be involved in the regulation of the stability of this RNA or its binding to structural elements in the cell.

  1. Broad-Scale Phosphoprotein Profiling of Beta Adrenergic Receptor (β-AR) Signaling Reveals Novel Phosphorylation and Dephosphorylation Events

    PubMed Central

    Chruscinski, Andrzej J.; Singh, Harvir; Chan, Steven M.; Utz, Paul J.

    2013-01-01

    β-adrenergic receptors (β-ARs) are model G-protein coupled receptors that mediate signal transduction in the sympathetic nervous system. Despite the widespread clinical use of agents that target β-ARs, the signaling pathways that operate downstream of β-AR stimulation have not yet been completely elucidated. Here, we utilized a lysate microarray approach to obtain a broad-scale perspective of phosphoprotein signaling downstream of β-AR. We monitored the time course of phosphorylation states of 54 proteins after β-AR activation mouse embryonic fibroblast (MEF) cells. In response to stimulation with the non-selective β-AR agonist isoproterenol, we observed previously described phosphorylation events such as ERK1/2(T202/Y204) and CREB(S133), but also novel phosphorylation events such as Cdc2(Y15) and Pyk2(Y402). All of these events were mediated through cAMP and PKA as they were reproduced by stimulation with the adenylyl cyclase activator forskolin and were blocked by treatment with H89, a PKA inhibitor. In addition, we also observed a number of novel isoproterenol-induced protein dephosphorylation events in target substrates of the PI3K/AKT pathway: GSK3β(S9), 4E-BP1(S65), and p70s6k(T389). These dephosphorylations were dependent on cAMP, but were independent of PKA and correlated with reduced PI3K/AKT activity. Isoproterenol stimulation also led to a cAMP-dependent dephosphorylation of PP1α(T320), a modification known to correlate with enhanced activity of this phosphatase. Dephosphorylation of PP1α coincided with the secondary decline in phosphorylation of some PKA-phosphorylated substrates, suggesting that PP1α may act in a feedback loop to return these phosphorylations to baseline. In summary, lysate microarrays are a powerful tool to profile phosphoprotein signaling and have provided a broad-scale perspective of how β-AR signaling can regulate key pathways involved in cell growth and metabolism. PMID:24340001

  2. Identification and Characterization of the Binding Site of the Respiratory Syncytial Virus Phosphoprotein to RNA-Free Nucleoprotein

    PubMed Central

    Galloux, Marie; Gabiane, Gaëlle; Sourimant, Julien; Richard, Charles-Adrien; England, Patrick; Moudjou, Mohammed; Aumont-Nicaise, Magali; Fix, Jenna; Rameix-Welti, Marie-Anne

    2015-01-01

    ABSTRACT The RNA genome of respiratory syncytial virus (RSV) is constitutively encapsidated by the viral nucleoprotein N, thus forming a helical nucleocapsid. Polymerization of N along the genomic and antigenomic RNAs is concomitant to replication and requires the preservation of an unassembled monomeric nucleoprotein pool. To this end, and by analogy with Paramyxoviridae and Rhabdoviridae, it is expected that the viral phosphoprotein P acts as a chaperone protein, forming a soluble complex with the RNA-free form of N (N0-P complex). Here, we have engineered a mutant form of N that is monomeric, is unable to bind RNA, still interacts with P, and could thus mimic the N0 monomer. We used this N mutant, designated Nmono, as a substitute for N0 in order to characterize the P regions involved in the N0-P complex formation. Using a series of P fragments, we determined by glutathione S-transferase (GST) pulldown assays that the N and C termini of P are able to interact with Nmono. We analyzed the functional role of amino-terminal residues of P by site-directed mutagenesis, using an RSV polymerase activity assay based on a human RSV minireplicon, and found that several residues were critical for viral RNA synthesis. Using GST pulldown and surface plasmon resonance assays, we showed that these critical residues are involved in the interaction between P[1-40] peptide and Nmono in vitro. Finally, we showed that overexpression of the peptide P[1-29] can inhibit the polymerase activity in the context of the RSV minireplicon, thus demonstrating that targeting the N0-P interaction could constitute a potential antiviral strategy. IMPORTANCE Respiratory syncytial virus (RSV) is the leading cause of lower respiratory tract illness in infants. Since no vaccine or efficient antiviral treatment is available against RSV, it is essential to better understand how the viral machinery functions in order to develop new antiviral strategies. RSV phosphoprotein P, the main RNA polymerase

  3. Crosslinking of Ribosomal Proteins to RNA in Maize Ribosomes by UV-B and Its Effects on Translation1[w

    PubMed Central

    Casati, Paula; Walbot, Virginia

    2004-01-01

    Ultraviolet-B (UV-B) photons can cause substantial cellular damage in biomolecules, as is well established for DNA. Because RNA has the same absorption spectrum for UV as DNA, we have investigated damage to this cellular constituent. In maize (Zea mays) leaves, UV-B radiation damages ribosomes by crosslinking cytosolic ribosomal proteins S14, L23a, and L32, and chloroplast ribosomal protein L29 to RNA. Ribosomal damage accumulated during a day of UV-B exposure correlated with a progressive decrease in new protein production; however, de novo synthesis of some ribosomal proteins is increased after 6 h of UV-B exposure. After 16 h without UV-B, damaged ribosomes were eliminated and translation was restored to normal levels. Ribosomal protein S6 and an S6 kinase are phosphorylated during UV-B exposure; these modifications are associated with selective translation of some ribosomal proteins after ribosome damage in mammalian fibroblast cells and may be an adaptation in maize. Neither photosynthesis nor pigment levels were affected significantly by UV-B, demonstrating that the treatment applied is not lethal and that maize leaf physiology readily recovers. PMID:15466230

  4. Identification on Membrane and Characterization of Phosphoproteins Using an Alkoxide-Bridged Dinuclear Metal Complex as a Phosphate-Binding Tag Molecule

    PubMed Central

    Nakanishi, Tsuyoshi; Ando, Eiji; Furuta, Masaru; Kinoshita, Eiji; Kinoshita-Kikuta, Emiko; Koike, Tohru; Tsunasawa, Susumu; Nishimura, Osamu

    2007-01-01

    We have developed a method for on-membrane direct identification of phosphoproteins, which are detected by a phosphate-binding tag (Phos-tag) that has an affinity to phosphate groups with a chelated Zn2+ ion. This rapid profiling approach for phosphoproteins combines chemical inkjet technology for microdispensing of reagents onto a tiny region of target proteins with mass spectrometry for on-membrane digested peptides. Using this method, we analyzed human epidermoid carcinoma cell lysates of A-431 cells stimulated with epidermal growth factor, and identified six proteins with intense signals upon affinity staining with the phosphate-binding tag. It was already known that these proteins are phosphorylated, and our new approach proved to be effective at rapid profiling of phosphoproteins. Furthermore, we tried to determine their phosphorylation sites by MS/MS analysis after in-gel digestion of the corresponding spots on the 2DE gel to the rapid on-membrane identifications. As one example of use of information gained from the rapid-profiling approach, we successfully characterized a phosphorylation site at Ser-113 on prostaglandin E synthase 3. PMID:18166671

  5. A novel phosphoprotein analysis scheme for assessing changes in premalignant and malignant breast cell lines using 2D liquid separations, protein microarrays and tandem mass spectrometry

    PubMed Central

    Patwa, Tasneem H.; Wang, Yanfei; Miller, Fred R.; Goodison, Steve; Pennathur, Subramaniam; Barder, Timothy J.; Lubman, David M.

    2008-01-01

    An analysis of phosphorylation changes that occur during cancer progression would provide insights into the molecular pathways responsible for a malignant phenotype. In this study we employed a novel coupling of 2D-liquid separations and protein microarray technology to reveal changes in phosphoprotein status between premalignant (AT1) and malignant (CA1a) cell lines derived from the human MCF10A breast cell lines. Intact proteins were first separated according to their isoelectric point and hydrophobicities, then arrayed on SuperAmine glass slides. Phosphoproteins were detected using the universal, inorganic phospho-sensor dye, ProQ Diamond. Using this dye, out of 140 spots that were positive for phosphorylation, a total of 85 differentially expressed spots were detected over a pH range of 7.2 to 4.0. Proteins were identified and their peptides sequenced by mass spectrometry. The strategy enabled the identification of 75 differentially expressed phosphoproteins, from which 51 phosphorylation sites in 27 unique proteins were confirmed. Interestingly, the majority of differentially expressed phosphorylated proteins observed were nuclear proteins. Three regulators of apoptosis, Bad, Bax and Acinus, were also differentially phosphorylated in the two cell lines. Further development of this strategy will facilitate an understanding of the mechanisms involved in malignancy progression and other disease-related phenotypes. PMID:19194518

  6. A novel phosphoprotein analysis scheme for assessing changes in premalignant and malignant breast cell lines using 2D liquid separations, protein microarrays and tandem mass spectrometry.

    PubMed

    Patwa, Tasneem H; Wang, Yanfei; Miller, Fred R; Goodison, Steve; Pennathur, Subramaniam; Barder, Timothy J; Lubman, David M

    2008-01-01

    An analysis of phosphorylation changes that occur during cancer progression would provide insights into the molecular pathways responsible for a malignant phenotype. In this study we employed a novel coupling of 2D-liquid separations and protein microarray technology to reveal changes in phosphoprotein status between premalignant (AT1) and malignant (CA1a) cell lines derived from the human MCF10A breast cell lines. Intact proteins were first separated according to their isoelectric point and hydrophobicities, then arrayed on SuperAmine glass slides. Phosphoproteins were detected using the universal, inorganic phospho-sensor dye, ProQ Diamond. Using this dye, out of 140 spots that were positive for phosphorylation, a total of 85 differentially expressed spots were detected over a pH range of 7.2 to 4.0. Proteins were identified and their peptides sequenced by mass spectrometry. The strategy enabled the identification of 75 differentially expressed phosphoproteins, from which 51 phosphorylation sites in 27 unique proteins were confirmed. Interestingly, the majority of differentially expressed phosphorylated proteins observed were nuclear proteins. Three regulators of apoptosis, Bad, Bax and Acinus, were also differentially phosphorylated in the two cell lines. Further development of this strategy will facilitate an understanding of the mechanisms involved in malignancy progression and other disease-related phenotypes.

  7. An Introduction to the Structure and Function of the Ribosome.

    PubMed

    Dunkle, Jack A; Cate, Jamie H D

    2013-02-01

    E. coli continues to serve as a key model for the structure and function of the ribosome, structures of ribosome from other organisms and domains of life have also greatly contributed to our knowledge of protein synthesis. Many structural models of the ribosome in a number of steps of the protein synthesis cycle have been solved by cryo-electron microscopy (cryo-EM) and x-ray crystallography. This chapter introduces the structure and dynamics of the ribosome based on these structures and ends with a brief discussion of the many questions that the structures leave unanswered. Protein synthesis is a multistep process, and the structural features of the ribosome along with the large number of cofactors reflect the complexity of translation. Numerous protein factors in addition to the ribosome contribute to translation in bacteria during the steps of initiation, elongation, termination, and recycling. These protein factors make intimate contacts to key regions of the ribosome, and this aspect is discussed in the chapter in light of our present understanding of the structure and function of the ribosome. The intact ribosome contains three binding sites for substrate tRNAs that are termed as the aminoacyl-tRNA site (A site), peptidyl-tRNA site (P site), and exit-tRNA site (E site). These three binding sites span the interface between the 30S and 50S subunits. The central activity of the ribosome is catalysis of peptide bond formation. The region of the ribosome responsible for catalyzing the reaction is called the peptidyl transferase center (PTC).

  8. Defining the bacteroides ribosomal binding site.

    PubMed

    Wegmann, Udo; Horn, Nikki; Carding, Simon R

    2013-03-01

    The human gastrointestinal tract, in particular the colon, hosts a vast number of commensal microorganisms. Representatives of the genus Bacteroides are among the most abundant bacterial species in the human colon. Bacteroidetes diverged from the common line of eubacterial descent before other eubacterial groups. As a result, they employ unique transcription initiation signals and, because of this uniqueness, they require specific genetic tools. Although some tools exist, they are not optimal for studying the roles and functions of these bacteria in the human gastrointestinal tract. Focusing on translation initiation signals in Bacteroides, we created a series of expression vectors allowing for different levels of protein expression in this genus, and we describe the use of pepI from Lactobacillus delbrueckii subsp. lactis as a novel reporter gene for Bacteroides. Furthermore, we report the identification of the 3' end of the 16S rRNA of Bacteroides ovatus and analyze in detail its ribosomal binding site, thus defining a core region necessary for efficient translation, which we have incorporated into the design of our expression vectors. Based on the sequence logo information from the 5' untranslated region of other Bacteroidales ribosomal protein genes, we conclude that our findings are relevant to all members of this order.

  9. Defining the Bacteroides Ribosomal Binding Site

    PubMed Central

    Horn, Nikki; Carding, Simon R.

    2013-01-01

    The human gastrointestinal tract, in particular the colon, hosts a vast number of commensal microorganisms. Representatives of the genus Bacteroides are among the most abundant bacterial species in the human colon. Bacteroidetes diverged from the common line of eubacterial descent before other eubacterial groups. As a result, they employ unique transcription initiation signals and, because of this uniqueness, they require specific genetic tools. Although some tools exist, they are not optimal for studying the roles and functions of these bacteria in the human gastrointestinal tract. Focusing on translation initiation signals in Bacteroides, we created a series of expression vectors allowing for different levels of protein expression in this genus, and we describe the use of pepI from Lactobacillus delbrueckii subsp. lactis as a novel reporter gene for Bacteroides. Furthermore, we report the identification of the 3′ end of the 16S rRNA of Bacteroides ovatus and analyze in detail its ribosomal binding site, thus defining a core region necessary for efficient translation, which we have incorporated into the design of our expression vectors. Based on the sequence logo information from the 5′ untranslated region of other Bacteroidales ribosomal protein genes, we conclude that our findings are relevant to all members of this order. PMID:23335775

  10. Phosphoprotein Keratin 23 accumulates in MSS but not MSI colon cancers in vivo and impacts viability and proliferation in vitro.

    PubMed

    Birkenkamp-Demtroder, Karin; Mansilla, Francisco; Sørensen, Flemming Brandt; Kruhøffer, Mogens; Cabezón, Teresa; Christensen, Lise Lotte; Aaltonen, Lauri A; Verspaget, Hein W; Ørntoft, Torben Falck

    2007-09-01

    Transcript profiling of 27 normal colon mucosas and 258 adenocarcinomas showed Keratin23 to be increased in 78% microsatellite-stable tumors, while microsatellite-instable tumors showed low transcript levels, comparable to normal mucosas. Immunohistochemical analyses demonstrated that 88% of microsatellite-instable tumors were negative for Keratin23 protein, while 70% of MSS tumors and metastases derived from MSS-tumors showed high Keratin23 levels. Immunofluorescence analysis localized Keratin23 in the Golgi-apparatus. Golgi accumulation was unique for gastrointestinal adenocarcinomas. Immunoprecipitation and 2D-blot analysis revealed Keratin23 to be a 46.8 kDa phosphoprotein. Keratin23 impaired the proliferation of human colon cancer cells significantly, leading to cell death in microsatellite-instable but not microsatellite-stable cell lines, while COS7 cells experienced multiple nuclei and apoptosis. Keratin23 expression correlated significantly with transcription factor CEBPB. In conclusion, Keratin23 expression is a novel and important difference between microsatellite-stable and microsatellite-instable colon cancers.

  11. Phosphoprotein network analysis of white adipose tissues unveils deregulated pathways in response to high-fat diet

    PubMed Central

    Asfa, Alli Shaik; Qiu, Beiying; Wee, Sheena; Choi, Hyungwon; Gunaratne, Jayantha; Tergaonkar, Vinay

    2016-01-01

    Despite efforts in the last decade, signaling aberrations associated with obesity remain poorly understood. To dissect molecular mechanisms that define this complex metabolic disorder, we carried out global phosphoproteomic analysis of white adipose tissue (WAT) from mice fed on low-fat diet (LFD) and high-fat diet (HFD). We quantified phosphorylation levels on 7696 peptides, and found significant differential phosphorylation levels in 282 phosphosites from 191 proteins, including various insulin-responsive proteins and metabolic enzymes involved in lipid homeostasis in response to high-fat feeding. Kinase-substrate prediction and integrated network analysis of the altered phosphoproteins revealed underlying signaling modulations during HFD-induced obesity, and suggested deregulation of lipogenic and lipolytic pathways. Mutation of the differentially-regulated novel phosphosite on cytoplasmic acetyl-coA forming enzyme ACSS2 (S263A) upon HFD-induced obesity led to accumulation of serum triglycerides and reduced insulin-responsive AKT phosphorylation as compared to wild type ACSS2, thus highlighting its role in obesity. Altogether, our study presents a comprehensive map of adipose tissue phosphoproteome in obesity and reveals many previously unknown candidate phosphorylation sites for future functional investigation. PMID:27180971

  12. Cloning of the VASP (Vasodilator-Stimulated Phosphoprotein) genes in human and mouse: Structure, sequence, and chromosomal localization

    SciTech Connect

    Zimmer, M.; Fischer, L.; Hauser, W.

    1996-09-01

    The genes encoding the vasodilator-stimulated phosphoprotein (VASP) in human and mouse were isolated, and major parts were sequenced. In both species the gene is composed of 13 exons with conserved exon-intron positions. The mouse VASP cDNA sequence was deduced from the genomic sequence. The predicted amino acid sequence is 89% identical to the human protein. The high nucleotide sequence homology extends not only over the coding regions but also into the 3{prime}-UTRs, indicating a possible function in mRNA targeting or regulation of translation. Prominent 5{prime} CpG islands including multiple SP1 sites indicate a housekeeping function of VASP. Using cosmid DNA as a probe for fluorescence in situ hybridization, the human VASP gene was assigned to chromosome 19q13.2-q13.3, an extended region with homology to mouse chromosome 7. A sequence overlap of the VASP 5{prime}-region with the telomeric end of a cosmid contig physically links the VASP gene with ERCC1. VASP is located about 92 kb distal to ERCC1 and about 300 kb proximal to the myotonic dystrophy protein kinase gene. 43 refs., 6 figs.

  13. [The highly expressed secreted phosphoprotein 1 gene in prostate cancer metastasis: a microarray-based bioinformatic analysis].

    PubMed

    Li, Tie-qiu; Teng, Yi-li; Zou, Ya-guang; Yang, Yu; Li, Qi; Mao, Xiang-ming

    2014-11-01

    To investigate the composition, function, and regulatory mechanisms of the secreted phosphoprotein 1 (SPP1) gene in metastatic prostate cancer. We obtained the data about the whole genomic expression profiles on prostate cancer metastasis from the GEO database, and performed data-mining and bioinformatic analysis using BRB-Array Tools and such softwares as Protparam, MotifScan, SignalP 4.0, TMHMM, NetPhos2.0, PredictProtein, GO, KEGG, and STRING. Totally, 73 co-expressed differential genes in prostate cancer metastasis were identified, 21 up-regulated and 52 down-regulated (P <0.01). Bioinformatic analysis indicated that the highly expressed SPP1 gene encoded 314 amino acids and contained 2 N-glycosylation sites, 8 casein kinase II phosphorylation sites and 3 protein kinase C phosphorylation sites, playing essential roles in extracellular matrix (ECM) binding, ossification, osteoblast differentiation, cell adhesion, PI3K-Akt signaling pathway, focal adhesion, Toll-like receptor signaling pathway, and ECM-receptor interaction. The bioinformatic method showed a high efficiency in analyzing microarray data and revealing internal biological information. SPP1 may play an important role in prostate cancer metastasis and become a novel biomarker for the diagnosis of prostate cancer metastasis and a new target for its treatment.

  14. Identification of isoforms of the exocytosis-sensitive phosphoprotein PP63/parafusin in Paramecium tetraurelia and demonstration of phosphoglucomutase activity.

    PubMed Central

    Hauser, K; Kissmehl, R; Linder, J; Schultz, J E; Lottspeich, F; Plattner, H

    1997-01-01

    PP63 (parafusin) is a 63 kDa phosphoprotein which is very rapidly (within 80 ms) dephosphorylated (to P63) during triggered trichocyst exocytosis; this occurs selectively in exocytosis-competent Paramecium tetraurelia strains. In the present work, two cDNAs coding for PP63/parafusin have been isolated, one of which is a new isoform. These isoforms are 99.6% identical and are derived from two different genes. Similarity searches revealed 43-51% identity of the deduced amino acid sequences with known phosphoglucomutases from yeast and mammals. The sequences of two proteolytic peptides obtained from PP63/parafusin isolated from Paramecium are identical to parts of the amino acid sequence deduced from the major cDNA. The major cDNA was mutated from the macronuclear ciliate genetic code into the universal genetic code and expressed in Escherichia coli. The recombinant protein shows the same biochemical and immunological characteristics as the (P)P63/parafusin originally isolated from Paramecium. It has the same specific phosphoglucomutase activity as phosphoglucomutase from chicken muscle. We also show that recombinant P63-1 parafusin 1 is a substrate of an endogenous casein kinase from Paramecium, as is the originally isolated P63/parafusin. Polyclonal antibodies against recombinant P63-1/parafusin 1 were raised which recognized phosphoglucomutases from different sources. Thus we show that PP63/parafusin and phosphoglucomutase in Paramecium are identical. PMID:9173895

  15. Spatial Phosphoprotein Profiling Reveals a Compartmentalized Extracellular Signal-regulated Kinase Switch Governing Neurite Growth and Retraction

    SciTech Connect

    Wang, Yingchun; Yang, Feng; Fu, Yi; Huang, Xiahe; Wang, Wei; Jiang, Xining; Gritsenko, Marina A.; Zhao, Rui; Monroe, Matthew E.; Pertz, Olivier C.; Purvine, Samuel O.; Orton, Daniel J.; Jacobs, Jon M.; Camp, David G.; Smith, Richard D.; Klemke, Richard L.

    2011-05-20

    Abstract - Brain development and spinal cord regeneration require neurite sprouting and growth cone navigation in response to extension and collapsing factors present in the extracellular environment. These external guidance cues control neurite growth cone extension and retraction processes through intracellular protein phosphorylation of numerous cytoskeletal, adhesion, and polarity complex signaling proteins. However, the complex kinase/substrate signaling networks that mediate neuritogenesis have not been investigated. Here, we compare the neurite phosphoproteome under growth and retraction conditions using neurite purification methodology combined with mass spectrometry. More than 4000 non-redundant phosphorylation sites from 1883 proteins have been annotated and mapped to signaling pathways that control kinase/phosphatase networks, cytoskeleton remodeling, and axon/dendrite specification. Comprehensive informatics and functional studies revealed a compartmentalized ERK activation/deactivation cytoskeletal switch that governs neurite growth and retraction, respectively. Our findings provide the first system-wide analysis of the phosphoprotein signaling networks that enable neurite growth and retraction and reveal an important molecular switch that governs neuritogenesis.

  16. Potential of liquid-isoelectric-focusing protein fractionation to improve phosphoprotein characterization of Pseudomonas aeruginosa PA14.

    PubMed

    Ouidir, Tassadit; Jarnier, Frédérique; Cosette, Pascal; Jouenne, Thierry; Hardouin, Julie

    2014-10-01

    Protein phosphorylation on serine, threonine, and tyrosine is known to be involved in a wide variety of cellular processes and signal transduction in bacteria. Bacterial-proteome analysis is required to determine which proteins have been conditionally expressed and whether any post-translational modifications are present. One of the greatest challenges of proteome analysis is the fractionation of these complex protein mixtures to detect low-abundance phosphoproteins. Liquid-phase isoelectric focusing (IEF) is a promising analytical tool in proteomics, but as far as we are aware no work has studied the reproducibility of this approach. In this study, we investigated the phosphoproteome of Pseudomonas aeruginosa strain PA14. We first tested in-solution IEF protein fractionation, and then used this technique to fractionate the proteins in the complex mixture. Next, phosphopeptides were enriched with titanium dioxide and analyzed by high-resolution, high-accuracy liquid chromatography-mass spectrometry. With this approach, we succeeded in characterizing 73 unique phosphorylated peptides belonging to 63 proteins. Interestingly, we observed a higher percentage of modified tyrosine, revealing the importance of this phosphorylated residue in bacteria.

  17. Viral vector vaccines expressing nucleoprotein and phosphoprotein genes of avian bornaviruses ameliorate homologous challenge infections in cockatiels and common canaries

    PubMed Central

    Olbert, Marita; Römer-Oberdörfer, Angela; Herden, Christiane; Malberg, Sara; Runge, Solveig; Staeheli, Peter; Rubbenstroth, Dennis

    2016-01-01

    Avian bornaviruses are causative agents of proventricular dilatation disease (PDD), an often fatal disease of parrots and related species (order Psittaciformes) which is widely distributed in captive psittacine populations and may affect endangered species. Here, we established a vaccination strategy employing two different well described viral vectors, namely recombinant Newcastle disease virus (NDV) and modified vaccinia virus Ankara (MVA) that were engineered to express the phosphoprotein and nucleoprotein genes of two avian bornaviruses, parrot bornavirus 4 (PaBV-4) and canary bornavirus 2 (CnBV-2). When combined in a heterologous prime/boost vaccination regime, NDV and MVA vaccine viruses established self-limiting infections and induced a bornavirus-specific humoral immune response in cockatiels (Nymphicus hollandicus) and common canaries (Serinus canaria forma domestica). After challenge infection with a homologous bornavirus, shedding of bornavirus RNA and viral loads in tissue samples were significantly reduced in immunized birds, indicating that vaccination markedly delayed the course of infection. However, cockatiels still developed signs of PDD if the vaccine failed to prevent viral persistence. Our work demonstrates that avian bornavirus infections can be repressed by vaccine-induced immunity. It represents a first crucial step towards a protective vaccination strategy to combat PDD in psittacine birds. PMID:27830736

  18. Phosphoprotein phosphatase 1CB (PPP1CB), a novel adipogenic activator, promotes 3T3-L1 adipogenesis.

    PubMed

    Cho, Young-Lai; Min, Jeong-Ki; Roh, Kyung Min; Kim, Won Kon; Han, Baek Soo; Bae, Kwang-Hee; Lee, Sang Chul; Chung, Sang J; Kang, Hyo Jin

    2015-11-13

    Understanding the molecular networks that regulate adipogenesis is crucial for gaining insight into obesity and identifying medicinal targets thereof is necessary for pharmacological interventions. However, the identity and molecular actions of activators that promote the early development of adipocytes are still largely unknown. Here, we demonstrate a novel role for phosphoprotein phosphatase 1CB (PPP1CB) as a potent adipogenic activator that promotes adipocyte differentiation. PPP1CB expression increased in vitro during the early phase of 3T3-L1 adipogenesis and in the murine model of high-fat diet-induced obesity. Depletion of PPP1CB dramatically suppressed the differentiation of 3T3-L1 cells into mature adipocytes, with a concomitant change in adipocyte marker genes and significantly inhibited clonal expansion. We also showed that knockdown of PPP1CB caused a significant decrease in C/EBPδ expression, which in turn resulted in attenuation of PPARγ, C/EBPα, adiponectin, and aP2. In addition, we elucidated the functional significance of PPP1CB by linking p38 activation to C/EBPδ expression in early adipogenesis. Overall, our findings demonstrate a novel function of PPP1CB in promoting adipogenesis and suggest that PPP1CB may be a promising therapeutic target for treatment of obesity and obesity-related diseases.

  19. Effects of thyrotropin on the phosphorylation of histones and nonhistone phosphoproteins in micrococcal nuclease-sensitive and resistant thyroid chromatin

    SciTech Connect

    Cooper, E.; Spaulding, S.W.

    1983-05-01

    Actively transcribed regions of chromatin are more susceptible than bulk chromatin to digestion by nucleases, and useful information about the composition and structure of active chromatin may be obtained by studying the chromatin fragments released from nuclei by limited nuclease digestion. In the present study, we have used micrococcal nuclease to investigate the effects of TSH on protein phosphorylation in nuclease-sensitive fractions of calf thyroid chromatin. Batches of calf thyroid slices were incubated for 2 h with /sup 32/Pi, with or without 50 mU/ml TSH. Nuclei were then prepared and the distribution of /sup 32/P-labeled histones, high mobility group (HMG) proteins, and other acid-soluble phosphoproteins between micrococcal nuclease-sensitive and resistant fractions of chromatin was examined. TSH increased the amount of /sup 32/P incorporated into HMG 14 and the histones H1 and H3. Hormone-dependent increases in the /sup 32/P-labeling of H1 and H3 were not selectively associated with micrococcal nuclease-sensitive chromatin. In contrast, (/sup 32/P) HMG-14 was preferentially solubilized from nuclei by micrococcal nuclease. This lends support to the view that TSH-induced effects on the structure and function of transcriptionally active chromatin may be mediated in part by phosphorylation of HMG 14.

  20. Viral vector vaccines expressing nucleoprotein and phosphoprotein genes of avian bornaviruses ameliorate homologous challenge infections in cockatiels and common canaries.

    PubMed

    Olbert, Marita; Römer-Oberdörfer, Angela; Herden, Christiane; Malberg, Sara; Runge, Solveig; Staeheli, Peter; Rubbenstroth, Dennis

    2016-11-10

    Avian bornaviruses are causative agents of proventricular dilatation disease (PDD), an often fatal disease of parrots and related species (order Psittaciformes) which is widely distributed in captive psittacine populations and may affect endangered species. Here, we established a vaccination strategy employing two different well described viral vectors, namely recombinant Newcastle disease virus (NDV) and modified vaccinia virus Ankara (MVA) that were engineered to express the phosphoprotein and nucleoprotein genes of two avian bornaviruses, parrot bornavirus 4 (PaBV-4) and canary bornavirus 2 (CnBV-2). When combined in a heterologous prime/boost vaccination regime, NDV and MVA vaccine viruses established self-limiting infections and induced a bornavirus-specific humoral immune response in cockatiels (Nymphicus hollandicus) and common canaries (Serinus canaria forma domestica). After challenge infection with a homologous bornavirus, shedding of bornavirus RNA and viral loads in tissue samples were significantly reduced in immunized birds, indicating that vaccination markedly delayed the course of infection. However, cockatiels still developed signs of PDD if the vaccine failed to prevent viral persistence. Our work demonstrates that avian bornavirus infections can be repressed by vaccine-induced immunity. It represents a first crucial step towards a protective vaccination strategy to combat PDD in psittacine birds.

  1. The nucleolar phosphoprotein B23 targets Newcastle disease virus matrix protein to the nucleoli and facilitates viral replication.

    PubMed

    Duan, Zhiqiang; Chen, Jian; Xu, Haixu; Zhu, Jie; Li, Qunhui; He, Liang; Liu, Huimou; Hu, Shunlin; Liu, Xiufan

    2014-03-01

    The cellular nucleolar proteins are reported to facilitate the replication cycles of some human and animal viruses by interaction with viral proteins. In this study, a nucleolar phosphoprotein B23 was identified to interact with Newcastle disease virus (NDV) matrix (M) protein. We found that NDV M protein accumulated in the nucleolus by binding B23 early in infection, but resulted in the redistribution of B23 from the nucleoli to the nucleoplasm later in infection. In vitro binding studies utilizing deletion mutants indicated that amino acids 30-60 of M and amino acids 188-245 of B23 were required for binding. Furthermore, knockdown of B23 by siRNA or overexpression of B23 or M-binding B23-derived polypeptides remarkably reduced cytopathic effect and inhibited NDV replication. Collectively, we show that B23 facilitates NDV replication by targeting M to the nucleolus, demonstrating for the first time a direct role for nucleolar protein B23 in a paramyxovirus replication process.

  2. Sequence analysis of the phosphoprotein gene of peste des petits ruminants (PPR) virus: editing of the gene transcript.

    PubMed

    Mahapatra, Madhuchhanda; Parida, Satya; Egziabher, Berhe G; Diallo, Adama; Barrett, Tom

    2003-10-01

    The gene encoding the phosphoprotein of the vaccine strain of Peste des petits ruminants (PPR) virus (Nigeria 75/1 vaccine strain) has been cloned and its nucleotide sequence been determined. This gene is 1655 nucleotides long and encodes two overlapping open reading frames (ORFs). Translation from the first AUG would produce a polypeptide of 509 amino acid residues with a predicted molecular mass of 54.9 kDa, the longest of the published morbillivirus P proteins. Translation from the second AUG would produce a protein of 177 amino acid residues with a predicted molecular mass of 20.3 kDa, analogous to the C proteins of other morbilliviruses. Evidence was found for the production of two types of P mRNA transcript, one a faithful transcript of the gene and the other with an extra G residue inserted at position 751. Translation from the first AUG of this second mRNA would produce a protein of 298 amino acids, with a predicted molecular mass 32.3 kDa, analogous to the V protein produced by other morbilliviruses. Sequences of the predicted P, C and V proteins were compared with those of the other morbillivirus sequences available to date. The P protein was found to be the most poorly conserved of the morbillivirus proteins, the amino acid identity ranging from 54% in case of Canine distemper virus (CDV) to 60% in the case of the Dolphin morbillivirus (DMV).

  3. Secreted phosphoprotein 24 kD (Spp24) inhibits growth of human pancreatic cancer cells caused by BMP-2

    SciTech Connect

    Li, Chen-Shuang; Tian, Haijun; Zou, Min; Zhao, Ke-Wei; Li, Yawei; Lao, Lifeng; Brochmann, Elsa J.; Duarte, M. Eugenia L.; Daubs, Michael D.; Zhou, Yan-Heng; Murray, Samuel S.; Wang, Jeffrey C.

    2015-10-16

    The emerging role of bone morphogenetic proteins (BMPs) in the initiation and progression of multiple cancers has drawn great attention in cancer research. In this study, we report that BMP-2 can promote the proliferation of the pancreatic tumor cell line, PANC-1. Secreted phosphoprotein 24 kD (Spp24), a BMP binding protein, did not affect the proliferation of the cells but promoted the apoptosis of the cells in vitro. In a xeneograft tumor model using PANC-1 cells, BMP-2 dramatically promoted tumor growth, while Spp24 not only abolished the effect of BMP-2, but also dramatically induced tumor shrinking when used alone. Activation of Smad1/5/8 participated in this process as demonstrated by immunohistochemical staining of phosphorylated Smad 1/5/8. We conclude that Spp24 can be developed into a therapeutic agent that could be employed in clinical situations where the inhibition of BMPs and related proteins is advantageous. - Highlights: • Spp24 effectively inhibited the in vivo tumor growth of PANC-1. • BMP-2 dramatically promoted tumor growth by promoting PANC-1 proliferation. • Spp24 abolished the tumor growth effect of BMP-2 by promoting PANC-1 apoptosis. • Spp24 may be a candidate as a therapeutic agent of pancreatic cancer.

  4. The co-chaperone Cdc37 regulates the rabies virus phosphoprotein stability by targeting to Hsp90AA1 machinery

    PubMed Central

    Xu, Yunbin; Liu, Fei; Liu, Juan; Wang, Dandan; Yan, Yan; Ji, Senlin; Zan, Jie; Zhou, Jiyong

    2016-01-01

    Cdc37, as a kinase-specific co-chaperone of the chaperone Hsp90AA1 (Hsp90), actively aids with the maturation, stabilization and activation of the cellular or viral kinase/kinase-like targets. Phosphoprotein (P) of rabies virus (RABV) is a multifunctional, non-kinase protein involved in interferon antagonism, viral transcription and replication. Here, we demonstrated that the RABV non-kinase P is chaperoned by Cdc37 and Hsp90 during infection. We found that Cdc37 and Hsp90 affect the RABV life cycle directly. Activity inhibition and knockdown of Cdc37 and Hsp90 increased the instability of the viral P protein. Overexpression of Cdc37 and Hsp90 maintained P’s stability but did not increase the yield of infectious RABV virions. We further demonstrated that the non-enzymatic polymerase cofactor P protein of all the genotypes of lyssaviruses is a target of the Cdc37/Hsp90 complex. Cdc37, phosphorylated or unphosphorylated on Ser13, aids the P protein to load onto the Hsp90 machinery, with or without Cdc37 binding to Hsp90. However, the interaction between Cdc37 and Hsp90 appears to have additional allosteric regulation of the conformational switch of Hsp90. Our study highlighted a novel mechanism in which Cdc37/Hsp90 chaperones a non-kinase target, which has significant implications for designing therapeutic targets against Rabies. PMID:27251758

  5. The non-pathogenic Henipavirus Cedar paramyxovirus phosphoprotein has a compromised ability to target STAT1 and STAT2.

    PubMed

    Lieu, Kim G; Marsh, Glenn A; Wang, Lin-Fa; Netter, Hans J

    2015-12-01

    Immune evasion by the lethal henipaviruses, Hendra (HeV) and Nipah virus, is mediated by its interferon (IFN) antagonist P gene products, phosphoprotein (P), and the related V and W proteins, which can target the signal transducer and activator of transcription 1 (STAT1) and STAT2 proteins to inhibit IFN/STAT signaling. However, it is not clear if the recently identified non-pathogenic Henipavirus, Cedar paramyxovirus (CedPV), is also able to antagonize the STAT proteins. We performed comparative studies between the HeV P gene products (P/V/W) and CedPV-P (CedPV does not encode V or W) and demonstrate that differences exist in their ability to engage the STAT proteins using immunoprecipitation and quantitative confocal microscopic analysis. In contrast to HeV-P gene encoded proteins, the ability of CedPV-P to interact with and relocalize STAT1 or STAT2 is compromised, correlating with a reduced capacity to inhibit the mRNA synthesis of IFN-inducible gene MxA. Furthermore, infection studies with HeV and CedPV demonstrate that HeV is more potent than CedPV in inhibiting the IFN-α-mediated nuclear accumulation of STAT1. These results strongly suggest that the ability of CedPV to counteract the IFN/STAT response is compromised compared to HeV.

  6. Inorganic-Organic Nanocomposite Assembly Using Collagen as Template and Sodium Tripolyphosphate as A Biomimetic Analog of Matrix Phosphoprotein

    PubMed Central

    Dai, Lin; Qi, Yi-Pin; Niu, Li-Na; Liu, Yan; Pucci, Cesar R.; Looney, Stephen W.; Ling, Jun-Qi; Pashley, David H.; Tay, Franklin R.

    2011-01-01

    Nanocomposites created with polycarboxylic acid alone as a stabilization agent for prenucleation clusters-derived amorphous calcium phosphate exhibit non-periodic apatite deposition. In the present study, we report the use of inorganic polyphosphate as a biomimetic analog of matrix phosphoprotein for directing polyacrylic acid-stabilized amorphous nanoprecursor phases to assemble into periodic apatite-collagen nanocomposites. The sorption and desorption characteristics of sodium tripolyphosphate to type I collagen was examined. Periodic nanocomposite assembly with collagen as a template was demonstrated with TEM and SEM using a Portland cement-based resin composite and a phosphate-containing simulated body fluid. Apatite was detected within the collagen at 24 hours and became more distinct at 48 hours, with prenucleation clusters attaching to the collagen fibril surface during the initial infiltration stage. Apatite-collagen nanocomposites at 72 hours were heavily mineralized with periodically-arranged intrafibrillar apatite platelets. Defect-containing nanocomposites caused by desorption of TPP from collagen fibrils were observed in regions lacking the inorganic phase. PMID:21857797

  7. Phosphoric acid esters cannot replace polyvinylphosphonic acid as phosphoprotein analogs in biomimetic remineralization of resin-bonded dentin

    PubMed Central

    Mai, Sui; Kim, Young Kyung; Toledano, Manuel; Breschi, Lorenzo; Ling, Jun Qi; Pashley, David H.; Tay, Franklin R.

    2009-01-01

    Polyvinylphosphonic acid (PVPA), a biomimetic analog of phosphoproteins, is crucial for recruiting polyacrylic acid (PAA)-stabilized amorphous calcium phosphate nanoprecursors during biomimetic remineralization of dentin collagen matrices. This study tested the null hypothesis that phosphoric acid esters of methacrylates in dentin adhesives cannot replace PVPA during bimimetic remineralization of resin-dentin interfaces. Human dentin specimens were bonded with: I) XP Bond, an etch-and-rinse adhesive using moist bonding; II) XP Bond using dry bonding; and III) Adper Prompt L-Pop, a self-etching adhesive. The control medium contained only set Portland cement and a simulated body fluid (SBF) without any biomimetic analog. Two experimental Portland cement/SBF remineralization media were evaluated: the first contained PAA as the sole biomimetic analog, the second contained PAA and PVPA as dual biomimetic analogs. No remineralization of the resin-dentin interfaces could be identified from specimens immersed in the control medium. After 2–4 months in the first experimental medium, specimens exhibited either no remineralization or large crystal formation within hybrid layers. Only specimens immersed in the second remineralization medium produced nanocrystals that accounted for intrafibrillar remineralization within hybrid layers. The null hypothesis could not be rejected; phosphoric acid esters in dentin adhesives cannot replace PVPA during biomimetic remineralization of adhesive-bonded dentin. PMID:19481792

  8. Positively Charged Residues Are the Major Determinants of Ribosomal Velocity

    PubMed Central

    Charneski, Catherine A.; Hurst, Laurence D.

    2013-01-01

    Both for understanding mechanisms of disease and for the design of transgenes, it is important to understand the determinants of ribosome velocity, as changes in the rate of translation are important for protein folding, error attenuation, and localization. While there is great variation in ribosomal occupancy along even a single transcript, what determines a ribosome's occupancy is unclear. We examine this issue using data from a ribosomal footprinting assay in yeast. While codon usage is classically considered a major determinant, we find no evidence for this. By contrast, we find that positively charged amino acids greatly retard ribosomes downstream from where they are encoded, consistent with the suggestion that positively charged residues interact with the negatively charged ribosomal exit tunnel. Such slowing is independent of and greater than the average effect owing to mRNA folding. The effect of charged amino acids is additive, with ribosomal occupancy well-predicted by a linear fit to the density of positively charged residues. We thus expect that a translated poly-A tail, encoding for positively charged lysines regardless of the reading frame, would act as a sandtrap for the ribosome, consistent with experimental data. PMID:23554576

  9. Regulating the Ribosome: A Spotlight on RNA Dark Matter

    PubMed Central

    Lintner, Nathanael G.; Cate, Jamie H.D.

    2014-01-01

    In this issue Pircher et al.(2014) show that an abundant ribosome-associated 18-nt noncoding RNA (ncRNA),derived from the open reading frame of an mRNA,acts directly on the ribosome and regulates global translation levels in response to hypertonic shock. PMID:24725592

  10. Ribonuclease Activity Associated With Ribosomes of Zea mays 1

    PubMed Central

    Hsiao, Theodore C.

    1968-01-01

    At pH 6.5, a ribonuclease(s) is associated with ribosomes isolated from corn (Zea mays L.) and cannot be removed by repeated differential centrifugation or by sedimenting through the sucrose gradient. The enzyme is active under conditions favoring the maintenance of integrity of the ribosomes. Little or no latent ribonuclease appears to be present. The activity of the enzyme at pH 5.8 is stimulated by KCl and inhibited by polyvinyl sulfate, zinc, and bentonite. Deoxyribonuclease is also found on the particles. The enzyme can be removed from ribosomes by adsorption onto bentonite. Ribosomes are also adsorbed but to a much lesser extent at low bentonite concentrations. The enzyme is easily dissociated from ribosomes by raising the pH to 8.5, and readsorbed when the pH is lowered. The ribonuclease activity on ribosomes shows a sharp increase with cell age that parallels closely the increase in total activity in the homogenate. The ratio of activities of deoxyribonuclease to ribonuclease on ribosomes also changes with cell age and again the changes appear to reflect changes in the homogenate. It is suggested that most of the association of ribonuclease with corn ribosomes may not be meaningful in vivo and occurs only after the cells are ruptured. Images PMID:16656919

  11. Role of ribosomal protein mutations in tumor development (Review)

    PubMed Central

    GOUDARZI, KAVEH M.; LINDSTRÖM, MIKAEL S.

    2016-01-01

    Ribosomes are cellular machines essential for protein synthesis. The biogenesis of ribosomes is a highly complex and energy consuming process that initiates in the nucleolus. Recently, a series of studies applying whole-exome or whole-genome sequencing techniques have led to the discovery of ribosomal protein gene mutations in different cancer types. Mutations in ribosomal protein genes have for example been found in endometrial cancer (RPL22), T-cell acute lymphoblastic leukemia (RPL10, RPL5 and RPL11), chronic lymphocytic leukemia (RPS15), colorectal cancer (RPS20), and glioma (RPL5). Moreover, patients suffering from Diamond-Blackfan anemia, a bone marrow failure syndrome caused by mutant ribosomal proteins are also at higher risk for developing leukemia, or solid tumors. Different experimental models indicate potential mechanisms whereby ribosomal proteins may initiate cancer development. In particular, deregulation of the p53 tumor suppressor network and altered mRNA translation are mechanisms likely to be involved. We envisage that changes in expression and the occurrence of ribosomal protein gene mutations play important roles in cancer development. Ribosome biology constitutes a re-emerging vital area of basic and translational cancer research. PMID:26892688

  12. 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…

  13. 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…

  14. Probing the mechanisms underlying human diseases in making ribosomes

    PubMed Central

    Farley, Katherine I.; Baserga, Susan J.

    2017-01-01

    Ribosomes are essential, highly complex machines responsible for protein synthesis in all growing cells. Because of their importance, the process of building these machines is intricately regulated. While the proteins involved in regulating ribosome biogenesis are just beginning to be understood, especially in human cells, the consequences for dysregulating this process have been even less studied. Such interruptions in ribosome synthesis result in a collection of human disorders known as ribosomopathies. Ribosomopathies, which occur due to mutations in proteins involved in the global process of ribosome biogenesis, result in tissue-specific defects. The questions posed by this dichotomy and the steps taken to address these questions are therefore the focus of this review: How can tissue-specific disorders result from alterations in global processes? Could ribosome specialization account for this difference? PMID:27528749

  15. Prediction of ribosome footprint profile shapes from transcript sequences

    PubMed Central

    Liu, Tzu-Yu; Song, Yun S.

    2016-01-01

    Motivation: Ribosome profiling is a useful technique for studying translational dynamics and quantifying protein synthesis. Applications of this technique have shown that ribosomes are not uniformly distributed along mRNA transcripts. Understanding how each transcript-specific distribution arises is important for unraveling the translation mechanism. Results: Here, we apply kernel smoothing to construct predictive features and build a sparse model to predict the shape of ribosome footprint profiles from transcript sequences alone. Our results on Saccharomyces cerevisiae data show that the marginal ribosome densities can be predicted with high accuracy. The proposed novel method has a wide range of applications, including inferring isoform-specific ribosome footprints, designing transcripts with fast translation speeds and discovering unknown modulation during translation. Availability and implementation: A software package called riboShape is freely available at https://sourceforge.net/projects/riboshape Contact: yss@berkeley.edu PMID:27307616

  16. Discovery of a small molecule that inhibits bacterial ribosome biogenesis.

    PubMed

    Stokes, Jonathan M; Davis, Joseph H; Mangat, Chand S; Williamson, James R; Brown, Eric D

    2014-09-18

    While small molecule inhibitors of the bacterial ribosome have been instrumental in understanding protein translation, no such probes exist to study ribosome biogenesis. We screened a diverse chemical collection that included previously approved drugs for compounds that induced cold sensitive growth inhibition in the model bacterium Escherichia coli. Among the most cold sensitive was lamotrigine, an anticonvulsant drug. Lamotrigine treatment resulted in the rapid accumulation of immature 30S and 50S ribosomal subunits at 15 °C. Importantly, this was not the result of translation inhibition, as lamotrigine was incapable of perturbing protein synthesis in vivo or in vitro. Spontaneous suppressor mutations blocking lamotrigine activity mapped solely to the poorly characterized domain II of translation initiation factor IF2 and prevented the binding of lamotrigine to IF2 in vitro. This work establishes lamotrigine as a widely available chemical probe of bacterial ribosome biogenesis and suggests a role for E. coli IF2 in ribosome assembly.

  17. Components of the macrolide binding site on the ribosome.

    PubMed

    Tejedor, F; Ballesta, J P

    1985-07-01

    Carbomycin A, niddamycin and tylosin, macrolide antibiotics that inhibit ribosomal activity, have alpha-beta unsaturated ketone groups in their structure that make them photoreactive. These drugs can also take part in thermic reactions, probably through an addition mechanism to the double bond. Given of the photoactivity and thermic reactivity of their molecules, affinity labeling of the macrolide binding site on the ribosome has been performed using radioactive derivatives of these drugs. After either irradiating or incubating samples containing antibiotics and ribosomal particles, radioactivity appears covalent associated to the proteins. Ribosomal protein L27 is the major labeled component, indicating that this polypeptide, which seems to be part of the peptidyl transferase centre of the ribosome, also plays an important role on the macrolide binding site.

  18. DExD/H-box RNA helicases in ribosome biogenesis

    PubMed Central

    Martin, Roman; Straub, Annika U.; Doebele, Carmen; Bohnsack, Markus T.

    2013-01-01

    Ribosome synthesis requires a multitude of cofactors, among them DExD/H-box RNA helicases. Bacterial RNA helicases involved in ribosome assembly are not essential, while eukaryotes strictly require multiple DExD/H-box proteins that are involved in the much more complex ribosome biogenesis pathway. Here, RNA helicases are thought to act in structural remodeling of the RNPs including the modulation of protein binding, and they are required for allowing access or the release of specific snoRNPs from pre-ribosomes. Interestingly, helicase action is modulated by specific cofactors that can regulate recruitment and enzymatic activity. This review summarizes the current knowledge and focuses on recent findings and open questions on RNA helicase function and regulation in ribosome synthesis. PMID:22922795

  19. Probing the mechanisms underlying human diseases in making ribosomes.

    PubMed

    Farley, Katherine I; Baserga, Susan J

    2016-08-15

    Ribosomes are essential, highly complex machines responsible for protein synthesis in all growing cells. Because of their importance, the process of building these machines is intricately regulated. Although the proteins involved in regulating ribosome biogenesis are just beginning to be understood, especially in human cells, the consequences for dysregulating this process have been even less studied. Such interruptions in ribosome synthesis result in a collection of human disorders known as ribosomopathies. Ribosomopathies, which occur due to mutations in proteins involved in the global process of ribosome biogenesis, result in tissue-specific defects. The questions posed by this dichotomy and the steps taken to address these questions are therefore the focus of this review: How can tissue-specific disorders result from alterations in global processes? Could ribosome specialization account for this difference? © 2016 The Author(s). published by Portland Press Limited on behalf of the Biochemical Society.

  20. The ribosome-recycling step: consensus or controversy?

    PubMed

    Hirokawa, Go; Demeshkina, Natalia; Iwakura, Nobuhiro; Kaji, Hideko; Kaji, Akira

    2006-03-01

    Ribosome recycling, the last step in translation, is now accepted as an essential process for prokaryotes. In 2005, three laboratories showed that ribosome-recycling factor (RRF) and elongation factor G (EF-G) cause dissociation of ribosomes into subunits, solving the long-standing problem of how this essential step of translation occurs. However, there remains ongoing controversy regarding the other actions of RRF and EF-G during ribosome recycling. We propose that the available data are consistent with the notion that RRF and EF-G not only split ribosomes into subunits but also participate directly in the release of deacylated tRNA and mRNA for the next round of translation.

  1. Structures of Eukaryotic Ribosomal Stalk Proteins and Its Complex with Trichosanthin, and Their Implications in Recruiting Ribosome-Inactivating Proteins to the Ribosomes

    PubMed Central

    Choi, Andrew K. H.; Wong, Eddie C. K.; Lee, Ka-Ming; Wong, Kam-Bo

    2015-01-01

    Ribosome-inactivating proteins (RIP) are RNA N-glycosidases that inactivate ribosomes by specifically depurinating a conserved adenine residue at the α-sarcin/ricin loop of 28S rRNA. Recent studies have pointed to the involvement of the C-terminal domain of the eukaryotic stalk proteins in facilitating the toxic action of RIPs. This review highlights how structural studies of eukaryotic stalk proteins provide insights into the recruitment of RIPs to the ribosomes. Since the C-terminal domain of eukaryotic stalk proteins is involved in specific recognition of elongation factors and some eukaryote-specific RIPs (e.g., trichosanthin and ricin), we postulate that these RIPs may have evolved to hijack the translation-factor-recruiting function of ribosomal stalk in reaching their target site of rRNA. PMID:25723321

  2. Structures of eukaryotic ribosomal stalk proteins and its complex with trichosanthin, and their implications in recruiting ribosome-inactivating proteins to the ribosomes.

    PubMed

    Choi, Andrew K H; Wong, Eddie C K; Lee, Ka-Ming; Wong, Kam-Bo

    2015-02-25

    Ribosome-inactivating proteins (RIP) are RNA N-glycosidases that inactivate ribosomes by specifically depurinating a conserved adenine residue at the α-sarcin/ricin loop of 28S rRNA. Recent studies have pointed to the involvement of the C-terminal domain of the eukaryotic stalk proteins in facilitating the toxic action of RIPs. This review highlights how structural studies of eukaryotic stalk proteins provide insights into the recruitment of RIPs to the ribosomes. Since the C-terminal domain of eukaryotic stalk proteins is involved in specific recognition of elongation factors and some eukaryote-specific RIPs (e.g., trichosanthin and ricin), we postulate that these RIPs may have evolved to hijack the translation-factor-recruiting function of ribosomal stalk in reaching their target site of rRNA.

  3. Regulation of ribosomal DNA amplification by the TOR pathway

    PubMed Central

    Jack, Carmen V.; Cruz, Cristina; Hull, Ryan M.; Keller, Markus A.; Ralser, Markus; Houseley, Jonathan

    2015-01-01

    Repeated regions are widespread in eukaryotic genomes, and key functional elements such as the ribosomal DNA tend to be formed of high copy repeated sequences organized in tandem arrays. In general, high copy repeats are remarkably stable, but a number of organisms display rapid ribosomal DNA amplification at specific times or under specific conditions. Here we demonstrate that target of rapamycin (TOR) signaling stimulates ribosomal DNA amplification in budding yeast, linking external nutrient availability to ribosomal DNA copy number. We show that ribosomal DNA amplification is regulated by three histone deacetylases: Sir2, Hst3, and Hst4. These enzymes control homologous recombination-dependent and nonhomologous recombination-dependent amplification pathways that act in concert to mediate rapid, directional ribosomal DNA copy number change. Amplification is completely repressed by rapamycin, an inhibitor of the nutrient-responsive TOR pathway; this effect is separable from growth rate and is mediated directly through Sir2, Hst3, and Hst4. Caloric restriction is known to up-regulate expression of nicotinamidase Pnc1, an enzyme that enhances Sir2, Hst3, and Hst4 activity. In contrast, normal glucose concentrations stretch the ribosome synthesis capacity of cells with low ribosomal DNA copy number, and we find that these cells show a previously unrecognized transcriptional response to caloric excess by reducing PNC1 expression. PNC1 down-regulation forms a key element in the control of ribosomal DNA amplification as overexpression of PNC1 substantially reduces ribosomal DNA amplification rate. Our results reveal how a signaling pathway can orchestrate specific genome changes and demonstrate that the copy number of repetitive DNA can be altered to suit environmental conditions. PMID:26195783

  4. Regulation of ribosome biogenesis in maize embryonic axes during germination.

    PubMed

    Villa-Hernández, J M; Dinkova, T D; Aguilar-Caballero, R; Rivera-Cabrera, F; Sánchez de Jiménez, E; Pérez-Flores, L J

    2013-10-01

    Ribosome biogenesis is a pre-requisite for cell growth and proliferation; it is however, a highly regulated process that consumes a great quantity of energy. It requires the coordinated production of rRNA, ribosomal proteins and non-ribosomal factors which participate in the processing and mobilization of the new ribosomes. Ribosome biogenesis has been studied in yeast and animals; however, there is little information about this process in plants. The objective of the present work was to study ribosome biogenesis in maize seeds during germination, a stage characterized for its fast growth, and the effect of insulin in this process. Insulin has been reported to accelerate germination and to induce seedling growth. It was observed that among the first events reactivated just after 3 h of imbibition are the rDNA transcription and the pre-rRNA processing and that insulin stimulates both of them (40-230%). The transcript of nucleolin, a protein which regulates rDNA transcription and pre-rRNA processing, is among the messages stored in quiescent dry seeds and it is mobilized into the polysomal fraction during the first hours of imbibition (6 h). In contrast, de novo ribosomal protein synthesis was low during the first hours of imbibition (3 and 6 h) increasing by 60 times in later stages (24 h). Insulin increased this synthesis (75%) at 24 h of imbibition; however, not all ribosomal proteins were similarly regulated. In this regard, an increase in RPS6 and RPL7 protein levels was observed, whereas RPL3 protein levels did not change even though its transcription was induced. Results show that ribosome biogenesis in the first stages of imbibition is carried out with newly synthesized rRNA and ribosomal proteins translated from stored mRNA.

  5. Crystal Structures of the uL3 Mutant Ribosome: Illustration of the Importance of Ribosomal Proteins for Translation Efficiency.

    PubMed

    Mailliot, Justine; Garreau de Loubresse, Nicolas; Yusupova, Gulnara; Meskauskas, Arturas; Dinman, Jonathan D; Yusupov, Marat

    2016-05-22

    The ribosome has been described as a ribozyme in which ribosomal RNA is responsible for peptidyl-transferase reaction catalysis. The W255C mutation of the universally conserved ribosomal protein uL3 has diverse effects on ribosome function (e.g., increased affinities for transfer RNAs, decreased rates of peptidyl-transfer), and cells harboring this mutation are resistant to peptidyl-transferase inhibitors (e.g., anisomycin). These observations beg the question of how a single amino acid mutation may have such wide ranging consequences. Here, we report the structure of the vacant yeast uL3 W255C mutant ribosome by X-ray crystallography, showing a disruption of the A-site side of the peptidyl-transferase center (PTC). An additional X-ray crystallographic structure of the anisomycin-containing mutant ribosome shows that high concentrations of this inhibitor restore a "WT-like" configuration to this region of the PTC, providing insight into the resistance mechanism of the mutant. Globally, our data demonstrate that ribosomal protein uL3 is structurally essential to ensure an optimal and catalytically efficient organization of the PTC, highlighting the importance of proteins in the RNA-centered ribosome.

  6. Crystal Structures of the uL3 Mutant Ribosome: Illustration of the Importance of Ribosomal Proteins for Translation Efficiency

    PubMed Central

    Mailliot, Justine; de Loubresse, Nicolas Garreau; Yusupova, Gulnara; Meskauskas, Arturas; Dinman, Jonathan D.; Yusupov, Marat

    2017-01-01

    The ribosome has been described as a ribozyme in which ribosomal RNA is responsible for peptidyl-transferase reaction catalysis. The W255C mutation of the universally conserved ribosomal protein uL3 has diverse effects on ribosome function (e.g., increased affinities for transfer RNAs, decreased rates of peptidyl-transfer), and cells harboring this mutation are resistant to peptidyl-transferase inhibitors (e.g., anisomycin). These observations beg the question of how a single amino acid mutation may have such wide ranging consequences. Here, we report the structure of the vacant yeast uL3 W255C mutant ribosome by X-ray crystallography, showing a disruption of the A-site side of the peptidyl-transferase center (PTC). An additional X-ray crystallographic structure of the anisomycin-containing mutant ribosome shows that high concentrations of this inhibitor restore a “WT-like” configuration to this region of the PTC, providing insight into the resistance mechanism of the mutant. Globally, our data demonstrate that ribosomal protein uL3 is structurally essential to ensure an optimal and catalytically efficient organization of the PTC, highlighting the importance of proteins in the RNA-centered ribosome. PMID:26906928

  7. Ribosome-Inactivating and Related Proteins

    PubMed Central

    Schrot, Joachim; Weng, Alexander; Melzig, Matthias F.

    2015-01-01

    Ribosome-inactivating proteins (RIPs) are toxins that act as N-glycosidases (EC 3.2.2.22). They are mainly produced by plants and classified as type 1 RIPs and type 2 RIPs. There are also RIPs and RIP related proteins that cannot be grouped into the classical type 1 and type 2 RIPs because of their different sizes, structures or functions. In addition, there is still not a uniform nomenclature or classification existing for RIPs. In this review, we give the current status of all known plant RIPs and we make a suggestion about how to unify those RIPs and RIP related proteins that cannot be classified as type 1 or type 2 RIPs. PMID:26008228

  8. Actinomadura Species: Laboratory Maintenance and Ribosome Engineering.

    PubMed

    Dhakal, Dipesh; Chung, Nguyen Thanh; Rayamajhi, Vijay; Sohng, Jae Kyung

    2017-02-06

    Actinomadura spp. are aerobic, Gram-positive, catalase-positive, non-acid fast, non-motile actinomycetes. Some species of Actinomadura are associated with opportunistic infections in humans. However, many bioactive compounds with pharmaceutical applications can be isolated from various Actinomadura spp. This unit includes general protocols for the laboratory maintenance of Actinomadura spp., including growth in liquid medium, growth on solid agar, long-term storage, and generation of a higher producing strain by ribosome engineering. Actinomadura hibisca P157-2 is used as a prototype for explaining the considerations for efficient laboratory maintenance of Actinomadura spp. © 2017 by John Wiley & Sons, Inc. Copyright © 2017 John Wiley & Sons, Inc.

  9. Ribosomally encoded cyclic peptide toxins from mushrooms.

    PubMed

    Walton, Jonathan D; Luo, Hong; Hallen-Adams, Heather

    2012-01-01

    The cyclic peptide toxins of poisonous Amanita mushrooms are chemically unique among known natural products. Furthermore, they differ from other fungal cyclic peptides in being synthesized on ribosomes instead of by nonribosomal peptide synthetases. Because of their novel structures and biogenic origins, elucidation of the biosynthetic pathway of the Amanita cyclic peptides presents both challenges and opportunities. In particular, a full understanding of the pathway should lead to the ability to direct synthesis of a large number of novel cyclic peptides based on the Amanita toxin scaffold by genetic engineering of the encoding genes. Here, we highlight some of the principal methods for working with the Amanita cyclic peptides and the known steps in their biosynthesis.

  10. Higher order structure in ribosomal RNA.

    PubMed

    Gutell, R R; Noller, H F; Woese, C R

    1986-05-01

    The only reliable general method currently available for determining precise higher order structure in the large ribosomal RNAs is comparative sequence analysis. The method is here applied to reveal 'tertiary' structure in the 16S-like rRNAs, i.e. structure more complex than simple double-helical, secondary structure. From a list of computer-generated potential higher order interactions within 16S rRNA one such interaction considered likely was selected for experimental test. The putative interaction involves a Watson-Crick one to one correspondence between positions 570 and 866 in the molecule (E. coli numbering). Using existing oligonucleotide catalog information several organisms were selected whose 16S rRNA sequences might test the proposed co-variation. In all of the (phylogenetically independent) cases selected, full sequence evidence confirms the predicted one to one (Watson-Crick) correspondence. An interaction between positions 570 and 866 is, therefore, considered proven phylogenetically.

  11. Ribosome Inactivating Proteins from an evolutionary perspective.

    PubMed

    Lapadula, Walter Jesús; Ayub, Maximiliano Juri

    2017-09-15

    Ribosome Inactivating Proteins (RIPs) are rRNA N-glycosidases that inhibit protein synthesis through the elimination of a single adenine residue from 28S rRNA. Many of these toxins have been characterized in depth from a biochemical and molecular point of view. In addition, their potential use in medicine as highly selective toxins is being explored. In contrast, the evolutionary history of RIP encoding genes has remained traditionally underexplored. In recent years, accumulation of large genomic data has fueled research on this issue and revealed unexpected information about the origin and evolution of RIP toxins. In this review we summarize the current evidence available on the occurrence of different evolutionary mechanisms (gene duplication and losses, horizontal gene transfer, synthesis de novo and domain combination) involved in the evolution of the RIP gene family. Finally, we propose a revised nomenclature for RIP genes based on their evolutionary history. Copyright © 2017 Elsevier Ltd. All rights reserved.

  12. 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

  13. Ribosomal protein uS19 mutants reveal its role in coordinating ribosome structure and function

    PubMed Central

    Bowen, Alicia M; Musalgaonkar, Sharmishtha; Moomau, Christine A; Gulay, Suna P; Mirvis, Mary; Dinman, Jonathan D

    2015-01-01

    Prior studies identified allosteric information pathways connecting functional centers in the large ribosomal subunit to the decoding center in the small subunit through the B1a and B1b/c intersubunit bridges in yeast. In prokaryotes a single SSU protein, uS13, partners with H38 (the A-site finger) and uL5 to form the B1a and B1b/c bridges respectively. In eukaryotes, the SSU component was split into 2 separate proteins during the course of evolution. One, also known as uS13, participates in B1b/c bridge with uL5 in eukaryotes. The other, called uS19 is the SSU partner in the B1a bridge with H38. Here, polyalanine mutants of uS19 involved in the uS19/uS13 and the uS19/H38 interfaces were used to elucidate the important amino acid residues involved in these intersubunit communication pathways. Two key clusters of amino acids were identified: one located at the junction between uS19 and uS13, and a second that appears to interact with the distal tip of H38. Biochemical analyses reveal that these mutations shift the ribosomal rotational equilibrium toward the unrotated state, increasing ribosomal affinity for tRNAs in the P-site and for ternary complex in the A-site, and inhibit binding of the translocase, eEF2. These defects in turn affect specific aspects of translational fidelity. These findings suggest that uS19 plays a critical role as a conduit of information exchange between the large and small ribosomal subunits directly through the B1a, and indirectly through the B1b/c bridges. PMID:26824029

  14. An extra-ribosomal function of ribosomal protein L13a in macrophage resolves inflammation

    PubMed Central

    Poddar, Darshana; Basu, Abhijit; Baldwin, William; Kondratov, Roman V; Barik, Sailen; Mazumder, Barsanjit

    2013-01-01

    Inflammation is an obligatory attempt of the immune system to protect the host from infections. However, unregulated synthesis of pro-inflammatory products can have detrimental effects. Although mechanisms that lead to inflammation are well appreciated, those that restrain it are not adequately understood. Creating macrophage-specific L13a-knockout (KO) mice here we report that depletion of ribosomal protein L13a abrogates the endogenous translation control of several chemokines in macrophages. Upon LPS-induced endotoxemia these animals displayed symptoms of severe inflammation caused by widespread infiltration of macrophages in major organs causing tissue injury and reduced survival rates. Macrophages from these KO animals show unregulated expression of several chemokines e.g. CXCL13, CCL22, CCL8 and CCR3. These macrophages failed to show L13a-dependent RNA binding complex formation on target mRNAs. In addition, increased polyribosomal abundance of these mRNAs shows a defect in translation control in the macrophages. Thus, our studies provide the first evidence of an essential extra-ribosomal function of ribosomal protein L13a in resolving physiological inflammation in a mammalian host. PMID:23460747

  15. Rli1/ABCE1 Recycles Terminating Ribosomes and Controls Translation Reinitiation in 3'UTRs In Vivo.

    PubMed

    Young, David J; Guydosh, Nicholas R; Zhang, Fan; Hinnebusch, Alan G; Green, Rachel

    2015-08-13

    To study the function of Rli1/ABCE1 in vivo, we used ribosome profiling and biochemistry to characterize its contribution to ribosome recycling. When Rli1 levels were diminished, 80S ribosomes accumulated both at stop codons and in the adjoining 3'UTRs of most mRNAs. Frequently, these ribosomes reinitiated translation without the need for a canonical start codon, as small peptide products predicted by 3'UTR ribosome occupancy in all three reading frames were confirmed by western analysis and mass spectrometry. Eliminating the ribosome-rescue factor Dom34 dramatically increased 3'UTR ribosome occupancy in Rli1 depleted cells, indicating that Dom34 clears the bulk of unrecycled ribosomes. Thus, Rli1 is crucial for ribosome recycling in vivo and controls ribosome homeostasis. 3'UTR translation occurs in wild-type cells as well, and observations of elevated 3'UTR ribosomes during stress suggest that modulating recycling and reinitiation is involved in responding to environmental changes.

  16. Cooperative binding of multimeric phosphoprotein (P) of vesicular stomatitis virus to polymerase (L) and template: pathways of assembly.

    PubMed Central

    Gao, Y; Lenard, J

    1995-01-01

    It was previously shown that the phosphoprotein (P) of vesicular stomatitis virus must undergo phosphorylation-dependent multimerization to become transcriptionally active. Phosphorylation at S-60 and/or T-62 by casein kinase II or substitution of these residues by D is required for multimer formation. We now find that substitution of either one of these residues by A prevents phosphorylation by casein kinase II and multimer formation. The binding of multimeric P to the other two transcriptional components of vesicular stomatitis virus (L protein and the N-RNA template) has been characterized by using P immobilized on beads through its poly(His) tag to facilitate recovery of bound complexes. Multimerization of P was absolutely required for binding to both L and template. Multimeric P combined with the polymerase enzyme (L) in a stoichiometric 1:1 complex, which bound to the N-RNA template much more strongly than multimeric P alone. Substitution of S-227 and S-233 by A residues had no effect on multimerization or binding of L to P but prevented binding of both P and L to template and abolished transcriptional activity. In contrast, substitution of these residues with D residues had no effect on template binding or activity. However, substitution at these sites by either D or A largely abolished phosphorylation by L-associated kinases, thus identifying S-227 and S-233 as the major sites targeted by these kinases and confirming that phosphorylation of P protein by L-associated kinases is without transcriptional effect. PMID:7494281

  17. Vasodilator-stimulated phosphoprotein restricts cell-to-cell spread of Shigella flexneri at the cell periphery

    PubMed Central

    Lee, Soo Young; Gertler, Frank B.

    2015-01-01

    Shigella spp. are intracellular bacterial pathogens that cause diarrhoeal disease in humans. Shigella utilize the host actin cytoskeleton to enter cells, move through the cytoplasm of cells and pass into adjacent cells. Ena/VASP family proteins are highly conserved proteins that participate in actin-dependent dynamic cellular processes. We tested whether Ena/VASP family members VASP (vasodilator-stimulated phosphoprotein), Mena (mammalian-enabled) or EVL (Ena-VASP-like) contribute to Shigella flexneri spread through cell monolayers. VASP and EVL restricted cell-to-cell spread without significantly altering actin-based motility, whereas Mena had no effect on these processes. Phosphorylation of VASP on Ser153, Ser235 and Thr274 regulated its subcellular distribution and function. VASP derivatives that lack the Ena/VASP homology 1 (EVH1) domain or contain a phosphoablative mutation of Ser153 were defective in restricting S. flexneri spread, indicating that the EVH1 domain and phosphorylation on Ser153 are required for this process. The EVH1 domain and Ser153 of VASP were required for VASP localization to focal adhesions, and localization of VASP to focal adhesions and/or the leading edge was required for restriction of spread. The contribution of the EVH1 domain was from both the donor and the recipient cell, whereas the contribution of Ser153 phosphorylation was only from the donor cell. Thus, unlike host proteins characterized in Shigella pathogenesis that promote bacterial spread, VASP and EVL function to limit it. The ability of VASP and EVL to limit spread highlights the critical role of focal adhesion complexes and/or the leading edge in bacterial passage between cells. PMID:26358985

  18. Carboxy terminus of secreted phosphoprotein-24 kDa (spp24) is essential for full inhibition of BMP-2 activity.

    PubMed

    Brochmann, Elsa J; Simon, Robert J; Jawien, Janusz; Behnam, Keyvan; Sintuu, Chananit; Wang, Jeffrey C; Murray, Samuel S

    2010-09-01

    Secreted phosphoprotein-24 kDa (spp24) is a bone morphogenetic protein (BMP)-binding protein isolated from bone. It exists in a number of size forms and is hypothesized to function as a BMP latency protein and/or a "slow release" mechanism for BMPs involved in bone turnover and repair. We have examined the hypothesis that proteolytic modification of the C-terminus of spp24 affects its BMP-2-binding properties and bioactivity in the BMP-2-stimulated ectopic bone forming bioassay. Three different size forms of recombinant spp24 that correspond to predicted 18.1 kDa, 16.0 kDa, and 14.5 kDa proteolytic products were compared to full-length (fl) spp24. One of these forms (spp18.1) we hypothesize to be the protein which Urist initially, but apparently inaccurately, called "BMP." Only full-length spp24 completely inhibited BMP-2-induced bone formation. The 18.1 kDa truncated isoform of spp24 which we hypothesize to be Urist's protein did not. The inhibitory capacity of the proteins was correlated with their kinetic constants, assessed by surface plasmon resonance. At the highest, inhibitory, dose of spp24 and its derivatives, k(d) ("stability") best predicted the extent of ectopic bone formation whereas at the lowest dose, which was not inhibitory, k(a) ("recognition") best predicted the extent of ectopic bone formation. We conclude that proteolytic processing of spp24 affects the interaction of this protein with BMP-2 and this affects the function of the protein. (c) 2010 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.

  19. Molecular inotropy mediated by cardiac miR-based PDE4D/PRKAR1α/phosphoprotein signaling

    PubMed Central

    Bedada, Fikru B.; Martindale, Joshua J.; Arden, Erik; Metzger, Joseph M.

    2016-01-01

    Molecular inotropy refers to cardiac contractility that can be modified to affect overall heart pump performance. Here we show evidence of a new molecular pathway for positive inotropy by a cardiac-restricted microRNA (miR). We report enhanced cardiac myocyte performance by acute titration of cardiac myosin-embedded miR-208a. The observed positive effect was independent of host gene myosin effects with evidence of negative regulation of cAMP-specific 3′,5′-cyclic phosphodiesterase 4D (PDE4D) and the regulatory subunit of PKA (PRKAR1α) content culminating in PKA-site dependent phosphorylation of cardiac troponin I (cTnI) and phospholamban (PLN). Further, acute inhibition of miR-208a in adult myocytes in vitro increased PDE4D expression causing reduced isoproterenol-mediated phosphorylation of cTnI and PLN. Next, rAAV-mediated miR-208a gene delivery enhanced heart contractility and relaxation parameters in vivo. Finally, acute inducible increases in cardiac miR-208a in vivo reduced PDE4D and PRKAR1α, with evidence of increased content of several complementary miRs harboring the PDE4D recognition sequence. Physiologically, this resulted in significant cardiac cTnI and PLN phosphorylation and improved heart performance in vivo. As phosphorylation of cTnI and PLN is critical to myocyte function, titration of miR-208a represents a potential new mechanism to enhance myocardial performance via the PDE4D/PRKAR1α/PKA phosphoprotein signaling pathway. PMID:27833092

  20. Vasodilator-stimulated phosphoprotein restricts cell-to-cell spread of Shigella flexneri at the cell periphery.

    PubMed

    Lee, Soo Young; Gertler, Frank B; Goldberg, Marcia B

    2015-11-01

    Shigella spp. are intracellular bacterial pathogens that cause diarrhoeal disease in humans. Shigella utilize the host actin cytoskeleton to enter cells, move through the cytoplasm of cells and pass into adjacent cells. Ena/VASP family proteins are highly conserved proteins that participate in actin-dependent dynamic cellular processes. We tested whether Ena/VASP family members VASP (vasodilator-stimulated phosphoprotein), Mena (mammalian-enabled) or EVL (Ena-VASP-like) contribute to Shigella flexneri spread through cell monolayers. VASP and EVL restricted cell-to-cell spread without significantly altering actin-based motility, whereas Mena had no effect on these processes. Phosphorylation of VASP on Ser153, Ser235 and Thr274 regulated its subcellular distribution and function. VASP derivatives that lack the Ena/VASP homology 1 (EVH1) domain or contain a phosphoablative mutation of Ser153 were defective in restricting S. flexneri spread, indicating that the EVH1 domain and phosphorylation on Ser153 are required for this process. The EVH1 domain and Ser153 of VASP were required for VASP localization to focal adhesions, and localization of VASP to focal adhesions and/or the leading edge was required for restriction of spread. The contribution of the EVH1 domain was from both the donor and the recipient cell, whereas the contribution of Ser153 phosphorylation was only from the donor cell. Thus, unlike host proteins characterized in Shigella pathogenesis that promote bacterial spread, VASP and EVL function to limit it. The ability of VASP and EVL to limit spread highlights the critical role of focal adhesion complexes and/or the leading edge in bacterial passage between cells.

  1. Regulation of Stress-Inducible Phosphoprotein 1 Nuclear Retention by Protein Inhibitor of Activated STAT PIAS1

    PubMed Central

    Soares, Iaci N.; Caetano, Fabiana A.; Pinder, Jordan; Rodrigues, Bruna Roz; Beraldo, Flavio H.; Ostapchenko, Valeriy G.; Durette, Chantal; Pereira, Grace Schenatto; Lopes, Marilene H.; Queiroz-Hazarbassanov, Nicolle; Cunha, Isabela W.; Sanematsu, Paulo I.; Suzuki, Sergio; Bleggi-Torres, Luiz F.; Schild-Poulter, Caroline; Thibault, Pierre; Dellaire, Graham; Martins, Vilma R.; Prado, Vania F.; Prado, Marco A. M.

    2013-01-01

    Stress-inducible phosphoprotein 1 (STI1), a cochaperone for Hsp90, has been shown to regulate multiple pathways in astrocytes, but its contributions to cellular stress responses are not fully understood. We show that in response to irradiation-mediated DNA damage stress STI1 accumulates in the nucleus of astrocytes. Also, STI1 haploinsufficiency decreases astrocyte survival after irradiation. Using yeast two-hybrid screenings we identified several nuclear proteins as STI1 interactors. Overexpression of one of these interactors, PIAS1, seems to be specifically involved in STI1 nuclear retention and in directing STI1 and Hsp90 to specific sub-nuclear regions. PIAS1 and STI1 co-immunoprecipitate and PIAS1 can function as an E3 SUMO ligase for STI. Using mass spectrometry we identified five SUMOylation sites in STI1. A STI1 mutant lacking these five sites is not SUMOylated, but still accumulates in the nucleus in response to increased expression of PIAS1, suggesting the possibility that a direct interaction with PIAS1 could be responsible for STI1 nuclear retention. To test this possibility, we mapped the interaction sites between PIAS1 and STI1 using yeast-two hybrid assays and surface plasmon resonance and found that a large domain in the N-terminal region of STI1 interacts with high affinity with amino acids 450–480 of PIAS1. Knockdown of PIAS1 in astrocytes impairs the accumulation of nuclear STI1 in response to irradiation. Moreover, a PIAS1 mutant lacking the STI1 binding site is unable to increase STI1 nuclear retention. Interestingly, in human glioblastoma multiforme PIAS1 expression is increased and we found a significant correlation between increased PIAS1 expression and STI1 nuclear localization. These experiments provide evidence that direct interaction between STI1 and PIAS1 is involved in the accumulation of nuclear STI1. This retention mechanism could facilitate nuclear chaperone activity. PMID:23938469

  2. Phosphoprotein Gene Contributes to the Enhanced Apoptosis Induced by Wild-Type Rabies Virus GD-SH-01 In Vitro

    PubMed Central

    Tian, Qin; Wang, Yifei; Zhang, Qiong; Luo, Jun; Jiang, He; Zhang, Boyue; Mei, Mingzhu; Wu, Fan; Wu, Yuting; Peng, Jiaojiao; Long, Teng; Luo, Yongwen; Guo, Xiaofeng

    2017-01-01

    Previous research demonstrated that the matrix protein (M) and glycoprotein (G) of attenuated rabies virus (RABV) strains are involved in the induction of host cell apoptosis. In this work, we show that wild-type (wt) RABV GD-SH-01 induces significantly greater apoptosis than the attenuated strain HEP-Flury. In order to identify the gene(s) accounting for this phenotype, five recombinant RABVs (rRABVs) were constructed by replacing each single gene of HEP-Flury with the corresponding gene of GD-SH-01. By using these rRABVs, we found that not only M and G, but also the phosphoprotein (P) plays an important role in inducing apoptosis. In order to figure out the different role of P gene in inducing apoptosis from the highly divergent background, another rRABV rGDSH-P, which carries the P gene of HEP-Flury in the background of the GD-SH-01 was generated. It was found that infection of NA cells with GD-SH-01 or the recombinant strain rHEP-shP, which carries P gene of GD-SH-01, induced significantly greater apoptosis than HEP-Flury or rGDSH-P in a caspase-dependent pathway that ultimately leads to the activation of the intrinsic apoptotic pathway, which is well characterized with the downregulation of bcl-2, the decrease of mitochondrial membrane potential, the release of mitochondrial cytochrome c, the activation of caspase-9 and caspase-3, and finally the cleavage of poly (ADP-ribose) polymerase. Our results imply that wt P from GD-SH-01 mediates this effect may partly by facilitating viral RNA synthesis but not by viral replication. In sum, we demonstrate a wt RABV strain GD-SH-01 to induce stronger apoptosis than an attenuated RABV HEP-Flury and propose that wt P from GD-SH-01 is involved in this process. PMID:28928726

  3. Phosphoprotein Gene Contributes to the Enhanced Apoptosis Induced by Wild-Type Rabies Virus GD-SH-01 In Vitro.

    PubMed

    Tian, Qin; Wang, Yifei; Zhang, Qiong; Luo, Jun; Jiang, He; Zhang, Boyue; Mei, Mingzhu; Wu, Fan; Wu, Yuting; Peng, Jiaojiao; Long, Teng; Luo, Yongwen; Guo, Xiaofeng

    2017-01-01

    Previous research demonstrated that the matrix protein (M) and glycoprotein (G) of attenuated rabies virus (RABV) strains are involved in the induction of host cell apoptosis. In this work, we show that wild-type (wt) RABV GD-SH-01 induces significantly greater apoptosis than the attenuated strain HEP-Flury. In order to identify the gene(s) accounting for this phenotype, five recombinant RABVs (rRABVs) were constructed by replacing each single gene of HEP-Flury with the corresponding gene of GD-SH-01. By using these rRABVs, we found that not only M and G, but also the phosphoprotein (P) plays an important role in inducing apoptosis. In order to figure out the different role of P gene in inducing apoptosis from the highly divergent background, another rRABV rGDSH-P, which carries the P gene of HEP-Flury in the background of the GD-SH-01 was generated. It was found that infection of NA cells with GD-SH-01 or the recombinant strain rHEP-shP, which carries P gene of GD-SH-01, induced significantly greater apoptosis than HEP-Flury or rGDSH-P in a caspase-dependent pathway that ultimately leads to the activation of the intrinsic apoptotic pathway, which is well characterized with the downregulation of bcl-2, the decrease of mitochondrial membrane potential, the release of mitochondrial cytochrome c, the activation of caspase-9 and caspase-3, and finally the cleavage of poly (ADP-ribose) polymerase. Our results imply that wt P from GD-SH-01 mediates this effect may partly by facilitating viral RNA synthesis but not by viral replication. In sum, we demonstrate a wt RABV strain GD-SH-01 to induce stronger apoptosis than an attenuated RABV HEP-Flury and propose that wt P from GD-SH-01 is involved in this process.

  4. Detecting Remote Sequence Homology in Disordered Proteins: Discovery of Conserved Motifs in the N-Termini of Mononegavirales phosphoproteins

    PubMed Central

    Karlin, David; Belshaw, Robert

    2012-01-01

    Paramyxovirinae are a large group of viruses that includes measles virus and parainfluenza viruses. The viral Phosphoprotein (P) plays a central role in viral replication. It is composed of a highly variable, disordered N-terminus and a conserved C-terminus. A second viral protein alternatively expressed, the V protein, also contains the N-terminus of P, fused to a zinc finger. We suspected that, despite their high variability, the N-termini of P/V might all be homologous; however, using standard approaches, we could previously identify sequence conservation only in some Paramyxovirinae. We now compared the N-termini using sensitive sequence similarity search programs, able to detect residual similarities unnoticeable by conventional approaches. We discovered that all Paramyxovirinae share a short sequence motif in their first 40 amino acids, which we called soyuz1. Despite its short length (11–16aa), several arguments allow us to conclude that soyuz1 probably evolved by homologous descent, unlike linear motifs. Conservation across such evolutionary distances suggests that soyuz1 plays a crucial role and experimental data suggest that it binds the viral nucleoprotein to prevent its illegitimate self-assembly. In some Paramyxovirinae, the N-terminus of P/V contains a second motif, soyuz2, which might play a role in blocking interferon signaling. Finally, we discovered that the P of related Mononegavirales contain similarly overlooked motifs in their N-termini, and that their C-termini share a previously unnoticed structural similarity suggesting a common origin. Our results suggest several testable hypotheses regarding the replication of Mononegavirales and suggest that disordered regions with little overall sequence similarity, common in viral and eukaryotic proteins, might contain currently overlooked motifs (intermediate in length between linear motifs and disordered domains) that could be detected simply by comparing orthologous proteins. PMID:22403617

  5. Differential Expression of Secreted Phosphoprotein 1 in the Motor Cortex among Primate Species and during Postnatal Development and Functional Recovery

    PubMed Central

    Yamamoto, Tatsuya; Oishi, Takao; Higo, Noriyuki; Murayama, Shigeo; Sato, Akira; Takashima, Ichiro; Sugiyama, Yoko; Nishimura, Yukio; Murata, Yumi; Yoshino-Saito, Kimika; Isa, Tadashi; Kojima, Toshio

    2013-01-01

    We previously reported that secreted phosphoprotein 1 (SPP1) mRNA is expressed in neurons whose axons form the corticospinal tract (CST) of the rhesus macaque, but not in the corresponding neurons of the marmoset and rat. This suggests that SPP1 expression is involved in the functional or structural specialization of highly developed corticospinal systems in certain primate species. To further examine this hypothesis, we evaluated the expression of SPP1 mRNA in the motor cortex from three viewpoints: species differences, postnatal development, and functional/structural changes of the CST after a lesion of the lateral CST (l-CST) at the mid-cervical level. The density of SPP1-positive neurons in layer V of the primary motor cortex (M1) was much greater in species with highly developed corticospinal systems (i.e., rhesus macaque, capuchin monkey, and humans) than in those with less developed corticospinal systems (i.e., squirrel monkey, marmoset, and rat). SPP1-positive neurons in the macaque monkey M1 increased logarithmically in layer V during postnatal development, following a time course consistent with the increase in conduction velocity of the CST. After an l-CST lesion, SPP1-positive neurons increased in layer V of the ventral premotor cortex, in which compensatory changes in CST function/structure may occur, which positively correlated with the extent of finger dexterity recovery. These results further support the concept that the expression of SPP1 may reflect functional or structural specialization of highly developed corticospinal systems in certain primate species. PMID:23741508

  6. The use of sodium trimetaphosphate as a biomimetic analog of matrix phosphoproteins for remineralization of artificial caries-like dentin

    PubMed Central

    Liu, Yan; Li, Nan; Qi, Yipin; Niu, Li-na; Elshafiy, Sally; Mao, Jing; Breschi, Lorenzo; Pashley, David H.; Tay, Franklin R.

    2011-01-01

    Objectives This study examined the use of sodium trimetaphosphate (STMP) as a biomimetic analog of matrix phosphoproteins for remineralization of artificial carious-affected dentin. Methods Artificial carious lesions with lesion depths of 300±30 µm were created by pH-cycling. 2.5% hydrolyzed STMP was applied to the artificial carious lesions to phosphorylate the partially-demineralized collagen matrix. Half of the STMP-treated specimens were bonded with One-Step. The adhesive and non-adhesive infiltrated specimens were remineralized in a Portland cement-simulated body fluid system containing polyacrylic acid (PAA) to stabilize amorphous calcium phosphate as nanoprecursors. Micro-computed tomography (micro-CT) and transmission electron microscopy (TEM) were used to evaluate the results of remineralization after a 4-month period. Results In absence of PAA and STMP as biomimetic analogs (control groups), there was no remineralization irrespective of whether the lesions were infiltrated with adhesive. For the STMP-treated experimental groups immersed in PAA-containing simulated body fluid, specimens without adhesive infiltration were more heavily remineralized than those infiltrated with adhesive. Statistical analysis of the 4-month micro-CT data revealed significant differences in the lesion depth, relative mineral content along the lesion surface and changes in ΔZ between the non-adhesive and adhesive experimental groups (p<0.05 for all the three parameters). TEM examination indicated that collagen degradation occurred in both the non-adhesive and adhesive control and experimental groups after 4 months of remineralization. Significance Biomimetic remineralization using STMP is a promising method to remineralize artificial carious lesions particularly in areas devoid of seed crystallites. Future studies should consider the incorporation of MMP-inhibitors within the partially-demineralized collagen matrix to prevent collagen degradation during remineralization. PMID

  7. Altered 40 S ribosomal subunits in omnipotent suppressors of yeast.

    PubMed

    Eustice, D C; Wakem, L P; Wilhelm, J M; Sherman, F

    1986-03-20

    The five suppressors SUP35, SUP43, SUP44, SUP45 and SUP46, each mapping at a different chromosomal locus in the yeast Saccharomyces cerevisiae, suppress a wide range of mutations, including representatives of all three types of nonsense mutations, UAA, UAG and UGA. We have demonstrated that ribosomes from the four suppressors SUP35, SUP44, SUP45 and SUP46 translate polyuridylate templates in vitro with higher errors than ribosomes from the normal stain, and that this misreading is substantially enhanced by the antibiotic paromomycin. Furthermore, ribosomal subunit mixing experiments established that the 40 S ribosomal subunit, and this subunit only, is responsible for the higher levels of misreading. Thus, the gene products of SUP35, SUP44, SUP45 and SUP46 are components of the 40 S subunit or are enzymes that modify the subunit. In addition, a protein from the 40 S subunit of the SUP35 suppressor has an altered electrophoretic mobility; this protein is distinct from the altered protein previously uncovered in the 40 S subunit of the SUP46 suppressor. In contrast to the ribosomes from the four suppressors SUP35, SUP44, SUP45 and SUP46, the ribosomes from the SUP43 suppressor do not significantly misread polyuridylate templates in vitro, suggesting that this locus may not encode a ribosomal component or that the misreading is highly specific.

  8. PURE ribosome display and its application in antibody technology.

    PubMed

    Kanamori, Takashi; Fujino, Yasuhiro; Ueda, Takuya

    2014-11-01

    Ribosome display utilizes formation of the mRNA-ribosome-polypeptide ternary complex in a cell-free protein synthesis system to link genotype (mRNA) to phenotype (polypeptide). However, the presence of intrinsic components, such as nucleases in the cell-extract-based cell-free protein synthesis system, reduces the stability of the ternary complex, which would prevent attainment of reliable results. We have developed an efficient and highly controllable ribosome display system using the PURE (Protein synthesis Using Recombinant Elements) system. The mRNA-ribosome-polypeptide ternary complex is highly stable in the PURE system, and the selected mRNA can be easily recovered because activities of nucleases and other inhibitory factors are very low in the PURE system. We have applied the PURE ribosome display to antibody engineering approaches, such as epitope mapping and affinity maturation of antibodies, and obtained results showing that the PURE ribosome display is more efficient than the conventional method. We believe that the PURE ribosome display can contribute to the development of useful antibodies. This article is part of a Special Issue entitled: Recent advances in molecular engineering of antibody.

  9. Genome-wide translational profiling by ribosome footprinting.

    PubMed

    Ingolia, Nicholas T

    2010-01-01

    We present a detailed protocol for ribosome profiling, an approach that we developed to make comprehensive and quantitative measurements of translation in yeast. In this technique, ribosome positions are determined from their nuclease footprint on their mRNA template and the footprints are quantified by deep sequencing. Ribosome profiling has already enabled highly reproducible measurements of translational control. Because this technique reports on the exact position of ribosomes, it also revealed the presence of ribosomes on upstream open reading frames and demonstrated that ribosome density was higher near the beginning of protein-coding genes. Here, we describe nuclease digestion conditions that produce uniform ~28 nucleotide (nt) protected fragments of mRNA templates that indicate the exact position of translating ribosomes. We also give a protocol for converting these RNA fragments into a DNA library that can be sequenced using the Illumina Genome Analyzer. Unbiased conversion of anonymous, small RNAs into a sequencing library is challenging, and we discuss standards that played a key role in optimizing library generation. Finally, we discuss how deep sequencing data can be used to quantify gene expression at the level of translation. Copyright © 2010 Elsevier Inc. All rights reserved.

  10. Bmi1 promotes erythroid development through regulating ribosome biogenesis

    PubMed Central

    Gao, Rui; Chen, Sisi; Kobayashi, Michihiro; Yu, Hao; Zhang, Yingchi; Wan, Yang; Young, Sara K.; Soltis, Anthony; Yu, Ming; Vemula, Sasidhar; Fraenkel, Ernest; Cantor, Alan; Antipin, Yevgeniy; Xu, Yang; Yoder, Mervin C.; Wek, Ronald C.; Ellis, Steven R.; Kapur, Reuben; Zhu, Xiaofan; Liu, Yan

    2015-01-01

    While Polycomb group protein Bmi1 is important for stem cell maintenance, its role in lineage commitment is largely unknown. We have identified Bmi1 as a novel regulator of erythroid development. Bmi1 is highly expressed in mouse erythroid progenitor cells and its deficiency impairs erythroid differentiation. BMI1 is also important for human erythroid development. Furthermore, we discovered that loss of Bmi1 in erythroid progenitor cells results in down-regulation of transcription of multiple ribosomal protein genes and impaired ribosome biogenesis. Bmi1 deficiency stabilizes p53 protein, leading to upregulation of p21 expression and subsequent G0/G1 cell cycle arrest. Genetic inhibition of p53 activity rescues the erythroid defects seen in the Bmi1 null mice, demonstrating that a p53-dependent mechanism underlies the pathophysiology of the anemia. Mechanistically, Bmi1 is associated with multiple ribosomal protein genes and may positively regulate their expression in erythroid progenitor cells. Thus, Bmi1 promotes erythroid development, at least in part through regulating ribosome biogenesis. Ribosomopathies are human disorders of ribosome dysfunction, including diamond blackfan anemia (DBA) and 5q- syndrome, in which genetic abnormalities cause impaired ribosome biogenesis, resulting in specific clinical phenotypes. We observed that BMI1 expression in human hematopoietic stem and progenitor cells (HSPCs) from patients with DBA is correlated with the expression of some ribosomal protein genes, suggesting that BMI1 deficiency may play a pathological role in DBA and other ribosomopathies. PMID:25385494

  11. Bmi1 promotes erythroid development through regulating ribosome biogenesis.

    PubMed

    Gao, Rui; Chen, Sisi; Kobayashi, Michihiro; Yu, Hao; Zhang, Yingchi; Wan, Yang; Young, Sara K; Soltis, Anthony; Yu, Ming; Vemula, Sasidhar; Fraenkel, Ernest; Cantor, Alan; Antipin, Yevgeniy; Xu, Yang; Yoder, Mervin C; Wek, Ronald C; Ellis, Steven R; Kapur, Reuben; Zhu, Xiaofan; Liu, Yan

    2015-03-01

    While Polycomb group protein Bmi1 is important for stem cell maintenance, its role in lineage commitment is largely unknown. We have identified Bmi1 as a novel regulator of erythroid development. Bmi1 is highly expressed in mouse erythroid progenitor cells and its deficiency impairs erythroid differentiation. BMI1 is also important for human erythroid development. Furthermore, we discovered that loss of Bmi1 in erythroid progenitor cells results in decreased transcription of multiple ribosomal protein genes and impaired ribosome biogenesis. Bmi1 deficiency stabilizes p53 protein, leading to upregulation of p21 expression and subsequent G0/G1 cell cycle arrest. Genetic inhibition of p53 activity rescues the erythroid defects seen in the Bmi1 null mice, demonstrating that a p53-dependent mechanism underlies the pathophysiology of the anemia. Mechanistically, Bmi1 is associated with multiple ribosomal protein genes and may positively regulate their expression in erythroid progenitor cells. Thus, Bmi1 promotes erythroid development, at least in part through regulating ribosome biogenesis. Ribosomopathies are human disorders of ribosome dysfunction, including Diamond-Blackfan anemia (DBA) and 5q- syndrome, in which genetic abnormalities cause impaired ribosome biogenesis, resulting in specific clinical phenotypes. We observed that BMI1 expression in human hematopoietic stem and progenitor cells from patients with DBA is correlated with the expression of some ribosomal protein genes, suggesting that BMI1 deficiency may play a pathological role in DBA and other ribosomopathies. © 2014 AlphaMed Press.

  12. DnaK-facilitated ribosome assembly in Escherichia coli revisited

    PubMed Central

    ALIX, JEAN-HERVÉ; NIERHAUS, KNUD H.

    2003-01-01

    Assembly helpers exist for the formation of ribosomal subunits. Such a function has been suggested for the DnaK system of chaperones (DnaK, DnaJ, GrpE). Here we show that 50S and 30S ribosomal subunits from an Escherichia coli dnaK-null mutant (containing a disrupted dnaK gene) grown at 30°C are physically and functionally identical to wild-type ribosomes. Furthermore, ribosomal components derived from mutant 30S and 50S subunits are fully competent for in vitro reconstitution of active ribosomal subunits. On the other hand, the DnaK chaperone system cannot circumvent the necessary heat-dependent activation step for the in vitro reconstitution of fully active 30S ribosomal subunits. It is therefore questionable whether the requirement for DnaK observed during in vivo ribosome assembly above 37°C implicates a direct or indirect role for DnaK in this process. PMID:12810912

  13. The DARC site: a database of aligned ribosomal complexes.

    PubMed

    Jarasch, Alexander; Dziuk, Philipp; Becker, Thomas; Armache, Jean-Paul; Hauser, Andreas; Wilson, Daniel N; Beckmann, Roland

    2012-01-01

    The ribosome is a highly dynamic machine responsible for protein synthesis within the cell. Cryo-electron microscopy (cryo-EM) and X-ray crystallography structures of ribosomal particles, alone and in complex with diverse ligands (protein factors, RNAs and small molecules), have revealed the dynamic nature of the ribosome and provided much needed insight into translation and its regulation. In the past years, there has been exponential growth in the deposition of cryo-EM maps into the Electron Microscopy Data Bank (EMDB) as well as atomic structures into the Protein Data Bank (PDB). Unfortunately, the deposited ribosomal particles usually have distinct orientations with respect to one another, which complicate the comparison of the available structures. To simplify this, we have developed a Database of Aligned Ribosomal Complexes, the DARC site (http://darcsite.genzentrum.lmu.de/darc/), which houses the available cryo-EM maps and atomic coordinates of ribosomal particles from the EMDB and PDB aligned within a common coordinate system. An easy-to-use, searchable interface allows users to access and download >130 cryo-EM maps and >300 atomic models in the format of brix and pdb files, respectively. The aligned coordinate system substantially simplifies direct visualization of conformational changes in the ribosome, such as subunit rotation and head-swiveling, as well as direct comparison of bound ligands, such as antibiotics or translation factors.

  14. Stochastic kinetics of ribosomes: Single motor properties and collective behavior

    NASA Astrophysics Data System (ADS)

    Garai, Ashok; Chowdhury, Debanjan; Chowdhury, Debashish; Ramakrishnan, T. V.

    2009-07-01

    Syntheses of protein molecules in a cell are carried out by ribosomes. A ribosome can be regarded as a molecular motor which utilizes the input chemical energy to move on a messenger RNA (mRNA) track that also serves as a template for the polymerization of the corresponding protein. The forward movement, however, is characterized by an alternating sequence of translocation and pause. Using a quantitative model, which captures the mechanochemical cycle of an individual ribosome, we derive an exact analytical expression for the distribution of its dwell times at the successive positions on the mRNA track. Inverse of the average dwell time satisfies a “Michaelis-Menten-type” equation and is consistent with the general formula for the average velocity of a molecular motor with an unbranched mechanochemical cycle. Extending this formula appropriately, we also derive the exact force-velocity relation for a ribosome. Often many ribosomes simultaneously move on the same mRNA track, while each synthesizes a copy of the same protein. We extend the model of a single ribosome by incorporating steric exclusion of different individuals on the same track. We draw the phase diagram of this model of ribosome traffic in three-dimensional spaces spanned by experimentally controllable parameters. We suggest new experimental tests of our theoretical predictions.

  15. RiboVision suite for visualization and analysis of ribosomes.

    PubMed

    Bernier, Chad R; Petrov, Anton S; Waterbury, Chris C; Jett, James; Li, Fengbo; Freil, Larry E; Xiong, Xiao; Wang, Lan; Migliozzi, Blacki L R; Hershkovits, Eli; Xue, Yuzhen; Hsiao, Chiaolong; Bowman, Jessica C; Harvey, Stephen C; Grover, Martha A; Wartell, Zachary J; Williams, Loren Dean

    2014-01-01

    RiboVision is a visualization and analysis tool for the simultaneous display of multiple layers of diverse information on primary (1D), secondary (2D), and three-dimensional (3D) structures of ribosomes. The ribosome is a macromolecular complex containing ribosomal RNA and ribosomal proteins and is a key component of life responsible for the synthesis of proteins in all living organisms. RiboVision is intended for rapid retrieval, analysis, filtering, and display of a variety of ribosomal data. Preloaded information includes 1D, 2D, and 3D structures augmented by base-pairing, base-stacking, and other molecular interactions. RiboVision is preloaded with rRNA secondary structures, rRNA domains and helical structures, phylogeny, crystallographic thermal factors, etc. RiboVision contains structures of ribosomal proteins and a database of their molecular interactions with rRNA. RiboVision contains preloaded structures and data for two bacterial ribosomes (Thermus thermophilus and Escherichia coli), one archaeal ribosome (Haloarcula marismortui), and three eukaryotic ribosomes (Saccharomyces cerevisiae, Drosophila melanogaster, and Homo sapiens). RiboVision revealed several major discrepancies between the 2D and 3D structures of the rRNAs of the small and large subunits (SSU and LSU). Revised structures mapped with a variety of data are available in RiboVision as well as in a public gallery (). RiboVision is designed to allow users to distill complex data quickly and to easily generate publication-quality images of data mapped onto secondary structures. Users can readily import and analyze their own data in the context of other work. This package allows users to import and map data from CSV files directly onto 1D, 2D, and 3D levels of structure. RiboVision has features in rough analogy with web-based map services capable of seamlessly switching the type of data displayed and the resolution or magnification of the display. RiboVision is available at .

  16. Novel role for p90 ribosomal S6 kinase in the regulation of cardiac myofilament phosphorylation.

    PubMed

    Cuello, Friederike; Bardswell, Sonya C; Haworth, Robert S; Ehler, Elisabeth; Sadayappan, Sakthivel; Kentish, Jonathan C; Avkiran, Metin

    2011-02-18

    In myocardium, the 90-kDa ribosomal S6 kinase (RSK) is activated by diverse stimuli and regulates the sarcolemmal Na(+)/H(+) exchanger through direct phosphorylation. Only limited information is available on other cardiac RSK substrates and functions. We evaluated cardiac myosin-binding protein C (cMyBP-C), a sarcomeric regulatory phosphoprotein, as a potential RSK substrate. In rat ventricular myocytes, RSK activation by endothelin 1 (ET1) increased cMyBP-C phosphorylation at Ser(282), which was inhibited by the selective RSK inhibitor D1870. Neither ET1 nor D1870 affected the phosphorylation status of Ser(273) or Ser(302), cMyBP-C residues additionally targeted by cAMP-dependent protein kinase (PKA). Complementary genetic gain- and loss-of-function experiments, through the adenoviral expression of wild-type or kinase-inactive RSK isoforms, confirmed RSK-mediated phosphorylation of cMyBP-C at Ser(282). Kinase assays utilizing as substrate wild-type or mutated (S273A, S282A, S302A) recombinant cMyBP-C fragments revealed direct and selective Ser(282) phosphorylation by RSK. Immunolabeling with a Ser(P)(282) antibody and confocal fluorescence microscopy showed RSK-mediated phosphorylation of cMyBP-C across the C-zones of sarcomeric A-bands. In chemically permeabilized mouse ventricular muscles, active RSK again induced selective Ser(282) phosphorylation in cMyBP-C, accompanied by significant reduction in Ca(2+) sensitivity of force development and significant acceleration of cross-bridge cycle kinetics, independently of troponin I phosphorylation at Ser(22)/Ser(23). The magnitudes of these RSK-induced changes were comparable with those induced by PKA, which phosphorylated cMyBP-C additionally at Ser(273) and Ser(302). We conclude that Ser(282) in cMyBP-C is a novel cardiac RSK substrate and its selective phosphorylation appears to regulate cardiac myofilament function.

  17. Mapping the messenger RNA within the elongating ribosome

    NASA Astrophysics Data System (ADS)

    Jünemann, R.; Wadzack, J.; Burkhardt, N.; Schmitt, M.; Zhao, J.; Stuhrmann, H. B.; Nierhaus, K. H.

    1997-02-01

    The method of proton-spin contrast-variation was applied for determining the position of the messenger RNA within the elongating ribosome. Using an artificial mRNA fragment the mass center of the mRNA sequence covered by the ribosome could be localized for the pre- and the post-translocational elongation states. The mass center moves about 12 ± 5 Å upon translocation. The radius of gyration was 12 ± e Å. The data give an independent contribution for refining a structural model including the RNA ligands of the elongating ribosome.

  18. Whither Ribosome Structure and Dynamics Research? (A Perspective).

    PubMed

    Frank, Joachim

    2016-09-11

    As high-resolution cryogenic electron microscopy (cryo-EM) structures of ribosomes proliferate, at resolutions that allow atomic interactions to be visualized, this article attempts to give a perspective on the way research on ribosome structure and dynamics may be headed, and particularly the new opportunities we have gained through recent advances in cryo-EM. It is pointed out that single-molecule FRET and cryo-EM form natural complements in the characterization of ribosome dynamics and transitions among equilibrating states of in vitro translational systems. Copyright © 2016 Elsevier Ltd. All rights reserved.

  19. Cut in translation: ribosome-dependent mRNA decay.

    PubMed

    Lalaouna, David; Massé, Eric

    2017-05-02

    Transcription and translation are two complex mechanisms that are tightly coupled in prokaryotic cells. Even before the completion of transcription, ribosomes attach to the nascent mRNA and initiate protein synthesis. Remarkably, recent publications have indicated an association between translation and decay of certain mRNAs. In this issue of The EMBO Journal, Leroy et al (2017) depicts a fascinating mechanism of mRNA degradation, which involves the ribosome-associated ribonuclease Rae1 in Bacillus subtilis In a translation-dependent manner, Rae1 binds the ribosomal aminoacylation (A)-site and cleaves between specific codons of the targeted mRNA. © 2017 The Authors.

  20. Quantitative assessment of ribosome drop-off in E. coli

    PubMed Central

    Sin, Celine; Chiarugi, Davide; Valleriani, Angelo

    2016-01-01

    Premature ribosome drop-off is one of the major errors in translation of mRNA by ribosomes. However, repeated analyses of Ribo-seq data failed to quantify its strength in E. coli. Relying on a novel highly sensitive data analysis method we show that a significant rate of ribosome drop-off is measurable and can be quantified also when cells are cultured under non-stressing conditions. Moreover, we find that the drop-off rate is highly variable, depending on multiple factors. In particular, under environmental stress such as amino acid starvation or ethanol intoxication, the drop-off rate markedly increases. PMID:26935582

  1. Evidence that Yih1 resides in a complex with ribosomes.

    PubMed

    Waller, Tracey; Lee, Su Jung; Sattlegger, Evelyn

    2012-05-01

    Adjusting protein synthesis by phosphorylating eukaryotic translation initiation factor 2 (eIF2α) is a major mechanism by which eukaryotes adapt to and overcome stress. The eIF2α kinase Gcn2 is essential for overcoming amino acid starvation in all eukaryotes. We have shown that to sense starvation, the Gcn2 RWD domain must directly contact its effector protein, Gcn1, and both must bind to the ribosome, suggesting that starvation is sensed within a Gcn1-Gcn2-ribosome complex. The mammalian protein IMPACT, highly expressed in neurons, and its yeast orthologue yeast IMPACT homologue (Yih1) harbour an RWD domain with Gcn1-binding activity. We have shown that Yih1 downregulates Gcn2 by competing with Gcn2 for Gcn1-binding. Here, we provide evidence that Yih1 forms a complex with ribosomes. In velocity sedimentation assays, overexpressed glutathione S-transferase (GST)-tagged Yih1 cosedimented with polyribosomes independently of Gcn1. Reduction of polyribosomes to monosomes concomitantly decreased GST-Yih1 sedimentation in the heavy fractions where polyribosomes are normally found. Furthermore, GST-Yih1 coprecipitated large ribosomal protein Rpl39 independently of Gcn1. GST-Yih1 overexpression did not significantly affect Gcn1-ribosome or Gcn2-ribosome cosedimentation. myc-tagged Yih1 expressed from its own promoter cosedimented with polyribosomes independently of Gcn1, indicating that Yih1-ribosome interaction occurs under physiological conditions. GST-IMPACT cosedimented with yeast ribosomes and coprecipitated Rpl39 in a Gcn1-independent fashion, suggesting that Yih1/IMPACT-ribosome association is evolutionarily conserved. Moreover, GST-IMPACT coprecipitated actin as found for GST-Yih1. Taken together, our findings strongly suggest that IMPACT/Yih1 associates with ribosomes and that these ribosomes may simultaneously carry Gcn1 and Gcn2. Close physical proximity of Yih1 to the Gcn1-Gcn2-ribosome complex would allow cells to quickly inhibit Gcn2 whenever or wherever

  2. Ribosomal RNA characterization in the leech Hirudo medicinalis.

    PubMed

    Macchi, M; Durante, M; Bernardi, R; Traina, G

    2008-09-01

    In the present study the ribosomal RNA of the leech Hirudo medicinalis has been characterized at the aim of identifying possible analogies with other invertebrates. Upon electrophoresis on denaturating gels, ribosomal RNA fraction of H. medicinalis exhibited a remarkable thermal instability by dissociating into two hydrogen-bonded components when heated at 60 degrees C, at variance with the behaviour of the rat rRNA, which does not show this process. This result suggests a functional role in leech ribosome organisation that requires deeper structural studies.

  3. Distinct Th1- and Th2-Type prenatal cytokine responses to Plasmodium falciparum erythrocyte invasion ligands.

    PubMed

    Malhotra, Indu; Mungai, Peter; Muchiri, Eric; Ouma, John; Sharma, Shobhona; Kazura, James W; King, Christopher L

    2005-06-01

    Prenatal immunity to Plasmodium falciparum merozoite proteins involved in erythrocyte invasion may contribute to the partial protection against malaria that is acquired during infancy in areas of stable malaria transmission. We examined newborn and maternal cytokine and antibody responses to merozoite surface protein-1 (MSP-1), ribosomal phosphoprotein P0 (PfP0), and region II of erythrocyte binding antigen-175 (EBA-175) in infant-mother pairs in Kenya. Overall, 82 of 167 (50%), 106 of 176 (60%), and 38 of 84 (45%) cord blood lymphocytes (CBL) from newborns produced one or more cytokines in response to MSP-1, PfP0, and EBA-175, respectively. Newborns of primigravid and/or malaria-infected women were more likely to have antigen-responsive CBL than were newborns of multigravid and/or uninfected women at delivery. Newborn cytokine responses did not match those of their mothers and fell into three distinct categories, Th1 (21 of 55 CBL donors produced only gamma interferon and/or interleukin 2 [IL-2]), Th2 (21 of 55 produced only IL-5 and/or IL-13), and mixed Th1/Th2 (13 of 55). Newborns produced more IL-10 than adults. High and low levels of cord blood IL-12 p70 production induced by anti-CD40 activation were associated with malaria-specific Th1 and Th2 responses, respectively. Antigen-responsive CBL in some newborns were detected only after depletion of IL-10-secreting CD8 cells with enrichment for CD4 cells. These data indicate that prenatal sensitization to blood-stage Plasmodium falciparum occurs frequently in areas where malaria is holoendemic. Modulation of this immunity, possibly by maternal parity and malaria, may affect the acquisition of protective immunity against malaria during infancy.

  4. Coordinated Ribosomal L4 Protein Assembly into the Pre-Ribosome Is Regulated by Its Eukaryote-Specific Extension.

    PubMed

    Stelter, Philipp; Huber, Ferdinand M; Kunze, Ruth; Flemming, Dirk; Hoelz, André; Hurt, Ed

    2015-06-04

    Eukaryotic ribosome biogenesis requires nuclear import and hierarchical incorporation of ∼80 ribosomal proteins (RPs) into the ribosomal RNA core. In contrast to prokaryotes, many eukaryotic RPs possess long extensions that interdigitate in the mature ribosome. RpL4 is a prime example, with an ∼80-residue-long surface extension of unknown function. Here, we identify assembly chaperone Acl4 that initially binds the universally conserved internal loop of newly synthesized RpL4 via its superhelical TPR domain, thereby restricting RpL4 loop insertion at its cognate nascent rRNA site. RpL4 release from Acl4 is orchestrated with pre-ribosome assembly, during which the eukaryote-specific RpL4 extension makes several distinct interactions with the 60S surface, including a co-evolved site on neighboring RpL18. Consequently, mutational inactivation of this contact site, on either RpL4 or RpL18, impairs RpL4-Acl4 disassembly and RpL4 pre-ribosome incorporation. We propose that hierarchical ribosome assembly can be achieved by eukaryotic RP extensions and dedicated assembly chaperones. Copyright © 2015 Elsevier Inc. All rights reserved.

  5. Post-translational Modification of Ribosomal Proteins

    PubMed Central

    Arragain, Simon; Garcia-Serres, Ricardo; Blondin, Geneviève; Douki, Thierry; Clemancey, Martin; Latour, Jean-Marc; Forouhar, Farhad; Neely, Helen; Montelione, Gaetano T.; Hunt, John F.; Mulliez, Etienne; Fontecave, Marc; Atta, Mohamed

    2010-01-01

    Post-translational modifications of ribosomal proteins are important for the accuracy of the decoding machinery. A recent in vivo study has shown that the rimO gene is involved in generation of the 3-methylthio derivative of residue Asp-89 in ribosomal protein S12 (Anton, B. P., Saleh, L., Benner, J. S., Raleigh, E. A., Kasif, S., and Roberts, R. J. (2008) Proc. Natl. Acad. Sci. U. S. A. 105, 1826–1831). This reaction is formally identical to that catalyzed by MiaB on the C2 of adenosine 37 near the anticodon of several tRNAs. We present spectroscopic evidence that Thermotoga maritima RimO, like MiaB, contains two [4Fe-4S] centers, one presumably bound to three invariant cysteines in the central radical S-adenosylmethionine (AdoMet) domain and the other to three invariant cysteines in the N-terminal UPF0004 domain. We demonstrate that holo-RimO can specifically methylthiolate the aspartate residue of a 20-mer peptide derived from S12, yielding a mixture of mono- and bismethylthio derivatives. Finally, we present the 2.0 Å crystal structure of the central radical AdoMet and the C-terminal TRAM (tRNA methyltransferase 2 and MiaB) domains in apo-RimO. Although the core of the open triose-phosphate isomerase (TIM) barrel of the radical AdoMet domain was conserved, RimO showed differences in domain organization compared with other radical AdoMet enzymes. The unusually acidic TRAM domain, likely to bind the basic S12 protein, is located at the distal edge of the radical AdoMet domain. The basic S12 protein substrate is likely to bind RimO through interactions with both the TRAM domain and the concave surface of the incomplete TIM barrel. These biophysical results provide a foundation for understanding the mechanism of methylthioation by radical AdoMet enzymes in the MiaB/RimO family. PMID:20007320

  6. Dual Role of a SAS10/C1D Family Protein in Ribosomal RNA Gene Expression and Processing Is Essential for Reproduction in Arabidopsis thaliana

    PubMed Central

    Chen, Ying-Jiun C.; Wang, Huei-Jing

    2016-01-01

    In eukaryotic cells, ribosomal RNAs (rRNAs) are transcribed, processed, and assembled with ribosomal proteins in the nucleolus. Regulatory mechanisms of rRNA gene (rDNA) transcription and processing remain elusive in plants, especially their connection to nucleolar organization. We performed an in silico screen for essential genes of unknown function in Arabidopsis thaliana and identified Thallo (THAL) encoding a SAS10/C1D family protein. THAL disruption caused enlarged nucleoli in arrested embryos, aberrant processing of precursor rRNAs at the 5’ External Transcribed Spacer, and repression of the major rDNA variant (VAR1). THAL overexpression lines showed de-repression of VAR1 and overall reversed effects on rRNA processing sites. Strikingly, THAL overexpression also induced formation of multiple nucleoli per nucleus phenotypic of mutants of heterochromatin factors. THAL physically associated with histone chaperone Nucleolin 1 (NUC1), histone-binding NUC2, and histone demethylase Jumonji 14 (JMJ14) in bimolecular fluorescence complementation assay, suggesting that it participates in chromatin regulation. Furthermore, investigation of truncated THAL proteins revealed that the SAS10 C-terminal domain is likely important for its function in chromatin configuration. THAL also interacted with putative Small Subunit processome components, including previously unreported Arabidopsis homologue of yeast M Phase Phosphoprotein 10 (MPP10). Our results uncovering the dual role of THAL in transcription and processing events critical for proper rRNA biogenesis and nucleolar organization during reproduction are the first to define the function of SAS10/C1D family members in plants. PMID:27792779

  7. Design, preparation and use of ligated phosphoproteins: a novel approach to study protein phosphatases by dot blot array, ELISA and Western blot assays.

    PubMed

    Sun, Luo; Ghosh, Inca; Barshevsky, Tanya; Kochinyan, Samvel; Xu, Ming-Qun

    2007-07-01

    The study of substrate specificity of protein phosphatases (PPs) is very challenging since it is difficult to prepare a suitable phosphorylated substrate. Phosphoproteins, phosphorylated by a protein kinase, or chemically synthesized phosphopeptides are commonly used substrates for PPs. Both types of these substrates have their advantages and limitations. Phosphoproteins mimic more closely the physiologically relevant PP substrates, but their preparation is technically demanding. Synthetic phosphopeptides present advantages over proteins because they can be easily produced in large quantity and their amino acid sequence can be designed to contain potential determinants of substrate specificity. However, short peptides are less optimal compared to in vivo PP substrates and often display poor and variable binding to different matrices, resulting in low sensitivity in analysis of PP activity on solid support. In this work we utilize the intein-mediated protein ligation (IPL) technique to generate substrates for PPs, combining the advantages of proteins and synthetic peptides in one molecule. The ligation of a synthetic phosphopeptide to an intein-generated carrier protein (CP) with a one-to-one stoichiometry results in the formation of a ligated phosphoprotein (LPP). Three widely used assays, dot blot array, Western blot and ELISA were employed to study the PP activity on LPP substrates. Dephosphorylation was measured by detection of the remaining phosphorylation, or lack of it, with a phospho-specific antibody. The data show the advantage of LPPs over free peptides in assays on solid supports. LPPs exhibited enhanced binding to the matrices used in the study, which significantly improved sensitivity and consistency of the assays. In addition, saturation of the signal was circumvented by serial dilution of the assay samples. This report describes detailed experimental procedures for preparation of LPP substrates and their use in PP assays based on immobilization on

  8. Enhanced expression of the Marek's disease virus-specific phosphoproteins after stable transfection of MSB-1 cells with the Marek's disease virus homologue of ICP4.

    PubMed

    Pratt, W D; Cantello, J; Morgan, R W; Schat, K A

    1994-05-15

    Phosphoprotein pp38, coded for by the BamHI-H fragment of the Marek's disease herpesvirus (MDV) genome is expressed in tumor cells and tumor cell lines. pp38 is associated with two other phosphoproteins, pp41 and pp24, and can be detected in a small percentage of tumor cells by indirect immunofluorescence assays (IIFA). The importance of MDV ICP4 for the regulation of pp38 expression was examined in the following MSB-1-derived cell lines stably transfected with the selection plasmid pNL1 [MDCC-CU221 (CU221)], pNL1 and the BamHI-A fragment of MDV DNA containing ICP4 (CU224), MDV ICP4 inserted in antisense direction in the eukaryotic expression vector pXT1 (CU222), or ICP4 in sense direction in pXT1 (CU223) or cotransfected with pNL1 and EcoRI-linearized BamHI-A MDV DNA (CU225, -237, -243, -244). IIFA analysis showed that CU223 had a markedly increased expression of pp38, while CU224 had a slightly increased expression. No changes were noted in CU221 or CU222, while expression of pp38 was decreased in CU225, -237, -243, and -244. Radioimmunoprecipitation assays demonstrated that the expression of all three phosphoproteins was enhanced in CU223. Steady-state transcriptional analysis showed that CU223 had increased levels of pp38-specific (1.9 and 3.3 kb) and ICP4-specific (10.0 kb) transcripts.

  9. Ecto-protein kinase substrate p120 revealed as the cell-surface-expressed nucleolar phosphoprotein Nopp140: a candidate protein for extracellular Ca2+-sensing.

    PubMed Central

    Kübler, D

    2001-01-01

    A variety of cell membrane proteins become phosphorylated in their ecto-domains by cell-surface protein kinase (ecto-PK) activities, as detected in a broad spectrum of cell types. This study reports the isolation and identification of a frequent ecto-PK substrate, ecto-p120, using HeLa cells as a model. Data from MS and further biochemical and immunochemical means identified ecto-p120 as a cell-surface homologue of human nucleolar phosphoprotein p140 (hNopp140), which belongs to the family of argyrophilic (AgNOR-stainable) proteins. The superposition of (32)P-labelled ecto-nucleolar phosphoprotein p140 (ecto-Nopp140) with anti-Nopp140 immunostaining could be demonstrated in a wide range of cell lines without any exceptions, suggesting a nearly universal occurrence of cell-surface Nopp140. A previous, tentative association of ecto-p120 with the nucleoplasmic pre-mRNA-binding protein hnRNP U has thus been supplanted, since improved purification techniques have allowed unambiguous identification of this ecto-PK cell-surface substrate. Furthermore, we have shown that rapid suppression of ecto-hNopp140 phosphorylation resulted upon a rise in the free extracellular calcium, while lowering the calcium concentrations returned ecto-Nopp140 phosphorylation to the original level. It is important to note that these Ca(2+)-dependent effects on ecto-Nopp140 phosphorylation are not accompanied by alterations in the phosphorylation of other ecto-PK substrates. Our results indicate that, in addition to nucleolin, a further nucleolar protein, which was considered initially to be strictly intracellular, is identified as a cell-surface phosphoprotein. PMID:11736647

  10. Ribosomes containing mutants of L4 ribosomal protein from Thermus thermophilus display multiple defects in ribosomal functions and sensitivity against erythromycin

    PubMed Central

    TSAGKALIA, AIKATERINI; LEONTIADOU, FOTINI; XAPLANTERI, MARIA A.; PAPADOPOULOS, GEORGIOS; KALPAXIS, DIMITRIOS L.; CHOLI-PAPADOPOULOU, THEODORA

    2005-01-01

    Protein L4 from Thermus thermophilus (TthL4) was heterologously overproduced in Escherichia coli cells. To study the implication of the extended loop of TthL4 in the exit-tunnel and peptidyltransferase functions, the highly conserved E56 was replaced by D or Q, while the semiconserved G55 was changed to E or S. Moreover, the sequence -G55E56- was inverted to -E55G56-. When we incorporated these mutants into E. coli ribosomes and investigated their impact on poly(Phe) synthesis, high variations in the synthetic activity and response to erythromycin of the resulting ribosomes were observed. In the absence of erythromycin, ribosomes harboring mutations G55E and E56D in TthL4 protein were characterized by low activity in synthesizing poly(Phe) and decreased capability in binding tRNA at the A site. On the other hand, ribosomes possessing mutations G55E, G55S, G55E-E56G, or E56Q in TthL4 protein were unexpectedly more sensitive to erythromycin. Evidence in support of these findings was drawn by in vivo experiments, assessing the erythromycin sensitivity of E. coli cells expressing wild-type or mutant TthL4 proteins. Our results emphasize the role of the extended loop of L4 ribosomal protein in the exit-tunnel and peptidyltransferase center functions. PMID:16244130

  11. A kinetic analysis of the dephosphorylation, by bovine spleen phosphoprotein phosphatase (EC 3.1.3.16) of a phosphopeptide derived from beta-casein.

    PubMed

    West, D W; Dalgleish, D G

    1976-06-07

    A peptide containing the four closely grouped phosphoseryl residues present in beta-casein has been enzymatically dephosphorylated with bovine spleen phosphoprotein phosphatase (EC 3.1.3.16). The course of the dephosphorylation reaction has been followed by cellulose acetate electrophoresis and the amount of partially phosphorylated peptides present at each stage quantified by the same method. The phosphate groups are shown to be removed in a sequential manner and the rate constants for each stage of the dephosphorylation have been computed from the data obtained. The rate constants indicate that interaction in the intact peptide results in an enhancement of the activity of the phosphoseryl cluster.

  12. Cotranslational Protein Folding inside the Ribosome Exit Tunnel.

    PubMed

    Nilsson, Ola B; Hedman, Rickard; Marino, Jacopo; Wickles, Stephan; Bischoff, Lukas; Johansson, Magnus; Müller-Lucks, Annika; Trovato, Fabio; Puglisi, Joseph D; O'Brien, Edward P; Beckmann, Roland; von Heijne, Gunnar

    2015-09-08

    At what point during translation do proteins fold? It is well established that proteins can fold cotranslationally outside the ribosome exit tunnel, whereas studies of folding inside the exit tunnel have so far detected only the formation of helical secondary structure and collapsed or partially structured folding intermediates. Here, using a combination of cotranslational nascent chain force measurements, inter-subunit fluorescence resonance energy transfer studies on single translating ribosomes, molecular dynamics simulations, and cryoelectron microscopy, we show that a small zinc-finger domain protein can fold deep inside the vestibule of the ribosome exit tunnel. Thus, for small protein domains, the ribosome itself can provide the kind of sheltered folding environment that chaperones provide for larger proteins. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

  13. Structural basis for the inhibition of the eukaryotic ribosome.

    PubMed

    Garreau de Loubresse, Nicolas; Prokhorova, Irina; Holtkamp, Wolf; Rodnina, Marina V; Yusupova, Gulnara; Yusupov, Marat

    2014-09-25

    The ribosome is a molecular machine responsible for protein synthesis and a major target for small-molecule inhibitors. Compared to the wealth of structural information available on ribosome-targeting antibiotics in bacteria, our understanding of the binding mode of ribosome inhibitors in eukaryotes is currently limited. Here we used X-ray crystallography to determine 16 high-resolution structures of 80S ribosomes from Saccharomyces cerevisiae in complexes with 12 eukaryote-specific and 4 broad-spectrum inhibitors. All inhibitors were found associated with messenger RNA and transfer RNA binding sites. In combination with kinetic experiments, the structures suggest a model for the action of cycloheximide and lactimidomycin, which explains why lactimidomycin, the larger compound, specifically targets the first elongation cycle. The study defines common principles of targeting and resistance, provides insights into translation inhibitor mode of action and reveals the structural determinants responsible for species selectivity which could guide future drug development.

  14. tmRNA on its way through the ribosome

    PubMed Central

    Fu, Jie; Hashem, Yaser; Wower, Jacek

    2011-01-01

    Trans-translation is a universal quality-control process eubacteria use to degrade incompletely synthesized proteins and rescue ribosome stalled on defective mRNAs. This process is facilitated by a ribonucleoprotein complex composed of transfer-messenger RNA (tmRNA)—a chimera made of a tRNA-like molecule and a short open reading frame (ORF)—and small protein B (SmpB). Determination of the structure of tmRNA and SmpB in complex with the ribosome, at the stage when translation has resumed on tmRNA, has provided an increased understanding of the structure of tmRNA as it transits the ribosome and unique insights into the complex mechanism of template switching on the ribosome and SmpB-driven selection of the correct reading frame on tmRNA's ORF. PMID:21593606

  15. Chaos and Hyperchaos in a Model of Ribosome Autocatalytic Synthesis

    PubMed Central

    Likhoshvai, Vitaly A.; Kogai, Vladislav V.; Fadeev, Stanislav I.; Khlebodarova, Tamara M.

    2016-01-01

    Any vital activities of the cell are based on the ribosomes, which not only provide the basic machinery for the synthesis of all proteins necessary for cell functioning during growth and division, but for biogenesis itself. From this point of view, ribosomes are self-replicating and autocatalytic structures. In current work we present an elementary model in which the autocatalytic synthesis of ribosomal RNA and proteins, as well as enzymes ensuring their degradation are described with two monotonically increasing functions. For certain parameter values, the model, consisting of one differential equation with delayed argument, demonstrates both stationary and oscillatory dynamics of the ribosomal protein synthesis, which can be chaotic and hyperchaotic dependent on the value of the delayed argument. The biological interpretation of the modeling results and parameter estimation suggest the feasibility of chaotic dynamics in molecular genetic systems of eukaryotes, which depends only on the internal characteristics of functioning of the translation system. PMID:27941909

  16. Inhibition of bacterial ribosome assembly: a suitable drug target?

    PubMed

    Maguire, Bruce A

    2009-03-01

    The assembly of bacterial ribosomes is viewed with increasing interest as a potential target for new antibiotics. The in vivo synthesis and assembly of ribosomes are briefly reviewed here, highlighting the many ways in which assembly can be perturbed. The process is compared with the model in vitro process from which much of our knowledge is derived. The coordinate synthesis of the ribosomal components is essential for their ordered and efficient assembly; antibiotics interfere with this coordination and therefore affect assembly. It has also been claimed that the binding of antibiotics to nascent ribosomes prevents their assembly. These two contrasting models of antibiotic action are compared and evaluated. Finally, the suitability and tractability of assembly as a drug target are assessed.

  17. 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

  18. Cooperation between translating ribosomes and RNA polymerase in transcription elongation.

    PubMed

    Proshkin, Sergey; Rahmouni, A Rachid; Mironov, Alexander; Nudler, Evgeny

    2010-04-23

    During transcription of protein-coding genes, bacterial RNA polymerase (RNAP) is closely followed by a ribosome that translates the newly synthesized transcript. Our in vivo measurements show that the overall elongation rate of transcription is tightly controlled by the rate of translation. Acceleration and deceleration of a ribosome result in corresponding changes in the speed of RNAP. Moreover, we found an inverse correlation between the number of rare codons in a gene, which delay ribosome progression, and the rate of transcription. The stimulating effect of a ribosome on RNAP is achieved by preventing its spontaneous backtracking, which enhances the pace and also facilitates readthrough of roadblocks in vivo. Such a cooperative mechanism ensures that the transcriptional yield is always adjusted to translational needs at different genes and under various growth conditions.

  19. Cotranslational Protein Folding inside the Ribosome Exit Tunnel

    PubMed Central

    Nilsson, Ola B.; Hedman, Rickard; Marino, Jacopo; Wickles, Stephan; Bischoff, Lukas; Johansson, Magnus; Müller-Lucks, Annika; Trovato, Fabio; Puglisi, Joseph D.; O’Brien, Edward P.; Beckmann, Roland; von Heijne, Gunnar

    2015-01-01

    Summary At what point during translation do proteins fold? It is well established that proteins can fold cotranslationally outside the ribosome exit tunnel, whereas studies of folding inside the exit tunnel have so far detected only the formation of helical secondary structure and collapsed or partially structured folding intermediates. Here, using a combination of cotranslational nascent chain force measurements, inter-subunit fluorescence resonance energy transfer studies on single translating ribosomes, molecular dynamics simulations, and cryoelectron microscopy, we show that a small zinc-finger domain protein can fold deep inside the vestibule of the ribosome exit tunnel. Thus, for small protein domains, the ribosome itself can provide the kind of sheltered folding environment that chaperones provide for larger proteins. PMID:26321634

  20. Structural features of the tmRNA-ribosome interaction.

    PubMed

    Bugaeva, Elizaveta Y; Surkov, Serhiy; Golovin, Andrey V; Ofverstedt, Lars-Göran; Skoglund, Ulf; Isaksson, Leif A; Bogdanov, Alexey A; Shpanchenko, Olga V; Dontsova, Olga A

    2009-12-01

    Trans-translation is a process which switches the synthesis of a polypeptide chain encoded by a nonstop messenger RNA to the mRNA-like domain of a transfer-messenger RNA (tmRNA). It is used in bacterial cells for rescuing the ribosomes arrested during translation of damaged mRNA and directing this mRNA and the product polypeptide for degradation. The molecular basis of this process is not well understood. Earlier, we developed an approach that allowed isolation of tmRNA-ribosomal complexes arrested at a desired step of tmRNA passage through the ribosome. We have here exploited it to examine the tmRNA structure using chemical probing and cryo-electron microscopy tomography. Computer modeling has been used to develop a model for spatial organization of the tmRNA inside the ribosome at different stages of trans-translation.

  1. Structural features of the tmRNA–ribosome interaction

    PubMed Central

    Bugaeva, Elizaveta Y.; Surkov, Serhiy; Golovin, Andrey V.; Öfverstedt, Lars-Göran; Skoglund, Ulf; Isaksson, Leif A.; Bogdanov, Alexey A.; Shpanchenko, Olga V.; Dontsova, Olga A.

    2009-01-01

    Trans-translation is a process which switches the synthesis of a polypeptide chain encoded by a nonstop messenger RNA to the mRNA-like domain of a transfer-messenger RNA (tmRNA). It is used in bacterial cells for rescuing the ribosomes arrested during translation of damaged mRNA and directing this mRNA and the product polypeptide for degradation. The molecular basis of this process is not well understood. Earlier, we developed an approach that allowed isolation of tmRNA–ribosomal complexes arrested at a desired step of tmRNA passage through the ribosome. We have here exploited it to examine the tmRNA structure using chemical probing and cryo-electron microscopy tomography. Computer modeling has been used to develop a model for spatial organization of the tmRNA inside the ribosome at different stages of trans-translation. PMID:19861420

  2. On the specificity of antibiotics targeting the large ribosomal subunit.

    PubMed

    Wilson, Daniel N

    2011-12-01

    The peptidyltransferase center of the large ribosomal subunit is responsible for catalyzing peptide bonds. This active site is the target of a variety of diverse antibiotics, many of which are used clinically. The past decade has seen a plethora of structures of antibiotics in complex with the large ribosomal subunit, providing unprecedented insight into the mechanism of action of these inhibitors. Ten distinct antibiotics (chloramphenicol, clindamycin, linezolid, tiamulin, sparsomycin, and five macrolides) have been crystallized in complex with four distinct ribosomal species, three bacterial, and one archaeal. This review aims to compare these structures in order to provide insight into the conserved and species-specific modes of interaction for particular members of each class of antibiotics. Coupled with the wealth of biochemical data, a picture is emerging defining the specific functional states of the ribosome that antibiotics preferentially target. Such mechanistic insight into antibiotic inhibition will be important for the development of the next generation of antimicrobial agents.

  3. 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.

  4. Cotranslational response to proteotoxic stress by elongation pausing of ribosomes.

    PubMed

    Liu, Botao; Han, Yan; Qian, Shu-Bing

    2013-02-07

    Translational control permits cells to respond swiftly to a changing environment. Rapid attenuation of global protein synthesis under stress conditions has been largely ascribed to the inhibition of translation initiation. Here we report that intracellular proteotoxic stress reduces global protein synthesis by halting ribosomes on transcripts during elongation. Deep sequencing of ribosome-protected messenger RNA (mRNA) fragments reveals an early elongation pausing, roughly at the site where nascent polypeptide chains emerge from the ribosomal exit tunnel. Inhibiting endogenous chaperone molecules by a dominant-negative mutant or chemical inhibitors recapitulates the early elongation pausing, suggesting a dual role of molecular chaperones in facilitating polypeptide elongation and cotranslational folding. Our results further support the chaperone "trapping" mechanism in promoting the passage of nascent chains. Our study reveals that translating ribosomes fine tune the elongation rate by sensing the intracellular folding environment. The early elongation pausing represents a cotranslational stress response to maintain the intracellular protein homeostasis.

  5. Sequence-dependent elongation dynamics on macrolide-bound ribosomes.

    PubMed

    Johansson, Magnus; Chen, Jin; Tsai, Albert; Kornberg, Guy; Puglisi, Joseph D

    2014-06-12

    The traditional view of macrolide antibiotics as plugs inside the ribosomal nascent peptide exit tunnel (NPET) has lately been challenged in favor of a more complex, heterogeneous mechanism, where drug-peptide interactions determine the fate of a translating ribosome. To investigate these highly dynamic processes, we applied single-molecule tracking of elongating ribosomes during inhibition of elongation by erythromycin of several nascent chains, including ErmCL and H-NS, which were shown to be, respectively, sensitive and resistant to erythromycin. Peptide sequence-specific changes were observed in translation elongation dynamics in the presence of a macrolide-obstructed NPET. Elongation rates were not severely inhibited in general by the presence of the drug; instead, stalls or pauses were observed as abrupt events. The dynamic pathways of nascent-chain-dependent elongation pausing in the presence of macrolides determine the fate of the translating ribosome stalling or readthrough.

  6. The SSU Processome in Ribosome Biogenesis – Progress and Prospects

    PubMed Central

    Phipps, Kathleen R.; Charette, J. Michael; Baserga, Susan J.

    2010-01-01

    The small subunit (SSU) processome is a 2.2 MDa ribonucleoprotein complex involved in the processing, assembly and maturation of the SSU of eukaryotic ribosomes. The identities of many of the factors involved in SSU biogenesis have been elucidated over the past 40 years. However, as our understanding increases, so do the number of questions about the nature of this complicated process. Cataloguing the components is the first step towards understanding the molecular workings of a system. This review will focus on how identifying components of ribosome biogenesis has led to the knowledge of how these factors, protein and RNA alike, associate with one another into sub-complexes, with a concentration on the small ribosomal subunit. We will also explore how this knowledge of sub-complex assembly has informed our understanding of the workings of the ribosome synthesis system as a whole. PMID:21318072

  7. Large Variations in Bacterial Ribosomal RNA Genes

    PubMed Central

    Lim, Kyungtaek; Furuta, Yoshikazu; Kobayashi, Ichizo

    2012-01-01

    Ribosomal RNA (rRNA) genes, essential to all forms of life, have been viewed as highly conserved and evolutionarily stable, partly because very little is known about their natural variations. Here, we explored large-scale variations of rRNA genes through bioinformatic analyses of available complete bacterial genomic sequences with an emphasis on formation mechanisms and biological significance. Interestingly, we found bacterial genomes in which no 16S rRNA genes harbor the conserved core of the anti–Shine-Dalgarno sequence (5′-CCTCC-3′). This loss was accompanied by elimination of Shine-Dalgarno–like sequences upstream of their protein-coding genes. Those genomes belong to 1 or 2 of the following categories: primary symbionts, hemotropic Mycoplasma, and Flavobacteria. We also found many rearranged rRNA genes and reconstructed their history. Conjecturing the underlying mechanisms, such as inversion, partial duplication, transposon insertion, deletion, and substitution, we were able to infer their biological significance, such as co-orientation of rRNA transcription and chromosomal replication, lateral transfer of rRNA gene segments, and spread of rRNA genes with an apparent structural defect through gene conversion. These results open the way to understanding dynamic evolutionary changes of rRNA genes and the translational machinery. PMID:22446745

  8. Molecular mechanisms of ribosomal protein gene coregulation

    PubMed Central

    Reja, Rohit; Vinayachandran, Vinesh; Ghosh, Sujana; Pugh, B. Franklin

    2015-01-01

    The 137 ribosomal protein genes (RPGs) of Saccharomyces provide a model for gene coregulation. We examined the positional and functional organization of their regulators (Rap1 [repressor activator protein 1], Fhl1, Ifh1, Sfp1, and Hmo1), the transcription machinery (TFIIB, TFIID, and RNA polymerase II), and chromatin at near-base-pair resolution using ChIP-exo, as RPGs are coordinately reprogrammed. Where Hmo1 is enriched, Fhl1, Ifh1, Sfp1, and Hmo1 cross-linked broadly to promoter DNA in an RPG-specific manner and demarcated by general minor groove widening. Importantly, Hmo1 extended 20–50 base pairs (bp) downstream from Fhl1. Upon RPG repression, Fhl1 remained in place. Hmo1 dissociated, which was coupled to an upstream shift of the +1 nucleosome, as reflected by the Hmo1 extension and core promoter region. Fhl1 and Hmo1 may create two regulatable and positionally distinct barriers, against which chromatin remodelers position the +1 nucleosome into either an activating or a repressive state. Consistent with in vitro studies, we found that specific TFIID subunits, in addition to cross-linking at the core promoter, made precise cross-links at Rap1 sites, which we interpret to reflect native Rap1–TFIID interactions. Our findings suggest how sequence-specific DNA binding regulates nucleosome positioning and transcription complex assembly >300 bp away and how coregulation coevolved with coding sequences. PMID:26385964

  9. Molecular mechanisms of ribosomal protein gene coregulation.

    PubMed

    Reja, Rohit; Vinayachandran, Vinesh; Ghosh, Sujana; Pugh, B Franklin

    2015-09-15

    The 137 ribosomal protein genes (RPGs) of Saccharomyces provide a model for gene coregulation. We examined the positional and functional organization of their regulators (Rap1 [repressor activator protein 1], Fhl1, Ifh1, Sfp1, and Hmo1), the transcription machinery (TFIIB, TFIID, and RNA polymerase II), and chromatin at near-base-pair resolution using ChIP-exo, as RPGs are coordinately reprogrammed. Where Hmo1 is enriched, Fhl1, Ifh1, Sfp1, and Hmo1 cross-linked broadly to promoter DNA in an RPG-specific manner and demarcated by general minor groove widening. Importantly, Hmo1 extended 20-50 base pairs (bp) downstream from Fhl1. Upon RPG repression, Fhl1 remained in place. Hmo1 dissociated, which was coupled to an upstream shift of the +1 nucleosome, as reflected by the Hmo1 extension and core promoter region. Fhl1 and Hmo1 may create two regulatable and positionally distinct barriers, against which chromatin remodelers position the +1 nucleosome into either an activating or a repressive state. Consistent with in vitro studies, we found that specific TFIID subunits, in addition to cross-linking at the core promoter, made precise cross-links at Rap1 sites, which we interpret to reflect native Rap1-TFIID interactions. Our findings suggest how sequence-specific DNA binding regulates nucleosome positioning and transcription complex assembly >300 bp away and how coregulation coevolved with coding sequences. © 2015 Reja et al.; Published by Cold Spring Harbor Laboratory Press.

  10. Rooting the ribosomal tree of life.

    PubMed

    Fournier, Gregory P; Gogarten, J Peter

    2010-08-01

    The origin of the genetic code and the rooting of the tree of life (ToL) are two of the most challenging problems in the study of life's early evolution. Although both have been the focus of numerous investigations utilizing a variety of methods, until now, each problem has been addressed independently. Typically, attempts to root the ToL have relied on phylogenies of genes with ancient duplications, which are subject to artifacts of tree reconstruction and horizontal gene transfer, or specific physiological characters believed to be primitive, which are often based on subjective criteria. Here, we demonstrate a unique method for rooting based on the identification of amino acid usage biases comprising the residual signature of a more primitive genetic code. Using a phylogenetic tree of concatenated ribosomal proteins, our analysis of amino acid compositional bias detects a strong and unique signal associated with the early expansion of the genetic code, placing the root of the translation machinery along the bacterial branch.

  11. Nonenzymatic microorganism identification based on ribosomal RNA

    NASA Astrophysics Data System (ADS)

    Ives, Jeffrey T.; Pierini, Alicia M.; Stokes, Jeffrey A.; Wahlund, Thomas M.; Read, Betsy; Bechtel, James H.; Bronk, Burt V.

    1999-11-01

    Effective defense against biological warfare (BW) agents requires rapid, fieldable and accurate systems. For micro- organisms like bacteria and viruses, ribosomal RNA (rRNA) provides a valuable target with multiple advantages of species specificity and intrinsic target amplification. Vegetative and spore forms of bacteria contain approximately 104 copies of rRNA. Direct detection of rRNA copies can eliminate some of the interference and preparation difficulties involved in enzymatic amplification methods. In order to apply the advantages of rRNA to BW defense, we are developing a fieldable system based on 16S rRNA, physical disruption of the micro-organism, solid phase hybridization, and fluorescence detection. Our goals include species-specific identification, complete operation from raw sample to identification in 15 minutes or less, and compact, fieldable instrumentation. Initial work on this project has investigated the lysis and hybridization steps, the species-specificity of oligonucleotides probes, and the development of a novel electromagnetic method to physically disrupt the micro- organisms. Target bacteria have been Escherichia coli (E. coli) and Bacillus subtilis (B. subtilis). Continuing work includes further development of methods to rapidly disrupt the micro-organisms and release the rRNA, improved integration and processing, and extension to bacterial and mammalian viruses like MS2 and vesicular stomatitis virus.

  12. The ribosomal gene spacer region in archaebacteria

    NASA Technical Reports Server (NTRS)

    Achenbach-Richter, L.; Woese, C. R.

    1988-01-01

    Sequences for the spacer regions that separate the 16S and 23S ribosomal RNA genes have been determined for four more (strategically placed) archaebacteria. These confirm the general rule that methanogens and extreme halophiles have spacers that contain a single tRNAala gene, while tRNA genes are not found in the spacer region of the true extreme thermophiles. The present study also shows that the spacer regions from the sulfate reducing Archaeglobus and the extreme thermophile Thermococcus (both of which cluster phylogenetically with the methanogens and extreme halophiles) contain each a tRNAala gene. Thus, not only all methanogens and extreme halophiles show this characteristic, but all organisms o