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PubMed2005

The extracellular and cytoplasmic proteomes of the non-virulent Bacillus anthracis strain UM23C1-2.

The recently published genome sequence of Bacillus anthracis Ames has facilitated the prediction of proteins associated with the virulence of this bacterium. The aim of this study was to define reference maps for the extracellular and cytoplasmic proteomes of the avirulent B. anthracis strain UM23C1-2 that are useful for physiological studies and the development of improved vaccines. Using 2-DE and subsequent MALDI-TOF-TOF MS, 64 proteins were identified in the extracellular proteome, only 29 of which were predicted to be exported into the culture medium. The latter included chitinases, proteases, nucleotidases, sulfatases, phosphatases and proteins of unknown function. Of the remaining proteins in the culture medium, 18 were predicted to be associated with the cell wall or anchored on the trans side of the cytoplasmic membrane while 17 other proteins lacked identifiable export signals and were predicted to be cytoplasmic proteins. Among the S-layer proteins, Sap and Eag account for 10% of the total extracellular proteome. Many of the proteins are predicted to contribute to the virulence and antigenic signature of B. anthracis. We have also studied the composition of the cytoplasmic proteome, identifying 300 distinct proteins. The most abundant cytoplasmic proteins are primarily those involved in glycolysis, amino acid metabolism, protein translation, protein folding and stress adaptation. The presence of a variety of proteases, peptidases, peptide binding proteins, as well as enzymes required for the metabolism of amino acids, suggests that B. anthracis is adapted to life in a protein-rich environment rather than the soil. We therefore speculate that proteases and peptidases could be useful targets for the development of improved vaccines. In addition, both of these B. anthracis compartment-specific proteomes can be used as reference maps to monitor changes in the production of secreted and cytosolic proteins that occur, for example, during growth in macrophages.

Amino Acid Sequence↗

PubMed2006

Recombination between elongation factor 1alpha genes from distantly related archaeal lineages.

Homologous recombination (HR) and lateral gene transfer are major processes in genome evolution. The combination of the two processes, HR between genes in different species, has been documented but is thought to be restricted to very similar sequences in relatively closely related organisms. Here we report two cases of interspecific HR in the gene encoding the core translational protein translation elongation factor 1alpha (EF-1alpha) between distantly related archaeal groups. Maximum-likelihood sliding window analyses indicate that a fragment of the EF-1alpha gene from the archaeal lineage represented by Methanopyrus kandleri was recombined into the orthologous gene in a common ancestor of the Thermococcales. A second recombination event appears to have occurred between the EF-1alpha gene of the genus Methanothermobacter and its ortholog in a common ancestor of the Methanosarcinales, a distantly related euryarchaeal lineage. These findings suggest that HR occurs across a much larger evolutionary distance than generally accepted and affects highly conserved essential "informational" genes. Although difficult to detect by standard whole-gene phylogenetic analyses, interspecific HR in highly conserved genes may occur at an appreciable frequency, potentially confounding deep phylogenetic inference and hypothesis testing.

Archaea↗

PubMed2007

Characterizing residual structure in disordered protein States using nuclear magnetic resonance.

The importance of disordered protein states in biology is gaining recognition, and can be attributed in part to the participation of unfolded and partially folded states of globular proteins in normal and abnormal biological functions, such as protein translation, protein translocation, protein degradation, protein assembly, and protein aggregation (1-5). There is also a growing awareness that a significant fraction of gene products from various genomes, including the human genome, fall into a category that includes low complexity, low globularity, or intrinsically unstructured proteins (6-9). Unlike native states of globular proteins, disordered protein states, by definition, do not adopt a fixed structure that can be determined using classical high-resolution methods. Nevertheless, there has long been evidence that many disordered states contain detectable and significant residual or nascent structure (10-16). This structure has been found to be important for nucleating local structure, as well as mediating long range contacts upon either intramolecular folding to the native state (17-21) or intermolecular folding with specific binding partners (22-24), and is also predicted to influence intermolecular folding into structured aggregates (25,26). The primary tool for the characterization of such structure is high-resolution solution state nuclear magnetic resonance (NMR) spectroscopy. Advances in NMR instrumentation and methods have greatly facilitated this task and in principle can now be accomplished by those without extensive prior experience in NMR spectroscopy. This chapter describes how this can be accomplished.

Nuclear Magnetic Resonance, Biomolecular↗

PubMed1994

Influenza virus NS1 protein stimulates translation of the M1 protein.

The influenza virus NS1 protein was shown to stimulate translation of the M1 protein. M-CAT RNA, which contains the chloramphenicol acetyltransferase (CAT) reporter gene and the terminal noncoding sequence of segment 7 (coding for the M1 and M2 proteins), was ribonucleoprotein transfected into clone 76 cells expressing the influenza virus RNA polymerase and NP proteins required for the transcription and replication of influenza virus ribonucleoproteins. When the cells were superinfected with a recombinant vaccinia virus which expresses the NS1 protein, CAT expression from the M-CAT RNA was significantly stimulated but transcription was not altered. The expression of NS-CAT RNA, which contains noncoding sequences of segment 8 (coding for the NS1 and NS2 proteins), was not altered by the NS1 protein. Site-directed mutagenesis showed that the sequence GGUAGAUA upstream of the initiation codon on segment 7 was required for stimulation.

Base Sequence↗

PubMed2005

Translation of an integral membrane protein in distal dendrites of hippocampal neurons.

Maintenance of synaptic plasticity requires protein translation. Because changes in synaptic strength are regulated at the level of individual synapses, a mechanism is required for newly translated proteins to specifically and persistently modify only a subset of synapses. Evidence suggests this may be accomplished through local translation of proteins at or near synapses in response to plasticity-inducing patterns of activity. A number of proteins important for synaptic function are integral membrane proteins, which require a specialized group of organelles, proteins and enzymatic activities for proper synthesis. Dendrites appear to contain machinery necessary for the proper production of these proteins, and mRNAs for integral membrane proteins have been found localized to dendrites. Experiments are described that investigate the local translation of membrane proteins in the dendrites of cultured rat hippocampal neurons, using fluorescence recovery after photobleaching. Neurons were transfected with cDNAs encoding a fluorescently labeled transmembrane protein, TGN-38. Under conditions where the transport of this reporter construct was inhibited, the appearance of newly synthesized protein was observed via fluorescent microscopy. The dendritic translation of this protein required activation of glutamate receptors. The results demonstrate a functional capacity for activity-dependent synthesis of integral membrane proteins for distal dendrites in hippocampal neurons.

Animals↗

PubMed1999

Phosphorylation of the cap-binding protein eukaryotic translation initiation factor 4E by protein kinase Mnk1 in vivo.

Eukaryotic translation initiation factor 4E (eIF4E) binds to the mRNA 5' cap and brings the mRNA into a complex with other protein synthesis initiation factors and ribosomes. The activity of mammalian eIF4E is important for the translation of capped mRNAs and is thought to be regulated by two mechanisms. First, eIF4E is sequestered by binding proteins, such as 4EBP1, in quiescent cells. Mitogens induce the release of eIF4E by stimulating the phosphorylation of 4EBP1. Second, mitogens and stresses induce the phosphorylation of eIF4E at Ser 209, increasing the affinity of eIF4E for capped mRNA and for an associated scaffolding protein, eIF4G. We previously showed that a mitogen- and stress-activated kinase, Mnk1, phosphorylates eIF4E in vitro at the physiological site. Here we show that Mnk1 regulates eIF4E phosphorylation in vivo. Mnk1 binds directly to eIF4G and copurifies with eIF4G and eIF4E. We identified activating phosphorylation sites in Mnk1 and developed dominant-negative and activated mutants. Expression of dominant-negative Mnk1 reduces mitogen-induced eIF4E phosphorylation, while expression of activated Mnk1 increases basal eIF4E phosphorylation. Activated mutant Mnk1 also induces extensive phosphorylation of eIF4E in cells overexpressing 4EBP1. This suggests that phosphorylation of eIF4E is catalyzed by Mnk1 or a very similar kinase in cells and is independent of other mitogenic signals that release eIF4E from 4EBP1.

3T3 Cells↗

PubMed2003

Low-energy laser irradiation enhances de novo protein synthesis via its effects on translation-regulatory proteins in skeletal muscle myoblasts.

Low-energy laser irradiation (LELI) drives quiescent skeletal muscle satellite cells into the cell cycle and enhances their proliferation, thereby promoting skeletal muscle regeneration. Ongoing protein synthesis is a prerequisite for these processes. Here, we studied the signaling pathways involved in the LELI regulation of protein synthesis. High levels of labeled [35S]methionine incorporation were detected in LELI cells as early as 20 min after irradiation, suggesting translation of pre-existing mRNAs. Induced levels of protein synthesis were detected up until 8 h after LELI implying a role for LELI in de novo protein synthesis. Elevated levels of cyclin D1, associated with augmented phosphorylation of the eukaryotic initiation factor 4E (eIF4E) and its inhibitory binding protein PHAS-I, suggested the involvement of LELI in the initiation steps of protein translation. In the presence of the MEK inhibitor, PD98059, eIF4E phosphorylation was abolished and levels of cyclin D1 were dramatically reduced. The LELI-induced PHAS-I phosphorylation was abolished after preincubation with the PI3K inhibitor, Wortmannin. Concomitantly, LELI enhanced Akt phosphorylation, which was attenuated in the presence of Wortmannin. Taken together, these results suggest that LELI induces protein translation via the PI3K/Akt and Ras/Raf/ERK pathways.

Adaptor Proteins, Signal Transducing↗

PubMed2003

Human homologue of ariadne promotes the ubiquitylation of translation initiation factor 4E homologous protein, 4EHP.

Human homologue of Drosophila ariadne (HHARI) is a RING-IBR-RING domain protein identified through its ability to bind the human ubiquitin-conjugating enzyme, UbcH7. We now demonstrate that HHARI also interacts with the eukaryotic mRNA cap binding protein, translation initiation factor 4E homologous protein (4EHP), via the N-terminal RING1 finger of HHARI. HHARI, 4EHP and UbcH7 do not form a stable heterotrimeric complex as 4EHP cannot immunoprecipitate UbcH7 even in the presence of HHARI. Overexpression of 4EHP and HHARI in mammalian cells leads to polyubiquitylation of 4EHP. By contrast, HHARI does not promote its own autoubiquitylation. Thus, by promoting the ubiquitin-mediated degradation of 4EHP, HHARI may have a role in both protein degradation and protein translation.

Carrier Proteins↗

PubMed1997

Fragile X mental retardation protein is translated near synapses in response to neurotransmitter activation.

Local translation of proteins in distal dendrites is thought to support synaptic structural plasticity. We have previously shown that metabotropic glutamate receptor (mGluR1) stimulation initiates a phosphorylation cascade, triggering rapid association of some mRNAs with translation machinery near synapses, and leading to protein synthesis. To determine the identity of these mRNAs, a cDNA library produced from distal nerve processes was used to screen synaptic polyribosome-associated mRNA. We identified mRNA for the fragile X mental retardation protein (FMRP) in these processes by use of synaptic subcellular fractions, termed synaptoneurosomes. We found that this mRNA associates with translational complexes in synaptoneurosomes within 1-2 min after mGluR1 stimulation of this preparation, and we observed increased expression of FMRP after mGluR1 stimulation. In addition, we found that FMRP is associated with polyribosomal complexes in these fractions. In vivo, we observed FMRP immunoreactivity in spines, dendrites, and somata of the developing rat brain, but not in nuclei or axons. We suggest that rapid production of FMRP near synapses in response to activation may be important for normal maturation of synaptic connections.

Amino Acid Sequence↗

PubMed1990

v-mos protein produced by in vitro translation has protein kinase activity.

The v-mos protein, termed p37v-mos, has a closely associated serine/threonine protein kinase activity. To provide further information about its protein kinase activity, we tested the activity of p37v-mos produced in a cell-free translation system from transcripts generated from a cloned v-mos gene. Anti-mos(37-55) immunoprecipitates of in vitro-produced p37v-mos were found to possess serine/threonine protein kinase activity, whereas those obtained with anti-mos(260-271), known to block v-mos autophosphorylation, lacked kinase activity. The phosphorylated products were identical in size to p37v-mos and p43v-mos produced in protein kinase assays from Moloney murine sarcoma virus-infected cells expressing authentic p37v-mos. These results provide further proof that the protein kinase activity associated with p37v-mos is an intrinsic property of the v-mos gene product. This translation system also provides a useful experimental model to study the activation of the mos protein kinase. Thus, protein kinase assays performed on [35S]methionine-labeled p37v-mos produced p43v-mos at the expense of p37v-mos. Phosphatase treatment removed the p43v-mos species, resulting in increase of the p37v-mos-sized protein, confirming our previous interpretation that p43v-mos is a hyperphosphorylated form of p37v-mos.

Animals↗

PubMed2001

Oral administration of leucine stimulates ribosomal protein mRNA translation but not global rates of protein synthesis in the liver of rats.

The objective of the current study was to examine the role of the branched-chain amino acid (BCAA) leucine in the regulation of hepatic protein synthesis and ribosomal protein (rp) mRNA translation in vivo. Food-deprived (18 h) male rats (200 g) were orally administered saline (control) or 270 mg leucine, isoleucine or valine and killed 1 h later. Administration of any BCAA resulted in enhanced phosphorylation of eukaryotic initiation factor (eIF) 4E-binding protein-1 (4E-BP1) compared with controls. However, leucine was the most effective at stimulating phosphorylation of 4E-BP1 as well as the 70-kDa ribosomal protein S6 kinase (S6K1). Despite these effects on components of the translation initiation process, there were no differences in total protein synthesis rates among treatment groups. The distribution of rp (S4, S8, L26) and non-rp (albumin, beta-actin) mRNAs across sucrose density gradients showed that the preponderance of hepatic rp mRNAs in control rats was unloaded from polysomes. Of the BCAA, only leucine was the most effective in causing a shift in the distribution of rp mRNA to polysomes compared with controls. Non-rp transcripts remained mainly polysome-associated under all conditions. These results suggest that leucine is most effective among the BCAA in its ability to stimulate translation of rp mRNA in liver. Furthermore, the translation of rp mRNA is disjointed from rates of total protein synthesis in liver and related to the degree of S6K1 phosphorylation.

Administration, Oral↗

PubMed2005

Hepatitis C virus internal ribosome entry site-dependent translation in Saccharomyces cerevisiae is independent of polypyrimidine tract-binding protein, poly(rC)-binding protein 2, and La protein.

Translation initiation of some viral and cellular mRNAs occurs by ribosome binding to an internal ribosome entry site (IRES). Internal initiation mediated by the hepatitis C virus (HCV) IRES in Saccharomyces cerevisiae was shown by translation of the second open reading frame in a bicistronic mRNA. Introduction of a single base change in the HCV IRES, known to abrogate internal initiation in mammalian cells, abolished translation of the second open reading frame. Internal initiation mediated by the HCV IRES was independent of the nonsense-mediated decay pathway and the cap binding protein eIF4E, indicating that translation is not a result of mRNA degradation or 5'-end-dependent initiation. Human La protein binds the HCV IRES and is required for efficient internal initiation. Disruption of the S. cerevisiae genes that encode La protein orthologs and synthesis of wild-type human La protein in yeast had no effect on HCV IRES-dependent translation. Polypyrimidine tract-binding protein (Ptb) and poly-(rC)-binding protein 2 (Pcbp2), which may be required for HCV IRES-dependent initiation in mammalian cells, are not encoded within the S. cerevisiae genome. HCV IRES-dependent translation in S. cerevisiae was independent of human Pcbp2 protein and stimulated by the presence of human Ptb protein. These findings demonstrate that the genome of S. cerevisiae encodes all proteins necessary for internal initiation of translation mediated by the HCV IRES.

Autoantigens↗

PubMed1996

Pseudoknot and translational control in the expression of the S15 ribosomal protein.

Translational autocontrol of the expression of the ribosomal protein S15 proceeds through the transitory formation of a pseudoknot. A synopsis of the known data is used to propose a molecular model of the mechanism involved and for the role of the pseudoknot. This latter structure is able to recruit 30S ribosomal subunits to initiate translation, but also to bind S15 and to stop translation by trapping the ribosome on its loading site. Information on the S15 protein recognition of the messenger RNA site was deduced from mutational analyses and chemical probing. A comparison of this messenger site with the S15 ribosomal binding site was conducted by analysing hydroxyl radical footprintings of these two sites. The existence of two subsites in 16S RNA suggests that the ribosomal protein S15 might present either two different binding sites or at least one common subsite. Clues for the presence of a common site between the messenger and 16S RNA are given which cannot rule out that recognition specificity is linked to a few other determinants. Whether these determinants are different or not remains an open question.

Base Sequence↗

PubMed1997

Functional recognition of fragmented operator sites by R17/MS2 coat protein, a translational repressor.

The R17/MS2 coat protein serves as a translational repressor of replicase by binding to a 19 nt RNA hairpin containing the Shine-Dalgarno sequence and the initiation codon of the replicase gene. We have explored the structural features of the RNA operator site that are necessary for efficient translational repression by the R17/MS2 coat protein in vivo . The R17/MS2 coat protein efficiently directs lysogen formation for P22 R17 , a bacteriophage P22 derivative that carries the R17/MS2 RNA operator site within the P22 phage ant mRNA. Phages were constructed that contain fragmented operator sites such that the Shine-Dalgarno sequence and the initiation codon of the affected gene are not located within the RNA hairpin. The wild-type coat protein directs efficient lysogen formation for P22 phages that carry several fragmented RNA operator sites, including one in which the Shine-Dalgarno sequence is positioned 4 nt outside the coat protein binding site. Neither the wild-type R17/MS2 coat protein nor super-repressor mutants induce lysogen formation for a P22 phage encoding an RNA hairpin at a distance of 9 nt from the Shine-Dalgarno sequence, implying that a discrete region of biological repression is defined by the coat protein-RNA hairpin interaction. The assembly of RNA species into capsid structures is not an efficient means whereby the coat protein achieves translational repression of target mRNA transcripts. The R17/MS2 coat protein exerts translational regulation that extends considerably beyond the natural biological RNA operator site structure; however, the coat protein still mediates repression in these constructs by preventing ribosome access to linear sequence determinants of the translational initiation region by the formation of a stable RNA secondary structure. An efficient translational regulatory mechanism in bacteria appears to reside in the ability of proteins to regulate RNA folding states for host cell and phage mRNAs.

Bacteriophage P22↗

PubMed1993

CCAAT/enhancer-binding protein mRNA is translated into multiple proteins with different transcription activation potentials.

The CCAAT/enhancer-binding protein (C/EBP) alpha is a leucine zipper protein that is preferentially expressed in certain cell types, such as adipocytes and hepatocytes. Here we show that C/EBP alpha mRNA is translated into two major proteins, C/EBP-42 and C/EBP-30, that differ in their content of N-terminal amino acid sequences. These results are best explained by a ribosome-scanning mechanism in which a fraction of ribosomes ignore the first two AUGs and initiate translation at an AUG located 351 nt downstream of the first one. Because C/EBP-30, the translation product initiated at the third AUG, is devoid of the potent transcription-activation domain contained in C/EBP-42, the former protein stimulates transcription from the mouse albumin promoter much less efficiently than the latter. The gene encoding the liver-enriched transcriptional-activator protein LAP (C/EBP-beta) has also been shown to issue two proteins, LAP and the liver-enriched transcriptional-inhibitory protein LIP, with different transcription-activation potentials. The production of multiple proteins from a single mRNA is not only shared between different C/EBP family members but also appears to be conserved in vertebrate evolution.

Affinity Labels↗

PubMed1993

In vitro translation of androgen receptor cRNA results in an activated androgen receptor protein.

Translation of androgen receptor (AR) cRNA in a reticulocyte lysate and subsequent analysis of the translation products by SDS/PAGE showed a protein with an apparent molecular mass of 108 kDa. Scatchard-plot analysis revealed a single binding component with high affinity for R1881 (Kd = 0.3 nM). All AR molecules synthesized specifically bound steroid. No evidence for AR phosphorylation during in vitro synthesis was found. When AR was labelled with [3H]R1881 and analysed on sucrose-density gradients, a complex of approx. 6 S was observed. The complex was shifted to a higher sedimentation coefficient after incubation with a monoclonal AR antibody directed against an epitope in the DNA-binding domain. In the presence as well as the absence of hormone, AR molecules were able to bind to DNA-cellulose without an activation step. Gel retardation assays revealed that the AR forms complexes with a DNA element containing glucocorticoid-responsive element/androgen-responsive element sequences. Receptor-DNA interactions were stabilized by different polyclonal antibodies directed against either the N- or C-terminal part of the AR and were abolished by an antibody directed against the DNA-binding domain of the receptor. In conclusion, translation of AR cRNA in vitro yields an activated AR protein which binds steroid with high affinity. It is proposed that AR antibodies enhance AR-DNA binding by stabilizing AR dimers when bound to DNA.

Adenosine Triphosphate↗

PubMed1997

Translational controls impinging on the 5'-untranslated region and initiation factor proteins.

Translation of eukaryotic mRNAs is generally initiated by the scanning ribosome mechanism. This can be downregulated by high affinity protein binding to cap-proximal RNA motifs. Translation can also be regulated by short open reading frames within the 5' -untranslated region. A key factor for initiation is elF4F, in which one of the polypeptide chains, elF4G, seems to have a bridging function and binds three other factors at separate sites: elF4E (the cap-binding factor), the helicase elF4A, and elF3, which also interacts with 40S ribosomal subunits. Initiation is regulated by the MAP kinase and rapamycin-sensitive signalling pathways, which control phosphorylation of elF4E and 4E-BP1, a protein which in the dephosphorylated form binds and sequesters elF4E.

Animals↗

PubMedDate not supplied

Self-assembling protein arrays using electronic semiconductor microchips and in vitro translation.

Protein arrays will greatly accelerate research and development in medical and biological sciences. We have used cell-free protein biosynthesis and a parallel immobilization strategy for producing protein biochips. We demonstrate a model two-protein microarray using luciferase and green fluorescent protein, both expressed in a cell-free system and specifically immobilized on CombiMatrix semiconductor oligonucleotide microarrays. This demonstration provides evidence for the appropriate folding, activity, robust presentation, and efficient flexible detection of proteins on the microscale.

Fluorescent Antibody Technique, Indirect↗