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At least 127 records · Page 7Linked to original sources
PubMed2005

VSV-tumor selective replication and protein translation.

The emergence of vesicular stomatatis virus (VSV) as a potent antitumor agent has made a dissection of the molecular determinants of host-cell permissiveness to this virus an important objective. Such insight would not only enable the intelligent design of future generations of recombinant VSV vectors to combat disease, but may also resolve general features of cellular transformation that may be exploited by this virus, and perhaps other oncolytic viruses. The defective pathways underlining the oncolytic activity of VSV remain to be fully determined but recent data indicates that flaws in innate immune responses, involving the interferon (IFN) system, may commonly occur in tumor cells and thus play a large role in facilitating oncolysis. Aside from the IFN system, however, it is almost certain that other key cellular pathways may be similarly defective and therefore cooperatively contribute towards mediating rapid oncolytic virus activity. Recent data have indicated that defects in cancer cell translational regulation could be one area that may be exploited by VSV. Certainly, all viruses require cellular protein synthesis pathways to facilitate their replication and many have devised numerous mechanisms to ensure that viral mRNAs become translated at the expense of the host. Using VSV as a model, this review will discuss some of the recent developments in the fields of innate immunity and translational regulation that may help explain mechanisms of viral oncolysis.

Immunity, Innate↗
PubMed2004

Immune recognition of a human renal cancer antigen through post-translational protein splicing.

Cytotoxic T lymphocytes (CTLs) detect and destroy cells displaying class I molecules of the major histocompatibility complex (MHC) that present oligopeptides derived from aberrant self or foreign proteins. Most class I peptide ligands are created from proteins that are degraded by proteasomes and transported, by the transporter associated with antigen processing, from the cytosol into the endoplasmic reticulum, where peptides bind MHC class I molecules and are conveyed to the cell surface. C2 CTLs, cloned from human CTLs infiltrating a renal cell carcinoma, kill cancer cells overexpressing fibroblast growth factor-5 (FGF-5). Here we show that C2 cells recognize human leukocyte antigen-A3 MHC class I molecules presenting a nine-residue FGF-5 peptide generated by protein splicing. This process, previously described strictly in plants and unicellular organisms, entails post-translational excision of a polypeptide segment followed by ligation of the newly liberated carboxy-terminal and amino-terminal residues. The occurrence of protein splicing in vertebrates has important implications for the complexity of the vertebrate proteome and for the immune recognition of self and foreign peptides.

Amino Acid Sequence↗
PubMed1986

Regulation of chloroplast-encoded chlorophyll-binding protein translation during higher plant chloroplast biogenesis.

Etioplasts of 5-day-old dark-grown barley seedlings synthesize most of the soluble and membrane proteins found in chloroplasts of illuminated plants. Prominent among these proteins are the large subunit of ribulose bisphosphate carboxylase and the alpha- and beta-subunits of the chloroplast ATPase. However, etioplasts do not synthesize four chloroplast-encoded proteins which are major constituents of the chloroplast thylakoid membrane: two chlorophyll apoproteins of photosystem I (68 and 65 kDa) and two chlorophyll apoproteins of photosystem II (47 and 43 kDa). Pulse-labeling experiments show that the lack of radiolabel accumulation in the chlorophyll apoproteins in etioplasts is due to inhibition of synthesis rather than apoprotein instability. Illumination of 5-day-old dark-grown barley selectively induces synthesis of the plastid-encoded chlorophyll apoproteins and proteins of 32, 23, and 21 kDa. Synthesis of the chlorophyll apoproteins was significant in plants illuminated for 15 min and was near maximum by 1 h. The induction of photosystem I chlorophyll apoprotein synthesis was not accompanied by an increase in mRNA for these proteins. These results demonstrate that the synthesis of the plastid-encoded photosystem I chlorophyll apoproteins is blocked at the translational level in dark-grown barley. Translation of the chlorophyll apoproteins is induced rapidly by light with a time course which is similar to the light-dependent formation of chlorophyll from protochlorophyllide.

Chlorophyll↗
PubMed2005

Insulin stimulates postsynaptic density-95 protein translation via the phosphoinositide 3-kinase-Akt-mammalian target of rapamycin signaling pathway.

Insulin receptors are highly enriched at neuronal synapses, but whose function remains unclear. Here we present evidence that brief incubations of rat hippocampal slices with insulin resulted in an increased protein expression of dendritic scaffolding protein postsynaptic density-95 (PSD-95) in area CA1. This insulin-induced increase in the PSD-95 protein expression was inhibited by the tyrosine kinase inhibitor, AG1024, phosphatidylinositol 3-kinase (PI3K) inhibitors, LY294002 and wortmannin, translational inhibitors, anisomycin and rapamycin, but not by LY303511 (an inactive analogue of LY294002), and transcriptional inhibitor, actinomycin D, suggesting that insulin regulates the translation of PSD-95 by activating the receptor tyrosine kinase-PI3K-mammalian target of rapamycin (mTOR) signaling pathway. A similar insulin-induced increase in the PSD-95 protein expression was detected after stimulation of the synaptic fractions isolated from the hippocampal neurons. Furthermore, insulin treatment did not affect the PSD-95 mRNA levels. In agreement, insulin rapidly induced the phosphorylation of 3-phosphoinositide-dependent protein kinase-1 (PDK1), protein kinase B (Akt), and mTOR, effects that were prevented by the AG1024 and LY294002. We also show that insulin stimulated the phosphorylation of 4E-binding protein 1 (4E-BP1) and p70S6 kinase (p70S6K) in a mTOR-dependent manner. Finally, we demonstrate the constitutive expression of PSD-95 mRNA in the synaptic fractions isolated from hippocampal neurons. Taken together, these findings suggest that activation of the PI3K-Akt-mTOR signaling pathway is essential for the insulin-induced up-regulation of local PSD-95 protein synthesis in neuronal dendrites and indicate a new molecular mechanism that may contribute to the modulation of synaptic function by insulin in hippocampal area CA1.

Animals↗
PubMed2003

Ribosome binding to the Oxa1 complex facilitates co-translational protein insertion in mitochondria.

The Oxa1 translocase of the mitochondrial inner membrane facilitates the insertion of both mitochondrially and nuclear-encoded proteins from the matrix into the inner membrane. Most mitochondrially encoded proteins are hydrophobic membrane proteins which are integrated into the lipid bilayer during their synthesis on mitochondrial ribosomes. The molecular mechanism of this co-translational insertion process is unknown. Here we show that the matrix-exposed C-terminus of Oxa1 forms an alpha-helical domain that has the ability to bind to mitochondrial ribosomes. Deletion of this Oxa1 domain strongly diminished the efficiency of membrane insertion of subunit 2 of cytochrome oxidase, a mitochondrially encoded substrate of the Oxa1 translocase. This suggests that co-translational membrane insertion of mitochondrial translation products is facilitated by a physical interaction of translation complexes with the membrane-bound translocase.

Chromatography, Affinity↗
PubMed1998

Branched-chain amino acids are essential in the regulation of PHAS-I and p70 S6 kinase by pancreatic beta-cells. A possible role in protein translation and mitogenic signaling.

Amino acids have been identified as important signaling molecules involved in pancreatic beta-cell proliferation, although the cellular mechanism responsible for this effect is not well defined. We previously reported that amino acids are required for glucose or exogenous insulin to stimulate phosphorylation of PHAS-I (phosphorylated heat- and acid-stable protein regulated by insulin), a recently discovered regulator of translation initiation during cell mitogenesis. Here we demonstrate that essential amino acids, in particular branched-chain amino acids (leucine, valine, and isoleucine), are largely responsible for mediating this effect. The transamination product of leucine, alpha-ketoisocaproic acid, also stimulates PHAS-I phosphorylation although the transamination products of isoleucine and valine are ineffective. Since amino acids are secretagogues for insulin secretion by beta-cells, we investigated whether endogenous insulin secreted by beta-cells is involved. Interestingly, branched-chain amino acids stimulate phosphorylation of PHAS-I independent of endogenous insulin secretion since genistein (10 microM) and herbimycin A (1 microM), two tyrosine kinase inhibitors in the insulin signaling pathway, exert no effect on amino acid-induced phosphorylation of PHAS-I. Furthermore, branched-chain amino acids retain their ability to induce phosphorylation of PHAS-I under conditions that block insulin secretion from beta-cells. In exploring the signaling pathway responsible for these effects, we find that rapamycin (25 nM) inhibits the ability of branched-chain amino acids to stimulate the phosphorylation of PHAS-I and p70(s6) kinase, suggesting that the mammalian target of rapamycin signaling pathway is involved. The branched-chain amino acid, leucine, also exerts similar effects on PHAS-I phosphorylation in isolated pancreatic islets. In addition, we find that amino acids are necessary for insulin-like growth factor (IGF-I) to stimulate the phosphorylation of PHAS-I indicating that a requirement for amino acids may be essential for other beta-cell growth factors in addition to insulin and IGF-I to activate this signaling pathway. We propose that amino acids, in particular branched-chain amino acids, may promote beta-cell proliferation either by stimulating phosphorylation of PHAS-I and p70(s6k) via the mammalian target of rapamycin pathway and/or by facilitating the proliferative effect mediated by growth factors such as insulin and IGF-I.

Amino Acids, Branched-Chain↗
PubMed1998

Two proteins translated by alternative usage of initiation codons in mRNA encoding a JunD transcriptional regulator.

The junD gene encodes one component of the transcription factor, AP-1. Since two forms of JunD protein have been reported, we analyzed here the molecular mechanisms involved in the isoform production. Immunochemical analysis indicated that the longer and shorter forms of mouse JunD (JunD-L and JunD-S, with apparent molecular weights of 44 and 39 kDa, respectively) differ in their content of an N-terminal peptide. Mutational analysis further indicated that JunD-S is the translational product initiated at the third AUG located 144 bp from the first AUG, at which JunD-L translation starts. Such production of two junD isoforms from a single mRNA using the same reading frame seems to be conserved in human, rat, and chicken. To examine the functional differences between the isoforms, each type of JunD was exclusively expressed by the use of retrovirus vectors harboring the mutated junD gene. The exogenous expression of either one of these forms did not cause cellular transformation of NIH3T3, but suppressed the anchorage-independent growth of NIH3T3 transfor-bold by the activated K-ras or v-src gene. These two isoforms were expressed in all the mouse tissues examined and in various cell lines established from human tumors, though the expression ratio between JunD-L and JunD-S varied, suggesting that some factor(s) modulate the alternative usage of the initiation codon of the junD gene.

Amino Acid Sequence↗
PubMed1994

Protein translation elongation factor-1 alpha from Trypanosoma brucei binds calmodulin.

The following study examines the calmodulin (CaM) branch of the calcium signal pathway in the protozoan parasite Trypanosoma brucei. To accomplish this goal, a subset of cytosolic CaM-binding proteins (CaMBPs) was partially purified by a combination of DE52 and CaM-Sepharose affinity chromatography. Monoclonal antibodies (CBP-KK1) were used to clone the cDNA for a 53-kDa CaMBP from a lambda ZAP expression library of the metacyclic stage of T. brucei. The deduced amino acid sequence of clone CaMBP-12B had 81% overall amino acid identity to the translation elongation factor-1 alpha (EF-1 alpha) from Euglena gracilis and 76% identity to the rabbit EF-1 alpha. Rabbit EF-1 alpha was recognized by CBP-KK1 and was shown to bind to CaM-Sepharose in a calcium-dependent manner. By contrast, the complex of EF-1 alpha beta gamma did not bind CaM-Sepharose. A heterobifunctional sulfhydryl derivative of CaM (N-succinimidyl 3-(2-pyridyldithio)propionate-CaM) formed reducible cross-links with EF-1 alpha in solution but not with the complex of EF-1 alpha beta gamma. Biotinylated CaM bound weakly to trypanosome and rabbit EF-1 alpha in a gel overlay assay. This report demonstrates the direct interaction between CaM and the translation elongation factor EF-1 alpha.

Amino Acid Sequence↗
PubMed2005

The 14-3-3 protein translates the NA+,K+-ATPase {alpha}1-subunit phosphorylation signal into binding and activation of phosphoinositide 3-kinase during endocytosis.

Clathrin-dependent endocytosis of Na(+),K(+)-ATPase molecules in response to G protein-coupled receptor signals is triggered by phosphorylation of the alpha-subunit and the binding of phosphoinositide 3-kinase. In this study, we describe a molecular mechanism linking phosphorylation of Na(+),K(+)-ATPase alpha-subunit to binding and activation of phosphoinositide 3-kinase. Co-immunoprecipitation studies, as well as experiments using confocal microscopy, revealed that dopamine favored the association of 14-3-3 protein with the basolateral plasma membrane and its co-localization with the Na(+),K(+)-ATPase alpha-subunit. The functional relevance of this interaction was established in opossum kidney cells expressing a 14-3-3 dominant negative mutant, where dopamine failed to decrease Na(+),K(+)-ATPase activity and to promote its endocytosis. The phosphorylated Ser-18 residue within the alpha-subunit N terminus is critical for 14-3-3 binding. Activation of phosphoinositide 3-kinase by dopamine during Na(+),K(+)-ATPase endocytosis requires the binding of the kinase to a proline-rich domain within the alpha-subunit, and this effect was blocked by the presence of a 14-3-3 dominant negative mutant. Thus, the 14-3-3 protein represents a critical linking mechanism for recruiting phosphoinositide 3-kinase to the site of Na(+),K(+)-ATPase endocytosis.

14-3-3 Proteins↗
PubMed1999

ASH1 mRNA localization in yeast involves multiple secondary structural elements and Ash1 protein translation.

Localization of ASH1 mRNA to the distal cortex of daughter but not mother cells at the end of anaphase is responsible for the two cells' differential mating-type switching during the subsequent cell cycle. This localization depends on actin filaments and a type V myosin (She1/Myo4). The 3' untranslated region (3' UTR) of ASH1 mRNA is reportedly capable of directing heterologous RNAs to a mother cell's bud [1] [2]. Surprisingly, however, its replacement has little or no effect on the localisation of ASH1 mRNA. We show here that, unlike all other known localization sequences that have been found in 3' UTRs, all the elements involved in ASH1 mRNA localization are located at least partly within its coding region. A 77 nucleotide region stretching from 7 nucleotides 5' to 67 nucleotides 3' of the stop codon of ASH1 mRNA is sufficient to localize mRNAs to buds; the secondary structure of this region, in particular two stems, is important for its localizing activity. Two regions entirely within coding sequences, both sufficient to localize green fluorescent protein (GFP) mRNA to growing buds, are necessary for ASH1 mRNA localization during anaphase. These three regions can anchor GFP mRNA to the distal cortex of daughter cells only inefficiently. The tight anchoring of ASH1 mRNA to the cortex of the daughter cell depends on translation of the carboxy-terminal sequences of Ash1 protein.

3' Untranslated Regions↗
PubMed1994

Storage of messenger RNA in eukaryotes: envelopment with protein, translational barrier at 5' side, or conformational masking by 3' side?

Messenger RNA can be stored in the cytoplasm of higher Eukaryotes in the form of masked messenger ribonucleoprotein particles (masked mRNPs, or informosomes). The typical example is the storage of mRNPs in germ cells (oocytes and spermatocytes). The masked mRNPs are inactive in translation, stable, i.e., protected against degradation, and unavailable for poly(A) tail processing, such as cytoplasmic polyadenylation and deadenylation. The major nonspecific mRNA-binding protein forming mRNPs and belonging to a special p50 family of basic, glycine-rich, phosphorylatable proteins seems to be necessary, but not sufficient for the masking. In some cases, mRNA-specific repressor proteins bound to the 5'-untranslated regions (5'-UTR) of mRNAs may be involved. Interactions of the 3'-untranslated regions (3'-UTR) with sequence-specific proteins seem to be of decisive importance for the masking of mRNPs. The hypothesis is proposed that the masking is achieved through a 3'-UTR-induced conformational rearrangement of mRNP; closing into a circle and condensation of mRNP are considered plausible.

Animals↗
PubMed1982

Expression of the split gene cob in yeast: evidence for a precursor of a "maturase" protein translated from intron 4 and preceding exons.

Intron 4 (14) of the split gene cob in mitochondrial DNA contains a long open reading frame in phase with the preceding exon. Mutations in this intron block the excision of the 14 sequence from the cob precursor RNA and, at the same time, generate a series of new polypeptides, parts of which apparently result from translation of 14 sequences. We sequenced six mutations clustered in the upstream part of the open reading frame, about 340 bp from the exon-intron boundary (box9 cluster). Four are base pair exchanges in the same triplet of this region; these form the polypeptides typical for 14 plus a trans-acting product encoded by 14, as shown by complementation studies. The other two mutations--a -2 bp deletion at the same site, causing frameshift with a chain-terminating codon within a few triplets, and a base pair exchange at a nearby site--affect both the formation of 14 typical translation products and the trans-acting function. These results on box9 mutants combined with results on box7 mutants suggest that an 14-encoded "maturase" protein (apparent molecular weight, 27,000) is cleaved off a precursor protein (apparent molecular weight, 55,000) encoded by exon sequences B1 to B4 and the intron open reading frame. We further discuss the role of the box9 nucleotide sequence in the maturation of cob-specific RNA.

Base Sequence↗
PubMed1995

Effects of a minor isoleucyl tRNA on heterologous protein translation in Escherichia coli.

In Escherichia coli, the isoleucine codon AUA occurs at a frequency of about 0.4% and is the fifth rarest codon in E. coli mRNA. Since there is a correlation between the frequency of codon usage and the level of its cognate tRNA, translational problems might be expected when the mRNA contains high levels of AUA codons. When a hemagglutinin from the influenza virus, a 304-amino-acid protein with 12 (3.9%) AUA codons and 1 tandem codon, and a mupirocin-resistant isoleucyl tRNA synthetase, a 1,024-amino-acid protein, with 33 (3.2%) AUA codons and 2 tandem codons, were expressed in E. coli, product accumulation was highly variable and dependent to some degree on the growth medium. In rich medium, the flu antigen represented about 16% of total cell protein, whereas in minimal medium, it was only 2 to 3% of total cell protein. In the presence of the cloned ileX, which encodes the cognate tRNA for AUA, however, the antigen was 25 to 30% of total cell protein in cells grown in minimal medium. Alternatively, the isoleucyl tRNA synthetase did not accumulate to detectable levels in cells grown in Luria broth unless the ileX tRNA was coexpressed when it accounted for 7 to 9% of total cell protein. These results indicate that the rare isoleucine AUA codon, like the rare arginine codons AGG and AGA, can interfere with the efficient expression of cloned proteins.

Bacterial Proteins↗
PubMed2006

Akt1 activation can augment hypoxia-inducible factor-1alpha expression by increasing protein translation through a mammalian target of rapamycin-independent pathway.

The phosphoinositide 3-kinase (PI3K)/Akt pathway is commonly activated in cancer; therefore, we investigated its role in hypoxia-inducible factor-1alpha (HIF-1alpha) regulation. Inhibition of PI3K in U87MG glioblastoma cells, which have activated PI3K/Akt activity secondary to phosphatase and tensin homologue deleted on chromosome 10 (PTEN) mutation, with LY294002 blunted the induction of HIF-1alpha protein and its targets vascular endothelial growth factor and glut1 mRNA in response to hypoxia. Introduction of wild-type PTEN into these cells also blunted HIF-1alpha induction in response to hypoxia and decreased HIF-1alpha accumulation in the presence of the proteasomal inhibitor MG132. Akt small interfering RNA (siRNA) also decreased HIF-1alpha induction under hypoxia and its accumulation in normoxia in the presence of dimethyloxallyl glycine, a prolyl hydroxylase inhibitor that prevents HIF-1alpha degradation. Metabolic labeling studies showed that Akt siRNA decreased HIF-1alpha translation in normoxia in the presence of dimethyloxallyl glycine and in hypoxia. Inhibition of mammalian target of rapamycin (mTOR) with rapamycin (10-100 nmol/L) had no significant effect on HIF-1alpha induction in a variety of cell lines, a finding that was confirmed using mTOR siRNA. Furthermore, neither mTOR siRNA nor rapamycin decreased HIF-1alpha translation as determined by metabolic labeling studies. Therefore, our results indicate that Akt can augment HIF-1alpha expression by increasing its translation under both normoxic and hypoxic conditions; however, the pathway we are investigating seems to be rapamycin insensitive and mTOR independent. These observations, which were made on cells grown in standard tissue culture medium (10% serum), were confirmed in PC3 prostate carcinoma cells. We did find that rapamycin could decrease HIF-1alpha expression when cells were cultured in low serum, but this seems to represent a different pathway.

Cell Line, Tumor↗
PubMed1996

Inhibition of retinoblastoma protein translation by UVB in human melanocytic cells and reduced cell cycle arrest following repeated irradiation.

In human melanocytes and a human melanoma cell line (MM96L), the level of the retinoblastoma gene product (pRB) detected by Western blotting transiently decreased to 55% and 70% of controls respectively 9-12 h after a noncytostatic exposure (75 Jm-2) to UVB (280-315 nm) and to 2% and 14% 48 h after a cytostatic exposure (300 Jm-2). The pRB levels in fibroblasts and HeLa showed minimal loss, and under some conditions increased compared with unirradiated cells. Equitoxic doses of gamma radiation, cisplatin or the antimetabolite deoxyinosine had little effect on pRB levels. UVC (254 nm) was less inhibitory compared with equitoxic UVB. No loss of pRB mRNA was found in MM96L after UVB, nor was pRB protein stability significantly affected. Synthesis of new pRB in MM96L 24 h after UVB was 16% of controls, suggesting that loss of pRB results from a UVB-specific inhibition of translation. Compared with HeLa cells and fibroblasts, MM96L cells exhibited reduced cycle arrest if irradiated when pRB was depleted by a previous UVB exposure. These results suggest a mechanism whereby down-regulation of pRB translation by UVB may play a role in genesis of melanoma.

Cell Cycle↗