AbstracteIF3 in mammals is the largest translation initiation factor (~800 kDa) and is composed of 13 nonidentical subunits designated eIF3a-m. The role of mammalian eIF3 in assembly of the 48 S complex occurs through high affinity binding to eIF4G. Interactions of eIF4G with eIF4E, eIF4A, eIF3, poly(A)-binding protein, and Mnk1/2 have been mapped to discrete domains on eIF4G, and conversely, the eIF4G-binding sites on all but one of these ligands have been determined. The only eIF4G ligand for which this has not been determined is eIF3. In this study, we have sought to identify the mammalian eIF3 subunit(s) that directly interact(s) with eIF4G. Established procedures for detecting protein-protein interactions gave ambiguous results. However, binding of partially proteolyzed HeLa eIF3 to the eIF3-binding domain of human eIF4G-1, followed by high throughput analysis of mass spectrometric data with a novel peptide matching algorithm, identified a single subunit, eIF3e (p48/Int-6). In addition, recombinant FLAG-eIF3e specifically competed with HeLa eIF3 for binding to eIF4G in vitro. Adding FLAG-eIF3e to a cell-free translation system (i) inhibited protein synthesis, (ii) caused a shift of mRNA from heavy to light polysomes, (iii) inhibited capdependent translation more severely than translation dependent on the HCV or CSFV internal ribosome entry sites, which do not require eIF4G, and (iv) caused a dramatic loss of eIF4G and eIF2α from complexes sedimenting at ~40 S. These data suggest a specific, direct, and functional interaction of eIF3e with eIF4G during the process of cap-dependent translation initiation, although they do not rule out participation of other eIF3 subunits.Eukaryotic translation initiation involves numerous initiation factors (eIFs)2 that participate in recruitment of initiator tRNA and mRNA to the 40 S ribosomal subunit, recognition of the initiator AUG codon, and joining of the 40 S and 60 S ribosomal subunits, culminating in formation of the first peptide bond (1). The factors required for recruitment of mRNA include eIF3, eIF4A, eIF4B, eIF4E, eIF4G, eIF4H, and PABP. eIF4E and PABP bind the 5′ cap and 3′ poly(A) tract of mRNA, respectively, whereas eIF4A unwinds 5′-terminal secondary structure in an ATP-dependent process that also involves the RNA-binding proteins eIF4B and eIF4H. eIF4G forms specific complexes with eIF4E, eIF4A, and PABP, thereby linking the processes of cap recognition, poly(A) binding, and secondary structure melting. eIF4G in turn is recruited to the 40 S ribosomal subunit via binding to the multisubunit complex eIF3.* This work was supported by National Institutes of Health Grants GM20818 (to R. E. R.) and GM22135 (to J. W. B. H.).1 To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Biology, Louisiana State University Health Sciences Center, 1501 Kings Hwy., Shreveport, LA 71130-3932. Tel.: 318-675-5161; Fax: 318-675-5180; E-mail: rrhoad@lsuhsc.edu. NIH Public Access NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript ...

The mRNA cap-binding protein eukaryotic translation initiation factor 4E (eIF4E) participates in protein synthesis initiation, translational repression of specific mRNAs, and nucleocytoplasmic shuttling. Multiple isoforms of eIF4E are expressed in a variety of organisms, but their specific roles are poorly understood. We investigated one Caenorhabditis elegans isoform, IFE-4, which has homologues in plants and mammals. IFE-4::green fluorescent protein (GFP) was expressed in pharyngeal and tail neurons, body wall muscle, spermatheca, and vulva. Knockout of ife-4 by RNA interference (RNAi) or a null mutation produced a pleiotropic phenotype that included egg-laying defects. Sedimentation analysis demonstrated that IFE-4, but not IFE-1, was present in 48S initiation complexes, indicating that it participates in protein synthesis initiation. mRNAs affected by ife-4 knockout were determined by DNA microarray analysis of polysomal distribution. Polysome shifts, in the absence of total mRNA changes, were observed for only 33 of the 18,967 C. elegans mRNAs tested, of which a disproportionate number were related to egg laying and were expressed in neurons and/or muscle. Translational regulation was confirmed by reduced levels of DAF-12, EGL-15, and KIN-29. The functions of these proteins can explain some phenotypes observed in ife-4 knockout mutants. These results indicate that translation of a limited subset of mRNAs is dependent on a specific isoform of eIF4E.The most highly regulated phase of protein synthesis is initiation (8, 39). A different class of initiation factors catalyzes each of the individual steps (12). A ternary complex of eukaryotic translation initiation factor 2 (eIF2)-GTP-Met-tRNA i binds to the 40S ribosomal subunit to form the 43S initiation complex. The next step, recruitment of mRNA to the 43S initiation complex to form the 48S initiation complex, is rate limiting for initiation and requires the recognition of the 5Ј-terminal m 7 G-containing cap by eIF4E and the 3Ј-terminal poly(A) tract by the poly(A)-binding protein. The complex of eIF4E, eIF4G, and eIF4A unwinds mRNA secondary structure at the expense of ATP. Global regulation of initiation involves modulation of the canonical initiation factor activities, whereas mRNA-specific regulation is often mediated through proteins that bind cis-regulatory sequences in the 5Ј-or 3Ј-untranslated regions (UTRs) (23

SummaryCaenorhabditis elegans expresses five family members of the translation initiation factor eIF4E whose individual physiological roles are only partially understood. We report a specific role for IFE-2 in a conserved temperature-sensitive meiotic process. ife-2 deletion mutants have severe temperature-sensitive chromosome-segregation defects. Mutant germ cells contain the normal six bivalents at diakinesis at 20°C but 12 univalents at 25°C, indicating a defect in crossover formation. Analysis of chromosome pairing in ife-2 mutants at the permissive and restrictive temperatures reveals no defects. The presence of RAD-51-marked early recombination intermediates and 12 well condensed univalents indicate that IFE-2 is not essential for formation of meiotic double-strand breaks or their repair through homologous recombination but is required for crossover formation. However, RAD-51 foci in ife-2 mutants persist into inappropriately late stages of meiotic prophase at 25°C, similar to mutants defective in MSH-4/HIM-14 and MSH-5, which stabilize a critical intermediate in crossover formation. In wild-type worms, mRNAs for msh-4/him-14 and msh-5 shift from free messenger ribonucleoproteins to polysomes at 25°C but not in ife-2 mutants, suggesting that IFE-2 translationally upregulates synthesis of MSH-4/HIM-14 and MSH-5 at elevated temperatures to stabilize Holliday junctions. This is confirmed by an IFE-2-dependent increase in MSH-5 protein levels.

BackgroundChronic opioid therapy for non-malignant pain conditions has significantly increased over the last 15 years. Recently, the correlation between opioid analgesics and alternations in brain structure, such as leukoencephalopathy, axon demyelination, and white matter lesions, has been demonstrated in patients with a history of long-term use of prescription opioids. The exact mechanisms underlying the neurotoxic effect of opioids on the central nervous system are still not fully understood. We investigated the effect of chronic opioids using an animal model in which female rats were orally gavaged with 15 mg/kg of oxycodone every 24 h for 30 days. In addition we tested oxycodone, morphine and DAMGO in breast adenocarcinoma MCF7 cells, which are known to express the μ-opioid receptor.ResultsWe observed several changes in the white matter of animals treated with oxycodone: deformation of axonal tracks, reduction in size of axonal fascicles, loss of myelin basic protein and accumulation of amyloid precursor protein beta (β-APP), suggesting axonal damages by chronic oxycodone. Moreover, we demonstrated activation of pro-apoptotic machinery amid suppression of anti-apoptotic signaling in axonal tracks that correlated with activation of biomarkers of the integrated stress response (ISR) in these structures after oxycodone exposure. Using MCF7 cells, we observed induction of the ISR and pro-apoptotic signaling after opioid treatment. We showed that the ISR inhibitor, ISRIB, suppresses opioid-induced Bax and CHOP expression in MCF7 cells.ConclusionsAltogether, our data suggest that chronic opioid administration may cause neuronal degeneration by activation of the integrated stress response leading to induction of apoptotic signaling in neurons and also by promoting demyelination in CNS.

Interaction of the mRNA cap with the translational machinery is a critical and early step in the initiation of protein synthesis. To better understand this process, we determined kinetic constants for the interaction of m 7 GpppG with human eIF4E by stopped-flow fluorescence quenching in the presence of a 90-amino acid fragment of human eIF4G that contains the eIF4E-binding domain (eIF4G(557-646)). The values obtained, k on ‫؍‬ 179 ؋ 10 6 M ؊1 s ؊1 and k off ‫؍‬ 79 s ؊1 , were the same as reported previously in the absence of an eIF4G-derived peptide. We also used surface plasmon resonance to determine kinetic constants for the binding of eIF4E to eIF4G(557-646), both in the presence and absence of m 7 GpppG. The results indicated that eIF4G(557-646) binds eIF4E and eIF4E⅐m 7 GpppG at the same rate, with k on ‫؍‬ 3 ؋ 10 6 M ؊1 s ؊1 and k off ‫؍‬ 0.01 s ؊1 . Our data represent the first full kinetic description of the interaction of eIF4E with its two specific ligands. The results demonstrate that the formation of the m 7 GpppG⅐eIF4E⅐eIF4G(557-646) complex obeys a sequential, random kinetic mechanism and that there is no preferential pathway for its formation. Thus, even though eIF4G(557-646) binds eIF4E tightly, it does not increase the affinity of eIF4E for m 7 GpppG, as has been claimed in several previous publications. We did, in fact, observe increased binding to m 7 GTP-Sepharose in the presence of eIF4G(557-646), but only with recombinant eIF4E that was prepared from inclusion bodies.Recruitment of mRNA to the 43 S translation initiation complex to form the 48 S initiation complex is a highly regulated process that is rate-limiting for protein synthesis under normal circumstances (in the absence of cellular stress, virus infection, etc.) and requires eIF3, 2 poly (A)-binding protein (PABP), and the eIF4 factors (1, 2). The eIF4 factors consist of: eIF4A, a 46-kDa ATP-dependent RNA helicase; eIF4B, a 70-kDa RNAbinding and -annealing protein that stimulates eIF4A activity; eIF4H, a 25-kDa protein that also stimulates eIF4A; eIF4E, a 25-kDa cap-binding protein; and eIF4G, a 185-kDa protein that specifically binds to and co-localizes all of the other proteins involved in mRNA recruitment on the 40 S subunit. A complex of eIF4G, eIF4E, and eIF4A can be isolated from the ribosomal high-salt wash and is termed eIF4F.A critical step in mRNA recruitment is binding of the cap to eIF4E. This occurs mainly by means of -stacking, H-bonding, and ionic interactions inside the narrow cap-binding slot in the concave surface of the protein (3-5). The binding reaction is electrostatically steered and is accompanied by a partial protonation of the m 7 G moiety at N1 and hydration of the complex (6, 7). The kinetics of cap analog binding to eIF4E have been studied by stopped-flow fluorescence quenching (8 -11). Authors of the earlier studies interpreted their kinetic data as indicating a two-step association reaction, in which an initial fast binding is followed by a slow conformational rearrangement (8, 9). However, subsequen...

The alpha/beta interferon (IFN-␣/␤) response is critical for host protection against disseminated replication of many viruses, primarily due to the transcriptional upregulation of genes encoding antiviral proteins. Previously, we determined that infection of mice with Sindbis virus (SB) could be converted from asymptomatic to rapidly fatal by elimination of this response (K. Pathogenesis studies with mice deficient in receptors for alpha/beta interferon (IFN-␣/␤) and IFN-␥ have indicated that IFN-mediated innate responses are vital for the protection of mammals from infections caused by diverse virus types, e.g., lymphocytic choriomeningitis virus (4, 50), Theiler's virus (14), influenza virus (17), measles virus (32), vesicular stomatitis virus (48), and several alphaviruses, including Semliki Forest virus (23, 33), Sindbis virus (SB) (41, 42), and Venezuelan equine encephalitis virus (VEEV) (52). Our results with SB indicate that antiviral activities upregulated by IFN-␣/␤ signaling so severely restrict replication of this apathogenic virus in adult mice that it is virtually undetectable, and disease is completely prevented. However, in the absence of a functional IFN-␣/␤ system, adult mice succumb rapidly to fatal SB disease primarily as a result of systemic infection of macrophages and dendritic cells (DCs) (41). The mechanisms through which the IFN response controls alphavirus replication have not been elucidated (8,43,44). Our results have shown that PKR, but not RNase L, plays an early role in limiting virus replication in DCs; however, IFN-␣/␤ signaling in PKR-deficient animals (PKR Ϫ/Ϫ ) can protect them from fatal SB infection and any manifestations of disease. Furthermore, IFN-mediated protection of DCs or murine embryo fibroblast (MEF) cultures derived from PKR Ϫ/Ϫ mice is as potent as in control cultures (44), and virus replication in these cultures is inhibited very early after infection (43).DThe canonical IFN-␣/␤ signaling pathway involves interaction with the heterodimeric IFN-␣/␤ receptor (IFNAR1 and IFNAR2 subunits), activation of Jak1 and Tyk2 kinases, and phosphorylation and heterodimerization of the STAT1 and STAT2 transcription factors. STAT1/2 heterodimers associate with IRF9, forming the ISGF3 complex which translocates to the nucleus and binds to IFN-stimulated response elements, resulting in transcriptional upregulation of genes, some of which possess antiviral activity. The IFN-␣/␤-upregulated antiviral responses have been thought to primarily involve activation of PKR and 2Ј-5Ј oligoadenylate synthetase (2Ј-5Ј OAS) by double-stranded RNA (dsRNA). PKR-mediated antiviral effects have been attributed to phosphorylation of eukaryotic translation initiation factor 2␣ (eIF2␣) and consequent inhibition of host cell and viral translation initiation. In addition, activated PKR plays a role in apoptosis of infected cells (see, for example, reference 54). The IFN-inducible 2Ј-5Ј OAS binds dsRNA and synthesizes 2Ј-5Ј-linked oligoadenylates that activate constitutive RNase L to cleave host and vi...

Within the framework of translational initiation, one of the major points of regulation involves the recruitment of the mRNA to the 43 S pre-initiation complex. Recruitment is mediated by members of the group four initiation factors (eIF4), 1 the most prominent member of which is the cap-binding complex of eIF4F. eIF4F is a heterotrimeric protein complex consisting of the 25-kDa cap-binding protein eIF4E, the 46-kDa bi-directional RNA helicase eIF4A, and the 220-kDa scaffold protein eIF4G. eIF4F (via eIF4E) binds to the m 7 G-cap of mRNAs along with eIF4B and eIF4H, positioning eIF4A to unwind mRNA secondary structures 5Ј to the AUG start codon (reviewed in Refs. 1-7). Unwinding of the secondary structural elements presumably facilitates the binding of the 43 S preinitiation complex to the eIF4⅐mRNA complex.Both eIF4E and eIF4G are phosphoproteins (reviewed in Refs. 8 and 9). While the phosphorylation of eIF4G has not been well characterized, the site in eIF4E phosphorylated in vitro and in vivo has been identified as Ser 209 (10). eIF4E is phosphorylated at this site in vitro by the mitogen-activated protein kinase-interacting kinases 1 and 2 (Mnk1 and -2) and by protein kinase C (11-15). Phosphorylation of eIF4E and eIF4G is stimulated in vivo by insulin, progesterone, tumor necrosis factor ␣, interleukin-1␤, and phorbol ester (PMA) (15)(16)(17)(18)(19)(20). eIF4G is phosphorylated in vitro by protein kinase C, multifunctional S6 kinase, and the p21-activated protein kinase Pak2/␥-PAK (11,21,22). Two gene products of eIF4G have been identified, eIF4GI and -II. Raught et al. (23) has identified three sites that are phosphorylated in response to serum within a putative hinge region (amino acids 1035-1190) in the C terminus of human eIF4GI and one site (Ser 274 ) in the N terminus. Two unidentified serum-repressed phosphorylation sites in eIF4G were also observed. Tuazon et al. (21) showed the rate of phosphorylation/dephosphorylation of eIF4E is significantly greater in the eIF4F complex than with purified eIF4E, suggesting that regulation of eIF4E by phosphorylation occurs primarily on eIF4F. Phosphorylation of eIF4F by protein kinase C or the eIF4G subunit of eIF4F by multifunctional S6 kinase stimulates translation in a reconstituted protein-synthesizing system dependent on eIF4F (22). However, overall the role of phosphorylation of 4E and 4G is not well understood.Mnk1 and -2 are activated by the MAP kinases Erk1 and -2 and p38 (18,19). Activation of Mnk occurs upon phosphorylation at two sites, Thr 197 and Thr 202 . An additional residue, Thr332 in mouse Mnk2 and Thr344 in human Mnk1, has been identified as a phosphorylation site for Erk2 (14,15,24). Ser 22 has also been shown to be phosphorylated in vitro, but the protein kinase has not been identified (24). Support for phosphorylation of eIF4E by Mnk1 and -2 in vivo comes from studies utilizing kinase-inactive and constitutively active mutants of Mnk1 (15). Kinase-inactive mutants of Mnk1 expressed in 293 cells inhibit the mitogen-induced phosphorylatio...

BackgroundOxycodone is an opioid that is prescribed to treat multiple types of pain, especially when other opioids are ineffective. Unfortunately, similar to other opioids, repetitive oxycodone administration has the potential to lead to development of analgesic tolerance, withdrawal, and addiction. Studies demonstrate that chronic opioid exposure, including oxycodone, alters gene expression profiles and that these changes contribute to opioid-induced analgesic effect, tolerance and dependence. However, very little is known about opioids altering the translational machinery of the central nervous system. Considering that opioids induce clinically significant levels of hypoxia, increase intracellular Ca2+ levels, and induce the production of nitric oxide and extracellular glutamate transmission, we hypothesize that opioids also trigger a defensive mechanism called the integrated stress response (ISR). The key event in the ISR activation, regardless of the trigger, is phosphorylation of translation initiation factor 2 alpha (eIF2α), which modulates expression and translational activation of specific mRNAs important for adaptation to stress. To test this hypothesis, we used an animal model in which female rats were orally gavaged with 15 mg/kg of oxycodone every 24 h for 30 days.ResultsWe demonstrated increased levels of hsp70 and BiP expression as well as phosphorylation of eIF2α in various rat brain areas after oxycodone administration. Polysomal analysis indicated oxycodone-induced translational stimulation of ATF4 and PDGFRα mRNAs, which have previously been shown to depend on the eIF2α kinase activation. Moreover, using breast adenocarcinoma MCF7 cells, which are known to express the μ-opioid receptor, we observed induction of the ISR pathway after one 24-h treatment with oxycodone.ConclusionsThe combined in vivo and in vitro data suggest that prolonged opioid treatment induces the integrated stress response in the central nervous system; it modulates translational machinery in favor of specific mRNA and this may contribute to the drug-induced changes in neuronal plasticity.