Increase in eukaryotic initiation factor 2B activity following fertilization reflects changes in redox potential.

One of the factors involved in the postfertilization activation of protein synthesis in the sea urchin, Strongylocentrotus purpuratus, is the activation of eIF-2B, the initiation factor responsible for guanine nucleotide exchange on eIF-2. Cell-free translation systems from unfertilized eggs are stimulated by added eIF-2B, although this dependency is rapidly lost in translation systems prepared at various times following fertilization. Cell-free translation systems prepared from unfertilized eggs show significantly lower eIF-2B activities than those prepared from 2-h embryos. However, the provision of an NADPH regeneration system significantly stimulates eIF-2B activity in egg extracts and, in addition, stimulates both binding of initiator tRNA to the small ribosomal subunit and protein synthetic activity. These data suggest that the activation of eIF-2B following fertilization reflects the fertilization-induced increase in NADPH levels.     

MicroRNA‐383 inhibits doxorubicin resistance in hepatocellular carcinoma by targeting eukaryotic translation initiation factor 5A2

Drug resistance occurs commonly in cancers, especially in hepatocellular carcinoma (HCC). Accumulating evidence has demonstrated that microRNAs (miRNAs) play a vital role in tumour chemoresistance. However, little is known about the role of miR‐383 in HCC chemoresistance. In the present study, RT‐PCR and western blotting were used to identify the expression profile of miR‐383 and eukaryotic translation initiation factor 5A2 (EIF5A2). The bioinformatics website Targetscan was used to predict the target genes of miR‐383. In vitro and in vivo loss‐ and gain‐of‐function studies were performed to reveal the effects and potential mechanism of the miR‐383/EIF5A2 axis in chemoresistance of HCC cells. The expression level of miR‐383 correlated negatively with doxorubicin (Dox) sensitivity. Overexpression of miR‐383 promoted HCC cells to undergo Dox‐induced cytotoxicity and apoptosis, whereas miR‐383 knockdown had the opposite effects. EIF5A2 was predicted as a target gene of miR‐383. EIF5A2 knockdown sensitized HCC cells to Dox. Moreover, miR‐383 inhibition‐mediated HCC Dox resistance could be reversed by silencing EIF5A2. Finally, we demonstrated that miR‐383 inhibition could enhance Dox sensitivity by targeting EIF5A2 in vivo. The results indicated that miR‐383 inhibited Dox resistance in HCC cells by targeting EIF5A2. Targeting the miR‐383/EIF5A2 axis might help to alleviate the chemoresistance of HCC cells.     

Effect of starvation and diabetes on the activity of the eukaryotic initiation factor eIF-2 in rat skeletal muscle

The ability of the initiation factor eIF-2 in skeletal muscle extracts to form ternary initiation complexes ([Met-tRNA(f).eIF-2.GDP]) is decreased by either starvation or diabetes. These conditions also impair the ability of muscle extracts to dissociate [eIF-2.GDP], suggesting inhibition of the guanine nucleotide exchange reaction essential for eIF-2 recycling. We could not, however, detect any change in the phosphorylation state of the alpha subunit of eIF-2. This suggests that eIF-2 activity may be regulated in this system by a mechanism not involving its phosphorylation.     

Granzyme B inhibits vaccinia virus production through proteolytic cleavage of eukaryotic initiation factor 4 gamma 3

Cytotoxic T lymphocytes (CTLs) are the major killer of virus-infected cells. Granzyme B (GrB) from CTLs induces apoptosis in target cells by cleavage and activation of substrates like caspase-3 and Bid. However, while undergoing apoptosis, cells are still capable of producing infectious viruses unless a mechanism exists to specifically inhibit viral production. Using proteomic approaches, we identified a novel GrB target that plays a major role in protein synthesis: eukaryotic initiation factor 4 gamma 3 (eIF4G3). We hypothesized a novel role for GrB in translation of viral proteins by targeting eIF4G3, and showed that GrB cleaves eIF4G3 specifically at the IESD(1408)S sequence. Both GrB and human CTL treatment resulted in degradation of eIF4G3 and reduced rates of translation. When Jurkat cells infected with vaccinia virus were treated with GrB, there was a halt in viral protein synthesis and a decrease in production of infectious new virions. The GrB-induced inhibition of viral translation was independent of the activation of caspases, as inhibition of protein synthesis still occurred with addition of the pan-caspase inhibitor zVAD-fmk. This demonstrated for the first time that GrB prevents the production of infectious vaccinia virus by targeting the host translational machinery.     

Nucleophosmin interacts with and inhibits the catalytic function of eukaryotic initiation factor 2 kinase PKR

In normal cells the protein kinase PKR effects apoptosis in response to various extra and intracellular cues and can also function to suppress the neoplastic phenotype. Because most neoplastic cells are resistant to certain apoptotic cues, we reasoned that an early molecular event in carcinogenesis or leukemogenesis might be the inactivation of PKR by expression or activation of intracellular PKR inhibitors. Seeking novel PKR-modulating proteins we report here that nucleophosmin (NPM), a protein frequently overexpressed in a variety of human malignancies, binds to PKR, and inhibits its activation. Co-immunoprecipitation and in vitro binding experiments showed that NPM associated with PKR. Kinase assays demonstrated that recombinant NPM inhibited PKR activation in a dose-dependent manner. In addition, purified recombinant NPM was phosphorylated by activated PKR. Most importantly, overexpression of NPM suppressed PKR activity, enhanced protein synthesis, and inhibited apoptosis. Lymphoblasts from patients with Fanconi anemia (FA) expressed low levels of NPM, which correlated with high ground-state activation of PKR and cellular hypersensitivity to apoptotic cues, but enforced expression of NPM in these mutant cells reduced aberrant apoptotic responses. Inhibition of PKR by NPM may be one mechanism by which neoplastic clones evolve in sporadic malignancies and in neoplastic cells arising in the context of the cancer predisposition syndrome, Fanconi anemia.     

Interaction of eukaryotic initiation factor eIF-4B with a picornavirus internal translation initiation site.

We studied the interaction of cellular proteins with the internal ribosome entry site (IRES) of foot-and-mouth disease virus by UV cross-linking and observed specific binding of a 80-kDa protein contained in cytosolic HeLa cell extract and in rabbit reticulocyte lysate. Binding of the protein was dependent on the presence of ATP. Immunoprecipitation with eIF-4B antiserum revealed that the protein is identical to the initiation factor eIF-4B. Deletions in the 3' part, but not in the 5' part, of the IRES interfered with UV cross-linking, indicating that the binding site of eIF-4B is located close to the end of the element. Attempts to separate ribosome-associated from non-ribosome-associated protein fractions of cytosolic cell extracts led to the loss of cross-linking activity. This finding suggests that additional protein factors contribute to this interaction of eIF-4B with the IRES of foot-and-mouth disease virus.     

Postribosomal complexes containing eukaryotic initiation factor eIF-2

Eukaryotic initiation factors are found in the postribosomal supernatant as well as bound to the 40S ribosomal subunits. We have analyzed the factor activities from the supernatant by means of zonal centrifugation followed by Sepharose-heparin affinity chromatography. They exist both as free factors, sedimenting in a broad range from 4 to 7S, and complexed with other protein(s) with a sedimentation value of 16–20S. This complexed fraction contains besides eIF-2 another activity which exhibits a profound stimulation on amino acid incorporation in crude lysates and appears to counteract the heme-regulated inhibitor.Abbreviations eIF-2, eIF-3, eIF-4A and eIF-4B are eukaryotic initiation factors, see FEBS Letters 76, 1-10 (1977).     

The activity of eukaryotic initiation factor eIF-2 in ternary complex formation with GTP and Met-tRNA.

Ternary complex formation between [3H]Met-tRNAf, [14C]H3-eIF-2, and GTP was measured on nitrocellulose filters. It is shown that [3H]Met-tRNAf and [14C]H3-eIF-2 are present on the filter in equimolar amounts when ATP, creatine phosphate, and creatine kinase are included in the reaction mixture. Under these conditions the factor is 100% active. With small amounts of factor significant losses occur due to adsorption to the wall of the reaction vessels, resulting in seemingly low activities of eIF-2. These losses can be prevented by the presence of "stimulatory" proteins, which enhance the recovery of both [3H]Met-tRNAf and [14C]H3-eIF-2 on the filter but do not alter their ratio.     

Identification and characterization of eukaryotic initiation factor 5A-2.

The phylogenetically conserved eukaryotic translation initiation factor 5A (eIF5A) is the only known cellular protein to contain the post-translationally derived amino acid hypusine [Nepsilon-(4-amino-2-hydroxybutyl)lysine]. Both eIF5A and its hypusine modification are essential for sustained cell proliferation. Normally only one eIF5A protein is expressed in human cells. Recently, we identified a second human EIF5A gene that would encode an isoform (eIF5A-2) of 84% sequence identity. Overexpression of eIF5A-2 mRNA in certain human cancer cells, in contrast to weak normal expression limited to human testis and brain, suggests EIF5A2 as a potential oncogene. However, eIF5A-2 protein has not been described in human or mammalian cells heretofore. Here, we describe the identification of eIF5A-2 protein in human colorectal and ovarian cancer lines, SW-480 and UACC-1598, that overexpress eIF5A-2 mRNAs. Functional characterization of the human isoforms revealed that either human EIF5A gene can complement growth of a yeast strain in which the yeast EIF5A genes were disrupted. This indicates functional similarity of the human isoforms in yeast and suggests that eIF5A-2 has an important role in eukaryotic cell survival similar to that of the ubiquitous eIF5A-1. Detectable structural differences were also noted, including lack of immunological cross-reactivity, formation of different complexes with deoxyhypusine synthase, and Km values (1.5 +/- 0.2 vs. 8.3 +/- 1.4 microm for eIF5A-1 and -2, respectively) as substrates for deoxyhypusine synthase in vitro. These physical characteristics and distinct amino acid sequences in the C-terminal domain together with differences in gene expression patterns imply differentiated, tissue-specific functions of the eIF5A-2 isoform in the mammalian organism and in cancer.     

Reduced amino acid availability inhibits muscle protein synthesis and decreases activity of initiation factor eIF2B

We have examined the effect of a hemodialysis-induced 40% reduction in plasma amino acid concentrations on rates of muscle protein synthesis and breakdown in normal swine. Muscle protein kinetics were measured by tracer methodology using [(2)H(5)]phenylalanine and [1-(13)C]leucine and analysis of femoral arterial and venous samples and tissue biopsies. Net amino acid release by muscle was accelerated during dialysis. Phenylalanine utilization for muscle protein synthesis was reduced from the basal value of 45 +/- 8 to 25 +/- 6 nmol x min(-1) x 100 ml leg(-1) between 30 and 60 min after start of dialysis and was stimulated when amino acids were replaced while dialysis continued. Muscle protein breakdown was unchanged. The signal for changes in synthesis appeared to be changes in plasma amino acid concentrations, as intramuscular concentrations remained constant throughout. The changes in muscle protein synthesis were accompanied by a reduction or stimulation, respectively, in the guanine nucleotide exchange activity of eukaryotic initiation factor (eIF)2B following hypoaminoacidemia vs. amino acid replacement. We conclude that a reduction in plasma amino acid concentrations below the normal basal value signals an inhibition of muscle protein synthesis and that corresponding changes in eIF2B activity suggest a possible role in mediating the response.     

Mutations causing childhood ataxia with central nervous system hypomyelination reduce eukaryotic initiation factor 2B complex formation and activity

Childhood ataxia with central nervous system hypomyelination (CACH), or vanishing white matter leukoencephalopathy (VWM), is a fatal brain disorder caused by mutations in eukaryotic initiation factor 2B (eIF2B). eIF2B is essential for protein synthesis and regulates translation in response to cellular stresses. We performed mutagenesis to introduce changes equivalent to 12 human CACH/VWM mutations in three subunits of the equivalent factor from yeast (Saccharomyces cerevisiae) and analyzed effects on cell growth, translation, and gene expression in response to stresses. None of the mutations is lethal or temperature sensitive, but almost all confer some defect in eIF2B function significant enough to alter growth or gene expression under normal or stress conditions. Biochemical analyses indicate that mutations analyzed in eIF2Balpha and -epsilon reduce the steady-state level of the affected subunit, while the most severe mutant tested, eIF2Bbeta(V341D) (human eIF2B(betaV316D)), forms complexes with reduced stability and lower eIF2B activity. eIF2Bdelta is excluded from eIF2Bbeta(V341D) complexes. eIF2B(betav341D) function can be rescued by overexpression of eIF2Bdelta alone. Our findings imply CACH/VWM mutations do not specifically impair responses to eIF2 phosphorylation, but instead cause protein structure defects that impair eIF2B activity. Altered protein folding is characteristic of other diseases, including cystic fibrosis and neurodegenerative disorders such as Huntington, Alzheimer's, and prion diseases.     

Relationship of eukaryotic initiation factor 3 to poliovirus-induced p220 cleavage activity.

The cleavage of the p220 subunit of eukaryotic initiation factor 4F (eIF-4F) that is induced by the poliovirus protease 2A has been shown previously to require another translation initiation factor, eIF-3. The role of eIF-3 in this cleavage reaction, however, is not known. An antiserum was raised against human eIF-3 and used to analyze the eIF-3 subunit composition in poliovirus-infected and uninfected HeLa cells and after incubation of eIF-3 in vitro with viral 2A protease. No evidence for 2Apro-dependent cleavage of any eIF-3 subunit was detected. Infected cells contain an activity that catalyzes the cleavage of p220 to a specific set of cleavage products. This activity is thought to be an activated form of a latent cellular protease. The p220-specific cleavage activity was partially purified. It was resolved from eIF-3 by both gel filtration and anion-exchange chromatography. Neither intact eIF-3 nor any detectable subunits of eIF-3 were found to copurify with the p220-specific cleavage activity. The latter activity behaves as a protein of 55,000 to 60,000 molecular weight and is inhibited by alkylating agents and metals, which indicates the presence of essential thiol groups. When this activity was incubated with partially purified p220, cleavage occurred only in the presence of eIF-3. Thus, eIF-3 appears to play a role in the p220 cleavage cascade which is subsequent to the 2Apro-induced activation of the p220-specific protease.     

The use of [14C]eukaryotic initiation factor 2 to measure the endogenous pool size of eukaryotic initiation factor 2 in rabbit reticulocyte lysate.

[14C]Eukaryotic initiation factor 2 (eIF-2), obtained by reductive methylation of the purified initiation factor, was shown to be active in the unfractionated reticulocyte lysate. This allowed a direct measurement of the endogenous pool size of eIF-2 in rabbit reticulocyte lysate according to the principle of isotope dilution. A value of 20 to 30 pmol/ml of lysate was obtained. Although translational inhibition resulting from hemin deficiency appears to be characterized by a change from catalytic to stoichiometric utilization of eIF-2, the pool size of eIF-2 is too small to account for the normal period of protein synthesis before the onset of translation inhibition. This suggests, therefore, that additional events to eIF-2 alpha phosphorylation may be required for translational inhibition.     

Phosphorothioated binary complex of eukaryotic initiation factor eIF-2 and GDP inhibits protein synthesis in hemin-supplemented reticulocyte lysates

The rabbit reticulocyte heme-regulated eIF-2 alpha kinase (HRI) utilizes adenosine-5'-0-(3-thiotriphosphate) (ATP-gamma-S) as a substrate for its autophosphorylation and activation, and for the phosphorylation of eIF-2. The phosphorothioated binary complex [eIF-2(alpha-[35S]P) . GDP], interacted with the reticulocyte reversing factor (RF) in in vitro assays, and inhibited the ability of RF to catalyze GDP exchange from (eIF-2 . [3H]GDP) complexes. The phosphorothioate residue in the binary complex was resistant to phosphatase action under protein synthesis conditions. eIF-2(alpha-[35S]P) . GDP inhibited protein synthesis in hemin-supplemented lysates with biphasic kinetics, but had no effect on protein synthesis in heme-deficient lysates. The data reported here indicate that phosphorylation of eIF-2 . GDP alone, through the ability of eIF-2(alpha-P) . GDP to bind and sequester RF, is sufficient to inhibit protein chain initiation in the reticulocyte lysate.     

Identification of intersubunit domain interactions within eukaryotic initiation factor (eIF) 2B, the nucleotide exchange factor for translation initiation

In eukaryotic translation initiation, eIF2B is the guanine nucleotide exchange factor (GEF) required for reactivation of the G protein eIF2 between rounds of protein synthesis initiation. eIF2B is unusually complex with five subunits (α-ε) necessary for GEF activity and its control by phosphorylation of eIF2α. In addition, inherited mutations in eIF2B cause a fatal leukoencephalopathy. Here we describe experiments examining domains of eIF2Bγ and ε that both share sequence and predicted tertiary structure similarity with a family of phospho-hexose sugar nucleotide pyrophosphorylases. Firstly, using a genetic approach, we find no evidence to support a significant role for a potential nucleotide-binding region within the pyrophosphorylase-like domain (PLD) of eIF2Bε for nucleotide exchange. These findings are at odds with one mechanism for nucleotide exchange proposed previously. By using a series of constructs and a co-expression and precipitation strategy, we find that the eIF2Bε and -γ PLDs and a shared second domain predicted to form a left-handed β helix are all critical for interprotein interactions between eIF2B subunits necessary for eIF2B complex formation. We have identified extensive interactions between the PLDs and left-handed β helix domains that form the eIF2Bγε subcomplex and propose a model for domain interactions between eIF2B subunits.     

A GDP/GTP exchange factor essential for eukaryotic initiation factor 2 cycling in Ehrlich ascites tumor cells and its regulation by eukaryotic initiation factor 2 phosphorylation

Formation of the ternary complex Met-tRNAi X eukaryotic initiation factor (eIF) 2 X GTP from eIF-2 X GDP requires exchange of GDP for GTP. However, at physiological Mg2+ concentrations, GDP is released from eIF-2 exceedingly slowly (Clemens, M.J., Pain, V.M., Wong, S.T., and Henshaw, E.C. (1982) Nature (Lond.) 296, 93-95). However, GDP is released rapidly from impure eIF-2 preparations, indicating the presence of a GDP/GTP exchange factor. We have now purified this factor from Ehrlich cells and refer to it as GEF. CM-Sephadex chromatography of ribosomal salt wash separated two peaks of eIF-2 activity. GEF was found in association with eIF-2 in the first peak and co-purified with eIF-2 under low salt conditions. It was separated from eIF-2 in high salt buffers and further purified on hydroxylapatite and phosphocellulose. Gel electrophoresis of our purest preparations showed major bands at 85, 67, 52, 37, 27, and 21 kDa. Purified GEF increased the rate of exchange of [32P] GDP for unlabeled GDP 25-fold but did not function with phosphorylated eIF-2 (alpha subunit). The factor also stimulated markedly the rate of ternary complex formation using eIF-2 X GDP as substrate with GTP and Met-tRNAi but not using phosphorylated eIF-2 X GDP as substrate. eIF-2 is released from the 80 S initiation complex with hydrolysis of GTP. If eIF-2 X GDP is actually the complex released, then GEF is absolutely required for eIF-2 to cycle and it is therefore a new eukaryotic initiation factor. Furthermore, the inability of GEF to utilize eIF-2 (alpha P) X GDP explains how phosphorylation of eIF-2 can inhibit polypeptide chain initiation.