scholarly journals Regulation of Antioxidant Metabolism by Translation Initiation Factor 2α

2001 ◽  
Vol 152 (5) ◽  
pp. 997-1006 ◽  
Author(s):  
Shirlee Tan ◽  
Nikunj Somia ◽  
Pamela Maher ◽  
David Schubert
Keyword(s):  
Oxidative Stress ◽  
Cell Death ◽  
Nerve Cell ◽  
Translation Initiation ◽  
Cell Model ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Expression Cloning ◽  
Wild Type ◽  
Antisense Gene

Oxidative stress and highly specific decreases in glutathione (GSH) are associated with nerve cell death in Parkinson's disease. Using an experimental nerve cell model for oxidative stress and an expression cloning strategy, a gene involved in oxidative stress–induced programmed cell death was identified which both mediates the cell death program and regulates GSH levels. Two stress-resistant clones were isolated which contain antisense gene fragments of the translation initiation factor (eIF)2α and express a low amount of eIF2α. Sensitivity is restored when the clones are transfected with full-length eIF2α; transfection of wild-type cells with the truncated eIF2α gene confers resistance. The phosphorylation of eIF2α also results in resistance to oxidative stress. In wild-type cells, oxidative stress results in rapid GSH depletion, a large increase in peroxide levels, and an influx of Ca2+. In contrast, the resistant clones maintain high GSH levels and show no elevation in peroxides or Ca2+ when stressed, and the GSH synthetic enzyme γ-glutamyl cysteine synthetase (γGCS) is elevated. The change in γGCS is regulated by a translational mechanism. Therefore, eIF2α is a critical regulatory factor in the response of nerve cells to oxidative stress and in the control of the major intracellular antioxidant, GSH, and may play a central role in the many neurodegenerative diseases associated with oxidative stress.

scholarly journals Disparate Phenotypes Resulting from Mutations of a Single Histidine in Switch II of Geobacillus stearothermophilus Translation Initiation Factor IF2

2020 ◽  
Vol 21 (3) ◽  
pp. 735
Author(s):  
Jerneja Tomsic ◽  
Arianna Smorlesi ◽  
Enrico Caserta ◽  
Anna Maria Giuliodori ◽  
Cynthia L. Pon ◽  
...  
Keyword(s):  
Amino Acids ◽  
Cell Viability ◽  
Translation Initiation ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Geobacillus Stearothermophilus ◽  
Wild Type ◽  
Initiation Complex ◽  
Structural Alterations

The conserved Histidine 301 in switch II of Geobacillus stearothermophilus IF2 G2 domain was substituted with Ser, Gln, Arg, Leu and Tyr to generate mutants displaying different phenotypes. Overexpression of IF2H301S, IF2H301L and IF2H301Y in cells expressing wtIF2, unlike IF2H301Q and IF2H301R, caused a dominant lethal phenotype, inhibiting in vivo translation and drastically reducing cell viability. All mutants bound GTP but, except for IF2H301Q, were inactive in ribosome-dependent GTPase for different reasons. All mutants promoted 30S initiation complex (30S IC) formation with wild type (wt) efficiency but upon 30S IC association with the 50S subunit, the fMet-tRNA reacted with puromycin to different extents depending upon the IF2 mutant present in the complex (wtIF2 ≥ to IF2H301Q > IF2H301R >>> IF2H301S, IF2H301L and IF2H301Y) whereas only fMet-tRNA 30S-bound with IF2H301Q retained some ability to form initiation dipeptide fMet-Phe. Unlike wtIF2, all mutants, regardless of their ability to hydrolyze GTP, displayed higher affinity for the ribosome and failed to dissociate from the ribosomes upon 50S docking to 30S IC. We conclude that different amino acids substitutions of His301 cause different structural alterations of the factor, resulting in disparate phenotypes with no direct correlation existing between GTPase inactivation and IF2 failure to dissociate from ribosomes.

scholarly journals TgIF2K-B is an eIF2α kinase in Toxoplasma gondii that responds to oxidative stress and optimizes pathogenicity

2020 ◽  
Author(s):  
Leonardo Augusto ◽  
Jennifer Martynowicz ◽  
Parth H. Amin ◽  
Kenneth R. Carlson ◽  
Ronald C. Wek ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Stress Response ◽  
Toxoplasma Gondii ◽  
Translation Initiation ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Eukaryotic Translation ◽  
Oxidative Stresses ◽  
Oxidative Balance

AbstractToxoplasma gondii is an obligate intracellular parasite that persists in its vertebrate hosts in the form of dormant tissue cysts, which facilitate transmission through predation. The parasite must strike a balance that allows it to disseminate throughout its host without killing it, which requires the ability to properly counter host cell defenses. For example, oxidative stress encountered by Toxoplasma is suggested to impair parasite replication and dissemination. However, the strategies by which Toxoplasma mitigates oxidative stress are not completely clear. Among eukaryotes, environmental stresses induce the integrated stress response via phosphorylation of a translation initiation factor, eIF2. Here, we show that the Toxoplasma eIF2 kinase, TgIF2K-B, is activated in response to oxidative stress and affords protection. Knockout of TgIF2K-B, Δtgif2k-b, disrupted parasite responses to oxidative stresses and enhanced replication, diminishing the ability of the parasite to differentiate into tissue cysts. In addition, parasites lacking TgIF2K-B exhibited resistance to activated macrophages and showed greater virulence in an in vivo model of infection. Our results establish that TgIF2K-B is essential for Toxoplasma responses to oxidative stress, which are important for the parasite’s ability to establish persistent infection in its host.IMPORTANCEToxoplasma gondii is a single-celled parasite that infects nucleated cells of warm-blooded vertebrates, including one-third of the human population. The parasites are not cleared by the immune response and persist in the host by converting into a latent tissue cyst form. Development of tissue cysts can be triggered by cellular stresses, which activate a family of TgIF2 kinases to phosphorylate the eukaryotic translation initiation factor, TgIF2α. Here, we establish that the TgIF2 kinase TgIF2K-B is activated by oxidative stress and is critical for maintaining oxidative balance in the parasite. Depletion of TgIF2K-B alters gene expression, leading to accelerated growth and a diminished ability to convert into tissue cysts. This study establishes that TgIF2K-B is essential for the parasite’s oxidative stress response and its ability to persist in the host as a latent infection.

scholarly journals The A1 x U72 base pair conserved in eukaryotic initiator tRNAs is important specifically for binding to the eukaryotic translation initiation factor eIF2.

1996 ◽  
Vol 16 (8) ◽  
pp. 4248-4256 ◽  
Author(s):  
D Farruggio ◽  
J Chaudhuri ◽  
U Maitra ◽  
U L RajBhandary
Keyword(s):  
Translation Initiation ◽  
Base Pair ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Trna Genes ◽  
Wild Type ◽  
80S Ribosome ◽  
Subsequent Formation ◽  
Initiator Trna

The formation of a specific ternary complex between eukaryotic initiation factor 2 (eIF2), the initiator methionyl-tRNA (Met-tRNA), and GTP is a critical step in translation initiation in the cytoplasmic protein-synthesizing system of eukaryotes. We show that the A1 x U72 base pair conserved at the end of the acceptor stem in eukaryotic and archaebacterial initiator methionine tRNAs plays an important role in this interaction. We changed the A1 x U72 base pair of the human initiator tRNA to G1 x C72 and expressed the wild-type and mutant tRNA genes in the yeast Saccharomyces cerevisiae by using constructs previously developed in our laboratory for expression of the human initiator tRNA gene in yeasts. We show that both the wild-type and mutant human initiator tRNAs are aminoacylated well in vivo. We have isolated the wild-type and mutant human initiator tRNAs in substantially pure form, free of the yeast initiator tRNA, and have analyzed their properties in vitro. The G1 x C72 mutation affects specifically the binding affinity of eIF2 for the initiator tRNA. It has no effect on the subsequent formation of 40S or 80S ribosome initiator Met-tRNA-AUG initiation complexes in vitro or on the puromycin reactivity of the Met-tRNA in the 80S initiation complex.

scholarly journals Regulation of Protein Synthesis by Hypoxia via Activation of the Endoplasmic Reticulum Kinase PERK and Phosphorylation of the Translation Initiation Factor eIF2α

2002 ◽  
Vol 22 (21) ◽  
pp. 7405-7416 ◽  
Author(s):  
Constantinos Koumenis ◽  
Christine Naczki ◽  
Marianne Koritzinsky ◽  
Sally Rastani ◽  
Alan Diehl ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Protein Synthesis ◽  
Translation Initiation ◽  
Prolonged Exposure ◽  
Response To Therapy ◽  
Dominant Negative ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Hypoxic Stress ◽  
Wild Type

ABSTRACT Hypoxia profoundly influences tumor development and response to therapy. While progress has been made in identifying individual gene products whose synthesis is altered under hypoxia, little is known about the mechanism by which hypoxia induces a global downregulation of protein synthesis. A critical step in the regulation of protein synthesis in response to stress is the phosphorylation of translation initiation factor eIF2α on Ser51, which leads to inhibition of new protein synthesis. Here we report that exposure of human diploid fibroblasts and transformed cells to hypoxia led to phosphorylation of eIF2α, a modification that was readily reversed upon reoxygenation. Expression of a transdominant, nonphosphorylatable mutant allele of eIF2α attenuated the repression of protein synthesis under hypoxia. The endoplasmic reticulum (ER)-resident eIF2α kinase PERK was hyperphosphorylated upon hypoxic stress, and overexpression of wild-type PERK increased the levels of hypoxia-induced phosphorylation of eIF2α. Cells stably expressing a dominant-negative PERK allele and mouse embryonic fibroblasts with a homozygous deletion of PERK exhibited attenuated phosphorylation of eIF2α and reduced inhibition of protein synthesis in response to hypoxia. PERK−/− mouse embryo fibroblasts failed to phosphorylate eIF2α and exhibited lower survival after prolonged exposure to hypoxia than did wild-type fibroblasts. These results indicate that adaptation of cells to hypoxic stress requires activation of PERK and phosphorylation of eIF2α and suggest that the mechanism of hypoxia-induced translational attenuation may be linked to ER stress and the unfolded-protein response.

scholarly journals DAP-5, a novel homolog of eukaryotic translation initiation factor 4G isolated as a putative modulator of gamma interferon-induced programmed cell death.

1997 ◽  
Vol 17 (3) ◽  
pp. 1615-1625 ◽  
Author(s):  
N Levy-Strumpf ◽  
L P Deiss ◽  
H Berissi ◽  
A Kimchi
Keyword(s):  
Cell Death ◽  
Programmed Cell Death ◽  
Translation Initiation ◽  
Hela Cells ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Terminal Part ◽  
Ifn Gamma ◽  
Eukaryotic Translation ◽  
Eukaryotic Translation Initiation

A functional approach to gene cloning was applied to HeLa cells in an attempt to isolate cDNA fragments which convey resistance to gamma interferon (IFN-gamma)-induced programmed cell death. One of the rescued cDNAs, described in this work, was a fragment of a novel gene, named DAP-5. Analysis of a DAP-5 full-length cDNA clone revealed that it codes for a 97-kDa protein that is highly homologous to eukaryotic translation initiation factor 4G (eIF4G, also known as p220). According to its deduced amino acid sequence, this novel protein lacks the N-terminal region of eIF4G responsible for association with the cap binding protein eIF4E. The N-terminal part of DAP-5 has 39% identity and 63% similarity to the central region of mammalian p220. Its C-terminal part is less homologous to the corresponding region of p220, suggesting that it may possess unique functional properties. The rescued DAP-5 cDNA fragment which conveyed resistance to IFN-gamma-induced cell death was expressed from the vector in the sense orientation. Intriguingly, it comprised part of the coding region which corresponds to the less conserved C-terminal part of DAP-5 and directed the synthesis of a 28-kDa miniprotein. The miniprotein exerted a dual effect on HeLa cells. Low levels of expression protected the cells from IFN-gamma-induced programmed cell death, while high levels of expression were not compatible with continuous cell growth. The relevance of DAP-5 protein to possible changes in a cell's translational machinery during programmed cell death and growth arrest is discussed.

scholarly journals Protein Kinase B Localization and Activation Differentially Affect S6 Kinase 1 Activity and Eukaryotic Translation Initiation Factor 4E-Binding Protein 1 Phosphorylation

1999 ◽  
Vol 19 (6) ◽  
pp. 4525-4534 ◽  
Author(s):  
Almut Dufner ◽  
Mirjana Andjelkovic ◽  
Boudewijn M. T. Burgering ◽  
Brian A. Hemmings ◽  
George Thomas
Keyword(s):  
Translation Initiation ◽  
Protein Kinase B ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Wild Type ◽  
S6 Kinase ◽  
Eukaryotic Translation ◽  
Highly Active ◽  
Eukaryotic Translation Initiation ◽  
Gsk 3Β

ABSTRACT Recent studies indicate that phosphatidylinositide-3OH kinase (PI3K)-induced S6 kinase (S6K1) activation is mediated by protein kinase B (PKB). Support for this hypothesis has largely relied on results obtained with highly active, constitutively membrane-localized alleles of wild-type PKB, whose activity is independent of PI3K. Here we set out to examine the importance of PKB signaling in S6K1 activation. In parallel, glycogen synthase kinase 3β (GSK-3β) inactivation and eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1) phosphorylation were monitored as markers of the rapamycin-insensitive and -sensitive branches of the PI3K signaling pathway, respectively. The results demonstrate that two activated PKBα mutants, whose basal activity is equivalent to that of insulin-induced wild-type PKB, inhibit GSK-3β to the same extent as a highly active, constitutively membrane-targeted wild-type PKB allele. However, of these two mutants, only the constitutively membrane-targeted allele of PKB induces S6K1 activation. Furthermore, an interfering mutant of PKB, which blocks insulin-induced PKB activation and GSK-3β inactivation, has no effect on S6K1 activation. Surprisingly, all the activated PKB mutants, regardless of constitutive membrane localization, induce 4E-BP1 phosphorylation and the interfering PKB mutant blocks insulin-induced 4E-BP1 phosphorylation. The results demonstrate that PKB mediates S6K1 activation only as a function of constitutive membrane localization, whereas the activation of PKB appears both necessary and sufficient to induce 4E-BP1 phosphorylation independently of its intracellular location.

scholarly journals Mutational analysis of the Prt1 protein subunit of yeast translation initiation factor 3.

1995 ◽  
Vol 15 (8) ◽  
pp. 4525-4535 ◽  
Author(s):  
D R Evans ◽  
C Rasmussen ◽  
P J Hanic-Joyce ◽  
G C Johnston ◽  
R A Singer ◽  
...  
Keyword(s):  
Translation Initiation ◽  
Mutational Analysis ◽  
Dominant Negative ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Temperature Sensitive ◽  
Gene Transcript ◽  
Missense Mutations ◽  
Wild Type ◽  
Protein Subunit

The Saccharomyces cerevisiae PRT1 gene product Prt1p is a component of translation initiation factor eIF-3, and mutations in PRT1 inhibit translation initiation. We have investigated structural and functional aspects of Prt1p and its gene. Transcript analysis and deletion of the PRT1 5' end revealed that translation of PRT1 mRNA is probably initiated at the second in-frame ATG in the open reading frame. The amino acid changes encoded by six independent temperature-sensitive prt1 mutant alleles were found to be distributed throughout the central and C-terminal regions of Prt1p. The temperature sensitivity of each mutant allele was due to a single missense mutation, except for the prt1-2 allele, in which two missense mutations were required. In-frame deletion of an N-terminal region of Prt1p generated a novel, dominant-negative form of Prt1p that inhibits translation initiation even in the presence of wild-type Prt1p. Subcellular fractionation suggested that the dominant-negative Prt1p competes with wild-type Prt1p for association with a component of large Prt1p complexes and as a result inhibits the binding of wild-type Prt1p to the 40S ribosome.

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