scholarly journals Post-translational modification of the protein-synthesis initiation factor eIF-4D by spermidine in rat hepatoma cells

1986 ◽  
Vol 239 (2) ◽  
pp. 379-386 ◽  
Author(s):  
E W Gerner ◽  
P S Mamont ◽  
A Bernhardt ◽  
M Siat
Keyword(s):  
Growth Rates ◽  
Exponential Phase ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Polyamine Metabolism ◽  
Synthesis Rate ◽  
Post Translational Modification ◽  
Irreversible Inhibitor ◽  
Rat Hepatoma ◽  
In Cells

The rates of synthesis and turnover of the rare amino acid hypusine [N6-(4-amino-2-hydroxybutyl)-2,6-diaminohexanoic acid] in protein were studied in relationship to polyamine metabolism and growth rates in rat hepatoma tissue-culture (HTC) cells. Hypusine is selectively formed in the eukaryotic translation initiation factor eIF-4D, by a post-translational mechanism involving spermidine [Cooper, Park, Folk, Safer & Braverman (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 1854-1857]. The half-life of the hypusine-containing protein was longer than 24 h. In cells whose intracellular spermidine pools had been initially depleted, by using DL-alpha-difluoromethylornithine (DFMO), maximum synthesis rates of hypusine in protein were 5-10 times higher, on restoration of endogenous spermidine contents by exogenous addition, than those observed in untreated exponential-phase cultures. In cells pretreated with DFMO, the rate of hypusine synthesis was constant for up to 1 h after the addition of 5 microM-spermidine, whereas endogenous spermidine contents varied from less than 1 to more than 10 nmol/mg of protein. However, the overall amount of hypusine formed, during the first 1 h after the addition of various concentrations of spermidine (0.05-10 microM) to the culture medium, was markedly dependent on the final endogenous spermidine content achieved at the end of the 1 h measurement interval. Early in exponential-phase growth, protein-bound hypusine was synthesized at a rate of 1-2 pmol/h per mg of protein. This rate decreased to less than 0.5 pmol/h per mg of protein when cell growth rates decreased as cultures reached high cell densities. Analysis of the polyamine substrate specificity for hypusine formation showed that N1-acetylspermidine did not compete with spermidine in the reaction, nor did N1-(buta-2,3-dienyl)-N2-methylbutane-1,4-diamine, and irreversible inhibitor of polyamine oxidase, block the reaction. On the basis of comparative radiolabelling experiments, spermine was either a poor substrate, or not a substrate, for hypusine formation. These results confirm that spermidine is the likely precursor of the aminohydroxybutyl moiety of hypusine, and show that overall hypusine formation, but not necessarily the synthesis rate, is dependent on the endogenous spermidine concentration, especially under conditions where spermidine concentrations are initially low, as is the case after DFMO treatment, and then increase.

scholarly journals Feedback Regulation of O-GlcNAc Transferase through Translation Control to Maintain Intracellular O-GlcNAc Homeostasis

2021 ◽  
Vol 22 (7) ◽  
pp. 3463
Author(s):  
Chia-Hung Lin ◽  
Chen-Chung Liao ◽  
Mei-Yu Chen ◽  
Teh-Ying Chou
Keyword(s):  
Molecular Mechanisms ◽  
Mrna Level ◽  
Feedback Regulation ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Cell Physiology ◽  
Hydroxyl Groups ◽  
Post Translational Modification ◽  
Translation Control ◽  
Glcnac Transferase

Protein O-GlcNAcylation is a dynamic post-translational modification involving the attachment of N-acetylglucosamine (GlcNAc) to the hydroxyl groups of Ser/Thr residues on numerous nucleocytoplasmic proteins. Two enzymes are responsible for O-GlcNAc cycling on substrate proteins: O-GlcNAc transferase (OGT) catalyzes the addition while O-GlcNAcase (OGA) helps the removal of GlcNAc. O-GlcNAcylation modifies protein functions; therefore, dysregulation of O-GlcNAcylation affects cell physiology and contributes to pathogenesis. To maintain homeostasis of cellular O-GlcNAcylation, there exists feedback regulation of OGT and OGA expression responding to fluctuations of O-GlcNAc levels; yet, little is known about the molecular mechanisms involved. In this study, we investigated the O-GlcNAc-feedback regulation of OGT and OGA expression in lung cancer cells. Results suggest that, upon alterations in O-GlcNAcylation, the regulation of OGA expression occurs at the mRNA level and likely involves epigenetic mechanisms, while modulation of OGT expression is through translation control. Further analyses revealed that the eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1) contributes to the downregulation of OGT induced by hyper-O-GlcNAcylation; the S5A/S6A O-GlcNAcylation-site mutant of 4E-BP1 cannot support this regulation, suggesting an important role of O-GlcNAcylation. The results provide additional insight into the molecular mechanisms through which cells may fine-tune intracellular O-GlcNAc levels to maintain homeostasis.

scholarly journals Mammalian orthoreovirus Infection is Enhanced in Cells Pre-Treated with Sodium Arsenite

Viruses ◽  
2019 ◽  
Vol 11 (6) ◽  
pp. 563 ◽  
Author(s):  
Michael M. Lutz ◽  
Megan P. Worth ◽  
Meleana M. Hinchman ◽  
John S.L. Parker ◽  
Emily D. Ledgerwood
Keyword(s):  
Protein Expression ◽  
Stress Responses ◽  
Viral Protein ◽  
Sodium Arsenite ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Viral Protein Expression ◽  
Viral Yield ◽  
Pre Treatment ◽  
In Cells

Following reovirus infection, cells activate stress responses that repress canonical translation as a mechanism to limit progeny virion production. Work by others suggests that these stress responses, which are part of the integrated stress response (ISR), may benefit rather than repress reovirus replication. Here, we report that compared to untreated cells, treating cells with sodium arsenite (SA) to activate the ISR prior to infection enhanced viral protein expression, percent infectivity, and viral titer. SA-mediated enhancement was not strain-specific, but was cell-type specific. While SA pre-treatment of cells offered the greatest enhancement, treatment within the first 4 h of infection increased the percent of cells infected. SA activates the heme-regulated eIF2α (HRI) kinase, which phosphorylates eukaryotic translation initiation factor 2 alpha (eIF2α) to induce stress granule (SG) formation. Heat shock (HS), another activator of HRI, also induced eIF2α phosphorylation and SGs in cells. However, HS had no effect on percent infectivity or viral yield but did enhance viral protein expression. These data suggest that SA pre-treatment perturbs the cell in a way that is beneficial for reovirus and that this enhancement is independent of SG induction. Understanding how to manipulate the cellular stress responses during infection to enhance replication could help to maximize the oncolytic potential of reovirus.

scholarly journals OGFOD1, a Novel Modulator of Eukaryotic Translation Initiation Factor 2α Phosphorylation and the Cellular Response to Stress

2010 ◽  
Vol 30 (8) ◽  
pp. 2006-2016 ◽  
Author(s):  
Karen A. Wehner ◽  
Sylvia Schütz ◽  
Peter Sarnow
Keyword(s):  
Translation Initiation ◽  
Stress Granule ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Eukaryotic Translation Initiation Factor ◽  
Eukaryotic Translation ◽  
Induced Stress ◽  
Eukaryotic Translation Initiation ◽  
Phosphorylated Eif2α ◽  
In Cells

ABSTRACT Cells possess mechanisms that permit survival and recovery from stress, several of which regulate the phosphorylation of eukaryotic translation initiation factor 2α (eIF2α). We identified the human OGFOD1 protein as a novel stress granule component that regulates the phosphorylation of eIF2α and the resumption of translation in cells recovering from arsenite-induced stress. Coimmunoprecipitation studies revealed that OGFOD1 associates with a small subset of stress granule proteins (G3BP1, USP10, Caprin1, and YB-1) and the ribosome in both unstressed and stressed cells. Overexpression of OGFOD1 led to increased abundance of phosphorylated eIF2α, both in unstressed cells and in cells exposed to arsenite-induced stress, and to accelerated apoptosis during stress. Conversely, knockdown of OGFOD1 resulted in smaller amounts of phosphorylated eIF2α and a faster accumulation of polyribosomes in cells recovering from stress. Finally, OGFOD1 interacted with both eIF2α and the eIF2α kinase heme-regulated inhibitor (HRI), which was identified as a novel stress granule resident. These findings argue that OGFOD1 plays important proapoptotic roles in the regulation of translation and HRI-mediated phosphorylation of eIF2α in cells subjected to arsenite-induced stress.

scholarly journals Cleavage of Eukaryotic Translation Initiation Factor 4G by Exogenously Added Hybrid Proteins Containing Poliovirus 2Apro in HeLa Cells: Effects on Gene Expression

1999 ◽  
Vol 19 (4) ◽  
pp. 2445-2454 ◽  
Author(s):  
Isabel Novoa ◽  
Luis Carrasco
Keyword(s):  
Heat Shock ◽  
Hela Cells ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Luciferase Gene ◽  
Normal Medium ◽  
Hypertonic Medium ◽  
Hybrid Proteins ◽  
Eukaryotic Initiation Factor 4G ◽  
In Cells

ABSTRACT Efficient cleavage of both forms of eukaryotic initiation factor 4G (eIF4G-1 and eIF4G-2) has been achieved in HeLa cells by incubation with hybrid proteins containing poliovirus 2Apro. Entry of these proteins into cells is promoted by adenovirus particles. Substantial levels of ongoing translation on preexisting cellular mRNAs still continue for several hours after eIF4G degradation. Treatment of control HeLa cells with hypertonic medium causes an inhibition of translation that is reversed upon restoration of cells to normal medium. Protein synthesis is not restored in cells lacking intact eIF4G after hypertonic treatment. Notably, induction of synthesis of heat shock proteins still occurs in cells pretreated with poliovirus 2Apro, suggesting that transcription and translation of these mRNAs takes place even in the presence of cleaved eIF4G. Finally, the synthesis of luciferase was examined in a HeLa cell line bearing the luciferase gene under control of a tetracycline-regulated promoter. Transcription of the luciferase gene and transport of the mRNA to the cytoplasm occurs at control levels in eIF4G-deficient cells. However, luciferase synthesis is strongly inhibited in these cells. These findings indicate that intact eIF4G is necessary for the translation of mRNAs not engaged in translation with the exception of heat shock mRNAs but is not necessary for the translation of mRNAs that are being translated.

scholarly journals Translational control in the tumor microenvironment promotes lung metastasis: Phosphorylation of eIF4E in neutrophils

2018 ◽  
Vol 115 (10) ◽  
pp. E2202-E2209 ◽  
Author(s):  
Nathaniel Robichaud ◽  
Brian E. Hsu ◽  
Roman Istomine ◽  
Fernando Alvarez ◽  
Julianna Blagih ◽  
...  
Keyword(s):  
Tumor Microenvironment ◽  
Cancer Progression ◽  
Translational Control ◽  
Lung Metastases ◽  
Phosphorylation Site ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Metastatic Progression ◽  
The Impact ◽  
In Cells

The translation of mRNAs into proteins serves as a critical regulatory event in gene expression. In the context of cancer, deregulated translation is a hallmark of transformation, promoting the proliferation, survival, and metastatic capabilities of cancer cells. The best-studied factor involved in the translational control of cancer is the eukaryotic translation initiation factor 4E (eIF4E). We and others have shown that eIF4E availability and phosphorylation promote metastasis in mouse models of breast cancer by selectively augmenting the translation of mRNAs involved in invasion and metastasis. However, the impact of translational control in cell types within the tumor microenvironment (TME) is unknown. Here, we demonstrate that regulatory events affecting translation in cells of the TME impact cancer progression. Mice bearing a mutation in the phosphorylation site of eIF4E (S209A) in cells comprising the TME are resistant to the formation of lung metastases in a syngeneic mammary tumor model. This is associated with reduced survival of prometastatic neutrophils due to decreased expression of the antiapoptotic proteins BCL2 and MCL1. Furthermore, we demonstrate that pharmacological inhibition of eIF4E phosphorylation prevents metastatic progression in vivo, supporting the development of phosphorylation inhibitors for clinical use.

scholarly journals Sendai Virus C Protein Plays a Role in Restricting PKR Activation by Limiting the Generation of Intracellular Double-Stranded RNA

2008 ◽  
Vol 82 (20) ◽  
pp. 10102-10110 ◽  
Author(s):  
Kenji Takeuchi ◽  
Takayuki Komatsu ◽  
Yoshinori Kitagawa ◽  
Kiyonao Sada ◽  
Bin Gotoh
Keyword(s):  
Protein Synthesis ◽  
Sendai Virus ◽  
Translation Initiation Factor ◽  
Immunofluorescent Staining ◽  
Synthesis Rate ◽  
Protein Synthesis Rate ◽  
Intracellular Protein ◽  
C Protein ◽  
Double Stranded Rna ◽  
In Cells

ABSTRACT Sendai virus (SeV) C protein is a multifunctional protein that plays important roles in regulating viral genome replication and transcription, antagonizing the host interferon system, suppressing virus-induced apoptosis, and facilitating virus assembly and budding. We here report a novel role of SeV C protein, the limitation of double-stranded RNA (dsRNA) generation for maintaining the rate of protein synthesis in infected cells. It was found that the intracellular protein synthesis rate was maintained even after wild-type (wt) SeV infection, but markedly suppressed following C-knockout SeV infection. This indicates the requirement of C protein for maintaining protein synthesis after infection. In contrast to wt SeV infection, C-knockout SeV infection caused phosphorylation of both the translation initiation factor eIF2α and dsRNA-dependent protein kinase (PKR). Phosphorylation of eIF2α occurred mainly due to the action of PKR, since knockdown of PKR by small interfering RNA limited eIF2α phosphorylation. C protein, however, could inhibit neither poly(I):poly(C)-activated nor Newcastle disease virus-induced phosphorylation of PKR and eIF2α, suggesting that C protein does not target common pathways leading to PKR activation. Immunofluorescent staining experiments with a monoclonal antibody specifically recognizing dsRNA revealed generation of a large amount of dsRNA in cells infected with C-knockout SeV but not wt SeV. The dsRNA generation as well as phosphorylation of PKR and eIF2α induced by C-knockout SeV was markedly suppressed in cells constitutively expressing C protein. Taken together, these results demonstrate that the SeV C protein limits generation of dsRNA, thereby keeping PKR inactive to maintain intracellular protein synthesis.

scholarly journals The Cellular Protein P58IPK Regulates Influenza Virus mRNA Translation and Replication through a PKR-Mediated Mechanism

2006 ◽  
Vol 81 (5) ◽  
pp. 2221-2230 ◽  
Author(s):  
Alan G. Goodman ◽  
Jennifer A. Smith ◽  
Siddharth Balachandran ◽  
Olivia Perwitasari ◽  
Sean C. Proll ◽  
...  
Keyword(s):  
Influenza Virus ◽  
Mammalian Cells ◽  
Mrna Translation ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Virus Protein ◽  
Mrna Levels ◽  
Viral Mrna ◽  
Eif2α Phosphorylation ◽  
In Cells

ABSTRACT We previously hypothesized that efficient translation of influenza virus mRNA requires the recruitment of P58IPK, the cellular inhibitor of PKR, an interferon-induced kinase that targets the eukaryotic translation initiation factor eIF2α. P58IPK also inhibits PERK, an eIF2α kinase that is localized in the endoplasmic reticulum (ER) and induced during ER stress. The ability of P58IPK to interact with and inhibit multiple eIF2α kinases suggests it is a critical regulator of both cellular and viral mRNA translation. In this study, we sought to definitively define the role of P58IPK during viral infection of mammalian cells. Using mouse embryo fibroblasts from P58IPK−/− mice, we demonstrated that the absence of P58IPK led to an increase in eIF2α phosphorylation and decreased influenza virus mRNA translation. The absence of P58IPK also resulted in decreased vesicular stomatitis virus replication but enhanced reovirus yields. In cells lacking the P58IPK target, PKR, the trends were reversed—eIF2α phosphorylation was decreased, and influenza virus mRNA translation was increased. Although P58IPK also inhibits PERK, the presence or absence of this kinase had little effect on influenza virus mRNA translation, despite reduced levels of eIF2α phosphorylation in cells lacking PERK. Finally, we showed that influenza virus protein synthesis and viral mRNA levels decrease in cells that express a constitutively active, nonphosphorylatable eIF2α. Taken together, our results support a model in which P58IPK regulates influenza virus mRNA translation and infection through a PKR-mediated mechanism which is independent of PERK.

scholarly journals The Ubiquitin-Proteasome System Plays an Important Role during Various Stages of the Coronavirus Infection Cycle

2010 ◽  
Vol 84 (15) ◽  
pp. 7869-7879 ◽  
Author(s):  
Matthijs Raaben ◽  
Clara C. Posthuma ◽  
Monique H. Verheije ◽  
Eddie G. te Lintelo ◽  
Marjolein Kikkert ◽  
...  
Keyword(s):  
Protein Expression ◽  
Ubiquitin Proteasome System ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Infection Cycle ◽  
Protein Ubiquitination ◽  
Ubiquitin Proteasome ◽  
The Impact ◽  
In Cells

ABSTRACT The ubiquitin-proteasome system (UPS) is a key player in regulating the intracellular sorting and degradation of proteins. In this study we investigated the role of the UPS in different steps of the coronavirus (CoV) infection cycle. Inhibition of the proteasome by different chemical compounds (i.e., MG132, epoxomicin, and Velcade) appeared to not only impair entry but also RNA synthesis and subsequent protein expression of different CoVs (i.e., mouse hepatitis virus [MHV], feline infectious peritonitis virus, and severe acute respiratory syndrome CoV). MHV assembly and release were, however, not appreciably affected by these compounds. The inhibitory effect on CoV protein expression did not appear to result from a general inhibition of translation due to induction of a cellular stress response by the inhibitors. Stress-induced phosphorylation of eukaryotic translation initiation factor 2α (eIF2α) generally results in impaired initiation of protein synthesis, but the sensitivity of MHV infection to proteasome inhibitors was unchanged in cells lacking a phosphorylatable eIF2α. MHV infection was affected not only by inhibition of the proteasome but also by interfering with protein ubiquitination. Viral protein expression was reduced in cells expressing a temperature-sensitive ubiquitin-activating enzyme E1 at the restrictive temperature, as well as in cells in which ubiquitin was depleted by using small interfering RNAs. Under these conditions, the susceptibility of the cells to virus infection was, however, not affected, excluding an important role of ubiquitination in virus entry. Our observations reveal an important role of the UPS in multiple steps of the CoV infection cycle and identify the UPS as a potential drug target to modulate the impact of CoV infection.

scholarly journals Deciphering the Translation Initiation Factor 5A Modification Pathway in Halophilic Archaea

Archaea ◽  
2016 ◽  
Vol 2016 ◽  
pp. 1-14 ◽  
Author(s):  
Laurence Prunetti ◽  
Michael Graf ◽  
Ian K. Blaby ◽  
Lauri Peil ◽  
Andrea M. Makkay ◽  
...  
Keyword(s):  
Translation Initiation ◽  
Halophilic Archaea ◽  
Arginine Decarboxylase ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Polyamine Metabolism ◽  
Metabolic Reconstruction ◽  
Deoxyhypusine Synthase

Translation initiation factor 5A (IF5A) is essential and highly conserved in Eukarya (eIF5A) and Archaea (aIF5A). The activity of IF5A requires hypusine, a posttranslational modification synthesized in Eukarya from the polyamine precursor spermidine. Intracellular polyamine analyses revealed that agmatine and cadaverine were the main polyamines produced inHaloferax volcaniiin minimal medium, raising the question of how hypusine is synthesized in this halophilic Archaea. Metabolic reconstruction led to a tentative picture of polyamine metabolism and aIF5A modification inHfx. volcaniithat was experimentally tested. Analysis of aIF5A fromHfx. volcaniiby LC-MS/MS revealed it was exclusively deoxyhypusinylated. Genetic studies confirmed the role of the predicted arginine decarboxylase gene(HVO_1958)in agmatine synthesis. The agmatinase-like gene(HVO_2299)was found to be essential, consistent with a role in aIF5A modification predicted by physical clustering evidence. Recombinant deoxyhypusine synthase (DHS) fromS. cerevisiaewas shown to transfer 4-aminobutyl moiety from spermidine to aIF5A fromHfx. volcanii in vitro.However, at least under conditions tested, this transfer was not observed with theHfx. volcaniiDHS. Furthermore, the growth ofHfx. volcaniiwas not inhibited by the classical DHS inhibitor GC7. We propose a model of deoxyhypusine synthesis inHfx. volcaniithat differs from the canonical eukaryotic pathway, paving the way for further studies.

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