Phylogenetic analysis of eIF4E-family members

PubMed Central

Joshi, Bhavesh; Lee, Kibwe; Maeder, Dennis L; Jagus, Rosemary

2005-01-01

Background Translation initiation in eukaryotes involves the recruitment of mRNA to the ribosome which is controlled by the translation factor eIF4E. eIF4E binds to the 5'-m7Gppp cap-structure of mRNA. Three dimensional structures of eIF4Es bound to cap-analogues resemble 'cupped-hands' in which the cap-structure is sandwiched between two conserved Trp residues (Trp-56 and Trp-102 of H. sapiens eIF4E). A third conserved Trp residue (Trp-166 of H. sapiens eIF4E) recognizes the 7-methyl moiety of the cap-structure. Assessment of GenBank NR and dbEST databases reveals that many organisms encode a number of proteins with homology to eIF4E. Little is understood about the relationships of these structurally related proteins to each other. Results By combining sequence data deposited in the Genbank databases, we have identified sequences encoding 411 eIF4E-family members from 230 species. These sequences have been deposited into an internet-accessible database designed for sequence comparisons of eIF4E-family members. Most members can be grouped into one of three classes. Class I members carry Trp residues equivalent to Trp-43 and Trp-56 of H. sapiens eIF4E and appear to be present in all eukaryotes. Class II members, possess Trp→Tyr/Phe/Leu and Trp→Tyr/Phe substitutions relative to Trp-43 and Trp-56 of H. sapiens eIF4E, and can be identified in Metazoa, Viridiplantae, and Fungi. Class III members possess a Trp residue equivalent to Trp-43 of H. sapiens eIF4E but carry a Trp→Cys/Tyr substitution relative to Trp-56 of H. sapiens eIF4E, and can be identified in Coelomata and Cnidaria. Some eIF4E-family members from Protista show extension or compaction relative to prototypical eIF4E-family members. Conclusion The expansion of sequenced cDNAs and genomic DNAs from all eukaryotic kingdoms has revealed a variety of proteins related in structure to eIF4E. Evolutionarily it seems that a single early eIF4E gene has undergone multiple gene duplications generating multiple

The eIF4AIII RNA helicase is a critical determinant of human cytomegalovirus replication

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ziehr, Ben; Lenarcic, Erik; Cecil, Chad

Human cytomegalovirus (HCMV) was recently shown to encode a large number of spliced mRNAs. While the nuclear export of unspliced viral transcripts has been extensively studied, the role of host mRNA export factors in HCMV mRNA trafficking remains poorly defined. We found that the eIF4AIII RNA helicase, a component of the exon junction complex, was necessary for efficient virus replication. Depletion of eIF4AIII limited viral DNA accumulation, export of viral mRNAs from the nucleus, and the production of progeny virus. However eIF4AIII was dispensable for the association of viral transcripts with ribosomes. We found that pateamine A, a natural compoundmore » that inhibits both eIF4AI/II and eIF4AIII, has potent antiviral activity and inhibits HCMV replication throughout the virus lytic cycle. Our results demonstrate that eIF4AIII is required for efficient HCMV replication, and suggest that eIF4A family helicases may be a new class of targets for the development of host-directed antiviral therapeutics. - Highlights: • The host eIF4AIII RNA helicase is required for efficient HCMV replication. • Depleting eIF4AIII inhibited the nuclear export of HCMV mRNAs. • HCMV mRNAs did not require eIF4AIII to associate with polyribosomes. • The eIF4A family helicases may be new targets for host-directed antiviral drugs.« less

Yeast eIF4A enhances recruitment of mRNAs regardless of their structural complexity

PubMed Central

Yourik, Paul; Aitken, Colin Echeverría; Zhou, Fujun; Gupta, Neha

2017-01-01

eIF4A is a DEAD-box RNA-dependent ATPase thought to unwind RNA secondary structure in the 5'-untranslated regions (UTRs) of mRNAs to promote their recruitment to the eukaryotic translation pre-initiation complex (PIC). We show that eIF4A's ATPase activity is markedly stimulated in the presence of the PIC, independently of eIF4E•eIF4G, but dependent on subunits i and g of the heteromeric eIF3 complex. Surprisingly, eIF4A accelerated the rate of recruitment of all mRNAs tested, regardless of their degree of structural complexity. Structures in the 5'-UTR and 3' of the start codon synergistically inhibit mRNA recruitment in a manner relieved by eIF4A, indicating that the factor does not act solely to melt hairpins in 5'-UTRs. Our findings that eIF4A functionally interacts with the PIC and plays important roles beyond unwinding 5'-UTR structure is consistent with a recent proposal that eIF4A modulates the conformation of the 40S ribosomal subunit to promote mRNA recruitment. PMID:29192585

Importin 8 mediates m7G cap-sensitive nuclear import of the eukaryotic translation initiation factor eIF4E

PubMed Central

Volpon, Laurent; Culjkovic-Kraljacic, Biljana; Osborne, Michael J.; Ramteke, Anup; Sun, Qingxiang; Niesman, Ashley; Chook, Yuh Min; Borden, Katherine L. B.

2016-01-01

Regulation of nuclear-cytoplasmic trafficking of oncoproteins is critical for growth homeostasis. Dysregulated trafficking contributes to malignancy, whereas understanding the process can reveal unique therapeutic opportunities. Here, we focus on eukaryotic translation initiation factor 4E (eIF4E), a prooncogenic protein highly elevated in many cancers, including acute myeloid leukemia (AML). Typically, eIF4E is localized to both the nucleus and cytoplasm, where it acts in export and translation of specific methyl 7-guanosine (m7G)–capped mRNAs, respectively. Nuclear accumulation of eIF4E in patients who have AML is correlated with increased eIF4E-dependent export of transcripts encoding oncoproteins. The subcellular localization of eIF4E closely correlates with patients’ responses. During clinical responses to the m7G-cap competitor ribavirin, eIF4E is mainly cytoplasmic. At relapse, eIF4E reaccumulates in the nucleus, leading to elevated eIF4E-dependent mRNA export. We have identified importin 8 as a factor that directly imports eIF4E into the nucleus. We found that importin 8 is highly elevated in untreated patients with AML, leading to eIF4E nuclear accumulation. Importin 8 only imports cap-free eIF4E. Cap-dependent changes to the structure of eIF4E underpin this selectivity. Indeed, m7G cap analogs or ribavirin prevents nuclear entry of eIF4E, which mirrors the trafficking phenotypes observed in patients with AML. Our studies also suggest that nuclear entry is important for the prooncogenic activity of eIF4E, at least in this context. These findings position nuclear trafficking of eIF4E as a critical step in its regulation and position the importin 8–eIF4E complex as a novel therapeutic target. PMID:27114554

Cooperative modulation by eIF4G of eIF4E-binding to the mRNA 5' cap in yeast involves a site partially shared by p20.

PubMed Central

Ptushkina, M; von der Haar, T; Vasilescu, S; Frank, R; Birkenhäger, R; McCarthy, J E

1998-01-01

Interaction between the mRNA 5'-cap-binding protein eIF4E and the multiadaptor protein eIF4G has been demonstrated in all eukaryotic translation assemblies examined so far. This study uses immunological, genetic and biochemical methods to map the surface amino acids on eIF4E that contribute to eIF4G binding. Cap-analogue chromatography and surface plasmon resonance (SPR) analyses demonstrate that one class of mutations in these surface regions disrupts eIF4E-eIF4G association, and thereby polysome formation and growth. The residues at these positions in wild-type eIF4E mediate positive cooperativity between the binding of eIF4G to eIF4E and the latter's cap-affinity. Moreover, two of the mutations confer temperature sensitivity in eIF4G binding to eIF4E which correlates with the formation of large numbers of inactive ribosome 80S couples in vivo and the loss of cellular protein synthesis activity. The yeast 4E-binding protein p20 is estimated by SPR to have a ten times lower binding affinity than eIF4G for eIF4E. Investigation of a second class of eIF4E mutations reveals that p20 shares only part of eIF4G's binding site on the cap-binding protein. The results presented provide a basis for understanding how cycling of eIF4E and eIF4G occurs in yeast translation and explains how p20 can act as a fine, but not as a coarse, regulator of protein synthesis. PMID:9707439

The eIF4F and eIFiso4F Complexes of Plants: An Evolutionary Perspective

PubMed Central

Patrick, Ryan M.; Browning, Karen S.

2012-01-01

Translation initiation in eukaryotes requires a number of initiation factors to recruit the assembled ribosome to mRNA. The eIF4F complex plays a key role in initiation and is a common target point for regulation of protein synthesis. Most work on the translation machinery of plants to date has focused on flowering plants, which have both the eIF4F complex (eIF4E and eIF4G) as well as the plant-specific eIFiso4F complex (eIFiso4E and eIFiso4G). The increasing availability of plant genome sequence data has made it possible to trace the evolutionary history of these two complexes in plants, leading to several interesting discoveries. eIFiso4G is conserved throughout plants, while eIFiso4E only appears with the evolution of flowering plants. The eIF4G N-terminus, which has been difficult to annotate, appears to be well conserved throughout the plant lineage and contains two motifs of unknown function. Comparison of eIFiso4G and eIF4G sequence data suggests conserved features unique to eIFiso4G and eIF4G proteins. These findings have answered some questions about the evolutionary history of the two eIF4F complexes of plants, while raising new ones. PMID:22611336

A Prospective Trial on Initiation Factor 4E (eIF4E) Overexpression and Cancer Recurrence in Node-Positive Breast Cancer

PubMed Central

McClusky, Derek R.; Chu, Quyen; Yu, Herbert; DeBenedetti, Arrigo; Johnson, Lester W.; Meschonat, Carol; Turnage, Richard; McDonald, John C.; Abreo, Fleurette; Li, Benjamin D. L.

2005-01-01

Objective: A previous study of patients with stage I to III breast cancer showed that those patients whose tumors were in the highest tertile of eIF4E overexpression experienced a higher risk for recurrence. This study was designed to determine whether high eIF4E overexpression predicts cancer recurrence independent of nodal status by specifically targeting patients with node-positive disease. Methods: The prospective trial was designed to accrue 168 patients with node-positive breast cancer to detect a 2.5-fold increase in risk for recurrence. eIF4E level was quantified by Western blots as x-fold elevated compared with breast tissues from noncancer patients. End points measured were disease recurrence and cancer-related death. Statistical analyses performed include survival analysis by the Kaplan-Meier method, log-rank test, and Cox proportional hazard model. Results: One hundred seventy-four patients with node-positive breast cancer were accrued. All patients fulfilled study inclusion and exclusion criteria, treatment protocol, and surveillance requirements, with a compliance rate >95%. The mean eIF4E elevation was 11.0 ± 7.0-fold (range, 1.4–34.3-fold). Based on previously published data, tertile distribution was as follow: 1) lowest tertile (<7.5-fold) = 67 patients, 2) intermediate tertile (7.5–14-fold) = 54 patients, and 3) highest tertile (>14-fold) = 53 patients. At a median follow up of 32 months, patients with the highest tertile had a statistically significant higher cancer recurrence rate (log-rank test, P = 0.002) and cancer-related death rate (P = 0.036) than the lowest group. Relative risk calculations demonstrated that high eIF4E patients had a 2.4-fold increase in relative risk increase for cancer recurrence (95% confidence interval, 1.2–4.1; P = 0.01). Conclusions: In this prospective study designed to specifically address risk for recurrence in patients with node-positive breast cancer, the patients whose tumors were in the highest tertile

Diversity of Eukaryotic Translational Initiation Factor eIF4E in Protists.

PubMed

Jagus, Rosemary; Bachvaroff, Tsvetan R; Joshi, Bhavesh; Place, Allen R

2012-01-01

The greatest diversity of eukaryotic species is within the microbial eukaryotes, the protists, with plants and fungi/metazoa representing just two of the estimated seventy five lineages of eukaryotes. Protists are a diverse group characterized by unusual genome features and a wide range of genome sizes from 8.2 Mb in the apicomplexan parasite Babesia bovis to 112,000-220,050 Mb in the dinoflagellate Prorocentrum micans. Protists possess numerous cellular, molecular and biochemical traits not observed in "text-book" model organisms. These features challenge some of the concepts and assumptions about the regulation of gene expression in eukaryotes. Like multicellular eukaryotes, many protists encode multiple eIF4Es, but few functional studies have been undertaken except in parasitic species. An earlier phylogenetic analysis of protist eIF4Es indicated that they cannot be grouped within the three classes that describe eIF4E family members from multicellular organisms. Many more protist sequences are now available from which three clades can be recognized that are distinct from the plant/fungi/metazoan classes. Understanding of the protist eIF4Es will be facilitated as more sequences become available particularly for the under-represented opisthokonts and amoebozoa. Similarly, a better understanding of eIF4Es within each clade will develop as more functional studies of protist eIF4Es are completed.

Diversity of Eukaryotic Translational Initiation Factor eIF4E in Protists

PubMed Central

Jagus, Rosemary; Bachvaroff, Tsvetan R.; Joshi, Bhavesh; Place, Allen R.

2012-01-01

The greatest diversity of eukaryotic species is within the microbial eukaryotes, the protists, with plants and fungi/metazoa representing just two of the estimated seventy five lineages of eukaryotes. Protists are a diverse group characterized by unusual genome features and a wide range of genome sizes from 8.2 Mb in the apicomplexan parasite Babesia bovis to 112,000-220,050 Mb in the dinoflagellate Prorocentrum micans. Protists possess numerous cellular, molecular and biochemical traits not observed in “text-book” model organisms. These features challenge some of the concepts and assumptions about the regulation of gene expression in eukaryotes. Like multicellular eukaryotes, many protists encode multiple eIF4Es, but few functional studies have been undertaken except in parasitic species. An earlier phylogenetic analysis of protist eIF4Es indicated that they cannot be grouped within the three classes that describe eIF4E family members from multicellular organisms. Many more protist sequences are now available from which three clades can be recognized that are distinct from the plant/fungi/metazoan classes. Understanding of the protist eIF4Es will be facilitated as more sequences become available particularly for the under-represented opisthokonts and amoebozoa. Similarly, a better understanding of eIF4Es within each clade will develop as more functional studies of protist eIF4Es are completed. PMID:22778692

The unique Leishmania EIF4E4 N-terminus is a target for multiple phosphorylation events and participates in critical interactions required for translation initiation.

PubMed

de Melo Neto, Osvaldo P; da Costa Lima, Tamara D C; Xavier, Camila C; Nascimento, Larissa M; Romão, Tatiany P; Assis, Ludmila A; Pereira, Mariana M C; Reis, Christian R S; Papadopoulou, Barbara

2015-01-01

The eukaryotic initiation factor 4E (eIF4E) recognizes the mRNA cap structure and, together with eIF4G and eIF4A, form the eIF4F complex that regulates translation initiation in eukaryotes. In trypanosomatids, 2 eIF4E homologues (EIF4E3 and EIF4E4) have been shown to be part of eIF4F-like complexes with presumed roles in translation initiation. Both proteins possess unique N-terminal extensions, which can be targeted for phosphorylation. Here, we provide novel insights on the Leishmania infantum EIF4E4 function and regulation. We show that EIF4E4 is constitutively expressed throughout the parasite development but is preferentially phosphorylated in exponentially grown promastigote and amastigote life stages, hence correlating with high levels of translation. Phosphorylation targets multiple serine-proline or threonine-proline residues within the N-terminal extension of EIF4E4 but does not require binding to the EIF4E4's partner, EIF4G3, or to the cap structure. We also report that EIF4E4 interacts with PABP1 through 3 conserved boxes at the EIF4E4 N-terminus and that this interaction is a prerequisite for efficient EIF4E4 phosphorylation. EIF4E4 is essential for Leishmania growth and an EIF4E4 null mutant was only obtained in the presence of an ectopically provided wild type gene. Complementation for the loss of EIF4E4 with several EIF4E4 mutant proteins affecting either phosphorylation or binding to mRNA or to EIF4E4 protein partners revealed that, in contrast to other eukaryotes, only the EIF4E4-PABP1 interaction but neither the binding to EIF4G3 nor phosphorylation is essential for translation. These studies also demonstrated that the lack of both EIF4E4 phosphorylation and EIF4G3 binding leads to a non-functional protein. Altogether, these findings further highlight the unique features of the translation initiation process in trypanosomatid protozoa.

The unique Leishmania EIF4E4 N-terminus is a target for multiple phosphorylation events and participates in critical interactions required for translation initiation

PubMed Central

de Melo Neto, Osvaldo P; da Costa Lima, Tamara D C; Xavier, Camila C; Nascimento, Larissa M; Romão, Tatiany P; Assis, Ludmila A; Pereira, Mariana M C; Reis, Christian R S; Papadopoulou, Barbara

2015-01-01

The eukaryotic initiation factor 4E (eIF4E) recognizes the mRNA cap structure and, together with eIF4G and eIF4A, form the eIF4F complex that regulates translation initiation in eukaryotes. In trypanosomatids, 2 eIF4E homologues (EIF4E3 and EIF4E4) have been shown to be part of eIF4F-like complexes with presumed roles in translation initiation. Both proteins possess unique N-terminal extensions, which can be targeted for phosphorylation. Here, we provide novel insights on the Leishmania infantum EIF4E4 function and regulation. We show that EIF4E4 is constitutively expressed throughout the parasite development but is preferentially phosphorylated in exponentially grown promastigote and amastigote life stages, hence correlating with high levels of translation. Phosphorylation targets multiple serine-proline or threonine-proline residues within the N-terminal extension of EIF4E4 but does not require binding to the EIF4E4's partner, EIF4G3, or to the cap structure. We also report that EIF4E4 interacts with PABP1 through 3 conserved boxes at the EIF4E4 N-terminus and that this interaction is a prerequisite for efficient EIF4E4 phosphorylation. EIF4E4 is essential for Leishmania growth and an EIF4E4 null mutant was only obtained in the presence of an ectopically provided wild type gene. Complementation for the loss of EIF4E4 with several EIF4E4 mutant proteins affecting either phosphorylation or binding to mRNA or to EIF4E4 protein partners revealed that, in contrast to other eukaryotes, only the EIF4E4-PABP1 interaction but neither the binding to EIF4G3 nor phosphorylation is essential for translation. These studies also demonstrated that the lack of both EIF4E4 phosphorylation and EIF4G3 binding leads to a non-functional protein. Altogether, these findings further highlight the unique features of the translation initiation process in trypanosomatid protozoa. PMID:26338184

Analysis of eIF4E and 4EBP1 mRNAs in head and neck cancer.

PubMed

Sunavala-Dossabhoy, Gulshan; Palaniyandi, Senthilnathan; Clark, Cheryl; Nathan, Cherie-Ann O; Abreo, Fleurette W; Caldito, Gloria

2011-10-01

The eukaryotic translation initiation factor 4E (eIF4E) in conjunction with its binding protein, 4EBP1, regulates the translation of cap-dependent mRNAs. An aberrant increase in eIF4E shifts the balance in favor of translation of transcripts that promote cell proliferation and malignancy. eIF4E protein is commonly elevated in head and neck squamous cell carcinomas (HNSCC), and its overexpression is associated with increased recurrence. An underlying mechanism for eIF4E overexpression is gene amplification, and we wanted to determine whether eIF4E mRNA could serve as a prognostic maker of HNSCC. Tumor specimens from 26 HNSCC patients and oral tissues from 17 control subjects were examined for eIF4E and 4EBP1 by semiquantitative RT-PCR and correlated with clinical and pathologic findings. Unlike eIF4E mRNA alone, expression of eIF4E relative to 4EBP1 was a more precise predictor of HNSCC and its progression (P < .01, Wilcoxon rank sum test). Eight of 26 patients (31%) had elevated eIF4E:4EBP1 (4E:4EBP1; >25), and 7 of these (87.5%) had recurrence. Alternately, from 18 patients with low 4E:4EBP1 (<25; 69%), only 5 patients had recurrence (30.1%). To determine the probability of no recurrence, Kaplan-Meier analysis showed significantly poor disease-free survival in patients with elevated 4E:4EBP1 than those with low ratios (P < .01, log rank test). Elevated 4E:4EBP1 significantly correlated with increased disease recurrence. Because 4EBP1 modulates eIF4E activity, our results highlight the importance of incorporating a joint analysis of eIF4E and 4EBP1 mRNAs in HNSCC patient care decisions. Copyright © 2011 The American Laryngological, Rhinological, and Otological Society, Inc.

EIF2AK4 Mutations in Pulmonary Capillary Hemangiomatosis

PubMed Central

Best, D. Hunter; Sumner, Kelli L.; Austin, Eric D.; Chung, Wendy K.; Brown, Lynette M.; Borczuk, Alain C.; Rosenzweig, Erika B.; Bayrak-Toydemir, Pinar; Mao, Rong; Cahill, Barbara C.; Tazelaar, Henry D.; Leslie, Kevin O.; Hemnes, Anna R.; Robbins, Ivan M.

2014-01-01

Background: Pulmonary capillary hemangiomatosis (PCH) is a rare disease of capillary proliferation of unknown cause and with a high mortality. Families with multiple affected individuals with PCH suggest a heritable cause although the genetic etiology remains unknown. Methods: We used exome sequencing to identify a candidate gene for PCH in a family with two affected brothers. We then screened 11 unrelated patients with familial (n = 1) or sporadic (n = 10) PCH for mutations. Results: Using exome sequencing, we identified compound mutations in eukaryotic translation initiation factor 2 α kinase 4 (EIF2AK4) (formerly known as GCN2) in both affected brothers. Both parents and an unaffected sister were heterozygous carriers. In addition, we identified two EIF2AK4 mutations in each of two of 10 unrelated individuals with sporadic PCH. EIF2AK4 belongs to a family of kinases that regulate angiogenesis in response to cellular stress. Conclusions: Mutations in EIF2AK4 are likely to cause autosomal-recessive PCH in familial and some nonfamilial cases. PMID:24135949

Complexity of the 5' Untranslated Region of EIF4A3, a Critical Factor for Craniofacial and Neural Development.

PubMed

Hsia, Gabriella S P; Musso, Camila M; Alvizi, Lucas; Brito, Luciano A; Kobayashi, Gerson S; Pavanello, Rita C M; Zatz, Mayana; Gardham, Alice; Wakeling, Emma; Zechi-Ceide, Roseli M; Bertola, Debora; Passos-Bueno, Maria Rita

2018-01-01

Repeats in coding and non-coding regions have increasingly been associated with many human genetic disorders, such as Richieri-Costa-Pereira syndrome (RCPS). RCPS, mostly characterized by midline cleft mandible, Robin sequence and limb defects, is an autosomal-recessive acrofacial dysostosis mainly reported in Brazilian patients. This disorder is caused by decreased levels of EIF4A3 , mostly due to an increased number of repeats at the EIF4A3 5'UTR. EIF4A3 5'UTR alleles are CG-rich and vary in size and organization of three types of motifs. An exclusive allelic pattern was identified among affected individuals, in which the CGCA-motif is the most prevalent, herein referred as "disease-associated CGCA-20nt motif." The origin of the pathogenic alleles containing the disease-associated motif, as well as the functional effects of the 5'UTR motifs on EIF4A3 expression, to date, are entirely unknown. Here, we characterized 43 different EIF4A3 5'UTR alleles in a cohort of 380 unaffected individuals. We identified eight heterozygous unaffected individuals harboring the disease-associated CGCA-20nt motif and our haplotype analyses indicate that there are more than one haplotype associated with RCPS. The combined analysis of number, motif organization and haplotypic diversity, as well as the observation of two apparently distinct haplotypes associated with the disease-associated CGCA-20nt motif, suggest that the RCPS alleles might have arisen from independent unequal crossing-over events between ancient alleles at least twice. Moreover, we have shown that the number and sequence of motifs in the 5'UTR region is associated with EIF4A3 repression, which is not mediated by CpG methylation. In conclusion, this study has shown that the large number of repeats in EIF4A3 does not represent a dynamic mutation and RCPS can arise in any population harboring alleles with the CGCA-20nt motif. We also provided further evidence that EIF4A3 5'UTR is a regulatory region and the size and

A biochemical framework for eIF4E-dependent mRNA export and nuclear recycling of the export machinery.

PubMed

Volpon, Laurent; Culjkovic-Kraljacic, Biljana; Sohn, Hye Seon; Blanchet-Cohen, Alexis; Osborne, Michael J; Borden, Katherine L B

2017-06-01

The eukaryotic translation initiation factor eIF4E acts in the nuclear export and translation of a subset of mRNAs. Both of these functions contribute to its oncogenic potential. While the biochemical mechanisms that underlie translation are relatively well understood, the molecular basis for eIF4E's role in mRNA export remains largely unexplored. To date, over 3000 transcripts, many encoding oncoproteins, were identified as potential nuclear eIF4E export targets. These target RNAs typically contain a ∼50-nucleotide eIF4E sensitivity element (4ESE) in the 3' UTR and a 7-methylguanosine cap on the 5' end. While eIF4E associates with the cap, an unknown factor recognizes the 4ESE element. We previously identified cofactors that functionally interacted with eIF4E in mammalian cell nuclei including the leucine-rich pentatricopeptide repeat protein LRPPRC and the export receptor CRM1/XPO1. LRPPRC simultaneously interacts with both eIF4E bound to the 5' mRNA cap and the 4ESE element in the 3' UTR. In this way, LRPPRC serves as a specificity factor to recruit 4ESE-containing RNAs within the nucleus. Further, we show that CRM1 directly binds LRPPRC likely acting as the export receptor for the LRPPRC-eIF4E-4ESE RNA complex. We also found that Importin 8, the nuclear importer for cap-free eIF4E, imports RNA-free LRPPRC, potentially providing both coordinated nuclear recycling of the export machinery and an important surveillance mechanism to prevent futile export cycles. Our studies provide the first biochemical framework for the eIF4E-dependent mRNA export pathway. © 2017 Volpon et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

The DEAD-box helicase eIF4A: paradigm or the odd one out?

PubMed

Andreou, Alexandra Z; Klostermeier, Dagmar

2013-01-01

DEAD-box helicases catalyze the ATP-dependent unwinding of RNA duplexes. They share a helicase core formed by two RecA-like domains that carries a set of conserved motifs contributing to ATP binding and hydrolysis, RNA binding and duplex unwinding. The translation initiation factor eIF4A is the founding member of the DEAD-box protein family, and one of the few examples of DEAD-box proteins that consist of a helicase core only. It is an RNA-stimulated ATPase and a non-processive helicase that unwinds short RNA duplexes. In the catalytic cycle, a series of conformational changes couples the nucleotide cycle to RNA unwinding. eIF4A has been considered a paradigm for DEAD-box proteins, and studies of its function have revealed the governing principles underlying the DEAD-box helicase mechanism. However, as an isolated helicase core, eIF4A is rather the exception, not the rule. Most helicase modules in other DEAD-box proteins are modified, some by insertions into the RecA-like domains, and the majority by N- and C-terminal appendages. While the basic catalytic function resides within the helicase core, its modulation by insertions, additional domains or a network of interaction partners generates the diversity of DEAD-box protein functions in the cell. This review summarizes the current knowledge on eIF4A and its regulation, and discusses to what extent eIF4A serves as a model DEAD-box protein.

Targeting the eIF4F translation initiation complex: a critical nexus for cancer development.

PubMed

Pelletier, Jerry; Graff, Jeremy; Ruggero, Davide; Sonenberg, Nahum

2015-01-15

Elevated protein synthesis is an important feature of many cancer cells and often arises as a consequence of increased signaling flux channeled to eukaryotic initiation factor 4F (eIF4F), the key regulator of the mRNA-ribosome recruitment phase of translation initiation. In many cellular and preclinical models of cancer, eIF4F deregulation results in changes in translational efficiency of specific mRNA classes. Importantly, many of these mRNAs code for proteins that potently regulate critical cellular processes, such as cell growth and proliferation, enhanced cell survival and cell migration that ultimately impinge on several hallmarks of cancer, including increased angiogenesis, deregulated growth control, enhanced cellular survival, epithelial-to-mesenchymal transition, invasion, and metastasis. By being positioned as the molecular nexus downstream of key oncogenic signaling pathways (e.g., Ras, PI3K/AKT/TOR, and MYC), eIF4F serves as a direct link between important steps in cancer development and translation initiation. Identification of mRNAs particularly responsive to elevated eIF4F activity that typifies tumorigenesis underscores the critical role of eIF4F in cancer and raises the exciting possibility of developing new-in-class small molecules targeting translation initiation as antineoplastic agents. ©2014 American Association for Cancer Research.

EIF2AK4 Mutations in Patients Diagnosed With Pulmonary Arterial Hypertension.

PubMed

Best, D Hunter; Sumner, Kelli L; Smith, Benjamin P; Damjanovich-Colmenares, Kristy; Nakayama, Ikue; Brown, Lynette M; Ha, Youna; Paul, Eleri; Morris, Ashley; Jama, Mohamed A; Dodson, Mark W; Bayrak-Toydemir, Pinar; Elliott, C Gregory

2017-04-01

Differentiating pulmonary venoocclusive disease (PVOD) and pulmonary capillary hemangiomatosis (PCH) from idiopathic pulmonary arterial hypertension (IPAH) or heritable pulmonary arterial hypertension (HPAH) is important clinically. Mutations in eukaryotic translation initiation factor 2 alpha kinase 4 (EIF2AK4) cause heritable PVOD and PCH, whereas mutations in other genes cause HPAH. The aim of this study was to describe the frequency of pathogenic EIF2AK4 mutations in patients diagnosed clinically with IPAH or HPAH. Sanger sequencing and deletion/duplication analysis were performed to detect mutations in the bone morphogenetic protein receptor type II (BMPR2) gene in 81 patients diagnosed at 30 North American medical centers with IPAH (n = 72) or HPAH (n = 9). BMPR2 mutation-negative patients (n = 67) were sequenced for mutations in four other genes (ACVRL1, ENG, CAV1, and KCNK3) known to cause HPAH. Patients negative for mutations in all known PAH genes (n = 66) were then sequenced for mutations in EIF2AK4. We assessed the pathogenicity of EIF2AK4 mutations and reviewed clinical characteristics of patients with pathogenic EIF2AK4 mutations. Pathogenic BMPR2 mutations were identified in 8 of 72 (11.1%) patients with IPAH and 6 of 9 (66.7%) patients with HPAH. A novel homozygous EIF2AK4 mutation (c.257+4A>C) was identified in 1 of 9 (11.1%) patients diagnosed with HPAH. The novel EIF2AK4 mutation (c.257+4A>C) was homozygous in two sisters with severe pulmonary hypertension. None of the 72 patients with IPAH had biallelic EIF2AK4 mutations. Pathogenic biallelic EIF2AK4 mutations are rarely identified in patients diagnosed with HPAH. Identification of pathogenic biallelic EIF2AK4 mutations can aid clinicians in differentiating HPAH from heritable PVOD or PCH. Copyright © 2016 American College of Chest Physicians. Published by Elsevier Inc. All rights reserved.

Structure of the exportin Xpo4 in complex with RanGTP and the hypusine-containing translation factor eIF5A

PubMed Central

Aksu, Metin; Trakhanov, Sergei; Görlich, Dirk

2016-01-01

Xpo4 is a bidirectional nuclear transport receptor that mediates nuclear export of eIF5A and Smad3 as well as import of Sox2 and SRY. How Xpo4 recognizes such a variety of cargoes is as yet unknown. Here we present the crystal structure of the RanGTP·Xpo4·eIF5A export complex at 3.2 Å resolution. Xpo4 has a similar structure as CRM1, but the NES-binding site is occluded, and a new interaction site evolved that recognizes both globular domains of eIF5A. eIF5A contains hypusine, a unique amino acid with two positive charges, which is essential for cell viability and eIF5A function in translation. The hypusine docks into a deep, acidic pocket of Xpo4 and is thus a critical element of eIF5A's complex export signature. This further suggests that Xpo4 recognizes other cargoes differently, and illustrates how Xpo4 suppresses – in a chaperone-like manner – undesired interactions of eIF5A inside nuclei. PMID:27306458

eIF4A RNA Helicase Associates with Cyclin-Dependent Protein Kinase A in Proliferating Cells and Is Modulated by Phosphorylation1[OPEN

PubMed Central

Bush, Maxwell S.; Pierrat, Olivier; Nibau, Candida; Mikitova, Veronika; Zheng, Tao; Corke, Fiona M. K.; Mayberry, Laura K.; Browning, Karen S.

2016-01-01

Eukaryotic initiation factor 4A (eIF4A) is a highly conserved RNA-stimulated ATPase and helicase involved in the initiation of messenger RNA translation. Previously, we found that eIF4A interacts with cyclin-dependent kinase A (CDKA), the plant ortholog of mammalian CDK1. Here, we show that this interaction occurs only in proliferating cells where the two proteins coassociate with 5′-cap-binding protein complexes, eIF4F or the plant-specific eIFiso4F. CDKA phosphorylates eIF4A on a conserved threonine residue (threonine-164) within the RNA-binding motif 1b TPGR. In vivo, a phospho-null (APGR) variant of the Arabidopsis (Arabidopsis thaliana) eIF4A1 protein retains the ability to functionally complement a mutant (eif4a1) plant line lacking eIF4A1, whereas a phosphomimetic (EPGR) variant fails to complement. The phospho-null variant (APGR) rescues the slow growth rate of roots and rosettes, together with the ovule-abortion and late-flowering phenotypes. In vitro, wild-type recombinant eIF4A1 and its phospho-null variant both support translation in cell-free wheat germ extracts dependent upon eIF4A, but the phosphomimetic variant does not support translation and also was deficient in ATP hydrolysis and helicase activity. These observations suggest a mechanism whereby CDK phosphorylation has the potential to down-regulate eIF4A activity and thereby affect translation. PMID:27388680

The Expression Alteration of BC1 RNA and its Interaction with Eukaryotic Translation Initiation Factor eIF4A Post-Status Epilepticus.

PubMed

Zeng, Xiangchang; Zong, Wenjing; Gao, Qing; Chen, Siyu; Chen, Lulu; Zeng, Guirong; Huang, Weihua; Li, Zhenyu; Zeng, Chang; Xie, Yuanyuan; Li, Xiaohui; Xiao, Bo; Dongsheng-Ouyang; Hu, Kai

2018-05-17

Abnormal dendritic sprouting and synaptic remodelling are important pathological features of temporal lobe epilepsy. BC1 RNA is a translation repressor involved in the regulation of the dendritic protein synthesis and mRNA transport, which is essential for dendritic development and plasticity. The expression alteration of BC1 RNA in the pilocarpine induced epilepsy model remains unknown. It is unclear if the interactions between BC1 RNA and eukaryotic initiation factor 4A (eIF4A) exists in this model. The purpose of this study was to investigate the expression changes of BC1 RNA and its interactions with eIF4A post-status epilepticus (SE). Chloride lithium and pilocarpine were used to induce the SE rat model. Either a whole brain or hippocampus tissues were collected at different time points after SE. The expression patterns of BC1 was detected by qPCR and in situ hybridization. The levels of eIF4AI/II protein expression were analyzed via western blotting and immunohistochemistry. The BC1 RNA-eIF4AI/II interaction was determined by electrophoretic mobility shift assay (EMSA). We found that the BC1 RNA levels decreased in hippocampus 3d, 1w and 2w post-SE before the levels recovered. The eIF4AI/II began to rise 3d post-SE and reached the maximum level 1w post-SE. After 1w post-SE the levels decreased in the hippocampal CA1, CA3 and DG subregions. EMSA analysis showed that BC1 RNA specifically interacted with the eIF4AI/II. The BC1 RNA-eIF4AI/II complex reduced to the lowest level 1w post-SE. Our results suggested that BC1 has a negative regulatory correlation with eIF4AI/II, where BC1 RNA could be involved in epileptogenesis by regulating dendritic protein synthesis.

Eukaryotic Initiation Factor (eIF) 4F Binding to Barley Yellow Dwarf Virus (BYDV) 3′-Untranslated Region Correlates with Translation Efficiency*

PubMed Central

Banerjee, Bidisha; Goss, Dixie J.

2014-01-01

Eukaryotic initiation factor (eIF) 4F binding to mRNA is the first committed step in cap-dependent protein synthesis. Barley yellow dwarf virus (BYDV) employs a cap-independent mechanism of translation initiation that is mediated by a structural BYDV translation element (BTE) located in the 3′-UTR of its mRNA. eIF4F bound the BTE and a translationally inactive mutant with high affinity, thus questioning the role of eIF4F in translation of BYDV. To examine the effects of eIF4F in BYDV translation initiation, BTE mutants with widely different in vitro translation efficiencies ranging from 5 to 164% compared with WT were studied. Using fluorescence anisotropy to obtain quantitative data, we show 1) the equilibrium binding affinity (complex stability) correlated well with translation efficiency, whereas the “on” rate of binding did not; 2) other unidentified proteins or small molecules in wheat germ extract prevented eIF4F binding to mutant BTE but not WT BTE; 3) BTE mutant-eIF4F interactions were found to be both enthalpically and entropically favorable with an enthalpic contribution of 52–90% to ΔG° at 25 °C, suggesting that hydrogen bonding contributes to stability; and 4) in contrast to cap-dependent and tobacco etch virus internal ribosome entry site interaction with eIF4F, poly(A)-binding protein did not increase eIF4F binding. Further, the eIF4F bound to the 3′ BTE with higher affinity than for either m7G cap or tobacco etch virus internal ribosome entry site, suggesting that the 3′ BTE may play a role in sequestering host cell initiation factors and possibly regulating the switch from replication to translation. PMID:24379412

Nuclear assortment of eIF4E coincides with shut-off of host protein synthesis upon poliovirus infection.

PubMed

Sukarieh, R; Sonenberg, N; Pelletier, J

2010-05-01

Eukaryotic initiation factor (eIF) 4E is a subunit of the cap-binding protein complex, eIF4F, which recognizes the cap structure of cellular mRNAs to facilitate translation initiation. eIF4E is assembled into the eIF4F complex via its interaction with eIF4G, an event that is under Akt/mTOR regulation. The eIF4E-eIF4G interaction is regulated by the eIF4E binding partners, eIF4E-binding proteins and eIF4E-transporter. Cleavage of eIF4G occurs upon poliovirus infection and is responsible for the shut-off of host-cell protein synthesis observed early in infection. Here, we document that relocalization of eIF4E to the nucleus occurs concomitantly with cleavage of eIF4G upon poliovirus infection. This event is not dependent upon virus replication, but is dependent on eIF4G cleavage. We postulate that eIF4E nuclear relocalization may contribute to the shut-off of host protein synthesis that is a hallmark of poliovirus infection by perturbing the circular status of actively translating mRNAs.

mTORC1 and CK2 coordinate ternary and eIF4F complex assembly

PubMed Central

Gandin, Valentina; Masvidal, Laia; Cargnello, Marie; Gyenis, Laszlo; McLaughlan, Shannon; Cai, Yutian; Tenkerian, Clara; Morita, Masahiro; Balanathan, Preetika; Jean-Jean, Olivier; Stambolic, Vuk; Trost, Matthias; Furic, Luc; Larose, Louise; Koromilas, Antonis E.; Asano, Katsura; Litchfield, David; Larsson, Ola; Topisirovic, Ivan

2016-01-01

Ternary complex (TC) and eIF4F complex assembly are the two major rate-limiting steps in translation initiation regulated by eIF2α phosphorylation and the mTOR/4E-BP pathway, respectively. How TC and eIF4F assembly are coordinated, however, remains largely unknown. We show that mTOR suppresses translation of mRNAs activated under short-term stress wherein TC recycling is attenuated by eIF2α phosphorylation. During acute nutrient or growth factor stimulation, mTORC1 induces eIF2β phosphorylation and recruitment of NCK1 to eIF2, decreases eIF2α phosphorylation and bolsters TC recycling. Accordingly, eIF2β mediates the effect of mTORC1 on protein synthesis and proliferation. In addition, we demonstrate a formerly undocumented role for CK2 in regulation of translation initiation, whereby CK2 stimulates phosphorylation of eIF2β and simultaneously bolsters eIF4F complex assembly via the mTORC1/4E-BP pathway. These findings imply a previously unrecognized mode of translation regulation, whereby mTORC1 and CK2 coordinate TC and eIF4F complex assembly to stimulate cell proliferation. PMID:27040916

High eIF4E, VEGF, and Microvessel Density in Stage I to III Breast Cancer

PubMed Central

Byrnes, Kerry; White, Stephen; Chu, Quyen; Meschonat, Carol; Yu, Herbert; Johnson, Lester W.; DeBenedetti, Arrigo; Abreo, Fleurette; Turnage, Richard H.; McDonald, John C.; Li, Benjamin D.

2006-01-01

Objective: In a prospective trial, to determine if eIF4E overexpression in breast cancer specimens is correlated with VEGF elevation, increased tumor microvessel density (MVD) counts, and a worse clinical outcome irrespective of nodal status. Summary and Background Data: In vitro, the overexpression of eukaryotic initiation factor 4E (eIF4E) up-regulates the translation of mRNAs with long 5′-untranslated regions (5′-UTRs). One such gene product is the vascular endothelial growth factor (VEGF). Methods: A total of 114 stage I to III breast cancer patients were prospectively accrued and followed with a standardized clinical surveillance protocol. Cancer specimens were quantified for eIF4E, VEGF, and MVD. Outcome endpoints were cancer recurrence and cancer-related death. Results: eIF4E overexpression was found in all cancer specimens (mean ± SD, 12.5 ± 7.6-fold). Increasing eIF4E overexpression correlated with increasing VEGF elevation (r = 0.24, P = 0.01, Spearman's coefficient), and increasing MVD counts (r = 0.35, P < 0.0002). Patients whose tumor had high eIF4E overexpression had shorter disease-free survival (P = 0.004, log-rank test) and higher cancer-related deaths (P = 0.002) than patients whose tumors had low eIF4E overexpression. Patients with high eIF4E had a hazard ratio for cancer recurrence and cancer-related death of 1.8 and 2.1 times that of patients with low eIF4E (respectively, P = 0.009 and P = 0.002, Cox proportional hazard model). Conclusions: In breast cancer patients, increasing eIF4E overexpression in the cancer specimens correlates with higher VEGF levels and MVD counts. Patients whose tumors had high eIF4E overexpression had a worse clinical outcome, independent of nodal status. Thus, eIF4E overexpression in breast cancer appears to predict increased tumor vascularity and perhaps cancer dissemination by hematogenous means. PMID:16633004

Autism-related deficits via dysregulated eIF4E-dependent translational control.

PubMed

Gkogkas, Christos G; Khoutorsky, Arkady; Ran, Israeli; Rampakakis, Emmanouil; Nevarko, Tatiana; Weatherill, Daniel B; Vasuta, Cristina; Yee, Stephanie; Truitt, Morgan; Dallaire, Paul; Major, François; Lasko, Paul; Ruggero, Davide; Nader, Karim; Lacaille, Jean-Claude; Sonenberg, Nahum

2013-01-17

Hyperconnectivity of neuronal circuits due to increased synaptic protein synthesis is thought to cause autism spectrum disorders (ASDs). The mammalian target of rapamycin (mTOR) is strongly implicated in ASDs by means of upstream signalling; however, downstream regulatory mechanisms are ill-defined. Here we show that knockout of the eukaryotic translation initiation factor 4E-binding protein 2 (4E-BP2)-an eIF4E repressor downstream of mTOR-or eIF4E overexpression leads to increased translation of neuroligins, which are postsynaptic proteins that are causally linked to ASDs. Mice that have the gene encoding 4E-BP2 (Eif4ebp2) knocked out exhibit an increased ratio of excitatory to inhibitory synaptic inputs and autistic-like behaviours (that is, social interaction deficits, altered communication and repetitive/stereotyped behaviours). Pharmacological inhibition of eIF4E activity or normalization of neuroligin 1, but not neuroligin 2, protein levels restores the normal excitation/inhibition ratio and rectifies the social behaviour deficits. Thus, translational control by eIF4E regulates the synthesis of neuroligins, maintaining the excitation-to-inhibition balance, and its dysregulation engenders ASD-like phenotypes.

Translation Initiation Factor eIF4B Interacts with a Picornavirus Internal Ribosome Entry Site in both 48S and 80S Initiation Complexes Independently of Initiator AUG Location

PubMed Central

Ochs, Kerstin; Rust, René C.; Niepmann, Michael

1999-01-01

Most eukaryotic initiation factors (eIFs) are required for internal translation initiation at the internal ribosome entry site (IRES) of picornaviruses. eIF4B is incorporated into ribosomal 48S initiation complexes with the IRES RNA of foot-and-mouth disease virus (FMDV). In contrast to the weak interaction of eIF4B with capped cellular mRNAs and its release upon entry of the ribosomal 60S subunit, eIF4B remains tightly associated with the FMDV IRES during formation of complete 80S ribosomes. Binding of eIF4B to the IRES is energy dependent, and binding of the small ribosomal subunit to the IRES requires the previous energy-dependent association of initiation factors with the IRES. The interaction of eIF4B with the IRES in 48S and 80S complexes is independent of the location of the initiator AUG and thus independent of the mechanism by which the small ribosomal subunit is placed at the actual start codon, either by direct internal ribosomal entry or by scanning. eIF4B does not greatly rearrange its binding to the IRES upon entry of the ribosomal subunits, and the interaction of eIF4B with the IRES is independent of the polypyrimidine tract-binding protein, which enhances FMDV translation. PMID:10438840

Plant growth and fertility requires functional interactions between specific PABP and eIF4G gene family members

PubMed Central

2018-01-01

The initiation of protein synthesis requires the involvement of the eukaryotic translation initiation factor (eIF) 4G to promote assembly of the factors needed to recruit a 40S ribosomal subunit to an mRNA. Although many eukaryotes express two eIF4G isoforms that are highly similar, those in plants, referred to as eIF4G and eIFiso4G, are highly divergent in size, sequence, and domain organization. Species of the Brassicaceae and the Cleomaceae also express a divergent eIFiso4G isoform, referred to as eIFiso4G2, not found elsewhere in the plant kingdom. Despite their divergence, eIF4G and eIFiso4G interact with eIF4A, eIF4B, and eIF4E isoforms needed for binding an mRNA. eIF4G and eIFiso4G also interact with the poly(A)-binding protein (PABP) which promotes ribosome recruitment to an mRNA. Increasing the complexity of such an interaction, however, Arabidopsis also expresses three PABP isoforms (PAB2, PAB4, and PAB8) in vegetative and reproductive tissues. In this study, the functional interactions among the eIF4G and the widely-expressed PABP isoforms were examined. Loss of PAB2 or PAB8 in combination with loss of eIF4G or eIFiso4G had little to no effect on growth or fertility whereas pab2 pab8 eif4g or pab2 pab8 eifiso4g1/2 mutants exhibited smaller stature and reduced fertility. Although the pab4 eifiso4g1 mutant grows normally and is fertile, pab4 eif4g or pab4 eifiso4g2 mutants could not be isolated. Even pab4/PAB4 eif4g/eIF4G heterozygous plants exhibited growth defects and low fertility. Mutant co-inheritance analysis in reciprocal crosses with wild-type plants revealed that most ovaries and pollen from pab4/PAB4 eif4g/eIF4G plants were PAB4 eif4g. Similarly, co-inheritance studies with pab4/PAB4 eifiso4g2/eIFiso4G2 plants suggested most ovaries were PAB4 eifiso4g2. These results suggest that a functional interaction between PAB4 and eIF4G and between PAB4 and eIFiso4G2 is required for growth and normal fertility. PMID:29381712

Characterization of a novel RNA-binding region of eIF4GI critical for ribosomal scanning

PubMed Central

Prévôt, Déborah; Décimo, Didier; Herbreteau, Cécile H.; Roux, Florence; Garin, Jérôme; Darlix, Jean-Luc; Ohlmann, Théophile

2003-01-01

The eukaryotic translation initiation factor eIF4GI binds several proteins and acts as a scaffold to promote preinitiation complex formation on the mRNA molecule (48S). Following mRNA attachment this complex scans along the messenger in a 5′ to 3′ direction until it locates and recognizes the initiation start codon. By using a combination of retroviral and picornaviral proteases (HIV-2 and L respectively) in the reticulocyte lysate system, we have characterized a 40 amino acid (aa) region of eIF4GI (aa 642–681) that exhibits general RNA-binding properties. Removal of this domain by proteolytic processing followed by translational assays showed virtually no inhibition of internal ribosome entry on the encephalomyocarditis virus, but resulted in drastic impairment of ribosome scanning as demonstrated by studying poliovirus and foot-and-mouth disease virus translation. Based on these findings, we propose that this 40 aa motif of eIF4GI is critical for ribosome scanning. PMID:12682023

Toward the mechanism of eIF4F-mediated ribosomal attachment to mammalian capped mRNAs.

PubMed

Kumar, Parimal; Hellen, Christopher U T; Pestova, Tatyana V

2016-07-01

Ribosomal attachment to mammalian capped mRNAs is achieved through the cap-eukaryotic initiation factor 4E (eIF4E)-eIF4G-eIF3-40S chain of interactions, but the mechanism by which mRNA enters the mRNA-binding channel of the 40S subunit remains unknown. To investigate this process, we recapitulated initiation on capped mRNAs in vitro using a reconstituted translation system. Formation of initiation complexes at 5'-terminal AUGs was stimulated by the eIF4E-cap interaction and followed "the first AUG" rule, indicating that it did not occur by backward scanning. Initiation complexes formed even at the very 5' end of mRNA, implying that Met-tRNAi (Met) inspects mRNA from the first nucleotide and that initiation does not have a "blind spot." In assembled initiation complexes, the cap was no longer associated with eIF4E. Omission of eIF4A or disruption of eIF4E-eIF4G-eIF3 interactions converted eIF4E into a specific inhibitor of initiation on capped mRNAs. Taken together, these results are consistent with the model in which eIF4E-eIF4G-eIF3-40S interactions place eIF4E at the leading edge of the 40S subunit, and mRNA is threaded into the mRNA-binding channel such that Met-tRNAi (Met) can inspect it from the first nucleotide. Before entering, eIF4E likely dissociates from the cap to overcome steric hindrance. We also found that the m(7)G cap specifically interacts with eIF3l. © 2016 Kumar et al.; Published by Cold Spring Harbor Laboratory Press.

Unphosphorylated HSP27 (HSPB1) regulates the translation initiation process via a direct association with eIF4E in osteoblasts.

PubMed

Kuroyanagi, Gen; Tokuda, Haruhiko; Yamamoto, Naohiro; Matsushima-Nishiwaki, Rie; Kozawa, Osamu; Otsuka, Takanobu

2015-09-01

Heat-shock protein 27 (HSP27/HSPB1) and its phosphorylation are implicated in multiple physiological and pathophysiological cell functions. Our previous study reported that unphosphorylated HSP27 has an inhibitory role in triiodothyronine (T(3))‑induced osteocalcin (OC) synthesis in osteoblasts. However, the mechanisms behind the HSP27‑mediated effects on osteoblasts remain to be clarified. In the present study, to investigate the exact mechanism of HSP27 and its phosphorylation in osteoblasts, the molecular targets of HSP27 were explored using osteoblast‑like MC3T3‑E1 cells. The levels of OC mRNA induced by T(3) in the HSP27‑overexpressing cells did not show any significant differences compared with those in the control empty vector‑transfected cells. Therefore, the interactions between HSP27 and translational molecules were focused on, including eukaryotic translation initiation factor 4E (eIF4E), eIF4G and 4E‑binding protein 1 (4E‑BP1). The HSP27 protein in the unstimulated cells co‑immunoprecipitated with eIF4E, but not eIF4G or 4E‑BP1. In addition, the association of eIF4E with 4E‑BP1 was observed in the HSP27‑overexpressing cells, as well as in the control cells. Under T(3) stimulation, the binding of eIF4E to eIF4G was markedly attenuated in the HSP27‑overexpressing cells compared with the control cells. In addition, the binding of HSP27 to eIF4E in the unstimulated cells was diminished by the phosphorylation of HSP27. In response to T(3) stimulation, the association of eIF4E with eIF4G in the unphosphorylatable HSP27‑overexpressing cells was markedly reduced compared with the phospho‑mimic HSP27‑overexpressing cells. Taken together, these findings strongly suggest that unphosphorylated HSP27 associates with eIF4E in osteoblasts and suppresses the translation initiation process.

Sapovirus Translation Requires an Interaction between VPg and the Cap Binding Protein eIF4E

PubMed Central

Hosmillo, Myra; Chaudhry, Yasmin; Kim, Deok-Song

2014-01-01

ABSTRACT Sapoviruses of the Caliciviridae family of small RNA viruses are emerging pathogens that cause gastroenteritis in humans and animals. Molecular studies on human sapovirus have been hampered due to the lack of a cell culture system. In contrast, porcine sapovirus (PSaV) can be grown in cell culture, making it a suitable model for understanding the infectious cycle of sapoviruses and the related enteric caliciviruses. Caliciviruses are known to use a novel mechanism of protein synthesis that relies on the interaction of cellular translation initiation factors with the virus genome-encoded viral protein genome (VPg) protein, which is covalently linked to the 5′ end of the viral genome. Using PSaV as a representative member of the Sapovirus genus, we characterized the role of the viral VPg protein in sapovirus translation. As observed for other caliciviruses, the PSaV genome was found to be covalently linked to VPg, and this linkage was required for the translation and the infectivity of viral RNA. The PSaV VPg protein was associated with the 4F subunit of the eukaryotic translation initiation factor (eIF4F) complex in infected cells and bound directly to the eIF4E protein. As has been previously demonstrated for feline calicivirus, a member of the Vesivirus genus, PSaV translation required eIF4E and the interaction between eIF4E and eIF4G. Overall, our study provides new insights into the novel mechanism of sapovirus translation, suggesting that sapovirus VPg can hijack the cellular translation initiation mechanism by recruiting the eIF4F complex through a direct eIF4E interaction. IMPORTANCE Sapoviruses, which are members of the Caliciviridae family, are one of the causative agents of viral gastroenteritis in humans. However, human sapovirus remains noncultivable in cell culture, hampering the ability to characterize the virus infectious cycle. Here, we show that the VPg protein from porcine sapovirus, the only cultivatable sapovirus, is essential for

Sapovirus translation requires an interaction between VPg and the cap binding protein eIF4E.

PubMed

Hosmillo, Myra; Chaudhry, Yasmin; Kim, Deok-Song; Goodfellow, Ian; Cho, Kyoung-Oh

2014-11-01

Sapoviruses of the Caliciviridae family of small RNA viruses are emerging pathogens that cause gastroenteritis in humans and animals. Molecular studies on human sapovirus have been hampered due to the lack of a cell culture system. In contrast, porcine sapovirus (PSaV) can be grown in cell culture, making it a suitable model for understanding the infectious cycle of sapoviruses and the related enteric caliciviruses. Caliciviruses are known to use a novel mechanism of protein synthesis that relies on the interaction of cellular translation initiation factors with the virus genome-encoded viral protein genome (VPg) protein, which is covalently linked to the 5' end of the viral genome. Using PSaV as a representative member of the Sapovirus genus, we characterized the role of the viral VPg protein in sapovirus translation. As observed for other caliciviruses, the PSaV genome was found to be covalently linked to VPg, and this linkage was required for the translation and the infectivity of viral RNA. The PSaV VPg protein was associated with the 4F subunit of the eukaryotic translation initiation factor (eIF4F) complex in infected cells and bound directly to the eIF4E protein. As has been previously demonstrated for feline calicivirus, a member of the Vesivirus genus, PSaV translation required eIF4E and the interaction between eIF4E and eIF4G. Overall, our study provides new insights into the novel mechanism of sapovirus translation, suggesting that sapovirus VPg can hijack the cellular translation initiation mechanism by recruiting the eIF4F complex through a direct eIF4E interaction. Sapoviruses, which are members of the Caliciviridae family, are one of the causative agents of viral gastroenteritis in humans. However, human sapovirus remains noncultivable in cell culture, hampering the ability to characterize the virus infectious cycle. Here, we show that the VPg protein from porcine sapovirus, the only cultivatable sapovirus, is essential for viral translation and

Two related trypanosomatid eIF4G homologues have functional differences compatible with distinct roles during translation initiation

PubMed Central

Moura, Danielle MN; Reis, Christian RS; Xavier, Camila C; da Costa Lima, Tamara D; Lima, Rodrigo P; Carrington, Mark; de Melo Neto, Osvaldo P

2015-01-01

In higher eukaryotes, eIF4A, eIF4E and eIF4G homologues interact to enable mRNA recruitment to the ribosome. eIF4G acts as a scaffold for these interactions and also interacts with other proteins of the translational machinery. Trypanosomatid protozoa have multiple homologues of eIF4E and eIF4G and the precise function of each remains unclear. Here, 2 previously described eIF4G homologues, EIF4G3 and EIF4G4, were further investigated. In vitro, both homologues bound EIF4AI, but with different interaction properties. Binding to distinct eIF4Es was also confirmed; EIF4G3 bound EIF4E4 while EIF4G4 bound EIF4E3, both these interactions required similar binding motifs. EIF4G3, but not EIF4G4, interacted with PABP1, a poly-A binding protein homolog. Work in vivo with Trypanosoma brucei showed that both EIF4G3 and EIF4G4 are cytoplasmic and essential for viability. Depletion of EIF4G3 caused a rapid reduction in total translation while EIF4G4 depletion led to changes in morphology but no substantial inhibition of translation. Site-directed mutagenesis was used to disrupt interactions of the eIF4Gs with either eIF4E or eIF4A, causing different levels of growth inhibition. Overall the results show that only EIF4G3, with its cap binding partner EIF4E4, plays a major role in translational initiation. PMID:25826663

A Case-Matched Gender Comparison Transcriptomic Screen Identifies eIF4E and eIF5 as Potential Prognostic Markers in Male Breast Cancer.

PubMed

Humphries, Matthew P; Sundara Rajan, Sreekumar; Droop, Alastair; Suleman, Charlotte A B; Carbone, Carmine; Nilsson, Cecilia; Honarpisheh, Hedieh; Cserni, Gabor; Dent, Jo; Fulford, Laura; Jordan, Lee B; Jones, J Louise; Kanthan, Rani; Litwiniuk, Maria; Di Benedetto, Anna; Mottolese, Marcella; Provenzano, Elena; Shousha, Sami; Stephens, Mark; Walker, Rosemary A; Kulka, Janina; Ellis, Ian O; Jeffery, Margaret; Thygesen, Helene H; Cappelletti, Vera; Daidone, Maria G; Hedenfalk, Ingrid A; Fjällskog, Marie-Louise; Melisi, Davide; Stead, Lucy F; Shaaban, Abeer M; Speirs, Valerie

2017-05-15

Purpose: Breast cancer affects both genders, but is understudied in men. Although still rare, male breast cancer (MBC) is being diagnosed more frequently. Treatments are wholly informed by clinical studies conducted in women, based on assumptions that underlying biology is similar. Experimental Design: A transcriptomic investigation of male and female breast cancer was performed, confirming transcriptomic data in silico Biomarkers were immunohistochemically assessed in 697 MBCs ( n = 477, training; n = 220, validation set) and quantified in pre- and posttreatment samples from an MBC patient receiving everolimus and PI3K/mTOR inhibitor. Results: Gender-specific gene expression patterns were identified. eIF transcripts were upregulated in MBC. eIF4E and eIF5 were negatively prognostic for overall survival alone (log-rank P = 0.013; HR = 1.77, 1.12-2.8 and P = 0.035; HR = 1.68, 1.03-2.74, respectively), or when coexpressed ( P = 0.01; HR = 2.66, 1.26-5.63), confirmed in the validation set. This remained upon multivariate Cox regression analysis [eIF4E P = 0.016; HR = 2.38 (1.18-4.8), eIF5 P = 0.022; HR = 2.55 (1.14-5.7); coexpression P = 0.001; HR = 7.04 (2.22-22.26)]. Marked reduction in eIF4E and eIF5 expression was seen post BEZ235/everolimus, with extended survival. Conclusions: Translational initiation pathway inhibition could be of clinical utility in MBC patients overexpressing eIF4E and eIF5. With mTOR inhibitors that target this pathway now in the clinic, these biomarkers may represent new targets for therapeutic intervention, although further independent validation is required. Clin Cancer Res; 23(10); 2575-83. ©2016 AACR . ©2016 American Association for Cancer Research.

Identification of a Kinase in Wheat Germ that Phosphorylates the Large Subunit of Initiation Factor 4F 1

PubMed Central

Humphreys, Jean; Browning, Karen S.; Ravel, Joanne M.

1988-01-01

A kinase has been isolated from wheat (Triticum aestivum) germ that phosphorylates the 220 kilodaltons (kD) subunit of wheat germ initiation factor (eIF) 4F, the 80 kD subunit of eIF-4B (an isozyme form of eIF-4F) and eIF-4G (the functional equivalent to mammalian eIF-4B). The kinase elutes from Sephacryl S-200 slightly in front of ovalbumin. The kinase phosphorylates casein and histone IIA to a small extent, but does not phosphorylate phosvitin. Of the wheat germ initiation factors, elongation factors, and small and large ribosomal subunits, only eIF-4F, eIF-4B, and eIF-4G are phosphorylated to a significant extent. The kinase phosphorylates eIF-4F to the extent of two phosphates per mole of the 220 kD subunit and phosphorylates eIF-4B to the extent of one phosphate per mole of the 80 kD subunit. The 26 kD subunit of eIF-4F and the 28 kD subunit of eIF-4B are not phosphorylated by the kinase. The kinase phosphorylates the 59 kD component of eIF-4G to the extent of 0.25 phosphate per mole of eIF-4G. Phosphorylation of eIF-4F and eIF-4B does not affect their ability to support the binding of mRNA to small ribosomal subunits in vitro. Images Fig. 2 Fig. 3 PMID:16666331

Geographical gradient of the eIF4E alleles conferring resistance to potyviruses in pea (Pisum) germplasm.

PubMed

Konečná, Eva; Šafářová, Dana; Navrátil, Milan; Hanáček, Pavel; Coyne, Clarice; Flavell, Andrew; Vishnyakova, Margarita; Ambrose, Mike; Redden, Robert; Smýkal, Petr

2014-01-01

The eukaryotic translation initiation factor 4E was shown to be involved in resistance against several potyviruses in plants, including pea. We combined our knowledge of pea germplasm diversity with that of the eIF4E gene to identify novel genetic diversity. Germplasm of 2803 pea accessions was screened for eIF4E intron 3 length polymorphism, resulting in the detection of four eIF4E(A-B-C-S) variants, whose distribution was geographically structured. The eIF4E(A) variant conferring resistance to the P1 PSbMV pathotype was found in 53 accessions (1.9%), of which 15 were landraces from India, Afghanistan, Nepal, and 7 were from Ethiopia. A newly discovered variant, eIF4E(B), was present in 328 accessions (11.7%) from Ethiopia (29%), Afghanistan (23%), India (20%), Israel (25%) and China (39%). The eIF4E(C) variant was detected in 91 accessions (3.2% of total) from India (20%), Afghanistan (33%), the Iberian Peninsula (22%) and the Balkans (9.3%). The eIF4E(S) variant for susceptibility predominated as the wild type. Sequencing of 73 samples, identified 34 alleles at the whole gene, 26 at cDNA and 19 protein variants, respectively. Fifteen alleles were virologically tested and 9 alleles (eIF4E(A-1-2-3-4-5-6-7), eIF4E(B-1), eIF4E(C-2)) conferred resistance to the P1 PSbMV pathotype. This work identified novel eIF4E alleles within geographically structured pea germplasm and indicated their independent evolution from the susceptible eIF4E(S1) allele. Despite high variation present in wild Pisum accessions, none of them possessed resistance alleles, supporting a hypothesis of distinct mode of evolution of resistance in wild as opposed to crop species. The Highlands of Central Asia, the northern regions of the Indian subcontinent, Eastern Africa and China were identified as important centers of pea diversity that correspond with the diversity of the pathogen. The series of alleles identified in this study provides the basis to study the co-evolution of potyviruses and the

Attacking a Nexus of the Oncogenic Circuitry by Reversing Aberrant eIF4F-Mediated Translation

PubMed Central

Bitterman, Peter B.; Polunovsky, Vitaly A.

2012-01-01

Notwithstanding their genetic complexity, different cancers share a core group of perturbed pathways converging upon a few regulatory nodes that link the intracellular signaling network with the basic metabolic machinery. The clear implication of this view for cancer therapy is that instead of targeting individual genetic alterations one-by-one, the next generation of cancer therapeutics will target critical hubs in the cancer network. One such hub is the translation initiation complex eIF4F, which integrates several cancer-related pathways into a self-amplifying signaling system. When hyperactivated by apical oncogenic signals, the eIF4F-driven translational apparatus selectively switches the translational repertoire of a cell towards malignancy. This central integrative role of pathologically activated eIF4F has motivated the development of small molecule inhibitors to correct its function. A genome-wide, systems-level means to objectively evaluate the pharmacological response to therapeutics targeting eIF4F remains an unmet challenge. PMID:22572598

A Noncoding Expansion in EIF4A3 Causes Richieri-Costa-Pereira Syndrome, a Craniofacial Disorder Associated with Limb Defects

PubMed Central

Favaro, Francine P.; Alvizi, Lucas; Zechi-Ceide, Roseli M.; Bertola, Debora; Felix, Temis M.; de Souza, Josiane; Raskin, Salmo; Twigg, Stephen R.F.; Weiner, Andrea M.J.; Armas, Pablo; Margarit, Ezequiel; Calcaterra, Nora B.; Andersen, Gregers R.; McGowan, Simon J.; Wilkie, Andrew O.M.; Richieri-Costa, Antonio; de Almeida, Maria L.G.; Passos-Bueno, Maria Rita

2014-01-01

Richieri-Costa-Pereira syndrome is an autosomal-recessive acrofacial dysostosis characterized by mandibular median cleft associated with other craniofacial anomalies and severe limb defects. Learning and language disabilities are also prevalent. We mapped the mutated gene to a 122 kb region at 17q25.3 through identity-by-descent analysis in 17 genealogies. Sequencing strategies identified an expansion of a region with several repeats of 18- or 20-nucleotide motifs in the 5′ untranslated region (5′ UTR) of EIF4A3, which contained from 14 to 16 repeats in the affected individuals and from 3 to 12 repeats in 520 healthy individuals. A missense substitution of a highly conserved residue likely to affect the interaction of eIF4AIII with the UPF3B subunit of the exon junction complex in trans with an expanded allele was found in an unrelated individual with an atypical presentation, thus expanding mutational mechanisms and phenotypic diversity of RCPS. EIF4A3 transcript abundance was reduced in both white blood cells and mesenchymal cells of RCPS-affected individuals as compared to controls. Notably, targeting the orthologous eif4a3 in zebrafish led to underdevelopment of several craniofacial cartilage and bone structures, in agreement with the craniofacial alterations seen in RCPS. Our data thus suggest that RCPS is caused by mutations in EIF4A3 and show that EIF4A3, a gene involved in RNA metabolism, plays a role in mandible, laryngeal, and limb morphogenesis. PMID:24360810

Modulation of tumor eIF4E by antisense inhibition: A phase I/II translational clinical trial of ISIS 183750-an antisense oligonucleotide against eIF4E-in combination with irinotecan in solid tumors and irinotecan-refractory colorectal cancer.

PubMed

Duffy, A G; Makarova-Rusher, O V; Ulahannan, S V; Rahma, O E; Fioravanti, S; Walker, M; Abdullah, S; Raffeld, M; Anderson, V; Abi-Jaoudeh, N; Levy, E; Wood, B J; Lee, S; Tomita, Y; Trepel, J B; Steinberg, S M; Revenko, A S; MacLeod, A R; Peer, C J; Figg, W D; Greten, T F

2016-10-01

The eukaryotic translation initiation factor 4E (eIF4E) is a potent oncogene that is found to be dysregulated in 30% of human cancer, including colorectal carcinogenesis (CRC). ISIS 183750 is a second-generation antisense oligonucleotide (ASO) designed to inhibit the production of the eIF4E protein. In preclinical studies we found that EIF4e ASOs reduced expression of EIF4e mRNA and inhibited proliferation of colorectal carcinoma cells. An additive antiproliferative effect was observed in combination with irinotecan. We then performed a clinical trial evaluating this combination in patients with refractory cancer. No dose-limiting toxicities were seen but based on pharmacokinetic data and tolerability the dose of irinotecan was reduced to 160 mg/m(2) biweekly. Efficacy was evaluated in 15 patients with irinotecan-refractory colorectal cancer. The median time of disease control was 22.1 weeks. After ISIS 183750 treatment, peripheral blood levels of eIF4E mRNA were decreased in 13 of 19 patients. Matched pre- and posttreatment tumor biopsies showed decreased eIF4E mRNA levels in five of nine patients. In tumor tissue, the intracellular and stromal presence of ISIS 183750 was detected by IHC in all biopsied patients. Although there were no objective responses stable disease was seen in seven of 15 (47%) patients who were progressing before study entry, six of whom were stable at the time of the week 16 CT scan. We were also able to confirm through mandatory pre- and posttherapy tumor biopsies penetration of the ASO into the site of metastasis. © 2016 UICC.

Geographical Gradient of the eIF4E Alleles Conferring Resistance to Potyviruses in Pea (Pisum) Germplasm

PubMed Central

Konečná, Eva; Šafářová, Dana; Navrátil, Milan; Hanáček, Pavel; Coyne, Clarice; Flavell, Andrew; Vishnyakova, Margarita; Ambrose, Mike; Redden, Robert; Smýkal, Petr

2014-01-01

Background The eukaryotic translation initiation factor 4E was shown to be involved in resistance against several potyviruses in plants, including pea. We combined our knowledge of pea germplasm diversity with that of the eIF4E gene to identify novel genetic diversity. Methodology/Principal findings Germplasm of 2803 pea accessions was screened for eIF4E intron 3 length polymorphism, resulting in the detection of four eIF4EA-B-C-S variants, whose distribution was geographically structured. The eIF4EA variant conferring resistance to the P1 PSbMV pathotype was found in 53 accessions (1.9%), of which 15 were landraces from India, Afghanistan, Nepal, and 7 were from Ethiopia. A newly discovered variant, eIF4EB, was present in 328 accessions (11.7%) from Ethiopia (29%), Afghanistan (23%), India (20%), Israel (25%) and China (39%). The eIF4EC variant was detected in 91 accessions (3.2% of total) from India (20%), Afghanistan (33%), the Iberian Peninsula (22%) and the Balkans (9.3%). The eIF4ES variant for susceptibility predominated as the wild type. Sequencing of 73 samples, identified 34 alleles at the whole gene, 26 at cDNA and 19 protein variants, respectively. Fifteen alleles were virologically tested and 9 alleles (eIF4EA-1-2-3-4-5-6-7, eIF4EB-1, eIF4EC-2) conferred resistance to the P1 PSbMV pathotype. Conclusions/Significance This work identified novel eIF4E alleles within geographically structured pea germplasm and indicated their independent evolution from the susceptible eIF4ES1 allele. Despite high variation present in wild Pisum accessions, none of them possessed resistance alleles, supporting a hypothesis of distinct mode of evolution of resistance in wild as opposed to crop species. The Highlands of Central Asia, the northern regions of the Indian subcontinent, Eastern Africa and China were identified as important centers of pea diversity that correspond with the diversity of the pathogen. The series of alleles identified in this study provides the basis

Gating by tryptophan 73 exposes a cryptic pocket at the protein-binding interface of the oncogenic eIF4E protein.

PubMed

Lama, Dilraj; Brown, Christopher J; Lane, David P; Verma, Chandra S

2015-10-27

Targeting protein-protein interacting sites for potential therapeutic applications is a challenge in the development of inhibitors, and this becomes more difficult when these interfaces are relatively planar, as in the eukaryotic translation initiation factor 4E (eIF4E) protein. eIF4E is an oncogene that is overexpressed in numerous forms of cancer, making it a prime target as a therapeutic molecule. We report here the presence of a cryptic pocket at the protein-binding interface of eIF4E, which opens transiently during molecular dynamics simulations of the protein in solvent water and is observed to be stable when solvent water is mixed with benzene molecules. This pocket can also be seen in the ensemble of structures available from the solution-state conformations of eIF4E. The accessibility of the pocket is gated by the side-chain transitions of an evolutionarily conserved tryptophan residue. It is found to be feasible for accommodating clusters of benzene molecules, which signify the plasticity and ligandability of the pocket. We also observe that the newly formed cavity provides a favorable binding environment for interaction of a well-recognized small molecule inhibitor of eIF4E. The occurrence of this transiently accessible cavity highlights the existence of a more pronounced binding groove in a region that has traditionally been considered to be planar. Together, the data suggest that an alternate binding cavity exists on eIF4E and could be exploited for the rational design and development of a new class of lead compounds against the protein.

A noncoding expansion in EIF4A3 causes Richieri-Costa-Pereira syndrome, a craniofacial disorder associated with limb defects.

PubMed

Favaro, Francine P; Alvizi, Lucas; Zechi-Ceide, Roseli M; Bertola, Debora; Felix, Temis M; de Souza, Josiane; Raskin, Salmo; Twigg, Stephen R F; Weiner, Andrea M J; Armas, Pablo; Margarit, Ezequiel; Calcaterra, Nora B; Andersen, Gregers R; McGowan, Simon J; Wilkie, Andrew O M; Richieri-Costa, Antonio; de Almeida, Maria L G; Passos-Bueno, Maria Rita

2014-01-02

Richieri-Costa-Pereira syndrome is an autosomal-recessive acrofacial dysostosis characterized by mandibular median cleft associated with other craniofacial anomalies and severe limb defects. Learning and language disabilities are also prevalent. We mapped the mutated gene to a 122 kb region at 17q25.3 through identity-by-descent analysis in 17 genealogies. Sequencing strategies identified an expansion of a region with several repeats of 18- or 20-nucleotide motifs in the 5' untranslated region (5' UTR) of EIF4A3, which contained from 14 to 16 repeats in the affected individuals and from 3 to 12 repeats in 520 healthy individuals. A missense substitution of a highly conserved residue likely to affect the interaction of eIF4AIII with the UPF3B subunit of the exon junction complex in trans with an expanded allele was found in an unrelated individual with an atypical presentation, thus expanding mutational mechanisms and phenotypic diversity of RCPS. EIF4A3 transcript abundance was reduced in both white blood cells and mesenchymal cells of RCPS-affected individuals as compared to controls. Notably, targeting the orthologous eif4a3 in zebrafish led to underdevelopment of several craniofacial cartilage and bone structures, in agreement with the craniofacial alterations seen in RCPS. Our data thus suggest that RCPS is caused by mutations in EIF4A3 and show that EIF4A3, a gene involved in RNA metabolism, plays a role in mandible, laryngeal, and limb morphogenesis. Copyright © 2014 The American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.

The Role of eIF4E Activity in Breast Cancer

DTIC Science & Technology

2010-08-01

marker with some success. Furthermore, eIF4E is an established target for cancer therapy [3] and clinical trials of the efficacy and safety of cancer...individual group was small, for overall survival (OS), disease-free survival (DFS) and disease- specific survival ( DSS ) (Figure 2). High eIF4E scores were...indicative of poor prognosis. Prognosis seemed to worsen with each increasing eIF4E score for OS, whereas patterns for DFS and DSS sug- gested weaker

Modulation of translation-initiation in CHO-K1 cells by rapamycin-induced heterodimerization of engineered eIF4G fusion proteins.

PubMed

Schlatter, Stefan; Senn, Claudia; Fussenegger, Martin

2003-07-20

Translation-initiation is a predominant checkpoint in mammalian cells which controls protein synthesis and fine-tunes the flow of information from gene to protein. In eukaryotes, translation-initiation is typically initiated at a 7-methyl-guanylic acid cap posttranscriptionally linked to the 5' end of mRNAs. Alternative cap-independent translation-initiation involves 5' untranslated regions (UTR) known as internal ribosome entry sites, which adopt a particular secondary structure. Translation-initiating ribosome assembly at cap or IRES elements is mediated by a multiprotein complex of which the initiation factor 4F (eIF4F) consisting of eIF4A (helicase), eIF4E (cap-binding protein), and eIF4G is a major constituent. eIF4G is a key target of picornaviral protease 2A, which cleaves this initiation factor into eIF4G(Delta) and (Delta)eIF4G to redirect the cellular translation machinery exclusively to its own IRES-containing transcripts. We have designed a novel translation control system (TCS) for conditional as well as adjustable translation of cap- and IRES-dependent transgene mRNAs in mammalian cells. eIF4G(Delta) and (Delta)eIF4G were fused C- and N-terminally to the FK506-binding protein (FKBP) and the FKBP-rapamycin-binding domain (FRB) of the human FKBP-rapamycin-associated protein (FRAP), respectively. Rapamycin-induced heterodimerization of eIF4G(Delta)-FKBP and FRB-(Delta)eIF4G fusion proteins reconstituted a functional chimeric elongation factor 4G in a dose-dependent manner. Rigorous quantitative expression analysis of cap- and IRES-dependent SEAP- (human placental secreted alkaline phosphatase) and luc- (Photinus pyralis luciferase) encoding reporter constructs confirmed adjustable translation control and revealed increased production of desired proteins in response to dimerization-induced heterologous eIF4G in Chinese hamster ovary (CHO-K1) cells. Copyright 2003 Wiley Periodicals, Inc. Biotechnol Bioeng 83: 210-225, 2003.

A Conserved Interaction between a C-Terminal Motif in Norovirus VPg and the HEAT-1 Domain of eIF4G Is Essential for Translation Initiation

PubMed Central

Leen, Eoin N.; Sorgeloos, Frédéric; Correia, Samantha; Chaudhry, Yasmin; Cannac, Fabien; Pastore, Chiara; Xu, Yingqi; Graham, Stephen C.; Matthews, Stephen J.; Goodfellow, Ian G.; Curry, Stephen

2016-01-01

Translation initiation is a critical early step in the replication cycle of the positive-sense, single-stranded RNA genome of noroviruses, a major cause of gastroenteritis in humans. Norovirus RNA, which has neither a 5´ m7G cap nor an internal ribosome entry site (IRES), adopts an unusual mechanism to initiate protein synthesis that relies on interactions between the VPg protein covalently attached to the 5´-end of the viral RNA and eukaryotic initiation factors (eIFs) in the host cell. For murine norovirus (MNV) we previously showed that VPg binds to the middle fragment of eIF4G (4GM; residues 652–1132). Here we have used pull-down assays, fluorescence anisotropy, and isothermal titration calorimetry (ITC) to demonstrate that a stretch of ~20 amino acids at the C terminus of MNV VPg mediates direct and specific binding to the HEAT-1 domain within the 4GM fragment of eIF4G. Our analysis further reveals that the MNV C terminus binds to eIF4G HEAT-1 via a motif that is conserved in all known noroviruses. Fine mutagenic mapping suggests that the MNV VPg C terminus may interact with eIF4G in a helical conformation. NMR spectroscopy was used to define the VPg binding site on eIF4G HEAT-1, which was confirmed by mutagenesis and binding assays. We have found that this site is non-overlapping with the binding site for eIF4A on eIF4G HEAT-1 by demonstrating that norovirus VPg can form ternary VPg-eIF4G-eIF4A complexes. The functional significance of the VPg-eIF4G interaction was shown by the ability of fusion proteins containing the C-terminal peptide of MNV VPg to inhibit in vitro translation of norovirus RNA but not cap- or IRES-dependent translation. These observations define important structural details of a functional interaction between norovirus VPg and eIF4G and reveal a binding interface that might be exploited as a target for antiviral therapy. PMID:26734730

Mextli proteins use both canonical bipartite and novel tripartite binding modes to form eIF4E complexes that display differential sensitivity to 4E-BP regulation

PubMed Central

Peter, Daniel; Weber, Ramona; Köne, Carolin; Chung, Min-Yi; Ebertsch, Linda; Truffault, Vincent; Weichenrieder, Oliver; Igreja, Cátia; Izaurralde, Elisa

2015-01-01

The eIF4E-binding proteins (4E-BPs) are a diverse class of translation regulators that share a canonical eIF4E-binding motif (4E-BM) with eIF4G. Consequently, they compete with eIF4G for binding to eIF4E, thereby inhibiting translation initiation. Mextli (Mxt) is an unusual 4E-BP that promotes translation by also interacting with eIF3. Here we present the crystal structures of the eIF4E-binding regions of the Drosophila melanogaster (Dm) and Caenorhabditis elegans (Ce) Mxt proteins in complex with eIF4E in the cap-bound and cap-free states. The structures reveal unexpected evolutionary plasticity in the eIF4E-binding mode, with a classical bipartite interface for Ce Mxt and a novel tripartite interface for Dm Mxt. Both interfaces comprise a canonical helix and a noncanonical helix that engage the dorsal and lateral surfaces of eIF4E, respectively. Remarkably, Dm Mxt contains a C-terminal auxiliary helix that lies anti-parallel to the canonical helix on the eIF4E dorsal surface. In contrast to the eIF4G and Ce Mxt complexes, the Dm eIF4E–Mxt complexes are resistant to competition by bipartite 4E-BPs, suggesting that Dm Mxt can bind eIF4E when eIF4G binding is inhibited. Our results uncovered unexpected diversity in the binding modes of 4E-BPs, resulting in eIF4E complexes that display differential sensitivity to 4E-BP regulation. PMID:26294658

Reducing eIF4E-eIF4G Interactions Restores the Balance Between Protein Synthesis and Actin Dynamics in Fragile X Syndrome Model Mice*

PubMed Central

Santini, Emanuela; Huynh, Thu N.; Longo, Francesco; Koo, So Yeon; Mojica, Edward; D’Andrea, Laura; Bagni, Claudia; Klann, Eric

2018-01-01

Fragile X syndrome (FXS) is the most common form of inherited intellectual disability and autism spectrum disorder. FXS is caused by silencing of the FMR1 gene, which encodes fragile X mental retardation protein (FMRP), an mRNA-binding protein that represses the translation of its target mRNAs. One mechanism by which FMRP represses translation is through its association with cytoplasmic FMRP-interacting protein 1 (CYFIP1), which binds to and sequesters eukaryotic initiation factor 4E (eIF4E). CYFIP1 shuttles between the FMRP–eIF4E complex and the Rac1–Wave regulatory complex, thereby connecting translation regulation to actin dynamics and dendritic spine morphology, which are dysregulated in FXS model mice that lack FMRP. Treating FXS mice with 4EGI-1, which blocks interactions between eIF4E and eukaryotic factor 4G (eIF4G), a critical interacting partner for protein synthesis, reversed defects in hippocampus-dependent memory and spine morphology. We also found that 4EGI-1 normalized the phenotypes of enhanced metabotropic glutamate receptor (mGluR)-mediated long-term depression (LTD), upregulated Rac1–p21-activated kinase (PAK)–cofilin signaling, altered actin dynamics, and dysregulated CYFIP1/eIF4E and CYFIP1/Rac1 interactions in FXS mice. Our findings are consistent with the idea that an imbalance of protein synthesis and actin dynamics contributes to pathophysiology in FXS mice, and suggest that targeting eIF4E may be a strategy for treating FXS. PMID:29114037

Antibiotic drug rifabutin is effective against lung cancer cells by targeting the eIF4E-β-catenin axis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Li, Ji; Huang, Yijiang; Gao, Yunsuo

The essential roles of overexpression of eukaryotic translation initiation factor 4E (eIF4E) and aberrant activation of β-catenin in lung cancer development have been recently identified. However, whether there is a direct connection between eIF4E overexpression and β-catenin activation in lung cancer cells is unknown. In this study, we show that antibiotic drug rifabutin targets human lung cancer cells via inhibition of eIF4E-β-catenin axis. Rifabutin is effectively against lung cancer cells in in vitro cultured cells and in vivo xenograft mouse model through inhibiting proliferation and inducing apoptosis. Mechanistically, eIF4E regulates β-catenin activity in lung cancer cells as shown by the increased β-cateninmore » phosphorylation and activity in cells overexpressing eIF4E, and furthermore that the regulation is dependent on phosphorylation at S209. Rifabutin suppresses eIF4E phosphorylation, leads to decreased β-catenin phosphorylation and its subsequent transcriptional activities. Depletion of eIF4E abolishes the inhibitory effects of rifabutin on β-catenin activities and overexpression of β-catenin reverses the inhibitory effects of rifabutin on cell growth and survival, further confirming that rifabutin acts on lung cancer cells via targeting eIF4E- β-catenin axis. Our findings identify the eIF4E- β-catenin axis as a critical regulator of lung cancer cell growth and survival, and suggest that its pharmacological inhibition may be therapeutically useful in lung cancer. - Highlights: • Rifabutin targets EGFR-mutated lung cancer cells in vitro and in vivo. • eIF4E phosphorylation regulates β-catenin activity in lung cancer cells. • Rifabutin acts on lung cancer cells via eIF4E- β-catenin axis. • Rifabutin can be repurposed for lung cancer treatment.« less

eIF4E Threshold Levels Differ in Governing Normal and Neoplastic Expansion of Mammary Stem and Luminal Progenitor cells

PubMed Central

Avdulov, Svetlana; Herrera, Jeremy; Smith, Karen; Peterson, Mark; Gomez-Garcia, Jose R.; Beadnell, Thomas C.; Schwertfeger, Kathryn L.; Benyumov, Alexey O.; Manivel, J. Carlos; Li, Shunan; Bielinsky, Anja-Katrin; Yee, Douglas; Bitterman, Peter B.; Polunovsky, Vitaly A.

2015-01-01

Translation initiation factor eIF4E mediates normal cell proliferation, yet induces tumorigenesis when overexpressed. The mechanisms by which eIF4E directs such distinct biological outputs remains unknown. We found that mouse mammary morphogenesis during pregnancy and lactation is accompanied by increased cap-binding capability of eIF4E and activation of the eIF4E-dependent translational apparatus, but only subtle oscillations in eIF4E abundance. Using a transgenic mouse model engineered so that lactogenic hormones stimulate a sustained increase in eIF4E abundance in stem/progenitor cells of lactogenic mammary epithelium during successive pregnancy/lactation cycles, eIF4E overexpression increased cell self-renewal, triggered DNA replication stress, and induced formation of pre-malignant and malignant lesions. Using complementary in vivo and ex vivo approaches, we found that increasing eIF4E levels rescued cells harboring oncogenic c-Myc or H-RasV12 from DNA replication stress and oncogene-induced replication catastrophe. Our findings indicate that distinct threshold levels of eIF4E govern its biological output in lactating mammary glands, and that eIF4E overexpression in the context of stem/progenitor cell population expansion can initiate malignant transformation by enabling cells to evade DNA damage checkpoints activated by oncogenic stimuli. Maintaining eIF4E levels below its pro-neoplastic threshold is an important anticancer defense in normal cells, with important implications for understanding pregnancy-associated breast cancer. PMID:25524901

eIF4E threshold levels differ in governing normal and neoplastic expansion of mammary stem and luminal progenitor cells.

PubMed

Avdulov, Svetlana; Herrera, Jeremy; Smith, Karen; Peterson, Mark; Gomez-Garcia, Jose R; Beadnell, Thomas C; Schwertfeger, Kathryn L; Benyumov, Alexey O; Manivel, J Carlos; Li, Shunan; Bielinsky, Anja-Katrin; Yee, Douglas; Bitterman, Peter B; Polunovsky, Vitaly A

2015-02-15

Translation initiation factor eIF4E mediates normal cell proliferation, yet induces tumorigenesis when overexpressed. The mechanisms by which eIF4E directs such distinct biologic outputs remain unknown. We found that mouse mammary morphogenesis during pregnancy and lactation is accompanied by increased cap-binding capability of eIF4E and activation of the eIF4E-dependent translational apparatus, but only subtle oscillations in eIF4E abundance. Using a transgenic mouse model engineered so that lactogenic hormones stimulate a sustained increase in eIF4E abundance in stem/progenitor cells of lactogenic mammary epithelium during successive pregnancy/lactation cycles, eIF4E overexpression increased self-renewal, triggered DNA replication stress, and induced formation of premalignant and malignant lesions. Using complementary in vivo and ex vivo approaches, we found that increasing eIF4E levels rescued cells harboring oncogenic c-Myc or H-RasV12 from DNA replication stress and oncogene-induced replication catastrophe. Our findings indicate that distinct threshold levels of eIF4E govern its biologic output in lactating mammary glands and that eIF4E overexpression in the context of stem/progenitor cell population expansion can initiate malignant transformation by enabling cells to evade DNA damage checkpoints activated by oncogenic stimuli. Maintaining eIF4E levels below its proneoplastic threshold is an important anticancer defense in normal cells, with important implications for understanding pregnancy-associated breast cancer. ©2014 American Association for Cancer Research.

Mutational Analysis of Plant Cap-Binding Protein eIF4E Reveals Key Amino Acids Involved in Biochemical Functions and Potyvirus Infection▿

PubMed Central

German-Retana, Sylvie; Walter, Jocelyne; Doublet, Bénédicte; Roudet-Tavert, Geneviève; Nicaise, Valérie; Lecampion, Cécile; Houvenaghel, Marie-Christine; Robaglia, Christophe; Michon, Thierry; Le Gall, Olivier

2008-01-01

The eukaryotic translation initiation factor 4E (eIF4E) (the cap-binding protein) is involved in natural resistance against several potyviruses in plants. In lettuce, the recessive resistance genes mo11 and mo12 against Lettuce mosaic virus (LMV) are alleles coding for forms of eIF4E unable, or less effective, to support virus accumulation. A recombinant LMV expressing the eIF4E of a susceptible lettuce variety from its genome was able to produce symptoms in mo11 or mo12 varieties. In order to identify the eIF4E amino acid residues necessary for viral infection, we constructed recombinant LMV expressing eIF4E with point mutations affecting various amino acids and compared the abilities of these eIF4E mutants to complement LMV infection in resistant plants. Three types of mutations were produced in order to affect different biochemical functions of eIF4E: cap binding, eIF4G binding, and putative interaction with other virus or host proteins. Several mutations severely reduced the ability of eIF4E to complement LMV accumulation in a resistant host and impeded essential eIF4E functions in yeast. However, the ability of eIF4E to bind a cap analogue or to fully interact with eIF4G appeared unlinked to LMV infection. In addition to providing a functional mutational map of a plant eIF4E, this suggests that the role of eIF4E in the LMV cycle might be distinct from its physiological function in cellular mRNA translation. PMID:18480444

Insulin Signaling Augments eIF4E-Dependent Nonsense-Mediated mRNA Decay in Mammalian Cells.

PubMed

Park, Jungyun; Ahn, Seyoung; Jayabalan, Aravinth K; Ohn, Takbum; Koh, Hyun Chul; Hwang, Jungwook

2016-07-01

Nonsense-mediated mRNA decay (NMD) modulates the level of mRNA harboring a premature termination codon (PTC) in a translation-dependent manner. Inhibition of translation is known to impair NMD; however, few studies have investigated the correlation between enhanced translation and increased NMD. Here, we demonstrate that insulin signaling events increase translation, leading to an increase in NMD of eIF4E-bound transcripts. We provide evidence that (i) insulin-mediated enhancement of translation augments NMD and rapamycin abrogates this enhancement; (ii) an increase in AKT phosphorylation due to inhibition of PTEN facilitates NMD; (iii) insulin stimulation increases the binding of up-frameshift factor 1 (UPF1), most likely to eIF4E-bound PTC-containing transcripts; and (iv) insulin stimulation induces the colocalization of UPF1 and eIF4E in processing bodies. These results illustrate how extracellular signaling promotes the removal of eIF4E-bound NMD targets. Copyright © 2015 Elsevier B.V. All rights reserved.

Role of p70S6K1-mediated phosphorylation of eIF4B and PDCD4 proteins in the regulation of protein synthesis.

PubMed

Dennis, Michael D; Jefferson, Leonard S; Kimball, Scot R

2012-12-14

Modulation of mRNA binding to the 40 S ribosomal subunit during translation initiation controls not only global rates of protein synthesis but also regulates the pattern of protein expression by allowing for selective inclusion, or exclusion, of mRNAs encoding particular proteins from polysomes. The mRNA binding step is modulated by signaling through a protein kinase known as the mechanistic target of rapamycin complex 1 (mTORC1). mTORC1 directly phosphorylates the translational repressors eIF4E binding proteins (4E-BP) 1 and 2, releasing them from the mRNA cap binding protein eIF4E, thereby promoting assembly of the eIF4E·eIF4G complex. mTORC1 also phosphorylates the 70-kDa ribosomal protein S6 kinase 1 (p70S6K1), which subsequently phosphorylates eIF4B, and programmed cell death 4 (PDCD4), which sequesters eIF4A from the eIF4E·eIF4G complex, resulting in repressed translation of mRNAs with highly structured 5'-untranslated regions. In the present study, we compared the role of the 4E-BPs in the regulation of global rates of protein synthesis to that of eIF4B and PDCD4. We found that maintenance of eIF4E interaction with eIF4G was not by itself sufficient to sustain global rates of protein synthesis in the absence of mTORC1 signaling to p70S6K1; phosphorylation of both eIF4B and PDCD4 was additionally required. We also found that the interaction of eIF4E with eIF4G was maintained in the liver of fasted rats as well as in serum-deprived mouse embryo fibroblasts lacking both 4E-BP1 and 4E-BP2, suggesting that the interaction of eIF4G with eIF4E is controlled primarily through the 4E-BPs.

Molecular Dynamics Simulation of the Allosteric Regulation of eIF4A Protein from the Open to Closed State, Induced by ATP and RNA Substrates

PubMed Central

Meng, Hongqing; Li, Chaoqun; Wang, Yan; Chen, Guangju

2014-01-01

Background Eukaryotic initiation factor 4A (eIF4A) plays a key role in the process of protein translation initiation by facilitating the melting of the 5′ proximal secondary structure of eukaryotic mRNA for ribosomal subunit attachment. It was experimentally postulated that the closed conformation of the eIF4A protein bound by the ATP and RNA substrates is coupled to RNA duplex unwinding to promote protein translation initiation, rather than an open conformation in the absence of ATP and RNA substrates. However, the allosteric process of eIF4A from the open to closed state induced by the ATP and RNA substrates are not yet fully understood. Methodology In the present work, we constructed a series of diplex and ternary models of the eIF4A protein bound by the ATP and RNA substrates to carry out molecular dynamics simulations, free energy calculations and conformation analysis and explore the allosteric properties of eIF4A. Results The results showed that the eIF4A protein completes the conformational transition from the open to closed state via two allosteric processes of ATP binding followed by RNA and vice versa. Based on cooperative allosteric network analysis, the ATP binding to the eIF4A protein mainly caused the relative rotation of two domains, while the RNA binding caused the proximity of two domains via the migration of RNA bases in the presence of ATP. The cooperative binding of ATP and RNA for the eIF4A protein plays a key role in the allosteric transition. PMID:24465900

A new eIF4E1 allele characterized by RNAseq data mining is associated with resistance to potato virus Y in tomato albeit with a low durability.

PubMed

Lebaron, Caroline; Rosado, Aurélie; Sauvage, Christopher; Gauffier, Camille; German-Retana, Sylvie; Moury, Benoît; Gallois, Jean-Luc

2016-11-01

Allele mining on susceptibility factors offers opportunities to find new sources of resistance among crop wild relatives for breeding purposes. As a proof of concept, we used available RNAseq data to investigate polymorphisms among the four tomato genes encoding translation initiation factors [eIF4E1 and eIF4E2, eIFiso4E and the related gene new cap-binding protein(nCBP)] to look for new potential resistance alleles to potyviruses. By analysing polymorphism among RNAseq data obtained for 20 tomato accessions, 10 belonging to the cultivated type Solanum lycopersicum and 10 belonging to the closest related wild species Solanum pimpinellifolium, we isolated one new eIF4E1 allele, in the S. pimpinellifolium LA0411 accession, which encodes a potential new resistance allele, mainly due to a polymorphism associated with an amino acid change within eIF4E1 region II. We confirmed that this new allele, pot12, is indeed associated with resistance to potato virus Y, although with a restricted resistance spectrum and a very low durability potential. This suggests that mutations occurring in eIF4E region II only may not be sufficient to provide efficient and durable resistance in plants. However, our study emphasizes the opportunity brought by RNAseq data to mine for new resistance alleles. Moreover, this approach could be extended to seek for putative new resistance alleles by screening for variant forms of susceptibility genes encoding plant host proteins known to interact with viral proteins.

Whole exome sequencing of rare variants in EIF4G1 and VPS35 in Parkinson disease

PubMed Central

Nuytemans, Karen; Bademci, Guney; Inchausti, Vanessa; Dressen, Amy; Kinnamon, Daniel D.; Mehta, Arpit; Wang, Liyong; Züchner, Stephan; Beecham, Gary W.; Martin, Eden R.; Scott, William K.

2013-01-01

Objective: Recently, vacuolar protein sorting 35 (VPS35) and eukaryotic translation initiation factor 4 gamma 1 (EIF4G1) have been identified as 2 causal Parkinson disease (PD) genes. We used whole exome sequencing for rapid, parallel analysis of variations in these 2 genes. Methods: We performed whole exome sequencing in 213 patients with PD and 272 control individuals. Those rare variants (RVs) with <5% frequency in the exome variant server database and our own control data were considered for analysis. We performed joint gene-based tests for association using RVASSOC and SKAT (Sequence Kernel Association Test) as well as single-variant test statistics. Results: We identified 3 novel VPS35 variations that changed the coded amino acid (nonsynonymous) in 3 cases. Two variations were in multiplex families and neither segregated with PD. In EIF4G1, we identified 11 (9 nonsynonymous and 2 small indels) RVs including the reported pathogenic mutation p.R1205H, which segregated in all affected members of a large family, but also in 1 unaffected 86-year-old family member. Two additional RVs were found in isolated patients only. Whereas initial association studies suggested an association (p = 0.04) with all RVs in EIF4G1, subsequent testing in a second dataset for the driving variant (p.F1461) suggested no association between RVs in the gene and PD. Conclusions: We confirm that the specific EIF4G1 variation p.R1205H seems to be a strong PD risk factor, but is nonpenetrant in at least one 86-year-old. A few other select RVs in both genes could not be ruled out as causal. However, there was no evidence for an overall contribution of genetic variability in VPS35 or EIF4G1 to PD development in our dataset. PMID:23408866

Stress Granule-Inducing Eukaryotic Translation Initiation Factor 4A Inhibitors Block Influenza A Virus Replication

PubMed Central

Slaine, Patrick D.; Kleer, Mariel; Smith, Nathan K.; Khaperskyy, Denys A.

2017-01-01

Eukaryotic translation initiation factor 4A (eIF4A) is a helicase that facilitates assembly of the translation preinitiation complex by unwinding structured mRNA 5′ untranslated regions. Pateamine A (PatA) and silvestrol are natural products that disrupt eIF4A function and arrest translation, thereby triggering the formation of cytoplasmic aggregates of stalled preinitiation complexes known as stress granules (SGs). Here we examined the effects of eIF4A inhibition by PatA and silvestrol on influenza A virus (IAV) protein synthesis and replication in cell culture. Treatment of infected cells with either PatA or silvestrol at early times post-infection resulted in SG formation, arrest of viral protein synthesis and failure to replicate the viral genome. PatA, which irreversibly binds to eIF4A, sustained long-term blockade of IAV replication following drug withdrawal, and inhibited IAV replication at concentrations that had minimal cytotoxicity. By contrast, the antiviral effects of silvestrol were fully reversible; drug withdrawal caused rapid SG dissolution and resumption of viral protein synthesis. IAV inhibition by silvestrol was invariably associated with cytotoxicity. PatA blocked replication of genetically divergent IAV strains, suggesting common dependence on host eIF4A activity. This study demonstrates that the core host protein synthesis machinery can be targeted to block viral replication. PMID:29258238

Brd4 modulates the innate immune response through Mnk2-eIF4E pathway-dependent translational control of IκBα.

PubMed

Bao, Yan; Wu, Xuewei; Chen, Jinjing; Hu, Xiangming; Zeng, Fuxing; Cheng, Jianjun; Jin, Hong; Lin, Xin; Chen, Lin-Feng

2017-05-16

Bromodomain-containing factor Brd4 has emerged as an important transcriptional regulator of NF-κB-dependent inflammatory gene expression. However, the in vivo physiological function of Brd4 in the inflammatory response remains poorly defined. We now demonstrate that mice deficient for Brd4 in myeloid-lineage cells are resistant to LPS-induced sepsis but are more susceptible to bacterial infection. Gene-expression microarray analysis of bone marrow-derived macrophages (BMDMs) reveals that deletion of Brd4 decreases the expression of a significant amount of LPS-induced inflammatory genes while reversing the expression of a small subset of LPS-suppressed genes, including MAP kinase-interacting serine/threonine-protein kinase 2 ( Mknk2 ). Brd4 -deficient BMDMs display enhanced Mnk2 expression and the corresponding eukaryotic translation initiation factor 4E (eIF4E) activation after LPS stimulation, leading to an increased translation of IκBα mRNA in polysomes. The enhanced newly synthesized IκBα reduced the binding of NF-κB to the promoters of inflammatory genes, resulting in reduced inflammatory gene expression and cytokine production. By modulating the translation of IκBα via the Mnk2-eIF4E pathway, Brd4 provides an additional layer of control for NF-κB-dependent inflammatory gene expression and inflammatory response.

Reducing eIF4E-eIF4G interactions restores the balance between protein synthesis and actin dynamics in fragile X syndrome model mice.

PubMed

Santini, Emanuela; Huynh, Thu N; Longo, Francesco; Koo, So Yeon; Mojica, Edward; D'Andrea, Laura; Bagni, Claudia; Klann, Eric

2017-11-07

Fragile X syndrome (FXS) is the most common form of inherited intellectual disability and autism spectrum disorder. FXS is caused by silencing of the FMR1 gene, which encodes fragile X mental retardation protein (FMRP), an mRNA-binding protein that represses the translation of its target mRNAs. One mechanism by which FMRP represses translation is through its association with cytoplasmic FMRP-interacting protein 1 (CYFIP1), which subsequently sequesters and inhibits eukaryotic initiation factor 4E (eIF4E). CYFIP1 shuttles between the FMRP-eIF4E complex and the Rac1-Wave regulatory complex, thereby connecting translational regulation to actin dynamics and dendritic spine morphology, which are dysregulated in FXS model mice that lack FMRP. Treating FXS mice with 4EGI-1, which blocks interactions between eIF4E and eIF4G, a critical interaction partner for translational initiation, reversed defects in hippocampus-dependent memory and spine morphology. We also found that 4EGI-1 normalized the phenotypes of enhanced metabotropic glutamate receptor (mGluR)-mediated long-term depression (LTD), enhanced Rac1-p21-activated kinase (PAK)-cofilin signaling, altered actin dynamics, and dysregulated CYFIP1/eIF4E and CYFIP1/Rac1 interactions in FXS mice. Our findings are consistent with the idea that an imbalance in protein synthesis and actin dynamics contributes to pathophysiology in FXS mice, and suggest that targeting eIF4E may be a strategy for treating FXS. Copyright © 2017 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.

Heat shock protein-27 (HSP27) regulates STAT3 and eIF4G levels in first trimester human placenta.

PubMed

Shochet, Gali Epstein; Komemi, Oded; Sadeh-Mestechkin, Dana; Pomeranz, Meir; Fishman, Ami; Drucker, Liat; Lishner, Michael; Matalon, Shelly Tartakover

2016-12-01

During placental implantation, cytotrophoblast cells differentiate to extravillous trophoblast (EVT) cells that invade from the placenta into the maternal uterine blood vessels. The heat shock protein-27 (HSP27), the signal transducer and activator of transcription-3 (STAT3) and the eukaryotic translation initiation factor 4E (EIF4E) are involved in regulating EVT cell differentiation/migration. EIF4E and EIF4G compose the translation initiation complex, which is a major control point in protein translation. The molecular chaperone distinctiveness of HSP27 implies that it directly interferes with many target proteins. STAT3, EIF4E, and EIF4G were found to be HSP27 client proteins in tumor cells. We aimed to analyze if HSP27 regulate STAT3 and EIF4G levels in first trimester human placenta. We found that like STAT3, EIF4G is highly expressed in the EVT cells (immunohistochemistry). Silencing HSP27 in HTR-8/SVneo cells (siRNA, EVT cell line) and in placental explants reduced STAT3 level (47 and 33 %, respectively, p < 0.05). HSP27 silencing reduced the levels of STAT3 phosphorylation (33 % reduction, p < 0.05) and targets (IRF1, MUC1, MMP2/9 and EIF4E, 30-49 % reduction, p < 0.05) in the HTR-8/SVneo cells. Moreover, HSP27 silencing significantly reduced EIF4G level and elevated the level of its fragments in HTR-8/SVneo cells and in the placental explants (p < 0.05). In conclusion, Placental implantation and development are accompanied by trophoblast cell proliferation and differentiation, which necessitates intense protein translation and STAT3 activation. HSP27 was found to be regulator of translation initiation and STAT3 level. Therefore, it suggests that HSP27 is a key protein during placental development and trophoblast cell differentiation.

The alveolate translation initiation factor 4E family reveals a custom toolkit for translational control in core dinoflagellates.

PubMed

Jones, Grant D; Williams, Ernest P; Place, Allen R; Jagus, Rosemary; Bachvaroff, Tsvetan R

2015-02-10

Dinoflagellates are eukaryotes with unusual cell biology and appear to rely on translational rather than transcriptional control of gene expression. The eukaryotic translation initiation factor 4E (eIF4E) plays an important role in regulating gene expression because eIF4E binding to the mRNA cap is a control point for translation. eIF4E is part of an extended, eukaryote-specific family with different members having specific functions, based on studies of model organisms. Dinoflagellate eIF4E diversity could provide a mechanism for dinoflagellates to regulate gene expression in a post-transcriptional manner. Accordingly, eIF4E family members from eleven core dinoflagellate transcriptomes were surveyed to determine the diversity and phylogeny of the eIF4E family in dinoflagellates and related lineages including apicomplexans, ciliates and heterokonts. The survey uncovered eight to fifteen (on average eleven) different eIF4E family members in each core dinoflagellate species. The eIF4E family members from heterokonts and dinoflagellates segregated into three clades, suggesting at least three eIF4E cognates were present in their common ancestor. However, these three clades are distinct from the three previously described eIF4E classes, reflecting diverse approaches to a central eukaryotic function. Heterokonts contain four clades, ciliates two and apicomplexans only a single recognizable eIF4E clade. In the core dinoflagellates, the three clades were further divided into nine sub-clades based on the phylogenetic analysis and species representation. Six of the sub-clades included at least one member from all eleven core dinoflagellate species, suggesting duplication in their shared ancestor. Conservation within sub-clades varied, suggesting different selection pressures. Phylogenetic analysis of eIF4E in core dinoflagellates revealed complex layering of duplication and conservation when compared to other eukaryotes. Our results suggest that the diverse eIF4E family in

Eukaryotic Initiation Factor eIFiso4G1 and eIFiso4G2 Are Isoforms Exhibiting Distinct Functional Differences in Supporting Translation in Arabidopsis*

PubMed Central

Gallie, Daniel R.

2016-01-01

The eukaryotic translation initiation factor (eIF) 4G is required during protein synthesis to promote the assembly of several factors involved in the recruitment of a 40S ribosomal subunit to an mRNA. Although many eukaryotes express two eIF4G isoforms that are highly similar, the eIF4G isoforms in plants, referred to as eIF4G and eIFiso4G, are highly divergent in size, sequence, and domain organization but both can interact with eIF4A, eIF4B, eIF4E isoforms, and the poly(A)-binding protein. Nevertheless, eIF4G and eIFiso4G from wheat exhibit preferences in the mRNAs they translate optimally. For example, mRNA containing the 5′-leader (called Ω) of tobacco mosaic virus preferentially uses eIF4G in wheat germ lysate. In this study, the eIF4G isoform specificity of Ω was used to examine functional differences of the eIF4G isoforms in Arabidopsis. As in wheat, Ω-mediated translation was reduced in an eif4g null mutant. Loss of the eIFiso4G1 isoform, which is similar in sequence to wheat eIFiso4G, did not substantially affect Ω-mediated translation. However, loss of the eIFiso4G2 isoform substantially reduced Ω-mediated translation. eIFiso4G2 is substantially divergent from eIFiso4G1 and is present only in the Brassicaceae, suggesting a recent evolution. eIFiso4G2 isoforms exhibit sequence-specific differences in regions representing partner protein and RNA binding sites. Loss of any eIF4G isoform also resulted in a substantial reduction in reporter transcript level. These results suggest that eIFiso4G2 appeared late in plant evolution and exhibits more functional similarity with eIF4G than with eIFiso4G1 during Ω-mediated translation. PMID:26578519

Oxidative stress increases eukaryotic initiation factor 4E phosphorylation in vascular cells.

PubMed Central

Duncan, Roger F; Peterson, Hazel; Hagedorn, Curt H; Sevanian, Alex

2003-01-01

Dysregulated cell growth can be caused by increased activity of protein synthesis eukaryotic initiation factor (eIF) 4E. Dysregulated cell growth is also characteristic of atherosclerosis. It is postulated that exposure of vascular cells, such as endothelial cells, smooth muscle cells and monocytes/macrophages, to oxidants, such as oxidized low-density lipoprotein (oxLDL), leads to the elaboration of growth factors and cytokines, which in turn results in smooth muscle cell hyperproliferation. To investigate whether activation of eIF4E might play a role in this hyperproliferative response, vascular cells were treated with oxLDL, oxidized lipid components of oxLDL and several model oxidants, including H(2)O(2) and dimethyl naphthoquinone. Exposure to each of these compounds led to a dose- and time-dependent increase in eIF4E phosphorylation in all three types of vascular cells, correlated with a modest increase in overall translation rate. No changes in eIF4EBP, eIF2 or eIF4B modification state were observed. Increased eIF4E phosphorylation was paralleled by increased presence of eIF4E in high-molecular-mass protein complexes characteristic of its most active form. Anti-oxidants at concentrations typically employed to block oxidant-induced cell signalling likewise promoted eIF4E phosphorylation. The results of this study indicate that increased eIF4E activity may contribute to the pathophysiological events in early atherogenesis by increasing the expression of translationally inefficient mRNAs encoding growth-promoting proteins. PMID:12215171

A novel mechanism for Bcr-Abl action: Bcr-Abl-mediated induction of the eIF4F translation initiation complex and mRNA translation.

PubMed

Prabhu, S; Saadat, D; Zhang, M; Halbur, L; Fruehauf, J P; Ong, S T

2007-02-22

The oncogenic kinase Bcr-Abl is thought to cause chronic myelogenous leukemia (CML) by altering the transcription of specific genes with growth- and survival-promoting functions. Recently, Bcr-Abl has also been shown to activate an important regulator of protein synthesis, the mammalian target of rapamycin (mTOR), which suggests that dysregulated translation may also contribute to CML pathogenesis. In this study, we found that both Bcr-Abl and the rapamycin-sensitive mTORC1 complex contribute to the phosphorylation (inactivation) of 4E-BP1, an inhibitor of the eIF4E translation initiation factor. Experiments with rapamycin and the Bcr-Abl inhibitor, imatinib mesylate, in Bcr-Abl-expressing cell lines and primary CML cells indicated that Bcr-Abl and mTORC1 induced formation of the translation initiation complex, eIF4F. This was characterized by reduced 4E-BP1 binding and increased eIF4G binding to eIF4E, two events that lead to the assembly of eIF4F. One target transcript is cyclin D3, which is regulated in Bcr-Abl-expressing cells by both Bcr-Abl and mTORC1 in a translational manner. In addition, the combination of imatinib and rapamycin was found to act synergistically against committed CML progenitors from chronic and blast phase patients. These experiments establish a novel mechanism of action for Bcr-Abl, and they provide insights into the modes of action of imatinib mesylate and rapamycin in treatment of CML. They also suggest that aberrant cap-dependent mRNA translation may be a therapeutic target in Bcr-Abl-driven malignancies.

Arginine methyltransferase inhibitor 1 inhibits gastric cancer by downregulating eIF4E and targeting PRMT5.

PubMed

Zhang, Baolai; Zhang, Su; Zhu, Lijuan; Chen, Xue; Zhao, Yunfeng; Chao, Li; Zhou, Juanping; Wang, Xing; Zhang, Xinyang; Ma, Nengqian

2017-12-01

Arginine methylation is carried out by protein arginine methyltransferase (PRMTs) family. Arginine methyltransferase inhibitor 1 (AMI-1) is mainly used to inhibit type I PRMT activity in vitro. However, the effects of AMI-1 on type II PRMT5 activity and gastric cancer (GC) remain unclear. In this study, we provided the first evidence that AMI-1 significantly inhibited GC cell proliferation and migration while induced GC cell apoptosis, and reduced the expression of PRMT5, eukaryotic translation initiation factor 4E (eIF4E), symmetric dimethylation of histone 3 (H3R8me2s) and histone 4 (H4R3me2s). In addition, AMI-1 inhibited tumor growth, downregulated eIF4E, H4R3me2s and H3R8me2s expression in mice xenografts model of GC. Collectively, our results suggest that AMI-1 inhibits GC by downregulating eIF4E and targeting type II PRMT5. Copyright © 2017 Elsevier Inc. All rights reserved.

Eukaryotic Translation Initiation Factor 4E Is a Feed-Forward Translational Coactivator of Transforming Growth Factor β Early Protransforming Events in Breast Epithelial Cells

PubMed Central

Decarlo, Lindsey; Mestel, Celine; Barcellos-Hoff, Mary-Helen

2015-01-01

Eukaryotic translation initiation factor 4E (eIF4E) is overexpressed early in breast cancers in association with disease progression and reduced survival. Much remains to be understood regarding the role of eIF4E in human cancer. We determined, using immortalized human breast epithelial cells, that elevated expression of eIF4E translationally activates the transforming growth factor β (TGF-β) pathway, promoting cell invasion, a loss of cell polarity, increased cell survival, and other hallmarks of early neoplasia. Overexpression of eIF4E is shown to facilitate the selective translation of integrin β1 mRNA, which drives the translationally controlled assembly of a TGF-β receptor signaling complex containing α3β1 integrins, β-catenin, TGF-β receptor I, E-cadherin, and phosphorylated Smad2/3. This receptor complex acutely sensitizes nonmalignant breast epithelial cells to activation by typically substimulatory levels of activated TGF-β. TGF-β can promote cellular differentiation or invasion and transformation. As a translational coactivator of TGF-β, eIF4E confers selective mRNA translation, reprogramming nonmalignant cells to an invasive phenotype by reducing the set point for stimulation by activated TGF-β. Overexpression of eIF4E may be a proinvasive facilitator of TGF-β activity. PMID:25986608

mTORC1 signalling and eIF4E/4E-BP1 translation initiation factor stoichiometry influence recombinant protein productivity from GS-CHOK1 cells.

PubMed

Jossé, Lyne; Xie, Jianling; Proud, Christopher G; Smales, C Mark

2016-12-15

Many protein-based biotherapeutics are produced in cultured Chinese hamster ovary (CHO) cell lines. Recent reports have demonstrated that translation of recombinant mRNAs and global control of the translation machinery via mammalian target of rapamycin (mTOR) signalling are important determinants of the amount and quality of recombinant protein such cells can produce. mTOR complex 1 (mTORC1) is a master regulator of cell growth/division, ribosome biogenesis and protein synthesis, but the relationship between mTORC1 signalling, cell growth and proliferation and recombinant protein yields from mammalian cells, and whether this master regulating signalling pathway can be manipulated to enhance cell biomass and recombinant protein production (rPP) are not well explored. We have investigated mTORC1 signalling and activity throughout batch culture of a panel of sister recombinant glutamine synthetase-CHO cell lines expressing different amounts of a model monoclonal IgG4, to evaluate the links between mTORC1 signalling and cell proliferation, autophagy, recombinant protein expression, global protein synthesis and mRNA translation initiation. We find that the expression of the mTORC1 substrate 4E-binding protein 1 (4E-BP1) fluctuates throughout the course of cell culture and, as expected, that the 4E-BP1 phosphorylation profiles change across the culture. Importantly, we find that the eIF4E/4E-BP1 stoichiometry positively correlates with cell productivity. Furthermore, eIF4E amounts appear to be co-regulated with 4E-BP1 amounts. This may reflect a sensing of either change at the mRNA level as opposed to the protein level or the fact that the phosphorylation status, as well as the amount of 4E-BP1 present, is important in the co-regulation of eIF4E and 4E-BP1. © 2016 The Author(s).

Boric acid induces cytoplasmic stress granule formation, eIF2α phosphorylation, and ATF4 in prostate DU-145 cells.

PubMed

Henderson, Kimberly A; Kobylewski, Sarah E; Yamada, Kristin E; Eckhert, Curtis D

2015-02-01

Dietary boron intake is associated with reduced prostate and lung cancer risk and increased bone mass. Boron is absorbed and circulated as boric acid (BA) and at physiological concentrations is a reversible competitive inhibitor of cyclic ADP ribose, the endogenous agonist of the ryanodine receptor calcium (Ca(+2)) channel, and lowers endoplasmic reticulum (ER) [Ca(2+)]. Low ER [Ca(2+)] has been reported to induce ER stress and activate the eIF2α/ATF4 pathway. Here we report that treatment of DU-145 prostate cells with physiological levels of BA induces ER stress with the formation of stress granules and mild activation of eIF2α, GRP78/BiP, and ATF4. Mild activation of eIF2α and its downstream transcription factor, ATF4, enables cells to reconfigure gene expression to manage stress conditions and mild activation of ATF4 is also required for the differentiation of osteoblast cells. Our results using physiological levels of boric acid identify the eIF2α/ATF pathway as a plausible mode of action that underpins the reported health effects of dietary boron.

Amino Acid Availability and Age Affect the Leucine Stimulation of Protein Synthesis and eIF4F Formation in Muscle

PubMed Central

Escobar, Jeffery; Frank, Jason W.; Suryawan, Agus; Nguyen, Hanh V.; Davis, Teresa A.

2009-01-01

We have previously shown that a physiological increase in plasma leucine for 60- and 120-min increases translation initiation factor activation in muscle of neonatal pigs. Although muscle protein synthesis is increased by leucine at 60 min, it is not maintained at 120 min, perhaps due to the decrease in plasma amino acids (AA). In the current study, 7- and 26-day-old pigs were fasted overnight and infused with leucine (0 or 400 µmol· kg−1· h−1) for 120 min to raise leucine within the postprandial range. The leucine was infused in the presence or absence of a replacement AA mixture (without leucine) to maintain baseline plasma AA levels. AA administration prevented the leucine-induced reduction in plasma AA in both age groups. At 7 days, leucine infusion alone increased eukaryotic initiation factor (eIF) 4E binding protein-1 (4E-BP1) phosphorylation, decreased inactive 4E-BP1·eIF4E complex abundance, and increased active eIF4G·eIF4E complex formation in skeletal muscle; leucine infusion with replacement AA also stimulated these, as well as S6K1, rpS6, and eIF4G phosphorylation. At 26 days, leucine infusion alone increased 4E-BP1 phosphorylation and decreased the inactive 4E-BP1·eIF4E complex only; leucine with AA also stimulated these, as well as S6K1 and rpS6 phosphorylation. Muscle protein synthesis was increased in 7-day-old (+60%) and 26-day-old (+40%) pigs infused with leucine and replacement AA, but not with leucine alone. Thus, the ability of leucine to stimulate eIF4F formation and protein synthesis in skeletal muscle is dependent on AA availability and age. PMID:17878223

Hippuristanol - A potent steroid inhibitor of eukaryotic initiation factor 4A

PubMed Central

Cencic, Regina; Pelletier, Jerry

2016-01-01

ABSTRACT Protein synthesis and its regulatory signaling pathways play essential roles in the initiation and maintenance of the cancer phenotype. Insight obtained over the last 3 decades on the mechanisms regulating translation in normal and transformed cells have revealed that perturbed control in cancer cells may offer an Achilles' heel for the development of novel anti-neoplastic agents. Several small molecule inhibitors have been identified and characterized that target translation initiation – more specifically, the rate-limiting step where ribosomes are recruited to mRNA templates. Among these, hippuristanol, a polyhydroxysteroid from the gorgonian Isis hippuris has been found to inhibit translation initiation by blocking the activity of eukaryotic initiation factor (eIF) 4A, an essential RNA helicase involved in this process. Herein, we highlight the biological properties of this compound, its potential development as an anti-cancer agent, and its use to validate eIF4A as an anti-neoplastic target. PMID:27335721

Activation of translation complex eIF4F is essential for the genesis and maintenance of the malignant phenotype in human mammary epithelial cells.

PubMed

Avdulov, Svetlana; Li, Shunan; Michalek, Van; Burrichter, David; Peterson, Mark; Perlman, David M; Manivel, J Carlos; Sonenberg, Nahum; Yee, Douglas; Bitterman, Peter B; Polunovsky, Vitaly A

2004-06-01

Common human malignancies acquire derangements of the translation initiation complex, eIF4F, but their functional significance is unknown. Hypophosphorylated 4E-BP proteins negatively regulate eIF4F assembly by sequestering its mRNA cap binding component eIF4E, whereas hyperphosphorylation abrogates this function. We found that breast carcinoma cells harbor increases in the eIF4F constituent eIF4GI and hyperphosphorylation of 4E-BP1 which are two alterations that activate eIF4F assembly. Ectopic expression of eIF4E in human mammary epithelial cells enabled clonal expansion and anchorage-independent growth. Transfer of 4E-BP1 phosphorylation site mutants into breast carcinoma cells suppressed their tumorigenicity, whereas loss of these 4E-BP1 phosphorylation site mutants accompanied spontaneous reversion to a malignant phenotype. Thus, eIF4F activation is an essential component of the malignant phenotype in breast carcinoma.

Serum-deprivation stimulates cap-binding by PARN at the expense of eIF4E, consistent with the observed decrease in mRNA stability

PubMed Central

Seal, Ruth; Temperley, Richard; Wilusz, Jeffrey; Lightowlers, Robert N.; Chrzanowska-Lightowlers, Zofia M. A.

2005-01-01

PARN, a poly(A)-specific ribonuclease, binds the 5′ cap-structure of mRNA and initiates deadenylation-dependent decay. Eukaryotic initiation factor 4E (eIF4E) also binds to the cap structure, an interaction that is critical for initiating cap-dependent translation. The stability of various mRNA transcripts in human cell lines is reduced under conditions of serum starvation as determined by both functional and chemical half-lives. Serum starvation also leads to enhanced cap association by PARN. In contrast, the 5′ cap occupancy by eIF4E decreases under serum-deprivation, as does the translation of reporter transcripts. Further, we show that PARN is a phosphoprotein and that this modification can be modulated by serum status. Taken together, these data are consistent with a natural competition existing at the 5′ cap structure between PARN and eIF4E that may be regulated by changes in post-translational modifications. These phosphorylation-induced changes in the interplay of PARN and eIF4E may determine whether the mRNA is translated or decayed. PMID:15653638

Effect of electroacupuncture on the expression of mTOR and eIF4E in hippocampus of rats with vascular dementia.

PubMed

Zhu, Yanzhen; Zeng, Yanjun; Wang, Xuan; Ye, Xiaobao

2013-07-01

Clinically, electroacupuncture is proved to be an effective therapy for vascular dementia; however, their mechanisms remain uncertain. The aim of the current study was to investigate the mechanism of electroacupuncture therapy for vascular dementia. One month after a vascular dementia animal model was established by bilateral occlusion of common carotid arteries, electroacupuncture treatment was given at "Baihui" (DU20), "Dazhui" (DU14), and "Shenshu" (BL23). Morris water maze was used to assess the learning and memory ability of rats. Western blot assay was performed to detect the expression of mammalian target of rapamycin (mTOR) and eukaryotic translation initiation factor 4E (eIF4E) in hippocampus of rats. Morris water maze test showed that electroacupuncture improved the learning ability of vascular dementia rats. Western blot assay revealed that the expression level of mTOR and eIF4E in the electroacupuncture group and sham-operated group was higher than that in the vascular dementia group (P < 0.05). In conclusion, the decreasing expression of mTOR and eIF4E plays important roles in the pathogenesis of vascular dementia. Electroacupuncture improves learning and memory ability by up-regulating expression of mTOR and eIF4E in the hippocampus of vascular dementia rats.

eIF4E/Fmr1 double mutant mice display cognitive impairment in addition to ASD-like behaviors.

PubMed

Huynh, Thu N; Shah, Manan; Koo, So Yeon; Faraud, Kirsten S; Santini, Emanuela; Klann, Eric

2015-11-01

Autism spectrum disorder (ASD) is a group of heritable disorders with complex and unclear etiology. Classic ASD symptoms include social interaction and communication deficits as well as restricted, repetitive behaviors. In addition, ASD is often comorbid with intellectual disability. Fragile X syndrome (FXS) is the leading genetic cause of ASD, and is the most commonly inherited form of intellectual disability. Several mouse models of ASD and FXS exist, however the intellectual disability observed in ASD patients is not well modeled in mice. Using the Fmr1 knockout mouse and the eIF4E transgenic mouse, two previously characterized mouse models of fragile X syndrome and ASD, respectively, we generated the eIF4E/Fmr1 double mutant mouse. Our study shows that the eIF4E/Fmr1 double mutant mice display classic ASD behaviors, as well as cognitive dysfunction. Importantly, the learning impairments displayed by the double mutant mice spanned multiple cognitive tasks. Moreover, the eIF4E/Fmr1 double mutant mice display increased levels of basal protein synthesis. The results of our study suggest that the eIF4E/Fmr1 double mutant mouse may be a reliable model to study cognitive dysfunction in the context of ASD. Copyright © 2015 Elsevier Inc. All rights reserved.

Essential role of eIF5-mimic protein in animal development is linked to control of ATF4 expression

USDA-ARS?s Scientific Manuscript database

Translational control of ATF4 through upstream ORFs (uORFs) plays an important role in eukaryotic gene regulation. While ATF4 translation is typically induced by inhibitory phosphorylation of eIF2, ATF4 translation can be also induced by expression of a new translational inhibitor protein, eIF5-mimi...

miR-139 is up-regulated in osteoarthritis and inhibits chondrocyte proliferation and migration possibly via suppressing EIF4G2 and IGF1R

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hu, Weihua; Zhang, Weikai; Li, Feng

Osteoarthritis (OA) is one of the most progressive articular cartilage erosions. microRNAs (miRNAs) play pivotal roles in OA modulation, but the role of miR-139 in OA remains elusive. This study aims to reveal the effects and possible mechanism of miR-139 in OA and chondrocytes. The levels of miR-139 and its possible targets eukaryotic translation initiation factor 4 gamma 2 (EIF4G2) and insulin-like growth factor 1 receptor (IGF1R) were detected by qRT-PCR in the articular cartilages of 20 OA patients and 20 non-OA patients. Human chondrocyte CHON-001 cells were transfected with miR-139 mimic or inhibitor, as well as the siRNAs of EIF4G2more » and IGF1R. Cell viability by MTT assay, proliferation by colony formation assay and migration by Transwell assay were performed. Results showed that miR-139 was up-regulated, while EIF4G2 and IGF1R mRNAs down-regulated in OA cartilages (P < 0.001), and negative correlations existed between the level of miR-139 and EIF4G2 or IGF1R. Overexpression of miR-139 in CHON-001 cells suppressed both mRNA and protein levels of EIF4G2 and IGF1R, and inhibited cell viability, colony formation number and cell migration, while miR-139 inhibitor induced the opposite effects. Knockdown of EIF4G2 or IGF1R in CHON-001 cells reversed the effects of miR-139 inhibitor on cell viability, colony formation and cell migration. These results indicate that miR-139 is capable of inhibiting chondrocyte proliferation and migration, thus being a possible therapeutic target for OA. The mechanism of miR-139 in chondrocytes may be related to its regulation on EIF4G2 and IGF1R.« less

Studying the Role of Eukaryotic Translation Initiation Factor 4E (eIF4E) Phosphorylation by MNK1/2 Kinases in Prostate Cancer Development and Progression

DTIC Science & Technology

2012-06-01

preclinical mouse model. J Clin Invest 118: 3051–3064. 49. Le Bacquer O, et al. (2007) Elevated sensitivity to diet-induced obesity and insulin resistance in...Elevated sensitivity to diet-induced obesity and insulin resistance in mice lacking 4E-BP1 and 4E-BP2. J Clin Invest 117:387–396. 7. Frederickson RM...that knock-in mice expressing a nonphosphorylatable form of eIF4E are resistant to tumorigenesis in a prostate cancer model. By using a genome-wide

A founder EIF2AK4 mutation causes an aggressive form of pulmonary arterial hypertension in Iberian Gypsies.

PubMed

Tenorio, J; Navas, P; Barrios, E; Fernández, L; Nevado, J; Quezada, C A; López-Meseguer, M; Arias, P; Mena, R; Lobo, J L; Alvarez, C; Heath, K; Escribano-Subías, P; Lapunzina, P

2015-12-01

Pulmonary arterial hypertension (PAH) is a pathological condition characterized by a persistent and progressive elevation of pulmonary vascular resistance with devastating consequences if untreated. In the past recent years, several genes have been related to PAH, however, the molecular defect remains unknown in a significant proportion of patients with familial PAH (∼20%). During the past few years, we have observed that PAH shows a particular behavior in Iberian Gypsies, with more aggressive course and frequently affecting multiple members of the same family. We studied five Gypsy families in whom at least one individual from each family developed a severe form of PAH and in whom no mutation had been identified in the common genes. We applied SNP-array-based homozygosity mapping in three families and obtained, among others, one of which included the gene EIF2AK4, recently reported in patients with PAH from group-1' pulmonary veno-occlusive disease (PVOD) and pulmonary capillary hemangiomatosis (PCH). Subsequently, we sequenced EIF2AK4 and found a homozygous mutation in all five families: c.3344C>T(p.P1115L). The majority of our patients required early lung transplantation. Hence, this mutation appeared with a more severe phenotype than previously reported for other EIF2AK4 mutations. The finding of this novel mutation is important for genetic counseling and calculation of population recurrence risks. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Arginine methylation promotes translation repression activity of eIF4G-binding protein, Scd6.

PubMed

Poornima, Gopalakrishna; Shah, Shanaya; Vignesh, Venkadasubramanian; Parker, Roy; Rajyaguru, Purusharth I

2016-11-02

Regulation of translation plays a critical role in determining mRNA fate. A new role was recently reported for a subset of RGG-motif proteins in repressing translation initiation by binding eIF4G1. However the signaling mechanism(s) that leads to spatial and temporal regulation of repression activity of RGG-motif proteins remains unknown. Here we report the role of arginine methylation in regulation of repression activity of Scd6, a conserved RGG-motif protein. We demonstrate that Scd6 gets arginine methylated at its RGG-motif and Hmt1 plays an important role in its methylation. We identify specific methylated arginine residues in the Scd6 RGG-motif in vivo We provide evidence that methylation augments Scd6 repression activity. Arginine methylation defective (AMD) mutant of Scd6 rescues the growth defect caused by overexpression of Scd6, a feature of translation repressors in general. Live-cell imaging of the AMD mutant revealed that it is defective in inducing formation of stress granules. Live-cell imaging and pull-down results indicate that it fails to bind eIF4G1 efficiently. Consistent with these results, a strain lacking Hmt1 is also defective in Scd6-eIF4G1 interaction. Our results establish that arginine methylation augments Scd6 repression activity by promoting eIF4G1-binding. We propose that arginine methylation of translation repressors with RGG-motif could be a general modulator of their repression activity. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

Defect in the GTPase activating protein (GAP) function of eIF5 causes repression of GCN4 translation.

PubMed

Antony A, Charles; Alone, Pankaj V

2017-05-13

In eukaryotes, the eIF5 protein plays an important role in translation start site selection by providing the GAP (GTPase activating protein) function. However, in yeast translation initiation fidelity defective eIF5 G31R mutant causes preferential utilization of UUG as initiation codon and is termed as Suppressor of initiation codon (Sui - ) phenotype due to its hyper GTPase activity. The eIF5 G31R mutant dominantly represses GCN4 expression and confers sensitivity to 3-Amino-1,2,4-Trizole (3AT) induced starvation. The down-regulation of the GCN4 expression (Gcn - phenotype) in the eIF5 G31R mutant was not because of leaky scanning defects; rather was due to the utilization of upUUG initiation codons at the 5' regulatory region present between uORF1 and the main GCN4 ORF. Copyright © 2017 Elsevier Inc. All rights reserved.

Structural modelling and phylogenetic analyses of PgeIF4A2 (Eukaryotic translation initiation factor) from Pennisetum glaucum reveal signature motifs with a role in stress tolerance and development

PubMed Central

Agarwal, Aakrati; Mudgil, Yashwanti; Pandey, Saurabh; Fartyal, Dhirendra; Reddy, Malireddy K

2016-01-01

Eukaryotic translation initiation factor 4A (eIF4A) is an indispensable component of the translation machinery and also play a role in developmental processes and stress alleviation in plants and animals. Different eIF4A isoforms are present in the cytosol of the cell, namely, eIF4A1, eIF4A2, and eIF4A3 and their expression is tightly regulated in cap-dependent translation. We revealed the structural model of PgeIF4A2 protein using the crystal structure of Homo sapiens eIF4A3 (PDB ID: 2J0S) as template by Modeller 9.12. The resultant PgeIF4A2 model structure was refined by PROCHECK, ProSA, Verify3D and RMSD that showed the model structure is reliable with 77 % amino acid sequence identity with template. Investigation revealed two conserved signatures for ATP-dependent RNA Helicase DEAD-box conserved site (VLDEADEML) and RNA helicase DEAD-box type, Q-motif in sheet-turn-helix and α-helical region respectively. All these conserved motifs are responsible for response during developmental stages and stress tolerance in plants. PMID:28358146

Structural modelling and phylogenetic analyses of PgeIF4A2 (Eukaryotic translation initiation factor) from Pennisetum glaucum reveal signature motifs with a role in stress tolerance and development.

PubMed

Agarwal, Aakrati; Mudgil, Yashwanti; Pandey, Saurabh; Fartyal, Dhirendra; Reddy, Malireddy K

2016-01-01

Eukaryotic translation initiation factor 4A (eIF4A) is an indispensable component of the translation machinery and also play a role in developmental processes and stress alleviation in plants and animals. Different eIF4A isoforms are present in the cytosol of the cell, namely, eIF4A1, eIF4A2, and eIF4A3 and their expression is tightly regulated in cap-dependent translation. We revealed the structural model of PgeIF4A2 protein using the crystal structure of Homo sapiens eIF4A3 (PDB ID: 2J0S) as template by Modeller 9.12. The resultant PgeIF4A2 model structure was refined by PROCHECK, ProSA, Verify3D and RMSD that showed the model structure is reliable with 77 % amino acid sequence identity with template. Investigation revealed two conserved signatures for ATP-dependent RNA Helicase DEAD-box conserved site (VLDEADEML) and RNA helicase DEAD-box type, Q-motif in sheet-turn-helix and α-helical region respectively. All these conserved motifs are responsible for response during developmental stages and stress tolerance in plants.

Enhance tumor radiosensitivity by intracellular delivery of eukaryotic translation initiation factor 4E binding proteins.

PubMed

Tian, Shuang; Li, Xiu-Li; Shi, Mei; Yao, Yuan-Qing; Li, Li-Wen; Xin, Xiao-Yan

2011-02-01

PTEN (phosphatase and tensin homologue deleted on chromosome ten)/PI3K (phosphatidylinositol 3-kinase)/Akt/mTOR (mammalian target of rapamycin) signaling pathway, which is commonly dysregulated in a broad array of human malignancies, controls the assembly of eukaryotic translation initiation factor 4F (eIF4F) complex through regulation of eIF4E binding proteins (4E-BPs) phosphorylation. And accumulated data over the past two decades implicated eIF4F complex as one of the promising targets for anticancer therapy. It has been confirmed that the translation initiation of mRNA coding for hypoxia-inducible factor-1α (HIF-1α) and survivin, which had been considered as the two major determinants of tumor radiosensitivity, are both controlled by eIF4F complex. Also, eIF4F complex controls the expression of VEGF and bFGF, the two well-known pro-angiogenic factors involved in developing radioresistance. Therefore eIF4F complex plays a pivotal role in regulation of radiosensitivity. In this article, we postulate that cell-permeable, phosphorylation-defective 4E-BP fusion proteins, which could be prepared by substituting the mTOR recognition motif located in N-terminal of 4E-BPs with protein transduction domain from HIV-1 TAT, HSV-1 VP22 or PTD4, could not only inhibit tumor growth but also enhance tumor response to radiation therapy through disruption of eIF4F complex assembly. In our opinion, the recombinant fusion proteins are superior to mTOR inhibitors for they do not cause immunosuppression, do not lead to Akt activation, and could be easily prepared by prokaryotic expression. If the hypothesis was proved to be practical, the cell-permeable, phosphorylation-defective 4E-BP fusion proteins would be widely used in clinical settings to improve tumor response to radiotherapy in the near future. Copyright © 2010 Elsevier Ltd. All rights reserved.

Influenza A virus-induced degradation of eukaryotic translation initiation factor 4B contributes to viral replication by suppressing IFITM3 protein expression.

PubMed

Wang, Song; Chi, Xiaojuan; Wei, Haitao; Chen, Yuhai; Chen, Zhilong; Huang, Shile; Chen, Ji-Long

2014-08-01

Although alteration in host cellular translation machinery occurs in virus-infected cells, the role of such alteration and the precise pathogenic processes are not well understood. Influenza A virus (IAV) infection shuts off host cell gene expression at transcriptional and translational levels. Here, we found that the protein level of eukaryotic translation initiation factor 4B (eIF4B), an integral component of the translation initiation apparatus, was dramatically reduced in A549 cells as well as in the lung, spleen, and thymus of mice infected with IAV. The decrease in eIF4B level was attributed to lysosomal degradation of eIF4B, which was induced by viral NS1 protein. Silencing eIF4B expression in A549 cells significantly promoted IAV replication, and conversely, overexpression of eIF4B markedly inhibited the viral replication. Importantly, we observed that eIF4B knockdown transgenic mice were more susceptible to IAV infection, exhibiting faster weight loss, shorter survival time, and more-severe organ damage. Furthermore, we demonstrated that eIF4B regulated the expression of interferon-induced transmembrane protein 3 (IFITM3), a critical protein involved in immune defense against a variety of RNA viruses, including influenza virus. Taken together, our findings reveal that eIF4B plays an important role in host defense against IAV infection at least by regulating the expression of IFITM3, which restricts viral entry and thereby blocks early stages of viral production. These data also indicate that influenza virus has evolved a strategy to overcome host innate immunity by downregulating eIF4B protein. Influenza A virus (IAV) infection stimulates the host innate immune system, in part, by inducing interferons (IFNs). Secreted IFNs activate the Janus kinase/signal transducers and activators of transcription (JAK/STAT) pathway, leading to elevated transcription of a large group of IFN-stimulated genes that have antiviral function. To circumvent the host innate

Anthelmintic drug niclosamide enhances the sensitivity of chronic myeloid leukemia cells to dasatinib through inhibiting Erk/Mnk1/eIF4E pathway

DOE Office of Scientific and Technical Information (OSTI.GOV)

Liu, Zhong; Li, Yong; Lv, Cao

Chronic myeloid leukemia (CML) responds well to BCR-ABL tyrosine kinase inhibitors (TKI), but becomes resistant to TKIs after it progresses to blast phase (BP). Here we show that niclosamide, a FDA-approved anthelmintic drug, enhances the sensitivity of BP-CML cells to dasatinib (2nd generation of BCR-ABL TKI) through inhibiting Erk/Mnk1/eIF4E signaling pathway. Niclosamide dose-dependently inhibits proliferation and induces apoptosis in a panel of CML cell lines. It also selectively targets BP-CML CD34 stem/progenitor cells through inducing apoptosis, inhibiting colony formation and self-renewal capacity while sparing normal bone marrow (NBM) counterparts. In addition, combination of niclosamide and dasatinib is synergistic in CMLmore » cell lines and BP-CML CD34 cells. Importantly, niclosamide inhibits phosphorylation of Erk, Mnk1 and eIF4E in CML cells. Overexpression of phosphomimetic but not nonphosphorylatable form of eIF4E reverses the inhibitory effects of niclosamide, suggesting that eIF4E inhibition is required for the action of niclosamide in CML. Compared to NBM, the increased levels of eIF4E and its activity in CML CD34 cells might explain the selective toxicity of niclosamide in CML versus NBM. We further show that dasatinib time-dependently induces eIF4E phosphorylation. The combination of eIF4E depletion and dasatinib results in similar effects as the combination of niclosamide and dasatinib, suggesting that niclosamide enhances dasatinib through targeting eIF4E. Our work is the first to demonstrate that niclosamide is a potential drug to overcome resistance to BCR-ABL TKI treatment in BP-CML. Our findings also suggest the therapeutic value of Erk/Mnk/eIF4E in CML treatment.« less

Eukaryotic Initiation Factor 4H Is under Transcriptional Control of p65/NF-κB

PubMed Central

Fiume, Giuseppe; Rossi, Annalisa; de Laurentiis, Annamaria; Falcone, Cristina; Pisano, Antonio; Vecchio, Eleonora; Pontoriero, Marilena; Scala, Iris; Scialdone, Annarita; Masci, Francesca Fasanella; Mimmi, Selena; Palmieri, Camillo; Scala, Giuseppe; Quinto, Ileana

2013-01-01

Protein synthesis is mainly regulated at the initiation step, allowing the fast, reversible and spatial control of gene expression. Initiation of protein synthesis requires at least 13 translation initiation factors to assemble the 80S ribosomal initiation complex. Loss of translation control may result in cell malignant transformation. Here, we asked whether translational initiation factors could be regulated by NF-κB transcription factor, a major regulator of genes involved in cell proliferation, survival, and inflammatory response. We show that the p65 subunit of NF-κB activates the transcription of eIF4H gene, which is the regulatory subunit of eIF4A, the most relevant RNA helicase in translation initiation. The p65-dependent transcriptional activation of eIF4H increased the eIF4H protein content augmenting the rate of global protein synthesis. In this context, our results provide novel insights into protein synthesis regulation in response to NF-κB activation signalling, suggesting a transcription-translation coupled mechanism of control. PMID:23776612

Cap-proximal nucleotides via differential eIF4E binding and alternative promoter usage mediate translational response to energy stress.

PubMed

Tamarkin-Ben-Harush, Ana; Vasseur, Jean-Jacques; Debart, Françoise; Ulitsky, Igor; Dikstein, Rivka

2017-02-08

Transcription start-site (TSS) selection and alternative promoter (AP) usage contribute to gene expression complexity but little is known about their impact on translation. Here we performed TSS mapping of the translatome following energy stress. Assessing the contribution of cap-proximal TSS nucleotides, we found dramatic effect on translation only upon stress. As eIF4E levels were reduced, we determined its binding to capped-RNAs with different initiating nucleotides and found the lowest affinity to 5'cytidine in correlation with the translational stress-response. In addition, the number of differentially translated APs was elevated following stress. These include novel glucose starvation-induced downstream transcripts for the translation regulators eIF4A and Pabp, which are also translationally-induced despite general translational inhibition. The resultant eIF4A protein is N-terminally truncated and acts as eIF4A inhibitor. The induced Pabp isoform has shorter 5'UTR removing an auto-inhibitory element. Our findings uncovered several levels of coordination of transcription and translation responses to energy stress.

Inhibition of Group I Metabotropic Glutamate Receptors Reverses Autistic-Like Phenotypes Caused by Deficiency of the Translation Repressor eIF4E Binding Protein 2

PubMed Central

Aguilar-Valles, Argel; Matta-Camacho, Edna; Khoutorsky, Arkady; Gkogkas, Christos; Nader, Karim

2015-01-01

Exacerbated mRNA translation during brain development has been linked to autism spectrum disorders (ASDs). Deletion of the eukaryotic initiation factor 4E (eIF4E)-binding protein 2 gene (Eif4ebp2), encoding the suppressor of mRNA translation initiation 4E-BP2, leads to an imbalance in excitatory-to-inhibitory neurotransmission and ASD-like behaviors. Inhibition of group I metabotropic glutamate receptors (mGluRs) mGluR1 and mGluR5 reverses the autistic phenotypes in several ASD mouse models. Importantly, these receptors control synaptic physiology via activation of mRNA translation. We investigated the potential reversal of autistic-like phenotypes in Eif4ebp2−/− mice by using antagonists of mGluR1 (JNJ16259685) or mGluR5 (fenobam). Augmented hippocampal mGluR-induced long-term depression (LTD; or chemically induced mGluR-LTD) in Eif4ebp2−/− mice was rescued by mGluR1 or mGluR5 antagonists. While rescue by mGluR5 inhibition occurs through the blockade of a protein synthesis-dependent component of LTD, normalization by mGluR1 antagonists requires the activation of protein synthesis. Synaptically induced LTD was deficient in Eif4ebp2−/− mice, and this deficit was not rescued by group I mGluR antagonists. Furthermore, a single dose of mGluR1 (0.3 mg/kg) or mGluR5 (3 mg/kg) antagonists in vivo reversed the deficits in social interaction and repetitive behaviors (marble burying) in Eif4ebp2−/− mice. Our results demonstrate that Eif4ebp2−/− mice serve as a relevant model to test potential therapies for ASD symptoms. In addition, we provide substantive evidence that the inhibition of mGluR1/mGluR5 is an effective treatment for physiological and behavioral alterations caused by exacerbated mRNA translation initiation. SIGNIFICANCE STATEMENT Exacerbated mRNA translation during brain development is associated with several autism spectrum disorders (ASDs). We recently demonstrated that the deletion of a negative regulator of mRNA translation initiation

Inhibition of Group I Metabotropic Glutamate Receptors Reverses Autistic-Like Phenotypes Caused by Deficiency of the Translation Repressor eIF4E Binding Protein 2.

PubMed

Aguilar-Valles, Argel; Matta-Camacho, Edna; Khoutorsky, Arkady; Gkogkas, Christos; Nader, Karim; Lacaille, Jean-Claude; Sonenberg, Nahum

2015-08-05

Exacerbated mRNA translation during brain development has been linked to autism spectrum disorders (ASDs). Deletion of the eukaryotic initiation factor 4E (eIF4E)-binding protein 2 gene (Eif4ebp2), encoding the suppressor of mRNA translation initiation 4E-BP2, leads to an imbalance in excitatory-to-inhibitory neurotransmission and ASD-like behaviors. Inhibition of group I metabotropic glutamate receptors (mGluRs) mGluR1 and mGluR5 reverses the autistic phenotypes in several ASD mouse models. Importantly, these receptors control synaptic physiology via activation of mRNA translation. We investigated the potential reversal of autistic-like phenotypes in Eif4ebp2(-/-) mice by using antagonists of mGluR1 (JNJ16259685) or mGluR5 (fenobam). Augmented hippocampal mGluR-induced long-term depression (LTD; or chemically induced mGluR-LTD) in Eif4ebp2(-/-) mice was rescued by mGluR1 or mGluR5 antagonists. While rescue by mGluR5 inhibition occurs through the blockade of a protein synthesis-dependent component of LTD, normalization by mGluR1 antagonists requires the activation of protein synthesis. Synaptically induced LTD was deficient in Eif4ebp2(-/-) mice, and this deficit was not rescued by group I mGluR antagonists. Furthermore, a single dose of mGluR1 (0.3 mg/kg) or mGluR5 (3 mg/kg) antagonists in vivo reversed the deficits in social interaction and repetitive behaviors (marble burying) in Eif4ebp2(-/-) mice. Our results demonstrate that Eif4ebp2(-/-) mice serve as a relevant model to test potential therapies for ASD symptoms. In addition, we provide substantive evidence that the inhibition of mGluR1/mGluR5 is an effective treatment for physiological and behavioral alterations caused by exacerbated mRNA translation initiation. Exacerbated mRNA translation during brain development is associated with several autism spectrum disorders (ASDs). We recently demonstrated that the deletion of a negative regulator of mRNA translation initiation, the eukaryotic initiation factor 4E

Characterisation of the biological response of Saccharomyces cerevisiae to the loss of an allele of the eukaryotic initiation factor 4A.

PubMed

Venturi, Veronica; Little, Richard; Bircham, Peter W; Rodigheri Brito, Juliana; Atkinson, Paul H; Maass, David R; Teesdale-Spittle, Paul H

2018-02-19

The translation initiation machinery is emerging as an important target for therapeutic intervention, with potential in the treatment of cancer, viral infections, and muscle wasting. Amongst the targets for pharmacological control of translation initiation is the eukaryotic initiation factor 4A (eIF4A), an RNA helicase that is essential for cap-dependent translation initiation. We set out to explore the system-wide impact of a reduction of functional eIF4A. To this end, we investigated the effect of deletion of TIF1, one of the duplicate genes that produce eIF4A in yeast, through synthetic genetic array interactions and system-wide changes in GFP-tagged protein abundances. We show that there is a biological response to deletion of the TIF1 gene that extends through the proteostasis network. Effects of the deletion are apparent in processes as distributed as chromatin remodelling, ribosome biogenesis, amino acid metabolism, and protein trafficking. The results from this study identify protein complexes and pathways that will make ideal targets for combination therapies with eIF4A inhibitors. Copyright © 2018 Elsevier Inc. All rights reserved.

Eukaryotic Translation Initiation Factor 4E Availability Controls the Switch between Cap-Dependent and Internal Ribosomal Entry Site-Mediated Translation†

PubMed Central

Svitkin, Yuri V.; Herdy, Barbara; Costa-Mattioli, Mauro; Gingras, Anne-Claude; Raught, Brian; Sonenberg, Nahum

2005-01-01

Translation of m7G-capped cellular mRNAs is initiated by recruitment of ribosomes to the 5′ end of mRNAs via eukaryotic translation initiation factor 4F (eIF4F), a heterotrimeric complex comprised of a cap-binding subunit (eIF4E) and an RNA helicase (eIF4A) bridged by a scaffolding molecule (eIF4G). Internal translation initiation bypasses the requirement for the cap and eIF4E and occurs on viral and cellular mRNAs containing internal ribosomal entry sites (IRESs). Here we demonstrate that eIF4E availability plays a critical role in the switch from cap-dependent to IRES-mediated translation in picornavirus-infected cells. When both capped and IRES-containing mRNAs are present (as in intact cells or in vitro translation extracts), a decrease in the amount of eIF4E associated with the eIF4F complex elicits a striking increase in IRES-mediated viral mRNA translation. This effect is not observed in translation extracts depleted of capped mRNAs, indicating that capped mRNAs compete with IRES-containing mRNAs for translation. These data explain numerous reported observations where viral mRNAs are preferentially translated during infection. PMID:16287867

Eukaryotic translation initiation factor 4E availability controls the switch between cap-dependent and internal ribosomal entry site-mediated translation.

PubMed

Svitkin, Yuri V; Herdy, Barbara; Costa-Mattioli, Mauro; Gingras, Anne-Claude; Raught, Brian; Sonenberg, Nahum

2005-12-01

Translation of m7G-capped cellular mRNAs is initiated by recruitment of ribosomes to the 5' end of mRNAs via eukaryotic translation initiation factor 4F (eIF4F), a heterotrimeric complex comprised of a cap-binding subunit (eIF4E) and an RNA helicase (eIF4A) bridged by a scaffolding molecule (eIF4G). Internal translation initiation bypasses the requirement for the cap and eIF4E and occurs on viral and cellular mRNAs containing internal ribosomal entry sites (IRESs). Here we demonstrate that eIF4E availability plays a critical role in the switch from cap-dependent to IRES-mediated translation in picornavirus-infected cells. When both capped and IRES-containing mRNAs are present (as in intact cells or in vitro translation extracts), a decrease in the amount of eIF4E associated with the eIF4F complex elicits a striking increase in IRES-mediated viral mRNA translation. This effect is not observed in translation extracts depleted of capped mRNAs, indicating that capped mRNAs compete with IRES-containing mRNAs for translation. These data explain numerous reported observations where viral mRNAs are preferentially translated during infection.

eIF2β is critical for eIF5-mediated GDP-dissociation inhibitor activity and translational control

PubMed Central

Jennings, Martin D.; Kershaw, Christopher J.; White, Christopher; Hoyle, Danielle; Richardson, Jonathan P.; Costello, Joseph L.; Donaldson, Ian J.; Zhou, Yu; Pavitt, Graham D.

2016-01-01

In protein synthesis translation factor eIF2 binds initiator tRNA to ribosomes and facilitates start codon selection. eIF2 GDP/GTP status is regulated by eIF5 (GAP and GDI functions) and eIF2B (GEF and GDF activities), while eIF2α phosphorylation in response to diverse signals is a major point of translational control. Here we characterize a growth suppressor mutation in eIF2β that prevents eIF5 GDI and alters cellular responses to reduced eIF2B activity, including control of GCN4 translation. By monitoring the binding of fluorescent nucleotides and initiator tRNA to purified eIF2 we show that the eIF2β mutation does not affect intrinsic eIF2 affinities for these ligands, neither does it interfere with eIF2 binding to 43S pre-initiation complex components. Instead we show that the eIF2β mutation prevents eIF5 GDI stabilizing nucleotide binding to eIF2, thereby altering the off-rate of GDP from eIF2•GDP/eIF5 complexes. This enables cells to grow with reduced eIF2B GEF activity but impairs activation of GCN4 targets in response to amino acid starvation. These findings provide support for the importance of eIF5 GDI activity in vivo and demonstrate that eIF2β acts in concert with eIF5 to prevent premature release of GDP from eIF2γ and thereby ensure tight control of protein synthesis initiation. PMID:27458202

eIF2β is critical for eIF5-mediated GDP-dissociation inhibitor activity and translational control.

PubMed

Jennings, Martin D; Kershaw, Christopher J; White, Christopher; Hoyle, Danielle; Richardson, Jonathan P; Costello, Joseph L; Donaldson, Ian J; Zhou, Yu; Pavitt, Graham D

2016-11-16

In protein synthesis translation factor eIF2 binds initiator tRNA to ribosomes and facilitates start codon selection. eIF2 GDP/GTP status is regulated by eIF5 (GAP and GDI functions) and eIF2B (GEF and GDF activities), while eIF2α phosphorylation in response to diverse signals is a major point of translational control. Here we characterize a growth suppressor mutation in eIF2β that prevents eIF5 GDI and alters cellular responses to reduced eIF2B activity, including control of GCN4 translation. By monitoring the binding of fluorescent nucleotides and initiator tRNA to purified eIF2 we show that the eIF2β mutation does not affect intrinsic eIF2 affinities for these ligands, neither does it interfere with eIF2 binding to 43S pre-initiation complex components. Instead we show that the eIF2β mutation prevents eIF5 GDI stabilizing nucleotide binding to eIF2, thereby altering the off-rate of GDP from eIF2•GDP/eIF5 complexes. This enables cells to grow with reduced eIF2B GEF activity but impairs activation of GCN4 targets in response to amino acid starvation. These findings provide support for the importance of eIF5 GDI activity in vivo and demonstrate that eIF2β acts in concert with eIF5 to prevent premature release of GDP from eIF2γ and thereby ensure tight control of protein synthesis initiation. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

Sox2 is translationally activated by eukaryotic initiation factor 4E in human glioma-initiating cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ge, Yuqing; Zhou, Fengbiao; Chen, Hong

2010-07-09

Sox2, a master transcription factor, contributes to the generation of induced pluripotent stem cells and plays significant roles in sustaining the self-renewal of neural stem cells and glioma-initiating cells. Understanding the functional differences of Sox2 between glioma-initiating cells and normal neural stem cells would contribute to therapeutic approach for treatment of brain tumors. Here, we first demonstrated that Sox2 could contribute to the self-renewal and proliferation of glioma-initiating cells. The following experiments showed that Sox2 was activated at translational level in a subset of human glioma-initiating cells compared with the normal neural stem cells. Further investigation revealed there was amore » positive correlation between Sox2 and eukaryotic initiation factor 4E (eIF4E) in glioma tissues. Down-regulation of eIF4E decreased Sox2 protein level without altering its mRNA level in glioma-initiating cells, indicating that Sox2 was activated by eIF4E at translational level. Furthermore, eIF4E was presumed to regulate the expression of Sox2 by its 5' untranslated region (5' UTR) sequence. Our results suggest that the eIF4E-Sox2 axis is a novel mechanism of unregulated self-renewal of glioma-initiating cells, providing a potential therapeutic target for glioma.« less

The RNA recognition motif of eukaryotic translation initiation factor 3g (eIF3g) is required for resumption of scanning of posttermination ribosomes for reinitiation on GCN4 and together with eIF3i stimulates linear scanning.

PubMed

Cuchalová, Lucie; Kouba, Tomás; Herrmannová, Anna; Dányi, István; Chiu, Wen-Ling; Valásek, Leos

2010-10-01

Recent reports have begun unraveling the details of various roles of individual eukaryotic translation initiation factor 3 (eIF3) subunits in translation initiation. Here we describe functional characterization of two essential Saccharomyces cerevisiae eIF3 subunits, g/Tif35 and i/Tif34, previously suggested to be dispensable for formation of the 48S preinitiation complexes (PICs) in vitro. A triple-Ala substitution of conserved residues in the RRM of g/Tif35 (g/tif35-KLF) or a single-point mutation in the WD40 repeat 6 of i/Tif34 (i/tif34-Q258R) produces severe growth defects and decreases the rate of translation initiation in vivo without affecting the integrity of eIF3 and formation of the 43S PICs in vivo. Both mutations also diminish induction of GCN4 expression, which occurs upon starvation via reinitiation. Whereas g/tif35-KLF impedes resumption of scanning for downstream reinitiation by 40S ribosomes terminating at upstream open reading frame 1 (uORF1) in the GCN4 mRNA leader, i/tif34-Q258R prevents full GCN4 derepression by impairing the rate of scanning of posttermination 40S ribosomes moving downstream from uORF1. In addition, g/tif35-KLF reduces processivity of scanning through stable secondary structures, and g/Tif35 specifically interacts with Rps3 and Rps20 located near the ribosomal mRNA entry channel. Together these results implicate g/Tif35 and i/Tif34 in stimulation of linear scanning and, specifically in the case of g/Tif35, also in proper regulation of the GCN4 reinitiation mechanism.

Pharmacogenetic Inhibition of eIF4E-Dependent Mmp9 mRNA Translation Reverses Fragile X Syndrome-like Phenotypes

PubMed Central

Gkogkas, Christos G.; Khoutorsky, Arkady; Cao, Ruifeng; Jafarnejad, Seyed Mehdi; Prager-Khoutorsky, Masha; Giannakas, Nikolaos; Kaminari, Archontia; Fragkouli, Apostolia; Nader, Karim; Price, Theodore J.; Konicek, Bruce W.; Graff, Jeremy R.; Tzinia, Athina K.; Lacaille, Jean-Claude; Sonenberg, Nahum

2015-01-01

SUMMARY Fragile X syndrome (FXS) is the leading genetic cause of autism. Mutations in Fmr1 (fragile X mental retardation 1 gene) engender exaggerated translation resulting in dendritic spine dysmorphogenesis, synaptic plasticity alterations, and behavioral deficits in mice, which are reminiscent of FXS pheno-types. Using postmortem brains from FXS patients and Fmr1 knockout mice (Fmr1 −/y), we show that phosphorylation of the mRNA 5′ cap binding protein, eukaryotic initiation factor 4E (eIF4E), is elevated concomitant with increased expression of matrix metalloproteinase 9 (MMP-9) protein. Genetic or pharmacological reduction of eIF4E phosphorylation rescued core behavioral deficits, synaptic plasticity alterations, and dendritic spine morphology defects via reducing exaggerated translation of Mmp9 mRNA in Fmr1 −/y mice, whereas MMP-9 overexpression produced several FXS-like phenotypes. These results uncover a mechanism of regulation of synaptic function by translational control of Mmp-9 in FXS, which opens the possibility of new treatment avenues for the diverse neurological and psychiatric aspects of FXS. PMID:25466251

Pharmacogenetic inhibition of eIF4E-dependent Mmp9 mRNA translation reverses fragile X syndrome-like phenotypes.

PubMed

Gkogkas, Christos G; Khoutorsky, Arkady; Cao, Ruifeng; Jafarnejad, Seyed Mehdi; Prager-Khoutorsky, Masha; Giannakas, Nikolaos; Kaminari, Archontia; Fragkouli, Apostolia; Nader, Karim; Price, Theodore J; Konicek, Bruce W; Graff, Jeremy R; Tzinia, Athina K; Lacaille, Jean-Claude; Sonenberg, Nahum

2014-12-11

Fragile X syndrome (FXS) is the leading genetic cause of autism. Mutations in Fmr1 (fragile X mental retardation 1 gene) engender exaggerated translation resulting in dendritic spine dysmorphogenesis, synaptic plasticity alterations, and behavioral deficits in mice, which are reminiscent of FXS phenotypes. Using postmortem brains from FXS patients and Fmr1 knockout mice (Fmr1(-/y)), we show that phosphorylation of the mRNA 5' cap binding protein, eukaryotic initiation factor 4E (eIF4E), is elevated concomitant with increased expression of matrix metalloproteinase 9 (MMP-9) protein. Genetic or pharmacological reduction of eIF4E phosphorylation rescued core behavioral deficits, synaptic plasticity alterations, and dendritic spine morphology defects via reducing exaggerated translation of Mmp9 mRNA in Fmr1(-/y) mice, whereas MMP-9 overexpression produced several FXS-like phenotypes. These results uncover a mechanism of regulation of synaptic function by translational control of Mmp-9 in FXS, which opens the possibility of new treatment avenues for the diverse neurological and psychiatric aspects of FXS. Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.

PRMT5 is essential for the eIF4E-mediated 5′-cap dependent translation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lim, Ji-Hong; Lee, Yoon-Mi; Lee, Gibok

2014-10-03

Highlights: • PRMT5 participates in syntheses of HIF-1α, c-Myc and cyclin D1 proteins. • PRMT5 promotes the 5′-cap dependent translation. • PRMT5 is required for eIF4E binding to mRNA 5′-cap. • PRMT5 is essential for eIF4E-dependent cell proliferation. - Abstract: It is becoming clear that PRMT5 plays essential roles in cell cycle progression, survival, and responses to external stresses. However, the precise mechanisms underlying such roles of PRMT5 have not been clearly understood. Previously, we have demonstrated that PRMT5 participates in cellular adaptation to hypoxia by ensuring 5′-cap dependent translation of HIF-1α. Given that c-Myc and cyclin D1 expressions aremore » also tightly regulated in 5′-cap dependent manner, we here tested the possibility that PRMT5 promotes cell proliferation by increasing de novo syntheses of the oncoproteins. c-Myc and cyclin D1 were found to be noticeably downregulated by PRMT5 knock-down. A RNA immunoprecipitation analysis, which can identify RNA–protein interactions, showed that PRMT5 is required for the interaction among eIF4E and 5′-UTRs of HIF-1α, c-Myc and cyclin D1 mRNAs. In addition, PRMT5 knock-down inhibited cell proliferation by inducing cell cycle arrest at the G1 phase. More importantly, ectopic expression of eIF4E significantly rescued the cell cycle progression and cell proliferation even in PRMT5-deficeint condition. Based on these results, we propose that PRMT5 determines cell fate by regulating 5′-cap dependent translation of proteins essential for proliferation and survival.« less

A chemical genetic screen for mTOR pathway inhibitors based on 4E-BP-dependent nuclear accumulation of eIF4E.

PubMed

Livingstone, Mark; Larsson, Ola; Sukarieh, Rami; Pelletier, Jerry; Sonenberg, Nahum

2009-12-24

The signal transduction pathway wherein mTOR regulates cellular growth and proliferation is an active target for drug discovery. The search for new mTOR inhibitors has recently yielded a handful of promising compounds that hold therapeutic potential. This search has been limited by the lack of a high-throughput assay to monitor the phosphorylation of a direct rapamycin-sensitive mTOR substrate in cells. Here we describe a novel cell-based chemical genetic screen useful for efficiently monitoring mTOR signaling to 4E-BPs in response to stimuli. The screen is based on the nuclear accumulation of eIF4E, which occurs in a 4E-BP-dependent manner specifically upon inhibition of mTOR signaling. Using this assay in a small-scale screen, we have identified several compounds not previously known to inhibit mTOR signaling, demonstrating that this method can be adapted to larger screens. Copyright 2009 Elsevier Ltd. All rights reserved.

The putative tumor suppressor microRNA-497 modulates gastric cancer cell proliferation and invasion by repressing eIF4E

DOE Office of Scientific and Technical Information (OSTI.GOV)

Li, Weidong; Jin, Xuejun; Deng, Xubin

2014-06-27

Highlights: • MiR-497 expression was down-regulated in GC patients and GC cell lines. • MiR-497 inhibited cell proliferation and invasion of GC cells in vitro. • MiR-497 modulated eIF4E expression in GC cells. • Restoration of miR-497 decreased tumor growth and metastasis in vivo. - Abstract: Accumulating evidence has shown that microRNAs are involved in multiple processes in gastric cancer (GC) development and progression. Aberrant expression of miR-497 has been frequently reported in cancer studies; however, the role and mechanism of its function in GC remains unknown. Here, we reported that miR-497 was frequently downregulated in GC tissues and associatedmore » with aggressive clinicopathological features of GC patients. Further in vitro observations showed that the enforced expression of miR-497 inhibited cell proliferation by blocking the G1/S transition and decreased the invasion of GC cells, implying that miR-497 functions as a tumor suppressor in the progression of GC. In vivo study indicated that restoration of miR-497 inhibited tumor growth and metastasis. Luciferase assays revealed that miR-497 inhibited eIF4E expression by targeting the binding sites in the 3′-untranslated region of eIF4E mRNA. qRT-PCR and Western blot assays verified that miR-497 reduced eIF4E expression at both the mRNA and protein levels. A reverse correlation between miR-497 and eIF4E expression was noted in GC tissues. Taken together, our results identify a crucial tumor suppressive role of miR-497 in the progression of GC and suggest that miR-497 might be an anticancer therapeutic target for GC patients.« less

Variable Expressivity of a Founder Mutation in the EIF2AK4 Gene in Hereditary Pulmonary Veno-occlusive Disease and Its Impact on Survival.

PubMed

Navas Tejedor, Paula; Palomino Doza, Julián; Tenorio Castaño, Jair Antonio; Enguita Valls, Ana Belén; Rodríguez Reguero, José Julián; Martínez Meñaca, Amaya; Hernández González, Ignacio; Bueno Zamora, Héctor; Lapunzina Badía, Pablo Daniel; Escribano Subías, Pilar

2018-02-01

Hereditary pulmonary veno-occlusive disease (PVOD) has been associated with biallelic mutations in EIF2AK4 with the recent discovery of a founder mutation in Iberian Romani patients with familial PVOD. The aims of this study were phenotypical characterization and survival analysis of Iberian Romani patients with familial PVOD carrying the founder p.Pro1115Leu mutation in EIF2AK4, according to their tolerance to pulmonary vasodilators (PVD). Familial genetic screening was conducted, as well as assessment of sociocultural determinants with a potential influence on disease course. Observational study of Romani patients with familial PVOD included in the Spanish Registry of Pulmonary Arterial Hypertension. Genetic screening of EIF2AK4 was performed in index cases and relatives between November 2011 and July 2016 and histological pulmonary examination was carried out in patients who received a lung transplant or died. The patients were divided into 2 groups depending on their tolerance to PVD, with comparison of baseline characteristics and survival free of death or lung transplant. Eighteen Romani patients were included: 9 index cases and 9 relatives. The biallelic founder mutation in EIF2AK4 was found in all affected cases and 2 unaffected relatives. Family screening showed 34.2% of healthy heterozygotes, high consanguinity, young age at childbirth, and frequent multiparity. Prognosis was bleak, with significant differences depending on tolerance to PVD. We describe 2 phenotypes of hereditary PVOD depending on tolerance to PVD, with prognostic impact and familial distribution. Consanguinity may have a negative impact on the transmission of PVOD, with familial genetic screening showing high effectiveness. Copyright © 2017 Sociedad Española de Cardiología. Published by Elsevier España, S.L.U. All rights reserved.

m6A and eIF2α-ⓟ Team Up to Tackle ATF4 Translation during Stress.

PubMed

Powers, Emily Nicole; Brar, Gloria Ann

2018-02-15

While m 6 A modification of mRNAs is now known to be widespread, the cellular roles of this modification remain largely mysterious. In this issue of Molecular Cell, Zhou et al. (2018) show that m 6 A modification unexpectedly contributes to the established uORF- and eIF2α-ⓟ-dependent mechanism of ATF4 translational regulation in response to stress. Copyright © 2018 Elsevier Inc. All rights reserved.

Eukaryotic translational initiation factor 4AII reduces the replication of infectious bursal disease virus by inhibiting VP1 polymerase activity.

PubMed

Gao, Li; Li, Kai; Zhong, Li; Zhang, Lizhou; Qi, Xiaole; Wang, Yongqiang; Gao, Yulong; Wang, Xiaomei

2017-03-01

Infectious bursal disease (IBD) is an acute, highly contagious, and immunosuppressive avian disease caused by IBD virus (IBDV). Although an interaction between eukaryotic translational initiation factor 4AII (eIF4AII) of the host and viral protein 1 (VP1), the RNA-dependent RNA polymerase (RdRp) of IBDV, has been established, the underlying effects of this interaction on IBDV and the molecular mechanism remain unclear. We here report that interaction of the host eIF4AII with VP1 inhibits the RNA polymerase activity of IBDV to reduce its replication in host cells. We found that ectopically expressed eIF4AII markedly inhibited IBDV growth in DF1 cells, and knockdown of eIF4AII by small interfering RNA significantly enhanced viral replication in CEF cells. Furthermore, IBDV infection led to an increase in host eIF4AII expression, suggesting a feedback mechanism between the host and virus infection both in vitro and in vivo, which further confirmed the involvement of the host eIF4AII in the IBDV life cycle. Thus, via the interaction with VP1, eIF4AII plays a critical role in the IBDV life cycle, by inhibiting viral RNA polymerase activity, leading to a reduction of IBDV replication in cells. Copyright © 2016 Elsevier B.V. All rights reserved.

Notch signaling sustains the expression of Mcl-1 and the activity of eIF4E to promote cell survival in CLL

PubMed Central

De Falco, Filomena; Sabatini, Rita; Del Papa, Beatrice; Falzetti, Franca; Di Ianni, Mauro; Sportoletti, Paolo; Baldoni, Stefano; Screpanti, Isabella; Marconi, Pierfrancesco; Rosati, Emanuela

2015-01-01

In chronic lymphocytic leukemia (CLL), Notch1 and Notch2 signaling is constitutively activated and contributes to apoptosis resistance. We show that genetic inhibition of either Notch1 or Notch2, through small-interfering RNA, increases apoptosis of CLL cells and is associated with decreased levels of the anti-apoptotic protein Mcl-1. Thus, Notch signaling promotes CLL cell survival at least in part by sustaining Mcl-1 expression. In CLL cells, an enhanced Notch activation also contributes to the increase in Mcl-1 expression and cell survival induced by IL-4. Mcl-1 downregulation by Notch targeting is not due to reduced transcription or degradation by caspases, but in part, to increased degradation by the proteasome. Mcl-1 downregulation by Notch targeting is also accompanied by reduced phosphorylation of eukaryotic translation initiation factor 4E (eIF4E), suggesting that this protein is another target of Notch signaling in CLL cells. Overall, we show that Notch signaling sustains CLL cell survival by promoting Mcl-1 expression and eIF4E activity, and given the oncogenic role of these factors, we underscore the therapeutic potential of Notch inhibition in CLL. PMID:26041884

The small molecule '1-(4-biphenylylcarbonyl)-4-(5-bromo-2-methoxybenzyl) piperazine oxalate' and its derivatives regulate global protein synthesis by inactivating eukaryotic translation initiation factor 2-alpha.

PubMed

Hong, Mi-Na; Nam, Ky-Youb; Kim, Kyung Kon; Kim, So-Young; Kim, InKi

2016-05-01

By environmental stresses, cells can initiate a signaling pathway in which eukaryotic translation initiation factor 2-alpha (eIF2-α) is involved to regulate the response. Phosphorylation of eIF2-α results in the reduction of overall protein neogenesis, which allows cells to conserve resources and to reprogram energy usage for effective stress control. To investigate the role of eIF2-α in cell stress responses, we conducted a viability-based compound screen under endoplasmic reticulum (ER) stress condition, and identified 1-(4-biphenylylcarbonyl)-4-(5-bromo-2-methoxybenzyl) piperazine oxalate (AMC-01) and its derivatives as eIF2-α-inactivating chemical. Molecular characterization of this signaling pathway revealed that AMC-01 induced inactivation of eIF2-α by phosphorylating serine residue 51 in a dose- and time-dependent manner, while the negative control compounds did not affect eIF2-α phosphorylation. In contrast with ER stress induction by thapsigargin, phosphorylation of eIF2-α persisted for the duration of incubation with AMC-01. By pathway analysis, AMC-01 clearly induced the activation of protein kinase RNA-activated (PKR) kinase and nuclear factor-κB (NF-κB), whereas it did not modulate the activity of PERK or heme-regulated inhibitor (HRI). Finally, we could detect a lower protein translation rate in cells incubated with AMC-01, establishing AMC-01 as a potent chemical probe that can regulate eIF2-α activity. We suggest from these data that AMC-01 and its derivative compounds can be used as chemical probes in future studies of the role of eIF2-α in protein synthesis-related cell physiology.

Eukaryotic Translation Initiation Factor eIFiso4G Is Required to Regulate Violaxanthin De-epoxidase Expression in Arabidopsis*

PubMed Central

Chen, Zhong; Jolley, Blair; Caldwell, Christian; Gallie, Daniel R.

2014-01-01

The eukaryotic translation initiation factor (eIF) 4G is a scaffold protein that organizes the assembly of those initiation factors needed to recruit the 40 S ribosomal subunit to an mRNA. Plants, like many eukaryotes, express two eIF4G isoforms. eIFiso4G, one of the isoforms specific to plants, is unique among eukaryotic eIF4G proteins in that it is highly divergent and unusually small in size, raising the possibility of functional specialization. In this study, the role of eIFiso4G in plant growth was investigated using null mutants for the eIF4G isoforms in Arabidopsis. eIFiso4G loss of function mutants exhibited smaller cell, leaf, plant size, and biomass accumulation that correlated with its reduced photosynthetic activity, phenotypes not observed with the eIF4G loss of function mutant. Although no change in photorespiration or dark respiration was observed in the eIFiso4G loss of function mutant, a reduction in chlorophyll levels and an increase in the level of nonphotochemical quenching were observed. An increase in xanthophyll cycle activity and the generation of reactive oxygen species contributed to the qE and qI components of nonphotochemical quenching, respectively. An increase in the transcript and protein levels of violaxanthin de-epoxidase in the eIFiso4G loss of function mutant and an increase in its xanthophyll de-epoxidation state correlated with the higher qE associated with loss of eIFiso4G expression. These observations indicate that eIFiso4G expression is required to regulate violaxanthin de-epoxidase expression and to support photosynthetic activity. PMID:24706761

Pharmacological dimerization and activation of the exchange factor eIF2B antagonizes the integrated stress response

PubMed Central

Sidrauski, Carmela; Tsai, Jordan C; Kampmann, Martin; Hearn, Brian R; Vedantham, Punitha; Jaishankar, Priyadarshini; Sokabe, Masaaki; Mendez, Aaron S; Newton, Billy W; Tang, Edward L; Verschueren, Erik; Johnson, Jeffrey R; Krogan, Nevan J; Fraser, Christopher S; Weissman, Jonathan S; Renslo, Adam R; Walter, Peter

2015-01-01

The general translation initiation factor eIF2 is a major translational control point. Multiple signaling pathways in the integrated stress response phosphorylate eIF2 serine-51, inhibiting nucleotide exchange by eIF2B. ISRIB, a potent drug-like small molecule, renders cells insensitive to eIF2α phosphorylation and enhances cognitive function in rodents by blocking long-term depression. ISRIB was identified in a phenotypic cell-based screen, and its mechanism of action remained unknown. We now report that ISRIB is an activator of eIF2B. Our reporter-based shRNA screen revealed an eIF2B requirement for ISRIB activity. Our results define ISRIB as a symmetric molecule, show ISRIB-mediated stabilization of activated eIF2B dimers, and suggest that eIF2B4 (δ-subunit) contributes to the ISRIB binding site. We also developed new ISRIB analogs, improving its EC50 to 600 pM in cell culture. By modulating eIF2B function, ISRIB promises to be an invaluable tool in proof-of-principle studies aiming to ameliorate cognitive defects resulting from neurodegenerative diseases. DOI: http://dx.doi.org/10.7554/eLife.07314.001 PMID:25875391

Yeast eIF4B binds to the head of the 40S ribosomal subunit and promotes mRNA recruitment through its N-terminal and internal repeat domains.

PubMed

Walker, Sarah E; Zhou, Fujun; Mitchell, Sarah F; Larson, Victoria S; Valasek, Leos; Hinnebusch, Alan G; Lorsch, Jon R

2013-02-01

Eukaryotic translation initiation factor (eIF)4B stimulates recruitment of mRNA to the 43S ribosomal pre-initiation complex (PIC). Yeast eIF4B (yeIF4B), shown previously to bind single-stranded (ss) RNA, consists of an N-terminal domain (NTD), predicted to be unstructured in solution; an RNA-recognition motif (RRM); an unusual domain comprised of seven imperfect repeats of 26 amino acids; and a C-terminal domain. Although the mechanism of yeIF4B action has remained obscure, most models have suggested central roles for its RRM and ssRNA-binding activity. We have dissected the functions of yeIF4B's domains and show that the RRM and its ssRNA-binding activity are dispensable in vitro and in vivo. Instead, our data indicate that the 7-repeats and NTD are the most critical domains, which mediate binding of yeIF4B to the head of the 40S ribosomal subunit via interaction with Rps20. This interaction induces structural changes in the ribosome's mRNA entry channel that could facilitate mRNA loading. We also show that yeIF4B strongly promotes productive interaction of eIF4A with the 43S•mRNA PIC in a manner required for efficient mRNA recruitment.

Yeast eIF4B binds to the head of the 40S ribosomal subunit and promotes mRNA recruitment through its N-terminal and internal repeat domains

PubMed Central

Walker, Sarah E.; Zhou, Fujun; Mitchell, Sarah F.; Larson, Victoria S.; Valasek, Leos; Hinnebusch, Alan G.; Lorsch, Jon R.

2013-01-01

Eukaryotic translation initiation factor (eIF)4B stimulates recruitment of mRNA to the 43S ribosomal pre-initiation complex (PIC). Yeast eIF4B (yeIF4B), shown previously to bind single-stranded (ss) RNA, consists of an N-terminal domain (NTD), predicted to be unstructured in solution; an RNA-recognition motif (RRM); an unusual domain comprised of seven imperfect repeats of 26 amino acids; and a C-terminal domain. Although the mechanism of yeIF4B action has remained obscure, most models have suggested central roles for its RRM and ssRNA-binding activity. We have dissected the functions of yeIF4B’s domains and show that the RRM and its ssRNA-binding activity are dispensable in vitro and in vivo. Instead, our data indicate that the 7-repeats and NTD are the most critical domains, which mediate binding of yeIF4B to the head of the 40S ribosomal subunit via interaction with Rps20. This interaction induces structural changes in the ribosome’s mRNA entry channel that could facilitate mRNA loading. We also show that yeIF4B strongly promotes productive interaction of eIF4A with the 43S•mRNA PIC in a manner required for efficient mRNA recruitment. PMID:23236192

The nematode eukaryotic translation initiation factor 4E/G complex works with a trans-spliced leader stem-loop to enable efficient translation of trimethylguanosine-capped RNAs.

PubMed

Wallace, Adam; Filbin, Megan E; Veo, Bethany; McFarland, Craig; Stepinski, Janusz; Jankowska-Anyszka, Marzena; Darzynkiewicz, Edward; Davis, Richard E

2010-04-01

Eukaryotic mRNA translation begins with recruitment of the 40S ribosome complex to the mRNA 5' end through the eIF4F initiation complex binding to the 5' m(7)G-mRNA cap. Spliced leader (SL) RNA trans splicing adds a trimethylguanosine (TMG) cap and a sequence, the SL, to the 5' end of mRNAs. Efficient translation of TMG-capped mRNAs in nematodes requires the SL sequence. Here we define a core set of nucleotides and a stem-loop within the 22-nucleotide nematode SL that stimulate translation of mRNAs with a TMG cap. The structure and core nucleotides are conserved in other nematode SLs and correspond to regions of SL1 required for early Caenorhabditis elegans development. These SL elements do not facilitate translation of m(7)G-capped RNAs in nematodes or TMG-capped mRNAs in mammalian or plant translation systems. Similar stem-loop structures in phylogenetically diverse SLs are predicted. We show that the nematode eukaryotic translation initiation factor 4E/G (eIF4E/G) complex enables efficient translation of the TMG-SL RNAs in diverse in vitro translation systems. TMG-capped mRNA translation is determined by eIF4E/G interaction with the cap and the SL RNA, although the SL does not increase the affinity of eIF4E/G for capped RNA. These results suggest that the mRNA 5' untranslated region (UTR) can play a positive and novel role in translation initiation through interaction with the eIF4E/G complex in nematodes and raise the issue of whether eIF4E/G-RNA interactions play a role in the translation of other eukaryotic mRNAs.

Crystallization and preliminary X-ray analysis of eukaryotic initiation factor 4E from Pisum sativum

PubMed Central

Ashby, Jamie A.; Stevenson, Clare E. M.; Maule, Andrew J.; Lawson, David M.

2009-01-01

Crystals of an N-terminally truncated 20 kDa fragment of Pisum sativum eIF4E (ΔN-eIF4E) were grown by vapour diffusion. X-ray data were recorded to a resolution of 2.2 Å from a single crystal in-house. Indexing was consistent with primitive monoclinic symmetry and solvent-content estimations suggested that between four and nine copies of the eIF4E fragment were possible per crystallographic asymmetric unit. eIF4E is an essential component of the eukaryotic translation machinery and recent studies have shown that point mutations of plant eIF4Es can confer resistance to potyvirus infection. PMID:19652353

Fusel Alcohols Regulate Translation Initiation by Inhibiting eIF2B to Reduce Ternary Complex in a Mechanism That May Involve Altering the Integrity and Dynamics of the eIF2B Body

PubMed Central

Taylor, Eleanor J.; Campbell, Susan G.; Griffiths, Christian D.; Reid, Peter J.; Slaven, John W.; Harrison, Richard J.; Sims, Paul F.G.; Pavitt, Graham D.; Delneri, Daniela

2010-01-01

Recycling of eIF2-GDP to the GTP-bound form constitutes a core essential, regulated step in eukaryotic translation. This reaction is mediated by eIF2B, a heteropentameric factor with important links to human disease. eIF2 in the GTP-bound form binds to methionyl initiator tRNA to form a ternary complex, and the levels of this ternary complex can be a critical determinant of the rate of protein synthesis. Here we show that eIF2B serves as the target for translation inhibition by various fusel alcohols in yeast. Fusel alcohols are endpoint metabolites from amino acid catabolism, which signal nitrogen scarcity. We show that the inhibition of eIF2B leads to reduced ternary complex levels and that different eIF2B subunit mutants alter fusel alcohol sensitivity. A DNA tiling array strategy was developed that overcame difficulties in the identification of these mutants where the phenotypic distinctions were too subtle for classical complementation cloning. Fusel alcohols also lead to eIF2α dephosphorylation in a Sit4p-dependent manner. In yeast, eIF2B occupies a large cytoplasmic body where guanine nucleotide exchange on eIF2 can occur and be regulated. Fusel alcohols impact on both the movement and dynamics of this 2B body. Overall, these results confirm that the guanine nucleotide exchange factor, eIF2B, is targeted by fusel alcohols. Moreover, they highlight a potential connection between the movement or integrity of the 2B body and eIF2B regulation. PMID:20444979

La-related protein 1 (LARP1) binds the mRNA cap, blocking eIF4F assembly on TOP mRNAs.

PubMed

Lahr, Roni M; Fonseca, Bruno D; Ciotti, Gabrielle E; Al-Ashtal, Hiba A; Jia, Jian-Jun; Niklaus, Marius R; Blagden, Sarah P; Alain, Tommy; Berman, Andrea J

2017-04-07

The 5'terminal oligopyrimidine (5'TOP) motif is a cis -regulatory RNA element located immediately downstream of the 7-methylguanosine [m 7 G] cap of TOP mRNAs, which encode ribosomal proteins and translation factors. In eukaryotes, this motif coordinates the synchronous and stoichiometric expression of the protein components of the translation machinery. La-related protein 1 (LARP1) binds TOP mRNAs, regulating their stability and translation. We present crystal structures of the human LARP1 DM15 region in complex with a 5'TOP motif, a cap analog (m 7 GTP), and a capped cytidine (m 7 GpppC), resolved to 2.6, 1.8 and 1.7 Å, respectively. Our binding, competition, and immunoprecipitation data corroborate and elaborate on the mechanism of 5'TOP motif binding by LARP1. We show that LARP1 directly binds the cap and adjacent 5'TOP motif of TOP mRNAs, effectively impeding access of eIF4E to the cap and preventing eIF4F assembly. Thus, LARP1 is a specialized TOP mRNA cap-binding protein that controls ribosome biogenesis.

eIF3a: A new anticancer drug target in the eIF family.

PubMed

Yin, Ji-Ye; Zhang, Jian-Ting; Zhang, Wei; Zhou, Hong-Hao; Liu, Zhao-Qian

2018-01-01

eIF3a is the largest subunit of eIF3, which is a key player in all steps of translation initiation. During the past years, eIF3a is recognized as a proto-oncogene, which is an important discovery in this field. It is widely reported to be correlated with cancer occurrence, metastasis, prognosis, and therapeutic response. Recently, the mechanisms of eIF3a action in the carcinogenesis are unveiled gradually. A number of cellular, physiological, and pathological processes involving eIF3a are identified. Most importantly, it is emerging as a new potential drug target in the eIF family, and some small molecule inhibitors are being developed. Thus, we perform a critical review of recent advances in understanding eIF3a physiological and pathological functions, with specific focus on its role in cancer and anticancer drug targets. Copyright © 2017 Elsevier B.V. All rights reserved.

The Nematode Eukaryotic Translation Initiation Factor 4E/G Complex Works with a trans-Spliced Leader Stem-Loop To Enable Efficient Translation of Trimethylguanosine-Capped RNAs ▿ †

PubMed Central

Wallace, Adam; Filbin, Megan E.; Veo, Bethany; McFarland, Craig; Stepinski, Janusz; Jankowska-Anyszka, Marzena; Darzynkiewicz, Edward; Davis, Richard E.

2010-01-01

Eukaryotic mRNA translation begins with recruitment of the 40S ribosome complex to the mRNA 5′ end through the eIF4F initiation complex binding to the 5′ m7G-mRNA cap. Spliced leader (SL) RNA trans splicing adds a trimethylguanosine (TMG) cap and a sequence, the SL, to the 5′ end of mRNAs. Efficient translation of TMG-capped mRNAs in nematodes requires the SL sequence. Here we define a core set of nucleotides and a stem-loop within the 22-nucleotide nematode SL that stimulate translation of mRNAs with a TMG cap. The structure and core nucleotides are conserved in other nematode SLs and correspond to regions of SL1 required for early Caenorhabditis elegans development. These SL elements do not facilitate translation of m7G-capped RNAs in nematodes or TMG-capped mRNAs in mammalian or plant translation systems. Similar stem-loop structures in phylogenetically diverse SLs are predicted. We show that the nematode eukaryotic translation initiation factor 4E/G (eIF4E/G) complex enables efficient translation of the TMG-SL RNAs in diverse in vitro translation systems. TMG-capped mRNA translation is determined by eIF4E/G interaction with the cap and the SL RNA, although the SL does not increase the affinity of eIF4E/G for capped RNA. These results suggest that the mRNA 5′ untranslated region (UTR) can play a positive and novel role in translation initiation through interaction with the eIF4E/G complex in nematodes and raise the issue of whether eIF4E/G-RNA interactions play a role in the translation of other eukaryotic mRNAs. PMID:20154140

Identification and Characterization of Functionally Critical, Conserved Motifs in the Internal Repeats and N-terminal Domain of Yeast Translation Initiation Factor 4B (yeIF4B)*

PubMed Central

Zhou, Fujun; Walker, Sarah E.; Mitchell, Sarah F.; Lorsch, Jon R.; Hinnebusch, Alan G.

2014-01-01

eIF4B has been implicated in attachment of the 43 S preinitiation complex (PIC) to mRNAs and scanning to the start codon. We recently determined that the internal seven repeats (of ∼26 amino acids each) of Saccharomyces cerevisiae eIF4B (yeIF4B) compose the region most critically required to enhance mRNA recruitment by 43 S PICs in vitro and stimulate general translation initiation in yeast. Moreover, although the N-terminal domain (NTD) of yeIF4B contributes to these activities, the RNA recognition motif is dispensable. We have now determined that only two of the seven internal repeats are sufficient for wild-type (WT) yeIF4B function in vivo when all other domains are intact. However, three or more repeats are needed in the absence of the NTD or when the functions of eIF4F components are compromised. We corroborated these observations in the reconstituted system by demonstrating that yeIF4B variants with only one or two repeats display substantial activity in promoting mRNA recruitment by the PIC, whereas additional repeats are required at lower levels of eIF4A or when the NTD is missing. These findings indicate functional overlap among the 7-repeats and NTD domains of yeIF4B and eIF4A in mRNA recruitment. Interestingly, only three highly conserved positions in the 26-amino acid repeat are essential for function in vitro and in vivo. Finally, we identified conserved motifs in the NTD and demonstrate functional overlap of two such motifs. These results provide a comprehensive description of the critical sequence elements in yeIF4B that support eIF4F function in mRNA recruitment by the PIC. PMID:24285537

Translation Initiation Factor AteIF(iso)4E Is Involved in Selective mRNA Translation in Arabidopsis Thaliana Seedlings

PubMed Central

Martínez-Silva, Ana Valeria; Aguirre-Martínez, César; Flores-Tinoco, Carlos E.; Alejandri-Ramírez, Naholi D.; Dinkova, Tzvetanka D.

2012-01-01

One of the most regulated steps of translation initiation is the recruitment of mRNA by the translation machinery. In eukaryotes, this step is mediated by the 5′end cap-binding factor eIF4E bound to the bridge protein eIF4G and forming the eIF4F complex. In plants, different isoforms of eIF4E and eIF4G form the antigenically distinct eIF4F and eIF(iso)4F complexes proposed to mediate selective translation. Using a microarray analysis of polyribosome- and non-polyribosome-purified mRNAs from 15 day-old Arabidopsis thaliana wild type [WT] and eIF(iso)4E knockout mutant [(iso)4E-1] seedlings we found 79 transcripts shifted from polyribosomes toward non-polyribosomes, and 47 mRNAs with the opposite behavior in the knockout mutant. The translationally decreased mRNAs were overrepresented in root-preferentially expressed genes and proteins from the endomembrane system, including several transporters such as the phosphate transporter PHOSPHATE1 (PHO1), Sucrose transporter 3 (SUC3), ABC transporter-like with ATPase activity (MRP11) and five electron transporters, as well as signal transduction-, protein modification- and transcription-related proteins. Under normal growth conditions, eIF(iso)4E expression under the constitutive promoter 35 S enhanced the polyribosomal recruitment of PHO1 supporting its translational preference for eIF(iso)4E. Furthermore, under phosphate deficiency, the PHO1 protein increased in the eIF(iso)4E overexpressing plants and decreased in the knockout mutant as compared to wild type. In addition, the knockout mutant had larger root, whereas the 35 S directed expression of eIF(iso)4E caused shorter root under normal growth conditions, but not under phosphate deficiency. These results indicate that selective translation mediated by eIF(iso)4E is relevant for Arabidopsis root development under normal growth conditions. PMID:22363683

La-related protein 1 (LARP1) binds the mRNA cap, blocking eIF4F assembly on TOP mRNAs

PubMed Central

Lahr, Roni M; Fonseca, Bruno D; Ciotti, Gabrielle E; Al-Ashtal, Hiba A; Jia, Jian-Jun; Niklaus, Marius R; Blagden, Sarah P; Alain, Tommy; Berman, Andrea J

2017-01-01

The 5’terminal oligopyrimidine (5’TOP) motif is a cis-regulatory RNA element located immediately downstream of the 7-methylguanosine [m7G] cap of TOP mRNAs, which encode ribosomal proteins and translation factors. In eukaryotes, this motif coordinates the synchronous and stoichiometric expression of the protein components of the translation machinery. La-related protein 1 (LARP1) binds TOP mRNAs, regulating their stability and translation. We present crystal structures of the human LARP1 DM15 region in complex with a 5’TOP motif, a cap analog (m7GTP), and a capped cytidine (m7GpppC), resolved to 2.6, 1.8 and 1.7 Å, respectively. Our binding, competition, and immunoprecipitation data corroborate and elaborate on the mechanism of 5’TOP motif binding by LARP1. We show that LARP1 directly binds the cap and adjacent 5’TOP motif of TOP mRNAs, effectively impeding access of eIF4E to the cap and preventing eIF4F assembly. Thus, LARP1 is a specialized TOP mRNA cap-binding protein that controls ribosome biogenesis. DOI: http://dx.doi.org/10.7554/eLife.24146.001 PMID:28379136

Non-synonymous single nucleotide polymorphisms in the watermelon eIF4E gene are closely associated with resistance to zucchini yellow mosaic virus.

PubMed

Ling, Kai-Shu; Harris, Karen R; Meyer, Jenelle D F; Levi, Amnon; Guner, Nihat; Wehner, Todd C; Bendahmane, Abdelhafid; Havey, Michael J

2009-12-01

Zucchini yellow mosaic virus (ZYMV) is one of the most economically important potyviruses infecting cucurbit crops worldwide. Using a candidate gene approach, we cloned and sequenced eIF4E and eIF(iso)4E gene segments in watermelon. Analysis of the nucleotide sequences between the ZYMV-resistant watermelon plant introduction PI 595203 (Citrullus lanatus var. lanatus) and the ZYMV-susceptible watermelon cultivar 'New Hampshire Midget' ('NHM') showed the presence of single nucleotide polymorphisms (SNPs). Initial analysis of the identified SNPs in association studies indicated that SNPs in the eIF4E, but not eIF(iso)4E, were closely associated to the phenotype of ZYMV-resistance in 70 F(2) and 114 BC(1R) progenies. Subsequently, we focused our efforts in obtaining the entire genomic sequence of watermelon eIF4E. Three SNPs were identified between PI 595203 and NHM. One of the SNPs (A241C) was in exon 1 and the other two SNPs (C309A and T554G) were in the first intron of the gene. SNP241 which resulted in an amino acid substitution (proline to threonine) was shown to be located in the critical cap recognition and binding area, similar to that of several plant species resistance to potyviruses. Analysis of a cleaved amplified polymorphism sequence (CAPS) marker derived from this SNP in F(2) and BC(1R) populations demonstrated a cosegregation between the CAPS-2 marker and their ZYMV resistance or susceptibility phenotype. When we investigated whether such SNP mutation in the eIF4E was also conserved in several other PIs of C. lanatus var. citroides, we identified a different SNP (A171G) resulting in another amino acid substitution (D71G) from four ZYMV-resistant C. lanatus var. citroides (PI 244018, PI 482261, PI 482299, and PI 482322). Additional CAPS markers were also identified. Availability of all these CAPS markers will enable marker-aided breeding of watermelon for ZYMV resistance.

The Eukaryotic Translation Initiation Factor 4E Controls Lettuce Susceptibility to the Potyvirus Lettuce mosaic virus1

PubMed Central

Nicaise, Valérie; German-Retana, Sylvie; Sanjuán, Raquel; Dubrana, Marie-Pierre; Mazier, Marianne; Maisonneuve, Brigitte; Candresse, Thierry; Caranta, Carole; LeGall, Olivier

2003-01-01

The eIF4E and eIF(iso)4E cDNAs from several genotypes of lettuce (Lactuca sativa) that are susceptible, tolerant, or resistant to infection by Lettuce mosaic virus (LMV; genus Potyvirus) were cloned and sequenced. Although Ls-eIF(iso)4E was monomorphic in sequence, three types of Ls-eIF4E differed by point sequence variations, and a short in-frame deletion in one of them. The amino acid variations specific to Ls-eIF4E1 and Ls-eIF4E2 were predicted to be located near the cap recognition pocket in a homology-based tridimensional protein model. In 19 lettuce genotypes, including two near-isogenic pairs, there was a strict correlation between these three allelic types and the presence or absence of the recessive LMV resistance genes mo11 and mo12. Ls-eIF4E1 and mo11 cosegregated in the progeny of two separate crosses between susceptible genotypes and an mo11 genotype. Finally, transient ectopic expression of Ls-eIF4E restored systemic accumulation of a green fluorescent protein-tagged LMV in LMV-resistant mo12 plants and a recombinant LMV expressing Ls-eIF4E° from its genome, but not Ls-eIF4E1 or Ls-eIF(iso)4E, accumulated and produced symptoms in mo11 or mo12 genotypes. Therefore, sequence correlation, tight genetic linkage, and functional complementation strongly suggest that eIF4E plays a role in the LMV cycle in lettuce and that mo11 and mo12 are alleles coding for forms of eIF4E unable or less effective to fulfill this role. More generally, the isoforms of eIF4E appear to be host factors involved in the cycle of potyviruses in plants, probably through a general mechanism yet to be clarified. PMID:12857809

Fission yeast translation initiation factor 3 subunit eIF3h is not essential for global translation initiation, but deletion of eif3h+ affects spore formation.

PubMed

Ray, Anirban; Bandyopadhyay, Amitabha; Matsumoto, Tomohiro; Deng, Haiteng; Maitra, Umadas

2008-11-01

The fission yeast Schizosaccharomyces pombe homologue of the p40/eIF3h subunit of mammalian translation initiation factor eIF3 has been characterized in this study. We show that this protein physically associates with the 40S ribosomal particles as a constituent of the multimeric eIF3 protein complex, which consists of all five known eIF3 core subunits (eIF3a, eIF3b, eIF3c, eIF3g and eIF3i) as well as the five non-core subunits (eIF3d, eIF3e, eIF3f, eIF3h and eIF3m) that constitute an eIF3 holocomplex in fission yeast. However, affinity purification of eIF3 from fission yeast cells expressing TAP-tagged eIF3h suggests the presence of distinct forms of eIF3 that differ in their composition of the non-core subunits. Further characterization of eIF3h shows that strains lacking eif3h(+) (eif3hDelta) are viable and show no gross defects, either in vegetative growth or in the rate of in vivo protein synthesis. Polysome profile analysis shows no apparent defects in translation initiation. Furthermore, deletion of eif3h(+) does not affect the ability of the other eIF3 subunits to remain associated with one another in a tight protein complex similar to the situation in wild-type cells. Additionally, we show that human eIF3h can functionally substitute fission yeast eIF3h in complementing in vivo a genetic deletion of eif3h(+). Interestingly, mutant eif3hDelta cells show several prominent phenotypic properties. They are hypersensitive to caffeine and highly defective in meiosis, producing either no spores or incomplete tetrads with a very high frequency. The implications of these results in relation to the functions of eIF3h in Sz. pombe are discussed. (c) 2008 John Wiley & Sons, Ltd.

Mitochondrial dysfunction enhances cisplatin resistance in human gastric cancer cells via the ROS-activated GCN2-eIF2α-ATF4-xCT pathway

PubMed Central

Wang, Sheng-Fan; Chen, Meng-Shian; Chou, Yueh-Ching; Ueng, Yune-Fang; Yin, Pen-Hui; Yeh, Tien-Shun; Lee, Hsin-Chen

2016-01-01

Mitochondrial DNA mutations and defects in mitochondrial enzymes have been identified in gastric cancers, and they might contribute to cancer progression. In previous studies, mitochondrial dysfunction was induced by oligomycin-enhanced chemoresistance to cisplatin. Herein, we dissected the regulatory mechanism for mitochondrial dysfunction-enhanced cisplatin resistance in human gastric cancer cells. Repeated cisplatin treatment-induced cisplatin-resistant cells exhibited high SLC7A11 (xCT) expression, and xCT inhibitors (sulfasalazine or erastin), xCT siRNA, or a GSH synthesis inhibitor (buthionine sulphoximine, BSO) could sensitize these cells to cisplatin. Clinically, the high expression of xCT was associated with a poorer prognosis for gastric cancer patients under adjuvant chemotherapy. Moreover, we found that mitochondrial dysfunction enhanced cisplatin resistance and up-regulated xCT expression, as well as intracellular glutathione (GSH). The xCT inhibitors, siRNA against xCT or BSO decreased mitochondrial dysfunction-enhanced cisplatin resistance. We further demonstrated that the upregulation of the eIF2α-ATF4 pathway contributed to mitochondrial dysfunction-induced xCT expression, and activated eIF2α kinase GCN2, but not PERK, stimulated the eIF2α-ATF4-xCT pathway in response to mitochondrial dysfunction-increased reactive oxygen species (ROS) levels. In conclusion, our results suggested that the ROS-activated GCN2-eIF2α-ATF4-xCT pathway might contribute to mitochondrial dysfunction-enhanced cisplatin resistance and could be a potential target for gastric cancer therapy. PMID:27708226

Mitochondrial dysfunction enhances cisplatin resistance in human gastric cancer cells via the ROS-activated GCN2-eIF2α-ATF4-xCT pathway.

PubMed

Wang, Sheng-Fan; Chen, Meng-Shian; Chou, Yueh-Ching; Ueng, Yune-Fang; Yin, Pen-Hui; Yeh, Tien-Shun; Lee, Hsin-Chen

2016-11-08

Mitochondrial DNA mutations and defects in mitochondrial enzymes have been identified in gastric cancers, and they might contribute to cancer progression. In previous studies, mitochondrial dysfunction was induced by oligomycin-enhanced chemoresistance to cisplatin. Herein, we dissected the regulatory mechanism for mitochondrial dysfunction-enhanced cisplatin resistance in human gastric cancer cells. Repeated cisplatin treatment-induced cisplatin-resistant cells exhibited high SLC7A11 (xCT) expression, and xCT inhibitors (sulfasalazine or erastin), xCT siRNA, or a GSH synthesis inhibitor (buthionine sulphoximine, BSO) could sensitize these cells to cisplatin. Clinically, the high expression of xCT was associated with a poorer prognosis for gastric cancer patients under adjuvant chemotherapy. Moreover, we found that mitochondrial dysfunction enhanced cisplatin resistance and up-regulated xCT expression, as well as intracellular glutathione (GSH). The xCT inhibitors, siRNA against xCT or BSO decreased mitochondrial dysfunction-enhanced cisplatin resistance. We further demonstrated that the upregulation of the eIF2α-ATF4 pathway contributed to mitochondrial dysfunction-induced xCT expression, and activated eIF2α kinase GCN2, but not PERK, stimulated the eIF2α-ATF4-xCT pathway in response to mitochondrial dysfunction-increased reactive oxygen species (ROS) levels. In conclusion, our results suggested that the ROS-activated GCN2-eIF2α-ATF4-xCT pathway might contribute to mitochondrial dysfunction-enhanced cisplatin resistance and could be a potential target for gastric cancer therapy.

Structure of the nucleotide exchange factor eIF2B reveals mechanism of memory-enhancing molecule.

PubMed

Tsai, Jordan C; Miller-Vedam, Lakshmi E; Anand, Aditya A; Jaishankar, Priyadarshini; Nguyen, Henry C; Renslo, Adam R; Frost, Adam; Walter, Peter

2018-03-30

Regulation by the integrated stress response (ISR) converges on the phosphorylation of translation initiation factor eIF2 in response to a variety of stresses. Phosphorylation converts eIF2 from a substrate to a competitive inhibitor of its dedicated guanine nucleotide exchange factor, eIF2B, thereby inhibiting translation. ISRIB, a drug-like eIF2B activator, reverses the effects of eIF2 phosphorylation, and in rodents it enhances cognition and corrects cognitive deficits after brain injury. To determine its mechanism of action, we solved an atomic-resolution structure of ISRIB bound in a deep cleft within decameric human eIF2B by cryo-electron microscopy. Formation of fully active, decameric eIF2B holoenzyme depended on the assembly of two identical tetrameric subcomplexes, and ISRIB promoted this step by cross-bridging a central symmetry interface. Thus, regulation of eIF2B assembly emerges as a rheostat for eIF2B activity that tunes translation during the ISR and that can be further modulated by ISRIB. Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.

Multiple Sclerosis and EIF2B5: A Paradox or a Missing Link.

PubMed

Zahoor, Insha; Haq, Ehtishamul; Asimi, Ravouf

2017-01-01

Multiple sclerosis (MS) is an encumbering inflammatory condition of the central nervous system (CNS) caused by axonal demyelination. There is sufficient evidence suggesting role of eukaryotic translation initiation factor 2B (EIF2B) gene family encoding the five subunits of eIF2B complex-α, β, γ, δ and ε respectively, in causing vanishing white matter (VWM) disease of the brain. Incidentally researchers have proposed overlapping between MS and VWM in terms of clinical, biochemical and genetic aspects, which incited us to write this chapter to explore the association between EIF2B5 and MS. eIF2B plays an essential role in translation initiation and its regulation in eukaryotes. Among EIF2B gene family, EIF2B5 gene encodes the catalytic and a crucial epsilon subunit of the eIF2B protein as most of the alterations have been found in this gene. The recent findings on the association between EIF2B5 and MS susceptibility point towards unfathomable and contentious role of EIF2B5 in MS development. This chapter briefly reviews the insights gleaned from recent studies conducted in understanding the association between EIF2B5 and MS risk. The need of hour is to conduct large scale conclusive studies aimed at expounding the mechanisms behind this relationship.

Functional equivalence of translation factor eIF5B from Candida albicans and Saccharomyces cerevisiae.

PubMed

Jun, Kyung Ok; Yang, Eun Ji; Lee, Byeong Jeong; Park, Jeong Ro; Lee, Joon H; Choi, Sang Ki

2008-04-30

Eukaryotic translation initiation factor 5B (eIF5B) plays a role in recognition of the AUG codon in conjunction with translation factor eIF2, and promotes joining of the 60S ribosomal subunit. To see whether the eIF5B proteins of other organisms function in Saccharomyces cerevisiae, we cloned the corresponding genes from Oryza sativa, Arabidopsis thaliana, Aspergillus nidulans and Candida albican and expressed them under the control of the galactose-inducible GAL promoter in the fun12Delta strain of Saccharomyces cerevisiae. Expression of Candida albicans eIF5B complemented the slow-growth phenotype of the fun12Delta strain, but that of Aspergillus nidulance did not, despite the fact that its protein was expressed better than that of Candida albicans. The Arabidopsis thaliana protein was also not functional in Saccharomyces. These results reveal that the eIF5B in Candida albicans has a close functional relationship with that of Sacharomyces cerevisiae, as also shown by a phylogenetic analysis based on the amino acid sequences of the eIF5Bs.

Disruption of Genes Encoding eIF4E Binding Proteins-1 And -2 Does Not Alter Basal or Sepsis-Induced Changes in Skeletal Muscle Protein Synthesis in Male or Female Mice

PubMed Central

Steiner, Jennifer L.; Pruznak, Anne M.; Deiter, Gina; Navaratnarajah, Maithili; Kutzler, Lydia; Kimball, Scot R.; Lang, Charles H.

2014-01-01

Sepsis decreases skeletal muscle protein synthesis in part by impairing mTOR activity and the subsequent phosphorylation of 4E-BP1 and S6K1 thereby controlling translation initiation; however, the relative importance of changes in these two downstream substrates is unknown. The role of 4E-BP1 (and -BP2) in regulating muscle protein synthesis was assessed in wild-type (WT) and 4E-BP1/BP2 double knockout (DKO) male mice under basal conditions and in response to sepsis. At 12 months of age, body weight, lean body mass and energy expenditure did not differ between WT and DKO mice. Moreover, in vivo rates of protein synthesis in gastrocnemius, heart and liver did not differ between DKO and WT mice. Sepsis decreased skeletal muscle protein synthesis and S6K1 phosphorylation in WT and DKO male mice to a similar extent. Sepsis only decreased 4E-BP1 phosphorylation in WT mice as no 4E-BP1/BP2 protein was detected in muscle from DKO mice. Sepsis decreased the binding of eIF4G to eIF4E in WT mice; however, eIF4E•eIF4G binding was not altered in DKO mice under either basal or septic conditions. A comparable sepsis-induced increase in eIF4B phosphorylation was seen in both WT and DKO mice. eEF2 phosphorylation was similarly increased in muscle from WT septic mice and both control and septic DKO mice, compared to WT control values. The sepsis-induced increase in muscle MuRF1 and atrogin-1 (markers of proteolysis) as well as TNFα and IL-6 (inflammatory cytokines) mRNA was greater in DKO than WT mice. The sepsis-induced decrease in myocardial and hepatic protein synthesis did not differ between WT and DKO mice. These data suggest overall basal protein balance and synthesis is maintained in muscle of mice lacking both 4E-BP1/BP2 and that sepsis-induced changes in mTOR signaling may be mediated by a down-stream mechanism independent of 4E-BP1 phosphorylation and eIF4E•eIF4G binding. PMID:24945486

Disruption of genes encoding eIF4E binding proteins-1 and -2 does not alter basal or sepsis-induced changes in skeletal muscle protein synthesis in male or female mice.

PubMed

Steiner, Jennifer L; Pruznak, Anne M; Deiter, Gina; Navaratnarajah, Maithili; Kutzler, Lydia; Kimball, Scot R; Lang, Charles H

2014-01-01

Sepsis decreases skeletal muscle protein synthesis in part by impairing mTOR activity and the subsequent phosphorylation of 4E-BP1 and S6K1 thereby controlling translation initiation; however, the relative importance of changes in these two downstream substrates is unknown. The role of 4E-BP1 (and -BP2) in regulating muscle protein synthesis was assessed in wild-type (WT) and 4E-BP1/BP2 double knockout (DKO) male mice under basal conditions and in response to sepsis. At 12 months of age, body weight, lean body mass and energy expenditure did not differ between WT and DKO mice. Moreover, in vivo rates of protein synthesis in gastrocnemius, heart and liver did not differ between DKO and WT mice. Sepsis decreased skeletal muscle protein synthesis and S6K1 phosphorylation in WT and DKO male mice to a similar extent. Sepsis only decreased 4E-BP1 phosphorylation in WT mice as no 4E-BP1/BP2 protein was detected in muscle from DKO mice. Sepsis decreased the binding of eIF4G to eIF4E in WT mice; however, eIF4E•eIF4G binding was not altered in DKO mice under either basal or septic conditions. A comparable sepsis-induced increase in eIF4B phosphorylation was seen in both WT and DKO mice. eEF2 phosphorylation was similarly increased in muscle from WT septic mice and both control and septic DKO mice, compared to WT control values. The sepsis-induced increase in muscle MuRF1 and atrogin-1 (markers of proteolysis) as well as TNFα and IL-6 (inflammatory cytokines) mRNA was greater in DKO than WT mice. The sepsis-induced decrease in myocardial and hepatic protein synthesis did not differ between WT and DKO mice. These data suggest overall basal protein balance and synthesis is maintained in muscle of mice lacking both 4E-BP1/BP2 and that sepsis-induced changes in mTOR signaling may be mediated by a down-stream mechanism independent of 4E-BP1 phosphorylation and eIF4E•eIF4G binding.

Regulation of eIF2α phosphorylation in hindlimb-unloaded and STS-135 space-flown mice

NASA Astrophysics Data System (ADS)

Zhao, Liming; Tanjung, Nancy; Swarnkar, Gaurav; Ledet, Eric; Yokota, Hiroki

2012-09-01

Various environmental stresses elevate the phosphorylation level of eukaryotic translation initiation factor 2 alpha (eIF2α) and induce transcriptional activation of a set of stress responsive genes such as activating transcription factors 3 and 6 (ATF3 and ATF6), CCAAT/enhancer-binding protein homologous protein (CHOP), and Xbp1 (X-box binding protein 1). These stress sources include radiation, oxidation, and stress to the endoplasmic reticulum, and it is recently reported that unloading by hindlimb unloading is such a stress source. No studies, however, have examined the phosphorylation level of eIF2α (eIF2α-p) using skeletal samples that have experienced microgravity in space. In this study we addressed a question: Does a mouse tibia flown in space show altered levels of eIF2α-p? To address this question, we obtained STS-135 flown samples that were harvested 4-7 h after landing. The tibia and femur isolated from hindlimb unloaded mice were employed as non-flight controls. The effects of loading were also investigated in non- flight controls. Results indicate that the level of eIF2α-p of the non-flight controls was elevated during hindlimb unloading and reduced after being released from unloading. Second, the eIF2α-p level of space-flown samples was decreased, and mechanical loading to the tibia caused the reduction of the eIF2α-p level. Third, the mRNA levels of ATF3, ATF6, and CHOP were lowered in space-flown samples as well as in the non-flight samples 4-7 h after being released from unloading. Collectively, the results herein indicated that a release from hindlimb unloading and a return to normal weight environment from space provided a suppressive effect to eIF2α-linked stress responses and that a period of 2-4 h is sufficient to induce this suppressive outcome.

Low-Intensity Extracorporeal Shock Wave Therapy Enhances Brain-Derived Neurotrophic Factor Expression through PERK/ATF4 Signaling Pathway.

PubMed

Wang, Bohan; Ning, Hongxiu; Reed-Maldonado, Amanda B; Zhou, Jun; Ruan, Yajun; Zhou, Tie; Wang, Hsun Shuan; Oh, Byung Seok; Banie, Lia; Lin, Guiting; Lue, Tom F

2017-02-16

Low-intensity extracorporeal shock wave therapy (Li-ESWT) is used in the treatment of erectile dysfunction, but its mechanisms are not well understood. Previously, we found that Li-ESWT increased the expression of brain-derived neurotrophic factor (BDNF). Here we assessed the underlying signaling pathways in Schwann cells in vitro and in penis tissue in vivo after nerve injury. The result indicated that BDNF were significantly increased by the Li-ESWT after nerve injury, as well as the expression of BDNF in Schwann cells (SCs, RT4-D6P2T) in vitro. Li-ESWT activated the protein kinase RNA-like endoplasmic reticulum (ER) kinase (PERK) pathway by increasing the phosphorylation levels of PERK and eukaryotic initiation factor 2a (eIF2α), and enhanced activating transcription factor 4 (ATF4) in an energy-dependent manner. In addition, GSK2656157-an inhibitor of PERK-effectively inhibited the effect of Li-ESWT on the phosphorylation of PERK, eIF2α, and the expression of ATF4. Furthermore, silencing ATF4 dramatically attenuated the effect of Li-ESWT on the expression of BDNF, but had no effect on hypoxia-inducible factor (HIF)1α or glial cell-derived neurotrophic factor (GDNF) in Schwann cells. In conclusion, our findings shed new light on the underlying mechanisms by which Li-ESWT may stimulate the expression of BDNF through activation of PERK/ATF4 signaling pathway. This information may help to refine the use of Li-ESWT to further improve its clinical efficacy.

Low-Intensity Extracorporeal Shock Wave Therapy Enhances Brain-Derived Neurotrophic Factor Expression through PERK/ATF4 Signaling Pathway

PubMed Central

Wang, Bohan; Ning, Hongxiu; Reed-Maldonado, Amanda B.; Zhou, Jun; Ruan, Yajun; Zhou, Tie; Wang, Hsun Shuan; Oh, Byung Seok; Banie, Lia; Lin, Guiting; Lue, Tom F.

2017-01-01

Low-intensity extracorporeal shock wave therapy (Li-ESWT) is used in the treatment of erectile dysfunction, but its mechanisms are not well understood. Previously, we found that Li-ESWT increased the expression of brain-derived neurotrophic factor (BDNF). Here we assessed the underlying signaling pathways in Schwann cells in vitro and in penis tissue in vivo after nerve injury. The result indicated that BDNF were significantly increased by the Li-ESWT after nerve injury, as well as the expression of BDNF in Schwann cells (SCs, RT4-D6P2T) in vitro. Li-ESWT activated the protein kinase RNA-like endoplasmic reticulum (ER) kinase (PERK) pathway by increasing the phosphorylation levels of PERK and eukaryotic initiation factor 2a (eIF2α), and enhanced activating transcription factor 4 (ATF4) in an energy-dependent manner. In addition, GSK2656157—an inhibitor of PERK—effectively inhibited the effect of Li-ESWT on the phosphorylation of PERK, eIF2α, and the expression of ATF4. Furthermore, silencing ATF4 dramatically attenuated the effect of Li-ESWT on the expression of BDNF, but had no effect on hypoxia-inducible factor (HIF)1α or glial cell-derived neurotrophic factor (GDNF) in Schwann cells. In conclusion, our findings shed new light on the underlying mechanisms by which Li-ESWT may stimulate the expression of BDNF through activation of PERK/ATF4 signaling pathway. This information may help to refine the use of Li-ESWT to further improve its clinical efficacy. PMID:28212323

The MNK–eIF4E Signaling Axis Contributes to Injury-Induced Nociceptive Plasticity and the Development of Chronic Pain

PubMed Central

Asiedu, Marina N.; Megat, Salim; Burton, Michael D.; Burgos-Vega, Carolina C.; Melemedjian, Ohannes K.; Boitano, Scott; Vagner, Josef; Pancrazio, Joseph J.; Mogil, Jeffrey S.; Dussor, Gregory

2017-01-01

Injury-induced sensitization of nociceptors contributes to pain states and the development of chronic pain. Inhibiting activity-dependent mRNA translation through mechanistic target of rapamycin and mitogen-activated protein kinase (MAPK) pathways blocks the development of nociceptor sensitization. These pathways convergently signal to the eukaryotic translation initiation factor (eIF) 4F complex to regulate the sensitization of nociceptors, but the details of this process are ill defined. Here we investigated the hypothesis that phosphorylation of the 5′ cap-binding protein eIF4E by its specific kinase MAPK interacting kinases (MNKs) 1/2 is a key factor in nociceptor sensitization and the development of chronic pain. Phosphorylation of ser209 on eIF4E regulates the translation of a subset of mRNAs. We show that pronociceptive and inflammatory factors, such as nerve growth factor (NGF), interleukin-6 (IL-6), and carrageenan, produce decreased mechanical and thermal hypersensitivity, decreased affective pain behaviors, and strongly reduced hyperalgesic priming in mice lacking eIF4E phosphorylation (eIF4ES209A). Tests were done in both sexes, and no sex differences were found. Moreover, in patch-clamp electrophysiology and Ca2+ imaging experiments on dorsal root ganglion neurons, NGF- and IL-6-induced increases in excitability were attenuated in neurons from eIF4ES209A mice. These effects were recapitulated in Mnk1/2−/− mice and with the MNK1/2 inhibitor cercosporamide. We also find that cold hypersensitivity induced by peripheral nerve injury is reduced in eIF4ES209A and Mnk1/2−/− mice and following cercosporamide treatment. Our findings demonstrate that the MNK1/2–eIF4E signaling axis is an important contributing factor to mechanisms of nociceptor plasticity and the development of chronic pain. SIGNIFICANCE STATEMENT Chronic pain is a debilitating disease affecting approximately one in three Americans. Chronic pain is thought to be driven by changes in

The translation initiation factor 3 subunit eIF3K interacts with PML and associates with PML nuclear bodies

DOE Office of Scientific and Technical Information (OSTI.GOV)

Salsman, Jayme; Pinder, Jordan; Tse, Brenda

2013-10-15

The promyelocytic leukemia protein (PML) is a tumor suppressor protein that regulates a variety of important cellular processes, including gene expression, DNA repair and cell fate decisions. Integral to its function is the ability of PML to form nuclear bodies (NBs) that serve as hubs for the interaction and modification of over 90 cellular proteins. There are seven canonical isoforms of PML, which encode diverse C-termini generated by alternative pre-mRNA splicing. Recruitment of specific cellular proteins to PML NBs is mediated by protein–protein interactions with individual PML isoforms. Using a yeast two-hybrid screen employing peptide sequences unique to PML isoformmore » I (PML-I), we identified an interaction with the eukaryotic initiation factor 3 subunit K (eIF3K), and in the process identified a novel eIF3K isoform, which we term eIF3K-2. We further demonstrate that eIF3K and PML interact both in vitro via pull-down assays, as well as in vivo within human cells by co-immunoprecipitation and co-immunofluorescence. In addition, eIF3K isoform 2 (eIF3K-2) colocalizes to PML bodies, particularly those enriched in PML-I, while eIF3K isoform 1 associates poorly with PML NBs. Thus, we report eIF3K as the first known subunit of the eIF3 translation pre-initiation complex to interact directly with the PML protein, and provide data implicating alternative splicing of both PML and eIF3K as a possible regulatory mechanism for eIF3K localization at PML NBs. - Highlights: • The PML-I C-terminus, encoded by exon 9, interacts with translation factor eIF3K. • We identify a novel eIF3K isoform that excludes exon 2 (eIF3K-2). • eIF3K-2 preferentially associates with PML bodies enriched in PML-I vs. PML-IV. • Alternative splicing of eIF3K regulates association with PML bodies.« less

Transcriptional Repression of ATF4 Gene by CCAAT/Enhancer-binding Protein β (C/EBPβ) Differentially Regulates Integrated Stress Response*

PubMed Central

Dey, Souvik; Savant, Sudha; Teske, Brian F.; Hatzoglou, Maria; Calkhoven, Cornelis F.; Wek, Ronald C.

2012-01-01

Different environmental stresses induce the phosphorylation of eIF2 (eIF2∼P), repressing global protein synthesis coincident with preferential translation of ATF4. ATF4 is a transcriptional activator of genes involved in metabolism and nutrient uptake, antioxidation, and regulation of apoptosis. Because ATF4 is a common downstream target that integrates signaling from different eIF2 kinases and their respective stress signals, the eIF2∼P/ATF4 pathway is collectively referred to as the integrated stress response. Although eIF2∼P elicits translational control in response to many different stresses, there are selected stresses, such as exposure to UV irradiation, that do not increase ATF4 expression despite robust eIF2∼P. The rationale for this discordant induction of ATF4 expression and eIF2∼P in response to UV irradiation is that transcription of ATF4 is repressed, and therefore ATF4 mRNA is not available for preferential translation. In this study, we show that C/EBPβ is a transcriptional repressor of ATF4 during UV stress. C/EBPβ binds to critical elements in the ATF4 promoter, resulting in its transcriptional repression. Expression of C/EBPβ increases in response to UV stress, and the liver-enriched inhibitory protein (LIP) isoform of C/EBPβ, but not the liver-enriched activating protein (LAP) version, represses ATF4 transcription. Loss of the liver-enriched inhibitory protein isoform results in increased ATF4 mRNA levels in response to UV irradiation and subsequent recovery of ATF4 translation, leading to enhanced expression of its target genes. Together these results illustrate how eIF2∼P and translational control combined with transcription factors regulated by alternative signaling pathways can direct programs of gene expression that are specifically tailored to each environmental stress. PMID:22556424

A MAP4 kinase related to Ste20 is a nutrient-sensitive regulator of mTOR signalling

PubMed Central

Findlay, Greg M.; Yan, Lijun; Procter, Julia; Mieulet, Virginie; Lamb, Richard F.

2007-01-01

The mTOR (mammalian target of rapamycin) signalling pathway is a key regulator of cell growth and is controlled by growth factors and nutrients such as amino acids. Although signalling pathways from growth factor receptors to mTOR have been elucidated, the pathways mediating signalling by nutrients are poorly characterized. Through a screen for protein kinases active in the mTOR signalling pathway in Drosophila we have identified a Ste20 family member (MAP4K3) that is required for maximal S6K (S6 kinase)/4E-BP1 [eIF4E (eukaryotic initiation factor 4E)-binding protein 1] phosphorylation and regulates cell growth. Importantly, MAP4K3 activity is regulated by amino acids, but not the growth factor insulin and is not regulated by the mTORC1 inhibitor rapamycin. Our results therefore suggest a model whereby nutrients signal to mTORC1 via activation of MAP4K3. PMID:17253963

NOX4 regulates autophagy during energy deprivation.

PubMed

Sciarretta, Sebastiano; Volpe, Massimo; Sadoshima, Junichi

2014-04-01

NADPH oxidase is a cellular enzyme devoted to the production of reactive oxygen species (ROS). NOX4 and NOX2 are the main isoforms of NADPH oxidase in the cardiovascular system. In our recent study, we demonstrated that NOX4, but not NOX2, is a critical mediator of the cardiomyocyte adaptive response to energy stress. NOX4 activity and protein levels are increased in the endoplasmic reticulum (ER) but not in mitochondria of cardiomyocytes during the early phase of energy deprivation. NOX4-derived production of ROS in the ER is a critical event that activates autophagy through stimulation of the EIF2AK3/PERK-EIF2S1/eIF-2α-ATF4 pathway. NOX4-dependent autophagy is an important mechanism to preserve cellular energy and limit cell death in energy-deprived cardiomyocytes. Aside from elucidating a crucial physiological function of NOX4 during cellular energy stress, our study dissects a novel signaling mechanism that regulates autophagy under this condition.

NOX4 regulates autophagy during energy deprivation

PubMed Central

Sciarretta, Sebastiano; Volpe, Massimo; Sadoshima, Junichi

2014-01-01

NADPH oxidase is a cellular enzyme devoted to the production of reactive oxygen species (ROS). NOX4 and NOX2 are the main isoforms of NADPH oxidase in the cardiovascular system. In our recent study, we demonstrated that NOX4, but not NOX2, is a critical mediator of the cardiomyocyte adaptive response to energy stress. NOX4 activity and protein levels are increased in the endoplasmic reticulum (ER) but not in mitochondria of cardiomyocytes during the early phase of energy deprivation. NOX4-derived production of ROS in the ER is a critical event that activates autophagy through stimulation of the EIF2AK3/PERK-EIF2S1/eIF-2α-ATF4 pathway. NOX4-dependent autophagy is an important mechanism to preserve cellular energy and limit cell death in energy-deprived cardiomyocytes. Aside from elucidating a crucial physiological function of NOX4 during cellular energy stress, our study dissects a novel signaling mechanism that regulates autophagy under this condition. PMID:24492492

Eukaryotic initiation factor 5A-1 (eIF5A-1) as a diagnostic marker for aberrant proliferation in intraepithelial neoplasia of the vulva.

PubMed

Cracchiolo, Bernadette M; Heller, Debra S; Clement, Paul M J; Wolff, Edith C; Park, Myung-Hee; Hanauske-Abel, Hartmut M

2004-07-01

The mature eukaryotic translation initiation factor 5A contains the unusual amino acid hypusine, formed post-translationally from a specific lysine residue and essential for proliferation of eukaryotic cells. We hypothesized that the major eIF5A isoform, eIF5A-1, is an in situ biomarker for proliferation. NIH-353, a polyclonal immunoreagent specific for hypusine-containing eIF5A-1, was used to test this proposal in biopsies of vulvar high-grade intraepithelial neoplasia (VIN), characterized by the presence of proliferating cells throughout the thickness of the epithelium. Methods. Formalin-fixed and paraffin-embedded archival samples with an independently established diagnosis of VIN 3 were stained immunohistochemically after antigen retrieval, employing NIH-353 and, for comparison, the standard Ki-67 antibody. NIH-353 labeled neoplastic keratinocytes throughout the thickness of the epithelium in all VIN 3 samples. Malignant cells in a case of focally invasive squamous cell carcinoma also stained strongly for mature, hypusine-containing eIF5A-1. Epithelium adjacent to these lesions, though still of apparently normal morphology, was immunoreactive throughout its full thickness. At inflammatory foci of lesional sites, solitary reactive lymphocytes were positive, as were individual proliferating cells within dermal appendages. The submucosal stroma lacked reactive cells. NIH-353 identifies mature eIF5A-1 as an in situ biomarker for proliferation. Like Ki-67, this immunoreagent promises broad applicability in histopathological diagnosis and may be helpful in outcome prediction. In contrast to Ki-67, NIH-353 visualizes a molecular target for antineoplastic therapy, and thus may guide the development and clinical testing of drugs that, like the fungicide ciclopirox, inhibit hypusine formation and cell proliferation.

Independent roles of eIF5A and polyamines in cell proliferation

PubMed Central

2004-01-01

To examine the roles of active hypusinated eIF5A (eukaryotic translation initiation factor 5A) and polyamines in cell proliferation, mouse mammary carcinoma FM3A cells were treated with an inhibitor of deoxyhypusine synthase, GC7 (N1-guanyl-1, 7-diaminoheptane), or with an inhibitor of ornithine decarboxylase, DFMO (α-difluoromethylornithine), or with DFMO plus an inhibitor of spermine synthase, APCHA [N1-(3-aminopropyl)-cyclohexylamine]. Treatment with GC7 decreased the level of active eIF5A on day 1 without affecting cellular polyamine content, and inhibition of cell growth occurred from day 2. This delay reflects the fact that eIF5A was present in excess and was very stable in these cells. Treatment with DFMO or with DFMO plus APCHA inhibited cell growth on day 1. DFMO considerably decreased the levels of putrescine and spermidine, and the formation of active eIF5A began to decrease when the level of spermidine fell below 8 nmol/mg of protein after 12 h of incubation with DFMO. The combination of DFMO and APCHA markedly decreased the levels of putrescine and spermine and significantly decreased the level of spermidine, but did not affect the level of active eIF5A until day 3 when spermidine level decreased to 7 nmol/mg of protein. The results show that a decrease in either active eIF5A or polyamines inhibits cell growth, indicating that eIF5A and polyamines are independently involved in cell growth. PMID:15377278

Cannabinoid Modulation of Eukaryotic Initiation Factors (eIF2α and eIF2B1) and Behavioral Cross-Sensitization to Cocaine in Adolescent Rats.

PubMed

Melas, Philippe A; Qvist, Johanna S; Deidda, Matteo; Upreti, Chirag; Wei, Ya Bin; Sanna, Fabrizio; Fratta, Walter; Scherma, Maria; Fadda, Paola; Kandel, Denise B; Kandel, Eric R

2018-03-13

Reduced eukaryotic Initiation Factor 2 (eIF2)α phosphorylation (p-eIF2α) enhances protein synthesis, memory formation, and addiction-like behaviors. However, p-eIF2α has not been examined with regard to psychoactive cannabinoids and cross-sensitization. Here, we find that a cannabinoid receptor agonist (WIN 55,212-2 mesylate [WIN]) reduced p-eIF2α in vitro by upregulating GADD34 (PPP1R15A), the recruiter of protein phosphatase 1 (PP1). The induction of GADD34 was linked to ERK/CREB signaling and to CREB-binding protein (CBP)-mediated histone hyperacetylation at the Gadd34 locus. In vitro, WIN also upregulated eIF2B1, an eIF2 activator subunit. We next found that WIN administration in vivo reduced p-eIF2α in the nucleus accumbens of adolescent, but not adult, rats. By contrast, WIN increased dorsal striatal levels of eIF2B1 and ΔFosB among both adolescents and adults. In addition, we found cross-sensitization between WIN and cocaine only among adolescents. These findings show that cannabinoids can modulate eukaryotic initiation factors, and they suggest a possible link between p-eIF2α and the gateway drug properties of psychoactive cannabinoids. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

A Transcript-Specific eIF3 Complex Mediates Global Translational Control of Energy Metabolism.

PubMed

Shah, Meera; Su, Dan; Scheliga, Judith S; Pluskal, Tomáš; Boronat, Susanna; Motamedchaboki, Khatereh; Campos, Alexandre Rosa; Qi, Feng; Hidalgo, Elena; Yanagida, Mitsuhiro; Wolf, Dieter A

2016-08-16

The multi-subunit eukaryotic translation initiation factor eIF3 is thought to assist in the recruitment of ribosomes to mRNA. The expression of eIF3 subunits is frequently disrupted in human cancers, but the specific roles of individual subunits in mRNA translation and cancer remain elusive. Using global transcriptomic, proteomic, and metabolomic profiling, we found a striking failure of Schizosaccharomyces pombe cells lacking eIF3e and eIF3d to synthesize components of the mitochondrial electron transport chain, leading to a defect in respiration, endogenous oxidative stress, and premature aging. Energy balance was maintained, however, by a switch to glycolysis with increased glucose uptake, upregulation of glycolytic enzymes, and strict dependence on a fermentable carbon source. This metabolic regulatory function appears to be conserved in human cells where eIF3e binds metabolic mRNAs and promotes their translation. Thus, via its eIF3d-eIF3e module, eIF3 orchestrates an mRNA-specific translational mechanism controlling energy metabolism that may be disrupted in cancer. Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.

Amino acid substrates impose polyamine, eIF5A, or hypusine requirement for peptide synthesis

PubMed Central

Shin, Byung-Sik; Katoh, Takayuki; Gutierrez, Erik; Kim, Joo-Ran; Suga, Hiroaki

2017-01-01

Abstract Whereas ribosomes efficiently catalyze peptide bond synthesis by most amino acids, the imino acid proline is a poor substrate for protein synthesis. Previous studies have shown that the translation factor eIF5A and its bacterial ortholog EF-P bind in the E site of the ribosome where they contact the peptidyl-tRNA in the P site and play a critical role in promoting the synthesis of polyproline peptides. Using misacylated Pro-tRNAPhe and Phe-tRNAPro, we show that the imino acid proline and not tRNAPro imposes the primary eIF5A requirement for polyproline synthesis. Though most proline analogs require eIF5A for efficient peptide synthesis, azetidine-2-caboxylic acid, a more flexible four-membered ring derivative of proline, shows relaxed eIF5A dependency, indicating that the structural rigidity of proline might contribute to the requirement for eIF5A. Finally, we examine the interplay between eIF5A and polyamines in promoting translation elongation. We show that eIF5A can obviate the polyamine requirement for general translation elongation, and that this activity is independent of the conserved hypusine modification on eIF5A. Thus, we propose that the body of eIF5A functionally substitutes for polyamines to promote general protein synthesis and that the hypusine modification on eIF5A is critically important for poor substrates like proline. PMID:28637321

Norovirus translation requires an interaction between the C Terminus of the genome-linked viral protein VPg and eukaryotic translation initiation factor 4G.

PubMed

Chung, Liliane; Bailey, Dalan; Leen, Eoin N; Emmott, Edward P; Chaudhry, Yasmin; Roberts, Lisa O; Curry, Stephen; Locker, Nicolas; Goodfellow, Ian G

2014-08-01

Viruses have evolved a variety of mechanisms to usurp the host cell translation machinery to enable translation of the viral genome in the presence of high levels of cellular mRNAs. Noroviruses, a major cause of gastroenteritis in man, have evolved a mechanism that relies on the interaction of translation initiation factors with the virus-encoded VPg protein covalently linked to the 5' end of the viral RNA. To further characterize this novel mechanism of translation initiation, we have used proteomics to identify the components of the norovirus translation initiation factor complex. This approach revealed that VPg binds directly to the eIF4F complex, with a high affinity interaction occurring between VPg and eIF4G. Mutational analyses indicated that the C-terminal region of VPg is important for the VPg-eIF4G interaction; viruses with mutations that alter or disrupt this interaction are debilitated or non-viable. Our results shed new light on the unusual mechanisms of protein-directed translation initiation. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Amino acid substrates impose polyamine, eIF5A, or hypusine requirement for peptide synthesis.

PubMed

Shin, Byung-Sik; Katoh, Takayuki; Gutierrez, Erik; Kim, Joo-Ran; Suga, Hiroaki; Dever, Thomas E

2017-08-21

Whereas ribosomes efficiently catalyze peptide bond synthesis by most amino acids, the imino acid proline is a poor substrate for protein synthesis. Previous studies have shown that the translation factor eIF5A and its bacterial ortholog EF-P bind in the E site of the ribosome where they contact the peptidyl-tRNA in the P site and play a critical role in promoting the synthesis of polyproline peptides. Using misacylated Pro-tRNAPhe and Phe-tRNAPro, we show that the imino acid proline and not tRNAPro imposes the primary eIF5A requirement for polyproline synthesis. Though most proline analogs require eIF5A for efficient peptide synthesis, azetidine-2-caboxylic acid, a more flexible four-membered ring derivative of proline, shows relaxed eIF5A dependency, indicating that the structural rigidity of proline might contribute to the requirement for eIF5A. Finally, we examine the interplay between eIF5A and polyamines in promoting translation elongation. We show that eIF5A can obviate the polyamine requirement for general translation elongation, and that this activity is independent of the conserved hypusine modification on eIF5A. Thus, we propose that the body of eIF5A functionally substitutes for polyamines to promote general protein synthesis and that the hypusine modification on eIF5A is critically important for poor substrates like proline. Published by Oxford University Press on behalf of Nucleic Acids Research 2017.

Mitotic MELK-eIF4B signaling controls protein synthesis and tumor cell survival

PubMed Central

Wang, Yubao; Begley, Michael; Li, Qing; Huang, Hai-Tsang; Lako, Ana; Eck, Michael J.; Gray, Nathanael S.; Mitchison, Timothy J.; Cantley, Lewis C.; Zhao, Jean J.

2016-01-01

The protein kinase maternal and embryonic leucine zipper kinase (MELK) is critical for mitotic progression of cancer cells; however, its mechanisms of action remain largely unknown. By combined approaches of immunoprecipitation/mass spectrometry and peptide library profiling, we identified the eukaryotic translation initiation factor 4B (eIF4B) as a MELK-interacting protein during mitosis and a bona fide substrate of MELK. MELK phosphorylates eIF4B at Ser406, a modification found to be most robust in the mitotic phase of the cell cycle. We further show that the MELK–eIF4B signaling axis regulates protein synthesis during mitosis. Specifically, synthesis of myeloid cell leukemia 1 (MCL1), an antiapoptotic protein known to play a role in cancer cell survival during cell division, depends on the function of MELK-elF4B. Inactivation of MELK or eIF4B results in reduced protein synthesis of MCL1, which, in turn, induces apoptotic cell death of cancer cells. Our study thus defines a MELK–eIF4B signaling axis that regulates protein synthesis during mitosis, and consequently influences cancer cell survival. PMID:27528663

Sevoflurane-Induced Endoplasmic Reticulum Stress Contributes to Neuroapoptosis and BACE-1 Expression in the Developing Brain: The Role of eIF2α.

PubMed

Liu, Bin; Xia, Junming; Chen, Yali; Zhang, Jun

2017-02-01

Neonatal exposure to volatile anesthetics causes apoptotic neurodegeneration in the developing brain, possibly leading to neurocognitive deficits in adulthood. Endoplasmic reticulum (ER) stress might be associated with sevoflurane (sevo)-induced neuroapoptosis. However, the signaling pathway regulating sevo-induced neuroapoptosis is not understood. We investigated the effects of neonatal sevo exposure on ER signaling pathway activation. Seven-day-old mouse pups were divided into control (C) and sevo (S; 3 % sevo exposure, 6 h) groups. ER stress marker [protein kinase RNA-like ER kinase (PERK), eukaryotic translation initiation factor 2α (eIF2α), activating transcription factor 4 (ATF4), CHOP, and caspase-12] levels were determined by western blotting. To understand the role of eIF2α in sevo-induced ER stress and caspase-3 activation, pups were pretreated with an eIF2α dephosphorylation inhibitor, salubrinal, and a potent and selective inhibitor of PERK, GSK2656157, before sevo exposure, and the effects on ER stress signaling and neuroapoptosis were examined. We investigated whether neonatal exposure to sevo increased β-site APP-cleaving enzyme 1 (BACE-1) expression. Neonatal sevo exposure elevated caspase-3 activation. ER stress signaling was activated, along with increased PERK and eIF2α phosphorylation, and upregulation of proapoptotic proteins (ATF4 and CHOP) in the cerebral cortex of the developing brain. Pretreatment with salubrinal augmented sevo-induced eIF2α phosphorylation, which inhibited ER stress-mediated ATF4 and caspase-3 activation. Inhibition of PERK phosphorylation due to GSK2656157 pretreatment reduced the sevo-induced increase in eIF2α phosphorylation. Sevo increased BACE-1 expression, which was attenuated by GSK2656157 and salubrinal pretreatment. Our data suggested that neonatal sevo exposure-induced neuroapoptosis is mediated via the PERK-eIF2α-ATF4-CHOP axis of the ER stress signaling pathway. Modulation of eIF2α phosphorylation

Novel characteristics of the biological properties of the yeast Saccharomyces cerevisiae eukaryotic initiation factor 2A.

PubMed

Komar, Anton A; Gross, Stephane R; Barth-Baus, Diane; Strachan, Ryan; Hensold, Jack O; Goss Kinzy, Terri; Merrick, William C

2005-04-22

Eukaryotic initiation factor 2A (eIF2A) has been shown to direct binding of the initiator methionyl-tRNA (Met-tRNA(i)) to 40 S ribosomal subunits in a codon-dependent manner, in contrast to eIF2, which requires GTP but not the AUG codon to bind initiator tRNA to 40 S subunits. We show here that yeast eIF2A genetically interacts with initiation factor eIF4E, suggesting that both proteins function in the same pathway. The double eIF2A/eIF4E-ts mutant strain displays a severe slow growth phenotype, which correlated with the accumulation of 85% of the double mutant cells arrested at the G(2)/M border. These cells also exhibited a disorganized actin cytoskeleton and elevated actin levels, suggesting that eIF2A might be involved in controlling the expression of genes involved in morphogenic processes. Further insights into eIF2A function were gained from the studies of eIF2A distribution in ribosomal fractions obtained from either an eIF5BDelta (fun12Delta) strain or a eIF3b-ts (prt1-1) strain. It was found that the binding of eIF2A to 40 and 80 S ribosomes was not impaired in either strain. We also found that eIF2A functions as a suppressor of Ure2p internal ribosome entry site-mediated translation in yeast cells. The regulation of expression from the URE2 internal ribosome entry site appears to be through the levels of eIF2A protein, which has been found to be inherently unstable with a half-life of approximately 17 min. It was hypothesized that this instability allows for translational control through the level of eIF2A protein in yeast cells.

Activation of the EIF2α/ATF4 and ATF6 Pathways in DU-145 Cells by Boric Acid at the Concentration Reported in Men at the US Mean Boron Intake.

PubMed

Kobylewski, Sarah E; Henderson, Kimberly A; Yamada, Kristin E; Eckhert, Curtis D

2017-04-01

Fruits, nuts, legumes, and vegetables are rich sources of boron (B), an essential plant nutrient with chemopreventive properties. Blood boric acid (BA) levels reflect recent B intake, and men at the US mean intake have a reported non-fasting level of 10 μM. Treatment of DU-145 prostate cancer cells with physiological concentrations of BA inhibits cell proliferation without causing apoptosis and activates eukaryotic initiation factor 2 (eIF2α). EIF2α induces cell differentiation and protects cells by redirecting gene expression to manage endoplasmic reticulum stress. Our objective was to determine the temporal expression of endoplasmic reticulum (ER) stress-activated genes in DU-145 prostate cells treated with 10 μM BA. Immunoblots showed post-treatment increases in eIF2α protein at 30 min and ATF4 and ATF6 proteins at 1 h and 30 min, respectively. The increase in ATF4 was accompanied by an increase in the expression of its downstream genes growth arrest and DNA damage-induced protein 34 (GADD34) and homocysteine-induced ER protein (Herp), but a decrease in GADD153/CCAAT/enhancer-binding protein homologous protein (CHOP), a pro-apoptotic gene. The increase in ATF6 was accompanied by an increase in expression of its downstream genes GRP78/BiP, calreticulin, Grp94, and EDEM. BA did not activate IRE1 or induce cleavage of XBP1 mRNA, a target of IRE1. Low boron status has been associated with increased cancer risk, low bone mineralization, and retinal degeneration. ATF4 and BiP/GRP78 function in osteogenesis and bone remodeling, calreticulin is required for tumor suppressor p53 function and mineralization of teeth, and BiP/GRP78 and EDEM prevent the aggregation of misfolded opsins which leads to retinal degeneration. The identification of BA-activated genes that regulate its phenotypic effects provides a molecular underpinning for boron nutrition and biology.

Infantile onset Vanishing White Matter disease associated with a novel EIF2B5 variant, remarkably long life span, severe epilepsy, and hypopituitarism.

PubMed

Woody, April L; Hsieh, David T; McIver, Harkirtin K; Thomas, Linda P; Rohena, Luis

2015-04-01

Vanishing White Matter disease (VWM) is an inherited progressive leukoencephalopathy caused by mutations in the genes EIF2B1-5, which encode for the 5 subunits of the eukaryotic initiation factor 2B (eIF2B), a regulator of protein synthesis. VWM typically presents with acute neurological decline following febrile infections or minor head trauma, and subsequent progressive neurological and cognitive regression. There is a varied clinical spectrum of VWM, with earlier onset associated with more severe phenotypes. Brain magnetic resonance imaging is usually diagnostic with diffusely abnormal white matter, progressing over time to cystic degeneration. We are reporting on a patient with infantile onset VWM associated with three heterozygous missense variants in EIF2B5, including a novel missense variant on exon 6 of EIF2B5 (D262N), as well as an interstitial duplication at 7q21.12. In addition, our case is unusual because of a severe epilepsy course, a novel clinical finding of hypopituitarism manifested by hypothyroidism and adrenal insufficiency, and a prolonged life span with current age of survival of 4 years and 11 months. © 2015 Wiley Periodicals, Inc.

Eukaryotic Translation Initiation Factor 5A (EIF5A) Regulates Pancreatic Cancer Metastasis by Modulating RhoA and Rho-associated Kinase (ROCK) Protein Expression Levels.

PubMed

Fujimura, Ken; Choi, Sunkyu; Wyse, Meghan; Strnadel, Jan; Wright, Tracy; Klemke, Richard

2015-12-11

Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest cancers with an overall survival rate of less than 5%. The poor patient outcome in PDAC is largely due to the high prevalence of systemic metastasis at the time of diagnosis and lack of effective therapeutics that target disseminated cells. The fact that the underlying mechanisms driving PDAC cell migration and dissemination are poorly understood have hindered drug development and compounded the lack of clinical success in this disease. Recent evidence indicates that mutational activation of K-Ras up-regulates eIF5A, a component of the cellular translational machinery that is critical for PDAC progression. However, the role of eIF5A in PDAC cell migration and metastasis has not been investigated. We report here that pharmacological inhibition or genetic knockdown of eIF5A reduces PDAC cell migration, invasion, and metastasis in vitro and in vivo. Proteomic profiling and bioinformatic analyses revealed that eIF5A controls an integrated network of cytoskeleton-regulatory proteins involved in cell migration. Functional interrogation of this network uncovered a critical RhoA/ROCK signaling node that operates downstream of eIF5A in invasive PDAC cells. Importantly, eIF5A mediates PDAC cell migration and invasion by modulating RhoA/ROCK protein expression levels. Together our findings implicate eIF5A as a cytoskeletal rheostat controlling RhoA/ROCK protein expression during PDAC cell migration and metastasis. Our findings also implicate the eIF5A/RhoA/ROCK module as a potential new therapeutic target to treat metastatic PDAC cells. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Separation of foot-and-mouth disease virus leader protein activities; identification of mutants that retain efficient self-processing activity but poorly induce eIF4G cleavage.

PubMed

Guan, Su Hua; Belsham, Graham J

2017-04-01

Foot-and-mouth disease virus is a picornavirus and its RNA genome encodes a large polyprotein. The N-terminal part of this polyprotein is the leader protein, a cysteine protease, termed Lpro. The virus causes the rapid inhibition of host cell cap-dependent protein synthesis within infected cells. This results from the Lpro-dependent cleavage of the cellular translation initiation factor eIF4G. Lpro also releases itself from the virus capsid precursor by cleaving the L/P1 junction. Using site-directed mutagenesis of the Lpro coding sequence, we have investigated the role of 51 separate amino acid residues in the functions of this protein. These selected residues either are highly conserved or are charged and exposed on the protein surface. Using transient expression assays, within BHK-21 cells, it was found that residues around the active site (W52, L53 and A149) of Lpro and others located elsewhere (K38, K39, R44, H138 and W159) are involved in the induction of eIF4G cleavage but not in the processing of the L/P1 junction. Modified viruses, encoding such amino acid substitutions within Lpro, can replicate in BHK-21 cells but did not grow well in primary bovine thyroid cells. This study characterizes mutant viruses that are deficient in blocking host cell responses to infection (e.g. interferon induction) and can assist in the rational design of antiviral agents targeting this process and in the production of attenuated viruses.

ONC201 kills solid tumor cells by triggering an integrated stress response dependent on ATF4 activation by specific eIF2α kinases

PubMed Central

Kline, C. Leah B.; Van den Heuvel, A. Pieter J.; Allen, Joshua E.; Prabhu, Varun V.; Dicker, David T.; El-Deiry, Wafik S.

2016-01-01

ONC201 (also called TIC10) is a small molecule that inactivates the cell proliferation- and cell survival-promoting kinases AKT and ERK and induces cell death through the pro-apoptotic protein TRAIL. ONC201 is currently in early phase clinical testing for various malignancies. Here, we found through gene expression and protein analyses that ONC201 triggered an increase in TRAIL abundance and cell death through an integrated stress response (ISR) involving the transcription factor ATF4, the transactivator CHOP, and the TRAIL receptor DR5. ATF4 was not activated in ONC201-resistant cancer cells, and in ONC201-sensitive cells, knockdown of ATF4 or CHOP partially abrogated ONC201-induced cytotoxicity and diminished the ONC201-stimulated increase in DR5 abundance. The activation of ATF4 in response to ONC201 required the kinases HRI and PKR, which phosphorylate and activate the translation initiation factor eIF2α. ONC201 rapidly triggered cell cycle arrest, which was associated with decreased abundance of cyclin D1, decreased activity of the kinase complex mTORC1, and dephosphorylation of the retinoblastoma (Rb) protein. The abundance of X-linked inhibitor of apoptosis protein (XIAP) negatively correlated with the extent of apoptosis in response to ONC201. These effects of ONC201 were independent of whether cancer cells had normal or mutant p53. Thus, ONC201 induces cell death through the coordinated induction of TRAIL by an ISR pathway. PMID:26884600

4E-BP is a target of the GCN2–ATF4 pathway during Drosophila development and aging

PubMed Central

Park, Jung-Eun; Zeng, Xiaomei

2017-01-01

Reduced amino acid availability attenuates mRNA translation in cells and helps to extend lifespan in model organisms. The amino acid deprivation–activated kinase GCN2 mediates this response in part by phosphorylating eIF2α. In addition, the cap-dependent translational inhibitor 4E-BP is transcriptionally induced to extend lifespan in Drosophila melanogaster, but through an unclear mechanism. Here, we show that GCN2 and its downstream transcription factor, ATF4, mediate 4E-BP induction, and GCN2 is required for lifespan extension in response to dietary restriction of amino acids. The 4E-BP intron contains ATF4-binding sites that not only respond to stress but also show inherent ATF4 activity during normal development. Analysis of the newly synthesized proteome through metabolic labeling combined with click chemistry shows that certain stress-responsive proteins are resistant to inhibition by 4E-BP, and gcn2 mutant flies have reduced levels of stress-responsive protein synthesis. These results indicate that GCN2 and ATF4 are important regulators of 4E-BP transcription during normal development and aging. PMID:27979906

Novel insights into the architecture and protein interaction network of yeast eIF3.

PubMed

Khoshnevis, Sohail; Hauer, Florian; Milón, Pohl; Stark, Holger; Ficner, Ralf

2012-12-01

Translation initiation in eukaryotes is a multistep process requiring the orchestrated interaction of several eukaryotic initiation factors (eIFs). The largest of these factors, eIF3, forms the scaffold for other initiation factors, promoting their binding to the 40S ribosomal subunit. Biochemical and structural studies on eIF3 need highly pure eIF3. However, natively purified eIF3 comprise complexes containing other proteins such as eIF5. Therefore we have established in vitro reconstitution protocols for Saccharomyces cerevisiae eIF3 using its five recombinantly expressed and purified subunits. This reconstituted eIF3 complex (eIF3(rec)) exhibits the same size and activity as the natively purified eIF3 (eIF3(nat)). The homogeneity and stoichiometry of eIF3(rec) and eIF3(nat) were confirmed by analytical size exclusion chromatography, mass spectrometry, and multi-angle light scattering, demonstrating the presence of one copy of each subunit in the eIF3 complex. The reconstituted and native eIF3 complexes were compared by single-particle electron microscopy showing a high degree of structural conservation. The interaction network between eIF3 proteins was studied by means of limited proteolysis, analytical size exclusion chromatography, in vitro binding assays, and isothermal titration calorimetry, unveiling distinct protein domains and subcomplexes that are critical for the integrity of the protein network in yeast eIF3. Taken together, the data presented here provide a novel procedure to obtain highly pure yeast eIF3, suitable for biochemical and structural analysis, in addition to a detailed picture of the network of protein interactions within this complex.

Fertility and polarized cell growth depends on eIF5A for translation of polyproline-rich formins in Saccharomyces cerevisiae.

PubMed

Li, Tianlu; Belda-Palazón, Borja; Ferrando, Alejandro; Alepuz, Paula

2014-08-01

eIF5A is an essential and evolutionary conserved translation elongation factor, which has recently been proposed to be required for the translation of proteins with consecutive prolines. The binding of eIF5A to ribosomes occurs upon its activation by hypusination, a modification that requires spermidine, an essential factor for mammalian fertility that also promotes yeast mating. We show that in response to pheromone, hypusinated eIF5A is required for shmoo formation, localization of polarisome components, induction of cell fusion proteins, and actin assembly in yeast. We also show that eIF5A is required for the translation of Bni1, a proline-rich formin involved in polarized growth during shmoo formation. Our data indicate that translation of the polyproline motifs in Bni1 is eIF5A dependent and this translation dependency is lost upon deletion of the polyprolines. Moreover, an exogenous increase in Bni1 protein levels partially restores the defect in shmoo formation seen in eIF5A mutants. Overall, our results identify eIF5A as a novel and essential regulator of yeast mating through formin translation. Since eIF5A and polyproline formins are conserved across species, our results also suggest that eIF5A-dependent translation of formins could regulate polarized growth in such processes as fertility and cancer in higher eukaryotes. Copyright © 2014 by the Genetics Society of America.

DYRK2 negatively regulates cardiomyocyte growth by mediating repressor function of GSK-3β on eIF2Bε.

PubMed

Weiss, Celine S; Ochs, Marco M; Hagenmueller, Marco; Streit, Marcus R; Malekar, Pratima; Riffel, Johannes H; Buss, Sebastian J; Weiss, Karl H; Sadoshima, Junichi; Katus, Hugo A; Hardt, Stefan E

2013-01-01

A prerequisite of hypertrophic response of the myocardium is an increase in protein synthesis. A central regulator of translation initiation is Eukaryotic initiation factor 2B (eIF2B). Here we assessed the hypothesis that regulation of protein synthesis via eIF2Bε is essential to cardiac hypertrophic response in vivo. Two transgenic mouse lines were generated with cardiac restricted overexpression of eIF2Bε or its mutant eIF2Bε-eIFS(535)A, which cannot be inactivated by phosphorylation through GSK-3β. (1) Under baseline conditions eIF2Bε transgenic mice showed no difference in cardiac phenotype compared to wild type, whereas in the mutant eIF2Bε-S(535)A an increase in LV/tibia length (7.5 ± 0.4 mg/mm vs. 6.2 ± 0.2 mg/mm, p<0.001) and cardiomyocyte cross sectional area (13004 ± 570 vs. 10843 ± 347 RU, p<0.01) was observed. (2) Cardiac overexpression of eIF2Bε did not change the response of the heart to pathologic stress induced by chronic isoproterenol treatment. (3) Cardiac overexpression of the eIF2Bε transgene was followed by overexpression of DYRK2 which is known to prime the inhibitory action of GSK-3β on eIF2Bε, while DYRK1A and GSK-3β itself were not increased. (4) In C57BL/6 mice after 48 h of isoproterenol-stimulation or aortic banding, eIF2Bε was increased and DYRK2 was concomitantly decreased. (5) In line with these in vivo findings, siRNA knockdown of DYRK2 in cultured cardiomyocytes resulted in decreased levels of p(S535)- eIF2Bε, (6) whereas adenoviral induced overexpression of DYRK2 was accompanied by clearly increased phosphorylation of eIF2Bε, indicating a coordinated response pattern (7) Adenoviral induced overexpression of DYRK2 leads to significantly reduced cardiomyocyte size and diminishes hypertrophic response to adrenergic stimulation. The interaction of GSK-3β and its priming kinase DYRK2 regulate the activity of eIF2Bε in cardiac myocytes. DYRK2 is a novel negative regulator of cardiomyocyte growth. DYRK2 could serve as

Guanabenz promotes neuronal survival via enhancement of ATF4 and parkin expression in models of Parkinson disease.

PubMed

Sun, Xiaotian; Aimé, Pascaline; Dai, David; Ramalingam, Nagendran; Crary, John F; Burke, Robert E; Greene, Lloyd A; Levy, Oren A

2018-05-01

Reduced function of parkin appears to be a central pathogenic event in Parkinson disease (PD). Increasing parkin levels enhances survival in models of PD-related neuronal death and is a promising therapeutic objective. Previously, we demonstrated that the transcription factor ATF4 promotes survival in response to PD-mimetic stressors by maintaining parkin levels. ATF4 translation is up-regulated by phosphorylation of the translation initiation factor eIF2α. The small molecule guanabenz enhances eIF2α phosphorylation by blocking the function of GADD34, a regulatory protein that promotes eIF2α dephosphorylation. We tested the hypothesis that guanabenz, by inhibiting GADD34 and consequently increasing eIF2α phosphorylation and elevating ATF4, would improve survival in models of PD by up-regulating parkin. We found that GADD34 is strongly induced by 6-OHDA, and that GADD34 localization is dramatically altered in dopaminergic substantia nigra neurons in PD cases. We further demonstrated that guanabenz attenuates 6-hydroxydopamine (6-OHDA) induced cell death of differentiated PC12 cells and primary ventral midbrain dopaminergic neurons in culture, and of dopaminergic neurons in the substantia nigra of mice. In culture models, guanabenz also increases eIF2α phosphorylation and ATF4 and parkin levels in response to 6-OHDA. Furthermore, if either ATF4 or parkin is silenced, then the protective effect of guanabenz is lost. We also found similar results in a distinct model of neuronal death: primary cultures of cortical neurons treated with the topoisomerase I inhibitor camptothecin, in which guanabenz limited camptothecin-induced neuronal death in an ATF4- and parkin-dependent manner. In summary, our data suggest that guanabenz and other GADD34 inhibitors could be used as therapeutic agents to boost parkin levels and thereby slow neurodegeneration in PD and other neurodegenerative conditions. Copyright © 2018 Elsevier Inc. All rights reserved.

Phosphorylation of Wheat Germ Initiation Factors and Ribosomal Proteins 1

PubMed Central

Browning, Karen S.; Yan, Tyan Fuh J.; Lauer, Stephen J.; Aquino, Lu Ann; Tao, Mariano; Ravel, Joanne M.

1985-01-01

The ability of the wheat germ initiation factors and ribosomes to serve as substrates for a wheat germ protein kinase (Yan and Tao 1982 J Biol Chem 257: 7037-7043) has been investigated. The wheat germ kinase catalyzes the phosphorylation of the 42,000 dalton subunit of eukaryotic initiation factor (eIF)-2 and the 107,000 dalton subunit of eIF-3. Other initiation factors, eIF-4B and eIF-4A, and elongation factors, EF-1 and EF-2, are not phosphorylated by the kinase. Quantitative analysis indicates that the kinase catalyzes the incorporation of about 0.5 to 0.6 mole of phosphate per mole of the 42,000 dalton subunit of eIF-2 and about 6 moles of phosphate per mole of the 107,000 dalton subunit of eIF-3. Three proteins (Mr = 38,000, 14,800, and 12,600) of the 60S ribosomal subunit are phosphorylated by the kinase, but none of the 40S ribosomal proteins are substrates of the kinase. No effects of phosphorylation on the activities of eIF-2, eIF-3, or 60S ribosomal subunits could be demonstrated in vitro. Images Fig. 1 Fig. 3 Fig. 4 PMID:16664060

ONC201 kills solid tumor cells by triggering an integrated stress response dependent on ATF4 activation by specific eIF2α kinases.

PubMed

Kline, C Leah B; Van den Heuvel, A Pieter J; Allen, Joshua E; Prabhu, Varun V; Dicker, David T; El-Deiry, Wafik S

2016-02-16

ONC201 (also called TIC10) is a small molecule that inactivates the cell proliferation- and cell survival-promoting kinases Akt and ERK and induces cell death through the proapoptotic protein TRAIL. ONC201 is currently in early-phase clinical testing for various malignancies. We found through gene expression and protein analyses that ONC201 triggered an increase in TRAIL abundance and cell death through an integrated stress response (ISR) involving the transcription factor ATF4, the transactivator CHOP, and the TRAIL receptor DR5. ATF4 was not activated in ONC201-resistant cancer cells, and in ONC201-sensitive cells, knockdown of ATF4 or CHOP partially abrogated ONC201-induced cytotoxicity and diminished the ONC201-stimulated increase in DR5 abundance. The activation of ATF4 in response to ONC201 required the kinases HRI and PKR, which phosphorylate and activate the translation initiation factor eIF2α. ONC201 rapidly triggered cell cycle arrest, which was associated with decreased abundance of cyclin D1, decreased activity of the kinase complex mTORC1, and dephosphorylation of the retinoblastoma (Rb) protein. The abundance of X-linked inhibitor of apoptosis protein (XIAP) negatively correlated with the extent of apoptosis in response to ONC201. These effects of ONC201 were independent of whether cancer cells had normal or mutant p53. Thus, ONC201 induces cell death through the coordinated induction of TRAIL by an ISR pathway. Copyright © 2016, American Association for the Advancement of Science.

Interaction of p190A RhoGAP with eIF3A and Other Translation Preinitiation Factors Suggests a Role in Protein Biosynthesis.

PubMed

Parasuraman, Prasanna; Mulligan, Peter; Walker, James A; Li, Bihua; Boukhali, Myriam; Haas, Wilhelm; Bernards, Andre

2017-02-17

The negative regulator of Rho family GTPases, p190A RhoGAP, is one of six mammalian proteins harboring so-called FF motifs. To explore the function of these and other p190A segments, we identified interacting proteins by tandem mass spectrometry. Here we report that endogenous human p190A, but not its 50% identical p190B paralog, associates with all 13 eIF3 subunits and several other translational preinitiation factors. The interaction involves the first FF motif of p190A and the winged helix/PCI domain of eIF3A, is enhanced by serum stimulation and reduced by phosphatase treatment. The p190A/eIF3A interaction is unaffected by mutating phosphorylated p190A-Tyr 308 , but disrupted by a S296A mutation, targeting the only other known phosphorylated residue in the first FF domain. The p190A-eIF3 complex is distinct from eIF3 complexes containing S6K1 or mammalian target of rapamycin (mTOR), and appears to represent an incomplete preinitiation complex lacking several subunits. Based on these findings we propose that p190A may affect protein translation by controlling the assembly of functional preinitiation complexes. Whether such a role helps to explain why, unique among the large family of RhoGAPs, p190A exhibits a significantly increased mutation rate in cancer remains to be determined. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

GIGYF1/2 proteins use auxiliary sequences to selectively bind to 4EHP and repress target mRNA expression

PubMed Central

Peter, Daniel; Weber, Ramona; Sandmeir, Felix; Wohlbold, Lara; Helms, Sigrun; Bawankar, Praveen; Valkov, Eugene; Igreja, Cátia; Izaurralde, Elisa

2017-01-01

The eIF4E homologous protein (4EHP) is thought to repress translation by competing with eIF4E for binding to the 5′ cap structure of specific mRNAs to which it is recruited through interactions with various proteins, including the GRB10-interacting GYF (glycine–tyrosine–phenylalanine domain) proteins 1 and 2 (GIGYF1/2). Despite its similarity to eIF4E, 4EHP does not interact with eIF4G and therefore fails to initiate translation. In contrast to eIF4G, GIGYF1/2 bind selectively to 4EHP but not eIF4E. Here, we present crystal structures of the 4EHP-binding regions of GIGYF1 and GIGYF2 in complex with 4EHP, which reveal the molecular basis for the selectivity of the GIGYF1/2 proteins for 4EHP. Complementation assays in a GIGYF1/2-null cell line using structure-based mutants indicate that 4EHP requires interactions with GIGYF1/2 to down-regulate target mRNA expression. Our studies provide structural insights into the assembly of 4EHP–GIGYF1/2 repressor complexes and reveal that rather than merely facilitating 4EHP recruitment to transcripts, GIGYF1/2 proteins are required for repressive activity. PMID:28698298

Defects in tRNA processing and nuclear export induce GCN4 translation independently of phosphorylation of the alpha subunit of eukaryotic translation initiation factor 2.

PubMed

Qiu, H; Hu, C; Anderson, J; Björk, G R; Sarkar, S; Hopper, A K; Hinnebusch, A G

2000-04-01

Induction of GCN4 translation in amino acid-starved cells involves the inhibition of initiator tRNA(Met) binding to eukaryotic translation initiation factor 2 (eIF2) in response to eIF2 phosphorylation by protein kinase GCN2. It was shown previously that GCN4 translation could be induced independently of GCN2 by overexpressing a mutant tRNA(AAC)(Val) (tRNA(Val*)) or the RNA component of RNase MRP encoded by NME1. Here we show that overexpression of the tRNA pseudouridine 55 synthase encoded by PUS4 also leads to translational derepression of GCN4 (Gcd(-) phenotype) independently of eIF2 phosphorylation. Surprisingly, the Gcd(-) phenotype of high-copy-number PUS4 (hcPUS4) did not require PUS4 enzymatic activity, and several lines of evidence indicate that PUS4 overexpression did not diminish functional initiator tRNA(Met) levels. The presence of hcPUS4 or hcNME1 led to the accumulation of certain tRNA precursors, and their Gcd(-) phenotypes were reversed by overexpressing the RNA component of RNase P (RPR1), responsible for 5'-end processing of all tRNAs. Consistently, overexpression of a mutant pre-tRNA(Tyr) that cannot be processed by RNase P had a Gcd(-) phenotype. Interestingly, the Gcd(-) phenotype of hcPUS4 also was reversed by overexpressing LOS1, required for efficient nuclear export of tRNA, and los1Delta cells have a Gcd(-) phenotype. Overproduced PUS4 appears to impede 5'-end processing or export of certain tRNAs in the nucleus in a manner remedied by increased expression of RNase P or LOS1, respectively. The mutant tRNA(Val*) showed nuclear accumulation in otherwise wild-type cells, suggesting a defect in export to the cytoplasm. We propose that yeast contains a nuclear surveillance system that perceives defects in processing or export of tRNA and evokes a reduction in translation initiation at the step of initiator tRNA(Met) binding to the ribosome.

Defects in tRNA Processing and Nuclear Export Induce GCN4 Translation Independently of Phosphorylation of the α Subunit of Eukaryotic Translation Initiation Factor 2

PubMed Central

Qiu, Hongfang; Hu, Cuihua; Anderson, James; Björk, Glenn R.; Sarkar, Srimonti; Hopper, Anita K.; Hinnebusch, Alan G.

2000-01-01

Induction of GCN4 translation in amino acid-starved cells involves the inhibition of initiator tRNAMet binding to eukaryotic translation initiation factor 2 (eIF2) in response to eIF2 phosphorylation by protein kinase GCN2. It was shown previously that GCN4 translation could be induced independently of GCN2 by overexpressing a mutant tRNAAACVal (tRNAVal*) or the RNA component of RNase MRP encoded by NME1. Here we show that overexpression of the tRNA pseudouridine 55 synthase encoded by PUS4 also leads to translational derepression of GCN4 (Gcd− phenotype) independently of eIF2 phosphorylation. Surprisingly, the Gcd− phenotype of high-copy-number PUS4 (hcPUS4) did not require PUS4 enzymatic activity, and several lines of evidence indicate that PUS4 overexpression did not diminish functional initiator tRNAMet levels. The presence of hcPUS4 or hcNME1 led to the accumulation of certain tRNA precursors, and their Gcd− phenotypes were reversed by overexpressing the RNA component of RNase P (RPR1), responsible for 5′-end processing of all tRNAs. Consistently, overexpression of a mutant pre-tRNATyr that cannot be processed by RNase P had a Gcd− phenotype. Interestingly, the Gcd− phenotype of hcPUS4 also was reversed by overexpressing LOS1, required for efficient nuclear export of tRNA, and los1Δ cells have a Gcd− phenotype. Overproduced PUS4 appears to impede 5′-end processing or export of certain tRNAs in the nucleus in a manner remedied by increased expression of RNase P or LOS1, respectively. The mutant tRNAVal* showed nuclear accumulation in otherwise wild-type cells, suggesting a defect in export to the cytoplasm. We propose that yeast contains a nuclear surveillance system that perceives defects in processing or export of tRNA and evokes a reduction in translation initiation at the step of initiator tRNAMet binding to the ribosome. PMID:10713174

Deletion of the eIF2α Kinase GCN2 fails to rescue the memory decline associated with Alzheimer's disease.

PubMed

Devi, Latha; Ohno, Masuo

2013-01-01

Emerging evidence suggests that dysregulated translation through phosphorylation of eukaryotic initiation factor-2α (eIF2α) may contribute to Alzheimer's disease (AD) and related memory impairments. However, the underlying mechanisms remain unclear. Here, we crossed knockout mice for an eIF2α kinase (GCN2: general control nonderepressible-2 kinase) with 5XFAD transgenic mice, and investigated whether GCN2 deletion affects AD-like traits in this model. As observed in AD brains, 5XFAD mice recapitulated significant elevations in the β-secretase enzyme BACE1 and the CREB repressor ATF4 concomitant with a dramatic increase of eIF2α phosphorylation. Contrary to expectation, we found that GCN2(-/-) and GCN2(+/-) deficiencies aggravate rather than suppress hippocampal BACE1 and ATF4 elevations in 5XFAD mice, failing to rescue memory deficits as tested by the contextual fear conditioning. The facilitation of these deleterious events resulted in exacerbated β-amyloid accumulation, plaque pathology and CREB dysfunction in 5XFAD mice with GCN2 mutations. Notably, GCN2 deletion caused overactivation of the PKR-endoplasmic reticulum-related kinase (PERK)-dependent eIF2α phosphorylation pathway in 5XFAD mice in the absence of changes in the PKR pathway. Moreover, PERK activation in response to GCN2 deficiency was specific to 5XFAD mice, since phosphorylated PERK levels were equivalent between GCN2(-/-) and wild-type control mice. Our findings suggest that GCN2 may be an important eIF2α kinase under the physiological condition, whereas blocking the GCN2 pathway under exposure to significant β-amyloidosis rather aggravates eIF2α phosphorylation leading to BACE1 and ATF4 elevations in AD.

The human insulin mRNA is partly translated via a cap- and eIF4A-independent mechanism

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fred, Rikard G., E-mail: Rikard.Fred@mcb.uu.se; Sandberg, Monica; Pelletier, Jerry

this binding corresponded well with rates of cap-independent insulin biosynthesis at the different conditions. In conclusion, our studies show that insulin biosynthesis is mainly cap-dependent at a high glucose concentration, but that the cap-independent biosynthesis of insulin can constitute as much as 40-100% of all insulin biosynthesis during conditions of nitrosative stress. These data suggest that the pancreatic {beta}-cell is able to uphold basal insulin synthesis at conditions of starvation and stress via a cap- and eIF4A-independent mechanism, possibly mediated by the binding of PTB to the 5'-UTR of the human insulin mRNA.« less

Inhibition of mTOR/eIF4E by anti-viral drug ribavirin effectively enhances the effects of paclitaxel in oral tongue squamous cell carcinoma

DOE Office of Scientific and Technical Information (OSTI.GOV)

Dai, Dehua; Chen, Hujie; Tang, Jing

Upregulation of eIF4E is associated with poor clinical outcome in many human cancers and represents a potential therapeutic target. However, the function of eIF4E remains unknown in oral tongue squamous cell carcinoma (OTSCC). In this work, we show that ribavirin, an anti-viral drug, effectively augments sensitivity of OTSCC cells to paclitaxel via inhibiting mTOR/eIF4E signaling pathway. Ribavirin dose-dependently inhibits proliferation and induces apoptosis in SCC-9 and CAL27 cells. Combination of ribavirin and paclitaxel are more effective in inhibiting proliferation and inducing apoptosis in OTSCC cells. Importantly, the in vivo efficacy of ribavirin and its synergism with paclitaxel is confirmed by two independentmore » OTSCC xenograft mouse models. Mechanistically, ribavirin significantly decreases mTOR/eIF4E signaling pathway in OTSCC cells via suppressing phosphorylation of Akt, mTOR, 4EBP1 and eIF4E. Overexpression of the phosphor-mimetic form of eIF4E (eIF4E S209D) but not the nonphosphorylatable form (eIF4E S209A) reverses the effects of ribavirin, confirming that eIF4E inhibition is the mechanism of action of ribavirin in OTSCC cells. In addition, eIF4E depletion significantly enhances the anti-proliferative and pro-apoptotic effects of paclitaxel, demonstrating the critical role of eIF4E in OTSCC cell response to paclitaxel. Our work is the first to demonstrate the efficacy of ribavirin as a single agent and synergism as combination with paclitaxel in OTSCC in vitro and in vivo. Our findings also demonstrate the therapeutic value of inhibiting eIF4E in OTSCC treatment. - Highlights: • Ribavirin effectively targets OTSCC in vitro and in vivo. • Ribavirin acts synergistically with paclitaxel in OTSCC cells. • Ribavirin inhibits Akt/mTOR/eIF4E signaling in OTSCC. • eIF4E inhibition sensitizes OTSCC cell response to paclitaxel.« less

Mechanism-based screen for G1/S checkpoint activators identifies a selective activator of EIF2AK3/PERK signalling.

PubMed

Stockwell, Simon R; Platt, Georgina; Barrie, S Elaine; Zoumpoulidou, Georgia; Te Poele, Robert H; Aherne, G Wynne; Wilson, Stuart C; Sheldrake, Peter; McDonald, Edward; Venet, Mathilde; Soudy, Christelle; Elustondo, Frédéric; Rigoreau, Laurent; Blagg, Julian; Workman, Paul; Garrett, Michelle D; Mittnacht, Sibylle

2012-01-01

Human cancers often contain genetic alterations that disable G1/S checkpoint control and loss of this checkpoint is thought to critically contribute to cancer generation by permitting inappropriate proliferation and distorting fate-driven cell cycle exit. The identification of cell permeable small molecules that activate the G1/S checkpoint may therefore represent a broadly applicable and clinically effective strategy for the treatment of cancer. Here we describe the identification of several novel small molecules that trigger G1/S checkpoint activation and characterise the mechanism of action for one, CCT020312, in detail. Transcriptional profiling by cDNA microarray combined with reverse genetics revealed phosphorylation of the eukaryotic initiation factor 2-alpha (EIF2A) through the eukaryotic translation initiation factor 2-alpha kinase 3 (EIF2AK3/PERK) as the mechanism of action of this compound. While EIF2AK3/PERK activation classically follows endoplasmic reticulum (ER) stress signalling that sets off a range of different cellular responses, CCT020312 does not trigger these other cellular responses but instead selectively elicits EIF2AK3/PERK signalling. Phosphorylation of EIF2A by EIF2A kinases is a known means to block protein translation and hence restriction point transit in G1, but further supports apoptosis in specific contexts. Significantly, EIF2AK3/PERK signalling has previously been linked to the resistance of cancer cells to multiple anticancer chemotherapeutic agents, including drugs that target the ubiquitin/proteasome pathway and taxanes. Consistent with such findings CCT020312 sensitizes cancer cells with defective taxane-induced EIF2A phosphorylation to paclitaxel treatment. Our work therefore identifies CCT020312 as a novel small molecule chemical tool for the selective activation of EIF2A-mediated translation control with utility for proof-of-concept applications in EIF2A-centered therapeutic approaches, and as a chemical starting point for

Translation of 5′ leaders is pervasive in genes resistant to eIF2 repression

PubMed Central

Fahey, Ciara; Kenny, Elaine M; Terenin, Ilya M; Dmitriev, Sergey E; Cormican, Paul; Morris, Derek W; Shatsky, Ivan N; Baranov, Pavel V

2015-01-01

Eukaryotic cells rapidly reduce protein synthesis in response to various stress conditions. This can be achieved by the phosphorylation-mediated inactivation of a key translation initiation factor, eukaryotic initiation factor 2 (eIF2). However, the persistent translation of certain mRNAs is required for deployment of an adequate stress response. We carried out ribosome profiling of cultured human cells under conditions of severe stress induced with sodium arsenite. Although this led to a 5.4-fold general translational repression, the protein coding open reading frames (ORFs) of certain individual mRNAs exhibited resistance to the inhibition. Nearly all resistant transcripts possess at least one efficiently translated upstream open reading frame (uORF) that represses translation of the main coding ORF under normal conditions. Site-specific mutagenesis of two identified stress resistant mRNAs (PPP1R15B and IFRD1) demonstrated that a single uORF is sufficient for eIF2-mediated translation control in both cases. Phylogenetic analysis suggests that at least two regulatory uORFs (namely, in SLC35A4 and MIEF1) encode functional protein products. DOI: http://dx.doi.org/10.7554/eLife.03971.001 PMID:25621764

O-GlcNAcylation of eIF2α regulates the phospho-eIF2α-mediated ER stress response.

PubMed

Jang, Insook; Kim, Han Byeol; Seo, Hojoong; Kim, Jin Young; Choi, Hyeonjin; Yoo, Jong Shin; Kim, Jae-woo; Cho, Jin Won

2015-08-01

O-GlcNAcylation is highly involved in cellular stress responses including the endoplasmic reticulum (ER) stress response. For example, glucosamine-induced flux through the hexosamine biosynthetic pathway can promote ER stress and ER stress inducers can change the total cellular level of O-GlcNAcylation. However, it is largely unknown which component(s) of the unfolded protein response (UPR) is directly regulated by O-GlcNAcylation. In this study, eukaryotic translation initiation factor 2α (eIF2α), a major branch of the UPR, was O-GlcNAcylated at Ser 219, Thr 239, and Thr 241. Upon ER stress, eIF2α is phosphorylated at Ser 51 by phosphorylated PKR-like ER kinase and this inhibits global translation initiation, except for that of specific mRNAs, including activating transcription factor 4, that induce stress-responsive genes such as C/EBP homologous protein (CHOP). Hyper-O-GlcNAcylation induced by O-GlcNAcase inhibitor (thiamet-G) treatment or O-GlcNAc transferase (OGT) overexpression hindered phosphorylation of eIF2α at Ser 51. The level of O-GlcNAcylation of eIF2α was changed by dithiothreitol treatment dependent on its phosphorylation at Ser 51. Point mutation of the O-GlcNAcylation sites of eIF2α increased its phosphorylation at Ser 51 and CHOP expression and resulted in increased apoptosis upon ER stress. These results suggest that O-GlcNAcylation of eIF2α affects its phosphorylation at Ser 51 and influences CHOP-mediated cell death. This O-GlcNAcylation of eIF2α was reproduced in thiamet-G-injected mouse liver. In conclusion, proper regulation of O-GlcNAcylation and phosphorylation of eIF2α is important to maintain cellular homeostasis upon ER stress. Copyright © 2015 Elsevier B.V. All rights reserved.

Biochemical quantitation of the eIF5A hypusination in Arabidopsis thaliana uncovers ABA-dependent regulation

PubMed Central

Belda-Palazón, Borja; Nohales, María A.; Rambla, José L.; Aceña, José L.; Delgado, Oscar; Fustero, Santos; Martínez, M. Carmen; Granell, Antonio; Carbonell, Juan; Ferrando, Alejandro

2014-01-01

The eukaryotic translation elongation factor eIF5A is the only protein known to contain the unusual amino acid hypusine which is essential for its biological activity. This post-translational modification is achieved by the sequential action of the enzymes deoxyhypusine synthase (DHS) and deoxyhypusine hydroxylase (DOHH). The crucial molecular function of eIF5A during translation has been recently elucidated in yeast and it is expected to be fully conserved in every eukaryotic cell, however the functional description of this pathway in plants is still sparse. The genetic approaches with transgenic plants for either eIF5A overexpression or antisense have revealed some activities related to the control of cell death processes but the molecular details remain to be characterized. One important aspect of fully understanding this pathway is the biochemical description of the hypusine modification system. Here we have used recombinant eIF5A proteins either modified by hypusination or non-modified to establish a bi-dimensional electrophoresis (2D-E) profile for the three eIF5A protein isoforms and their hypusinated or unmodified proteoforms present in Arabidopsis thaliana. The combined use of the recombinant 2D-E profile together with 2D-E/western blot analysis from whole plant extracts has provided a quantitative approach to measure the hypusination status of eIF5A. We have used this information to demonstrate that treatment with the hormone abscisic acid produces an alteration of the hypusine modification system in Arabidopsis thaliana. Overall this study presents the first biochemical description of the post-translational modification of eIF5A by hypusination which will be functionally relevant for future studies related to the characterization of this pathway in Arabidopsis thaliana. PMID:24904603

p97/DAP5 is a ribosome-associated factor that facilitates protein synthesis and cell proliferation by modulating the synthesis of cell cycle proteins

PubMed Central

Lee, Sang Hyun; McCormick, Frank

2006-01-01

p97 (also referred to as DAP5, NAT1 or eIF4G2) has been proposed to act as a repressor of protein synthesis. However, we found that p97 is abundantly expressed in proliferating cells and p97 is recruited to ribosomes following growth factor stimulation. We also report that p97 binds eIF2β through its C-terminal domain and localizes to ribosome through its N-terminal MIF4G domain. When overexpressed, p97 increases reporter luciferase activity. In contrast, overexpression of the C-terminal two-thirds of eukaryotic initiation factor 4GI (eIF4GI), a region that shares significant homology with p97, or the N-terminal MIF4G domain of p97 markedly inhibits reporter activity, the rate of global translation and cell proliferation. Conversely, downregulation of p97 levels by RNA interference also decreases the rate of global translation and inhibits cell proliferation. This coincides with an increase in p27/Kip1 protein levels and a marked decrease in CDK2 kinase activity. Taken together, our results demonstrate that p97 is functionally different from the closely related C-terminal two-thirds of eIF4GI and it can positively promote protein synthesis and cell proliferation. PMID:16932749

EphA4 is a prognostic factor in gastric cancer

PubMed Central

2013-01-01

Background Erythropoietin-producing hepatocellular (Eph) receptor, consisting of a family of receptor tyrosine kinases, plays critical roles in tumour development and is considered an attractive target for cancer therapy. Methods Tumour samples were obtained from 222 patients with gastric adenocarcinoma who underwent gastrectomy. The expressions of EphA2, EphA4, and ephrinA1 were evaluated immunohistochemically. Results High expressions of EphA2, EphA4, and ephrinA1 significantly correlated with variables related to tumour progression, including the depth of invasion, metastatic lymph nodes, pathological stage, and distant metastasis or recurrent disease. High expressions of EphA2, EphA4, and ephrinA1 were significantly associated with poorer disease-specific survival (DSS; p < 0.001, p < 0.001, p = 0.026). On multivariate analysis, EphA4 was an independent prognostic factor of DSS (hazard ratio [HR], 2.3; 95% confidence interval [CI], 1.1-4.8; p = 0.028), and EphA2 tended to be a prognostic factor (HR, 2.4; 95% CI, 1.0-5.8; p = 0.050). In stage II and III cancer, EphA4 and EphA2 were both significantly associated with shorter survival (p = 0.007 and 0.019), but only EphA2 was an independent prognostic factor (HR, 2.6; 95% CI, 1.1-6.3; p = 0.039). Conclusion EphA4 may play important roles in tumor progression and outcomes in patients with gastric cancer. PMID:23738943

Plant Translation Factors and Virus Resistance

PubMed Central

Sanfaçon, Hélène

2015-01-01

Plant viruses recruit cellular translation factors not only to translate their viral RNAs but also to regulate their replication and potentiate their local and systemic movement. Because of the virus dependence on cellular translation factors, it is perhaps not surprising that many natural plant recessive resistance genes have been mapped to mutations of translation initiation factors eIF4E and eIF4G or their isoforms, eIFiso4E and eIFiso4G. The partial functional redundancy of these isoforms allows specific mutation or knock-down of one isoform to provide virus resistance without hindering the general health of the plant. New possible targets for antiviral strategies have also been identified following the characterization of other plant translation factors (eIF4A-like helicases, eIF3, eEF1A and eEF1B) that specifically interact with viral RNAs and proteins and regulate various aspects of the infection cycle. Emerging evidence that translation repression operates as an alternative antiviral RNA silencing mechanism is also discussed. Understanding the mechanisms that control the development of natural viral resistance and the emergence of virulent isolates in response to these plant defense responses will provide the basis for the selection of new sources of resistance and for the intelligent design of engineered resistance that is broad-spectrum and durable. PMID:26114476

Platelet factor-4 (CXCL4/PF-4): an angiostatic chemokine for cancer therapy.

PubMed

Wang, Zhe; Huang, He

2013-05-01

Platelet factor-4 (CXCL4/PF-4) is the first chemokine identified to have several biological functions. Notably, CXCL4/PF-4 inhibits endothelial cell proliferation and migration, leading to suppression of angiogenesis. Since angiogenesis is essential for the growth of most primary tumors and their subsequent metastases, it is a target for cancer therapy; due to its multiple functions, CXCL4/PF-4 is a potential clinical anti-tumor agent. This report reviews the mechanisms of CXCL4/PF-4 angiostatic activity, including interference with angiogenic growth factors bFGF-2 and VEGF165, activation of CXCR3B, interactions with integrins, interference with cell cycle, interactions with factors such as VEGF121 and CXCL8/IL-8, and derived molecules of CXCL4/PF-4 with angiostatic and anti-tumoral activities in different models in vivo or in vitro. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

Rotenone Induction of Hydrogen Peroxide Inhibits mTOR-mediated S6K1 and 4E-BP1/eIF4E Pathways, Leading to Neuronal Apoptosis

PubMed Central

Zhou, Qian; Liu, Chunxiao; Liu, Wen; Zhang, Hai; Zhang, Ruijie; Liu, Jia; Zhang, Jinfei; Xu, Chong; Liu, Lei; Huang, Shile; Chen, Long

2015-01-01

Rotenone, a common pesticide and inhibitor of mitochondrial complex I, induces loss of dopaminergic neurons and consequential aspects of Parkinson’s disease (PD). However, the exact mechanism of rotenone neurotoxicity is not fully elucidated. Here, we show that rotenone induced reactive oxygen species (ROS), leading to apoptotic cell death in PC12 cells and primary neurons. Pretreatment with catalase (CAT), a hydrogen peroxide-scavenging enzyme, attenuated rotenone-induced ROS and neuronal apoptosis, implying hydrogen peroxide (H2O2) involved, which was further verified by imaging intracellular H2O2 using a peroxide-selective probe H2DCFDA. Using thenoyltrifluoroacetone (TTFA), antimycin A, or Mito-TEMPO, we further demonstrated rotenone-induced mitochondrial H2O2-dependent neuronal apoptosis. Rotenone dramatically inhibited mTOR-mediated phosphorylation of S6K1 and 4E-BP1, which was also attenuated by CAT in the neuronal cells. Of interest, ectopic expression of wild-type mTOR or constitutively active S6K1, or downregulation of 4E-BP1 partially prevented rotenone-induced H2O2 and cell apoptosis. Furthermore, we noticed that rotenone-induced H2O2 was linked to the activation of caspase-3 pathway. This was evidenced by the finding that pretreatment with CAT partially blocked rotenone-induced cleavages of caspase-3 and poly (ADP-ribose) polymerase. Of note, zVAD-fmk, a pan caspase inhibitor, only partially prevented rotenone-induced apoptosis in PC12 cells and primary neurons. Expression of mTOR-wt, S6K1-ca, or silencing 4E-BP1 potentiated zVAD-fmk protection against rotenone-induced apoptosis in the cells. The results indicate that rotenone induction of H2O2 inhibits mTOR-mediated S6K1 and 4E-BP1/eIF4E pathways, resulting in caspase-dependent and -independent apoptosis in neuronal cells. Our findings suggest that rotenone-induced neuronal loss in PD may be prevented by activating mTOR signaling and/or administering antioxidants. PMID:25304210

The delta-subunit of murine guanine nucleotide exchange factor eIF-2B. Characterization of cDNAs predicts isoforms differing at the amino-terminal end.

PubMed

Henderson, R A; Krissansen, G W; Yong, R Y; Leung, E; Watson, J D; Dholakia, J N

1994-12-02

Protein synthesis in mammalian cells is regulated at the level of the guanine nucleotide exchange factor, eIF-2B, which catalyzes the exchange of eukaryotic initiation factor 2-bound GDP for GTP. We have isolated and sequenced cDNA clones encoding the delta-subunit of murine eIF-2B. The cDNA sequence encodes a polypeptide of 544 amino acids with molecular mass of 60 kDa. Antibodies against a synthetic polypeptide of 30 amino acids deduced from the cDNA sequence specifically react with the delta-subunit of mammalian eIF-2B. The cDNA-derived amino acid sequence shows significant homology with the yeast translational regulator Gcd2, supporting the hypothesis that Gcd2 may be the yeast homolog of the delta-subunit of mammalian eIF-2B. Primer extension studies and anchor polymerase chain reaction analysis were performed to determine the 5'-end of the transcript for the delta-subunit of eIF-2B. Results of these experiments demonstrate two different mRNAs for the delta-subunit of eIF-2B in murine cells. The isolation and characterization of two different full-length cDNAs also predicts the presence of two alternate forms of the delta-subunit of eIF-2B in murine cells. These differ at their amino-terminal end but have identical nucleotide sequences coding for amino acids 31-544.

Induction of cap-independent BiP (hsp-3) and Bcl-2 (ced-9) translation in response to eIF4G (IFG-1) depletion in C. elegans

PubMed Central

Morrison, J Kaitlin; Friday, Andrew J; Henderson, Melissa A; Hao, Enhui; Keiper, Brett D

2014-01-01

During apoptosis, activated caspases cleave the translation initiation factor eIF4G. This cleavage disrupts cap-dependent mRNA translation initiation within the cell. However, a specific subset of mRNAs can still be recruited for protein synthesis in a cap-independent manner by the residual initiation machinery. Many of these mRNAs, including cell death related mRNAs, contain internal ribosome entry sites (IRESes) that promote their enhanced translation during apoptosis. Still other mRNAs have little dependence on the cap recognition mechanism. The expression of the encoded proteins, both anti- and pro-apoptotic, allows for an initial period of attempted cell survival, then commitment to cell death when damage is extensive. In this study we address the translational regulation of the stress and apoptosis-related mRNAs in C. elegans: BiP (hsp-3) (hsp-4), Hif-1 (hif-1), p53 (cep-1), Bcl-2 (ced-9) and Apaf-1 (ced-4). Altered translational efficiency of these messages was observed upon depletion of cap-dependent translation and induction of apoptosis within the C. elegans gonad. Our findings suggest a physiological link between the cap-independent mechanism and the enhanced translation of hsp-3 and ced-9. This increase in the efficiency of translation may be integral to the stress response during the induction of physiological apoptosis. PMID:26779406

Amino Acid Availability Controls TRB3 Transcription in Liver through the GCN2/eIF2α/ATF4 Pathway

PubMed Central

Carraro, Valérie; Maurin, Anne-Catherine; Lambert-Langlais, Sarah; Averous, Julien; Chaveroux, Cédric; Parry, Laurent; Jousse, Céline; Örd, Daima; Örd, Tõnis; Fafournoux, Pierre; Bruhat, Alain

2010-01-01

In mammals, plasma amino acid concentrations are markedly affected by dietary or pathological conditions. It has been well established that amino acids are involved in the control of gene expression. Up to now, all the information concerning the molecular mechanisms involved in the regulation of gene transcription by amino acid availability has been obtained in cultured cell lines. The present study aims to investigate the mechanisms involved in transcriptional activation of the TRB3 gene following amino acid limitation in mice liver. The results show that TRB3 is up-regulated in the liver of mice fed a leucine-deficient diet and that this induction is quickly reversible. Using transient transfection and chromatin immunoprecipitation approaches in hepatoma cells, we report the characterization of a functional Amino Acid Response Element (AARE) in the TRB3 promoter and the binding of ATF4, ATF2 and C/EBPβ to this AARE sequence. We also provide evidence that only the binding of ATF4 to the AARE plays a crucial role in the amino acid-regulated transcription of TRB3. In mouse liver, we demonstrate that the GCN2/eIF2α/ATF4 pathway is essential for the induction of the TRB3 gene transcription in response to a leucine-deficient diet. Therefore, this work establishes for the first time that the molecular mechanisms involved in the regulation of gene transcription by amino acid availability are functional in mouse liver. PMID:21203563

Functional Characterization of the Role of the N-terminal Domain of the c/Nip1 Subunit of Eukaryotic Initiation Factor 3 (eIF3) in AUG Recognition*

PubMed Central

Karásková, Martina; Gunišová, Stanislava; Herrmannová, Anna; Wagner, Susan; Munzarová, Vanda; Valášek, Leoš Shivaya

2012-01-01

In eukaryotes, for a protein to be synthesized, the 40 S subunit has to first scan the 5′-UTR of the mRNA until it has encountered the AUG start codon. Several initiation factors that ensure high fidelity of AUG recognition were identified previously, including eIF1A, eIF1, eIF2, and eIF5. In addition, eIF3 was proposed to coordinate their functions in this process as well as to promote their initial binding to 40 S subunits. Here we subjected several previously identified segments of the N-terminal domain (NTD) of the eIF3c/Nip1 subunit, which mediates eIF3 binding to eIF1 and eIF5, to semirandom mutagenesis to investigate the molecular mechanism of eIF3 involvement in these reactions. Three major classes of mutant substitutions or internal deletions were isolated that affect either the assembly of preinitiation complexes (PICs), scanning for AUG, or both. We show that eIF5 binds to the extreme c/Nip1-NTD (residues 1–45) and that impairing this interaction predominantly affects the PIC formation. eIF1 interacts with the region (60–137) that immediately follows, and altering this contact deregulates AUG recognition. Together, our data indicate that binding of eIF1 to the c/Nip1-NTD is equally important for its initial recruitment to PICs and for its proper functioning in selecting the translational start site. PMID:22718758

Structure of a human cap-dependent 48S translation pre-initiation complex

PubMed Central

Eliseev, Boris; Yeramala, Lahari; Leitner, Alexander; Karuppasamy, Manikandan; Raimondeau, Etienne; Huard, Karine; Alkalaeva, Elena; Aebersold, Ruedi

2018-01-01

Abstract Eukaryotic translation initiation is tightly regulated, requiring a set of conserved initiation factors (eIFs). Translation of a capped mRNA depends on the trimeric eIF4F complex and eIF4B to load the mRNA onto the 43S pre-initiation complex comprising 40S and initiation factors 1, 1A, 2, 3 and 5 as well as initiator-tRNA. Binding of the mRNA is followed by mRNA scanning in the 48S pre-initiation complex, until a start codon is recognised. Here, we use a reconstituted system to prepare human 48S complexes assembled on capped mRNA in the presence of eIF4B and eIF4F. The highly purified h-48S complexes are used for cross-linking/mass spectrometry, revealing the protein interaction network in this complex. We report the electron cryo-microscopy structure of the h-48S complex at 6.3 Å resolution. While the majority of eIF4B and eIF4F appear to be flexible with respect to the ribosome, additional density is detected at the entrance of the 40S mRNA channel which we attribute to the RNA-recognition motif of eIF4B. The eight core subunits of eIF3 are bound at the 40S solvent-exposed side, as well as the subunits eIF3d, eIF3b and eIF3i. elF2 and initiator-tRNA bound to the start codon are present at the 40S intersubunit side. This cryo-EM structure represents a molecular snap-shot revealing the h-48S complex following start codon recognition. PMID:29401259

Recombinant platelet factor 4: a therapeutic, anti-neoplastic chimera?

PubMed

Lippi, Giuseppe; Favaloro, Emmanuel J

2010-07-01

Angiogenesis plays a pivotal role in many serious and life-threatening disorders (e.g., cancer, atherosclerosis, diabetes, arthritis, psoriasis, nephropathy, and retinopathy) and is regulated by a delicate equilibrium between a variety of pro- and anti-angiogenic factors. Although recombinant platelet factor 4 (PF4) was originally developed and evaluated as a clinical alternative to protamine for heparin neutralization, the current scientific evidence supports a role for this protein and derivative peptides in inhibiting tumor growth and spread, by suppression of tumor-induced neovascularization in many different types of solid tumors. As a heparin-binding tetramer, recombinant PF4 interferes with several steps of endothelial cell proliferation, migration, and angiogenesis, regulates apoptotic death through activation of distinct signal transduction pathways, inhibits growth factor receptor binding, amplifies the inflammatory response of natural killer cells through regulation of cytokines production, and induces and maintains a nonspecific immune response to cancer cells. These biological evidences paved the way for the development and marketing of novel PF4-based angiostatic agents characterized by reduced toxicity and improved bioavailability, thus raising the possibility of an alternative approach for preventing and treating growth and metastasis of tumors. Some PF4-derived molecules such as carboxyl-terminal fragments of recombinant human PF4 and modified and chimeric peptides have already been developed that exhibit stronger anti-angiogenic properties than the parent molecule and may serve as leads for further therapeutic developments. Newer means of delivering of this anti-angiogenic agent are also being attempted, including PF4-bearing polymeric microspheres, vector-mediated PF4 transduction, transgene transfection into oncolytic viruses, and molecular targeting therapy against PF4 and rHuPF4 conjugates. These delivery systems aim to produce high

Stimulation of translation by human Unr requires cold shock domains 2 and 4, and correlates with poly(A) binding protein interaction.

PubMed

Ray, Swagat; Anderson, Emma C

2016-03-03

The RNA binding protein Unr, which contains five cold shock domains, has several specific roles in post-transcriptional control of gene expression. It can act as an activator or inhibitor of translation initiation, promote mRNA turnover, or stabilise mRNA. Its role depends on the mRNA and other proteins to which it binds, which includes cytoplasmic poly(A) binding protein 1 (PABP1). Since PABP1 binds to all polyadenylated mRNAs, and is involved in translation initiation by interaction with eukaryotic translation initiation factor 4G (eIF4G), we investigated whether Unr has a general role in translational control. We found that Unr strongly stimulates translation in vitro, and mutation of cold shock domains 2 or 4 inhibited its translation activity. The ability of Unr and its mutants to stimulate translation correlated with its ability to bind RNA, and to interact with PABP1. We found that Unr stimulated the binding of PABP1 to mRNA, and that Unr was required for the stable interaction of PABP1 and eIF4G in cells. siRNA-mediated knockdown of Unr reduced the overall level of cellular translation in cells, as well as that of cap-dependent and IRES-dependent reporters. These data describe a novel role for Unr in regulating cellular gene expression.

Inhibition of human megakaryocytopoiesis in vitro by platelet factor 4 (PF4) and a synthetic COOH-terminal PF4 peptide.

PubMed Central

Gewirtz, A M; Calabretta, B; Rucinski, B; Niewiarowski, S; Xu, W Y

1989-01-01

We report that highly purified human platelet factor 4 (PF4) inhibits human megakaryocytopoiesis in vitro. At greater than or equal to 25 micrograms/ml, PF4 inhibited megakaryocyte colony formation approximately 80% in unstimulated cultures, and approximately 58% in cultures containing recombinant human IL 3 and granulocyte-macrophage colony-stimulating factor. Because PF4 (25 micrograms/ml) had no effect on either myeloid or erythroid colony formation lineage specificity of this effect was suggested. A synthetic COOH-terminal PF4 peptide of 24, but not 13 residues, also inhibited megakaryocyte colony formation, whereas a synthetic 18-residue beta-thromboglobulin (beta-TG) peptide and native beta-TG had no such effect when assayed at similar concentrations. The mechanism of PF4-mediated inhibition was investigated. First, we enumerated total cell number, and examined cell maturation in control colonies (n = 200) and colonies (n = 100) that arose in PF4-containing cultures. Total cells per colony did not differ dramatically in the two groups (6.1 +/- 3.0 vs. 4.2 +/- 1.6, respectively), but the numbers of mature large cells per colony was significantly decreased in the presence of PF4 when compared with controls (1.6 +/- 1.5 vs. 3.9 +/- 2.3; P less than 0.001). Second, by using the human leukemia cell line HEL as a model for primitive megakaryocytic cells, we studied the effect of PF4 on cell doubling time, on the expression of both growth-regulated (H3, p53, c-myc,and c-myb), and non-growth-regulated (beta 2-microglobulin) genes. At high concentrations of native PF4 (50 micrograms/ml), no effect on cell doubling time, or H3 or p53 expression was discerned. In contrast, c-myc and c-myb were both upregulated. These results suggested the PF4 inhibited colony formation by impeding cell maturation, as opposed to cell proliferation, perhaps by inducing expression of c-myc and c-myb. The ability of PF4 to inhibit a normal cell maturation function was then tested

Requirement for the eIF4E binding proteins for the synergistic down-regulation of protein synthesis by hypertonic conditions and mTOR inhibition.

PubMed

Clemens, Michael J; Elia, Androulla; Morley, Simon J

2013-01-01

The protein kinase mammalian target of rapamycin (mTOR) regulates the phosphorylation and activity of several proteins that have the potential to control translation, including p70S6 kinase and the eIF4E binding proteins 4E-BP1 and 4E-BP2. In spite of this, in exponentially growing cells overall protein synthesis is often resistant to mTOR inhibitors. We report here that sensitivity of wild-type mouse embryonic fibroblasts (MEFs) to mTOR inhibitors can be greatly increased when the cells are subjected to the physiological stress imposed by hypertonic conditions. In contrast, protein synthesis in MEFs with a double knockout of 4E-BP1 and 4E-BP2 remains resistant to mTOR inhibitors under these conditions. Phosphorylation of p70S6 kinase and protein kinase B (Akt) is blocked by the mTOR inhibitor Ku0063794 equally well in both wild-type and 4E-BP knockout cells, under both normal and hypertonic conditions. The response of protein synthesis to hypertonic stress itself does not require the 4E-BPs. These data suggest that under certain stress conditions: (i) translation has a greater requirement for mTOR activity and (ii) there is an absolute requirement for the 4E-BPs for regulation by mTOR. Importantly, dephosphorylation of p70S6 kinase and Akt is not sufficient to affect protein synthesis acutely.

No evidence for a role of Ile587Val polymorphism of EIF2B5 gene in multiple sclerosis in Kashmir Valley of India.

PubMed

Zahoor, Insha; Asimi, Ravouf; Haq, Ehtishamul

2015-12-15

Multiple sclerosis (MS) is an inflammatory neurodegenerative disease of the nervous system with a profound genetic element. It is already known that alterations in Eukaryotic Translation Initiation Factor 2B (EIF2B) gene encoding the five subunits of eIF2B complex cause Vanishing White Matter (VWM) disease of the brain and emerging evidences have advocated certain resemblances between MS and VWM in terms of clinical and epidemiological characteristics, thus validating the association study between EIF2B and MS. Moreover, a recent study has implicated EIF2B5 Ile587Val (rs843358) polymorphism as a susceptibility factor for MS. In order to investigate the association of EIF2B5 Ile587Val polymorphism with MS susceptibility in Kashmir region in India, we screened EIF2B5 Exon 13 in 30 MS patients and 65 controls (a total of 95 participants). During the present course of study, we could not find statistically significant difference in the frequency of Ile587Val between MS patients and controls, thus indicating that such alteration does not appear to influence MS development in Kashmiri population. Our results provide evidence against a major role for Ile587Val polymorphism in MS susceptibility. Copyright © 2015 Elsevier B.V. All rights reserved.

Platelets release CXCL4L1, a nonallelic variant of the chemokine platelet factor-4/CXCL4 and potent inhibitor of angiogenesis.

PubMed

Struyf, Sofie; Burdick, Marie D; Proost, Paul; Van Damme, Jo; Strieter, Robert M

2004-10-29

Platelet factor-4 (PF-4)/CXCL4 was the first chemokine described to inhibit neovascularization. Here, the product of the nonallelic variant gene of CXCL4, PF-4var1/PF-4alt, designated CXCL4L1, was isolated for the first time from thrombin-stimulated human platelets and purified to homogeneity. Although secreted CXCL4 and CXCL4L1 differ in only three amino acids, CXCL4L1 was more potent in inhibiting chemotaxis of human microvascular endothelial cells toward interleukin-8 (IL-8)/CXCL8 or basic fibroblast growth factor (bFGF). In vivo, CXCL4L1 was also more effective than CXCL4 in inhibiting bFGF-induced angiogenesis in rat corneas. Thus, activated platelets release CXCL4L1, a potent regulator of endothelial cell biology, which affects angiogenesis and vascular diseases.

Translation initiation on mRNAs bound by nuclear cap-binding protein complex CBP80/20 requires interaction between CBP80/20-dependent translation initiation factor and eukaryotic translation initiation factor 3g.

PubMed

Choe, Junho; Oh, Nara; Park, Sungjin; Lee, Ye Kyung; Song, Ok-Kyu; Locker, Nicolas; Chi, Sung-Gil; Kim, Yoon Ki

2012-05-25

In the cytoplasm of mammalian cells, either cap-binding proteins 80 and 20 (CBP80/20) or eukaryotic translation initiation factor (eIF) 4E can direct the initiation of translation. Although the recruitment of ribosomes to mRNAs during eIF4E-dependent translation (ET) is well characterized, the molecular mechanism for CBP80/20-dependent translation (CT) remains obscure. Here, we show that CBP80/20-dependent translation initiation factor (CTIF), which has been shown to be preferentially involved in CT but not ET, specifically interacts with eIF3g, a component of the eIF3 complex involved in ribosome recruitment. By interacting with eIF3g, CTIF serves as an adaptor protein to bridge the CBP80/20 and the eIF3 complex, leading to efficient ribosome recruitment during CT. Accordingly, down-regulation of CTIF using a small interfering RNA causes a redistribution of CBP80 from polysome fractions to subpolysome fractions, without significant consequence to eIF4E distribution. In addition, down-regulation of eIF3g inhibits the efficiency of nonsense-mediated mRNA decay, which is tightly coupled to CT but not to ET. Moreover, the artificial tethering of CTIF to an intercistronic region of dicistronic mRNA results in translation of the downstream cistron in an eIF3-dependent manner. These findings support the idea that CT mechanistically differs from ET.

Inhibition of Mitogen-activated Protein Kinase (MAPK)-interacting Kinase (MNK) Preferentially Affects Translation of mRNAs Containing Both a 5'-Terminal Cap and Hairpin.

PubMed

Korneeva, Nadejda L; Song, Anren; Gram, Hermann; Edens, Mary Ann; Rhoads, Robert E

2016-02-12

The MAPK-interacting kinases 1 and 2 (MNK1 and MNK2) are activated by extracellular signal-regulated kinases 1 and 2 (ERK1/2) or p38 in response to cellular stress and extracellular stimuli that include growth factors, cytokines, and hormones. Modulation of MNK activity affects translation of mRNAs involved in the cell cycle, cancer progression, and cell survival. However, the mechanism by which MNK selectively affects translation of these mRNAs is not understood. MNK binds eukaryotic translation initiation factor 4G (eIF4G) and phosphorylates the cap-binding protein eIF4E. Using a cell-free translation system from rabbit reticulocytes programmed with mRNAs containing different 5'-ends, we show that an MNK inhibitor, CGP57380, affects translation of only those mRNAs that contain both a cap and a hairpin in the 5'-UTR. Similarly, a C-terminal fragment of human eIF4G-1, eIF4G(1357-1600), which prevents binding of MNK to intact eIF4G, reduces eIF4E phosphorylation and inhibits translation of only capped and hairpin-containing mRNAs. Analysis of proteins bound to m(7)GTP-Sepharose reveals that both CGP and eIF4G(1357-1600) decrease binding of eIF4E to eIF4G. These data suggest that MNK stimulates translation only of mRNAs containing both a cap and 5'-terminal RNA duplex via eIF4E phosphorylation, thereby enhancing the coupled cap-binding and RNA-unwinding activities of eIF4F. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Occupational exposure to organic solvents: a risk factor for pulmonary veno-occlusive disease.

PubMed

Montani, David; Lau, Edmund M; Descatha, Alexis; Jaïs, Xavier; Savale, Laurent; Andujar, Pascal; Bensefa-Colas, Lynda; Girerd, Barbara; Zendah, Inès; Le Pavec, Jerome; Seferian, Andrei; Perros, Frédéric; Dorfmüller, Peter; Fadel, Elie; Soubrier, Florent; Sitbon, Oliver; Simonneau, Gérald; Humbert, Marc

2015-12-01

Pulmonary veno-occlusive disease (PVOD) is a rare form of pulmonary hypertension characterised by predominant remodelling of pulmonary venules. Bi-allelic mutations in the eukaryotic translation initiation factor 2α kinase 4 (EIF2AK4) gene were recently described as the major cause of heritable PVOD, but risk factors associated with PVOD remain poorly understood. Occupational exposures have been proposed as a potential risk factor for PVOD, but epidemiological studies are lacking.A case-control study was conducted in consecutive PVOD (cases, n=33) and pulmonary arterial hypertension patients (controls, n=65). Occupational exposure was evaluated via questionnaire interview with blinded assessments using an expert consensus approach and a job exposure matrix (JEM).Using the expert consensus approach, PVOD was significantly associated with occupational exposure to organic solvents (adjusted OR 12.8, 95% CI 2.7-60.8), with trichloroethylene being the main agent implicated (adjusted OR 8.2, 95% CI 1.4-49.4). JEM analysis independently confirmed the association between PVOD and trichloroethylene exposure. Absence of significant trichloroethylene exposure was associated with a younger age of disease (54.8±21.4 years, p=0.037) and a high prevalence of harbouring bi-allelic EIF2AK4 mutations (41.7% versus 0%, p=0.015).Occupational exposure to organic solvents may represent a novel risk factor for PVOD. Genetic background and environmental exposure appear to influence the phenotypic expression of the disease. Copyright ©ERS 2015.

Kinases of eIF2a Switch Translation of mRNA Subset during Neuronal Plasticity

PubMed Central

Chesnokova, Ekaterina; Bal, Natalia

2017-01-01

Compared to other types of cells, neurons express the largest number of diverse mRNAs, including neuron-specific ones. This mRNA diversity is required for neuronal function, memory storage, maintenance and retrieval. Regulation of translation in neurons is very complicated and involves various proteins. Some proteins, implementing translational control in other cell types, are used by neurons for synaptic plasticity. In this review, we discuss the neuron-specific activity of four kinases: protein kinase R (PKR), PKR-like endoplasmic reticulum kinase (PERK), general control nonderepressible 2 kinase (GCN2), and heme-reguated eIF2α kinase (HRI), the substrate for which is α-subunit of eukaryotic initiation factor 2 (eIF2α). Phosphorylation of eIF2α is necessary for the cell during stress conditions, such as lack of amino acids, energy stress or viral infection. We propose that, during memory formation, neurons use some mechanisms similar to those involved in the cellular stress. The four eIF2α kinases regulate translation of certain mRNAs containing upstream open reading frames (uORFs). These mRNAs encode proteins involved in the processes of long-term potentiation (LTP) or long-term depression (LTD). The review examines some neuronal proteins for which translation regulation by eIF2 was suggested and checked experimentally. Of such proteins, we pay close attention to protein kinase Mζ, which is involved in memory storage and regulated at the translational level. PMID:29065505

C/EBPβ-LAP*/LAP Expression Is Mediated by RSK/eIF4B-Dependent Signalling and Boosted by Increased Protein Stability in Models of Monocytic Differentiation

PubMed Central

Christmann, Martin; Friesenhagen, Judith; Westphal, Andreas; Pietsch, Daniel; Brand, Korbinian

2015-01-01

The transcription factor C/EBPβ plays a key role in monocytic differentiation and inflammation. Its small isoform LIP is associated with proliferation at early premonocytic developmental stages and regulated via mTOR-dependent signalling. During later stages of (pre)monocytic differentiation there is a considerable increase in the large C/EBPβ isoforms LAP*/LAP which inhibit proliferation thus supporting terminal differentiation. Here, we showed in different models of monocytic differentiation that this dramatic increase in the LAP*/LAP protein and LAP/LIP ratio was accompanied by an only modest/retarded mRNA increase suggesting an important role for (post)translational mechanisms. We found that LAP*/LAP formation was induced via MEK/RSK-dependent cascades, whereas mTOR/S6K1 were not involved. Remarkably, LAP*/LAP expression was dependent on phosphorylated eIF4B, an acceleratory protein of RNA helicase eIF4A. PKR inhibition reduced the expression of eIF4B and C/EBPβ in an eIF2α-independent manner. Furthermore, under our conditions a marked stabilisation of LAP*/LAP protein occurred, accompanied by reduced chymotrypsin-like proteasome/calpain activities and increased calpastatin levels. Our study elucidates new signalling pathways inducing LAP*/LAP expression and indicates new alternative PKR functions in monocytes. The switch from mTOR- to RSK-mediated signalling to orchestrate eIF4B-dependent LAP*/LAP translation, accompanied by increased protein stability but only small mRNA changes, may be a prototypical example for the regulation of protein expression during selected processes of differentiation/proliferation. PMID:26646662

Two highly similar DEAD box proteins, OsRH2 and OsRH34, homologous to eukaryotic initiation factor 4AIII, play roles of the exon junction complex in regulating growth and development in rice.

PubMed

Huang, Chun-Kai; Sie, Yi-Syuan; Chen, Yu-Fu; Huang, Tian-Sheng; Lu, Chung-An

2016-04-12

The exon junction complex (EJC), which contains four core components, eukaryotic initiation factor 4AIII (eIF4AIII), MAGO/NASHI (MAGO), Y14/Tsunagi/RNA-binding protein 8A, and Barentsz/Metastatic lymph node 51, is formed in both nucleus and cytoplasm, and plays important roles in gene expression. Genes encoding core EJC components have been found in plants, including rice. Currently, the functional characterizations of MAGO and Y14 homologs have been demonstrated in rice. However, it is still unknown whether eIF4AIII is essential for the functional EJC in rice. This study investigated two DEAD box RNA helicases, OsRH2 and OsRH34, which are homologous to eIF4AIII, in rice. Amino acid sequence analysis indicated that OsRH2 and OsRH34 had 99 % identity and 100 % similarity, and their gene expression patterns were similar in various rice tissues, but the level of OsRH2 mRNA was about 58-fold higher than that of OsRH34 mRNA in seedlings. From bimolecular fluorescence complementation results, OsRH2 and OsRH34 interacted physically with OsMAGO1 and OsY14b, respectively, which indicated that both of OsRH2 and OsRH34 were core components of the EJC in rice. To study the biological roles of OsRH2 and OsRH34 in rice, transgenic rice plants were generated by RNA interference. The phenotypes of three independent OsRH2 and OsRH34 double-knockdown transgenic lines included dwarfism, a short internode distance, reproductive delay, defective embryonic development, and a low seed setting rate. These phenotypes resembled those of mutants with gibberellin-related developmental defects. In addition, the OsRH2 and OsRH34 double-knockdown transgenic lines exhibited the accumulation of unspliced rice UNDEVELOPED TAPETUM 1 mRNA. Rice contains two eIF4AIII paralogous genes, OsRH2 and OsRH34. The abundance of OsRH2 mRNA was about 58-fold higher than that of OsRH34 mRNA in seedlings, suggesting that the OsRH2 is major eIF4AIII in rice. Both OsRH2 and OsRH34 are core components of the EJC

Deletion of eIF2β lysine stretches creates a dominant negative that affects the translation and proliferation in human cell line: A tool for arresting the cell growth.

PubMed

Salton, Gabrielle Dias; Laurino, Claudia Cilene Fernandes Correia; Mega, Nicolás Oliveira; Delgado-Cañedo, Andrés; Setterblad, Niclas; Carmagnat, Maryvonnick; Xavier, Ricardo Machado; Cirne-Lima, Elizabeth; Lenz, Guido; Henriques, João Antonio Pêgas; Laurino, Jomar Pereira

2017-08-03

Eukaryote initiation factor 2 subunit β (eIF2β) plays a crucial role in regulation protein synthesis, which mediates the interaction of eIF2 with mRNA. eIF2β contains evolutionarily conserved polylysine stretches in amino-terminal region and a zinc finger motif in the carboxy-terminus. The gene eIF2β was cloned under tetracycline transcription control and the polylysine stretches were deleted by site-directed mutagenesis (eIF2βΔ3K). The plasmid was transfected into HEK 293 TetR cells. These cells were analyzed for their proliferative and translation capacities as well as cell death rate. Experiments were performed using gene reporter assays, western blotting, flow cytometry, cell sorting, cell proliferation assays and confocal immunofluorescence. eIF2βΔ3K affected negatively the protein synthesis, cell proliferation and cell survival causing G2 cell cycle arrest and increased cell death, acting in a negative dominant manner against the native protein. Polylysine stretches are also essential for eIF2β translocated from the cytoplasm to the nucleus, accumulating in the nucleolus and eIF2βΔ3K did not make this translocation. eIF2β is involved in the protein synthesis process and should act in nuclear processes as well. eIF2βΔ3K reduces cell proliferation and causes cell death. Since translation control is essential for normal cell function and survival, the development of drugs or molecules that inhibit translation has become of great interest in the scenario of proliferative disorders. In conclusion, our results suggest the dominant negative eIF2βΔ3K as a therapeutic strategy for the treatment of proliferative disorders and that eIF2β polylysine stretch domains are promising targets for this.

Differential activation of eIF2 kinases in response to cellular stresses in Schizosaccharomyces pombe.

PubMed

Zhan, Ke; Narasimhan, Jana; Wek, Ronald C

2004-12-01

Phosphorylation of eukaryotic initiation factor-2 (eIF2) is an important mechanism mitigating cellular injury in response to diverse environmental stresses. While all eukaryotic organisms characterized to date contain an eIF2 kinase stress response pathway, the composition of eIF2 kinases differs, with mammals containing four distinct family members and the well-studied lower eukaryote Saccharomyces cerevisiae expressing only a single eIF2 kinase. We are interested in the mechanisms by which multiple eIF2 kinases interface with complex stress signals and elicit response pathways. In this report we find that in addition to two previously described eIF2 kinases related to mammalian HRI, designated Hri1p and Hri2p, the yeast Schizosaccharomyces pombe expresses a third eIF2 kinase, a Gcn2p ortholog. To delineate the roles of each eIF2 kinase, we constructed S. pombe strains expressing only a single eIF2 kinase gene or deleted for the entire eIF2 kinase family. We find that Hri2p is the primary activated eIF2 kinase in response to exposure to heat shock, arsenite, or cadmium. Gcn2p serves as the primary eIF2 kinase induced during a nutrient downshift, treatment with the amino acid biosynthetic inhibitor 3-aminotriazole, or upon exposure to high concentrations of sodium chloride. In one stress example, exposure to H(2)O(2), there is early tandem activation of both Hri2p and Gcn2p. Interestingly, with extended stress conditions there is activation of alternative secondary eIF2 kinases, suggesting that eukaryotes have mechanisms of coordinate activation of eIF2 kinase in their stress remediation responses. Deletion of these eIF2 kinases renders S. pombe more sensitive to many of these stress conditions.

Excessive Cap-dependent Translation as a Molecular Mechanism Underlying ASD

DTIC Science & Technology

2012-08-01

SUPPLEMENTARY NOTES 14. ABSTRACT We hypothesize that excessive cap-dependent translation is a causative factor in autism spectrum disorder...compounds to therapeutically target eIF4E-eIF4G interactions and eIF4A for treating patients with ASD. 15. SUBJECT TERMS autism spectrum...Introduction We hypothesize that excessive cap-dependent translation is a causative factor in autism spectrum disorder (ASD). To test this

Genome-Wide Analyses and Functional Classification of Proline Repeat-Rich Proteins: Potential Role of eIF5A in Eukaryotic Evolution

PubMed Central

Mandal, Ajeet; Mandal, Swati; Park, Myung Hee

2014-01-01

The eukaryotic translation factor, eIF5A has been recently reported as a sequence-specific elongation factor that facilitates peptide bond formation at consecutive prolines in Saccharomyces cerevisiae, as its ortholog elongation factor P (EF-P) does in bacteria. We have searched the genome databases of 35 representative organisms from six kingdoms of life for PPP (Pro-Pro-Pro) and/or PPG (Pro-Pro-Gly)-encoding genes whose expression is expected to depend on eIF5A. We have made detailed analyses of proteome data of 5 selected species, Escherichia coli, Saccharomyces cerevisiae, Drosophila melanogaster, Mus musculus and Homo sapiens. The PPP and PPG motifs are low in the prokaryotic proteomes. However, their frequencies markedly increase with the biological complexity of eukaryotic organisms, and are higher in newly derived proteins than in those orthologous proteins commonly shared in all species. Ontology classifications of S. cerevisiae and human genes encoding the highest level of polyprolines reveal their strong association with several specific biological processes, including actin/cytoskeletal associated functions, RNA splicing/turnover, DNA binding/transcription and cell signaling. Previously reported phenotypic defects in actin polarity and mRNA decay of eIF5A mutant strains are consistent with the proposed role for eIF5A in the translation of the polyproline-containing proteins. Of all the amino acid tandem repeats (≥3 amino acids), only the proline repeat frequency correlates with functional complexity of the five organisms examined. Taken together, these findings suggest the importance of proline repeat-rich proteins and a potential role for eIF5A and its hypusine modification pathway in the course of eukaryotic evolution. PMID:25364902

Multi-functional regulation of 4E-BP gene expression by the Ccr4-Not complex.

PubMed

Okada, Hirokazu; Schittenhelm, Ralf B; Straessle, Anna; Hafen, Ernst

2015-01-01

The mechanistic target of rapamycin (mTOR) signaling pathway is highly conserved from yeast to humans. It senses various environmental cues to regulate cellular growth and homeostasis. Deregulation of the pathway has been implicated in many pathological conditions including cancer. Phosphorylation cascades through the pathway have been extensively studied but not much is known about the regulation of gene expression of the pathway components. Here, we report that the mRNA level of eukaryotic translation initiation factor (eIF) subunit 4E-binding protein (4E-BP) gene, one of the key mTOR signaling components, is regulated by the highly conserved Ccr4-Not complex. RNAi knockdown of Not1, a putative scaffold protein of this protein complex, increases the mRNA level of 4E-BP in Drosophila Kc cells. Examination of the gene expression mechanism using reporter swap constructs reveals that Not1 depletion increases reporter mRNAs with the 3'UTR of 4E-BP gene, but decreases the ones with the 4E-BP promoter region, suggesting that Ccr4-Not complex regulates both degradation and transcription of 4E-BP mRNA. These results indicate that the Ccr4-Not complex controls expression of a single gene at multiple levels and adjusts the magnitude of the total effect. Thus, our study reveals a novel regulatory mechanism of a key component of the mTOR signaling pathway at the level of gene expression.

MUC4: a novel prognostic factor of oral squamous cell carcinoma.

PubMed

Hamada, Tomofumi; Wakamatsu, Tsunenobu; Miyahara, Mayumi; Nagata, Satoshi; Nomura, Masahiro; Kamikawa, Yoshiaki; Yamada, Norishige; Batra, Surinder K; Yonezawa, Suguru; Sugihara, Kazumasa

2012-04-15

MUC4 mucin is now known to be expressed in various normal and cancer tissues. We have previously reported that MUC4 expression is a novel prognostic factor in several malignant tumors; however, it has not been investigated in oral squamous cell carcinoma (OSCC). The aim of our study is to evaluate the prognostic significance of MUC4 expression in OSCC. We examined the expression profile of MUC4 in OSCC tissues from 150 patients using immunohistochemistry. Its prognostic significance in OSCC was statistically analyzed. MUC4 was expressed in 61 of the 150 patients with OSCC. MUC4 expression was significantly correlated with higher T classification (p = 0.0004), positive nodal metastasis (p = 0.049), advanced tumor stage (p = 0.002), diffuse invasion of cancer cells (p = 0.004) and patient's death (p = 0.004) in OSCC. Multivariate analysis showed that MUC4 expression (p = 0.011), tumor location (p = 0.032) and diffuse invasion (p = 0.009) were statistically significant risk factors. Backward stepwise multivariate analysis demonstrated MUC4 expression (p = 0.0015) and diffuse invasion (p = 0.018) to be statistically significant independent risk factors of poor survival in OSCC. The disease-free and overall survival of patients with MUC4 expression was significantly worse than those without MUC4 expression (p < 0.0001 and p = 0.0001). In addition, the MUC4 expression was a significant risk factor for local recurrence and subsequent nodal metastasis in OSCC (p = 0.017 and p = 0.0001). We first report MUC4 overexpression is an independent factor for poor prognosis of patients with OSCC; therefore, patients with OSCC showing positive MUC4 expression should be followed up carefully. Copyright © 2011 UICC.

Inhibition of ribosome recruitment induces stress granule formation independently of eukaryotic initiation factor 2alpha phosphorylation.

PubMed

Mazroui, Rachid; Sukarieh, Rami; Bordeleau, Marie-Eve; Kaufman, Randal J; Northcote, Peter; Tanaka, Junichi; Gallouzi, Imed; Pelletier, Jerry

2006-10-01

Cytoplasmic aggregates known as stress granules (SGs) arise as a consequence of cellular stress and contain stalled translation preinitiation complexes. These foci are thought to serve as sites of mRNA storage or triage during the cell stress response. SG formation has been shown to require induction of eukaryotic initiation factor (eIF)2alpha phosphorylation. Herein, we investigate the potential role of other initiation factors in this process and demonstrate that interfering with eIF4A activity, an RNA helicase required for the ribosome recruitment phase of translation initiation, induces SG formation and that this event is not dependent on eIF2alpha phosphorylation. We also show that inhibition of eIF4A activity does not impair the ability of eIF2alpha to be phosphorylated under stress conditions. Furthermore, we observed SG assembly upon inhibition of cap-dependent translation after poliovirus infection. We propose that SG modeling can occur via both eIF2alpha phosphorylation-dependent and -independent pathways that target translation initiation.

Inhibition of Ribosome Recruitment Induces Stress Granule Formation Independently of Eukaryotic Initiation Factor 2α Phosphorylation

PubMed Central

Mazroui, Rachid; Sukarieh, Rami; Bordeleau, Marie-Eve; Kaufman, Randal J.; Northcote, Peter; Tanaka, Junichi; Gallouzi, Imed

2006-01-01

Cytoplasmic aggregates known as stress granules (SGs) arise as a consequence of cellular stress and contain stalled translation preinitiation complexes. These foci are thought to serve as sites of mRNA storage or triage during the cell stress response. SG formation has been shown to require induction of eukaryotic initiation factor (eIF)2α phosphorylation. Herein, we investigate the potential role of other initiation factors in this process and demonstrate that interfering with eIF4A activity, an RNA helicase required for the ribosome recruitment phase of translation initiation, induces SG formation and that this event is not dependent on eIF2α phosphorylation. We also show that inhibition of eIF4A activity does not impair the ability of eIF2α to be phosphorylated under stress conditions. Furthermore, we observed SG assembly upon inhibition of cap-dependent translation after poliovirus infection. We propose that SG modeling can occur via both eIF2α phosphorylation-dependent and -independent pathways that target translation initiation. PMID:16870703

EIF3C-enhanced exosome secretion promotes angiogenesis and tumorigenesis of human hepatocellular carcinoma

PubMed Central

Lee, Hsin-Yi; Chen, Chi-Kuan; Ho, Chun-Ming; Lee, Szu-Shuo; Chang, Chieh-Yu; Chen, Kuan-Ju; Jou, Yuh-Shan

2018-01-01

Targeting tumor angiogenesis is a common strategy against human hepatocellular carcinoma (HCC). However, identification of molecular targets as biomarker for elevating therapeutic efficacy is critical to prolong HCC patient survival. Here, we showed that EIF3C (eukaryotic translation initiation factor 3 subunit C) is upregulated during HCC tumor progression and associated with poor patient survival. Expression of EIF3C did not alter proliferation and expression of other tumor progressive genes such as HIF1A, TGFβ1 and VEGF, but reduced cell migration in HCC cells. Nevertheless, expression of EIF3C in HCC cells significantly increase secretion of extracellular exosomes confirmed by increased exosomes labelling by PKH26 fluorescent dye, vesicles in exosome size detected by electronic microscopy and nanoparticle tracking analysis, and expression of divergent exosome markers. The EIF3C-increased exosomes were oncogenic to potentiate tumor angiogenesis via tube formation of HUVEC cells and growth of vessels by plugs assays on nude mice. Subcutaneous inoculation of EIF3C-exosomes mixed with Huh7 HCC cells not only promoted growth of vessels but also increased expression of EIF3C in tumors. Conversely, treatment of exosome inhibitor GW4869 reversed aforementioned oncogenic assays. We identified EIF3C activated expression of S100A11 involved in EIF3C-exosome increased tube formation in angiogenesis. Simultaneous high expression of EIF3C and S100A11 in human HCC tumors for RNA level in TCGA and protein level by IHC are associated with poor survival of HCC patients. Collectively, our results demonstrated that EIF3C overexpression is a potential target of angiogenesis for treatment with exosome inhibitor or S100A11 reduction to suppress HCC angiogenesis and tumorigenesis. PMID:29568350

MiR-21 is enriched in the RNA-induced silencing complex and targets COL4A1 in human granulosa cell lines.

PubMed

Mase, Yuri; Ishibashi, Osamu; Ishikawa, Tomoko; Takizawa, Takami; Kiguchi, Kazushige; Ohba, Takashi; Katabuchi, Hidetaka; Takeshita, Toshiyuki; Takizawa, Toshihiro

2012-10-01

MicroRNAs (miRNAs) are noncoding small RNAs that play important roles in a variety of physiological and pathological events. In this study, we performed large-scale profiling of EIF2C2-bound miRNAs in 3 human granulosa-derived cell lines (ie, KGN, HSOGT, and GC1a) by high-throughput sequencing and found that miR-21 accounted for more than 80% of EIF2C2-bound miRNAs, suggesting that it was enriched in the RNA-induced silencing complex (RISC) and played a functional role in human granulosa cell (GC) lines. We also found high expression levels of miR-21 in primary human GCs. Assuming that miR-21 target mRNAs are enriched in RISC, we performed cDNA cloning of EIF2C2-bound mRNAs in KGN cells. We identified COL4A1 mRNA as a miR-21 target in the GC lines. These data suggest that miR-21 is involved in the regulation of the synthesis of COL4A1, a component of the basement membrane surrounding the GC layer and granulosa-embedded extracellular structure.

Economic Impact Forecast System (EIFS). Version 2.0. Users Manual. Supplement II. European Economic Impact Forecast System (EEIFS), Phase 1, (FRG/EIFS Pilot Model).

DTIC Science & Technology

1982-05-01

Chmpip. tL : Construction engineering Research Laboratory ; available from NTIS. 1982. 71 p. (Technical report / Construction Engineering Researsh ...AD-Al17 661 CONSTRUCTION ENGINEERING RESEARCH LAB (ARMY) CHAMPAIGN IL F/G 5/3 ECONOMIC IMPACT FORECAST SYSTEM (EIFS). VERSION 2.0. USERS MANU--ETC(u...CONSTRUCTION ENGINEERING RESEARCH LABORATORY 4A762720A896-C-004 P.O. BOX 4005, CHAMPAIGN, IL 61820 I. CONTROLLING OFFICE NAME AND ADDRESS It. REPORT

Trypanosoma brucei Metacaspase 4 Is a Pseudopeptidase and a Virulence Factor*

PubMed Central

Proto, William R.; Castanys-Munoz, Esther; Black, Alana; Tetley, Laurence; Moss, Catherine X.; Juliano, Luiz; Coombs, Graham H.; Mottram, Jeremy C.

2011-01-01

Metacaspases are caspase family cysteine peptidases found in plants, fungi, and protozoa but not mammals. Trypanosoma brucei is unusual in having five metacaspases (MCA1–MCA5), of which MCA1 and MCA4 have active site substitutions, making them possible non-enzymatic homologues. Here we demonstrate that recombinant MCA4 lacks detectable peptidase activity despite maintaining a functional peptidase structure. MCA4 is expressed primarily in the bloodstream form of the parasite and associates with the flagellar membrane via dual myristoylation/palmitoylation. Loss of function phenotyping revealed critical roles for MCA4; rapid depletion by RNAi caused lethal disruption to the parasite's cell cycle, yet the generation of MCA4 null mutant parasites (Δmca4) was possible. Δmca4 had normal growth in axenic culture but markedly reduced virulence in mice. Further analysis revealed that MCA4 is released from the parasite and is specifically processed by MCA3, the only metacaspase that is both palmitoylated and enzymatically active. Accordingly, we have identified that the multiple metacaspases in T. brucei form a membrane-associated proteolytic cascade to generate a pseudopeptidase virulence factor. PMID:21949125

Vascular Endothelial Growth Factor (VEGF) and Platelet (PF-4) Factor 4 Inputs Modulate Human Microvascular Endothelial Signaling in a Three-Dimensional Matrix Migration Context*

PubMed Central

Hang, Ta-Chun; Tedford, Nathan C.; Reddy, Raven J.; Rimchala, Tharathorn; Wells, Alan; White, Forest M.; Kamm, Roger D.; Lauffenburger, Douglas A.

2013-01-01

The process of angiogenesis is under complex regulation in adult organisms, particularly as it often occurs in an inflammatory post-wound environment. As such, there are many impacting factors that will regulate the generation of new blood vessels which include not only pro-angiogenic growth factors such as vascular endothelial growth factor, but also angiostatic factors. During initial postwound hemostasis, a large initial bolus of platelet factor 4 is released into localized areas of damage before progression of wound healing toward tissue homeostasis. Because of its early presence and high concentration, the angiostatic chemokine platelet factor 4, which can induce endothelial anoikis, can strongly affect angiogenesis. In our work, we explored signaling crosstalk interactions between vascular endothelial growth factor and platelet factor 4 using phosphotyrosine-enriched mass spectrometry methods on human dermal microvascular endothelial cells cultured under conditions facilitating migratory sprouting into collagen gel matrices. We developed new methods to enable mass spectrometry-based phosphorylation analysis of primary cells cultured on collagen gels, and quantified signaling pathways over the first 48 h of treatment with vascular endothelial growth factor in the presence or absence of platelet factor 4. By observing early and late signaling dynamics in tandem with correlation network modeling, we found that platelet factor 4 has significant crosstalk with vascular endothelial growth factor by modulating cell migration and polarization pathways, centered around P38α MAPK, Src family kinases Fyn and Lyn, along with FAK. Interestingly, we found EphA2 correlational topology to strongly involve key migration-related signaling nodes after introduction of platelet factor 4, indicating an influence of the angiostatic factor on this ambiguous but generally angiogenic signal in this complex environment. PMID:24023389

Excessive Cap-dependent Translation as a Molecular Mechanism Underlying ASD

DTIC Science & Technology

2013-08-01

dependent translation is a causative factor in autism spectrum disorder (ASD). To test this hypothesis, we have been studying transgenic mice that...determine whether eIF4E transgenic mice display cellular and molecular abnormalities due to excessive cap-dependent translation. mice. Our studies ...will provide information concerning whether overexpression of eIF4E is a biological risk factor for ASD. Our studies also will provide important

Hepatocyte Nuclear Factor 4α (HNF4α) Is a Transcription Factor of Vertebrate Fatty Acyl Desaturase Gene as Identified in Marine Teleost Siganus canaliculatus

PubMed Central

Dong, Yewei; Wang, Shuqi; Chen, Junliang; Zhang, Qinghao; Liu, Yang; You, Cuihong; Monroig, Óscar; Tocher, Douglas R.; Li, Yuanyou

2016-01-01

Rabbitfish Siganus canaliculatus was the first marine teleost demonstrated to have the capability of biosynthesizing long-chain polyunsaturated fatty acids (LC-PUFA) from C18 precursors, and to possess a Δ4 fatty acyl desaturase (Δ4 Fad) which was the first report in vertebrates, and is a good model for studying the regulatory mechanisms of LC-PUFA biosynthesis in teleosts. In order to understand regulatory mechanisms of transcription of Δ4 Fad, the gene promoter was cloned and characterized in the present study. An upstream sequence of 1859 bp from the initiation codon ATG was cloned as the promoter candidate. On the basis of bioinformatic analysis, several binding sites of transcription factors (TF) including GATA binding protein 2 (GATA-2), CCAAT enhancer binding protein (C/EBP), nuclear factor 1 (NF-1), nuclear factor Y (NF-Y), hepatocyte nuclear factor 4α (HNF4α) and sterol regulatory element (SRE), were identified in the promoter by site-directed mutation and functional assays. HNF4α and NF-1 were confirmed to interact with the core promoter of Δ4 Fad by gel shift assay and mass spectrometry. Moreover, over-expression of HNF4α increased promoter activity in HEK 293T cells and mRNA level of Δ4 Fad in rabbitfish primary hepatocytes, respectively. The results indicated that HNF4α is a TF of rabbitfish Δ4 Fad. To our knowledge, this is the first report on promoter structure of a Δ4 Fad, and also the first demonstration of HNF4α as a TF of vertebrate Fad gene involved in transcription regulation of LC-PUFA biosynthesis. PMID:27472219

Role of eIF2α Kinases in Translational Control and Adaptation to Cellular Stress.

PubMed

Wek, Ronald C

2018-02-12

A central mechanism regulating translation initiation in response to environmental stress involves phosphorylation of the α subunit of eukaryotic initiation factor 2 (eIF2α). Phosphorylation of eIF2α causes inhibition of global translation, which conserves energy and facilitates reprogramming of gene expression and signaling pathways that help to restore protein homeostasis. Coincident with repression of protein synthesis, many gene transcripts involved in the stress response are not affected or are even preferentially translated in response to increased eIF2α phosphorylation by mechanisms involving upstream open reading frames (uORFs). This review highlights the mechanisms regulating eIF2α kinases, the role that uORFs play in translational control, and the impact that alteration of eIF2α phosphorylation by gene mutations or small molecule inhibitors can have on health and disease. Copyright © 2018 Cold Spring Harbor Laboratory Press; all rights reserved.

Canonical Initiation Factor Requirements of the Myc Family of Internal Ribosome Entry Segments▿ †

PubMed Central

Spriggs, Keith A.; Cobbold, Laura C.; Jopling, Catherine L.; Cooper, Rebecca E.; Wilson, Lindsay A.; Stoneley, Mark; Coldwell, Mark J.; Poncet, Didier; Shen, Ya-Ching; Morley, Simon J.; Bushell, Martin; Willis, Anne E.

2009-01-01

Initiation of protein synthesis in eukaryotes requires recruitment of the ribosome to the mRNA and its translocation to the start codon. There are at least two distinct mechanisms by which this process can be achieved; the ribosome can be recruited either to the cap structure at the 5′ end of the message or to an internal ribosome entry segment (IRES), a complex RNA structural element located in the 5′ untranslated region (5′-UTR) of the mRNA. However, it is not well understood how cellular IRESs function to recruit the ribosome or how the 40S ribosomal subunits translocate from the initial recruitment site on the mRNA to the AUG initiation codon. We have investigated the canonical factors that are required by the IRESs found in the 5′-UTRs of c-, L-, and N-myc, using specific inhibitors and a tissue culture-based assay system, and have shown that they differ considerably in their requirements. The L-myc IRES requires the eIF4F complex and the association of PABP and eIF3 with eIF4G for activity. The minimum requirements of the N- and c-myc IRESs are the C-terminal domain of eIF4G to which eIF4A is bound and eIF3, although interestingly this protein does not appear to be recruited to the IRES RNA via eIF4G. Finally, our data show that all three IRESs require a ternary complex, although in contrast to c- and L-myc IRESs, the N-myc IRES has a lesser requirement for a ternary complex. PMID:19124605