Translation is regulated predominantly at the initiation phase by several signal transduction pathways that are often usurped in human cancers, including the PI3K/Akt/mTOR axis. mTOR exerts unique administration over translation by regulating assembly of eukaryotic initiation factor (eIF) 4F, a heterotrimeric complex responsible for recruiting 40S ribosomes (and associated factors) to mRNA 5' cap structures. Hence, there is much interest in targeted therapies that block eIF4F activity to assess the consequences on tumor cell growth and chemotherapy response. We report here that hippuristanol (Hipp), a translation initiation inhibitor that selectively inhibits the eIF4F RNA helicase subunit, eIF4A, resensitizes Eμ-Myc lymphomas to DNA damaging agents, including those that overexpress eIF4E-a modifier of rapamycin responsiveness. As Mcl-1 levels are significantly affected by Hipp, combining its use with the Bcl-2 family inhibitor, ABT-737, leads to a potent synergistic response in triggering cell death in mouse and human lymphoma and leukemia cells. Suppression of eIF4AI using RNA interference also synergized with ABT-737 in murine lymphomas, highlighting eIF4AI as a therapeutic target for modulating tumor cell response to chemotherapy.

Breast cancer stem cells (BCSCs) are intrinsically chemoresistant and capable of self-renewal. Following chemotherapy, patients can develop minimal residual disease due to BCSCs which can repopulate into a relapsed tumor. Therefore, it is imperative to co-target BCSCs along with the bulk tumor cells to achieve therapeutic success and prevent recurrence. So, it is vital to identify actionable molecular targets against both BCSCs and bulk tumor cells. Previous findings from our lab and others have demonstrated that inhibition of the emerging drug target eIF4A with Rocaglamide A (RocA) was efficacious against triple-negative breast cancer cells (TNBC). RocA specifically targets the pool of eIF4A bound to the oncogenic mRNAs that requires its helicase activity for their translation. This property enables specific targeting of tumor cells. The efficacy of RocA against BCSCs is unknown. In this study, we postulated that eIF4A could be a vulnerable node in BCSCs. In order to test this, we generated a paclitaxel-resistant TNBC cell line which demonstrated an elevated level of eIF4A along with increased levels of cancer stemness markers (ALDH activity and CD44), pluripotency transcription factors (SOX2, OCT4, and NANOG) and drug transporters (ABCB1, ABCG2, and ABCC1). Furthermore, genetic ablation of eIF4A resulted in reduced expression of ALDH1A1, pluripotency transcription factors and drug transporters. This pointed out that eIF4A is likely associated with selected set of proteins that are critical to BCSCs, and hence targeting eIF4A may eliminate BCSCs. Therefore, we isolated BCSCs from two TNBC cell lines: MDA-Bone-Un and SUM-159PT. Following RocA treatment, the self-renewal ability of the BCSCs was significantly reduced as determined by the efficiency of the formation of primary and secondary mammospheres. This was accompanied by a reduction in the levels of NANOG, OCT4, and drug transporters. Exposure to RocA also induced cell death of the BCSCs as evaluated by DRAQ7 and cell viability assays. RocA treatment induced apoptosis with increased levels of cleaved caspase-3. Overall, we identified that RocA is effective in targeting BCSCs, and eIF4A is an actionable molecular target in both BCSCs and bulk tumor cells. Therefore, anti-eIF4A inhibitors could potentially be combined synergistically with existing chemo-, radio- and/or immunotherapies.

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.

Dysregulated activity of helicase eIF4A drives transformation to and maintenance of cancer cell phenotype by reprogramming cellular translation. Interleukin 24 (IL-24) is a tumor-suppressing protein, which has the ability to inhibit angiogenesis, sensitize cancer cells to chemotherapy, and induce cancer cell-specific apoptosis. In this study, we found that eIF4A is inhibited by IL-24. Consequently, selective reduction of translation was observed for mRNAs harboring strong secondary structures in their 5'-untranslated regions (5'UTRs). These mRNAs encode proteins, which function in cell survival and proliferation. Consistently, overexpression of eIF4A conferred cancer cells with resistance to IL-24-induced cell death. It has been established that inhibition of eIF4A triggers mitochondrial-mediated apoptosis. We showed that IL-24 induces eIF4A-dependent mitochondrial depolarization. We also showed that IL-24 induces Sigma 1 Receptor-dependent eIF4A down-regulation and mitochondrial depolarization. Thus, the progress of apoptosis triggered by IL-24 is characterized by a complex program of changes in regulation of several initiation factors, including the eIF4A.

Leishmaniases are neglected parasitic diseases in spite of the major burden they inflict on public health. The identification of novel drugs and targets constitutes a research priority. For that purpose we used Leishmania infantum initiation factor 4A (LieIF), an essential translation initiation factor that belongs to the DEAD-box proteins family, as a potential drug target. We modeled its structure and identified two potential binding sites. A virtual screening of a diverse chemical library was performed for both sites. The results were analyzed with an in-house version of the Self-Organizing Maps algorithm combined with multiple filters, which led to the selection of 305 molecules. Effects of these molecules on the ATPase activity of LieIF permitted the identification of a promising hit (208) having a half maximal inhibitory concentration (IC50) of 150 ± 15 μM for 1 μM of protein. Ten chemical analogues of compound 208 were identified and two additional inhibitors were selected (20 and 48). These compounds inhibited the mammalian eIF4I with IC50 values within the same range. All three hits affected the viability of the extra-cellular form of L. infantum parasites with IC50 values at low micromolar concentrations. These molecules showed non-significant toxicity toward THP-1 macrophages. Furthermore, their anti-leishmanial activity was validated with experimental assays on L. infantum intramacrophage amastigotes showing IC50 values lower than 4.2 μM. Selected compounds exhibited selectivity indexes between 19 to 38, which reflects their potential as promising anti-Leishmania molecules.

Eukaryotic initiation factor 4A (eIF-4A) is an essential component for protein translation in eukaryotes. The eIF-4A gene (ApeIF-4A) was isolated and characterized from Antheraea pernyi (Guérin-Méneville) (Lepidoptera: Saturniidae). The obtained cDNA sequence was 1,435-bp long with an open reading frame of 1,266 bp encoding 421 amino acids. The predicted amino acid sequence shared several conserved features as found in known eIF-4As and revealed 74 and 78% identities with eIF-4As of Homo sapiens L. and Drosophila melanogaster (Meigen), respectively. Reverse transcription-polymerase chain reaction (RT-PCR) analysis showed that ApeIF-4A was transcribed at four developmental stages and in all tissues tested, suggesting that it plays an important role in development of A. pernyi. Homologous alignment suggested that eIF-4As are highly conserved throughout evolution of eukaryote organisms. Phylogenetic trees based on the amino acid and nucleotide sequences of eIF-4A demonstrated a similar topology with the classical systematics, suggesting that it has the potential value in phylogenetic inference of eukaryotes.

Purpose: Eukaryotic initiation factor 4A-3 (EIF4A3) is an RNA-binding protein (RBP) that is a core component of the exon junction complex (EJC). It has been identified as an important player in post-transcriptional regulation processes. Recently, investigations have focused on EIF4A3 dysfunction in carcinogenesis. The present study aims to determine whether EIF4A3 can serve as a prognostic marker and potential regulatory mechanism in human cancers. Materials and methods: EIF4A3 expression in various cancers was assessed using Oncomine. The Correlation between EIF4A3 expression and patient survival was evaluated using PrognoScan. EIF4A3 mutations in various cancers were investigated using cBioPortal. EIF4A3 co-expression networks in various cancers were established using Coexpedia. Finally, we analyzed potential functional roles of EIF4A3 using Gene Ontology and pathway enrichment analyses by FunRich V3. Results: EIF4A3 was overexpressed in common malignancies at the transcription levels. High incidences of the breast, lung, and urinary cancers were closely related to the prognostic index for survival. The most prevalent mutation in EIF4A3 was E59K/Q. The tumor necrosis factor-α (TNF-α)/nuclear factor-κB (NF-κB) signaling pathway was affected by these mutations. Co-expression networks showed that EIF4A3 regulates apoptosis and cell cycle via several cancer-related signal pathways, and promotes tumor cell migration, invasion and drug resistance. Conclusion: Our results suggest the potential role for EIF4A3 to serve as a diagnostic marker or therapeutic target for certain types of cancers.

Eukaryotic translational initiation factor 4A belong to family of helicases, involved in multifunctional activities during stress and non-stress conditions. The eIF4A gene was isolated and cloned from semi-arid cereal crop of Pennisetum glaucum. In present study, the PgeIF4A gene was expressed under the regulation of stress inducible Arabidopsis rd29A promoter in groundnut (cv JL-24) with bar as a selectable marker. The de-embryonated cotyledons were infected with Agrobacterium tumefaciens (LBA4404) carrying rd29A:PgeIF4A construct and generated high frequency of multiple shoots in phosphinothricin medium. Twenty- four T0 plants showed integration of both nos-bar and rd29A-PgeIF4A gene cassettes in genome with expected amplification products of 429 and 654 bps, respectively. Transgene copy number integration was observed in five T0 transgenic plants through Southern blot analysis. Predicted Mendelian ratio of segregation (3:1) was noted in transgenic plants at T1 generation. The T2 homozygous lines (L1-5, L8-2, and L16-2) expressing PgeIF4A gene were exhibited superior growth performance with respect to phenotypic parameters like shoot length, tap root length, and lateral root formation under simulated drought and salinity stresses compared to the wild type. In addition, the chlorophyll retention was found to be higher in these plants compared to the control plants. The quantitative real time-PCR results confirmed higher expression of PgeIF4A gene in L1-5, L8-3, and L16-2 plants imposed with drought/salt stress. Further, the salt stress tolerance was associated with increase in oxidative stress markers, such as superoxide dismutase accumulation, reactive oxygen species scavenging, and membrane stability in transgenic plants. Taken together our results confirmed that the PgeIF4A gene expressing transgenic groundnut plants exhibited better adaptation to stress conditions.

As an adult tumor with the most invasion and the highest mortality rate, the inherent heterogeneity of glioblastoma (GBM) is the main factor that causes treatment failure. Therefore, it is important to have a deeper understanding of the pathology of GBM. Some studies have shown that Eukaryotic Initiation Factor 4A-3 (EIF4A3) can promote the growth of many people's tumors, and the role of specific molecules in GBM remains unclear.

Cellular responses to environmental stress are frequently mediated by RNA-binding proteins (RBPs). Here, we examined global RBP dynamics in Saccharomyces cerevisiae in response to glucose starvation and heat shock. Each stress induced rapid remodeling of the RNA-protein interactome without corresponding changes in RBP abundance. Consistent with general translation shutdown, ribosomal proteins contacting the mRNA showed decreased RNA association. Among translation components, RNA association was most reduced for initiation factors involved in 40S scanning (eukaryotic initiation factor 4A [eIF4A], eIF4B, and Ded1), indicating a common mechanism of translational repression. In unstressed cells, eIF4A, eIF4B, and Ded1 primarily targeted the 5' ends of mRNAs. Following glucose withdrawal, 5' binding was abolished within 30 s, explaining the rapid translation shutdown, but mRNAs remained stable. Heat shock induced progressive loss of 5' RNA binding by initiation factors over ∼16 min and provoked mRNA degradation, particularly for translation-related factors, mediated by Xrn1. Taken together, these results reveal mechanisms underlying translational control of gene expression during stress.

Immunotoxins are being investigated as anti-cancer therapies and consist of a cytotoxic enzyme fused to a cancer targeting antibody. All currently used toxins function via the inhibition of protein synthesis, making them highly potent in both healthy and transformed cells. This non-specific cell killing mechanism causes dose-limiting side effects that can severely limit the potential of immunotoxin therapy. In this study, the recently characterised bacterial toxin Burkholderia lethal factor 1 (BLF1) is investigated as a possible alternative payload for targeted toxin therapy in the treatment of neuroblastoma. BLF1 inhibits translation initiation by inactivation of eukaryotic initiation translation factor 4A (eIF4A), a putative anti-cancer target that has been shown to regulate a number of oncogenic proteins at the translational level. We show that cellular delivery of BLF1 selectively induces apoptosis in neuroblastoma cells that display MYCN amplification but has little effect on non-transformed cells. Future immunotoxins based on this enzyme may therefore have higher specificity towards MYCN-amplified cancer cells than more conventional ribosome-inactivating proteins, leading to an increased therapeutic window and decreased side effects.

Cells employ several adaptive mechanisms under conditions of accelerated cell division, such as the unfolded protein response (UPR). The UPR is composed of a tripartite signaling system that involves ATF6, PERK, and IRE1, which maintain protein homeostasis (proteostasis). However, deregulation of protein translation initiation could be associated with breast cancer (BC) chemoresistance. Specifically, eukaryotic initiation factor-4A (eIF4A) is involved in the unfolding of the secondary structures of several mRNAs at the 5' untranslated region (5'-UTR), as well as in the regulation of targets involved in chemoresistance. Importantly, the tumor suppressor gene PDCD4 could modulate this process. This regulation might be disrupted in chemoresistant triple negative-BC (TNBC) cells. Therefore, we characterized the effect of doxorubicin (Dox), a commonly used anthracycline medication, on human breast carcinoma MDA-MB-231 cells. Here, we generated and characterized models of Dox chemoresistance, and chemoresistant cells exhibited lower Dox internalization levels followed by alteration of the IRE1 and PERK arms of the UPR and triggering of the antioxidant Nrf2 axis. Critically, chemoresistant cells exhibited PDCD4 downregulation, which coincided with a reduction in eIF4A interaction, suggesting a sophisticated regulation of protein translation. Likewise, Dox-induced chemoresistance was associated with alterations in cellular migration and invasion, which are key cancer hallmarks, coupled with changes in focal adhesion kinase (FAK) activation and secretion of matrix metalloproteinase-9 (MMP-9). Moreover, eIF4A knockdown via siRNA and its overexpression in chemoresistant cells suggested that eIF4A regulates FAK. Pro-atherogenic low-density lipoproteins (LDL) promoted cellular invasion in parental and chemoresistant cells in an MMP-9-dependent manner. Moreover, Dox only inhibited parental cell invasion. Significantly, chemoresistance was modulated by cryptotanshinone (Cry), a natural terpene purified from the roots of Salvia brandegeei. Cry and Dox co-exposure induced chemosensitization, connected with the Cry effect on eIF4A interaction. We further demonstrated the Cry binding capability on eIF4A and in silico assays suggest Cry inhibition on the RNA-processing domain. Therefore, strategic disruption of protein translation initiation is a druggable pathway by natural compounds during chemoresistance in TNBC. However, plasmatic LDL levels should be closely monitored throughout treatment.

Sulfonylurea (SU) herbicides are effective because they inhibit acetolactate synthase (ALS), a key enzyme in branched-chain amino acid synthesis required for plant growth. A soybean line known as W4-4 was developed through rounds of seed mutagenesis and was demonstrated to have a high degree of ALS-based resistance to both post-emergence and pre-emergence applications of a variety of SU herbicides. This report describes the molecular and phenotypic characterization of the Als1 and Als2 mutations that confer herbicide resistance to SUs and other ALS inhibitors.

Domestication of silkworm has led to alterations in various gene expression patterns. For instance, many protease inhibitors were significantly downregulated in the domestic silkworm cocoon compared to its wild progenitor. Considering that SPI51 is the most abundant protease inhibitor in silkworm cocoons, herein, we compared the gene structures and sequences of SPI51 from B. mori (BmoSPI51) and B. mandarina (BmaSPI51). Comparing to the "RGGFR" active site in BmaSPI51, that of BmoPI51 is "KGSFP" and the C-terminal "YNTCECSCP" tail sequence is lost in the latter. To investigate the effect elicited by the active site and tail sequences on the function of SPI51, we expressed two mutated forms of BmoSPI51, namely, BmoSPI51 + tail and BmoSPI51M. BmoSPI51, BmoSPI51 + tail and BmoSPI51M were compared and found to have similar levels of inhibitory activity against trypsin. However, the BmoSPI51 + tail and BmoSPI51M proteins exhibited significantly stronger capacities to inhibit fungi growth, compared to BmoSPI51. We concluded that the specific amino acid sequence of the active site, as well as its the disulfide bond formed by C-terminal sequence in the BmaSPI51, represent the key factors responsible for its higher antifungal activity. This study provided new insights into the antifungal mechanisms elicited by protease inhibitors in the cocoons of silkworms.

30K proteins are a major group of nutrient storage proteins in the silkworm hemolymph. Previous studies have shown that 30K proteins are involved in the anti-fungal immunity; however, the molecular mechanism involved in this immunity remains unclear.

This study is aimed at exploring the regulatory mechanism of 73HOXC-AS1 overexpression plasmid-activated Wntβ-catenin classic signaling pathway and eukaryotic initiation factor 4A (eIF4AIII) expression increased by lentivirus-eIF4AIII-RNAi (44682-1) (LV-eIF4AIII-RNAi (44682-1)).

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.

Although hepatocellular cancers (HCC) frequently arise in the setting of fibrosis and a hepatic regenerative response requiring new cell growth, therapeutic strategies for these cancers have not targeted protein synthesis. Silvestrol, a rocaglate isolated from Aglaiafoveolata, can inhibit protein synthesis by modulating the initiation of translation through the eukaryotic initiation factor 4A. In this study, we evaluated the therapeutic efficacy of silvestrol for HCC.

Leucine can affect intestinal protein expressions, and improve mucosal immune function. However, little study has been conducted to determine the change of protein component by leucine treatment in intestine epithelial cells. The present study was to cover the key proteins and cell pathways that could be regulated by leucine treatment in porcine intestinal epithelial cell line (IPEC-J2) cells with the approach of proteome analysis. A total number of 3,211 proteins were identified in our approach by searching the database of Uniprot sus scrofa. Among identified proteins, there were 101 proteins expressed differently between control group and leucine group. Compared with the control group, there were 50 up-regulated proteins and 51 down-regulated proteins in leucine group. In these proteins, leucine treatment decreased the expression of some proteins including pyruvate kinase, glyceraldehyde-3-phosphate dehydrogenase, E3 ubiquitin ligase, cathepsin D, caspase 3 and caspase 6, and increased the levels of some proteins, such as some eukaryotic translation initiation factors, ubiquitin carboxyl-terminal hydrolase, DNA-related RNA polymerase II, urokinase plasminogen activator, cyclin-dependent kinase inhibitor 2b, MutL homolog 1, 5-methylcytosine binding domain 4, polymerase δ, α-tubulin, syntaxin 18, Ras homolog D, actin related protein 2/3 complex and cofilin. Via the analysis of Gene Ontology and pathways, these proteins in IPEC-J2 cells were related with some physiological functions, such as protein metabolism, glycolysis, cell proliferation, apoptosis and phagocytosis. Thus, these results suggest that leucine affects gut barrier function possibly via regulating cell proliferation and apoptosis, metabolism and phagocytosis.

S-phase kinase-associated protein 2 (SKP2) is an oncogene and cell cycle regulator that specifically recognizes phosphorylated cell cycle regulator proteins and mediates their ubiquitination. Programmed cell death protein 4 (PDCD4) is a tumor suppressor gene that plays a role in cell apoptosis and DNA-damage response via interacting with eukaryotic initiation factor-4A (eIF4A) and P53. Previous research showed SKP2 may interact with PDCD4, however the relationship between SKP2 and PDCD4 is unclear.