LeishIF4E1 Deletion Affects the Promastigote Proteome, Morphology, and Infectivity.

Leishmania parasites cycle between sand-fly vectors and mammalian hosts, adapting to changing environmental conditions by driving a stage-specific program of gene expression, which is tightly regulated by translation processes. Leishmania encodes six eIF4E orthologs (LeishIF4Es) and five eIF4G candidates, forming different cap-binding complexes with potentially varying functions. Most LeishIF4E paralogs display temperature sensitivity in their cap-binding activity, except for LeishIF4E1, which maintains its cap-binding activity under all conditions. We used the CRISPR-Cas9 system to successfully generate a null mutant of LeishIF4E1 and examine how its elimination affected parasite physiology. Although the LeishIF4E1-/- null mutant was viable, its growth was impaired, in line with a reduction in global translation. As a result of the mutation, the null LeishIF4E1-/- mutant had a defective morphology, as the cells were round and unable to grow a normal flagellum. This was further emphasized when the LeishIF4E1-/- cells failed to develop the promastigote morphology once they shifted from conditions that generate axenic amastigotes (33°C, pH 5.5) back to neutral pH and 25°C, and they maintained their short flagellum and circular structure. Finally, the LeishIF4E1-/- null mutant displayed difficulty in infecting cultured macrophages. The morphological changes and reduced infectivity of the mutant may be related to differences in the proteomic profile of LeishIF4E1-/- cells from that of controls. All defects monitored in the LeishIF4E1-/- null mutant were reversed in the add-back strain, in which expression of LeishIF4E1 was reconstituted, establishing a strong link between the cellular defects and the absence of LeishIF4E1 expression.IMPORTANCELeishmania parasites are the causative agents of a broad spectrum of diseases. The parasites migrate between sand-fly vectors and mammalian hosts, adapting to changing environments by driving a regulated program of gene expression, with translation regulation playing a key role. The leishmanias encode six different paralogs of eIF4E, the cap-binding translation initiation factor. Since these vary in function, expression profile, and assemblage, it is assumed that each is assigned a specific role throughout the life cycle. Using the CRISPR-Cas9 system for Leishmania, we generated a null mutant of LeishIF4E1, eliminating both alleles. Although the mutant cells were viable, their morphology was altered and their ability to synthesize the flagellum was impaired. Elimination of LeishIF4E1 affected their protein expression profile and decreased their ability to infect cultured macrophages. Restoring LeishIF4E1 expression restored the affected features. This study highlights the importance of LeishIF4E1 in diverse cellular events during the life cycle of Leishmania.

4EGI-1 represses cap-dependent translation and regulates genome-wide translation in malignant pleural mesothelioma.

Deregulation of cap-dependent translation has been implicated in the malignant transformation of numerous human tissues. 4EGI-1, a novel small-molecule inhibitor of cap-dependent translation, disrupts formation of the eukaryotic initiation factor 4F (eIF4F) complex. The effects of 4EGI-1-mediated inhibition of translation initiation in malignant pleural mesothelioma (MPM) were examined. 4EGI-1 preferentially inhibited cell viability and induced apoptosis in MPM cells compared to normal mesothelial (LP9) cells. This effect was associated with hypophosphorylation of 4E-binding protein 1 (4E-BP1) and decreased protein levels of the cancer-related genes, c-myc and osteopontin. 4EGI-1 showed enhanced cytotoxicity in combination with pemetrexed or gemcitabine. Translatome-wide polysome microarray analysis revealed a large cohort of genes that were translationally regulated upon treatment with 4EGI-1. The 4EGI-1-regulated translatome was negatively correlated to a previously published translatome regulated by eIF4E overexpression in human mammary epithelial cells, which is in agreement with the notion that 4EGI-1 inhibits the eIF4F complex. These data indicate that inhibition of the eIF4F complex by 4EGI-1 or similar translation inhibitors could be a strategy for treating mesothelioma. Genome wide translational profiling identified a large cohort of promising target genes that should be further evaluated for their potential significance in the treatment of MPM.

Depletion of elongation initiation factor 4E binding proteins by CRISPR/Cas9 enhances the antiviral response in porcine cells.

Type I interferons (IFNs) are key mediators of the innate antiviral response in mammalian cells. Elongation initiation factor 4E binding proteins (4E-BPs) are translational controllers of interferon regulatory factor 7 (IRF-7), the "master regulator" of IFN transcription. Previous studies have suggested that mouse cells depleted of 4E-BPs are more sensitive to IFNß treatment and had lower viral loads as compared to wild type (WT) cells. However, such approach has not been tested as an antiviral strategy in livestock species. In this study, we tested the antiviral activity of porcine cells depleted of 4E-BP1 by a Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated protein-9 nuclease (Cas9) genome engineering system. We found that 4E-BP1 knockout (KO) porcine cells had increased expression of IFNα and ß, IFN stimulated genes, and significant reduction in vesicular stomatitis virus titer as compare to WT cells. No phenotypical changes associated with CRISPR/Cas9 manipulation were observed in 4E-BP1 KO cells. This work highlights the use of the CRISPR/Cas9 system to enhance the antiviral response in porcine cells.

Upstream open reading frames regulate translation of the long isoform of SLAMF1 mRNA that encodes costimulatory receptor CD150.

More than 40% of human genes contain upstream open reading frames (uORF) in their 5'-untranslated regions (5'-UTRs) and at the same time express at least one truncated mRNA isoform containing no uORF. We studied translational regulation by four uORFs found in the 5'-UTR of full-length mRNA for SLAMF1, the gene encoding CD150 membrane protein. CD150 is a member of the CD2 superfamily, a costimulatory lymphocyte receptor, a receptor for measles virus, and a microbial sensor on macrophages. The SLAMF1 gene produces at least two mRNA isoforms that differ in their 5'-UTRs. In the long isoform of the SLAMF1 mRNA that harbors four uORFs in the 5'-UTR, the stop codon of uORF4 overlaps with the AUG codon of the main ORF forming a potential termination-reinitiation site UGAUG, while uORF2 and uORF3 start codons flank a sequence identical to Motif 1 from the TURBS regulatory element. TURBS was shown to be required for a coupled termination-reinitiation event during translation of polycistronic RNAs of some viruses. In a model cell system, reporter mRNA based on the 5'-UTR of SLAMF1 short isoform, which lacks any uORF, is translated 5-6 times more efficiently than the mRNA with 5'-UTR from the long isoform. Nucleotide substitutions disrupting start codons in either uORF2-4 result in significant increase in translation efficiency, while substitution of two nucleotides in TURBS Motif 1 leads to a 2-fold decrease in activity. These data suggest that TURBS-like elements can serve for translation control of certain cellular mRNAs containing uORFs.

Suppression of AKT phosphorylation restores rapamycin-based synthetic lethality in SMAD4-defective pancreatic cancer cells.

mTOR has been implicated in survival signals for many human cancers. Rapamycin and TGF-ß synergistically induce G1 cell-cycle arrest in several cell lines with intact TGF-ß signaling pathway, which protects cells from the apoptotic effects of rapamycin during S-phase of the cell cycle. Thus, rapamycin is cytostatic in the presence of serum/TGF-ß and cytotoxic in the absence of serum. However, if TGF-ß signaling is defective, rapamycin induced apoptosis in both the presence and absence of serum/TGF-ß in colon and breast cancer cell lines. Because genetic dysregulation of TGF-ß signaling is commonly observed in pancreatic cancers-with defects in the Smad4 gene being most prevalent, we hypothesized that pancreatic cancers would display a synthetic lethality to rapamycin in the presence of serum/TGF-ß. We report here that Smad4-deficient pancreatic cancer cells are killed by rapamycin in the absence of serum; however, in the presence of serum, we did not observe the predicted synthetic lethality with rapamycin. Rapamycin also induced elevated phosphorylation of the survival kinase Akt at Ser473. Suppression of rapamycin-induced Akt phosphorylation restored rapamycin sensitivity in Smad4-null, but not Smad4 wild-type pancreatic cancer cells. This study shows that the synthetic lethality to rapamycin in pancreatic cancers with defective TGF-ß signaling is masked by rapamycin-induced increases in Akt phosphorylation. The implication is that a combination of approaches that suppress both Akt phosphorylation and mTOR could be effective in targeting pancreatic cancers with defective TGF-ß signaling.

eIF4E knockdown decreases breast cancer cell growth without activating Akt signaling.

Activation of translation initiation is essential for the malignant phenotype and is emerging as a potential therapeutic target. Translation is regulated by the expression of translation initiation factor 4E (eIF4E) as well as the interaction of eIF4E with eIF4E-binding proteins (e.g., 4E-BP1). Rapamycin inhibits translation initiation by decreasing the phosphorylation of 4E-BP1, increasing eIF4E/4E-BP1 interaction. However, rapamycin also inhibits S6K phosphorylation, leading to feedback loop activation of Akt. We hypothesized that targeting eIF4E directly would inhibit breast cancer cell growth without activating Akt. We showed that eIF4E is ubiquitously expressed in breast cancer cell lines. eIF4E knockdown by small interfering RNA inhibited growth in different breast cancer cell subtypes including triple-negative (estrogen receptor/progesterone receptor/HER-2-negative) cancer cells. eIF4E knockdown inhibited the growth of cells with varying total and phosphorylated 4E-BP1 levels and inhibited rapamycin-insensitive as well as rapamycin-sensitive cell lines. eIF4E knockdown led to a decrease in expression of cyclin D1, Bcl-2, and Bcl-xL. eIF4E knockdown did not lead to Akt phosphorylation but did decrease 4E-BP1 expression. We conclude that eIF4E is a promising target for breast cancer therapy. eIF4E-targeted therapy may be efficacious in a variety of breast cancer subtypes including triple-negative tumors for which currently there are no targeted therapies. Unlike rapamycin and its analogues, eIF4E knockdown is not associated with Akt activation.

Protein synthesis and aging: eIF4E and the soma vs. germline distinction.

Classic studies in diverse organisms, including humans, have demonstrated that aging is accompanied by marked alterations in both general and specific protein synthesis. These early observations established a link between the aging process and the regulation of protein synthesis. However, two important questions remained. First, what are the molecular mechanisms underlying the changes in protein synthesis during aging? Second, are these changes simply a consequence of aging or do they actually have a causative role in senescent decline? We have recently shown that elimination of a specific isoform of the eukaryotic mRNA translation initiation factor 4E (eIF4E) that functions in somatic cells, reduces protein synthesis and extends lifespan in the nematode Caenorhabditis elegans. Depletion of eIF4E in the soma extends lifespan via a mechanism independent of the insulin/IGF pathway that modulates aging in diverse species. Our findings suggest that regulation of protein synthesis is an important determinant of longevity and provide a framework for elucidating the mechanisms by which the rate of protein synthesis influences the process of aging.

eIF4E function in somatic cells modulates ageing in Caenorhabditis elegans.

Regulation of protein synthesis is critical for cell growth and maintenance. Ageing in many organisms, including humans, is accompanied by marked alterations in both general and specific protein synthesis. Whether these alterations are simply a corollary of the ageing process or have a causative role in senescent decline remains unclear. An array of protein factors facilitates the tight control of messenger RNA translation initiation. The eukaryotic initiation factor 4E (eIF4E), which binds the 7-monomethyl guanosine cap at the 5' end of all nuclear mRNAs, is a principal regulator of protein synthesis. Here we show that loss of a specific eIF4E isoform (IFE-2) that functions in somatic tissues reduces global protein synthesis, protects from oxidative stress and extends lifespan in Caenorhabditis elegans. Lifespan extension is independent of the forkhead transcription factor DAF-16, which mediates the effects of the insulin-like signalling pathway on ageing. Furthermore, IFE-2 deficiency further extends the lifespan of long-lived age and daf nematode mutants. Similarly, lack of IFE-2 enhances the long-lived phenotype of clk and dietary-restricted eat mutant animals. Knockdown of target of rapamycin (TOR), a phosphatidylinositol kinase-related kinase that controls protein synthesis in response to nutrient cues, further increases the longevity of ife-2 mutants. Thus, signalling via eIF4E in the soma is a newly discovered pathway influencing ageing in C. elegans.