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Nonalcoholic fatty liver disease (NAFLD) is characterized by the accumulation of lipids in the liver. Given the high prevalence of NAFLD, its evolution to nonalcoholic steatohepatitis (NASH) and hepatocellular carcinoma (HCC) is of global concern. Therapies for managing NASH-driven HCC can benefit from targeting factors that play a continuous role in NAFLD evolution to HCC. Recent work has shown that postprandial liver translation exacerbates lipid accumulation through the activity of a translation factor, eukaryotic initiation factor 6 (eIF6). Here, we test the effect of eIF6 inhibition on the progression of HCC. Mice heterozygous for eIF6 express half the level of eIF6 compared to wt mice and are resistant to the formation of HCC nodules upon exposure to a high fat/high sugar diet combined with liver damage. Histology showed that nodules in eIF6 het mice were smaller with reduced proliferation compared to wt nodules. By using an in vitro model of human HCC, we confirm that eIF6 depletion reduces the growth of HCC spheroids. We also tested three pharmacological inhibitors of eIF6 activity-eIFsixty-1, eIFsixty-4, and eIFsixty-6-and all three reduced eIF6 binding to 60S ribosomes and limited the growth of HCC spheroids. Thus, inhibition of eIF6 activity is feasible and limits HCC formation.

A postprandial increase of translation mediated by eukaryotic Initiation Factor 6 (eIF6) occurs in the liver. Its contribution to steatosis and disease is unknown. In this study we address whether eIF6-driven translation contributes to disease progression. eIF6 levels increase throughout the progression from Non-Alcoholic Fatty Liver Disease (NAFLD) to hepatocellular carcinoma. Reduction of eIF6 levels protects the liver from disease progression. eIF6 depletion blunts lipid accumulation, increases fatty acid oxidation (FAO) and reduces oncogenic transformation in vitro. In addition, eIF6 depletion delays the progression from NAFLD to hepatocellular carcinoma, in vivo. Mechanistically, eIF6 depletion reduces the translation of transcription factor C/EBPβ, leading to a drop in biomarkers associated with NAFLD progression to hepatocellular carcinoma and preserves mitochondrial respiration due to the maintenance of an alternative mTORC1-eIF4F translational branch that increases the expression of transcription factor YY1. We provide proof-of-concept that in vitro pharmacological inhibition of eIF6 activity recapitulates the protective effects of eIF6 depletion. We hypothesize the existence of a targetable, evolutionarily conserved translation circuit optimized for lipid accumulation and tumor progression., (© 2021. The Author(s).)

Metastasis is the leading cause of mortality in patients with solid tumors. In this regard, we previously reported that Pseudopodium-Enriched Atypical Kinase One (PEAK1) is necessary for non-canonical Transforming Growth Factor β (TGFβ) signaling and TGFβ/fibronectin-induced metastasis. Here, we demonstrate that inhibition of DHPS-dependent eIF5A1/2 hypusination blocks PEAK1 and E-Cadherin expression, breast cancer cell viability and TGFβ/fibronectin-induced PEAK1-dependent breast cancer metastasis. Interestingly, TGFβ stimulation of high-grade metastatic breast cancer cells increases and sustains eIF5A1/2 hypusination. We used a suite of bioinformatics platforms to search biochemical/functional interactions and clinical databases for additional control points in eIF5A1/2 and PEAK1-Epithelial to Mesenchymal Transition (EPE) pathways. This effort revealed that interacting EPE genes were enriched for TP53 transcriptional targets and were commonly co-amplified in breast cancer patients harboring inactivating TP53 mutations. Taken together, these results suggest that combinatorial therapies targeting DHPS and protein activities elevated in TP53-mutant breast cancers may reduce systemic tumor burden and improve patient outcomes., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Background: Pancreatic cancer (PC) has a very poor prognosis and comparatively short survival. Eukaryotic translation initiation factor 5A (EIF5A) promotes cancer metastasis. Here, we exploited the biological role of EIF5A in PC chemoresistance., Methods: Expression of EIF5A was analysed in PC cells and tissues by real-time PCR, Western blotting, immunohistochemistry and immunofluorescent. EIF5A expression was specifically suppressed by transfection, and subsequently the alterations of growth behaviour and resistance to anticancer treatment were tested in an orthotopic tumour model., Results: The results showed EIF5A was increased in human PC tissues and PC cells. We found EIF5A knockdown reduced the PC proliferation ability in vivo and in vitro. In addition, sonic hedgehog (sHH) signalling pathway may be a downstream of EIF5A in PC cells. Inhibition of EIF5A and sHH signalling pathway could suppress PC cells proliferation and tumour growth. Importantly, EIF5A played an important role in gemcitabine sensitivity for PC., Conclusion: Taken together, our results revealed that EIF5A regulated the proliferation of PC through the sHH signalling pathway and decreased the Gem sensitivity in PC, which provided a novel therapeutic strategy for PC patients., (© 2019 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.)

We have developed a transgenic mouse model of Type 1 Diabetes (T1D) in which human GAD65 is expressed in pancreatic β-cells, and human MHC-II is expressed on antigen presenting cells. Induced GAD65 antigen presentation activates T-cells, which initiates the downstream events leading to diabetes. In our humanized mice, we have shown downregulation of eukaryotic translation initiation factor 5 A (elF5A), expressed only in actively dividing mammalian cells. In-vivo inhibition of elF5A hypusination by deoxyhypusine synthase (DHS) inhibitor "GC7" was studied; DHS inhibitor alters the pathophysiology in our mouse model by catalyzing the crucial hypusination and the rate-limiting step of elF5A activation. In our mouse model, we have shown that inhibition of eIF5A resets the pro-inflammatory bias in the pancreatic microenvironment. There was: (a) reduction of Th1/Th17 response, (b) an increase in Treg numbers, (c) debase in IL17 and IL21 cytokines levels in serum, (d) lowering of anti-GAD65 antibodies, and (e) ablation of the ER stress that improved functionality of the β-cells, but minimal effect on the cytotoxic CD8 T-cell (CTL) mediated response. Conclusively, immune modulation, in the case of T1D, may help to manipulate inflammatory responses, decreasing disease severity, and may help manage T1D in early stages of disease. Our study also demonstrates that without manipulating the CTLs mediated response extensively, it is difficult to treat T1D.

Epithelial ovarian cancer (EOC) is one of the most common gynecological cancers and has the highest mortality rate thereof. We found abundant eukaryotic translation initiation factor 5A1 (EIF5A1) in 54 EOC tissues, and high EIF5A1 levels predicted poor survival. EIF5A1 ectopic expression enhanced EOC cell proliferative, migration, and invasive capabilities, while EIF5A1 knockdown suppressed them. Most importantly, GC7 (N1-guanyl-1,7-diaminoheptane, an EIF5A1 hypusination inhibitor) could reverse the effect of EIF5A1 upregulation on EOC cell proliferation, migration, and invasion and mutant type EIF5A1 K50A plasmid [bearing a single point mutation (K50 → A50) that prevents hypusination] had no effects on these malignant behaviors. Our findings imply that EIF5A1 is a vital regulator of EOC proliferation and progression and is a potential prognostic marker and therapeutic target in EOC., (Copyright © 2018 Elsevier Masson SAS. All rights reserved.)

Eukaryotic initiation factor 5A2 (eIF5A2), a newly identified oncogene, promotes cell survival, proliferation and motility in tumorigenesis. Drug resistance and dose-related adverse side-effects greatly reduce the efficiency and safety of cisplatin-based chemotherapy in advanced or recurrent oral squamous cell carcinoma (OSCC) patients. The present study investigated the effect of eIF5A2 combined with N1-guanyl-1,7-diaminoheptane (GC7, a novel eIF5A2 inhibitor) or siRNA. We found that low concentrations of GC7 (≤5 µM) had little effect on OSCC cell viability, but significantly enhanced cisplatin cytotoxicity. Compared with cisplatin, GC7/cisplatin had little effect on cisplatin-promoted mesenchymal-epithelial transition in mesenchymal phenotype Tca8113 and HN30 cells, or on cisplatin-induced epithelial-mesenchymal transition (EMT) in epithelial phenotype Cal27 and HN4 cells. Further research revealed that the upregulation of p-STAT3 and c-Myc which was induced by the single treatment with either cisplatin or GC7 was significantly reversed by the GC7/cisplatin combination in mesenchymal phenotype Tca8113 and HN30 cells. In in vivo treatment, we revealed that the GC7/cisplatin combination presented significant tumor volume reduction without distinct body weight loss. In conclusion, our data indicated that eIF5A2 is a potent therapeutic target in OSCC treatment. Our results revealed a novel mechanism by which GC7/cisplatin combination therapy may offer an efficient and safe therapeutic alternative to advanced or recurrent OSCC patients.

The eukaryotic initiation factor 5A (eIF5A), which contributes to several crucial processes during protein translation, is the only protein that requires activation by a unique post-translational hypusine modification. eIF5A hypusination controls cell proliferation and has been linked to cancer. eIF5A hypusination requires the enzymes deoxyhypusine synthase (DHPS) and deoxyhypusine hydroxylase and uniquely depends on the polyamine (PA) spermidine as the sole substrate. Ornithine decarboxylase (ODC) is the rate-limiting enzyme in PA biosynthesis. Both ODC and PAs control cell proliferation and are frequently dysregulated in cancer. Since only spermidine can activate eIF5A, we chose the hypusine-PA nexus as a rational target to identify new drug combinations with synergistic antiproliferative effects. We show that elevated mRNA levels of the two target enzymes DHPS and ODC correlate with poor prognosis in a large cohort of neuroblastoma (NB) tumors. The DHPS inhibitor GC7 ( N 1 -guanyl-1,7-diaminoheptane) and the ODC inhibitor α-difluoromethylornithine (DFMO) are target-specific and in combination induced synergistic effects in NB at concentrations that were not individually cytotoxic. Strikingly, while each drug alone at higher concentrations is known to induce p21/Rb- or p27/Rb-mediated G 1 cell cycle arrest, we found that the drug combination induced caspase 3/7/9, but not caspase 8-mediated apoptosis, in NB cells. Hypusinated eIF5A levels and intracellular spermidine levels correlated directly with drug treatments, signifying specific drug targeting effects. This two-pronged GC7/DFMO combination approach specifically inhibits both spermidine biosynthesis and post-translational, spermidine-dependent hypusine-eIF5A activation, offering an exciting clue for improved NB drug therapy., (© 2018 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

N1-guanyl-1,7-diaminoheptane (GC7), a deoxyhypusine synthase inhibitor, has been shown to exert antiproliferation effects in many solid tumors by regulating eukaryotic translation initiation factor 5a2 (eif5a-2). However, little is known about the role of GC7 and eif5a-2 in drug resistance in acute lymphoblastic leukemia (ALL). In the present study, we investigated the effect of GC7 on drug-resistant ALL and its potential mechanism. We found that using the CCK-8 assay that combined treatment with GC7 and vincristine (VCR) significantly inhibited the cell viability of two ALL cell lines. Using EdU incorporation assays and flow cytometry, we also showed that GC7 could markedly enhance the VCR sensitivity of ALL cells by suppressing cell proliferation and promoting apoptosis. Furthermore, we showed that GC7 could downregulate eif5a-2 and myeloid cell leukemia-1 (Mcl-1) expression. Knockdown of eif5a-2 inhibited the expression of Mcl-1 and significantly enhanced the VCR sensitivity. Moreover, eif5a-2 knockdown decreased the regulatory role of GC7 in increasing VCR sensitivity. Thus, our findings indicate that combined treatment with GC7 could enhance VCR sensitivity of ALL cells by regulating the eif5a-2/Mcl-1 axis. Together, our results highlight the potential clinical application of GC7 in VCR-based chemotherapy for the treatment of ALL.

Spinal cord injury (SCI) is frequently accompanied by a degree of spontaneous functional recovery. The underlying mechanisms through which such recovery is generated remain elusive. In this study, we observed a significant spontaneous motor function recovery 14 to 28 days after spinal cord transection (SCT) in rats. Using a comparative proteomics approach, caudal to the injury, we detected difference in 20 proteins. Two of these proteins, are eukaryotic translation initiation factor 5A1 (eIF5A1) that is involved in cell survival and proliferation, and Rho GDP dissociation inhibitor alpha (RhoGDIα), a member of Rho GDI family that is involved in cytoskeletal reorganization. After confirming the changes in expression levels of these two proteins following SCT, we showed that in vivo eIF5A1 up-regulation and down-regulation significantly increased and decreased, respectively, motor function recovery. In vitro, eIF5A1 overexpression in primary neurons increased cell survival and elongated neurite length while eIF5A1 knockdown reversed these results. We found that RhoGDIα up-regulation and down-regulation rescues the effect of eIF5A1 down-regulation and up-regulation both in vivo and in vitro. Therefore, we have identified eIF5A1/RhoGDIα pathway as a new therapeutic target for treatment of spinal cord injured patients.

Chronic exposure to trichothecenes is known to disturb insulin-like growth factor 1 and signaling of insulin and leptin hormones and causes considerable growth retardation in animals. However, limited information was available on mechanisms underlying trichothecene-induced growth retardation. In this study, we employed an integrated transcriptomics, proteomics, and RNA interference (RNAi) approach to study the molecular mechanisms underlying trichothecene cytotoxicity in rat pituitary adenoma GH3 cells. Our results showed that trichothecenes suppressed the synthesis of growth hormone 1 (Gh1) and inhibited the eukaryotic transcription and translation initiation by suppressing aminoacyl-tRNA synthetases transcription, inducing eukaryotic translation initiation factor 2-alpha kinase 2 (EIF2AK2) and reducing eukaryotic translation initiation factor 5 a. The sulfhydryl oxidases , protein disulfide isomerase,and heat shock protein 90 (were greatly reduced, which resulted in adverse regulation of protein processing and folding. Differential genes and proteins associated with a decline in energy metabolism and cell cycle arrest were also found in our study. However, use of RNAi to interfere with hemopoietic cell kinase (Hck) and EIF2AK2 transcriptions or use of chemical inhibitors of MAPK, p38, Ras, and JNK partially reversed the reduction of Gh1 levels induced by trichothecenes. It indicated that the activation of MAPKs, Hck, and EIF2AK2 were important for trichothecene-induced growth hormone suppression. Considering the potential hazards of exposure to trichothecenes, our findings could help to improve our understanding regarding human and animal health implications., (© The Author 2015. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

Oxidative stress plays a critical role in ischemia/reperfusion-injury, atherosclerosis, and aging. It causes cell damage that leads to apoptosis via uncertain mechanisms. Because conditioned medium from cardiac myocytes subjected to hypoxia/reoxygenation induces extensive apoptosis of cardiac myocytes under normoxia, we hypothesized that a humoral factor released from the hypoxic/reoxygenated cardiac myocytes mediates apoptosis. We identified an apoptosis-inducing humoral factor in the hypoxia/reoxygenation-conditioned medium. Here, we found that eIF5A undergoes tyrosine sulfation in the trans-Golgi and is rapidly secreted from cardiac myocytes in response to hypoxia/reoxygenation; then, eIF5A induces apoptosis by acting as a pro-apoptotic ligand. The apoptosis of cardiac myocytes induced by hypoxia/reoxygenation or ultraviolet irradiation was suppressed by anti-eIF5A neutralizing monoclonal antibodies (mAbs) in vitro. Myocardial ischemia/reperfusion (but not ischemia alone) markedly increased the plasma levels of eIF5A, and treatment with anti-eIF5A neutralizing mAbs significantly reduced myocardial injury. These results identify an important, novel specific biomarker and a critical therapeutic target for oxidative stress-induced cell injury.

The eukaryotic protozoan parasite Trypanosoma brucei is the causative agent of human African trypanosomiasis. Polyamine biosynthesis is essential in T. brucei, and the polyamine spermidine is required for synthesis of a novel cofactor called trypanothione and for deoxyhypusine modification of eukaryotic translation initiation factor 5A (eIF5A). eIF5A promotes translation of proteins containing polyprolyl tracts in mammals and yeast. To evaluate the function of eIF5A in T. brucei, we used RNA interference (RNAi) to knock down eIF5A levels and found that it is essential for T. brucei growth. The RNAi-induced growth defect was complemented by expression of wild-type human eIF5A but not by a Lys-50 mutant that blocks modification by deoxyhypusine. Bioinformatics analysis showed that 15% of the T. brucei proteome contains 3 or more consecutive prolines and that actin-related proteins and cysteine proteases were highly enriched in the group. Steady-state protein levels of representative proteins containing 9 consecutive prolines that are involved in actin assembly (formin and CAP/Srv2p) were significantly reduced by knockdown of eIF5A. Several T. brucei polyprolyl proteins are involved in flagellar assembly. Knockdown of TbeIF5A led to abnormal cell morphologies and detached flagella, suggesting that eIF5A is important for translation of proteins needed for these processes. Potential specialized functions for eIF5A in T. brucei in translation of variable surface glycoproteins were also uncovered. Inhibitors of deoxyhypusination would be expected to cause a pleomorphic effect on multiple cell processes, suggesting that deoxyhypusine/hypusine biosynthesis could be a promising drug target in not just T. brucei but in other eukaryotic pathogens., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

Background: Epithelial-mesenchymal transition (EMT) has been believed to be related with chemotherapy resistance in non-small cell lung cancer (NSCLC). Recent studies have suggested eIF5A-2 may function as a proliferation-related oncogene in tumorigenic processes., Methods: We used cell viability assays, western blotting, immunofluorescence, transwell-matrigel invasion assay, wound-healing assay combined with GC7 (a novel eIF5A-2 inhibitor) treatment or siRNA interference to investigate the role of eIF5A-2 playing in NSCLC chemotherapy., Results: We found low concentrations of GC7 have little effect on NSCLC viability, but could enhance cisplatin cytotoxicity in NSCLC cells. GC7 also could reverse mesenchymal phenotype in NCI-H1299 and prevented A549 cells undergoing EMT after TGF-β1 inducement. eIF5A-2 knockdown resulted in EMT inhibition., Conclusion: Our data indicated GC7 enhances cisplatin cytotoxicity and prevents the EMT in NSCLC cells by inhibiting eIF5A-2.

The high rates of recurrence and low median survival in many B-cell cancers highlight a need for new targeted therapeutic modalities. In dividing cells, eukaryotic translation initiation factor 5A (eIF5A) is hypusinated and involved in regulation of protein synthesis and proliferation, whereas the non-hypusinated form of eIF5A is a potent inducer of cell death in malignant cells. Here, we demonstrate the potential of modulating eIF5A expression as a novel approach to treating B-cell cancers. SNS01-T is a nonviral polyethylenimine-based nanoparticle, designed to induce apoptosis selectively in B-cell cancers by small interfering RNA-mediated suppression of hypusinated eIF5A and plasmid-based overexpression of a non-hypusinable eIF5A mutant. In this study, we show that SNS01-T is preferentially taken up by malignant B cells, inhibits tumor growth in multiple animal models of B-cell cancers without damaging normal tissues, and synergizes with the current therapies bortezomib and lenalidomide to inhibit tumor progression. The results collectively demonstrate the potential of SNS01-T as a novel therapeutic for treatment of a diverse range of B-cell malignancies.

Therapeutic options for treatment of type 1 diabetes (T1D) are still missing. New avenues for immune modulation need to be developed. Here we attempted at altering the diabetes outcome of our humanized model of T1D by inhibiting translation-initiation factor eIF5A hypusination in vivo. Double-transgenic (DQ8-GAD65) mice were immunized with adenoviral vectors carrying GAD65 for diabetes induction. Animals were subsequently treated with deoxyhypusine synthase (DHS) inhibitor GC7 and monitored for diabetes development over time. On one hand, helper CD4(+) T cells were clearly affected by the downregulation of the eIF5A not just at the pancreas level but overall. On the other hand, the T regulatory cell component of CD4 responded with activation and proliferation significantly higher than in the non-GC7-treated controls. Female mice seemed to be more susceptible to these effects. All together, our results show for the first time that downregulation of eIF5A through inhibition of DHS altered the physiopathology and observed immune outcome of diabetes in an animal model that closely resembles human T1D. Although the development of diabetes could not be abrogated by DHS inhibition, the immunomodulatory capacity of this approach may supplement other interventions directed at increasing regulation of autoreactive T cells in T1D.

Problem: The role of eukaryotic initiation factor 5A (eIF5A) in feto-maternal immunotolerance is poorly understood., Methods of Study: The effects of N1-guanyl-1,7-diaminoheptane (GC7), an inhibitor of eIF5A, on the proportion and function of natural killer (NK) cell subsets were investigated using flow cytometry, immunofluorescence, CCK8 assay, TUNEL assay, DNA fragmentation analysis, mitochondrial membrane potential assay, and Western blotting., Results: Inhibition of eIF5A by GC7 increased embryo loss and reduced the percentage of NK cells in the uterus and spleen. GC7 treatment caused inhibition of NK cell proliferation in a time- and dose-dependent manner. GC7 also induced apoptosis of NK cells. GC7 treatment increased the protein levels of FasL, bax, p53, and cleaved caspase-3. Moreover, GC7 caused loss of mitochondrial membrane potential in NK cells., Conclusion: Inhibition of eIF5A results in aberrant NK cell function and increased embryo loss., (© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Drug resistance greatly reduces the efficacy of doxorubicin-based chemotherapy in bladder cancer treatment; however, the underlying mechanisms are poorly understood. We aimed to investigate whether N1-guanyl-1,7-diaminoheptane (GC7), which inhibits eukaryotic translation initiation factor 5A2 (eIF5A2) activation, exerts synergistic cytotoxicity with doxorubicin in bladder cancer, and whether eIF5A2 is involved in chemoresistance to doxorubicin-based bladder cancer treatment. BIU-87, J82, and UM-UC-3 bladder cancer cells were transfected with eIF5A2 siRNA or negative control siRNA before incubation with doxorubicin alone or doxorubicin plus GC7 for 48 h. Doxorubicin cytotoxicity was enhanced by GC7 in BIU-87, J82, and UM-UC-3 cells. It significantly inhibited activity of eIF5A2, suppressed doxorubicin-induced epithelial-mesenchymal transition in BIU-87 cells, and promoted mesenchymal-epithelial transition in J82 and UM-UC-3 cells. Knockdown of eIF5A2 sensitized bladder cancer cells to doxorubicin, prevented doxorubicin-induced EMT in BIU-87 cells, and encouraged mesenchymal-epithelial transition in J82 and UM-UC-3 cells. Combination therapy with GC7 may enhance the therapeutic efficacy of doxorubicin in bladder cancer by inhibiting eIF5A2 activation and preventing epithelial-mesenchymal transition., (© 2013 The Authors. Cancer Science published by Wiley Publishing Asia Pty Ltd on behalf of Japanese Cancer Association.)

Cancer etiology is influenced by alterations in protein synthesis that are not fully understood. In this study, we took a novel approach to investigate the role of the eukaryotic translation initiation factor eIF5A in human cervical cancers, where it is widely overexpressed. eIF5A contains the distinctive amino acid hypusine, which is formed by a posttranslational modification event requiring deoxyhypusine hydroxylase (DOHH), an enzyme that can be inhibited by the drugs ciclopirox and deferiprone. We found that proliferation of cervical cancer cells can be blocked by DOHH inhibition with either of these pharmacologic agents, as well as by RNA interference-mediated silencing of eIF5A, DOHH, or another enzyme in the hypusine pathway. Proteomic and RNA analyses in HeLa cervical cancer cells identified two groups of proteins in addition to eIF5A that were coordinately affected by ciclopirox and deferiprone. Group 1 proteins (Hsp27, NM23, and DJ-1) were downregulated at the translational level, whereas group 2 proteins (TrpRS and PRDX2) were upregulated at the mRNA level. Further investigations confirmed that eIF5A and DOHH are required for Hsp27 expression in cervical cancer cells and for regulation of its key target IκB and hence NF-κB. Our results argue that mature eIF5A controls a translational network of cancer-driving genes, termed the eIF5A regulon, at the levels of mRNA abundance and translation. In coordinating cell proliferation, the eIF5A regulon can be modulated by drugs such as ciclopirox or deferiprone, which might be repositioned to control cancer cell growth.

Hepatocellular carcinoma (HCC) cells undergo the epithelial-mesenchymal transition (EMT) during chemotherapy, which reduces the efficacy of doxorubicin-based chemotherapy. We investigated N1-guanyl-1,7-diaminoheptane (GC7) which inhibits eukaryotic translation initiation factor 5A2 (eIF5A2) activation; eIF5A2 is associated with chemoresistance. GC7 enhanced doxorubicin cytotoxicity in epithelial HCC cells (Huh7, Hep3B and HepG2) but had little effect in mesenchymal HCC cells (SNU387, SNU449). GC7 suppressed the doxorubicin-induced EMT in epithelial HCC cells; knockdown of eIF5A2 inhibited the doxorubicin-induced EMT and enhanced doxorubicin cytotoxicity. GC7 combination therapy may enhance the therapeutic efficacy of doxorubicin in HCC by inhibiting eIF5A2 activation and preventing the EMT., (© 2013 Elsevier Inc. All rights reserved.)

The MAPKs are transducers of extracellular signals such as proinflammatory cytokines. In islet β-cells, cytokines acutely activate expression of the Nos2 gene encoding inducible nitric oxide synthase (iNOS), which ultimately impairs insulin release. Because iNOS production can also be regulated posttranscriptionally, we asked whether MAPKs participate in posttranscriptional regulatory events in β-cells and primary islets in response to cytokine signaling. We show that cytokines acutely reduce cellular oxygen consumption rate and impair aconitase activity. Inhibition of iNOS with l-NMMA or inhibition of Nos2 mRNA translation with GC7 [an inhibitor of eukaryotic translation initiation factor 5A (eIF5A) activity] reversed these defects, as did inhibition of p38 MAPK by PD169316. Although inhibition of p38 had no effect on the nuclear translocation of nuclear factor κB or the abundance of Nos2 transcripts during the immediate period after cytokine exposure, its inhibition or knockdown resulted in significant reduction in iNOS protein, a finding suggestive of a permissive role for p38 in Nos2 translation. Polyribosomal profiling experiments using INS-1 β-cells revealed that Nos2 mRNA remained associated with polyribosomes in the setting of p38 inhibition, in a manner similar to that seen with blockade of translational elongation by cycloheximide. Consistent with a role in translational elongation, p38 activity is required in part for the activation of the translational factor eIF5A by promoting its hypusination. Our results suggest a novel signaling pathway in β-cells in which p38 MAPK promotes translation elongation of Nos2 mRNA via regulation of eIF5A hypusination.

Background: Deoxyhypusine synthase (DHS) catalyzes the first step in hypusine biosynthesis of eukaryotic initiation factor 5A (eIF-5A) in Plasmodium falciparum. Target evaluation of parasitic DHS has recently been performed with CNI-1493, a novel selective pro-inflammatory cytokine inhibitor used in clinical phase II for the treatment of Crohn's disease. CNI-1493 prevented infected mice from experimental cerebral malaria by decreasing the levels in hypusinated eIF-5A and serum TNF, implicating a link between cytokine signaling and the hypusine pathway.Therefore we addressed the question whether either DHS itself or eIF-5A is required for the outcome of severe malaria. In a first set of experiments we performed an in vitro knockdown of the plasmodial eIF-5A and DHS proteins by RNA interference (RNAi) in 293 T cells. Secondly, transfection of siRNA constructs into murine Plasmodium schizonts was performed which, in turn, were used for infection., Results: 293 T cells treated with plasmodial DHS- and eIF-5A specific siRNAs or control siRNAs were analyzed by RT-PCR to determine endogenous dhs -and eIF-5A mRNA levels. The expressed DHS-shRNA and EIF-5A-shRNA clearly downregulated the corresponding transcript in these cells. Interestingly, mice infected with transgenic schizonts expressing either the eIF-5A or dhs shRNA showed an elevated parasitemia within the first two days post infection which then decreased intermittently. These results were obtained without drug selection. Blood samples, which were taken from the infected mice at day 5 post infection with either the expressed EIF-5A-shRNA or the DHS-shRNA were analyzed by RT-PCR and Western blot techniques, demonstrating the absence of either the hypusinated form of eIF-5A or DHS., Conclusions: Infection of NMRI mice with schizonts from the lethal P. berghei ANKA wildtype strain transgenic for plasmodial eIF-5A-specific shRNA or DHS-specific shRNA resulted in low parasitemia 2-9 days post infection before animals succumbed to hyperparasitemia similar to infections with the related but non-lethal phenotype P. berghei strain NK65. RT-PCR and Western blot experiments performed with blood from the transfected erythrocytic stages showed that both genes are important for the proliferation of the parasite. Moreover, these experiments clearly demonstrate that the hypusine pathway in Plasmodium is linked to human iNos induction.

The translation factor eIF5 is an important partner of eIF2, directly modulating its function in several critical steps. First, eIF5 binds eIF2/GTP/Met-tRNA(i)(Met) ternary complex (TC), promoting its recruitment to 40S ribosomal subunits. Secondly, its GTPase activating function promotes eIF2 dissociation for ribosomal subunit joining. Finally, eIF5 GDP dissociation inhibition (GDI) activity can antagonize eIF2 reactivation by competing with the eIF2 guanine exchange factor (GEF), eIF2B. The C-terminal domain (CTD) of eIF5, a W2-type HEAT domain, mediates its interaction with eIF2. Here, we characterize a related human protein containing MA3- and W2-type HEAT domains, previously termed BZW2 and renamed here as eIF5-mimic protein 1 (5MP1). Human 5MP1 interacts with eIF2 and eIF3 and inhibits general and gene-specific translation in mammalian systems. We further test whether 5MP1 is a mimic or competitor of the GEF catalytic subunit eIF2Bε or eIF5, using yeast as a model. Our results suggest that 5MP1 interacts with yeast eIF2 and promotes TC formation, but inhibits TC binding to the ribosome. Moreover, 5MP1 is not a GEF but a weak GDI for yeast eIF2. We propose that 5MP1 is a partial mimic and competitor of eIF5, interfering with the key steps by which eIF5 regulates eIF2 function.

Eukaryotic translation initiation factor 5A (eIF5A) has a unique character: the presence of an unusual amino acid, hypusine, which is formed by post-translational modifications. Even before the identification of hypusination in eIF5A, the correlation between hypusine formation and protein synthesis, shifting cell proliferation rates, had already been observed. Embryogenesis is a complex process in which cellular proliferation and differentiation are intense. In spite of the fact that many studies have described possible functions for eIF5A, its precise role is under investigation, and to date nothing has been reported about its participation in embryonic development. In this study we show that eIF5A is expressed at all mouse embryonic post-implantation stages with increase in eIF5A mRNA and protein expression levels between embryonic days E10.5 and E13.5. Immunohistochemistry revealed the ubiquitous presence of eIF5A in embryonic tissues and organs at E13.5 day. Interestingly, stronger immunoreactivity to eIF5A was observed in the stomodeum, liver, ectoderm, heart, and eye, and the central nervous system; regions which are known to undergo active differentiation at this stage, suggesting a role of eIF5A in differentiation events. Expression analyses of MyoD, a myogenic transcription factor, revealed a significantly higher expression from day E12.5 on, both at the mRNA and the protein levels suggesting a possible correlation to eIF5A. Accordingly, we next evidenced that inhibiting eIF5A hypusination in mouse myoblast C2C12 cells impairs their differentiation into myotubes and decreases MyoD transcript levels. Those results point to a new functional role for eIF5A, relating it to embryogenesis, development, and cell differentiation., ((c) 2010 Wiley-Liss, Inc.)

Using comparative proteomic and genomic approaches, the authors identified eukaryotic translation initiation factor 5A (eIF5A) as an oncofetal molecule highly abundant in mouse embryonic livers and human hepatocellular carcinoma (HCC) cell lines. To evaluate the oncogenic role and prognostic significance of eIF5A in HCC, we investigate the expression patterns of the two isoforms (eIF5A1 and eIF5A2) in a cohort of 258 HCC cases by cDNA microarray. Both eIF5A isoforms were expressed in the tumors, and clinically correlated eIF5A1 with more numbers of tumor nodules and eIF5A2 with tumor venous infiltration in HCC. In a separate cohort of 50 HCCs, high level of eIF5A2, but not eIF5A1, was associated with elevated levels of deoxyhypusine synthase and deoxyhypusine hydroxylase that catalyze post-translational hypusination of eIF5A protein. Interestingly, N1-guanyl-1,7-diaminoheptane (GC7), which is an inhibitor for the first step of eIF5A hypusination, was shown to significantly impair the cell proliferation and invasion of primary HCC cells (HepG2 and Hep3B). To further demonstrate the tumorigenic role associated with eIF5A, a drastic reduction of cell proliferation was associated with suppression of eIF5A2 by transfecting Hep3B, H2-P and H2-M HCC cells expressing high level of this isoform using small interfering RNA (siRNA) against eIF5A2. For these assays, a milder response was usually observed in normal hepatocyte cell line. Therefore, these findings suggest that eIF5A plays an important role in HCC tumorigenesis and metastasis, and targeting eIF5A hypusination by GC7 inhibitor or eIF5A2 by RNA interference (RNAi) may offer new therapeutic alternatives to HCC patients.

We have previously shown that inhibition of translation initiation, using the small molecule inhibitor silvestrol, induces apoptosis in a pre-clinical murine lymphoma model when combined with daunorubicin. Silvestrol blocks ribosome recruitment by targeting the RNA helicase, eIF4A, which is required for this process. Here we investigate the sensitivity of acute myelogenous leukemia (AML) cell lines to protein synthesis inhibition in combination with the standard cytotoxic agents daunorubicin, etoposide, and cytarabine. Silvestrol shows synergy with standard-of-care agents in AML cell lines and synergizes with ABT-737, a small molecule inhibitor of Bcl-X(L) and Bcl-2. The in vitro synergy between silvestrol and the cytotoxic drugs used in AML therapy provides a basis for in vivo evaluation of these combinations., (Copyright (c) 2009 Elsevier Ltd. All rights reserved.)

Stress granules (SGs) are cytoplasmic foci at which untranslated mRNAs accumulate in cells exposed to environmental stress. We have identified ornithine decarboxylase (ODC), an enzyme required for polyamine synthesis, and eIF5A, a polyamine (hypusine)-modified translation factor, as proteins required for arsenite-induced SG assembly. Knockdown of deoxyhypusine synthase (DHS) or treatment with a deoxyhypusine synthase inhibitor (GC7) prevents hypusine modification of eIF5A as well as arsenite-induced polysome disassembly and stress granule assembly. Time-course analysis reveals that this is due to a slowing of stress-induced ribosome run-off in cells lacking hypusine-eIF5A. Whereas eIF5A only marginally affects protein synthesis under normal conditions, it is required for the rapid onset of stress-induced translational repression. Our results reveal that hypusine-eIF5A-facilitated translation elongation promotes arsenite-induced polysome disassembly and stress granule assembly in cells subjected to adverse environmental conditions.

Unlabelled: A high incidence of tumor recurrence and metastasis has been reported in hepatocellular carcinoma (HCC) patients; however, the underlying molecular mechanisms are largely unknown. In the present study a novel metastasis-related gene, eukaryotic initiation factor 5A2 (EIF5A2), was characterized for its role in HCC metastasis and underlying molecular mechanisms. Overexpression of EIF5A2 messenger RNA (mRNA) was detected in 50/81 (61.7%) of HCCs, which was significantly higher than those in nontumorous liver tissues. Compared with matched primary HCC, higher expression of EIF5A2 protein was observed in 25/47 (53.2%) of metastatic tumors. Functional studies found that ectopic expression of EIF5A2 could enhance cancer cell migration and invasion in vitro and tumor metastasis in vivo in an experimental mouse model. Moreover, inhibition of EIF5A by small interfering RNA (siRNA) or deoxyhypusine synthase (DHPS) inhibitor GC7, which inhibits EIF5A2 maturation, could effectively decrease cell motility. Further study found that EIF5A2 was able to induce epithelial-mesenchymal transition (EMT), a key event in tumor invasion and metastasis, characterized by down-regulation of epithelial markers (E-cadherin and beta-catenin) and up-regulation of mesenchymal markers (fibronectin, N-cadherin, alpha-SMA, and vimentin). In addition, EIF5A2 could also activate RhoA/Rac1 to stimulate the formation of stress fiber and lamellipodia., Conclusion: EIF5A2 plays an important role in HCC invasion and metastasis by inducing EMT, as well as stimulating cytoskeleton rearrangement through activation of RhoA and Rac1.

Background: Eukaryotic translation initiation factor eIF5A has been implicated in HIV-1 replication. This protein contains the apparently unique amino acid hypusine that is formed by the post-translational modification of a lysine residue catalyzed by deoxyhypusine synthase and deoxyhypusine hydroxylase (DOHH). DOHH activity is inhibited by two clinically used drugs, the topical fungicide ciclopirox and the systemic medicinal iron chelator deferiprone. Deferiprone has been reported to inhibit HIV-1 replication in tissue culture., Results: Ciclopirox and deferiprone blocked HIV-1 replication in PBMCs. To examine the underlying mechanisms, we investigated the action of the drugs on eIF5A modification and HIV-1 gene expression in model systems. At early times after drug exposure, both drugs inhibited substrate binding to DOHH and prevented the formation of mature eIF5A. Viral gene expression from HIV-1 molecular clones was suppressed at the RNA level independently of all viral genes. The inhibition was specific for the viral promoter and occurred at the level of HIV-1 transcription initiation. Partial knockdown of eIF5A-1 by siRNA led to inhibition of HIV-1 gene expression that was non-additive with drug action. These data support the importance of eIF5A and hypusine formation in HIV-1 gene expression., Conclusion: At clinically relevant concentrations, two widely used drugs blocked HIV-1 replication ex vivo. They specifically inhibited expression from the HIV-1 promoter at the level of transcription initiation. Both drugs interfered with the hydroxylation step in the hypusine modification of eIF5A. These results have profound implications for the potential therapeutic use of these drugs as antiretrovirals and for the development of optimized analogs.

The downregulation of many mRNAs has been observed through bioinformatic analysis of microarray results following transfection of short interfering RNAs (siRNAs). Many of these mRNA changes are due to the interaction of the siRNA guide strand with partially complementary sites and thus are considered "off-target" effects. To examine the mRNA:siRNA interactions important for off-target effects, we generated a panel of mRNA:siRNA combinations containing single and double mismatches, bulges, and noncanonical base-pairing interactions in the 9th, 10th, and 11th positions of two siRNA binding sites located in the 3' UTR of an integrated reporter gene. Approximately half of the mRNA:siRNA combinations containing mismatches in positions 9-11 result in a twofold or more mRNA decrease with varying degrees of protein knockdown. However, mRNA and protein analysis of the various mRNA:siRNA combinations reveals instances in which mRNA and protein levels do not correlate. Analysis of the resulting degradation products recovered from an imperfectly complementary siRNA interaction with an endogenous gene reveals a small fraction of products that map to the canonical siRNA cleavage site. Furthermore, downregulation of ARGONAUTE 2 (AGO2), the only AGO family protein known to catalyze canonical siRNA-mediated cleavage, did not significantly affect the degree of mRNA knockdown observed for one of the stably expressed reporters after transfection of an imperfectly complementary siRNA. These results indicate that although some degree of canonical siRNA cleavage can take place between a siRNA and an off-target transcript, most off-target mRNA reductions are likely attributable to AGO2-independent degradation processes.

RNA interference and the microRNA (miRNA) pathway can induce sequence-specific mRNA degradation and/or translational repression. The human genome encodes hundreds of miRNAs that can post-transcriptionally repress thousands of genes. Using reporter constructs, we observed that degradation of mRNAs bearing sites imperfectly complementary to the endogenous let-7 miRNA is considerably stronger in human HEK293 than HeLa cells. The degradation did not result from the Ago2-mediated endonucleolytic cleavage but it was Dicer- and Ago2-dependent. We used this feature of HEK293 to address the size of a pool of transcripts regulated by RNA silencing in a single cell type. We generated HEK293 cell lines depleted of Dicer or individual Ago proteins. The cell lines were used for microarray analyses to obtain a comprehensive picture of RNA silencing. The 3'-untranslated region sequences of a few hundred transcripts that were commonly up-regulated upon Ago2 and Dicer knock-downs showed a significant enrichment of putative miRNA-binding sites. The up-regulation upon Ago2 and Dicer knock-downs was moderate and we found no evidence, at the mRNA level, for activation of silenced genes. Taken together, our data suggest that, independent of the effect on translation, miRNAs affect levels of a few hundred mRNAs in HEK293 cells.

The hypoxic genes of Saccharomyces cerevisiae are transcriptionally repressed during aerobic growth through recruitment of the Ssn6/Tup1 general repression complex by the DNA binding protein Rox1. A second DNA binding protein Mot3 enhances repression of some hypoxic genes. Previous studies characterized the role of Mot3 at the hypoxic ANB1 gene as promoting synergy among one Mot3 site and two Rox1 sites comprising operator A of that gene. Here we studied the role of Mot3 in enhancing repression by Rox1 at another hypoxic gene, HEM13, which is less strongly regulated than ANB1 and has a very different arrangement of Rox1 and Mot3 binding sites. By assessing the effects of deleting Rox1 and Mot3 sites individually and in combination, we found that the major repression of HEM13 occurred through three Mot3 sites closely spaced with a single Rox1 site. While the Mot3 sites functioned additively, they enhanced repression by the single Rox1 site, and the presence of Rox1 enhanced the additive effects of the Mot3 sites. In addition, using a Rox1-Ssn6 fusion protein, we demonstrated that Mot3 enhances Rox1 repression through helping recruit the Ssn6/Tup1 complex. Chromatin immunoprecipitation assays indicated that Rox1 stabilized Mot3 binding to DNA. Integrating these results, we were able to devise a set of rules that govern the combinatorial interactions between Rox1 and Mot3 to achieve differential repression.

Amplification of 3q26 is one of the most frequent chromosomal alterations in many solid tumors, including ovarian, lung, esophageal, prostate, breast, and nasopharyngeal cancers. A candidate oncogene to eukaryotic initiation factor 5A2 (eIF-5A2), a member of eukaryotic initiation factor 5A subfamily, has been isolated from a frequently amplified region at 3q26.2. In this work, the tumorigenic ability of eIF-5A2 was demonstrated by anchorage-independent growth in soft agar and tumor formation in nude mice. Furthermore, antisense DNA against eIF-5A2 could inhibit cell growth in ovarian cancer cell line UACC-1598 with amplification of eIF-5A2 in form of double minutes. Cell growth rate in UACC-1598 was also inhibited when the expression level of EIF-5A2 was decreased by the reduction of the copy number of double minutes. The correlation of EIF-5A2 overexpression and clinical features of ovarian cancer was investigated using tissue microarray, and the result showed that eIF-5A2 overexpression was significantly associated with the advanced stage of ovarian cancer. These findings suggest that eIF-5A2 plays important roles in ovarian pathogenesis.

Interferon-alpha (IFNalpha) can induce apoptosis, a process regulated by a complex network of cell factors. Among these, eukaryotic initiation factor-5A (eIF-5A) is peculiar because its activity is modulated by the post-translational formation of the amino acid hypusine. Here we report the effects of IFNalpha and epidermal growth factor (EGF) on apoptosis and eIF-5A activity in human epidermoid oropharyngeal KB and lung H1355 cancer cells. We found that 48-h exposure to 1000 and 2000 IU/ml IFNalpha induced about 50% growth inhibition and apoptosis in H1355 and KB cells, respectively, and the addition of EGF completely antagonized this effect. When IFNalpha induced apoptosis, a hyperactivation of MEK-1 and ERK signalling and a decrease of the hypusine-containing form and, thus, of eIF-5A activity were recorded. The latter effect was again antagonized by the addition of EGF to IFNalpha-pretreated cells, probably through the activation of the EGF-->ERK-dependent pathway, since the addition of the specific MEK-1 inhibitor PD098059 abrogated the recovery of intracellular hypusine content induced by EGF in IFNalpha-pretreated cancer cells. Subsequently, we evaluated if the hypusine synthesis inhibitor (and eIF-5A inactivator) N1-guanyl-1,7-diaminoheptane (GC7) synergized with IFNalpha in the induction of cell growth inhibition and apoptosis. The analysis of the isobologram of IFNalpha and GC7 demonstrated a strong synergism between the two drugs in inducing cell growth inhibition. We also found that GC7 and IFNalpha had a synergistic effect on apoptosis. These data suggest that the apoptosis induced by IFNalpha could be regulated by eIF-5A that, therefore, could represent a useful target for the potentiation of IFNalpha antitumor activity.

The mechanism of inhibition of cell growth by deoxyspergualin was studied using mouse mammary carcinoma FM3A cells. Results of studies using deoxyspergualin analogues showed that both the guanidinoheptanate amide and glyoxyspermidine moieties of deoxyspergualin were necessary to cause inhibition of cell growth. When deoxyspergualin was added to the medium, there was a strong inhibition of cell growth and formation of active eukaryotic translation initiation factor 5A (eIF5A) at the third day of culture. There was also a marked decrease in cellular putrescine content and a small decrease in spermidine content. Accumulation of decapped mRNA, which is typically associated with eIF5A deficiency in yeast, was also observed. The inhibition of cell growth and the formation of active eIF5A was not reversed by addition of spermidine. The activity of deoxyhypusine synthase, the first enzyme in the formation of active eIF5A, was inhibited by deoxyspergualin in a cell-free system. These results, taken together, indicate that inhibition of active eIF5A formation is strongly involved in the inhibition of cell growth by deoxyspergualin.

The promyelocytic leukemia protein (PML) is a mammalian regulator of cell growth which is characteristically disrupted in acute promyelocytic leukemia and by a variety of viruses. PML contains a RING domain which is required for its growth-suppressive and antiviral properties. Although normally nuclear, in certain pathogenic conditions, including arenaviral infection, PML is relocated to the cytoplasm, where its functions are poorly understood. Here, we observe that PML and arenavirus protein Z use regions around the first zinc-binding site of their respective RING domains to directly interact, with sub-micromolar affinity, with the dorsal surface of translation initiation factor eIF4E, representing a novel mode of eIF4E recognition. PML and Z profoundly reduce the affinity of eIF4E for its substrate, the 5' 7-methyl guanosine cap of mRNA, by over 100-fold. Association with the dorsal surface of eIF4E and direct antagonism of mRNA cap binding by PML and Z lead to direct inhibition of translation. These activities of the RING domains of PML and Z do not involve ubiquitin-mediated protein degradation, in contrast to many RINGs which have been observed to do so. Although PML and Z have well characterized physiological functions in regulation of growth and apoptosis, this work establishes the first discrete biochemical mechanism which underlies the biological activities of their RING domains. Thus, we establish PML and Z as translational repressors, with potential contributions to the pathogenesis of acute promyelocytic leukemia and variety of viral infections., (Copyright 2001 Academic Press.)

Eukaryotic initiation factor (eIF) 4B interacts with several components of the initiation pathway and is targeted for cleavage during apoptosis. In a cell-free system, cleavage of eIF4B by caspase-3 coincides with a general inhibition of protein synthetic activity. Affinity chromatography demonstrates that mammalian eIF4B interacts with the poly(A)-binding protein and that a region consisting of the N-terminal 80 amino acids of eIF4B is both necessary and sufficient for such binding. This interaction is lost when eIF4B is cleaved by caspase-3, which removes the N-terminal 45 amino acids. Similarly, the association of eIF4B with the poly(A)-binding protein in vivo is reduced when cells are induced to undergo apoptosis. Cleavage of the poly(A)-binding protein itself, using human rhinovirus 3C protease, also eliminates the interaction with eIF4B. Thus, disruption of the association between mammalian eIF4B and the poly(A)-binding protein can occur during both apoptosis and picornaviral infection and is likely to contribute to the inhibition of translation observed under these conditions.

The catabolic properties of glucocorticoid hormones are largely attributable to dual regulation of protein degradation and synthesis. With regard to the latter, glucocorticoids modulate the translational machinery, namely that component functional in translation initiation. This investigation revealed that in L6 myoblasts, dexamethasone, a synthetic glucocorticoid, deactivated the ribosomal protein S6 kinase (p70(S6k)) within 4 h, as evidenced by diminished phosphorylation of its physiological substrate, the 40S ribosomal protein S6. This deactivation correlated with dephosphorylation of p70(S6k) at Thr(389), whereas phosphorylation of Ser(411) was unaffected. Furthermore, glucocorticoid administration induced dephosphorylation of the cap-dependent translational repressor, eukaryotic initiation factor 4E (eIF4E) binding protein 1 (4E-BP1), thereby facilitating conjunction of the inhibitor and eIF4E. The mechanism of action is reminiscent of classical transcriptional regulation by steroid hormone receptors in that these effects were preceded by a temporal lag and were sensitive to inhibitors of glucocorticoid receptor function as well as transcriptional and translational inhibition. Okadaic acid and calyculin A corrected the dexamethasone-induced dephosphorylation of p70(S6k) and 4E-BP1, implicating a PP1- and/or PP2A-like protein phosphatase(s) in the observed phenomena. Hence, glucocorticoids attenuate distal constituents of the phosphatidylinositol-3 kinase signaling pathway and thereby encumber the protein synthetic apparatus.

Eukaryotic translation initiation involves recognition of the 5' end of cellular mRNA by the cap-binding complex known as eukaryotic initiation factor 4F (eIF4F). Initiation is a key point of regulation in gene expression in response to mechanisms mediated by signal transduction pathways. We have investigated the molecular interactions underlying inhibition of human eIF4E function by regulatable repressors called 4E-binding proteins (4E-BPs). Two essential components of eIF4F are the cap-binding protein eIF4E, and eIF4G, a multi-functional protein that binds both eIF4E and other essential eIFs. We show that the 4E-BPs 1 and 2 block the interaction between eIF4G and eIF4E by competing for binding to a dorsal site on eIF4E. Remarkably, binding of the 4E-BPs at this dorsal site enhances cap-binding via the ventral cap-binding slot, thus trapping eIF4E in inactive complexes with high affinity for capped mRNA. The binding contacts and affinities for the interactions between 4E-BP1/2 and eIF4E are distinct (estimated K(d) values of 10(-8) and 3x10(-9) for 4E-BP1 and 2, respectively), and the differences in these properties are determined by three amino acids within an otherwise conserved motif. These data provide a quantitative framework for a new molecular model of translational regulation.

Fifty-eight analogues of the 5'-terminal 7-methylguanosine-containing cap of eukaryotic messenger RNA were synthesized and tested for their ability to inhibit in vitro protein synthesis. A new algorithm was developed for extracting KI, the dissociation constant for the cap analogue.eIF4E complex, from protein synthesis data. The results indicated that addition of a methyl group to the N2 of guanine produced more inhibitory compounds, but addition of a second methyl group to N2 decreased the level of inhibition dramatically. Aryl substitution at N7 improved the efficacy of guanine nucleoside monophosphate analogues. Substitution of the aromatic ring at the para position with methyl or NO2 groups abolished this effect, but substitution with Cl or F enhanced it. By contrast, aryl substitution at N7 in nucleoside di- or triphosphate analogues produced only minor effects, both positive and negative. By far the strongest determinants of inhibitory activity for cap analogues were phosphate residues. The beneficial effect of more phosphate residues was related more to anionic charge than to the number of phosphate groups per se. The second nucleotide residue in analogues of the form m7GpppN affected inhibitory activity in the order G > C > U > A, but there was no effect of 2'-O-modification. Opening the first ribose ring of m7GpppG analogues dramatically decreased activity, but alterations at the 2'-position of this ribose had no effect. Non-nucleotide-based cap analogues containing benzimidazole derivatives were inhibitory, though less so than those containing 7-methylguanine.

eIF4G uses a conserved Tyr-X-X-X-X-Leu-phi segment (where X is variable and phi is hydrophobic) to recognize eIF4E during cap-dependent translation initiation in eukaryotes. High-resolution X-ray crystallography and complementary biophysical methods have revealed that this eIF4E recognition motif undergoes a disorder-to-order transition, adopting an L-shaped, extended chain/alpha-helical conformation when it interacts with a phylogenetically invariant portion of the convex surface of eIF4E. Inhibitors of translation initiation known as eIF4E-binding proteins (4E-BPs) contain similar eIF4E recognition motifs. These molecules are molecular mimics of eIF4G, which act by occupying the same binding site on the convex dorsum of eIF4E and blocking assembly of the translation machinery. The implications of our results for translation initiation are discussed in detail, and a molecular mechanism for relief of translation inhibition following phosphorylation of the 4E-BPs is proposed.

Eukaryotic translation initiation factor 5A (eIF-5A) is the only cell protein that contains the unusual basic amino acid hypusine [N epsilon-(4-amino-2-hydroxybutyl)lysine]. Hypusine is formed by the transfer of the butylamine portion from spermidine to the epsilon-amino group of a specific lysine residue of eIF-5A precursor and the subsequent hydroxylation at carbon 2 of the incoming 4-aminobutyl moiety. Agents that reduce cell hypusine levels inhibit the growth of mammalian cells. These observations suggest that hypusine is crucial for proliferation and transformation of eukaryotic cells. Here we have studied whether the inhibition of hypusine synthesis can potentiate the anti-cancer activity of the anti-tumour agents interferon-alpha (IFN alpha) and cytosine arabinoside (ara-C). We have found that IFN alpha increased epidermal growth factor receptor (EGF-R) expression, but reduced S phase and proliferative marker expression in human epidermoid KB cells and that this effect was antagonised by epidermal growth factor (EGF). Growth inhibition induced by IFN alpha was paralleled by decreased hypusine synthesis and, when EGF counteracted anti-proliferative effects, a reconstitution of hypusine levels was recorded. We also studied the effects of IFN alpha on the cytotoxicity of the recombinant toxin TP40 which inhibits elongation factor 2, another step of protein synthesis, through EGF-R binding and internalisation; IFN alpha induced an about 27-fold increase of TP40 cytotoxicity in KB cells. Ara-C, another antineoplastic agent commonly used in haematologic malignancies, induced both apoptosis and iron depletion in human acute myeloid leukaemic cells. The combination of ara-C and of the iron chelator desferioxamine, a strong inhibitor of hypusine synthesis, had a synergistic activity on apoptosis in these cells. The data strongly suggest that the post-translational modifications of eIF-5A could be a suitable target for the potentiation of the activity of anti-cancer agents.

The primary site in PHAS-I for phosphorylation by protein kinase CK2 in vitro was identified as Ser111. A relatively small amount of phosphorylation of Ser99 was also detected, and mutating Ser99 to Ala in PHAS-I slightly decreased phosphorylation by CK2 in vitro. In contrast, mutating Ser111 to Ala almost abolished phosphorylation, confirming Ser111 as the preferred site for CK2. Phosphorylation of Ser111 did not decrease binding of PHAS-I to eIF4E, and results of peptide mapping experiments with PHAS-I immunoprecipitated from 32P-labeled adipocytes indicated that Ser111 was not phosphorylated in cells. These results support the conclusion that CK2 is not involved in the control of PHAS-I.

The HIV-1 protein Rev, critical for translation of incompletely spliced retroviral mRNAs encoding capsid elements, requires a host cell protein termed "eukaryotic initiation factor 5A" (eIF-5A). This is the only protein containing hypusine, a lysine-derived hydroxylated residue that determines its proposed bioactivity, the translation of a subset of cellular mRNAs controlling G1-to-S transit of the cell cycle. We postulated that inhibiting the hypusine-forming deoxyhypusyl hydroxylase (DOHH) should, by depleting eukaryotic initiation factor 5A, compromise Rev function and thus reduce HIV-1 multiplication. We now report that the alpha-hydroxypyridones, specifically mimosine, a natural product, and deferiprone, an experimental drug, inhibited deoxyhypusyl hydroxylase in T-lymphocytic and promonocytic cell lines and, in a concentration-dependent manner, suppressed replication of HIV-1. However, the alpha-hydroxypyridones did not affect the formation of unspliced or multiply spliced HIV-1 transcripts. Rather, these agents caused Rev-dependent incompletely spliced HIV-1 mRNA such as gag, but not cellular "housekeeping" mRNAs, to disappear from polysomes. Consequently, alpha-hydroxypyridone-mediated depletion of eIF-5A decreased biosynthesis of structural HIV-1 protein encoded by gag, measured as p24, whereas the induced formation of cellular protein like tumor necrosis factor alpha remained unaffected. By interfering with the translation of incompletely spliced retroviral mRNAs, these compounds restrict HIV-1 to the early, nongenerative phase of its reproductive cycle. In the inducibly HIV-1 expressing T-cell line ACH-2, the deoxyhypusyl hydroxylase inhibitors triggered extensive apoptosis, particularly of cells that actively produce HIV-1. Selective suppression of retroviral protein biosynthesis and preferential apoptosis of retrovirally infected cells by alpha-hydroxypyridones point to a novel mode of antiretroviral action.

The 4E binding proteins (4E-BP1 and 4E-BP2) inhibit translation by binding to the limiting, proto-oncogenic initiation factor eIF4E. 4E-BPs produced in Escherichia coli had little or no folded structure, measured by NMR and CD. However, these proteins inhibited translation in reticulocyte lysate. Furthermore, they bound to isolated mouse eIF4E, showing a few broader, dispersed new NMR signals but no general increase in chemical shift dispersion. A peptide with the sequence of 4E-BP1 residues 49-68 was sufficient to bind eIF4E and to inhibit translation in reticulocyte lysate. These results suggest that a short central region of the 4E-BPs is responsible for eIF4E binding and translation inhibition while the remainder is unfolded and flexible.

Poliovirus induces a shut-off of messenger RNA (mRNA) translation by the proteolysis of a 220 kDa protein from the eukaryotic initiation factor eIF-4F, and by the phosphorylation of eIF-2. The absence of eIF-4F inhibits the initiation of translation dependent on cap structure recognition. Poliovirus RNA lacks cap structure and translates by a cap-independent mechanism which requires internal ribosomal entry. The poliovirus 5' untranslated region (5'UTR) contains the structural elements for cap-independent translation called internal ribosomal entry site (IRES element). Several cellular proteins have been described interacting with different segments from poliovirus 5'UTR. We have studied the specific interaction between 57/60, 52, and 35 kDa cellular proteins with poliovirus nt 275 to 636 and full length 5'UTR. By Western blot assay the 57/60 protein was identified as a pyrimidine tract binding protein (PTB), and the 52 kDa protein as a La-autoantigen. La and PTB are nuclear proteins involved in RNA polymerase III transcription termination and splicing, respectively.

An important aspect of the regulation of gene expression is the modulation of translation rates in response to growth factors, hormones and mitogens. Most of this control is at the level of translation initiation. Recent studies have implicated the MAP kinase pathway in the regulation of translation by insulin and growth factors. MAP kinase phosphorylates a repressor of translation initiation [4E-binding protein (BP) 1] that binds to the mRNA 5' cap binding protein eukaryotic initiation factor (eIF)-4E and inhibits cap-dependent translation. Phosphorylation of the repressor decreases its affinity for eIF-4E, and thus relieves translational inhibition. eIF-4E forms a complex with two other polypeptides, eIF-4A and p220, that promote 40S ribosome binding to mRNA. Here, we have studied the mechanism by which 4E-BP1 inhibits translation. We show that 4E-BP1 inhibits 48S pre-initiation complex formation. Furthermore, we demonstrate that 4E-BP1 competes with p220 for binding to eIF-4E. Mutants of 4E-BP1 that are deficient in their binding to eIF-4E do not inhibit the interaction between p220 and eIF-4E, and do not repress translation. Thus, translational control by growth factors, insulin and mitogens is affected by changes in the relative affinities of 4E-BP1 and p220 for eIF-4E.

To investigate whether preferential translation of the heat-shock mRNAs occurs via cap-independent translation, the requirement for the m7GpppG cap structure for efficient translation of 70-kDa heat-shock-protein (Hsp70) mRNA was quantified by in vitro translation and by in vivo translation following electroporation. Hsp70 mRNA was transcribed in vitro with and without a cap structure. Translation in the rabbit reticulocyte or wheat germ lysate was reduced about 70% when the cap was absent. For comparison, translation of uncapped encephalomyocarditis virus 5'-untranslated-region-containing mRNA was equal to or greater than capped mRNA, whereas translation of several non-heat-shock mRNAs was reduced by 85-95% when capping was omitted. Cap-dependent translational stimulation of Hsp70 is not due to increased stability, is not a kinetic effect, and requires the methylated GpppG. To confirm the in vitro analyses, capped and uncapped mRNA were introduced into Drosophila tissue culture cells by electroporation, followed by heat shock. Paralleling the in vitro results, uncapped Hsp70 mRNA translation was 70-80% reduced relative to the capped form. Complementary experiments in which eIF-4 was inactivated in vitro using either m7GTP cap analogue or foot-and-mouth-disease virus L protease expression likewise indicated that the cap-dependent translation pathway is required for optimal Hsp mRNA translation. Since cellular Hsp70 mRNA translation during heat shock is very efficient, it is unlikely that translation via a cap-independent pathway is the principal basis for preferential translation.

Restenosis occurs in 35% of patients within months after balloon angioplasty, due to a fibroproliferative response to vascular injury. These studies describe a combined fibrosuppressive/antiproliferative strategy on smooth muscle cells cultured from human primary atherosclerotic and restenotic coronary arteries and from normal rat aortas. L-Mimosine suppressed the posttranslational hydroxylation of the precursors for collagen and for eukaryotic initiation factor-5A (eIF-5A) by directly inhibiting the specific protein hydroxylases involved, prolyl 4-hydroxylase (E.C. 1.14.11.2) and deoxyhypusyl hydroxylase (E.C. 1.14.99.29), respectively. Inhibition of deoxyhypusyl hydroxylation correlated with a dose-dependent inhibition of DNA synthesis. Inhibition of prolyl hydroxylation caused a dose-dependent reduction in the secretion of hydroxyproline-containing protein and decreased the formation of procollagen types I and III. The antifibroproliferative action could not be attributed to nonspecific or toxic effects of mimosine, appeared to be selective for the hydroxylation step in the biosynthesis of the procollagens and of eIF-5A, and was reversible upon removal of the compound. The strategy of targeting these two protein hydroxylases has important implications for the pathophysiology of restenosis and for the development of agents to control fibroproliferative diseases.

The developmentally regulated inhibitor of eIF-4 function found in unfertilized sea urchin eggs has been partially purified and its mechanism of action studied in vitro using purified recombinant eIF-4 alpha and cell-free translation systems. The results demonstrate that although the phosphorylation of eIF-4 alpha is necessary to promote protein synthesis, it is not sufficient to maintain all aspects of eIF-4 function. The egg inhibitor does not change eIF-4 alpha phosphorylation state. During the blockage of initiation caused by the egg inhibitor, eIF-4 alpha remains phosphorylated but accumulates in a 48S initiation intermediate. This suggests that the egg inhibitor functions by preventing the release of eIF-4 alpha from the small ribosomal subunit. The characteristics of the inhibitor in a reticulocyte translation system demonstrate that eIF-4 activity is inhibited within 3-6 min. However, the inhibitor's characteristics in a mRNA-dependent translation system contrast with this. Preincubation with the inhibitor for 5-25 min prior to the addition of mRNA does not prevent endogenous eIF-4 from participating in translation but diminishes its ability to be reutilized, consistent with the accumulation of eIF-4 alpha on the small ribosomal subunit. The ribosomal localization of the inhibitor suggests that it could prevent eIF-4 alpha release by direct binding. The gradual inactivation of the inhibitor following fertilization indicates that it represents a component of a novel regulatory cascade that modulates eIF-4 activity.