Biomedical subjects

Ivan Topisirovic

Publications and source records attributed to Ivan Topisirovic.

13 recordsLinked to original sources

PubMed2006

eIF4E is a central node of an RNA regulon that governs cellular proliferation.

This study demonstrates that the eukaryotic translation initiation factor eIF4E is a critical node in an RNA regulon that impacts nearly every stage of cell cycle progression. Specifically, eIF4E coordinately promotes the messenger RNA (mRNA) export of several genes involved in the cell cycle. A common feature of these mRNAs is a structurally conserved, approximately 50-nucleotide element in the 3' untranslated region denoted as an eIF4E sensitivity element. This element is sufficient for localization of capped mRNAs to eIF4E nuclear bodies, formation of eIF4E-specific ribonucleoproteins in the nucleus, and eIF4E-dependent mRNA export. The roles of eIF4E in translation and mRNA export are distinct, as they rely on different mRNA elements. Furthermore, eIF4E-dependent mRNA export is independent of ongoing RNA or protein synthesis. Unlike the NXF1-mediated export of bulk mRNAs, eIF4E-dependent mRNA export is CRM1 dependent. Finally, the growth-suppressive promyelocytic leukemia protein (PML) inhibits this RNA regulon. These data provide novel perspectives into the proliferative and oncogenic properties of eIF4E.

Animals↗

PubMed2006

Cap-free structure of eIF4E suggests a basis for conformational regulation by its ligands.

The activity of the eukaryotic translation initiation factor eIF4E is modulated through conformational response to its ligands. For example, eIF4G and eIF4E-binding proteins (4E-BPs) modulate cap affinity, and thus physiological activity of eIF4E, by binding a site distal to the 7-methylguanosine cap-binding site. Further, cap binding substantially modulates eIF4E's affinity for eIF4G and the 4E-BPs. To date, only cap-bound eIF4E structures were reported. In the absence of structural information on the apo form, the molecular underpinnings of this conformational response mechanism cannot be established. We report here the first cap-free eIF4E structure. Apo-eIF4E exhibits structural differences in the cap-binding site and dorsal surface relative to cap-eIF4E. Analysis of structure and dynamics of apo-eIF4E, and changes observed upon ligand binding, reveal a molecular basis for eIF4E's conformational response to these ligands. In particular, alterations in the S4-H4 loop, distal to either the cap or eIF4G binding sites, appear key to modulating these effects. Mutation in this loop mimics these effects. Overall, our studies have important implications for the regulation of eIF4E.

Binding Sites↗

PubMed2006

Regulation of p53 localization and activity by Ubc13.

The abundance and activity of p53 are regulated largely by ubiquitin ligases. Here we demonstrate a previously undisclosed regulation of p53 localization and activity by Ubc13, an E2 ubiquitin-conjugating enzyme. While increasing p53 stability, Ubc13 decreases p53 transcriptional activity and increases its localization to the cytoplasm, changes that require its ubiquitin-conjugating activity. Ubc13 elicits K63-dependent ubiquitination of p53, which attenuates Hdm2-induced polyubiquitination of p53. Ubc13 association with p53 requires an intact C-terminal domain of p53 and is markedly stronger with a p53 mutant that cannot tetramerize. Expression of Ubc13 in vivo increases the pool of monomeric p53, indicating that Ubc13 affects tetramerization of p53. Significantly, wild-type but not mutant Ubc13 is associated with polysomes and enriches p53 within this fraction. In response to DNA damage, Ubc13 is no longer capable of facilitating p53 monomerization, in part due to a decrease in its own levels which is p53 dependent. Our findings point to a newly discerned mechanism important in the regulation of p53 organization, localization, and activity by Ubc13.

Animals↗

PubMed2006

Arenavirus Z protein as an antiviral target: virus inactivation and protein oligomerization by zinc finger-reactive compounds.

Several disulfide-based and azoic compounds have shown antiviral and virucidal properties against arenaviruses in virus yield-inhibition and inactivation assays, respectively. The most effective virucidal agent, the aromatic disulfide NSC20625, was able to inactivate two strains of the prototype arenavirus species Lymphocytic choriomeningitis virus (LCMV). Inactivated viral particles retained the biological functions of the virion envelope glycoproteins in virus binding and uptake, but were unable to perform viral RNA replication. Furthermore, in inactivated virions, the electrophoretic profile of the Z protein was altered when analysed under non-reducing conditions, whereas the patterns of the proteins NP and GP1 remained unaffected. Treatment of a recombinant LCMV Z protein with the virucidal agents induced unfolding and oligomerization of Z to high-molecular-mass aggregates, probably due to metal-ion ejection and the formation of intermolecular disulfide bonds through the cysteine residues of the Z RING finger. NSC20625 also exhibited antiviral properties in LCMV-infected cells without affecting other cellular RING-motif proteins, such as the promyelocytic leukaemia protein PML. Altogether, the investigations described here illustrate the potential of the Z protein as a promising target for therapy and the prospects of the Z-reactive compounds to prevent arenavirus dissemination.

Animals↗

PubMed2005

Further evidence that ribavirin interacts with eIF4E.

This commentary discusses the recent reports in RNA by Yan and colleagues and Westman and colleagues of the apparent failure of ribavirin to bind to recombinant eIF4E and inhibit 7-methyl guanosine cap-dependent exogenous mRNA translation of cell extracts in vitro. Measuring binding by using affinity chromatography of matrix-immobilized proteins and by using protein emission fluorescence spectroscopy in the presence of nucleotide ligands, as well as limitations of using cell extracts for the assessment of mechanisms of mRNA translation are discussed. Possible reasons for the discordant findings of Yan and colleagues and Westman and colleagues are suggested, and direct observation of the specific binding of ribavirin to eIF4E by using mass spectrometry is presented.

Eukaryotic Initiation Factor-4E↗

PubMed2005

eIF4E promotes nuclear export of cyclin D1 mRNAs via an element in the 3'UTR.

The eukaryotic translation initiation factor eIF4E is a critical modulator of cellular growth with functions in the nucleus and cytoplasm. In the cytoplasm, recognition of the 5' m(7)G cap moiety on all mRNAs is sufficient for their functional interaction with eIF4E. In contrast, we have shown that in the nucleus eIF4E associates and promotes the nuclear export of cyclin D1, but not GAPDH or actin mRNAs. We determined that the basis of this discriminatory interaction is an approximately 100-nt sequence in the 3' untranslated region (UTR) of cyclin D1 mRNA, we refer to as an eIF4E sensitivity element (4E-SE). We found that cyclin D1 mRNA is enriched at eIF4E nuclear bodies, suggesting these are functional sites for organization of specific ribonucleoproteins. The 4E-SE is required for eIF4E to efficiently transform cells, thereby linking recognition of this element to eIF4E mediated oncogenic transformation. Our studies demonstrate previously uncharacterized fundamental differences in eIF4E-mRNA recognition between the nuclear and cytoplasmic compartments and further a novel level of regulation of cellular proliferation.

3' Untranslated Regions↗

PubMed2005

The proline-rich homeodomain (PRH/HEX) protein is down-regulated in liver during infection with lymphocytic choriomeningitis virus.

The proline-rich homeodomain protein, PRH/HEX, participates in the early development of the brain, thyroid, and liver and in the later regenerative processes of damaged liver, vascular endothelial, and hematopoietic cells. A virulent strain of lymphocytic choriomeningitis virus (LCMV-WE) that destroys hematopoietic, vascular, and liver functions also alters the transcription and subcellular localization of PRH. A related virus (LCMV-ARM) that does not cause disease in primates can infect cells without affecting PRH. Biochemical experiments demonstrated the occurrence of binding between the viral RING protein (Z) and PRH, and genetic experiments mapped the PRH-suppressing phenotype to the large (L) segment of the viral genome, which encodes the Z and polymerase genes. The Z protein is clearly involved with PRH, but other viral determinants are needed to relocate PRH and to promote disease. By down-regulating PRH, the arenavirus is able to eliminate the antiproliferative effects of PRH and to promote liver cell division. The interaction of an arenavirus with a homeodomain protein suggests a mechanism for viral teratogenic effects and for the tissue-specific manifestations of arenavirus disease.

Animals↗

PubMed2005

Eukaryotic translation initiation factor 4E activity is modulated by HOXA9 at multiple levels.

The eukaryotic translation initiation factor 4E (eIF4E) alters gene expression on multiple levels. In the cytoplasm, eIF4E acts in the rate-limiting step of translation initiation. In the nucleus, eIF4E facilitates nuclear export of a subset of mRNAs. Both of these functions contribute to eIF4E's ability to oncogenically transform cells. We report here that the homeodomain protein, HOXA9, is a positive regulator of eIF4E. HOXA9 stimulates eIF4E-dependent export of cyclin D1 and ornithine decarboxylase (ODC) mRNAs in the nucleus, as well as increases the translation efficiency of ODC mRNA in the cytoplasm. These activities depend on direct interactions of HOXA9 with eIF4E and are independent of the role of HOXA9 in transcription. At the biochemical level, HOXA9 mediates these effects by competing with factors that repress eIF4E function, in particular the proline-rich homeodomain PRH/Hex. This competitive mechanism of eIF4E regulation is disrupted in a subset of leukemias, where HOXA9 displaces PRH from eIF4E, thereby contributing to eIF4E's dysregulation. In regard to these results and our previous finding that approximately 200 homeodomain proteins contain eIF4E binding sites, we propose that homeodomain modulation of eIF4E activity is a novel means through which this family of proteins implements their effects on growth and development.

Active Transport, Cell Nucleus↗

PubMed2004

Ribavirin suppresses eIF4E-mediated oncogenic transformation by physical mimicry of the 7-methyl guanosine mRNA cap.

The eukaryotic translation initiation factor eIF4E is deregulated in many human cancers, and its overexpression in cells leads to malignant transformation. Oncogenic properties of eIF4E are directly linked to its ability to bind 7-methyl guanosine of the 5' mRNA. Here, we observe that the antiviral guanosine analogue ribavirin binds to eIF4E with micromolar affinity at the functional site used by 7-methyl guanosine mRNA cap, competes with eIF4E:mRNA binding, and, at low micromolar concentrations, selectively disrupts eIF4E subcellular organization and transport and translation of mRNAs posttranscriptionally regulated by eIF4E, thereby reducing levels of oncogenes such as cyclin D1. Ribavirin potently suppresses eIF4E-mediated oncogenic transformation of murine cells in vitro, of tumor growth of a mouse model of eIF4E-dependent human squamous cell carcinoma in vivo, and of colony formation of eIF4E-dependent acute myelogenous leukemia cells derived from human patients. These findings describe a specific, potent, and unforeseen mechanism of action of ribavirin. Quantum mechanical and NMR structural studies offer directions for the development of derivatives with improved cytostatic and antiviral properties. In all, ribavirin's association with eIF4E may provide a pharmacologic means for the interruption of posttranscriptional networks of oncogenes that maintain and enhance neoplasia and malignancy in human cancer.

Animals↗

PubMed2004

Phosphorylation of the eukaryotic translation initiation factor eIF4E contributes to its transformation and mRNA transport activities.

The eukaryotic translation initiation factor eIF4E is dysregulated in a wide variety of human cancers. In the cytoplasm, eIF4E acts in the rate-limiting step of translation initiation whereas in the nucleus, eIF4E forms nuclear bodies and promotes the nucleo-cytoplasmic export of a subset of growth-promoting mRNAs including cyclin D1. The only known post-translational modification of eIF4E is its phosphorylation at S209. Many studies have examined the role of phosphorylation on cap-dependent translation. However, no studies to date have explored the role of phosphorylation on the ability of eIF4E to transform cells. Using mutagenesis and separately a small molecular inhibitor of eIF4E phosphorylation, we show that eIF4E phosphorylation enhances both its mRNA transport function and its transformation activity in cell culture. Thus, phosphorylation of nuclear eIF4E seems to be an important step in control of the mRNA transport and thus the transforming properties of eIF4E.

Aniline Compounds↗

PubMed2003

The proline-rich homeodomain protein, PRH, is a tissue-specific inhibitor of eIF4E-dependent cyclin D1 mRNA transport and growth.

The translation initiation factor eIF4E is involved in the modulation of cellular growth. In the nucleus, where eIF4E is associated with PML nuclear bodies, eIF4E mediates nucleocytoplasmic transport of specific transcripts, and this contributes to its transformation activity. Surprisingly, we found that a trans cription factor, the proline-rich homeodomain protein PRH, is a negative regulator of eIF4E in myeloid cells, interacting with eIF4E through a conserved binding site typically found in translational regulators. Through this interaction, PRH inhibits eIF4E-dependent mRNA transport and subsequent transformation. These activities of PRH are independent of its transcriptional functions. Further, we found that 199 homeodomain proteins contain potential eIF4E-binding sites. Thus, there could be many tissue-specific regulators of eIF4E. These findings provide a model for regulation of a general factor, eIF4E, in tissue- specific contexts, and suggest that its regulation is important in differentiation and development.

Active Transport, Cell Nucleus↗

PubMed2003

Aberrant eukaryotic translation initiation factor 4E-dependent mRNA transport impedes hematopoietic differentiation and contributes to leukemogenesis.

The eukaryotic translation initiation factor 4E (eIF4E) acts as both a key translation factor and as a promoter of nucleocytoplasmic transport of specific transcripts. Traditionally, its transformation capacity in vivo is attributed to its role in translation initiation in the cytoplasm. Here, we demonstrate that elevated eIF4E impedes granulocytic and monocytic differentiation. Our subsequent mutagenesis studies indicate that this block is a result of dysregulated eIF4E-dependent mRNA transport. These studies indicate that the RNA transport function of eIF4E could contribute to leukemogenesis. We extended our studies to provide the first evidence that the nuclear transport function of eIF4E contributes to human malignancy, specifically in a subset of acute and chronic myelogenous leukemia patients. We observe an increase in eIF4E-dependent cyclin D1 mRNA transport and a concomitant increase in cyclin D1 protein levels. The aberrant nuclear function of eIF4E is due to abnormally large eIF4E bodies and the loss of regulation by the proline-rich homeodomain PRH. We developed a novel tool to modulate this transport activity. The introduction of IkappaB, the repressor of NF-kappaB, leads to suppression of eIF4E, elevation of PRH, reorganization of eIF4E nuclear bodies, and subsequent downregulation of eIF4E-dependent mRNA transport. Thus, our findings indicate that this nuclear function of eIF4E can contribute to leukemogenesis by promoting growth and by impeding differentiation.

Active Transport, Cell Nucleus↗

PubMed2002

Gamma interferon and cadmium treatments modulate eukaryotic initiation factor 4E-dependent mRNA transport of cyclin D1 in a PML-dependent manner.

The eukaryotic initiation factor 4E (eIF4E), when dysregulated, transforms cells. A substantial fraction of eIF4E forms nuclear bodies that colocalize with those associated with the promyelocytic leukemia protein PML. Overexpression studies indicate that nuclear eIF4E promotes the transport of cyclin D1 mRNA from the nucleus to the cytoplasm and that PML is a key negative regulator of this function. Since previous studies used overexpression methods, the physiological relevance of eIF4E mRNA transport function or its interaction with PML remained unknown. Therefore, we monitored whether eIF4E-dependent transport could be modulated in response to environmental conditions. Here we report that cadmium treatment, which disperses PML nuclear bodies, leaves eIF4E bodies intact, leading to increased transport of cyclin D1 mRNA and increased cyclin D1 protein levels. Removal of cadmium allows PML to reassociate with eIF4E nuclear bodies, leading to decreased cyclin D1 transport and reduced cyclin D1 protein levels. In contrast, we show that treating cells with interferon increased the levels of PML protein at the PML-eIF4E nuclear body, leading to nuclear retention of cyclin D1 transcripts and reduced cyclin D1 protein levels. Neither interferon nor cadmium treatment altered cyclin D1 levels in PML(-/-) cells. Consistently, overexpression of a series of PML and eIF4E mutant proteins established that PML eIF4E interaction is required for the observed effects of cadmium and interferon treatment. The present study provides the first evidence that physiological factors modulate the mRNA transport functions of eIF4E and that this regulation is PML dependent.

3T3 Cells↗