Cap-dependent Protein Synthesis Research Articles

Dual targeting of glutamine and serine metabolism in acute myeloid leukemia.

Acute myeloid leukemia (AML) is a heterogeneous hematological malignancy characterized by disrupted blood cell production and function. Recent investigations have highlighted the potential of targeting glutamine metabolism as a promising therapeutic approach for AML. Asparaginases, enzymes that deplete circulating glutamine and asparagine, are approved for the treatment of acute lymphoblastic leukemia, but are also under investigation in AML, with promising results. We previously reported an elevation in plasma serine levels following treatment with Erwinia-derived asparaginase (also called crisantaspase). This led us to hypothesize that AML cells initiate the de novo serine biosynthesis pathway in response to crisantaspase treatment and that inhibiting this pathway in combination with crisantaspase would enhance AML cell death. Here we report that in AML cell lines, treatment with the clinically available crisantaspase, Rylaze, upregulates the serine biosynthesis enzymes phosphoglycerate dehydrogenase (PHGDH) and phosphoserine aminotransferase (PSAT1) through activation of the Amino Acid Response (AAR) pathway, a cellular stress response mechanism that regulates amino acid metabolism and protein synthesis under conditions of nutrient limitation. Inhibition of serine biosynthesis through CRISPR-Cas9-mediated knockout of PHGDH resulted in a ~250-fold reduction in the half-maximal inhibitory concentration (IC50) for Rylaze, indicating heightened sensitivity to crisantaspase therapy. Treatment of AML cells with a combination of Rylaze and a small molecule inhibitor of PHGDH (BI4916) revealed synergistic anti-proliferative effects in both cell lines and primary AML patient samples. Rylaze-BI4916 treatment in AML cell lines led to the inhibition of cap-dependent mRNA translation and protein synthesis, as well as a marked decrease in intracellular glutathione levels, a critical cellular antioxidant. Collectively, our results highlight the clinical potential of targeting serine biosynthesis in combination with crisantaspase as a novel therapeutic strategy for AML.

C8ORF88: A Novel eIF4E-Binding Protein.

Translation initiation in eukaryotes is regulated at several steps, one of which involves the availability of the cap binding protein to participate in cap-dependent protein synthesis. Binding of eIF4E to translational repressors (eIF4E-binding proteins [4E-BPs]) suppresses translation and is used by cells to link extra- and intracellular cues to protein synthetic rates. The best studied of these interactions involves repression of translation by 4E-BP1 upon inhibition of the PI3K/mTOR signaling pathway. Herein, we characterize a novel 4E-BP, C8ORF88, whose expression is predominantly restricted to early spermatids. C8ORF88:eIF4E interaction is dependent on the canonical eIF4E binding motif (4E-BM) present in other 4E-BPs. Whereas 4E-BP1:eIF4E interaction is dependent on the phosphorylation of 4E-BP1, these sites are not conserved in C8ORF88 indicating a different mode of regulation.

Alkaline intracellular pH (pHi) increases PI3K activity to promote mTORC1 and mTORC2 signaling and function during growth factor limitation

The conserved protein kinase mTOR (mechanistic target of rapamycin) responds to diverse environmental cues to control cell metabolism and promote cell growth, proliferation, and survival as part of two multiprotein complexes, mTOR complex 1 (mTORC1) and mTORC2. Our prior work demonstrated that an alkaline intracellular pH (pHi) increases mTORC2 activity and cell survival in complete media in part by activating AMP-activated protein kinase, a kinase best known to sense energetic stress. It is important to note that an alkaline pHi represents an underappreciated hallmark of cancer cells that promotes their oncogenic behaviors. In addition, mechanisms that control mTORC1 and mTORC2 signaling and function remain incompletely defined, particularly in response to stress conditions. Here, we demonstrate that an alkaline pHi increases phosphatidylinositide 3-kinase (PI3K) activity to promote mTORC1 and mTORC2 signaling in the absence of serum growth factors. Alkaline pHi increases mTORC1 activity through PI3K-Akt signaling, which mediates inhibitory phosphorylation of the upstream proteins tuberous sclerosis complex 2 and proline-rich Akt substrate of 40 kDa and dissociates tuberous sclerosis complex from lysosomal membranes, thus enabling Rheb-mediated activation of mTORC1. Thus, alkaline pHi mimics growth factor-PI3K signaling. Functionally, we also demonstrate that an alkaline pHi increases cap-dependent protein synthesis through inhibitory phosphorylation of eIF4E binding protein 1 and suppresses apoptosis in a PI3K- and mTOR-dependent manner. We speculate that an alkaline pHi promotes a low basal level of cell metabolism (e.g., protein synthesis) that enables cancer cells within growing tumors to proliferate and survive despite limiting growth factors and nutrients, in part through elevated PI3K-mTORC1 and/or PI3K-mTORC2 signaling.

A p53-dependent translational program directs tissue-selective phenotypes in a model of ribosomopathies.

In ribosomopathies, perturbed expression of ribosome components leads to tissue-specific phenotypes. What accounts for such tissue-selective manifestations as a result of mutations in the ribosome, a ubiquitous cellular machine, has remained a mystery. Combining mouse genetics and invivo ribosome profiling, we observe limb-patterning phenotypes in ribosomal protein (RP) haploinsufficient embryos, and we uncover selective translational changes of transcripts that controlling limb development. Surprisingly, both loss of p53, which is activated by RP haploinsufficiency, and augmented protein synthesis rescue these phenotypes. These findings are explained by the finding that p53 functions as a master regulator of protein synthesis, at least in part, through transcriptional activation of 4E-BP1. 4E-BP1, a key translational regulator, in turn, facilitates selective changes in the translatome downstream of p53, and this thereby explains how RP haploinsufficiency may elicit specificity to gene expression. These results provide an integrative model to help understand how invivo tissue-specific phenotypes emerge in ribosomopathies.

A biphenyl inhibitor of eIF4E targeting an internal binding site enables the design of cell-permeable PROTAC-degraders.

The eukaryotic translation initiation factor 4E (eIF4E) is the master regulator of cap-dependent protein synthesis. Overexpression of eIF4E is implicated in diseases such as cancer, where dysregulation of oncogenic protein translation is frequently observed. eIF4E has been an attractive target for cancer treatment. Here we report a high-resolution X-ray crystal structure of eIF4E in complex with a novel inhibitor (i4EG-BiP) that targets an internal binding site, in contrast to the previously described inhibitor, 4EGI-1, which binds to the surface. We demonstrate that i4EG-BiP is able to displace the scaffold protein eIF4G and inhibit the proliferation of cancer cells. We provide insights into how i4EG-BiP is able to inhibit cap-dependent translation by increasing the eIF4E-4E-BP1 interaction while diminishing the interaction of eIF4E with eIF4G. Leveraging structural details, we designed proteolysis targeted chimeras (PROTACs) derived from 4EGI-1 and i4EG-BiP and characterized these on biochemical and cellular levels. We were able to design PROTACs capable of binding eIF4E and successfully engaging Cereblon, which targets proteins for proteolysis. However, these initial PROTACs did not successfully stimulate degradation of eIF4E, possibly due to competitive effects from 4E-BP1 binding. Our results highlight challenges of targeted proteasomal degradation of eIF4E that must be addressed by future efforts.

An In Vitro Single-Molecule Imaging Assay for the Analysis of Cap-Dependent Translation Kinetics.

Cap-dependent protein synthesis is the predominant translation pathway in eukaryotic cells. While various biochemical and genetic approaches have allowed extensive studies of cap-dependent translation and its regulation, high resolution kinetic characterization of this translation pathway is still lacking. Recently, we developed an in vitro assay to measure cap-dependent translation kinetics with single-molecule resolution. The assay is based on fluorescently labeled antibody binding to nascent epitope-tagged polypeptide. By imaging the binding and dissociation of antibodies to and from nascent peptide-ribosome-mRNA complexes, the translation progression on individual mRNAs can be tracked. Here, we present a protocol for establishing this assay, including mRNA and PEGylated slide preparations, real-time imaging of translation, and analysis of single molecule trajectories. This assay enables tracking of individual cap-dependent translation events and resolves key translation kinetics, such as initiation and elongation rates. The assay can be widely applied to distinct translation systems and should broadly benefit in vitro studies of cap-dependent translation kinetics and translational control mechanisms.

Design of Development Candidate eFT226, a First in Class Inhibitor of Eukaryotic Initiation Factor 4A RNA Helicase

Dysregulation of protein translation is a key driver for the pathogenesis of many cancers. Eukaryotic initiation factor 4A (eIF4A), an ATP-dependent DEAD-box RNA helicase, is a critical component of the eIF4F complex, which regulates cap-dependent protein synthesis. The flavagline class of natural products (i.e., rocaglamide A) has been shown to inhibit protein synthesis by stabilizing a translation-incompetent complex for select messenger RNAs (mRNAs) with eIF4A. Despite showing promising anticancer phenotypes, the development of flavagline derivatives as therapeutic agents has been hampered because of poor drug-like properties as well as synthetic complexity. A focused effort was undertaken utilizing a ligand-based design strategy to identify a chemotype with optimized physicochemical properties. Also, detailed mechanistic studies were undertaken to further elucidate mRNA sequence selectivity, key regulated target genes, and the associated antitumor phenotype. This work led to the design of eFT226 (Zotatifin), a compound with excellent physicochemical properties and significant antitumor activity that supports clinical development.

Mammalian cell growth dynamics in mitosis.

The extent and dynamics of animal cell biomass accumulation during mitosis are unknown, primarily because growth has not been quantified with sufficient precision and temporal resolution. Using the suspended microchannel resonator and protein synthesis assays, we quantify mass accumulation and translation rates between mitotic stages on a single-cell level. For various animal cell types, growth rates in prophase are commensurate with or higher than interphase growth rates. Growth is only stopped as cells approach metaphase-to-anaphase transition and growth resumes in late cytokinesis. Mitotic arrests stop growth independently of arresting mechanism. For mouse lymphoblast cells, growth in prophase is promoted by CDK1 through increased phosphorylation of 4E-BP1 and cap-dependent protein synthesis. Inhibition of CDK1-driven mitotic translation reduces daughter cell growth. Overall, our measurements counter the traditional dogma that growth during mitosis is negligible and provide insight into antimitotic cancer chemotherapies.

Oxygen-Sensitive Remodeling of Central Carbon Metabolism by Archaic eIF5B

SUMMARYThe eukaryotic translation initiation factor 5B (eIF5B) is a homolog of IF2, an ancient translation factor that enables initiator methionine-tRNAiMet (met-tRNAiMet) loading on prokaryotic ribosomes. While it can be traced back to the last universal common ancestor, eIF5B is curiously dispensable in modern aerobic yeast and mammalian cells. Here, we show that eIF5B is an essential element of the cellular hypoxic cap-dependent protein synthesis machinery. System-wide interrogation of dynamic translation machineries by MATRIX (mass spectrometry analysis of active translation factors using ribosome density fractionation and isotopic labeling experiments) demonstrated augmented eIF5B activity in hypoxic translating ribosomes. Global translatome studies revealed central carbon metabolism, cellular hypoxic adaptation, and ATF4-mediated stress response as major eIF5B-dependent pathways. These primordial processes rely on eIF5B even in the presence of oxygen and active eIF2, the canonical recruiter of met-tRNAiMet in eukaryotes. We suggest that aerobic eukarya retained eIF5B/IF2 to remodel anaerobic pathways during episodes of oxygen deficiency.

Protein synthesis inhibition enhances paraptotic death induced by inhibition of cyclophilins in glioblastoma cells

Treatment of cancer is frequently unsuccessful related to the loss of apoptotic signaling in malignant cells. This is a particular problem for high-grade gliomas, such as Glioblastoma Multiforme (GBM), which are almost universally fatal within a year or so of diagnosis. Novel therapies that capitalize on non-apoptotic cell death pathways may yield more effective outcomes, if their underlying mechanisms can be more completely deciphered. In a recent publication (ref 10), the mechanisms by which cellular cyclophilins support GBM cell survival have been identified. Inhibition of cyclophilins activated paraptosis, which relied on a combination of endoplasmic reticulum (ER) stress and transient activation of autophagy. An important aspect of this effect was the relative rates of cap-dependent versus cap-independent protein synthesis, which were differentially modulated by protein synthesis inhibitors or mTOR inhibition. Although cycloheximide has previously been characterized as an inhibitor of paraptosis, in the case of cyclophilin inhibition, it appears to significantly enhance stress-related paraptosis and cell death. This work reveals an important role for cap-independent protein translation and autophagy in the ability of GBM cells to resist non-apoptotic death, and adds to our understanding of the events that underlie paraptosis.

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

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

Presynaptic Protein Synthesis Is Required for Long-Term Plasticity of GABA Release

Long-term changes of neurotransmitter release are critical for proper brain function. However, the molecular mechanisms underlying these changes are poorly understood. While protein synthesis is crucial for the consolidation of postsynaptic plasticity, whether and how protein synthesis regulates presynaptic plasticity in the mature mammalian brain remain unclear. Here, using paired whole-cell recordings in rodent hippocampal slices, we report that presynaptic protein synthesis is required for long-term, but not short-term, plasticity of GABA release from type 1 cannabinoid receptor (CB1)-expressing axons. This long-term depression of inhibitory transmission (iLTD) involves cap-dependent protein synthesis in presynaptic interneuron axons, but not somata. Translation is required during the induction, but not maintenance, of iLTD. Mechanistically, CB1 activation enhances protein synthesis via the mTORpathway. Furthermore, using super-resolution STORM microscopy, we revealed eukaryotic ribosomes in CB1-expressing axon terminals. These findings suggest that presynaptic local protein synthesis controls neurotransmitter release during long-term plasticity in the mature mammalian brain.

MRNA capping: biological functions and applications.

The 5′ m7G cap is an evolutionarily conserved modification of eukaryotic mRNA. Decades of research have established that the m7G cap serves as a unique molecular module that recruits cellular proteins and mediates cap-related biological functions such as pre-mRNA processing, nuclear export and cap-dependent protein synthesis. Only recently has the role of the cap 2′O methylation as an identifier of self RNA in the innate immune system against foreign RNA has become clear. The discovery of the cytoplasmic capping machinery suggests a novel level of control network. These new findings underscore the importance of a proper cap structure in the synthesis of functional messenger RNA. In this review, we will summarize the current knowledge of the biological roles of mRNA caps in eukaryotic cells. We will also discuss different means that viruses and their host cells use to cap their RNA and the application of these capping machineries to synthesize functional mRNA. Novel applications of RNA capping enzymes in the discovery of new RNA species and sequencing the microbiome transcriptome will also be discussed. We will end with a summary of novel findings in RNA capping and the questions these findings pose.

Systemic Reprogramming of Translation Efficiencies on Oxygen Stimulus.

Protein concentrations evolve under greater evolutionary constraint than mRNA levels. Translation efficiency of mRNA represents the chief determinant of basal protein concentrations. This raises a fundamental question of how mRNA and protein levels are coordinated in dynamic systems responding to physiological stimuli. This report examines the contributions of mRNA abundance and translation efficiency to protein output in cells responding to oxygen stimulus. We show that changes in translation efficiencies, and not mRNA levels, represent the major mechanism governing cellular responses to [O2] perturbations. Two distinct cap-dependent protein synthesis machineries select mRNAs for translation: the normoxic eIF4F and the hypoxic eIF4F(H). O2-dependent remodeling of translation efficiencies enables cells to produce adaptive translatomes from preexisting mRNA pools. Differences in mRNA expression observed under different [O2] are likely neutral, given that they occur during evolution. We propose that mRNAs contain translation efficiency determinants for their triage by the translation apparatus on [O2] stimulus.

Abstract 2262: Evidence that the translational initiation factor DAP5 plays a critical role in breast cancer metastasis

Abstract The metastatic process is highly inefficient. Among the thousands cells that escape daily from the primary tumor only a few survive and posses the ability of developing overt metastases in a distant organ. Yet, metastasis is the cause of 90% of cancer-related deaths. Despite its clinical importance, little is known about genetic and biochemical determinants of metastasis and new therapeutic targets are desperately needed. During their journey to colonize a distant organ, cancer cells undergo a wide variety of stresses (hypoxia, anoikis or colonization) that down-regulate global cap-dependent protein synthesis. In order to produce the proteins needed to overcome these stresses cancer cells rely on alternative mechanisms for translation initiation, typically through non-cap-dependent means such as internal ribosome entry (IRES). DAP5 is a poorly studied translation initiation factor known to mediate IRES-driven translation of cellular mRNAs that include anti-apoptotic factors and others factors likely involved in stress responses. Therefore, we hypothesized that DAP5 is crucial for the expression of the disseminated cancer cell proteome and cell survival during metastasis. We engineered highly metastatic murine 4T1 cancer cells to express a doxycycline (dox) inducible system shRNA silencing of DAP5 (4T1 shDAP5). These cells or the non silencing (NS) shRNA control cells were grown s.c. in immunocompetent syngeneic BALB-c mice. Dox induction was started by drinking water addition 12 days post cell injection and tumors measured twice weekly. Animals were sacrificed 18 days later, lungs collected and fixed in 4% PFA. While no significant effect was observed on cell proliferation in vitro, silencing DAP5 significantly decreased primary tumor growth and lung metastatic colonization. Importantly, when the same tumor study was performed in immunodeficient animals (NOD SCID γ) the inhibition of tumor growth with DAP5 silencing was completely abolished. These data suggest that DAP5 likely enables cancer cells to elude the immune response. However in this model, DAP5 silencing impaired the metastatic process and significantly decreased the number of lung metastases observed, implying that at least one of the mechanisms by which DAP5 prevents metastasis is immune system-independent. These results have been preliminarily confirmed in human metastatic breast cancer cells that efficiently disseminate to lungs. We engineered them with the same dox inducible system. When tested on NOD SCID mice, a significant decrease in the metastatic burden was also observed upon DAP5 silencing. Our results suggest that the translation initiation factor DAP5 likely plays a critical role in breast cancer metastasis and provides new concepts for therapeutic strategies involving translational regulation and immunotherapeutics. Indeed, inhibiting DAP5 might be a powerful approach, as it impacts the whole proteome of the disseminated cancer cell. Citation Format: Amandine Alard, Fernanda Musa, Robert Schneider. Evidence that the translational initiation factor DAP5 plays a critical role in breast cancer metastasis. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2262. doi:10.1158/1538-7445.AM2015-2262

Amyloid precursor protein (APP) affects global protein synthesis in dividing human cells.

Hypoxic non‐small cell lung cancer (NSCLC) is dependent on Notch‐1 signaling for survival. Targeting Notch‐1 by means of γ‐secretase inhibitors (GSI) proved effective in killing hypoxic NSCLC. Post‐mortem analysis of GSI‐treated, NSCLC‐burdened mice suggested enhanced phosphorylation of 4E‐BP1 at threonines 37/46 in hypoxic tumor tissues. In vitro dissection of this phenomenon revealed that Amyloid Precursor Protein (APP) inhibition was responsible for a non‐canonical 4E‐BP1 phosphorylation pattern rearrangement—a process, in part, mediated by APP regulation of the pseudophosphatase Styx. Upon APP depletion we observed modifications of eIF‐4F composition indicating increased recruitment of eIF‐4A to the mRNA cap. This phenomenon was supported by the observation that cells with depleted APP were partially resistant to silvestrol, an antibiotic that interferes with eIF‐4A assembly into eIF‐4F complexes. APP downregulation in dividing human cells increased the rate of global protein synthesis, both cap‐ and IRES‐dependent. Such an increase seemed independent of mTOR inhibition. After administration of Torin‐1, APP downregulation and Mechanistic Target of Rapamycin Complex 1 (mTORC‐1) inhibition affected 4E‐BP1 phosphorylation and global protein synthesis in opposite fashions. Additional investigations indicated that APP operates independently of mTORC‐1. Key phenomena described in this study were reversed by overexpression of the APP C‐terminal domain. The presented data suggest that APP may be a novel regulator of protein synthesis in dividing human cells, both cancerous and primary. Furthermore, APP appears to affect translation initiation using mechanisms seemingly dissimilar to mTORC‐1 regulation of cap‐dependent protein synthesis. J. Cell. Physiol. 230: 1064–1074, 2015. © 2014 The Authors. Journal of Cellular Physiology Published by Wiley Periodicals, Inc.

92 Role of ERK nuclear translocation in cisplatin-sensitive and -resistant ovarian cancer cells

Background: In contrast to other breast cancer subtypes, triple negative breast cancers are characterized by an absence of recurrent genetic alterations. However, alterations in the PI3K pathway are frequently observed, either through mutation or amplification of PIK3CA or the loss of the negative regulator PTEN. As a result, inhibitors of this pathway, including inhibitors of mTOR, the downstream target of the PI3K pathway, have been explored in the clinic. It has been found that mutational activation of PI3K represents an important determinant of sensitivity to mTOR inhibitors. Nevertheless, a number of PI3K wild-type cell lines also show sensitivity to these inhibitors, although the regulators of this response are still unknown. We set out to find such modulators of response to mTOR inhibitors in a panel of triple negative breast cancer cell lines initially using high throughput approaches. Material and Methods: We determined the sensitivity of a panel of triple negative breast cancer cell lines to AZD8055 (mTOR inhibitor) and BEZ235 (mTOR/PI3K dual inhibitor). This data was then used together with additional datasets generated for this panel, including RNA expression (by RNA-seq), (phospho-)protein levels (by Reverse Phase Protein Arrays) and mutational status (by post-capture DNA-seq), to carry out regression analysis to identify features correlated with response. Results: We found that the protein expression levels of 4E-BP1, a downstream target of mTOR involved in regulating cap-dependent protein synthesis, correlated with response to both inhibitors. In poorly responding cell lines expressing low levels of 4E-BP1, overexpression of the protein increased sensitivity. Conversely, in sensitive cell lines expressing high levels of 4E-BP1, knock-down of 4E-BP1 led to reduced sensitivity to mTOR inhibition. Conclusions: These results indicate that effective inhibition of protein synthesis downstream of PI3K/mTOR is an important determinant of mTOR inhibitor activity. Given that approximately 20% of breast cancer tumors annotated in The Cancer Genome Atlas show an amplification of the 4E-BP1 locus, such patients would likely benefit from mTOR inhibitor treatment. Furthermore, we hypothesize that mTOR inhibitor response can be improved even in patients expressing low 4E-BP1 by co-treating with agents that induce its expression. To this end, we are currently screening for genes and drugs that modulate 4E-BP1 levels.

Abstract 1405: Knockdown ARID5A suppresses proliferation of LNCaP prostate cancer cell through the inhibition of global protein synthesis

Abstract AT-Rich Interaction Domain-containing protein 5a (ARID5A; also known as Modulator Recognition Factor 1(MRF1)) was first cloned in our laboratory by screening a cDNA library of Tera-2 mRNA with an oligonucleotide probe from the major immediate early gene of human cytomegalovirus. It has been reported that ARID5A forms a complex with androgen receptor (AR) in yeast two hybrid assays and suppresses the transcriptional activity of AR in transient transfection assays. In this study, we report that the knockdown of Arid5a expression inhibits proliferation of LNCaP cells on the contrary to the expectation from the transient transfection assay. We found that proliferation of cells stimulated by the treatment with dihydro-testosterone (DHT) was suppressed when Arid5a expression was down-regulated by siRNA technology. Flow cytometer analysis showed that DHT-stimulated cell cycle was arrested at G1 phase after the knockdown of Arid5a expression. Oil Red O staining assays showed that the inhibition of Arid5a expression led to decreased DHT-induced lipid accumulation. Our data further proved that the knockdown of Arid5a expression inhibited the protein expression of DHT-induced sterol regulatory element binding transcription factor 1 (SREBP-1). In addition, Western blot analysis revealed that the down-regulation of Arid5a decreased the expression of cell survival/proliferation and cell cycle regulation proteins, such as hypoxia inducible factor 1 alpha (HIF1a), cyclin D1 and cyclin D3. Real time PCR data revealed that the knockdown of Arid5a expression did not affect the steady level of HIF1a, cyclin D1 and cyclin D3 mRNA. These suggested that knockdown of Arid5a expression affected protein expression at translational and/or post-translational level. Mammalian target of rapamycin complex 1 (mTORC1) is a well-known factor in regulating the cap-dependent protein synthesis. Western blot analysis indicated that the suppression of Arid5a expression had no effects on the activity of mTORC1. However, knockdown of Arid5a expression increased the phosphorylation of eukaryotic translation initiation factor 2a (eIF2a) which inhibited translation initiation. S35 labeling assay confirmed that the knockdown of Arid5a expression suppressed the global protein synthesis. In order to find out the mechanisms of Arid5a-dependent phosphorylation of eIF2a, we tested four known kinases which were responsible for the phosphorylation of eIF2a. Two of the four eIF2a kinases, general control non-derepressible 2 (GCN2) and RNA-dependent protein kinase (PKR)-like endoplasmic reticulum kinase (PERK), were activated by the suppression of Arid5a expression. We are currently studying on the mechanisms by which ARID5A regulates GCN2 and PERK kinase. Citation Format: Chao Sun, Viktor Chesnokov, Keiichi Itakura. Knockdown ARID5A suppresses proliferation of LNCaP prostate cancer cell through the inhibition of global protein synthesis. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1405. doi:10.1158/1538-7445.AM2014-1405

Cellular adaptation to low oxygen availability by a switch in the protein synthesis machinery

Protein synthesis is a classic molecular mechanism of cell biology that is taught in introductory biology classrooms. It involves the translation of messenger ribonucleic acid (mRNA) information into proteins, the building blocks of life. The initial step of protein synthesis consists of the eu¬karyotic translation initiation factor 4E (eIF4E) binding to the 5’cap of mRNAs. A variety of stress¬es repress translation to conserve energy because protein synthesis requires over half of a cell’s energy supply. An important stress for multicellular animals is low oxygen availability (hypoxia). This causes a repression of cap-directed translation by inhibiting eIF4E. This raises a fundamental question in cell biology as to how proteins are synthesized in periods of oxygen scarcity and eIF4E inhibition. Here, we describe an oxygen-regulated translation initiation complex that mediates selective cap-dependent protein synthesis. Hypoxia stimulates the formation of a complex that in¬cludes the oxygen-regulated hypoxia-inducible factor 2α (HIF-2α), the RNA binding protein RBM4 and the cap-binding eIF4E2, an eIF4E homologue. We also identified an RNA hypoxia response ele¬ment (rHRE) that recruits this complex to a wide array of mRNAs, including the epidermal growth factor receptor (EGFR), which plays a role in growth signaling and proliferation. Once assembled at the rHRE, HIF-2α/RBM4/eIF4E2 captures the 5’cap and targets mRNAs for active translation thereby evading hypoxia-induced repression of protein synthesis. These findings demonstrate that cells have evolved a program whereby oxygen availability switches the basic translation initiation machinery.

West nile virus-induced activation of mammalian target of rapamycin complex 1 supports viral growth and viral protein expression.

Since its introduction in New York City, NY, in 1999, West Nile virus (WNV) has spread to all 48 contiguous states of the United States and is now the leading cause of epidemic encephalitis in North America. As a member of the family Flaviviridae, WNV is part of a group of clinically important human pathogens, including dengue virus and Japanese encephalitis virus. The members of this family of positive-sense, single-stranded RNA viruses have limited coding capacity and are therefore obligated to co-opt a significant amount of cellular factors to translate their genomes effectively. Our previous work has shown that WNV growth was independent of macroautophagy activation, but the role of the evolutionarily conserved mammalian target of rapamycin (mTOR) pathway during WNV infection was not well understood. mTOR is a serine/threonine kinase that acts as a central cellular censor of nutrient status and exercises control of vital anabolic and catabolic cellular responses such as protein synthesis and autophagy, respectively. We now show that WNV activates mTOR and cognate downstream activators of cap-dependent protein synthesis at early time points postinfection and that pharmacologic inhibition of mTOR (KU0063794) significantly reduced WNV growth. We used an inducible Raptor and Rictor knockout mouse embryonic fibroblast (MEF) system to further define the role of mTOR complexes 1 and 2 in WNV growth and viral protein synthesis. Following inducible genetic knockout of the major mTOR cofactors raptor (TOR complex 1 [TORC1]) and rictor (TORC2), we now show that TORC1 supports flavivirus protein synthesis via cap-dependent protein synthesis pathways and supports subsequent WNV growth. Since its introduction in New York City, NY, in 1999, West Nile virus (WNV) has spread to all 48 contiguous states in the United States and is now the leading cause of epidemic encephalitis in North America. Currently, the mechanism by which flaviviruses such as WNV translate their genomes in host cells is incompletely understood. Elucidation of the host mechanisms required to support WNV genome translation will provide broad understanding for the basic mechanisms required to translate capped viral RNAs. We now show that WNV activates mTOR and cognate downstream activators of cap-dependent protein synthesis at early time points postinfection. Following inducible genetic knockout of the major mTOR complex cofactors raptor (TORC1) and rictor (TORC2), we now show that TORC1 supports WNV growth and protein synthesis. This study demonstrates the requirement for TORC1 function in support of WNV RNA translation and provides insight into the mechanisms underlying flaviviral RNA translation in mammalian cells.