RESUMEN
Eukaryotic translation initiation involves recruitment of the 43S pre-initiation complex to the 5' end of mRNA by the cap-binding complex eIF4F, forming the 48S translation initiation complex (48S), which then scans along the mRNA until the start codon is recognized. We have previously shown that eIF4F binds near the mRNA exit channel of the 43S, leaving open the question of how mRNA secondary structure is removed as it enters the mRNA channel on the other side of the 40S subunit. Here we report the structure of a human 48S that shows that, in addition to the eIF4A that is part of eIF4F, there is a second eIF4A helicase bound at the mRNA entry site, which could unwind RNA secondary structures as they enter the 48S. The structure also reveals conserved interactions between eIF4F and the 43S, probaby explaining how eIF4F can promote mRNA recruitment in all eukaryotes.
Asunto(s)
Factor 4F Eucariótico de Iniciación , Iniciación de la Cadena Peptídica Traduccional , Humanos , Factor 4F Eucariótico de Iniciación/genética , Factor 4F Eucariótico de Iniciación/metabolismo , ARN Mensajero/metabolismo , Codón Iniciador/metabolismo , Ribosomas/metabolismo , ADN Helicasas/metabolismo , Biosíntesis de Proteínas , Factor 4A Eucariótico de Iniciación/química , Factor 4A Eucariótico de Iniciación/genética , Factor 4A Eucariótico de Iniciación/metabolismoRESUMEN
The canonical DEAD-box helicase, eukaryotic initiation factor (eIF) 4A, unwinds 5' UTR secondary structures to promote mRNA translation initiation. Growing evidence has indicated that other helicases, such as DHX29 and DDX3/ded1p, also function to promote the scanning of the 40S subunit on highly structured mRNAs. It is unknown how the relative contributions of eIF4A and other helicases regulate duplex unwinding on an mRNA to promote initiation. Here, we have adapted a real-time fluorescent duplex unwinding assay to monitor helicase activity precisely in the 5' UTR of a reporter mRNA that can be translated in a cell-free extract in parallel. We monitored the rate of 5' UTR-dependent duplex unwinding in the absence or presence of an eIF4A inhibitor (hippuristanol), a dominant negative eIF4A (eIF4A-R362Q), or a mutant eIF4E (eIF4E-W73L) that can bind the m7G cap but not eIF4G. Our experiments reveal that the duplex unwinding activity in the cell-free extract is roughly evenly split between eIF4A-dependent and eIF4A-independent mechanisms. Importantly, we show that the robust eIF4A-independent duplex unwinding is not sufficient for translation. We also show that the m7G cap structure, and not the poly(A) tail, is the primary mRNA modification responsible for promoting duplex unwinding in our cell-free extract system. Overall, the fluorescent duplex unwinding assay provides a precise method to investigate how eIF4A-dependent and eIF4A-independent helicase activity regulates translation initiation in cell-free extracts. We anticipate that potential small molecule inhibitors could be tested for helicase inhibition using this duplex unwinding assay.
Asunto(s)
Factor 4A Eucariótico de Iniciación , Factor 4E Eucariótico de Iniciación , Procesamiento Postranscripcional del ARN , Humanos , Regiones no Traducidas 5' , ADN Helicasas/metabolismo , Factor 4A Eucariótico de Iniciación/química , Factor 4E Eucariótico de Iniciación/genética , Factor 4E Eucariótico de Iniciación/metabolismo , Factor 4G Eucariótico de Iniciación/metabolismo , Biosíntesis de Proteínas , ARN Helicasas/genética , ARN Helicasas/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismoRESUMEN
Inhibition of eukaryotic translation initiation through unscheduled RNA clamping of the DEAD-box (DDX) RNA helicases eIF4A1 and eIF4A2 has been documented for pateamine A (PatA) and rocaglates-two structurally different classes of compounds that share overlapping binding sites on eIF4A. Clamping of eIF4A to RNA causes steric blocks that interfere with ribosome binding and scanning, rationalizing the potency of these molecules since not all eIF4A molecules need to be engaged to elicit a biological effect. In addition to targeting translation, PatA and analogs have also been shown to target the eIF4A homolog, eIF4A3-a helicase necessary for exon junction complex (EJC) formation. EJCs are deposited on mRNAs upstream of exon-exon junctions and, when present downstream from premature termination codons (PTCs), participate in nonsense-mediated decay (NMD), a quality control mechanism aimed at preventing the production of dominant-negative or gain-of-function polypeptides from faulty mRNA transcripts. We find that rocaglates can also interact with eIF4A3 to induce RNA clamping. Rocaglates also inhibit EJC-dependent NMD in mammalian cells, but this does not appear to be due to induced eIF4A3-RNA clamping, but rather a secondary consequence of translation inhibition incurred by clamping eIF4A1 and eIF4A2 to mRNA.
Asunto(s)
Degradación de ARNm Mediada por Codón sin Sentido , ARN , Animales , ARN/metabolismo , ARN Mensajero/metabolismo , Codón sin Sentido , Exones , Factor 4A Eucariótico de Iniciación/química , Mamíferos/genéticaRESUMEN
Cells employ several adaptive mechanisms under conditions of accelerated cell division, such as the unfolded protein response (UPR). The UPR is composed of a tripartite signaling system that involves ATF6, PERK, and IRE1, which maintain protein homeostasis (proteostasis). However, deregulation of protein translation initiation could be associated with breast cancer (BC) chemoresistance. Specifically, eukaryotic initiation factor-4A (eIF4A) is involved in the unfolding of the secondary structures of several mRNAs at the 5' untranslated region (5'-UTR), as well as in the regulation of targets involved in chemoresistance. Importantly, the tumor suppressor gene PDCD4 could modulate this process. This regulation might be disrupted in chemoresistant triple negative-BC (TNBC) cells. Therefore, we characterized the effect of doxorubicin (Dox), a commonly used anthracycline medication, on human breast carcinoma MDA-MB-231 cells. Here, we generated and characterized models of Dox chemoresistance, and chemoresistant cells exhibited lower Dox internalization levels followed by alteration of the IRE1 and PERK arms of the UPR and triggering of the antioxidant Nrf2 axis. Critically, chemoresistant cells exhibited PDCD4 downregulation, which coincided with a reduction in eIF4A interaction, suggesting a sophisticated regulation of protein translation. Likewise, Dox-induced chemoresistance was associated with alterations in cellular migration and invasion, which are key cancer hallmarks, coupled with changes in focal adhesion kinase (FAK) activation and secretion of matrix metalloproteinase-9 (MMP-9). Moreover, eIF4A knockdown via siRNA and its overexpression in chemoresistant cells suggested that eIF4A regulates FAK. Pro-atherogenic low-density lipoproteins (LDL) promoted cellular invasion in parental and chemoresistant cells in an MMP-9-dependent manner. Moreover, Dox only inhibited parental cell invasion. Significantly, chemoresistance was modulated by cryptotanshinone (Cry), a natural terpene purified from the roots of Salvia brandegeei. Cry and Dox co-exposure induced chemosensitization, connected with the Cry effect on eIF4A interaction. We further demonstrated the Cry binding capability on eIF4A and in silico assays suggest Cry inhibition on the RNA-processing domain. Therefore, strategic disruption of protein translation initiation is a druggable pathway by natural compounds during chemoresistance in TNBC. However, plasmatic LDL levels should be closely monitored throughout treatment.
Asunto(s)
Neoplasias de la Mama Triple Negativas , Humanos , Neoplasias de la Mama Triple Negativas/tratamiento farmacológico , Neoplasias de la Mama Triple Negativas/genética , Factor 4A Eucariótico de Iniciación/química , Factor 4A Eucariótico de Iniciación/genética , Factor 4A Eucariótico de Iniciación/metabolismo , Metaloproteinasa 9 de la Matriz/metabolismo , Resistencia a Antineoplásicos , Proteínas de Unión al ARN/metabolismo , Proteínas Reguladoras de la Apoptosis/metabolismo , Doxorrubicina/farmacología , Proteínas Serina-Treonina Quinasas/metabolismoRESUMEN
Previous investigations of the Leishmania infantum eIF4A-like protein (LieIF4A) as a potential drug target delivered cholestanol derivatives inhibitors. Here, we investigated the mode of action of cholesterol derivatives as a novel scaffold structure of LieIF4A inhibitors on the RNA-dependent ATPase activity of LieIF4A and its mammalian ortholog (eIF4AI). We compared their biochemical effects on RNA-dependent ATPase activities of both proteins and investigated if rocaglamide, a known inhibitor of eIF4A, could affect LieIF4A as well. Kinetic measurements were conducted at different concentrations of ATP, of the compound and in the presence of saturating whole yeast RNA concentrations. Kinetic analyses showed different ATP binding affinities for the two enzymes as well as different sensitivities to 7-α-aminocholesterol and rocaglamide. The 7-α-aminocholesterol inhibited LieIF4A with a higher binding affinity relative to cholestanol analogs. Cholesterol, another tested sterol, had no effect on the ATPase activity of LieIF4A or eIF4AI. The 7-α-aminocholesterol demonstrated an anti-Leishmania activity on L. infantum promastigotes. Additionally, docking simulations explained the importance of the double bond between C5 and C6 in 7-α-aminocholesterol and the amino group in the C7 position. In conclusion, Leishmania and mammalian eIF4A proteins appeared to interact differently with effectors, thus making LieIF4A a potential drug against leishmaniases.
Asunto(s)
Factor 4A Eucariótico de Iniciación , Leishmania infantum , Adenosina Trifosfatasas/metabolismo , Adenosina Trifosfato/metabolismo , Animales , Colestanoles/metabolismo , Colesterol/metabolismo , Factor 4A Eucariótico de Iniciación/química , Factor 4A Eucariótico de Iniciación/genética , Factor 4A Eucariótico de Iniciación/metabolismo , Mamíferos/metabolismo , Ratones , Proteínas/metabolismo , ARN/metabolismo , Esteroles/metabolismo , Esteroles/farmacologíaRESUMEN
BACKGROUND: Deregulated translation initiation is implicated extensively in cancer initiation and progression. It is actively pursued as a viable target that circumvents the dependency on oncogenic signaling, a significant factor in current strategies. Eukaryotic translation initiation factor (eIF) 4A plays an essential role in translation initiation by unwinding the secondary structure of messenger RNA (mRNA) upstream of the start codon, enabling active ribosomal recruitment on the downstream genes. Several natural product molecules with similar scaffolds, such as Rocaglamide A (RocA), targeting eIF4A have been reported in the last decade. However, their clinical utilization is still elusive due to several pharmacological limitations. In this study we identified new eIF4A1 inhibitors and their possible mechanisms. METHODS: In this report, we conducted a pharmacophore-based virtual screen of RocA complexed with eIF4A and a polypurine RNA strand for novel eIF4A inhibitors from commercially available compounds in the MolPort Database. We performed target-based screening and optimization of active pharmacophores. We assessed the effects of novel compounds on biochemical and cell-based assays for efficacy and mechanistic evaluation. RESULTS: We validated three new potent eIF4A inhibitors, RBF197, RBF 203, and RBF 208, which decreased diffuse large B-cell lymphoma (DLBCL) cell viability. Biochemical and cellular studies, molecular docking, and functional assays revealed that thosenovel compounds clamp eIF4A into mRNA in an ATP-independent manner. Moreover, we found that RBF197 and RBF208 significantly depressed eIF4A-dependent oncogene expression as well as the colony formation capacity of DLBCL. Interestingly, exposure of these compounds to non-malignant cells had only minimal impact on their growth and viability. CONCLUSIONS: Identified compounds suggest a new strategy for designing novel eIF4A inhibitors.
Asunto(s)
Linfoma , Neoplasias , Factor 4A Eucariótico de Iniciación/química , Factor 4A Eucariótico de Iniciación/genética , Factor 4A Eucariótico de Iniciación/metabolismo , Humanos , Linfoma/tratamiento farmacológico , Simulación del Acoplamiento Molecular , ARN Mensajero/metabolismoRESUMEN
During translation initiation, the underlying mechanism by which the eukaryotic initiation factor (eIF) 4E, eIF4A, and eIF4G components of eIF4F coordinate their binding activities to regulate eIF4F binding to mRNA is poorly defined. Here, we used fluorescence anisotropy to generate thermodynamic and kinetic frameworks for the interaction of uncapped RNA with human eIF4F. We demonstrate that eIF4E binding to an autoinhibitory domain in eIF4G generates a high-affinity binding conformation of the eIF4F complex for RNA. In addition, we show that the nucleotide-bound state of the eIF4A component further regulates uncapped RNA binding by eIF4F, with a four-fold decrease in the equilibrium dissociation constant observed in the presence versus the absence of ATP. Monitoring uncapped RNA dissociation in real time reveals that ATP reduces the dissociation rate constant of RNA for eIF4F by â¼4-orders of magnitude. Thus, release of ATP from eIF4A places eIF4F in a dynamic state that has very fast association and dissociation rates from RNA. Monitoring the kinetic framework for eIF4A binding to eIF4G revealed two different rate constants that likely reflect two conformational states of the eIF4F complex. Furthermore, we determined that the eIF4G autoinhibitory domain promotes a more stable, less dynamic, eIF4A-binding state, which is overcome by eIF4E binding. Overall, our data support a model whereby eIF4E binding to eIF4G/4A stabilizes a high-affinity RNA-binding state of eIF4F and enables eIF4A to adopt a more dynamic interaction with eIF4G. This dynamic conformation may contribute to the ability of eIF4F to rapidly bind and release mRNA during scanning.
Asunto(s)
Factor 4A Eucariótico de Iniciación , Factor 4E Eucariótico de Iniciación , Humanos , Adenosina Trifosfato/metabolismo , Factor 4A Eucariótico de Iniciación/química , Factor 4E Eucariótico de Iniciación/química , Factor 4F Eucariótico de Iniciación/química , Factor 4G Eucariótico de Iniciación/química , Nucleótidos/química , Unión Proteica , ARN Mensajero/metabolismoRESUMEN
Ribonucleoprotein granules are ubiquitous features of eukaryotic cells. Several observations argue that the formation of at least some RNP granules can be considered analogous to the formation of unfolded protein aggregates. First, unfolded protein aggregates form from the exposure of promiscuous protein interaction surfaces, while some mRNP granules form, at least in part, by promiscuous intermolecular RNA-RNA interactions due to exposed RNA surfaces when mRNAs are not engaged with ribosomes. Second, analogous to the role of protein chaperones in preventing misfolded protein aggregation, cells contain abundant "RNA chaperones" to limit inappropriate RNA-RNA interactions and prevent mRNP granule formation. Third, analogous to the role of protein aggregates in diseases, situations where RNA aggregation exceeds the capacity of RNA chaperones to disaggregate RNAs may contribute to human disease. Understanding that RNP granules can be considered as promiscuous, reversible RNA aggregation events allow insight into their composition and how cells have evolved functions for RNP granules.
Asunto(s)
Condensados Biomoleculares/química , Proteínas de Choque Térmico/química , Chaperonas Moleculares/química , ARN Mensajero/química , Ribonucleoproteínas/química , Gránulos de Estrés/química , Condensados Biomoleculares/metabolismo , Eucariontes , Células Eucariotas/metabolismo , Factor 4A Eucariótico de Iniciación/química , Factor 4A Eucariótico de Iniciación/genética , Factor 4A Eucariótico de Iniciación/metabolismo , Floculación , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Iniciación de la Cadena Peptídica Traduccional , Agregado de Proteínas , Pliegue de Proteína , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ribonucleoproteínas/genética , Ribonucleoproteínas/metabolismo , Ribosomas/genética , Ribosomas/metabolismo , Gránulos de Estrés/genética , Gránulos de Estrés/metabolismoRESUMEN
Interfacial inhibitors exert their biological effects through co-association with two macromolecules. The pateamine A (PatA) class of molecules function by stabilizing eukaryotic initiation factor (eIF) 4A RNA helicase onto RNA, resulting in translation initiation inhibition. Here, we present the crystal structure of an eIF4A1:RNA complex bound to an analog of the marine sponge-derived natural product PatA, C5-desmethyl PatA (DMPatA). One end of this small molecule wedges itself between two RNA bases while the other end is cradled by several protein residues. Strikingly, DMPatA interacts with the eIF4A1:RNA complex in an almost identical fashion as rocaglamide A (RocA), despite being completely unrelated from a structural standpoint. The structural data rationalize the ability of PatA analogs to target a wider range of RNA substrates compared to RocA. We define the molecular basis of how DMPatA is able to clamp eIF4A1 onto RNA, imparting potent inhibitory properties to this molecule.
Asunto(s)
Compuestos Epoxi/química , Factor 4A Eucariótico de Iniciación/química , Macrólidos/química , ARN/química , Tiazoles/química , Línea Celular , Cristalografía por Rayos X , Humanos , Modelos Moleculares , Conformación MolecularRESUMEN
Enrichment of angiomotin (AMOT) in the ectoplacental cone of E7.5 murine placenta prompted our investigation on the role of AMOT in trophoblast differentiation. We show here that AMOT levels increased in mouse placenta during gestation and also upon induction of differentiation in trophoblast stem cell ex vivo. Proteomic data unravelling AMOT-interactome in trophoblast cells indicated a majority of AMOT interactors to be involved in protein translation. In-depth analysis of AMOT-interactome led to identification of eukaryotic translation initiation factor 4A (eIF4A) as the most plausible AMOT interactor. Loss of function of AMOT enhanced, whereas, gain in function resulted in decline of global protein synthesis in trophoblast cells. Bioinformatics analysis evaluating the potential energy of AMOT-eIF4A binding suggested a strong AMOT-eIF4A interaction using a distinct groove encompassing amino acid residue positions 238 to 255 of AMOT. Co-immunoprecipitation of AMOT with eIF4A reaffirmed AMOT-eIF4A association in trophoblast cells. Deletion of 238 to 255 amino acids of AMOT resulted in abrogation of AMOT-eIF4A interaction. In addition, 238 to 255 amino acid deletion of AMOT was ineffective in eliciting AMOT's function in reducing global protein synthesis. Interestingly, AMOT-dependent sequestration of eIF4A dampened its loading to the m7 -GTP cap and hindered its interaction with eIF4G. Furthermore, enhanced AMOT expression in placenta was associated with intrauterine growth restriction in both rats and humans. These results not only highlight a hitherto unknown novel function of AMOT in trophoblast cells but also have broad biological implications as AMOT might be an inbuilt switch to check protein synthesis in developmentally indispensable trophoblast cells.
Asunto(s)
Angiomotinas/biosíntesis , Factor 4A Eucariótico de Iniciación/biosíntesis , Biosíntesis de Proteínas/fisiología , Trofoblastos/metabolismo , Angiomotinas/química , Angiomotinas/genética , Animales , Células Cultivadas , Factor 4A Eucariótico de Iniciación/química , Factor 4A Eucariótico de Iniciación/genética , Femenino , Células Hep G2 , Humanos , Ratones , Placenta/citología , Placenta/metabolismo , Embarazo , Estructura Terciaria de Proteína , Ratas , Ratas Sprague-DawleyRESUMEN
Protein-protein interactions (PPIs) are an essential part of correct cellular functionality, making them increasingly interesting drug targets. While Förster resonance energy transfer-based methods have traditionally been widely used for PPI studies, label-free techniques have recently drawn significant attention. These methods are ideal for studying PPIs, most importantly as there is no need for labeling of either interaction partner, reducing potential interferences and overall costs. Already, several different label-free methods are available, such as differential scanning calorimetry and surface plasmon resonance, but these biophysical methods suffer from low to medium throughput, which reduces suitability for high-throughput screening (HTS) of PPI inhibitors. Differential scanning fluorimetry, utilizing external fluorescent probes, is an HTS compatible technique, but high protein concentration is needed for experiments. To improve the current concepts, we have developed a method based on time-resolved luminescence, enabling PPI monitoring even at low nanomolar protein concentrations. This method, called the protein probe technique, is based on a peptide conjugated with Eu3+ chelate, and it has already been applied to monitor protein structural changes and small molecule interactions at elevated temperatures. Here, the applicability of the protein probe technique was demonstrated by monitoring single-protein pairing and multiprotein complexes at room and elevated temperatures. The concept functionality was proven by using both artificial and multiple natural protein pairs, such as KRAS and eIF4A together with their binding partners, and C-reactive protein in a complex with its antibody.
Asunto(s)
Quelantes/química , Factor 4A Eucariótico de Iniciación/química , Europio/química , Péptidos/química , Proteínas Proto-Oncogénicas p21(ras)/química , Calorimetría , Transferencia Resonante de Energía de Fluorescencia , Humanos , Unión Proteica , Conformación Proteica , Resonancia por Plasmón de SuperficieRESUMEN
Human CWC27 is an uncharacterized splicing factor and mutations in its gene are linked to retinal degeneration and other developmental defects. We identify the splicing factor CWC22 as the major CWC27 partner. Both CWC27 and CWC22 are present in published Bact spliceosome structures, but no interacting domains are visible. Here, the structure of a CWC27/CWC22 heterodimer bound to the exon junction complex (EJC) core component eIF4A3 is solved at 3Å-resolution. According to spliceosomal structures, the EJC is recruited in the C complex, once CWC27 has left. Our 3D structure of the eIF4A3/CWC22/CWC27 complex is compatible with the Bact spliceosome structure but not with that of the C complex, where a CWC27 loop would clash with the EJC core subunit Y14. A CWC27/CWC22 building block might thus form an intermediate landing platform for eIF4A3 onto the Bact complex prior to its conversion into C complex. Knock-down of either CWC27 or CWC22 in immortalized retinal pigment epithelial cells affects numerous common genes, indicating that these proteins cooperate, targeting the same pathways. As the most up-regulated genes encode factors involved in inflammation, our findings suggest a possible link to the retinal degeneration associated with CWC27 deficiencies.
Asunto(s)
Ciclofilinas/química , Factor 4A Eucariótico de Iniciación/química , Proteínas de Unión al ARN/química , Empalmosomas/química , Línea Celular , Ciclofilinas/genética , Ciclofilinas/metabolismo , Factor 4A Eucariótico de Iniciación/metabolismo , Exones , Técnicas de Silenciamiento del Gen , Células HeLa , Humanos , Inflamación/genética , Modelos Moleculares , Dominios Proteicos , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Epitelio Pigmentado de la Retina/metabolismo , Empalmosomas/metabolismoRESUMEN
The PI3K/Akt/mTOR kinase pathway is extensively deregulated in human cancers. One critical node under regulation of this signaling axis is eukaryotic initiation factor (eIF) 4F, a complex involved in the control of translation initiation rates. eIF4F-dependent addictions arise during tumor initiation and maintenance due to increased eIF4F activity-generally in response to elevated PI3K/Akt/mTOR signaling flux. There is thus much interest in exploring eIF4F as a small molecule target for the development of new anticancer drugs. The DEAD-box RNA helicase, eIF4A, is an essential subunit of eIF4F, and several potent small molecules (rocaglates, hippuristanol, pateamine A) affecting its activity have been identified and shown to demonstrate anticancer activity in vitro and in vivo in preclinical models. Recently, a number of new small molecules have been reported as having the capacity to target and inhibit eIF4A. Here, we undertook a comparative analysis of their biological activity and specificity relative to the eIF4A inhibitor, hippuristanol.
Asunto(s)
Antineoplásicos/química , Factor 4A Eucariótico de Iniciación/química , Neoplasias/tratamiento farmacológico , Bibliotecas de Moléculas Pequeñas/química , Esteroles/química , Antineoplásicos/farmacología , Benzofuranos/química , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Compuestos Epoxi/química , Factor 4A Eucariótico de Iniciación/antagonistas & inhibidores , Factor 4F Eucariótico de Iniciación/antagonistas & inhibidores , Factor 4F Eucariótico de Iniciación/química , Humanos , Macrólidos/química , Neoplasias/genética , Fosfatidilinositol 3-Quinasas/genética , Biosíntesis de Proteínas/efectos de los fármacos , Proteínas Proto-Oncogénicas c-akt/genética , Bibliotecas de Moléculas Pequeñas/farmacología , Esteroles/farmacología , Serina-Treonina Quinasas TOR/genética , Tiazoles/químicaRESUMEN
Translation initiation factor eIF4F is essential for cap-dependent translation initiation in eukaryotes. eIF4F is a trimeric complex consisting of a scaffold protein eIF4G, cap-binding protein eIF4E and DEAD-box RNA helicase eIF4A. eIF4F binds to the 5' cap structure of the mRNA through eIF4E and facilitates the binding of the preinitiation complex (PIC) via protein-protein interactions of eIF4G with eIF3 in mammals or with eIF5 in yeast. Initiation factor eIF4A is known to unwind the secondary structures of the 5'UTRs encountered by the PIC during its initial binding to the mRNA and while scanning for the initiation codon. In Giardia, homologs for eIF4E (GleIF4E2) and eIF4A (GleIF4A) have been identified but not for eIF4G. To address how PIC is recruited to the 5' end of mRNA in the absence of eIF4G homolog, we have used yeast two-hybrid assays to identify potential interactions of GleIF4E2 with the components of the PIC. The results show that GleIF4E2 can interact with the ß subunit of the initiation factor GleIF2, a component of the PIC. ZDOCK modeling of the GleIF4E2-GleIF2ß complex revealed that the dorsal side of GleIF4E2 is likely involved in binding to GleIF2ß, which mimics the interaction of mammalian eIF4E with eIF4G, and with eIF4E binding proteins. These results suggest that GleIF4E2 can facilitate the recruitment of the PIC to the 5'end of the mRNA by binding directly to the components of the PIC. The role of GleIF4A in translation initiation in Giardia is not clearly understood as the short 5' UTRs of the mRNA are unlikely to form secondary structures. Interestingly, Pateamine A, a specific inhibitor of human eIF4A, inhibited the growth of Giardia in a dose-dependent manner, suggesting that the activity of GleIF4A is probably required for translation. Using yeast two-hybrid assays, we have identified a novel interaction of GleIF4A with i subunit of the initiation factor GleIF3 (GleIF3i), another component of the PIC. These results indicate that the GleIF4A can also interact directly with the components of the PIC. ZDOCK modeling of the GleIF3i-GleIF4A complex suggests that GleIF3i could serve as a stimulator of GleIF4A activity.
Asunto(s)
Factor 4A Eucariótico de Iniciación , Factor 4E Eucariótico de Iniciación , Biosíntesis de Proteínas/fisiología , Línea Celular Transformada , Codón Iniciador/metabolismo , Factor 4A Eucariótico de Iniciación/química , Factor 4A Eucariótico de Iniciación/metabolismo , Factor 4E Eucariótico de Iniciación/química , Factor 4E Eucariótico de Iniciación/metabolismo , Factor 4G Eucariótico de Iniciación/química , Factor 4G Eucariótico de Iniciación/metabolismo , Giardia lamblia/genética , Humanos , Modelos Estructurales , Unión Proteica , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismoRESUMEN
During gene expression, RNA export factors are mainly known for driving nucleo-cytoplasmic transport. While early studies suggested that the exon junction complex (EJC) provides a binding platform for them, subsequent work proposed that they are only recruited by the cap binding complex to the 5' end of RNAs, as part of TREX. Using iCLIP, we show that the export receptor Nxf1 and two TREX subunits, Alyref and Chtop, are recruited to the whole mRNA co-transcriptionally via splicing but before 3' end processing. Consequently, Alyref alters splicing decisions and Chtop regulates alternative polyadenylation. Alyref is recruited to the 5' end of RNAs by CBC, and our data reveal subsequent binding to RNAs near EJCs. We demonstrate that eIF4A3 stimulates Alyref deposition not only on spliced RNAs close to EJC sites but also on single-exon transcripts. Our study reveals mechanistic insights into the co-transcriptional recruitment of mRNA export factors and how this shapes the human transcriptome.
Asunto(s)
ARN Helicasas DEAD-box/genética , Factor 4A Eucariótico de Iniciación/genética , Proteínas Nucleares/genética , Proteínas de Transporte Nucleocitoplasmático/genética , Proteínas de Unión al ARN/genética , Factores de Transcripción/genética , Transcripción Genética , Transporte Activo de Núcleo Celular/genética , Sitios de Unión , ARN Helicasas DEAD-box/química , Factor 4A Eucariótico de Iniciación/química , Exones/genética , Regulación de la Expresión Génica/genética , Humanos , Proteínas Nucleares/química , Proteínas de Transporte Nucleocitoplasmático/química , Poliadenilación , Transporte de ARN/genética , ARN Mensajero/química , ARN Mensajero/genética , Proteínas de Unión al ARN/química , Factores de Transcripción/química , Transcriptoma/genéticaRESUMEN
The viability of chronic lymphocytic leukemia (CLL) is critically dependent upon staving off death by apoptosis, a hallmark of CLL pathophysiology. The recognition that Mcl-1, a major component of the anti-apoptotic response, is intrinsically short-lived and must be continually resynthesized suggested a novel therapeutic approach. Pateamine A (PatA), a macrolide marine natural product, inhibits cap-dependent translation by binding to the initiation factor eIF4A. In this study, we demonstrated that a synthetic derivative of PatA, des-methyl des-amino PatA (DMDAPatA), blocked mRNA translation, reduced Mcl-1 protein and initiated apoptosis in CLL cells. This action was synergistic with the Bcl-2 antagonist ABT-199. However, avid binding to human plasma proteins limited DMDAPatA potency, precluding further development. To address this, we synthesized a new series of PatA analogs and identified three new leads with potent inhibition of translation. They exhibited less plasma protein binding and increased cytotoxic potency toward CLL cells than DMDAPatA, with greater selectivity towards CLL cells over normal lymphocytes. Computer modeling analysis correlated their structure-activity relationships and suggested that these compounds may act by stabilizing the closed conformation of eIF4A. Thus, these novel PatA analogs hold promise for application to cancers within the appropriate biological context, such as CLL.
Asunto(s)
Apoptosis/efectos de los fármacos , Compuestos Bicíclicos Heterocíclicos con Puentes/farmacología , Compuestos Epoxi/farmacología , Leucemia Linfocítica Crónica de Células B/tratamiento farmacológico , Macrólidos/farmacología , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/antagonistas & inhibidores , Biosíntesis de Proteínas/efectos de los fármacos , Proteínas Proto-Oncogénicas c-bcl-2/antagonistas & inhibidores , Sulfonamidas/farmacología , Tiazoles/farmacología , Anciano , Anciano de 80 o más Años , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/metabolismo , Sinergismo Farmacológico , Quimioterapia Combinada , Factor 4A Eucariótico de Iniciación/química , Femenino , Estudios de Seguimiento , Regulación Neoplásica de la Expresión Génica , Humanos , Leucemia Linfocítica Crónica de Células B/metabolismo , Leucemia Linfocítica Crónica de Células B/patología , Masculino , Persona de Mediana Edad , Modelos Moleculares , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/genética , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/metabolismo , Pronóstico , Conformación Proteica , ARN Mensajero/antagonistas & inhibidores , ARN Mensajero/genética , ARN Mensajero/metabolismo , Células Tumorales CultivadasRESUMEN
Exon junction complexes (EJCs) loaded onto spliced mRNAs during splicing serve as molecular markers for various post-transcriptional gene-regulatory processes, including nonsense-mediated mRNA decay (NMD). Although the composition and structure of EJCs are well characterized, the mechanism regulating EJC deposition remains unknown. Here we find that threonine 163 (T163) within the RNA-binding motif of eIF4A3 (a core EJC component) is phosphorylated by cyclin-dependent protein kinases 1 and 2 in a cell cycle-dependent manner. T163 phosphorylation hinders binding of eIF4A3 to spliced mRNAs and other EJC components. Instead, it promotes association of eIF4A3 with CWC22, which guides eIF4A3 to an active spliceosome. These molecular events ensure the fidelity of specific deposition of the EJC â¼20-24 nt upstream of an exon-exon junction. Accordingly, NMD is affected by T163 phosphorylation. Collectively, our data provide evidence that T163 phosphorylation affects EJC formation and, consequently, NMD efficiency in a cell cycle-dependent manner.
Asunto(s)
Proteína Quinasa CDC2/metabolismo , Ciclo Celular , Quinasa 2 Dependiente de la Ciclina/metabolismo , ARN Helicasas DEAD-box/metabolismo , Factor 4A Eucariótico de Iniciación/metabolismo , Degradación de ARNm Mediada por Codón sin Sentido , Sitios de Unión , ARN Helicasas DEAD-box/química , ARN Helicasas DEAD-box/genética , Factor 4A Eucariótico de Iniciación/química , Factor 4A Eucariótico de Iniciación/genética , Células HEK293 , Células HeLa , Humanos , Mutación , Fosforilación , Unión Proteica , ARN Mensajero/metabolismoRESUMEN
A class of translation inhibitors, exemplified by the natural product rocaglamide A (RocA), isolated from Aglaia genus plants, exhibits antitumor activity by clamping eukaryotic translation initiation factor 4A (eIF4A) onto polypurine sequences in mRNAs. This unusual inhibitory mechanism raises the question of how the drug imposes sequence selectivity onto a general translation factor. Here, we determined the crystal structure of the human eIF4A1â ATP analogâ RocAâ polypurine RNA complex. RocA targets the "bi-molecular cavity" formed characteristically by eIF4A1 and a sharply bent pair of consecutive purines in the RNA. Natural amino acid substitutions found in Aglaia eIF4As changed the cavity shape, leading to RocA resistance. This study provides an example of an RNA-sequence-selective interfacial inhibitor fitting into the space shaped cooperatively by protein and RNA with specific sequences.
Asunto(s)
Benzofuranos/metabolismo , Factor 4A Eucariótico de Iniciación/metabolismo , Biosíntesis de Proteínas , Inhibidores de la Síntesis de la Proteína/metabolismo , ARN/metabolismo , Ribosomas/metabolismo , Adenilil Imidodifosfato/química , Adenilil Imidodifosfato/metabolismo , Aglaia/química , Aglaia/genética , Aglaia/metabolismo , Sustitución de Aminoácidos , Benzofuranos/química , Benzofuranos/aislamiento & purificación , Benzofuranos/farmacología , Sitios de Unión , Resistencia a Medicamentos/genética , Factor 4A Eucariótico de Iniciación/química , Factor 4A Eucariótico de Iniciación/genética , Células HEK293 , Humanos , Modelos Moleculares , Estructura Molecular , Mutación , Proteínas de Plantas/química , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Unión Proteica , Biosíntesis de Proteínas/efectos de los fármacos , Biosíntesis de Proteínas/genética , Dominios y Motivos de Interacción de Proteínas , Inhibidores de la Síntesis de la Proteína/química , Inhibidores de la Síntesis de la Proteína/aislamiento & purificación , Inhibidores de la Síntesis de la Proteína/farmacología , ARN/química , Ribosomas/química , Ribosomas/efectos de los fármacos , Ribosomas/genética , Relación Estructura-ActividadRESUMEN
The antifungal agent 6-aminocholestanol targets the production of ergosterol, which is the principle sterol in many fungi and protozoans; ergosterol serves many of the same roles as cholesterol in animals. We found that it also is an effective inhibitor of the translation-initiation factor eIF4AI from mouse (eIF4AIMus) and the Trypanosomatid parasite Leishmania (LieIF4A). The eIF4A proteins belong to the DEAD-box family of RNA helicases, which are ATP-dependent RNA-binding proteins and RNA-dependent ATPases. DEAD-box proteins contain a commonly-shared core structure consisting of two linked domains with structural homology to that of recombinant protein A (RecA) and that contain conserved motifs that are involved in RNA and ATP binding, and in the enzymatic activity. The compound inhibits both the ATPase and helicase activities by perturbing ATP and RNA binding, and it is capable of binding other proteins containing nucleic acid-binding sites as well. We undertook kinetic analyses and found that the Leishmania LieIF4A protein binds 6-aminocholestanol with a higher apparent affinity than for ATP, although multiple binding sites were probably involved. Competition experiments with the individual RecA-like domains indicate that the primary binding sites are on RecA-like domain 1, and they include a cavity that we previously identified by molecular modeling of LieIF4A that involve conserved RNA-binding motifs. The compound affects the mammalian and Leishmania proteins differently, which indicates the binding sites and affinities are not the same. Thus, it is possible to develop drugs that target DEAD-box proteins from different organisms even when they are implicated in the same biological process.
Asunto(s)
Adenosina Trifosfato/antagonistas & inhibidores , Colesterol/análogos & derivados , Factor 4A Eucariótico de Iniciación/química , Leishmania infantum/efectos de los fármacos , Proteínas Protozoarias/química , ARN de Helminto/antagonistas & inhibidores , Tripanocidas/química , Adenosina Trifosfato/química , Adenosina Trifosfato/metabolismo , Secuencia de Aminoácidos , Animales , Antifúngicos/química , Antifúngicos/farmacología , Sitios de Unión , Unión Competitiva , Colesterol/química , Colesterol/farmacología , Clonación Molecular , Secuencia Conservada , Reposicionamiento de Medicamentos , Escherichia coli/genética , Escherichia coli/metabolismo , Factor 4A Eucariótico de Iniciación/genética , Factor 4A Eucariótico de Iniciación/metabolismo , Expresión Génica , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Humanos , Cinética , Leishmania infantum/enzimología , Leishmania infantum/genética , Leishmania infantum/crecimiento & desarrollo , Ratones , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Estructura Secundaria de Proteína , Proteínas Protozoarias/genética , Proteínas Protozoarias/metabolismo , ARN de Helminto/química , ARN de Helminto/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Tripanocidas/farmacologíaRESUMEN
The marine natural product pateamine A (1) and its somewhat simplified designer analogue DMDA-Pat A (2) (DMDA = desmethyl-desamino) are potently cytotoxic compounds; most notably, 2 had previously been found to exhibit a promising differential in vivo activity in xenograft melanoma models, even though the ubiquitous eukaryotic initiation factor 4A (eIF4A) constitutes its primary biological target. In addition, 1 had also been identified as a possible lead in the quest for medication against cachexia, an often lethal muscle wasting syndrome affecting many immunocompromised or cancer patients. The short supply of these macrodiolides, however, rendered a more detailed biological assessment difficult. Therefore, a new synthetic approach to 1 and 2 has been devised, which centers on an unorthodox strategy for the formation of the highly isomerization-prone but essential Z, E-configured dienoate substructure embedded into the macrocyclic core. This motif was encoded in the form of a 2-pyrone ring and unveiled only immediately before macrocyclization by an unconventional iron-catalyzed ring opening/cross-coupling reaction, in which the enol ester entity of the pyrone gains the role of a leaving group. Since the required precursor was readily available by gold catalysis, this strategy rendered the overall sequence short, robust, and scalable. A surprisingly easy protecting group management together with a much improved end game for the formation of the trienyl side chain via a modern Stille coupling protocol also helped to make the chosen route practical. Change of a single building block allowed the synthesis to be redirected from the natural lead compound 1 toward its almost equipotent analogue 2. Isolation and reactivity profiling of pyrone tricarbonyliron complexes provide mechanistic information as well as insights into the likely origins of the observed chemoselectivity.