RESUMO
Oncogenic intercellular signaling is regulated by extracellular vesicles (EVs), but the underlying mechanisms remain mostly unclear. Since TCTP (translationally controlled tumor protein) is an EV component, we investigated whether it has a role in genotoxic stress signaling and malignant transformation. By generating a Tctp-inducible knockout mouse model (Tctp-/f-), we report that Tctp is required for genotoxic stress-induced apoptosis signaling via small EVs (sEVs). Human breast cancer cells knocked-down for TCTP show impaired spontaneous EV secretion, thereby reducing sEV-dependent malignant growth. Since Trp53-/- mice are prone to tumor formation, we derived tumor cells from Trp53-/-;Tctp-/f- double mutant mice and describe a drastic decrease in tumori-genicity with concomitant decrease in sEV secretion and content. Remarkably, Trp53-/-;Tctp-/f- mice show highly prolonged survival. Treatment of Trp53-/- mice with sertraline, which inhibits TCTP function, increases their survival. Mechanistically, TCTP binds DDX3, recruiting RNAs, including miRNAs, to sEVs. Our findings establish TCTP as an essential protagonist in the regulation of sEV-signaling in the context of apoptosis and tumorigenicity.
Assuntos
Biomarcadores Tumorais , Neoplasias , Camundongos , Humanos , Animais , Biomarcadores Tumorais/metabolismo , Neoplasias/patologia , Apoptose , Transdução de SinaisRESUMO
The DEAD-box RNA helicase Ded1 is an essential yeast protein involved in translation initiation that belongs to the DDX3 subfamily. The purified Ded1 protein is an ATP-dependent RNA-binding protein and an RNA-dependent ATPase, but it was previously found to lack substrate specificity and enzymatic regulation. Here we demonstrate through yeast genetics, yeast extract pull-down experiments, in situ localization, and in vitro biochemical approaches that Ded1 is associated with, and regulated by, the signal recognition particle (SRP), which is a universally conserved ribonucleoprotein complex required for the co-translational translocation of polypeptides into the endoplasmic reticulum lumen and membrane. Ded1 is physically associated with SRP components in vivo and in vitro. Ded1 is genetically linked with SRP proteins. Finally, the enzymatic activity of Ded1 is inhibited by SRP21 in the presence of SCR1 RNA. We propose a model where Ded1 actively participates in the translocation of proteins during translation. Our results provide a new understanding of the role of Ded1 during translation.
Assuntos
RNA Helicases DEAD-box , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Partícula de Reconhecimento de Sinal , Partícula de Reconhecimento de Sinal/metabolismo , Partícula de Reconhecimento de Sinal/genética , RNA Helicases DEAD-box/metabolismo , RNA Helicases DEAD-box/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Ligação Proteica , Biossíntese de Proteínas , Transporte ProteicoRESUMO
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.
Assuntos
Fator de Iniciação 4A em Eucariotos , Leishmania infantum , Adenosina Trifosfatases/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Colestanóis/metabolismo , Colesterol/metabolismo , Fator de Iniciação 4A em Eucariotos/química , Fator de Iniciação 4A em Eucariotos/genética , Fator de Iniciação 4A em Eucariotos/metabolismo , Mamíferos/metabolismo , Camundongos , Proteínas/metabolismo , RNA/metabolismo , Esteróis/metabolismo , Esteróis/farmacologiaRESUMO
DEAD-box helicases are involved in all steps of RNA metabolism. They are ATP-dependent RNA binding proteins and RNA-dependent ATPases. They can displace short duplexes, but they lack processivity. Their mechanism and functioning are not clearly understood; classical or bulk biochemical assays are not sufficient to answer these questions. Single-molecule techniques provide useful tools, but they are limited in cases where the proteins are nonprocessive and give weak signals. We present here a new, magnetic-tweezers-based, single-molecule assay that is simple and that can sensitively measure the displacement time of a small, hybridized, RNA oligonucleotide. Tens of molecules can be analyzed at the same time. Comparing the displacement times with and without a helicase gives insights into the enzymatic activity of the protein. We used this assay to study yeast Ded1, which is orthologous to human DDX3. Although Ded1 acts on a variety of substrates, we find that Ded1 requires an RNA substrate for its ATP-dependent unwinding activity and that ATP hydrolysis is needed to see this activity. Further, we find that only intramolecular single-stranded RNA extensions enhance this activity. We propose a model where ATP-bound Ded1 stabilizes partially unwound duplexes and where multiple binding events may be needed to see displacement.
Assuntos
RNA Helicases DEAD-box/química , RNA/química , Proteínas de Saccharomyces cerevisiae/química , Adenosina Trifosfatases/química , Adenosina Trifosfatases/genética , Trifosfato de Adenosina/química , Trifosfato de Adenosina/genética , Sequência de Aminoácidos/genética , RNA Helicases DEAD-box/genética , Humanos , Fenômenos Mecânicos , RNA/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
The DEAD-box helicase Ded1 is an essential yeast protein that is closely related to mammalian DDX3 and to other DEAD-box proteins involved in developmental and cell cycle regulation. Ded1 is considered to be a translation-initiation factor that helps the 40S ribosome scan the mRNA from the 5' 7-methylguanosine cap to the AUG start codon. We used IgG pull-down experiments, mass spectrometry analyses, genetic experiments, sucrose gradients, in situ localizations and enzymatic assays to show that Ded1 is a cap-associated protein that actively shuttles between the cytoplasm and the nucleus. NanoLC-MS/MS analyses of purified complexes show that Ded1 is present in both nuclear and cytoplasmic mRNPs. Ded1 physically interacts with purified components of the nuclear CBC and the cytoplasmic eIF4F complexes, and its enzymatic activity is stimulated by these factors. In addition, we show that Ded1 is genetically linked to these factors. Ded1 comigrates with these proteins on sucrose gradients, but treatment with rapamycin does not appreciably alter the distribution of Ded1; thus, most of the Ded1 is in stable mRNP complexes. We conclude that Ded1 is an mRNP cofactor of the cap complex that may function to remodel the different mRNPs and thereby regulate the expression of the mRNAs.
Assuntos
Núcleo Celular/enzimologia , Citoplasma/enzimologia , RNA Helicases DEAD-box/metabolismo , Capuzes de RNA/metabolismo , Ribonucleoproteínas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Transporte Ativo do Núcleo Celular , Adenosina Trifosfatases/metabolismo , RNA Helicases DEAD-box/genética , Guanosina/análogos & derivados , Guanosina/metabolismo , Biossíntese de Proteínas , Ribonucleoproteínas/genética , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
DEAD-box RNA helicases are ATP-dependent RNA binding proteins and RNA-dependent ATPases that possess weak, nonprocessive unwinding activity in vitro, but they can form long-lived complexes on RNAs when the ATPase activity is inhibited. Ded1 is a yeast DEAD-box protein, the functional ortholog of mammalian DDX3, that is considered important for the scanning efficiency of the 48S pre-initiation complex ribosomes to the AUG start codon. We used a modified PAR-CLIP technique, which we call quicktime PAR-CLIP (qtPAR-CLIP), to crosslink Ded1 to 4-thiouridine-incorporated RNAs in vivo using UV light centered at 365 nm. The irradiation conditions are largely benign to the yeast cells and to Ded1, and we are able to obtain a high efficiency of crosslinking under physiological conditions. We find that Ded1 forms crosslinks on the open reading frames of many different mRNAs, but it forms the most extensive interactions on relatively few mRNAs, and particularly on mRNAs encoding certain ribosomal proteins and translation factors. Under glucose-depletion conditions, the crosslinking pattern shifts to mRNAs encoding metabolic and stress-related proteins, which reflects the altered translation. These data are consistent with Ded1 functioning in the regulation of translation elongation, perhaps by pausing or stabilizing the ribosomes through its ATP-dependent binding.
Assuntos
Ribossomos , Saccharomyces cerevisiae , Animais , Saccharomyces cerevisiae/genética , Ribossomos/genética , Proteínas Ribossômicas , RNA , RNA Mensageiro , Proteínas Fúngicas , Proteínas de Choque Térmico , RNA Helicases DEAD-box/genética , Trifosfato de Adenosina/genética , MamíferosRESUMO
DEAD-box RNA helicases are ubiquitous proteins found in all kingdoms of life and that are associated with all processes involving RNA. Their central roles in biology make these proteins potential targets for therapeutic or prophylactic drugs. The Ded1/DDX3 subfamily of DEAD-box proteins is of particular interest because of their important role(s) in translation. In this paper, we identified and aligned the protein sequences of 28 different DEAD-box proteins from the kinetoplast-protozoan parasite Leishmania infantum, which is the cause of the visceral form of leishmaniasis that is often lethal if left untreated, and compared them with the consensus sequence derived from DEAD-box proteins in general, and from the Ded1/DDX3 subfamily in particular, from a wide variety of other organisms. We identified three potential homologs of the Ded1/DDX3 subfamily and the equivalent proteins from the related protozoan parasite Trypanosoma brucei, which is the causative agent of sleeping sickness. We subsequently tested these proteins for their ability to complement a yeast strain deleted for the essential DED1 gene. We found that the DEAD-box proteins from Trypanosomatids are highly divergent from other eukaryotes, and consequently they are suitable targets for protein-specific drugs.
Assuntos
RNA Helicases DEAD-box/genética , Proteínas de Saccharomyces cerevisiae/genética , Trypanosoma brucei brucei/genética , Tripanossomíase Africana/genética , Sequência de Aminoácidos/genética , Simulação por Computador , Humanos , Leishmania infantum/genética , Leishmania infantum/patogenicidade , Biossíntese de Proteínas/genética , RNA/genética , Saccharomyces cerevisiae/genética , Trypanosoma brucei brucei/patogenicidade , Tripanossomíase Africana/parasitologiaRESUMO
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.
Assuntos
Trifosfato de Adenosina/antagonistas & inibidores , Colesterol/análogos & derivados , Fator de Iniciação 4A em Eucariotos/química , Leishmania infantum/efeitos dos fármacos , Proteínas de Protozoários/química , RNA de Helmintos/antagonistas & inibidores , Tripanossomicidas/química , Trifosfato de Adenosina/química , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Animais , Antifúngicos/química , Antifúngicos/farmacologia , Sítios de Ligação , Ligação Competitiva , Colesterol/química , Colesterol/farmacologia , Clonagem Molecular , Sequência Conservada , Reposicionamento de Medicamentos , Escherichia coli/genética , Escherichia coli/metabolismo , Fator de Iniciação 4A em Eucariotos/genética , Fator de Iniciação 4A em Eucariotos/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Humanos , Cinética , Leishmania infantum/enzimologia , Leishmania infantum/genética , Leishmania infantum/crescimento & desenvolvimento , Camundongos , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Estrutura Secundária de Proteína , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , RNA de Helmintos/química , RNA de Helmintos/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Tripanossomicidas/farmacologiaRESUMO
Leishmaniases are neglected parasitic diseases in spite of the major burden they inflict on public health. The identification of novel drugs and targets constitutes a research priority. For that purpose we used Leishmania infantum initiation factor 4A (LieIF), an essential translation initiation factor that belongs to the DEAD-box proteins family, as a potential drug target. We modeled its structure and identified two potential binding sites. A virtual screening of a diverse chemical library was performed for both sites. The results were analyzed with an in-house version of the Self-Organizing Maps algorithm combined with multiple filters, which led to the selection of 305 molecules. Effects of these molecules on the ATPase activity of LieIF permitted the identification of a promising hit (208) having a half maximal inhibitory concentration (IC50) of 150 ± 15 µM for 1 µM of protein. Ten chemical analogues of compound 208 were identified and two additional inhibitors were selected (20 and 48). These compounds inhibited the mammalian eIF4I with IC50 values within the same range. All three hits affected the viability of the extra-cellular form of L. infantum parasites with IC50 values at low micromolar concentrations. These molecules showed non-significant toxicity toward THP-1 macrophages. Furthermore, their anti-leishmanial activity was validated with experimental assays on L. infantum intramacrophage amastigotes showing IC50 values lower than 4.2 µM. Selected compounds exhibited selectivity indexes between 19 to 38, which reflects their potential as promising anti-Leishmania molecules.
Assuntos
Antiprotozoários/isolamento & purificação , Antiprotozoários/farmacologia , Avaliação Pré-Clínica de Medicamentos/métodos , Fator de Iniciação 4A em Eucariotos/antagonistas & inibidores , Leishmania infantum/efeitos dos fármacos , Leishmania infantum/enzimologia , Adenosina Trifosfatases/análise , Adenosina Trifosfatases/antagonistas & inibidores , Sítios de Ligação , Fator de Iniciação 4A em Eucariotos/química , Concentração Inibidora 50 , Modelos Moleculares , Simulação de Acoplamento Molecular , Testes de Sensibilidade ParasitáriaRESUMO
The yeast DEAD-box protein Has1p is required for the maturation of 18S rRNA, the biogenesis of 40S r-subunits and for the processing of 27S pre-rRNAs during 60S r-subunit biogenesis. We purified recombinant Has1p and characterized its biochemical activities. We show that Has1p is an RNA-dependent ATPase in vitro and that it is able to unwind RNA/DNA duplexes in an ATP-dependent manner. We also report a mutational analysis of the conserved residues in motif I (86AKTGSGKT93), motif III (228SAT230) and motif VI (375HRVGRTARG383). The in vivo lethal K92A substitution in motif I abolishes ATPase activity in vitro. The mutations S228A and T230A partially dissociate ATPase and helicase activities, and they have cold-sensitive and lethal growth phenotypes, respectively. The H375E substitution in motif VI significantly decreased helicase but not ATPase activity and was lethal in vivo. These results suggest that both ATPase and unwinding activities are required in vivo. Has1p possesses a Walker A-like motif downstream of motif VI (383GTKGKGKS390). K389A substitution in this motif significantly increases the Has1p activity in vitro, which indicates it potentially plays a role as a negative regulator. Finally, rRNAs and poly(A) RNA serve as the best stimulators of the ATPase activity of Has1p among the tested RNAs.
Assuntos
Adenosina Trifosfatases/química , Adenosina Trifosfatases/metabolismo , RNA Helicases/química , RNA Helicases/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimologia , Adenosina Trifosfatases/genética , Motivos de Aminoácidos , Sequência Conservada , RNA Helicases DEAD-box , DNA/química , Análise Mutacional de DNA , Mutação , RNA/química , RNA/metabolismo , RNA Helicases/genética , RNA Ribossômico/metabolismo , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/crescimento & desenvolvimento , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
RNA helicases of the DEAD-box protein family have been shown to participate in every aspect of RNA metabolism. They are present in most organisms where they work as RNA helicases or RNPases. The properties of these enzymes in vivo remains poorly described, however some were extensively characterized in vitro, and the solved crystal structures of a few are now available. Taken together, this information gives insight into the regulation of ATP and RNA binding as well as in the ATPase and helicase activities. This review will focus on the description of the molecular characteristics of members of the DEAD-box protein family and on the enzymatic activities they possess.
Assuntos
RNA Helicases/genética , RNA Helicases/metabolismo , Difosfato de Adenosina/metabolismo , Adenosina Trifosfatases/metabolismo , Processamento Alternativo , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Sítios de Ligação , Sequência Conservada , Cristalografia por Raios X , Humanos , Modelos Biológicos , Modelos Moleculares , Dados de Sequência Molecular , Ligação Proteica , Conformação Proteica , Processamento de Proteína Pós-Traducional , Estrutura Terciária de Proteína , RNA Helicases/química , RNA Mensageiro/metabolismo , Homologia de Sequência de Aminoácidos , Transcrição GênicaRESUMO
DEAD-box RNA helicases have core structures consisting of two, tandemly linked, RecA-like domains that contain all of the conserved motifs involved in binding ATP and RNA, and that are needed for the enzymatic activities. The conserved sequence motifs and structural homology indicate that these proteins share common origins and underlining functionality. Indeed, the purified proteins generally act as ATP-dependent RNA-binding proteins and RNA-dependent ATPases in vitro, but for the most part without the substrate specificity or enzymatic regulation that exists in the cell. We are interested in understanding the relationships between the conserved motifs and structures that confer the commonly shared features, and we are interested in understanding how modifications of the core structure alter the enzymatic properties. We use sequence alignments and structural modeling to reveal regions of interest, which we modify by classical molecular biological techniques (mutations and deletions). We then use various biochemical techniques to characterize the purified proteins and their variants for their ATPase, RNA binding, and RNA unwinding activities to determine the functional roles of the different elements. In this chapter, we describe the methods we use to design our constructs and to determine their enzymatic activities in vitro.
Assuntos
Biologia Computacional/métodos , RNA Helicases DEAD-box/metabolismo , Adenosina Trifosfatases/metabolismo , Especificidade por SubstratoRESUMO
The DEAD-box family of putative RNA helicases is composed of ubiquitous proteins that are found in nearly all organisms and that are involved in virtually all processes involving RNA. They are characterized by two tandemly linked, RecA-like domains that contain 11 conserved motifs and highly variable amino- and carboxy-terminal flanking sequences. For this reason, they are often considered to be modular multi-domain proteins. We tested this by making extensive BLASTs and sequence alignments to elucidate the minimal functional unit in nature. We then used this information to construct chimeras and deletions of six essential yeast proteins that were assayed in vivo. We purified many of the different constructs and characterized their biochemical properties in vitro. We found that sequence elements can only be switched between closely related proteins and that the carboxy-terminal sequences are important for high ATPase and strand displacement activities and for high RNA binding affinity. The amino-terminal elements were often toxic when overexpressed in vivo, and they may play regulatory roles. Both the amino and the carboxyl regions have a high frequency of sequences that are predicted to be intrinsically disordered, indicating that the flanking regions do not form distinct modular domains but probably assume an ordered structure with ligand binding. Finally, the minimal functional unit of the DEAD-box core starts two amino acids before the isolated phenylalanine of the Q motif and extends to about 35 residues beyond motif VI. These experiments provide evidence for how a highly conserved structural domain can be adapted to different cellular needs.
Assuntos
RNA Helicases DEAD-box/química , Proteínas Mutantes Quiméricas/metabolismo , Saccharomyces cerevisiae/crescimento & desenvolvimento , Saccharomyces cerevisiae/genética , Deleção de Sequência , Adenosina Trifosfatases/metabolismo , Trifosfato de Adenosina/metabolismo , Motivos de Aminoácidos , Sequência Conservada , RNA Helicases DEAD-box/genética , RNA Helicases DEAD-box/metabolismo , Primers do DNA/química , Primers do DNA/genética , Técnicas In Vitro , Proteínas Mutantes Quiméricas/genética , Mutação/genética , Estrutura Terciária de Proteína , RNA Fúngico/genética , Saccharomyces cerevisiae/metabolismoAssuntos
Trifosfato de Adenosina/metabolismo , Células Eucarióticas/enzimologia , RNA Helicases/metabolismo , Motivos de Aminoácidos/genética , Sequência de Aminoácidos/genética , Animais , Sítios de Ligação/genética , DNA Helicases/genética , DNA Helicases/metabolismo , Humanos , RNA Helicases/genéticaRESUMO
Motif III in the putative helicases of superfamily 2 is highly conserved in both its sequence and its structural context. It typically consists of the sequence alcohol-alanine-alcohol (S/T-A-S/T). Historically, it was thought to link ATPase activity with a "helicase" strand displacement activity that disrupts RNA or DNA duplexes. DEAD-box proteins constitute the largest family of superfamily 2; they are RNA-dependent ATPases and ATP-dependent RNA binding proteins that, in some cases, are able to disrupt short RNA duplexes. We made mutations of motif III (S-A-T) in the yeast DEAD-box protein Ded1 and analyzed in vivo phenotypes and in vitro properties. Moreover, we made a tertiary model of Ded1 based on the solved structure of Vasa. We used Ded1 because it has relatively high ATPase and RNA binding activities; it is able to displace moderately stable duplexes at a large excess of substrate. We find that the alanine and the threonine in the second and third positions of motif III are more important than the serine, but that mutations of all three residues have strong phenotypes. We purified the wild-type and various mutants expressed in Escherichia coli. We found that motif III mutations affect the RNA-dependent hydrolysis of ATP (k(cat)), but not the affinity for ATP (K(m)). Moreover, mutations alter and reduce the affinity for single-stranded RNA and subsequently reduce the ability to disrupt duplexes. We obtained intragenic suppressors of the S-A-C mutant that compensate for the mutation by enhancing the affinity for ATP and RNA. We conclude that motif III and the binding energy of gamma-PO(4) of ATP are used to coordinate motifs I, II, and VI and the two RecA-like domains to create a high-affinity single-stranded RNA binding site. It also may help activate the beta,gamma-phosphoanhydride bond of ATP.
Assuntos
RNA Helicases DEAD-box/química , RNA Helicases DEAD-box/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Adenosina Trifosfatases/química , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Trifosfato de Adenosina/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Sítios de Ligação , Sequência Conservada , RNA Helicases DEAD-box/genética , Genes Fúngicos , Cinética , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Fenótipo , Estrutura Terciária de Proteína , RNA Fúngico/genética , RNA Fúngico/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Homologia de Sequência de AminoácidosRESUMO
We have identified a highly conserved phenylalanine in motif IV of the DEAD-box helicases that is important for their enzymatic activities. In vivo analyses of essential proteins in yeast showed that mutants of this residue had severe growth phenotypes. Most of the mutants also were temperature sensitive, which suggested that the mutations altered the conformational stability. Intragenic suppressors of the F405L mutation in yeast Ded1 mapped close to regions of the protein involved in ATP or RNA binding in DEAD-box crystal structures, which implicated a defect at this level. In vitro experiments showed that these mutations affected ATP binding and hydrolysis as well as strand displacement activity. However, the most pronounced effect was the loss of the ATP-dependent cooperative binding of the RNA substrates. Sequence analyses and an examination of the Protein Data Bank showed that the motif IV phenylalanine is conserved among superfamily 2 helicases. The phenylalanine appears to be an anchor that maintains the rigidity of the RecA-like domain. For DEAD-box proteins, the phenylalanine also aligns a highly conserved arginine of motif VI through van der Waals and cation-pi interactions, thereby helping to maintain the network of interactions that exist between the different motifs involved in ATP and RNA binding.
Assuntos
RNA Helicases DEAD-box/química , RNA Helicases DEAD-box/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Trifosfato de Adenosina/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Arginina/química , Sequência de Bases , Sequência Conservada , RNA Helicases DEAD-box/genética , Cinética , Modelos Moleculares , Mutagênese Sítio-Dirigida , Fenótipo , Fenilalanina/química , RNA Fúngico/genética , RNA Fúngico/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Homologia de Sequência de Aminoácidos , Especificidade por Substrato , Supressão GenéticaRESUMO
DEAD-box proteins are the most common RNA helicases, and they are associated with virtually all processes involving RNA. They have nine conserved motifs that are required for ATP and RNA binding, and for linking phosphoanhydride cleavage of ATP with helicase activity. The Q motif is the most recently identified conserved element, and it occurs approximately 17 amino acids upstream of motif I. There is a highly conserved, but isolated, aromatic group approximately 17 amino acids upstream of the Q motif. These two elements are involved in adenine recognition and in ATPase activity of DEAD-box proteins. We made extensive analyses of the Q motif and upstream aromatic residue in the yeast translation-initiation factor Ded1. We made site-specific mutations and tested them for viability in yeast. Moreover, we purified various mutant proteins and obtained the Michaelis-Menten parameters for the ATPase activities. We also measured RNA affinities and strand-displacement activities. We find that the Q motif not only regulates ATP binding and hydrolysis but also regulates the affinity of the protein for RNA substrates and ultimately the helicase activity.
Assuntos
Proteínas Fúngicas/metabolismo , RNA Helicases/química , RNA Helicases/metabolismo , RNA Fúngico/metabolismo , Adenina/química , Adenina/metabolismo , Adenosina Trifosfatases/metabolismo , Trifosfato de Adenosina/metabolismo , Alanina/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Substituição de Aminoácidos , Aminoácidos Aromáticos/química , Sequência Consenso , Ensaio de Desvio de Mobilidade Eletroforética , Escherichia coli/genética , Fator de Iniciação 4A em Eucariotos/genética , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Teste de Complementação Genética , Glicina/química , Cinética , Leucina/metabolismo , Dados de Sequência Molecular , Estrutura Secundária de Proteína , RNA Helicases/genética , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
SF1 and SF2 helicases have structurally conserved cores containing seven to eight distinctive motifs and variable amino- and carboxyl-terminal flanking sequences. We have discovered a motif upstream of motif I that is unique to and characteristic of the DEAD box family of RNA helicases. It consists of a 9 amino acid sequence containing an invariant glutamine. A conserved phenylalanine occurs 17 aa further upstream. Sequence alignments, site-specific mutagenesis, and ATPase assays show that this motif and the upstream phenylalanine are highly conserved, that they are essential for viability in the yeast Saccharomyces cerevisiae, and that they control ATP binding and hydrolysis in the yeast translation-initiation factor eIF4A. These results are consistent with computer studies of the solved crystal structures.