RESUMO
The interaction between the stem-loop structure of the Alzheimer's amyloid precursor protein IRE mRNA and iron regulatory protein was examined by employing molecular docking and multi-spectroscopic techniques. A detailed molecular docking analysis of APP IRE mRNAâIRP1 reveals that 11 residues are involved in hydrogen bonding as the main driving force for the interaction. Fluorescence binding results revealed a strong interaction between APP IRE mRNA and IRP1 with a binding affinity and an average binding sites of 31.3 × 106 M-1 and 1.0, respectively. Addition of Fe2+(anaerobic) showed a decreased (3.3-fold) binding affinity of APP mRNAâIRP1. Further, thermodynamic parameters of APP mRNAâIRP1 interactions were an enthalpy-driven and entropy-favored event, with a large negative ΔH (-25.7 ± 2.5 kJ/mol) and a positive ΔS (65.0 ± 3.7 J/mol·K). A negative ΔH value for the complex formation suggested the contribution of hydrogen bonds and van der Waals forces. The addition of iron increased the enthalpic contribution by 38% and decreased the entropic influence by 97%. Furthermore, the stopped-flow kinetics of APP IRE mRNAâIRP1 also confirmed the complex formation, having the rate of association (kon) and the rate of dissociation (koff) as 341 µM-1 s-1, and 11 s-1, respectively. The addition of Fe2+ has decreased the rate of association (kon) by ~ three-fold, whereas the rate of dissociation (koff) has increased by ~ two-fold. The activation energy for APP mRNAâIRP1 complex was 52.5 ± 2.1 kJ/mol. The addition of Fe2+ changed appreciably the activation energy for the binding of APP mRNA with IRP1. Moreover, circular dichroism spectroscopy has confirmed further the APP mRNAâIRP1 complex formation and IRP1 secondary structure change with the addition of APP mRNA. In the interaction between APP mRNA and IRP1, iron promotes structural changes in the APP IRE mRNAâIRP1 complexes by changing the number of hydrogen bonds and promoting a conformational change in the IRP1 structure when it is bound to the APP IRE mRNA. It further illustrates how IRE stem-loop structure influences selectively the thermodynamics and kinetics of these protein-RNA interactions.
Assuntos
Doença de Alzheimer , Ferro , Humanos , Doença de Alzheimer/metabolismo , Precursor de Proteína beta-Amiloide/genética , Precursor de Proteína beta-Amiloide/metabolismo , Ferro/metabolismo , Proteína 1 Reguladora do Ferro/metabolismo , Proteína 2 Reguladora do Ferro/genética , Proteínas Reguladoras de Ferro/genética , Simulação de Acoplamento Molecular , Ligação Proteica , Elementos de Resposta , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Análise EspectralRESUMO
Interaction of iron responsive elements (IRE) mRNA with the translational machinery is an early step critical in the initiation of protein synthesis. To investigate the binding specificity of IRE mRNA for eIF4F, kinetic rates for the eIF4F·IRE RNA interactions were determined and correlated with the translational efficiency. The observed rate of eIF4F·FRT IRE RNA interactions was 2-fold greater as compared to eIF4F·ACO2 IRE RNA binding. Addition of iron enhanced the association rates and lowered the dissociation rates for the eIF4F binding to both IRE RNAs, with having higher preferential binding to the FRT IRE RNA. The binding rates of both eIF4F·IRE RNA complexes correlated with the enhancement of protein synthesis in vitro. Presence of iron and eIF4F in the depleted WGE significantly enhanced translation for both IRE RNAs. This suggests that iron promotes translation by enhancing the binding rates of the eIF4FâIRE RNA complex. eIF4F·IRE RNA binding is temperature-dependent; raising the temperature from 5 to 25°C, enhanced the binding rates of eIF4F·FRT IRE (4-fold) and eIF4F·ACO2 IRE (5-fold). Presence of Fe2+ caused reduction in the activation energy for the binding of FRT IRE and ACO2 IRE to eIF4F, suggesting a more stable platform for initiating protein synthesis. In the presence of iron, lowered energy barrier has leads to the faster association rate and slower rate of dissociation for the protein-RNA complex, thus favoring efficient protein synthesis. Our results correlate well with the observed translational efficiency of IRE RNA, thereby suggesting that the presence of iron leads to a rapid, favorable, and stable complex formation that directs regulatory system to respond efficiently to cellular iron levels.
Assuntos
Fator de Iniciação 4F em Eucariotos/metabolismo , Ferritinas/metabolismo , Proteínas Reguladoras de Ferro/genética , RNA Mensageiro/metabolismo , Animais , Cinética , Conformação de Ácido Nucleico , Ligação Proteica , Biossíntese de ProteínasRESUMO
The interaction of ferritin iron responsive element (IRE) mRNA with eIF4F was examined by fluorescence and circular dichroism spectroscopy. Fluorescence quenching data indicated that eIF4F contains one high affinity binding site for ferritin IRE RNA. The Scatchard analysis revealed strong binding affinity (Ka = 11.1 × 107 M-1) and binding capacity (n = 1.0) between IRE RNA and eIF4F. The binding affinity of IRE RNA for eIF4F decreased (~4-fold) as temperature increased (from 5 °C to 30 °C). The van't Hoff analysis revealed that IRE RNA binding to eIF4F is enthalpy-driven (ΔH = -47.1 ± 3.4 kJ/mol) and entropy-opposed (ΔS = -30.1 ± 1.5 J/mol/K). The addition of iron increased the enthalpic, while decreasing the entropic contribution towards the eIF4Fâ¢IRE RNA complex, resulting in favorable free energy (ΔG = -49.8 ± 2.8 kJ/mol). Thermodynamic values and ionic strength data suggest that the presence of iron increases hydrogen bonding and decreases hydrophobic interactions, leading to formation of a more stable complex. The interaction of IRE RNA with eIF4F at higher concentrations produced significant changes in the secondary structure of the protein, as revealed from the far-UV CD results, clearly illustrating the structural alterations resulted from formation of the eIF4Fâ¢IRE RNA complex. A Lineweaver-Burk plot showed an uncompetitive binding behavior between IRE RNA and m7G cap for the eIF4F, indicating that there are different binding sites on the eIF4F for the IRE RNA and the cap analog; molecular docking analysis further supports this notion. Our findings suggest that the eIF4Fâ¢IRE RNA complex formation is accompanied by an elevated hydrogen bonding and weakened hydrophobic interactions, leading to an overall conformational change, favored in terms of its free energy. The conformational change in the eIF4F structure, caused by the IRE RNA binding, provides a more stable platform for effective IRE translation in iron homeostasis.
Assuntos
Fator de Iniciação 4F em Eucariotos , Ferritinas , Fator de Iniciação 4F em Eucariotos/metabolismo , Ferritinas/genética , Ferro/metabolismo , Simulação de Acoplamento Molecular , Ligação Proteica , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , TermodinâmicaRESUMO
Ricin is a plant derived protein toxin produced by the castor bean plant (Ricinus communis). The Centers for Disease Control (CDC) classifies ricin as a Category B biological agent. Currently, there is neither an effective vaccine that can be used to protect against ricin exposure nor a therapeutic to reverse the effects once exposed. Here we quantitatively characterize interactions between catalytic ricin A-chain (RTA) and a viral genome-linked protein (VPg) from turnip mosaic virus (TuMV). VPg and its N-terminal truncated variant, VPg1-110, bind to RTA and abolish ricin's catalytic depurination of 28S rRNA in vitro and in a cell-free rabbit reticulocyte translational system. RTA and VPg bind in a 1 to 1 stoichiometric ratio, and their binding affinity increases ten-fold as temperature elevates (5⯰C to 37⯰C). RTA-VPg binary complex formation is enthalpically driven and favored by entropy, resulting in an overall favorable energy, ΔGâ¯=â¯-136.8â¯kJ/mol. Molecular modeling supports our experimental observations and predicts a major contribution of electrostatic interactions, suggesting an allosteric mechanism of downregulation of RTA activity through conformational changes in RTA structure, and/or disruption of binding with the ribosomal stalk. Fluorescence anisotropy studies show that heat affects the rate constant and the activation energy for the RTA-VPg complex, Eaâ¯=â¯-62.1 kJ/mol. The thermodynamic and kinetic findings presented here are an initial lead study with promising results and provides a rational approach for synthesis of therapeutic peptides that successfully eliminate toxicity of ricin, and other cytotoxic RIPs.
Assuntos
Potyvirus/metabolismo , Ricina/antagonistas & inibidores , Ricinus/metabolismo , Proteínas Virais/farmacologia , Animais , Sistema Livre de Células , Modelos Moleculares , Ligação Proteica , RNA Ribossômico 28S/química , Coelhos , Reticulócitos/química , Reticulócitos/efeitos dos fármacos , Ricina/toxicidade , Deleção de Sequência , Termodinâmica , Proteínas Virais/química , Proteínas Virais/genéticaRESUMO
Pokeweed antiviral protein (PAP) is a ribosome inactivating protein (RIP) that depurinates the sarcin/ricin loop (SRL) of rRNA, inhibiting protein synthesis. PAP depurinates viral RNA, and in doing so, lowers the infectivity of many plant viruses. The mechanism by which PAP accesses uncapped viral RNA is not known, impeding scientists from developing effective antiviral agents for the prevention of the diseases caused by uncapped RNA viruses. Kinetic rates of PAP interacting with tobacco etch virus (TEV) RNA, in the presence and absence of eIFiso4F, were examined, addressing how the eIF affects selective PAP targeting and depurination of the uncapped viral RNA. PAP-eIFs copurification assay and fluorescence resonance energy transfer demonstrate that PAP forms a ternary complex with the eIFiso4G and eIFiso4E, directing the depurination of uncapped viral RNA. eIFiso4F selectively targets PAP to depurinate TEV RNA by increasing PAP's specificity constant for uncapped viral RNA 12-fold, when compared to the depurination of an oligonucleotide RNA that mimics the SRL of large rRNA, and cellular capped luciferase mRNA. This explains how PAP is able to lower infectivity of pokeweed viruses, while preserving its own ribosomes and cellular RNA from depurination: PAP utilizes cellular eIFiso4F in a novel strategy to target uncapped viral RNA. It may be possible to modulate and utilize these PAP-eIFs interactions for their public health benefit; by repurposing them to selectively target PAP to depurinate uncapped viral RNA, many plant and animal diseases caused by these viruses could be alleviated.
Assuntos
Fatores de Iniciação de Peptídeos/metabolismo , Proteínas de Plantas/metabolismo , Potyvirus/metabolismo , RNA Viral/metabolismo , Proteínas Inativadoras de Ribossomos Tipo 1/metabolismo , Traqueófitas/virologia , Iniciação Traducional da Cadeia Peptídica , Fatores de Iniciação de Peptídeos/genética , Proteínas de Plantas/genética , Potyvirus/genética , Purinas/química , Capuzes de RNA/genética , Capuzes de RNA/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Viral/genética , Proteínas Inativadoras de Ribossomos Tipo 1/antagonistas & inibidores , Ribossomos/genética , Ribossomos/metabolismoRESUMO
Fluorescent mRNA molecules offer a wide range of applications for studying capping/decapping reactions, translation, and other biophysical studies. Furthermore, fluorescent tags prove invaluable for tracking RNA molecules in cells. Here, we describe an efficient synthesis of a fluorescent cap analog, anthranioyl-GTP, its purification, and in vitro cap labeling of transcribed mRNA catalyzed by the recombinant vaccinia capping enzyme to produce anthranioyl-m(7)GpppG-capped RNA.
Assuntos
Análogos de Capuz de RNA/síntese química , RNA Mensageiro/química , Guanosina/análogos & derivados , Guanosina/química , Estrutura Molecular , Biossíntese de Proteínas , Análogos de Capuz de RNA/química , RNA Mensageiro/genética , Espectrometria de Fluorescência , Transcrição GênicaRESUMO
Viruses employ an array of elaborate strategies to overcome plant defense mechanisms and must adapt to the requirements of the host translational systems. Pokeweed antiviral protein (PAP) from Phytolacca americana is a ribosome inactivating protein (RIP) and is an RNA N-glycosidase that removes specific purine residues from the sarcin/ricin (S/R) loop of large rRNA, arresting protein synthesis at the translocation step. PAP is thought to play an important role in the plant's defense mechanism against foreign pathogens. This review focuses on the structure, function, and the relationship of PAP to other RIPs, discusses molecular aspects of PAP antiviral activity, the novel inhibition of this plant toxin by a virus counteraction-a peptide linked to the viral genome (VPg), and possible applications of RIP-conjugated immunotoxins in cancer therapeutics.
Assuntos
Proteínas Inativadoras de Ribossomos Tipo 1 , Animais , Sítios de Ligação , Endorribonucleases/química , Proteínas Fúngicas/química , Genoma Viral , Humanos , Isoformas de Proteínas , Capuzes de RNA/química , Capuzes de RNA/genética , Capuzes de RNA/metabolismo , RNA de Plantas/química , RNA de Plantas/genética , RNA de Plantas/metabolismo , RNA Ribossômico/química , RNA Ribossômico/genética , RNA Ribossômico/metabolismo , RNA Viral/química , RNA Viral/genética , RNA Viral/metabolismo , Proteínas Inativadoras de Ribossomos Tipo 1/química , Proteínas Inativadoras de Ribossomos Tipo 1/genética , Proteínas Inativadoras de Ribossomos Tipo 1/metabolismo , Proteínas Inativadoras de Ribossomos Tipo 1/farmacologia , Ribossomos/química , Ribossomos/metabolismo , Ricina/químicaRESUMO
A method has been developed for synthesising fluorescently labeled capped mRNA. The method incorporates a single fluorescent molecule as part of the 5'-mRNA or oligonucleotide cap site. The fluorescent molecule, Ant-m(7)GTP is specifically incorporated into the cap site to yield Ant-m(7)GpppG-capped mRNA or oligonucleotide. Efficient capping was observed with 60-100% of the RNA transcripts capped with the fluorescent molecule. The Ant-m(7)G derivative, which has been previously shown to interact with the eukaryotic cap binding protein eIF4E, is shown in this paper to be a substrate for the Vaccinia capping enzyme and the DCP2 decapping enzyme from Arabidopsis. Further, the Ant-m(7)GTP-capped RNA is readily translated. This Ant-m(7)GTP-capped RNA provides an important tool for monitoring capping reactions, translation, and biophysical studies.
RESUMO
Pokeweed antiviral protein (PAP) from Phytolacca americana is a ribosome-inactivating protein (RIP) and an RNA N-glycosidase that removes specific purine residues from the sarcin/ricin loop of large rRNA, arresting protein synthesis at the translocation step. PAP is also a cap-binding protein and is a potent antiviral agent against many plant, animal, and human viruses. To elucidate the mechanism of RNA depurination, and to understand how PAP recognizes and targets various RNAs, the interactions between PAP and turnip mosaic virus genome-linked protein (VPg) were investigated. VPg can function as a cap analog in cap-independent translation and potentially target PAP to uncapped IRES-containing RNA. In this work, fluorescence spectroscopy and HPLC techniques were used to quantitatively describe PAP depurination activity and PAP-VPg interactions. PAP binds to VPg with high affinity (29.5 nm); the reaction is enthalpically driven and entropically favored. Further, VPg is a potent inhibitor of PAP depurination of RNA in wheat germ lysate and competes with structured RNA derived from tobacco etch virus for PAP binding. VPg may confer an evolutionary advantage by suppressing one of the plant defense mechanisms and also suggests the possible use of this protein against the cytotoxic activity of ribosome-inactivating proteins.