RESUMEN
The nucleoprotein (NP) of influenza A virus (IAV) required for IAV replication is a promising target for new antivirals. We previously identified by in silico screening naproxen being a dual inhibitor of NP and cyclooxygenase COX2, thus combining antiviral and anti-inflammatory effects. However, the recently shown strong COX2 antiviral potential makes COX2 inhibition undesirable. Here we designed and synthesized two new series of naproxen analogues called derivatives 2, 3, and 4 targeting highly conserved residues of the RNA binding groove, stabilizing NP monomer without inhibiting COX2. Derivative 2 presented improved antiviral effects in infected cells compared to that of naproxen and afforded a total protection of mice against a lethal viral challenge. Derivative 4 also protected infected cells challenged with circulating 2009-pandemic and oseltamivir-resistant H1N1 virus. This improved antiviral effect likely results from derivatives 2 and 4 inhibiting NP-RNA and NP-polymerase acidic subunit PA N-terminal interactions.
Asunto(s)
Antivirales/química , Antivirales/farmacología , Inhibidores de la Ciclooxigenasa 2/farmacología , Virus de la Influenza A/efectos de los fármacos , Naproxeno/análogos & derivados , Células A549 , Animales , Sitios de Unión , Inhibidores de la Ciclooxigenasa 2/química , Perros , Diseño de Fármacos , Reposicionamiento de Medicamentos , Farmacorresistencia Viral/efectos de los fármacos , Femenino , Humanos , Virus de la Influenza A/patogenicidad , Gripe Humana/tratamiento farmacológico , Gripe Humana/patología , Células de Riñón Canino Madin Darby , Ratones Endogámicos C57BL , Simulación del Acoplamiento Molecular , Naproxeno/farmacología , Proteínas de la Nucleocápside , Oseltamivir/farmacología , Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/metabolismo , Resonancia por Plasmón de Superficie , Proteínas del Núcleo Viral/química , Proteínas del Núcleo Viral/metabolismoRESUMEN
The two hallmarks of Alzheimer's disease (AD) are the presence of neurofibrillary tangles (NFT) made of aggregates of the hyperphosphorylated tau protein and of amyloid plaques composed of amyloid-ß (Aß) peptides, primarily Aß1-40 and Aß1-42. Targeting the production, aggregation, and toxicity of Aß with small molecule drugs or antibodies is an active area of AD research due to the general acceptance of the amyloid cascade hypothesis, but thus far all drugs targeting Aß have failed. From a review of the recent literature and our own experience based on in vitro, in silico, and in vivo studies, we present some reasons to explain this repetitive failure.
Asunto(s)
Enfermedad de Alzheimer/tratamiento farmacológico , Enfermedad de Alzheimer/metabolismo , Péptidos beta-Amiloides/metabolismo , Fármacos Neuroprotectores/farmacología , Fármacos Neuroprotectores/uso terapéutico , Animales , Descubrimiento de Drogas , HumanosRESUMEN
UNLABELLED: Presently, respiratory syncytial virus (RSV), the main cause of severe respiratory infections in infants, cannot be treated efficiently with antivirals. However, its RNA-dependent polymerase complex offers potential targets for RSV-specific drugs. This includes the recognition of its template, the ribonucleoprotein complex (RNP), consisting of genomic RNA encapsidated by the RSV nucleoprotein, N. This recognition proceeds via interaction between the phosphoprotein P, which is the main polymerase cofactor, and N. The determinant role of the C terminus of P, and more particularly of the last residue, F241, in RNP binding and viral RNA synthesis has been assessed previously. Here, we provide detailed structural insight into this crucial interaction for RSV polymerase activity. We solved the crystallographic structures of complexes between the N-terminal domain of N (N-NTD) and C-terminal peptides of P and characterized binding by biophysical approaches. Our results provide a rationale for the pivotal role of F241, which inserts into a well-defined N-NTD pocket. This primary binding site is completed by transient contacts with upstream P residues outside the pocket. Based on the structural information of the N-NTD:P complex, we identified inhibitors of this interaction, selected by in silico screening of small compounds, that efficiently bind to N and compete with P in vitro. One of the compounds displayed inhibitory activity on RSV replication, thereby strengthening the relevance of N-NTD for structure-based design of RSV-specific antivirals. IMPORTANCE: Respiratory syncytial virus (RSV) is a widespread pathogen that is a leading cause of acute lower respiratory infections in infants worldwide. RSV cannot be treated efficiently with antivirals, and no vaccine is presently available, with the development of pediatric vaccines being particularly challenging. Therefore, there is a need for new therapeutic strategies that specifically target RSV. The interaction between the RSV phosphoprotein P and the ribonucleoprotein complex is critical for viral replication. In this study, we identified the main structural determinants of this interaction, and we used them to screen potential inhibitors in silico. We found a family of molecules that were efficient competitors of P in vitro and showed inhibitory activity on RSV replication in cellular assays. These compounds provide a basis for a pharmacophore model that must be improved but that holds promises for the design of new RSV-specific antivirals.
Asunto(s)
Antivirales/química , Modelos Moleculares , Nucleocápside/química , Fosfoproteínas/química , Infecciones por Virus Sincitial Respiratorio/tratamiento farmacológico , Virus Sincitial Respiratorio Humano/química , Calorimetría , Cristalografía por Rayos X , Diseño de Fármacos , Humanos , Proteínas Luminiscentes , Espectroscopía de Resonancia Magnética , Nucleocápside/metabolismo , Fosfoproteínas/metabolismo , Conformación Proteica , Virus Sincitial Respiratorio Humano/metabolismo , Difracción de Rayos X , Proteína Fluorescente RojaRESUMEN
We have studied the dimer of amyloid beta peptide Aß of 40 residues by means of all-atom replica exchange molecular dynamics. The Aß-dimers have been found to be the smallest toxic species in Alzheimer's disease, but their inherent flexibilities have precluded structural characterization by experimental methods. Though the 24-µs-scale simulation reveals a mean secondary structure of 18% ß-strand and 10% α helix, we find transient configurations with an unstructured N-terminus and multiple ß-hairpins spanning residues 17-21 and 30-36, but the antiparallel and perpendicular peptide orientations are preferred over the parallel organization. Short-lived conformational states also consist of all α topologies, and one compact peptide with ß-sheet structure stabilized by a rather extended peptide with α-helical content. Overall, this first all-atom study provides insights into the equilibrium structure of the Aß1-40 dimer in aqueous solution, opening a new avenue for a comprehensive understanding of the impact of pathogenic and protective mutations in early-stage Alzheimer's disease on a molecular level.
Asunto(s)
Péptidos beta-Amiloides/química , Simulación de Dinámica Molecular , Fragmentos de Péptidos/química , Multimerización de Proteína , Estructura Secundaria de Proteína , Termodinámica , Agua/químicaRESUMEN
Inhibition of the aggregation of the monomeric peptide ß-amyloid (Aß) into oligomers is a widely studied therapeutic approach in Alzheimer's disease (AD). Many small molecules have been reported to work in this way, including 1,4-naphthoquinon-2-yl-L-tryptophan (NQ-Trp). NQ-Trp has been reported to inhibit aggregation, to rescue cells from Aß toxicity, and showed complete phenotypic recovery in an in vivo AD model. In this work we investigated its molecular mechanism by using a combined approach of experimental and theoretical studies, and obtained converging results. NQ-Trp is a relatively weak inhibitor and the fluorescence data obtained by employing the fluorophore widely used to monitor aggregation into fibrils can be misinterpreted due to the inner filter effect. Simulations and NMR experiments showed that NQ-Trp has no specific "binding site"-type interaction with mono- and dimeric Aß, which could explain its low inhibitory efficiency. This suggests that the reported anti-AD activity of NQ-Trp-type molecules in in vivo models has to involve another mechanism. This study has revealed the potential pitfalls in the development of aggregation inhibitors for amyloidogenic peptides, which are of general interest for all the molecules studied in the context of inhibiting the formation of toxic aggregates.
Asunto(s)
Enfermedad de Alzheimer/tratamiento farmacológico , Péptidos beta-Amiloides/antagonistas & inhibidores , Péptidos beta-Amiloides/química , Naftoquinonas/química , Naftoquinonas/farmacología , Fragmentos de Péptidos/antagonistas & inhibidores , Fragmentos de Péptidos/química , Triptófano/análogos & derivados , Humanos , Espectroscopía de Resonancia Magnética , Simulación de Dinámica Molecular , Triptófano/química , Triptófano/farmacologíaRESUMEN
INTRODUCTION: Influenza viruses are a threat to human health. There are presently only two methods for treating influenza: vaccines, which require yearly updates, and two classes of antivirals that suffer with the problem of resistance by current human influenza viruses; this is especially the case with amantadine and rimantadine. Consequently, there is an urgent need for the development of new antivirals with new mechanisms of action. AREAS COVERED: In this review, the authors focus on viral protein domains, their associated activity and their inhibition by small molecules defined by a structure-based design with a special emphasis on the ribonucleoprotein complex and its inhibitors. Several new classes of antiviral candidates targeting viral replication through individual domains of the polymerase and the nucleoprotein (NP) have been developed through structure-based design. EXPERT OPINION: To date, the antivirals targeting neuraminidase are by far the most developed and potent. Antiviral candidates targeting the NP and polymerase domains are in the pipeline but their pharmacokinetics needs further studies. The recently published structures of the polymerase expand the possibilities for development of new antivirals. Combination therapies targeting conserved viral targets and new cellular proteins or exploiting drug promiscuity hold promises to fight against the emergence of resistance.
Asunto(s)
Antivirales/uso terapéutico , Diseño de Fármacos , Gripe Humana/tratamiento farmacológico , Animales , Antivirales/farmacocinética , Antivirales/farmacología , Farmacorresistencia Viral , Humanos , Gripe Humana/virología , Ribonucleoproteínas/antagonistas & inhibidores , Ribonucleoproteínas/metabolismo , Relación Estructura-Actividad , Replicación Viral/efectos de los fármacosAsunto(s)
Enfermedad de Alzheimer , Péptidos beta-Amiloides , Simulación por Computador , Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/patología , Secuencia de Aminoácidos , Péptidos beta-Amiloides/antagonistas & inhibidores , Péptidos beta-Amiloides/química , Péptidos beta-Amiloides/genética , Péptidos beta-Amiloides/metabolismo , Animales , Membrana Celular/metabolismo , Humanos , Datos de Secuencia Molecular , Multimerización de Proteína , Agua/químicaRESUMEN
The self-assembly of the amyloid-ß (Aß) peptide of various amino acid lengths into senile plaques is one hallmark of Alzheimer's disease pathology. In the past decade, many small molecules, including NQTrp, have been identified to reduce aggregation and toxicity. However, due to the heterogeneity of the conformational ensemble of Aß with drugs, we lack detailed structures of the transient complexes. Following our previous simulation of the monomer of Aß1-28, here we characterize the equilibrium ensemble of the Aß1-28 monomer with NQTrp by means of extensive atomistic replica exchange molecular dynamics simulations using a force field known to fold diverse proteins correctly. While the secondary structure content and the intrinsic disorder of the whole peptides are very similar and the lifetimes of the salt-bridges remain constant, the population of ß-hairpin is reduced by a factor of 1.5 and the population of α-helix in the region 17-24 is increased by a factor of two upon NQTrp binding. These two factors, which impact the free energy barrier for nucleation, provide a first explanation for the reported reduced Aß1-40/1-42 aggregation kinetics in the presence of NQTrp. Backbone and side-chain interactions of Aß with NQTrp may also inhibit Aß-Aß contacts. The fraction of free Aß1-28 monomer is, however, on the order of 20-25% at 17.5 mM, and this shows that the affinity of NQTrp is low and hence its inhibitory activity is not very strong. This inhibitor can be improved to reduce the formation of dimer, a critical step in aggregation and toxicity.
Asunto(s)
Péptidos beta-Amiloides/química , Simulación de Dinámica Molecular , Naftoquinonas/química , Fragmentos de Péptidos/química , Triptófano/análogos & derivados , Péptidos beta-Amiloides/antagonistas & inhibidores , Humanos , Cinética , Fragmentos de Péptidos/antagonistas & inhibidores , Unión Proteica , Pliegue de Proteína , Multimerización de Proteína , Estructura Secundaria de Proteína , Termodinámica , Triptófano/químicaRESUMEN
The nucleoprotein (NP) binds the viral RNA genome as oligomers assembled with the polymerase in a ribonucleoprotein complex required for transcription and replication of influenza A virus. Novel antiviral candidates targeting the nucleoprotein either induced higher order oligomers or reduced NP oligomerization by targeting the oligomerization loop and blocking its insertion into adjacent nucleoprotein subunit. In this study, we used a different structure-based approach to stabilize monomers of the nucleoprotein by drugs binding in its RNA-binding groove. We recently identified naproxen as a drug competing with RNA binding to NP with antiinflammatory and antiviral effects against influenza A virus. Here, we designed novel derivatives of naproxen by fragment extension for improved binding to NP. Molecular dynamics simulations suggested that among these derivatives, naproxen A and C0 were most promising. Their chemical synthesis is described. Both derivatives markedly stabilized NP monomer against thermal denaturation. Naproxen C0 bound tighter to NP than naproxen at a binding site predicted by MD simulations and shown by competition experiments using wt NP or single-point mutants as determined by surface plasmon resonance. MD simulations suggested that impeded oligomerization and stabilization of monomeric NP is likely to be achieved by drugs binding in the RNA grove and inducing close to their binding site conformational changes of key residues hosting the oligomerization loop as observed for the naproxen derivatives. Naproxen C0 is a potential antiviral candidate blocking influenza nucleoprotein function.
Asunto(s)
Antivirales/química , Virus de la Influenza A/química , Gripe Humana/virología , Naproxeno/química , Nucleoproteínas/química , Antivirales/uso terapéutico , Sitios de Unión , Humanos , Virus de la Influenza A/efectos de los fármacos , Gripe Humana/tratamiento farmacológico , Simulación de Dinámica Molecular , Naproxeno/análogos & derivados , Naproxeno/uso terapéutico , Multimerización de Proteína , ARN Viral/químicaRESUMEN
The self-assembly of the amyloid-ß (Aß) peptide of 39-43 amino acids into senile plaques is one hallmark of Alzheimer's disease (AD) pathology. While A2 V carriers remain healthy in the heterozygous state, they suffer from early onset AD in the homozygous state. As a first toward understanding the impact of A2 V on Aß at its earlier stage, we characterized the equilibrium ensemble of the Aß1-28 wild type and Aß1-28 A2 V monomers by means of extensive atomistic replica exchange molecular dynamics simulations. While global conformational properties such as the radius of gyration and the average secondary structure content of the whole peptides are very similar, the population of ß-hairpins is increased by a factor of 4 in A2 V, and this may explain the enhanced Aß1-40 A2 V aggregation kinetics with respect to Aß1-40 wild type. Both peptides display a non-negligible population of extended metastable conformations differing however in their atomic details that represent ideal seeds for polymerization. Remarkably, upon A2 V mutation, the intrinsic disorder of Aß1-28 monomer is reduced by a factor of 2, and the free energy landscape is completely different. This difference in the conformational ensembles of the two peptides may explain in part why the mixture of the Aß40 WT and A2 V peptides protects against AD.
Asunto(s)
Péptidos beta-Amiloides/química , Fragmentos de Péptidos/química , Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/patología , Secuencia de Aminoácidos , Péptidos beta-Amiloides/genética , Péptidos beta-Amiloides/metabolismo , Humanos , Cinética , Luz , Simulación de Dinámica Molecular , Datos de Secuencia Molecular , Mutación , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Análisis de Componente Principal , Estructura Secundaria de Proteína , Dispersión de Radiación , TermodinámicaRESUMEN
The nucleoprotein (NP) binds the viral RNA genome and associates with the polymerase in a ribonucleoprotein complex (RNP) required for transcription and replication of influenza A virus. NP has no cellular counterpart, and the NP sequence is highly conserved, which led to considering NP a hot target in the search for antivirals. We report here that monomeric nucleoprotein can be inhibited by a small molecule binding in its RNA binding groove, resulting in a novel antiviral against influenza A virus. We identified naproxen, an anti-inflammatory drug that targeted the nucleoprotein to inhibit NP-RNA association required for NP function, by virtual screening. Further docking and molecular dynamics (MD) simulations identified in the RNA groove two NP-naproxen complexes of similar levels of interaction energy. The predicted naproxen binding sites were tested using the Y148A, R152A, R355A, and R361A proteins carrying single-point mutations. Surface plasmon resonance, fluorescence, and other in vitro experiments supported the notion that naproxen binds at a site identified by MD simulations and showed that naproxen competed with RNA binding to wild-type (WT) NP and protected active monomers of the nucleoprotein against proteolytic cleavage. Naproxen protected Madin-Darby canine kidney (MDCK) cells against viral challenges with the H1N1 and H3N2 viral strains and was much more effective than other cyclooxygenase inhibitors in decreasing viral titers of MDCK cells. In a mouse model of intranasal infection, naproxen treatment decreased the viral titers in mice lungs. In conclusion, naproxen is a promising lead compound for novel antivirals against influenza A virus that targets the nucleoprotein in its RNA binding groove.
Asunto(s)
Antiinflamatorios no Esteroideos/farmacología , Antivirales/farmacología , Naproxeno/farmacología , Nucleoproteínas/antagonistas & inhibidores , ARN Viral/antagonistas & inhibidores , Proteínas Virales/antagonistas & inhibidores , Animales , Antiinflamatorios no Esteroideos/química , Antivirales/química , Sitios de Unión , Perros , Descubrimiento de Drogas , Reposicionamiento de Medicamentos , Subtipo H1N1 del Virus de la Influenza A/química , Subtipo H1N1 del Virus de la Influenza A/efectos de los fármacos , Subtipo H1N1 del Virus de la Influenza A/metabolismo , Subtipo H3N2 del Virus de la Influenza A/química , Subtipo H3N2 del Virus de la Influenza A/efectos de los fármacos , Subtipo H3N2 del Virus de la Influenza A/metabolismo , Células de Riñón Canino Madin Darby , Ratones , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Naproxeno/química , Nucleoproteínas/química , Nucleoproteínas/metabolismo , Infecciones por Orthomyxoviridae/tratamiento farmacológico , Infecciones por Orthomyxoviridae/virología , Mutación Puntual , Unión Proteica , ARN Viral/química , ARN Viral/metabolismo , Proteínas Virales/química , Proteínas Virales/metabolismoRESUMEN
We report the structure-based design and synthesis of a unique NOS inhibitor, called nanoshutter NS1, with two-photon absorption properties. NS1 targets the NADPH site of NOS by a nucleotide moiety mimicking NADPH linked to a conjugated push-pull chromophore with nonlinear absorption properties. Because NS1 could not provide reducing equivalents to the protein and competed with NADPH binding, it efficiently inhibited NOS catalysis. NS1 became fluorescent once bound to NOS with an excellent signal-to-noise ratio because of two-photon excitation avoiding interference from the flavin-autofluorescence and because free NS1 was not fluorescent in aqueous solutions. NS1 fluorescence enhancement was selective for constitutive NOS in vitro, in particular for endothelial NOS (eNOS). Molecular dynamics simulations suggested that two variable residues among NOS isoforms induced differences in binding of NS1 and in local solvation around NS1 nitro group, consistent with changes of NS1 fluorescence yield. NS1 colocalized with eNOS in living human umbilical vein endothelial cells. Thus, NS1 constitutes a unique class of eNOS probe with two-photon excitation in the 800-950-nm range, with great perspectives for eNOS imaging in living tissues.
Asunto(s)
Colorantes Fluorescentes , Células Endoteliales de la Vena Umbilical Humana/enzimología , Microscopía de Fluorescencia por Excitación Multifotónica/métodos , NADP , Óxido Nítrico Sintasa de Tipo III , Catálisis , Colorantes Fluorescentes/síntesis química , Colorantes Fluorescentes/química , Colorantes Fluorescentes/farmacología , Células Endoteliales de la Vena Umbilical Humana/química , Células Endoteliales de la Vena Umbilical Humana/citología , Humanos , Isoenzimas/química , Isoenzimas/metabolismo , Simulación de Dinámica Molecular , NADP/análogos & derivados , NADP/síntesis química , NADP/química , NADP/farmacología , Óxido Nítrico/biosíntesis , Óxido Nítrico/química , Óxido Nítrico Sintasa de Tipo III/química , Óxido Nítrico Sintasa de Tipo III/metabolismoRESUMEN
The human respiratory syncytial virus (HRSV) genome is composed of a negative-sense single-stranded RNA that is tightly associated with the nucleoprotein (N). This ribonucleoprotein (RNP) complex is the template for replication and transcription by the viral RNA-dependent RNA polymerase. RNP recognition by the viral polymerase involves a specific interaction between the C-terminal domain of the phosphoprotein (P) (P(CTD)) and N. However, the P binding region on N remains to be identified. In this study, glutathione S-transferase (GST) pulldown assays were used to identify the N-terminal core domain of HRSV N (N(NTD)) as a P binding domain. A biochemical characterization of the P(CTD) and molecular modeling of the N(NTD) allowed us to define four potential candidate pockets on N (pocket I [PI] to PIV) as hydrophobic sites surrounded by positively charged regions, which could constitute sites complementary to the P(CTD) interaction domain. The role of selected amino acids in the recognition of the N-RNA complex by P was first screened for by site-directed mutagenesis using a polymerase activity assay, based on an HRSV minigenome containing a luciferase reporter gene. When changed to Ala, most of the residues of PI were found to be critical for viral RNA synthesis, with the R132A mutant having the strongest effect. These mutations also reduced or abolished in vitro and in vivo P-N interactions, as determined by GST pulldown and immunoprecipitation experiments. The pocket formed by these residues is critical for P binding to the N-RNA complex, is specific for pneumovirus N proteins, and is clearly distinct from the P binding sites identified so far for other nonsegmented negative-strand viruses.
Asunto(s)
Nucleoproteínas/metabolismo , Mapeo de Interacción de Proteínas , Virus Sincitial Respiratorio Humano/metabolismo , Proteínas Estructurales Virales/metabolismo , Sustitución de Aminoácidos , Animales , Sitios de Unión , Línea Celular , Cricetinae , Inmunoprecipitación , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Unión Proteica , Virus Sincitial Respiratorio Humano/genéticaRESUMEN
The influenza viruses contain a segmented, negative stranded RNA genome. Each RNA segment is covered by multiple copies of the nucleoprotein (NP). X-ray structures have shown that NP contains well-structured domains juxtaposed with regions of missing electron densities corresponding to loops. In this study, we tested if these flexible loops gated or promoted RNA binding and RNA-induced oligomerization of NP. We first performed molecular dynamics simulations of wt NP monomer and trimer in comparison with the R361A protein mutated in the RNA binding groove, using the H1N1 NP as the initial structure. Calculation of the root-mean-square fluctuations highlighted the presence of two flexible loops in NP trimer: loop 1 (73-90), loop 2 (200-214). In NP, loops 1 and 2 formed a 10-15 Å-wide pinch giving access to the RNA binding groove. Loop 1 was stabilized by interactions with K113 of the adjacent ß-sheet 1 (91-112) that interacted with the RNA grove (linker 360-373) via multiple hydrophobic contacts. In R361A, a salt bridge formed between E80 of loop 1 and R208 of loop 2 driven by hydrophobic contacts between L79 and W207, due to a decreased flexibility of loop 2 and loop 1 unfolding. Thus, RNA could not access its binding groove in R361A; accordingly, R361A had a much lower affinity for RNA than NP. Disruption of the E80-R208 interaction in the triple mutant R361A-E80A-E81A increased its RNA binding affinity and restored its oligomerization back to wt levels in contrast with impaired levels of R361A. Our data suggest that the flexibility of loops 1 and 2 is required for RNA sampling and binding which likely involve conformational change(s) of the nucleoprotein.
Asunto(s)
Subtipo H1N1 del Virus de la Influenza A/metabolismo , Simulación de Dinámica Molecular , Proteínas de Unión al ARN/metabolismo , Proteínas del Núcleo Viral/metabolismo , Sustitución de Aminoácidos , Sitios de Unión/genética , Unión Competitiva , Cristalografía por Rayos X , Interacciones Hidrofóbicas e Hidrofílicas , Subtipo H1N1 del Virus de la Influenza A/genética , Modelos Moleculares , Mutación , Proteínas de la Nucleocápside , Unión Proteica , Conformación Proteica , Multimerización de Proteína , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , ARN/genética , ARN/metabolismo , Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/genética , Proteínas del Núcleo Viral/química , Proteínas del Núcleo Viral/genéticaRESUMEN
Picobirnaviruses possess a bisegmented double-stranded RNA genome. While the segment 2 encodes the RNA-dependent RNA polymerase, the segment 1 displays two open reading frames (ORFs). ORF2 was recently shown to code the capsid precursor and ORF1 product has not been characterized. In this study, we show that the three ORF1 sequences available in databases and representing three phylogenetically distant picobirnaviruses (two from human and one from rabbit hosts) encode proteins of various sizes (106-224 residues and without proline and cysteine) harbouring a particular sequence motif (ExxRxNxxxE) repeated four to ten times, depending on the virus species. Several algorithms predicted the three proteins to be mainly unfolded in the domains containing the repeats. The glycine-rich 25-40 amino acid long C-terminal domains containing hydrophobic residues with a periodicity of 3-4 residues are predicted structurally different of the upstream domains containing the motif repetitions. The ExxRxNxxxE sequence was not previously identified as a short linear motif in eukaryotic and prokaryotic proteins. Its function remains elusive.
Asunto(s)
Picobirnavirus/genética , Secuencias Repetitivas de Aminoácido , Proteínas Virales/genética , Secuencias de Aminoácidos , Animales , Biología Computacional/métodos , Humanos , ARN Viral/genética , Conejos , Homología de Secuencia de AminoácidoRESUMEN
Achieving molecular recognition of NADPH binding sites is a compelling strategy to control many redox biological processes. The NADPH sites recognize the ubiquitous NADPH cofactor via highly conserved binding interactions, despite differences in the regulation of the hydride transfer in redox active proteins. We recently developed a photoactive NADPH substitute, called nanotrigger NT synchronizing the initiation of enzymatic catalysis of the endothelial NO-synthase (eNOS) with a laser pulse. Spatial and temporal control of enzymatic activity by such a designed light-driven activator would benefit from achieving molecular selectivity, i.e. activation of a single NADPH-mediated enzyme.In this work, we probe the ability of NT to discriminate between two NADPH sites with light. The selected NADPH sites belong to dihydrofolate reductase dihydrofolate reductase enzyme (DHFR) and endothelial NO-synthase (eNOS). Ultrafast kinetics showed that NT could not activate DHFR catalysis with a laser pulse in contrast with the observed trigger of eNOS catalysis leading to NO formation. Homology modelling, molecular dynamics simulations showed that NT discriminated between the two NADPH sites by different donor to acceptor distances and by local steric effects hindering light activation of DHFR catalysis. The data suggested that the narrow NADPH site required a tight fit of the nanotrigger at a suitable distance/angle to the electron acceptor for a specific activation of the catalysis. The ability of the nanotrigger to activate eNOS combined with a low reactivity in unfavourable NADPH sites makes NT a highly promising tool for targeting eNOS in endothelial cells with a laser pulse.
Asunto(s)
Luz , NADP/metabolismo , Tetrahidrofolato Deshidrogenasa/metabolismo , Sitios de Unión , Catálisis/efectos de la radiación , Cinética , Simulación de Dinámica Molecular , Estructura Molecular , Óxido Nítrico Sintasa de Tipo III/química , Óxido Nítrico Sintasa de Tipo III/metabolismo , Estructura Secundaria de Proteína , Tetrahidrofolato Deshidrogenasa/químicaRESUMEN
The initial events in protein aggregation involve fluctuations that populate monomer conformations, which lead to oligomerization and fibril assembly. The highly populated structures, driven by a balance between hydrophobic and electrostatic interactions in the protease-resistant wild-type Abeta(21-30) peptide and mutants E22Q (Dutch), D23N (Iowa), and K28N, are analyzed using molecular dynamics simulations. Intrapeptide electrostatic interactions were connected to calculated pK(a) values that compare well with the experimental estimates. The pK(a) values of the titratable residues show that E22 and D23 side chains form salt bridges only infrequently with the K28 side chain. Contacts between E22-K28 are more probable in "dried" salt bridges, whereas D23-K28 contacts are more probable in solvated salt bridges. The strength of the intrapeptide hydrophobic interactions increases as D23NAsunto(s)
Péptidos beta-Amiloides/química
, Péptidos beta-Amiloides/genética
, Fragmentos de Péptidos/química
, Fragmentos de Péptidos/genética
, Enfermedad de Alzheimer/genética
, Enfermedad de Alzheimer/metabolismo
, Sustitución de Aminoácidos
, Simulación por Computador
, Humanos
, Concentración de Iones de Hidrógeno
, Interacciones Hidrofóbicas e Hidrofílicas
, Modelos Moleculares
, Complejos Multiproteicos/química
, Complejos Multiproteicos/genética
, Mutagénesis Sitio-Dirigida
, Mutación Puntual
, Pliegue de Proteína
, Cloruro de Sodio/química
, Electricidad Estática
, Termodinámica
RESUMEN
In the amyloid fibrils formed from long fragments of the amyloid beta-protein (Abeta-protein), the monomers are arranged in parallel and lie perpendicular to the fibril axis. The structure of the monomers satisfies the amyloid self-organization principle; namely, the low free energy state of the monomer maximizes the number of intra- and interpeptide contacts and salt bridges. The formation of the intramolecular salt bridge between Asp(D)23 and Lys(K)28 ensures that unpaired charges are not buried in the low-dielectric interior. We have investigated, using all-atom molecular dynamics simulations in explicit water, whether the D23-K28 interaction forms spontaneously in the isolated Abeta10-35 monomer. To validate the simulation protocol, we show, using five independent trajectories spanning a total of 100 ns, that the pKa values of the titratable groups are in good agreement with experimental measurements. The computed free energy disconnectvity graph shows that broadly the ensemble of compact random coil conformations can be clustered into four basins that are separated by free energy barriers ranging from 0.3 to 2.7 kcal/mol. There is significant residual structure in the conformation of the peptide in each of the basins. Due to the desolvation penalty, the structural motif with a stable turn involving the residues VGSN and a preformed D23-K28 contact is a minor component of the simulated structures. The extent of solvation of the peptides in the four basins varies greatly, which underscores the dynamical fluctuations in the monomer. Our results suggest that the early event in the oligomerization process must be the expulsion of discrete water molecules that facilitates the formation of interpeptide-interaction-driven stable structures with an intramolecular D23-K28 salt bridge and an intact VGSN turn.
Asunto(s)
Ácido Aspártico/química , Lisina/química , Péptidos beta-Amiloides/química , Simulación por Computador , Enlace de Hidrógeno , Interacciones Hidrofóbicas e Hidrofílicas , Modelos Moleculares , Estructura Terciaria de Proteína , Agua/químicaRESUMEN
Characterization of the early stages of peptide aggregation is of fundamental importance in elucidating the mechanism of the formation of deposits associated with amyloid disease. The initial step in the pathway of aggregation of the Abeta-protein, whose monomeric NMR structure is known, was studied through the simulation of the structure and stability of the peptide dimer in aqueous solution. A protocol based on shape complementarity was used to generate an assortment of possible dimer structures. The structures generated based on shape complementarity were evaluated using rapidly computed estimates of the desolvation and electrostatic interaction energies to identify a putative stable dimer structure. The potential of mean force associated with the dimerization of the peptides in aqueous solution was computed for both the hydrophobic and the electrostatic driven forces using umbrella sampling and classical molecular dynamics simulation at constant temperature and pressure with explicit solvent and periodic boundary conditions. The comparison of the two free energy profiles suggests that the structure of the peptide dimer is determined by the favorable desolvation of the hydrophobic residues at the interface. Molecular dynamics trajectories originating from two putative dimer structures indicate that the peptide dimer is stabilized primarily through hydrophobic interactions, while the conformations of the peptide monomers undergo substantial structural reorganization in the dimerization process. The finding that the phi-dimer may constitute the ensemble of stable Abeta(10-35) dimer has important implications for fibril formation. In particular, the expulsion of water molecules at the interface might be a key event, just as in the oligomerization of Abeta(16-22) fragments. We conjecture that events prior to the nucleation process themselves might involve crossing free energy barriers which depend on the peptide-peptide and peptide-water interactions. Consistent with existing experimental studies, the peptides within the ensemble of aggregated states show no signs of formation of secondary structure.
Asunto(s)
Péptidos beta-Amiloides/química , Péptidos beta-Amiloides/metabolismo , Fragmentos de Péptidos/química , Fragmentos de Péptidos/metabolismo , Simulación por Computador , Dimerización , Modelos Moleculares , Estructura Cuaternaria de Proteína , Estructura Secundaria de Proteína , Electricidad Estática , Factores de TiempoRESUMEN
Aberrant protein oligomerization is an important pathogenetic process in vivo. In Alzheimer's disease (AD), the amyloid beta-protein (Abeta) forms neurotoxic oligomers. The predominant in vivo Abeta alloforms, Abeta40 and Abeta42, have distinct oligomerization pathways. Abeta42 monomers oligomerize into pentamer/hexamer units (paranuclei) which self-associate to form larger oligomers. Abeta40 does not form these paranuclei, a fact which may explain the particularly strong linkage of Abeta42 with AD. Here, we sought to determine the structural elements controlling paranucleus formation as a first step toward the development of strategies for treating AD. Because oxidation of Met(35) is associated with altered Abeta assembly, we examined the role of Met(35) in controlling Abeta oligomerization. Oxidation of Met(35) in Abeta42 blocked paranucleus formation and produced oligomers indistinguishable in size and morphology from those produced by Abeta40. Systematic structural alterations of the C(gamma)(35)-substituent group revealed that its electronic nature, rather than its size (van der Waals volume), was the factor controlling oligomerization pathway choice. Preventing assembly of toxic Abeta42 paranuclei through selective oxidation of Met(35) thus represents a potential therapeutic approach for AD.