RESUMEN
Fused in sarcoma (FUS) is an abundant RNA-binding protein, which drives phase separation of cellular condensates and plays multiple roles in RNA regulation. The RNA-binding ability of FUS protein is crucial to its cellular function. Here, our molecular simulation study on the FUS-RNA complex provides atomic resolution insights into the observations from biochemical studies and also illuminates our understanding of molecular driving forces that mediate the structure, stability, and interaction of the RNA recognition motif (RRM) and RGG domains of FUS with a stem-loop junction RNA. We observe clear cooperativity and division of labor among the ordered (RRM) and disordered domains (RGG1 and RGG2) of FUS that leads to an organized and tighter RNA binding. Irrespective of the length of RGG2, the RGG2-RNA interaction is confined to the stem-loop junction and the proximal stem regions. On the other hand, the RGG1 interactions are primarily with the longer RNA stem. We find that the C terminus of RRM, which make up the "boundary residues" that connect the folded RRM with the long disordered RGG2 stretch of the protein, plays a critical role in FUS-RNA binding. Our study provides high-resolution molecular insights into the FUS-RNA interactions and forms the basis for understanding the molecular origins of full-length FUS interaction with RNA.
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Motivo de Reconocimiento de ARN , ARN , Dominios Proteicos , ARN/metabolismo , Motivo de Reconocimiento de ARN/genética , Proteína FUS de Unión a ARN/metabolismo , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , HumanosRESUMEN
HIV-1 protease is an essential enzyme in the life cycle of human immunodeficiency virus (HIV) and hence is one of the most important targets for antiviral drug design. Although there are ten FDA approved drugs against HIV protease (PR), their long term usage elicits mutations leading to drug resistance. As a result, novel therapeutic approaches are being explored including synthetic antibodies. Recently, a murine monoclonal antibody, mAB1696 (mAB) was reported to inhibit PR by preventing dimerization. Crystallographic data could reveal only six protease residues that interact with mAB. The present study employs a range of computational techniques, starting from protein-protein docking to all-atomic molecular dynamics simulations to generate plausible 3D structures of PR-mAB complex. Results show that mAB interacts very strongly with several PR dimer interface residues, such as Gln7, Arg8 (N-terminal), Cys95, Leu97 (C-terminal), Thr26, Gly27 (active site), Gly49, Ile50 (flap), apart from its interactions with the PR epitope region, Pro1-Trp6 (N-terminal). These observations support the hypothesis that binding of mAB prevents the dimerization of PR. The interactions and binding conformations identified in this study could form the basis for designing allosteric inhibitors preventing the dimerization of HIV-1 Protease.
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Inhibidores de la Proteasa del VIH , VIH-1 , Animales , Anticuerpos Monoclonales , Proteasa del VIH , Humanos , Ratones , Péptido HidrolasasRESUMEN
G-quadruplexes (GQs) are highly stable noncanonical forms of nucleic acids that are present in important genomic regions. The central core of the GQ is lined up by four closely spaced carbonyl groups from the G-quartets, and the resulting electrostatic repulsion is neutralized by the coordinating cations. In spite of several reports on GQ structure and cation-GQ interactions, the atomic- to molecular-level understanding of the ion dynamics and ion exchange in the GQ core is quite poor. Here, we attempt to elucidate the mechanism of Na+ and K+ binding to the GQ core and trace the exchange of these ions with the ions in bulk by means of all-atomic molecular dynamics (MD) simulations. One of the most studied GQs, Oxytricha nova telomeric G-quadruplex (OxyGQ), is taken as the representative GQ. Subsequently, umbrella sampling MD simulations were performed to elucidate the energetics of ion translocation from one end to the other end of the GQ central core. Our study highlights the importance of ion hydration for the uptake and correct positioning of the cations in the core. The free-energy landscape of ion transport has shown favorable in-plane binding of Na+ ions with GQ quartets, which matches very well with the crystal structure. The binding of K+ ions, on the other hand, was out-of-plane and its translocation required a larger barrier to cross.
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G-Cuádruplex , Oxytricha , ADN , Conformación de Ácido Nucleico , Oxytricha/genética , Potasio , SodioRESUMEN
Metastasis is the major hindrance in the treatment of all cancers, including laryngeal squamous cell carcinoma. Intensive researches are under way to identify the effective natural polyphenols with anti-metastatic ability for cancer treatment. Wheatgrass, an herbal plant has been reported to show anticancer effects. Hence, in this study, we aimed to analyze the anti-metastatic effect of methanol extract of wheatgrass (MEWG). The levels of metastatic marker proteins were determined by western blot. PI3K and AKT levels were determined by real time (RT)-PCR analysis. In silico molecular docking was done to check the interaction of the 14 components (identified by HPLC/GCMS) of MEWG with PI3K and AKT. MEWG effectively decreased the metastatic protein expressions, namely VEGF, MMP-9 and COX-2 and increased TIMP-2. RT-PCR results showed reduced m-RNA levels of both PI3K and AKT when compared to control. Molecular docking studies revealed interaction of most of the identified compounds of the extract with the important residues of PI3K and AKT. These findings indicate that MEWG inhibits metastasis and angiogenesis in Hep-2 cells possibly via PI3K/AKT due to the cumulative effect of polyphenols and other constituent present in extract. The compounds of the extract were also found to be directly involved in inhibition of AKT/PI3K, thus could help to restrain metastasis.
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Inhibidores de la Angiogénesis/metabolismo , Anticarcinógenos/metabolismo , Carcinoma de Células Escamosas/prevención & control , Metástasis de la Neoplasia/prevención & control , Neovascularización Patológica/prevención & control , Extractos Vegetales/metabolismo , Triticum/química , Inhibidores de la Angiogénesis/análisis , Inhibidores de la Angiogénesis/química , Inhibidores de la Angiogénesis/uso terapéutico , Anticarcinógenos/análisis , Anticarcinógenos/química , Anticarcinógenos/uso terapéutico , Antineoplásicos Fitogénicos/análisis , Antineoplásicos Fitogénicos/química , Antineoplásicos Fitogénicos/metabolismo , Antineoplásicos Fitogénicos/uso terapéutico , Biomarcadores de Tumor/metabolismo , Carcinoma de Células Escamosas/dietoterapia , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patología , Línea Celular Tumoral , Movimiento Celular , Biología Computacional , Suplementos Dietéticos , Etnofarmacología , Sistemas Especialistas , Regulación Neoplásica de la Expresión Génica , Humanos , India , Neoplasias Laríngeas/dietoterapia , Neoplasias Laríngeas/metabolismo , Neoplasias Laríngeas/patología , Neoplasias Laríngeas/prevención & control , Medicina Tradicional , Conformación Molecular , Simulación del Acoplamiento Molecular , Metástasis de la Neoplasia/patología , Metástasis de la Neoplasia/terapia , Proteínas de Neoplasias/química , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Neovascularización Patológica/dietoterapia , Neovascularización Patológica/metabolismo , Extractos Vegetales/química , Extractos Vegetales/uso terapéuticoRESUMEN
Biomolecular recognition of proteins and nucleic acids is mainly mediated by their structural features and the molecular dynamics simulations approach has been used to explore this recognition processes at the atomic level. L1-Endonuclease, an enzyme involved in L1 retrotransposition, cleaves the TA junction DNA (5'-TTTT/AA-3') and expresses high specificity for target site recognition. The present study highlights the structural features of L1-endonuclease as well as DNA responsible for such specific recognition. Especially, the importance of ßB6-B5 hairpin loop in DNA recognition has been elucidated by analyzing the dynamics of Thr192 mutated L1-endonuclease. In addition, simulations of the endonuclease complexed with DNA substrates (sequences having TA and CG junctions) revealed the specificity of L1 endonuclease towards TA junction. Molecular dynamics simulations revealed that the ßB6-B5 hairpin loop protrudes well into the minor groove of DNA having TA junction and induces DNA bending such that the width of minor groove is increased. Such endonuclease induced bending of TA junction DNA sequence positions the scissile phosphodiester bond of DNA for cleavage. The innate property of minor groove widening in TA junction than in CG junction is utilized by the ßB6-ßB5 hairpin loop of endonuclease while recognizing the DNA sequences. The present study also highlights the role of Mg2+ cation in catalysis and attempts to explore the possible target site DNA cleavage mechanism.
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Endonucleasas/metabolismo , Ácidos Nucleicos/metabolismo , Sitios de Unión/fisiología , ADN/metabolismo , Humanos , Simulación de Dinámica Molecular , Conformación de Ácido Nucleico , Especificidad por SustratoRESUMEN
The recognition and binding of nucleic acids by ORF1p, an L1 retrotransposon protein, have not yet been clearly understood due to the lack of structural knowledge. The present study attempts to identify the probable single-stranded RNA binding pathway of trimeric ORF1p using computational methods like ligand mapping methodology combined with molecular dynamics simulations. Using the ligand mapping methodology, the possible RNA interacting sites on the surface of the trimeric ORF1p were identified. The crystal structure of the ORF1p timer and an RNA molecule of 29 nucleotide bases in length were used to generate the structure of the ORF1p complex based on information on predicted binding sites as well as the functional states of the CTD. The various complexes of ORF1p-RNA were generated using polyU, polyA and L1RNA sequences and were simulated for a period of 75 ns. The observed stable interaction pattern was used to propose the possible binding pathway. Based on the binding free energy for complex formation, both polyU and L1RNA complexes were identified as stable complexes, while the complex formed with polyA was the least stable one. Furthermore, the importance of the residues in the CC domain (Lys137 and Arg141), the RRM loop (Arg206, Arg210 and Arg211) and the CTD (Arg 261 and Arg262) of all three chains in stabilizing the wrapped RNA has been highlighted in this study. The presence of several electrostatic interactions including H-bond interactions increases the affinity towards RNA and hence plays a vital role in retaining the wrapped position of RNA around ORF1p. Altogether, this study presents one of the possible RNA binding pathways of ORF1p and clearly highlights the functional state of ORF1p visited during RNA binding.
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Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Proteínas de Unión al ARN/química , ARN/química , Algoritmos , Humanos , Enlace de Hidrógeno , Conformación de Ácido Nucleico , Análisis de Componente Principal , Conformación Proteica , Multimerización de Proteína , ARN/metabolismo , Proteínas de Unión al ARN/metabolismo , Electricidad Estática , Relación Estructura-ActividadRESUMEN
Post translational modifications have a profound role in the regulation of several biological processes such as transcription, replication, and DNA repair. Acetylation and phosphorylation form a major class of post translational modifications involved in nucleosomal regulation by modifying its structure. The effect of post translational modifications on nucleosome structure could be better explored when the molecular trajectories explaining the time dependent structural evolution over a period of time is examined at the atomic level. The present study attempts to highlight the importance of acetylation, especially at entry-exit (Lys56) and dyad (Lys115 and Lys122) regions in regulating the nucleosome accessibility and mobility using all atom simulations. It is evident from this study that acetylation at Lys56, Lys115, and Lys122 introduces local changes in the electrostatic nature of the lateral surface and thereby weakens the histone-DNA interactions. In addition, simulations also reveal significant changes in the dynamics of superhelical DNA. The acetylation at Lys56 promotes a high amplitude out-of-planar movement of entry-exit termini. Whereas, acetylation at Lys115 and Lys122 increases the flexibility of the superhelical DNA to facilitate the rolling of the superhelical DNA around the octameric histone. In essence, the present study highlights the role of acetylation at Lys56, Lys115, and Lys122 in transcriptional regulation by promoting high amplitude dynamics of superhelical DNA for a possible unwrapping as well as mobility of nucleosome.
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Histonas/química , Histonas/metabolismo , Lisina/metabolismo , Nucleosomas/metabolismo , Acetilación , Simulación de Dinámica Molecular , Multimerización de Proteína , Estabilidad Proteica , Estructura Cuaternaria de Proteína , TermodinámicaRESUMEN
Retroviral integrases are reported to form alternate dimer assemblies like the core-core dimer and reaching dimer. The core-core dimer is stabilized predominantly by an extensive interface between two catalytic core domains. The reaching dimer is stabilized by N-terminal domains that reach to form intermolecular interfaces with the other subunit's core and C-terminal domains (CTD), as well as CTD-CTD interactions. In this study, molecular dynamics (MD), Brownian dynamics (BD) simulations, and free energy analyses, were performed to elucidate determinants for the stability of the reaching dimer forms of full-length Avian Sarcoma Virus (ASV) and Human Immunodeficiency Virus (HIV) IN, and to examine the role of the C-tails (the last ~16-18 residues at the C-termini) in their structural dynamics. The dynamics of an HIV reaching dimer derived from small angle X-ray scattering and protein crosslinking data, was compared with the dynamics of a core-core dimer model derived from combining the crystal structures of two-domain fragments. The results showed that the core domains in the ASV reaching dimer express free dynamics, whereas those in the HIV reaching dimer are highly stable. BD simulations suggest a higher rate of association for the HIV core-core dimer than the reaching dimer. The predicted stability of these dimers was therefore ranked in the following order: ASV reaching dimer < HIV reaching dimer < composite core-core dimer. Analyses of MD trajectories have suggested residues that are critical for intermolecular contacts in each reaching dimer. Tests of these predictions and insights gained from these analyses could reveal a potential pathway for the association and dissociation of full-length IN multimers.
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Virus del Sarcoma Aviar/química , Integrasa de VIH/química , VIH-1/química , Simulación de Dinámica Molecular , Multimerización de Proteína , Secuencias de Aminoácidos , Virus del Sarcoma Aviar/enzimología , Dominio Catalítico , Cristalografía por Rayos X , VIH-1/enzimología , Cinética , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , TermodinámicaRESUMEN
BACKGROUND: Deregs been implicated in the malignancy of cancer. Since many years investigation on the traditional herbs has been the focus to develop novel and effective drug for cancer remedies. Wheatgrass is a medicinal plant, used in folk medicine to cure various diseases. The present study was undertaken to gain insights into antiproliferative effect of methanol extract of wheatgrass. MATERIALS METHODS: Cell viability was assessed via 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and Lactate Dehydrogenase assays. Cell cycle was analyzed by flow cytometry. Western blot was performed to determine the p53 and cyclin D1 levels. In silico docking interaction of the 14 active components (identified by high-performance liquid chromatography/gas chromatography-mass spectroscopy) of the methanol extract was tested with cyclin D1 (Protein Data Bank ID: 2W96) and compared with the reference cyclin D1/Cdk4 inhibitor. RESULTS: Methanol extract of wheatgrass effectively reduced the cell viability. The cell cycle analysis showed that the extract treatment caused G1 arrest. The level of cyclin D1 was decreased, whereas p53 level was increased. Molecular docking studies revealed interaction of seven active compounds of the extract with the vital residues (Lys112/Glu141) of cyclin D1. CONCLUSION: These findings indicate that the methanol extract of wheatgrass inhibits human laryngeal cancer cell proliferation via cell cycle G1 arrest and p53 induction. The seven active compounds of the extract were also found to be directly involved in the inhibition of cyclin D1/Cdk4 binding, thus inhibiting the cell proliferation.
RESUMEN
VEGFR-2, a transmembrane tyrosine kinase receptor is responsible for angiogenesis and has been an attractive target in treating cancers. The inhibition mechanism of structurally diverse urea derivatives, reported as VEGFR-2 inhibitors, was explored by pharmacophore modeling, QSAR, and molecular dynamics based free energy analysis.The pharmacophore hypothesis AADRR, resulted in a highly significant atom based 3D-QSAR model (r(2) = 0.94 and q(2) = 0.84). Binding free energy analysis of the docked complexes of highly active and inactive compounds, after 7 ns MD simulation, revealed the importance of van der Waals interaction in VEGFR-2 inhibition. The decomposition of binding free energy on a per residue basis disclosed that the residues in hinge region and hydrophobic pocket play a role in discriminating the active and inactive inhibitors. Thus, the present study proposes a pharmacophore hypothesis representing the identified interactions pattern and its further application as a template in screening databases to identify novel VEGFR-2 inhibitor scaffolds.
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Oximas/química , Oximas/farmacología , Relación Estructura-Actividad Cuantitativa , Urea/análogos & derivados , Urea/farmacología , Receptor 2 de Factores de Crecimiento Endotelial Vascular/antagonistas & inhibidores , Relación Dosis-Respuesta a Droga , Modelos Moleculares , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Relación Estructura-Actividad , Termodinámica , Urea/química , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismoRESUMEN
HIV integrase catalyzes the integration between host and viral DNA and is considered as an interesting target for treating HIV. Knowledge of the complete structure of integrase is inevitable to describe the communicative inter-domain interactions affecting the HIV integration and disintegration process and hence the study on full-length integrase turns out to be an essential task. In this investigation, a structure of full-length integrase is designed to analyze the global dynamics of integrase dimer and monomers (with and without the C-terminal, 270-288 amino acids) for a period of 20 ns. The molecular dynamics analysis and the subsequent DynDom analysis reveal (i) a stable dynamics of dimeric CCD and NTD domains and (ii) CCD-α11-mediated rotational-cum-translational CTD motion as the functional dynamics of IN dimer. This observation supports that (i) aggregation enhances the integrase activity and (ii) flexible CTD for its cis and trans coordination with CCD. The role of C-loop over the dynamics of integrase is also explored, which unveils that the spatial arrangement of integrase domains is changed during dynamics in the absence of C-loop. In essence, here we report a C-loop-dependent structural dynamics of integrase and the active dynamics of integrase in dimer. Further studies on C-loop sensing mechanism and the multimerization of integrase would provide insight into HIV integration and disintegration processes. Supplementary material. Movies generated from molecular dynamics trajectory showing the CTD dynamics of IN structures (monomers with & without C-loop and dimer) are linked online to this article. The remaining supplementary data can be downloaded from the author's server at the URL http://ramutha.bicpu.edu.in .
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Integrasa de VIH/química , Simulación de Dinámica Molecular , Retroviridae/enzimología , Dimerización , Enlace de Hidrógeno , Modelos Moleculares , Conformación Proteica , Retroviridae/químicaRESUMEN
BACKGROUND: Patients with congestive heart failure, decreased left ventricular function, and debilitation are frequently maintained on anti-coagulants, including heparin. As such, these patients are at high risk for developing heparin-induced thrombocytopenia (HIT). Some of these HIT-positive individuals will ultimately undergo urgent heart transplantation or placement of a mechanical circulatory support device (MCSD). Such procedures require cardiopulmonary bypass (CPB) and full anti-coagulation. The safety of re-exposure to heparin during CPB despite the presence of recent-onset thrombocytopenia and Hep/PF4 antibodies has not been studied in the transplant or MCSD populations. METHODS: Over a 24-month period, 75 heart transplants and 55 MCSD implants were performed. Fourteen patients with acute HIT (thrombocytopenia and positive Hep/PF4 antibody by enzyme-linked immunosorbent assay [ELISA]) and 3 patients with a history of remote HIT underwent cardiac transplantation (n = 9) and/or MCSD placement (n = 8) using heparin as the anti-coagulant during CPB. The median time from diagnosis to CPB was 4.5 days in patients with acute HIT. RESULTS: Thirty-day survival was 100% in transplant patients and 75% in MCSD patients. Post-transplant ELISA testing was found to be negative in 4 patients with acute HIT and 3 with remote HIT. CONCLUSIONS: This series demonstrates that short-term re-exposure to heparin during urgent CPB for heart transplantation or MCSD placement is safe despite the presence of thrombocytopenia and Hep/PF4 antibodies. Moreover, the rapid clearance of Hep/PF4 antibodies in our transplant patients suggests a potential therapeutic role for immunosuppressive therapy in the management of HIT in the acute setting.