RESUMO
Several attempts have been made to identify antiviral genes against Tomato leaf curl New Delhi virus (ToLCNDV) and related viruses. This has led to the recognition of Ty genes (Ty1-Ty6), which have been successful in developing virus-resistant crops to some extent. Owing to the regular appearance of resistance-breaking strains of these viruses, it is important to identify genes related to resistance. In the present study, we identified a ToLCNDV resistance (R) gene, SlSw5a, in a ToLCNDV-resistant tomato cultivar, H-88-78-1, which lacks the known Ty genes. The expression of SlSw5a is controlled by the transcription factor SlMyb33, which in turn is regulated by microRNA159 (sly-miR159). Virus-induced gene silencing of either SlSw5a or SlMyb33 severely increases the disease symptoms and viral titer in leaves of resistant cultivar. Moreover, in SlMyb33-silenced plants, the relative messenger RNA level of SlSw5a was reduced, suggesting SlSw5a is downstream of the sly-miR159-SlMyb33 module. We also demonstrate that SlSw5a interacts physically with ToLCNDV-AC4 (viral suppressor of RNA silencing) to trigger a hypersensitive response (HR) and generate reactive oxygen species at infection sites to limit the spread of the virus. The "RTSK" motif in the AC4 C terminus is important for the interaction, and its mutation completely abolishes the interaction with Sw5a and HR elicitation. Overall, our research reports an R gene against ToLCNDV and establishes a connection between the upstream miR159-Myb33 module and its downstream target Sw5a to activate HR in the tomato, resulting in geminivirus resistance.
Assuntos
Begomovirus/fisiologia , Regulação da Expressão Gênica de Plantas/imunologia , Predisposição Genética para Doença , Doenças das Plantas/virologia , Solanum lycopersicum/genética , Solanum lycopersicum/virologia , Inativação Gênica , MicroRNAs , RNA de Plantas , Transcriptoma , Regulação para CimaRESUMO
The recurrent and recent global outbreak of the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has turned into a global concern which has infected more than 42 million people all over the globe, and this number is increasing in hours. Unfortunately, no vaccine or specific treatment is available, which makes it more deadly. A vaccine-informatics approach has shown significant breakthrough in peptide-based epitope mapping and opens the new horizon in vaccine development. In this study, we have identified a total of 15 antigenic peptides [including thymus cells (T-cells) and bone marrow or bursa-derived cells] in the surface glycoprotein (SG) of SARS-CoV-2 which is nontoxic and nonallergenic in nature, nonallergenic, highly antigenic and non-mutated in other SARS-CoV-2 virus strains. The population coverage analysis has found that cluster of differentiation 4 (CD4+) T-cell peptides showed higher cumulative population coverage over cluster of differentiation 8 (CD8+) peptides in the 16 different geographical regions of the world. We identified 12 peptides ((LTDEMIAQY, WTAGAAAYY, WMESEFRVY, IRASANLAA, FGAISSVLN, VKQLSSNFG, FAMQMAYRF, FGAGAALQI, YGFQPTNGVGYQ, LPDPSKPSKR, QTQTNSPRRARS and VITPGTNTSN) that are $80\hbox{--} 90\%$ identical with experimentally determined epitopes of SARS-CoV, and this will likely be beneficial for a quick progression of the vaccine design. Moreover, docking analysis suggested that the identified peptides are tightly bound in the groove of human leukocyte antigen molecules which can induce the T-cell response. Overall, this study allows us to determine potent peptide antigen targets in the SG on intuitive grounds, which opens up a new horizon in the coronavirus disease (COVID-19) research. However, this study needs experimental validation by in vitro and in vivo.
Assuntos
COVID-19/prevenção & controle , Epitopos de Linfócito B/imunologia , Epitopos de Linfócito T/imunologia , SARS-CoV-2/imunologia , Vacinas de Subunidades Antigênicas/imunologia , Sequência de Aminoácidos , COVID-19/imunologia , Biologia Computacional , Epitopos de Linfócito B/química , Epitopos de Linfócito T/química , Antígenos HLA/química , Humanos , Simulação de Acoplamento Molecular , Vacinas de Subunidades Antigênicas/químicaRESUMO
Appropriate amino acid substitutions are critical for protein engineering to redesign catalytic properties of industrially important enzymes like lipases. The present study aimed for improving the environmental stability of lipase from Pseudomonas plecoglossicida S7 through site-directed mutagenesis driven by computational studies. lipA gene was amplified and sequenced. Both wild type (WT) and mutant type (MT) lipase genes were expressed into the pET SUMO system. The expressed proteins were purified and characterized for pH and thermostability. The lipase gene belonged to subfamily I.1 lipase. Molecular dynamics revealed that Y12F-palmitic acid complex had a greater binding affinity (-6.3 Kcal/mol) than WT (-6.0 Kcal/mol) complex. Interestingly, MDS showed that the binding affinity of WT-complex (-130.314 ± 15.11 KJ/mol) was more than mutant complex (-108.405 ± 69.376 KJ/mol) with a marked increase in the electrostatic energy of mutant (-26.969 ± 12.646 KJ/mol) as compared to WT (-15.082 ± 13.802 KJ/mol). Y12F mutant yielded 1.27 folds increase in lipase activity at 55 °C as compared to the purified WT protein. Also, Y12F mutant showed increased activity (~ 1.2 folds each) at both pH 6 and 10. P. plecoglossicida S7. Y12F mutation altered the kinetic parameters of MT (Km- 1.38 mM, Vmax- 22.32 µM/min) as compared to WT (Km- 1.52 mM, Vmax- 29.76 µM/min) thus increasing the binding affinity of mutant lipase. Y12F mutant lipase with better pH and thermal stability can be used in biocatalysis.
Assuntos
Lipase , Lipase/metabolismo , Mutação , Mutagênese Sítio-Dirigida , Concentração de Íons de Hidrogênio , Estabilidade EnzimáticaRESUMO
Understanding of mechanistic details of Mediator functioning in plants is impeded as the knowledge of subunit organization and structure is lacking. In this study, an interaction map of Arabidopsis Mediator complex was analyzed to understand the arrangement of the subunits in the core part of the complex. Combining this interaction map with homology-based modeling, probable structural topology of core part of the Arabidopsis Mediator complex was deduced. Though the overall topology of the complex was similar to that of yeast, several differences were observed. Many interactions discovered in this study are not yet reported in other systems. AtMed14 and AtMed17 emerged as the key component providing important scaffold for the whole complex. AtMed6 and AtMed10 were found to be important for linking head with middle and middle with tail, respectively. Some Mediator subunits were found to form homodimers and some were found to possess transactivation property. Subcellular localization suggested that many of the Mediator subunits might have functions beyond the process of transcription. Overall, this study reveals role of individual subunits in the organization of the core complex, which can be an important resource for understanding the molecular mechanism of functioning of Mediator complex and its subunits in plants.
Assuntos
Proteínas de Arabidopsis/química , Arabidopsis/genética , Regulação da Expressão Gênica de Plantas , Complexo Mediador/química , Mapeamento de Interação de Proteínas , Subunidades Proteicas/química , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Sítios de Ligação , Clonagem Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Humanos , Complexo Mediador/genética , Complexo Mediador/metabolismo , Modelos Moleculares , Cebolas/genética , Cebolas/metabolismo , Células Vegetais/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Plântula/genética , Plântula/metabolismo , Homologia Estrutural de ProteínaRESUMO
The receptor for advanced glycation end products (RAGEs) was first illustrated in the year 1992. RAGE is a single-transmembrane and multi-ligand component of the immunoglobulin protein super family. The engagement of RAGE turns out to an establishment of numerous intracellular signalling mechanisms resulting in the progression and perpetuation of many types of cancer including, the pancreatic cancer. The present review primarily focuses on the multi-ligand activation of RAGEs leading to the downstream signalling cascade activation. The kick start of the RAGEs activation leads to the several anomalies and includes multiple types of cancers. The RAGE expression correlates well with the survival of pancreatic cancer cells leading to the myeloid response. RAGEs assist in the tumourogenesis which enhance and thrive to its fullest in the stressed tumour microenvironment. An improved perceptive of its involvement in pancreatic cancer may offer novel targets for tumour supervision and risk measurement.
Assuntos
Neoplasias Pancreáticas/metabolismo , Receptor para Produtos Finais de Glicação Avançada/metabolismo , Animais , Sobrevivência Celular , Humanos , Inflamação/metabolismo , Ligantes , Células Supressoras Mieloides/imunologia , Células Supressoras Mieloides/patologia , Estresse Oxidativo , Neoplasias Pancreáticas/imunologia , Neoplasias Pancreáticas/patologia , Receptor para Produtos Finais de Glicação Avançada/genética , Transdução de Sinais , Microambiente Tumoral , Proteína Supressora de Tumor p53/antagonistas & inibidores , Proteína Supressora de Tumor p53/metabolismoRESUMO
The YacK gene from Yersinia enterocolitica strain 7, cloned in pET28a vector and expressed in Escherichia coli BL21 (DE3), showed laccase activity when oxidized with 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) and guaiacol. The recombinant laccase protein was purified and characterized biochemically with a molecular mass of ≈58 KDa on SDS-PAGE and showed positive zymogram with ABTS. The protein was highly robust with optimum pH 9.0 and stable at 70 °C upto 12 h with residual activity of 70%. Kinetic constants, Km values, for ABTS and guaiacol were 675 µM and 2070 µM, respectively, with corresponding Vmax values of 0.125 µmol/ml/min and 6500 µmol/ml/min. It also possess antioxidative property against BSA and Cu(2+)/H2O2 model system. Constant pH MD simulation studies at different protonation states of the system showed ABTS to be most stable at acidic pH, whereas, diclofenac at neutral pH. Interestingly, aspirin drifted out of the binding pocket at acidic and neutral pH, but showed stable binding at alkaline pH. The biotransformation of diclofenac and aspirin by laccase also corroborated the in silico results. This is the first report on biotransformation of non-steroidal anti-inflammatory drugs (NSAIDs) using recombinant laccase from gut bacteria, supported by in silico simulation studies.
Assuntos
Anti-Inflamatórios não Esteroides/química , Anti-Inflamatórios não Esteroides/metabolismo , Lacase/química , Lacase/metabolismo , Simulação de Acoplamento Molecular , Yersinia enterocolitica/enzimologia , Sítios de Ligação , Biotransformação , Simulação por Computador , Ativação Enzimática , Estabilidade Enzimática , Escherichia coli/fisiologia , Lacase/genética , Modelos Químicos , Ligação Proteica , Conformação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/ultraestrutura , Yersinia enterocolitica/genéticaRESUMO
The leishmaniases are NDTs (neglected tropical diseases) that affect people all over the world. They are brought on by protozoans from the genus Leishmania and disseminated by phlebotomine flies that are afflicted with the disease. The best option to manage and lower the incidence of these diseases has been thought by the creation of a safe and effective vaccination. This research used an in silico based mining approach to look for high potential epitopes that might bind to MHC Class I and MHC Class II molecules (mainly; HLA-A*02:01 & HLA-DRB1*03:01) from human population in order to promote vaccine development. Based on the presence of signal peptides, GPI anchors, antigenicity predictions, and a subtractive proteomic technique, we have screened 17 putative antigenic proteins from the 8083 total proteins of L. major. After that thorough immunogenic epitope prediction were done using IEDB-AR tools. We isolated five immunogenic epitopes (three 9-mer & two 15-mer) from five antigenic proteins through docking and MD simulation analysis. Finally, these five anticipated epitopes, viz., TLPEIPVNV, ELMAPVFGL, TLAAAVALL, NSINIRLDGVTSAGF and NVPLVVDASSLFRVA have considerably stronger binding potential with their respective alleles and may trigger immunological responses. The goal of this work was to identify MHC restricted epitopes for CD8+ and CD4+ T cells activation using immunoinformatics in order to identify potential vaccine candidates against L. major parasites.
Assuntos
Epitopos de Linfócito T , Leishmania major , Humanos , Epitopos de Linfócito T/química , Leishmania major/metabolismo , Proteoma , Imunoinformática , Proteômica , Linfócitos T CD8-Positivos/metabolismo , Linfócitos T CD4-Positivos/metabolismo , Biologia ComputacionalRESUMO
The protein-protein interaction (PPI) network of the Mediator complex is very tightly regulated and depends on different developmental and environmental cues. Here, we present an interactive platform for comparative analysis of the Mediator subunits from humans, baker's yeast Saccharomyces cerevisiae, and model plant Arabidopsis thaliana in a user-friendly web-interface database called MediatorWeb. MediatorWeb provides an interface to visualize and analyze the PPI network of Mediator subunits. The database facilitates downloading the untargeted and unweighted network of Mediator complex, its submodules, and individual Mediator subunits to better visualize the importance of individual Mediator subunits or their submodules. Further, MediatorWeb offers network visualization of the Mediator complex and interacting proteins that are functionally annotated. This feature provides clues to understand functions of Mediator subunits in different processes. In an additional tab, MediatorWeb provides quick access to secondary and tertiary structures, as well as residue-level contact information for Mediator subunits in each of the three model organisms. Another useful feature of MediatorWeb is detection of interologs based on orthologous analyses, which can provide clues to understand the functions of Mediator complex in less explored kingdoms. Thus, MediatorWeb and its features can help the user to understand the role of Mediator complex and its subunits in the transcription regulation of gene expression.
RESUMO
In the present study, we generated a ligand-based scaffold model from a known bioactive datasets of mur enzymes of other species to identify multi-targeting inhibitors as antitubercular agents. Compounds in the ChEMBL database were first filtered to screen for substructure molecules ofMtb's multi-target enzymes. 5'-O-(5-Amino-5-deoxy-ß-D-ribofuranosyl)uridine has been identified as scaffold to develop compounds targeting Mtb's mur enzymes. A library of Murcko scaffolds was extracted and evaluated for their in-silico antitubercular activity against Mtb's mur enzymes. The screened compounds were subjected to molecular docking, molecular dynamics simulations, MM/PBSA calculation with Mtb's mur enzymes to evaluate the mechanism of interaction to assess inhibitory activity against the target protein. The results revealed that 15 compounds have higher docking scores and good interactions with multiple mur enzymes of Mtb. From the docking analysis, compound HPT had the best score and binding affinity with the all mur enzymes. Further, protein-ligand interactions were evaluated by molecular dynamics simulations to assess their stability throughout 100 ns period. From the MD trajectory, we calculated RMSD, RMSF, Rg, PCA, DCCM, FEL, hydrogen bonding, and vector motion. Furthermore, the binding free energies of the all nine mur enzymes with compound HPT exhibited good binding affinity might show the anti-mycobacterial activity. The compound HPT revealed from this computational study could act as potent anti-mycobacterial inhibitors and further serve as lead scaffolds to develop more potent pharmaceutical molecules targeting multiple mur enzymes of Mtb based on 5'-O-(5-Amino-5-deoxy-ß-D-ribofuranosyl)uridine in the future.Communicated by Ramaswamy H. Sarma.
Assuntos
Antituberculosos , Inibidores Enzimáticos , Sítios de Ligação , Simulação de Acoplamento Molecular , Ligação Proteica , Ligantes , Antituberculosos/farmacologia , Inibidores Enzimáticos/química , UridinaRESUMO
Lanosterol 14-α demethylase (LDM) is one of the promising drug targets of azoles antifungal. In this study, we have screened a large number of small molecules from different chemical databases (ZINC, DrugBank, ChEMBL, and ChemDiv) to find out novel and potential inhibitors of LDM. As a result, from more than a hundred thousand molecules, the two best candidates, C1 (ZINC000299817826) and C3 (ZINC000095786149), were selected from the top-scoring compounds and further validated in Molecular Dynamic (MD) simulation. The Glide scores of C1 and C3 were -19.33 kcal/mol and -19.13 kcal/mol, suggesting that these compounds bind with LDM with higher binding affinity than the benchmark compound (itraconazole), which has a Glide score of -6.85 kcal/mol. Docking poses reveal that the compounds C1 and C3 bind to the outermost region of the LDM binding site, which can prevent the lanosterol from getting into the catalytic pocket. Furthermore, MD simulation studies were performed to assess the stability of C1 and C3 in complex with LDM and were found to be stable over the 100 nanosecond simulation time. Binding free energy calculated by the MMPBSA method suggested that the C3 forms a more stable complex with the LDM as close to the benchmark compounds. Among the top selected molecules, C1 and C3 were predicted to be the significant inhibitors of LDM.Communicated by Ramaswamy H. Sarma.
Assuntos
Antifúngicos , Lanosterol , Lanosterol/farmacologia , Lanosterol/metabolismo , Esterol 14-Desmetilase/metabolismo , Antifúngicos/farmacologia , Sítios de Ligação , Itraconazol/farmacologia , Simulação de Dinâmica Molecular , Simulação de Acoplamento MolecularRESUMO
Candida glabrata, the second most common cause of invasive fungal infections, exhibits multi-drug resistance to commonly used antifungal drugs. To counter this resistance, there is a critical need for novel antifungals. This study identifies small molecule inhibitors that target a three-helix bundle KIX domain in the Med15a Mediator subunit of Candida glabrata (CgMed15a KIX). This domain plays a crucial role by interacting with the Pleiotropic Drug Resistance transcription factor Pdr1, a key regulator of the multidrug resistance pathway in Candida glabrata. We performed high throughput computational screening of large chemical datasets against the binding sites of the CgMed15a KIX domain to identify novel inhibitors. We selected six potential candidates with high affinity and confirmed their binding with the CgMed15a KIX domain. A phytochemical compound, Chebulinic acid binds to the CgMed15a KIX domain with a KD value of 0.339 µM and shows significant inhibitory effects on the growth of Candida glabrata. Molecular dynamics simulation studies further revealed the structural stability of the CgMed15a KIX-Chebulinic acid complex. Thus, in conclusion, this study highlights Chebulinic acid as a novel potential antifungal compound against Candida glabrata.
Assuntos
Antifúngicos , Candida glabrata , Antifúngicos/farmacologia , Antifúngicos/metabolismo , Candida glabrata/metabolismo , Fatores de Transcrição/metabolismo , Taninos Hidrolisáveis/farmacologia , Farmacorresistência FúngicaRESUMO
The interaction of lysozyme with cefoperazone was studied by means of spectroscopic and computational approaches. The change in the UV-visible spectrum of lysozyme in presence of cefoperazone was an indication of the complex formation between them. Fluorescence spectroscopy suggested that there was a fair interaction between the protein and drug which was taken place via dynamic quenching mechanism and the binding ratio was approximately 1:1. The binding was energetically feasible and principally supported by the hydrophobic forces. CD spectroscopic studies have shown that cefoperazone induced the secondary structure of lysozyme by increasing the α-helical contents of the latter. In silico studies revealed that the large nonpolar cavity was the preferred binding site of cefoperazone within lysozyme and the interaction was taken place mainly through hydrophobic forces with small involvement of hydrogen bonding and electrostatic interactions which is in good agreement with the experimental analyses. Effect of paracetamol was also seen on the binding and it was found that paracetamol had a negative influence on the binding between cefoperazone and lysozyme.
Assuntos
Acetaminofen , Cefoperazona , Cefoperazona/farmacologia , Acetaminofen/farmacologia , Dicroísmo Circular , Muramidase/química , Cefalosporinas , Simulação de Acoplamento Molecular , Termodinâmica , Sítios de Ligação , Espectrometria de Fluorescência , Ligação ProteicaRESUMO
Background : Titanium dioxide (TiO2) nanoparticles are among the largely manmade nanomaterials worldwide and are broadly used as both industrial and user products. The primary target site for several nanoparticles is the liver, including TiO2 nanoparticles (TNPs), exposed directly or indirectly through ingestion of contaminated water, food, or animals and elevated environmental contamination. Oxidative stress is a known facet of nanoparticle-induced toxicity, including TNPs. Mitochondria are potential targets for nanoparticles in several types of toxicity, such as hepatotoxicity. Nevertheless, its causal mechanism is still controversial due to scarcity of literature linking the role of mitochondria-mediated TNP-induced hepatotoxicity. Aim : The objective of the current study was to evaluate the relation of mitochondrial oxidative stress and respiratory chain mechanisms with TNP-induced mitochondrial dysfunction in vitro, and explore the hepatoprotective effect of quercetin (QR), which is a polyphenolic flavonoid abundant in fruits and vegetables with known antioxidant properties, on TNP-induced mitochondrial oxidative stress and disturbance in respiratory chain complex enzymes in the liver of rats. Results: Enzymatic and non-enzymatic antioxidant levels, oxidative stress markers, and mitochondrial complexes were assessed with regard to TNP-induced hepatotoxicity. The depleted lipid peroxidation levels and protein carbonyl content, in mitochondria, induced by TNPs were restored significantly by pretreatment with QR. QR modulated the altered non-enzymatic and enzymatic antioxidants and mitochondrial complex enzymes. Conclusion : Based on the findings, we conclude that QR, which mitigates oxidative stress caused by mitochondrial dysfunction, holds promising capability to potentially diminish TNP-induced adverse effects in the liver.
RESUMO
Herein we have studied the noncovalent molecular interactions between hen egg white lysozyme (HEWL) and the commonly employed antineoplastic drug gemcitabine through the cumulative implementation of spectroscopic techniques and in silico approaches. The formation of a complex between HEWL and gemcitabine was made evident by the differences between the UV-visible spectra of the protein and protein-gemcitabine complex. Fluorescence quenching of HEWL by gemcitabine was hardly detectable at room temperature, but it became prominent at higher temperatures. Very low values for the bimolecular quenching constant and the non-reciprocal dependence of quenching on temperature indicated that dynamic quenching was taking place. Analysis of experimental data indicated that the interaction was dominated by hydrophobic forces, while the results of a computational investigation suggested the concomitant contribution of hydrogen bonding. Gemcitabine binding induced modifications of the secondary structure of HEWL by slightly increasing the α-helical content of the protein. Finally, gemcitabine binding site was inferred to be located in HEWL big hydrophobic cavity.
Assuntos
Antineoplásicos/química , Desoxicitidina/análogos & derivados , Simulação de Acoplamento Molecular , Muramidase/química , Antineoplásicos/farmacologia , Sítios de Ligação , Desoxicitidina/química , Desoxicitidina/farmacologia , Muramidase/metabolismo , Ligação Proteica , GencitabinaRESUMO
In recent years, there has been a progress in the study of glycation reaction which is one the possible reason for multiple metabolic disorders. Glycation is a nonenzymatic reaction between nucleic acids, lipids, and proteins resulting into the formation of early glycation products that may further lead to the accumulation of advanced glycation end products (AGEs). The precipitation of AGEs in various cells, tissues, and organs is one of the factors for the initiation and progression of various metabolic derangements including the cancer. The AGE interaction with its receptor "RAGE" activates the inflammatory pathway; yet, the downregulation of RAGE and its role in these pathways are not clear. We explore the effect of anticancer novel nanoassemblies on AGEs to determine its role in the regulation of the expression of RAGE, NFÆB, TNF-α, and IFN-γ. This paper is based on the in vivo and in vitro study in glycation and lung cancer model systems. Upon the treatment of nanoassemblies in both the model systems, we observed a protective effect of nanoassemblies over the inhibition of glycative and oxidative stress via mRNA expression analysis. The mRNA expression results corroborated with the reactive oxygen species (ROS), carboxy-methyl-lysine (CML), and fluorescence studies. In this study, we found that the presence of common factors for glycation and lung cancer is oxidative and glycative stress. This oxidation and glycation might be responsible for the initiation of inflammation which may further lead to uncontrolled growth of cells leading to cancer. This can be a strong association between lung cancer and glycation reaction. The intervention of the anticancer and antiglycation effects of multimodal nanoassemblies throughout the study promises a new pathway for cancer research.
Assuntos
Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Nanopartículas/química , Estresse Oxidativo , Células A549 , Animais , Benzotiazóis , Glicemia/metabolismo , Carbocianinas , Proliferação de Células , Modelos Animais de Doenças , Glicosilação , Humanos , Interferon gama/metabolismo , Neoplasias Pulmonares/sangue , Células MCF-7 , Camundongos , Modelos Biológicos , NF-kappa B/metabolismo , Ratos Sprague-Dawley , Espécies Reativas de Oxigênio/metabolismo , Receptor para Produtos Finais de Glicação Avançada/metabolismo , Albumina Sérica Humana/metabolismo , Espectrofotometria , Trifluridina/química , Fator de Necrose Tumoral alfa/metabolismoRESUMO
The HEP II (Heparin-binding site II) region of fibronectin (FN) containing domain III14 plays a crucial role in cell adhesion and migration through heparin-binding on the cell surface. There are two such fibronectin heparin interacting peptide (FHIP I and FHIP II) sequences present in HEP II. However, the molecular principles by which these sites orchestrate heparin-binding processes are poorly understood. Such knowledge would have great implications in the therapeutic targeting of FN. With this aim, we have explored the binding studies of FHIP I and FHIP II with heparin using various biophysical methods. A fluorescence melting study specifically revealed the preference of heparin for domain III in FN, indicating the key contribution of FHIP I and FHIP II in heparin binding. In isothermal titration calorimetry (ITC), the higher binding affinity observed for FHIP II (â¼107 mol-1) compared to FHIP I (â¼106 mol-1) is expected due to the presence of a superior cluster of Arg and Lys residues in FHIP II, which can facilitate specific H-bonding interactions with heparin. Based on heat capacity changes, the key role of H-bonding, electrostatic and hydrophobic interactions was demonstrated in binding. Finally, the molecular docking and MD simulation results reinforced that the interaction of heparin (dodecasaccharide) is stronger and stable with the FHIP II peptide. The results described here suggest that these peptides provide all the structural and thermodynamic elements necessary for heparin-binding of HEP II of FN. Subsequently, it can be concluded that FHIP II could be a better location for therapeutic intervention in cell adhesion activity by FN.
RESUMO
Although several transcription factors (TFs) that regulate seed size/weight in plants are known, the molecular landscape regulating this important trait is unclear. Here, we report that a Mediator subunit, OsMED15a, links rice grain size/weight-regulating TFs to their target genes. Expression analysis and high-resolution quantitative trait loci (QTL) mapping suggested that OsMED15a is involved in rice seed development. OsMED15a has an N-terminal, three-helical KIX domain. Two of these helices, α1 and α3, and three amino acids, 76LRC78, within OsMED15a helix α3 were important for its interaction with several proteins, including interactions with the transactivation domains of two NAC-type TFs, OsNAC024 and OsNAC025. Moreover, OsMED15a, OsNAC024, and OsNAC025 all exhibited increased expression during seed development, and we identified several grain size/weight-associated SNPs in these genes in 509 low- and high-grain-weight rice genotypes. RNAi-mediated repression of OsMED15a expression down-regulated the expression of the grain size/weight regulating genes GW2, GW5 and DR11 and reduced grain length, weight, and yield. Of note, both OsNAC024 and OsNAC025 bound to the promoters of these three genes. We conclude that the transactivation domains of OsNAC024 and OsNAC025 target the KIX domain of OsMED15a in the regulation of grain size/weight-associated genes such as GW2, GW5, and D11. We propose that the integrated molecular-genetics approach used here could help identify networks of functional alleles of other regulator and co-regulator genes and thereby inform efforts for marker-assisted introgression of useful alleles in rice crop improvement.
Assuntos
Oryza/genética , Proteínas de Plantas/genética , Fatores de Transcrição/genética , Transcrição Gênica , Alelos , Regulação da Expressão Gênica de Plantas , Oryza/crescimento & desenvolvimento , Fenótipo , Locos de Características Quantitativas/genética , Sementes/genética , Sementes/crescimento & desenvolvimentoRESUMO
AIM: To evaluate the serum paraoxonase 1 activity and determine its association with duration in type 2 Diabetes mellitus patients. METHODS: A total of 80 cases from type 2 diabetes mellitus and healthy controls were enrolled in the present case control study. Human serum PON1 concentration was measured by ELISA and western blotting and it activity was determined spectrophotometrically using 4-nitrophenyle acetate. Diagnostic accuracy of serum PON1 to identify type 2 Diabetes mellitus was calculated with ROC analysis. RESULT: Serum concentration of LDL, VLDL, TG, A1C, FBS and TC levels showed significantly higher levels in type 2 diabetes patients as compared to healthy controls, however there were no significant differences found in the level of HDL. Serum PON1 concentration and activity monitored in patients with >1â¯year diabetes showed higher level (75.1⯱â¯6.8â¯ng/mL) as compared to patients with >3â¯years diabetes (65.24⯱â¯1.6â¯ng/mL), its level was further decreased in patients with >5 (53.8⯱â¯2.6â¯ng/mL) and >7â¯years (48.1⯱â¯2.7â¯ng/mL) of diabetes. PON1 concentration decreased as the duration of diabetes increased. PON1 level was further decreased due to habits like smoking and alcohol consumption. CONCLUSION: Serum PON1 levels decrease in states of high oxidative stress like metabolic syndrome, obesity, uncontrolled diabetes, and dyslipidemia. It can be used as diagnostic marker for diabetes mellitus along with increased TG, LDL, VLDL and FBG.
Assuntos
Arildialquilfosfatase/sangue , HDL-Colesterol/sangue , Diabetes Mellitus Tipo 2/sangue , Adulto , Biomarcadores/sangue , Estudos de Casos e Controles , Diabetes Mellitus Tipo 2/enzimologia , Diabetes Mellitus Tipo 2/epidemiologia , Humanos , Índia/epidemiologia , Pessoa de Meia-IdadeRESUMO
Med15 is an important subunit of Mediator Tail module and is characterized by a KIX domain present towards amino terminal. In yeast and metazoans, Med15 KIX domain has been found to interact with various transcription factors regulating several processes including carbohydrate metabolism, lipogenesis, stress response and multidrug resistance. Mechanism of Med15 functioning in Arabidopsis is largely unknown. In this study, interactome of KIX domain of Arabidopsis Med15, AtMed15a, was characterized. We found 45 proteins that interact with AtMed15a KIX domain, including 11 transcription factors, 3 single strand nucleic acid-binding proteins and 1 splicing factor. The third helix of the KIX domain was found to be involved in most of the interactions. Mapping of the regions participating in the interactions revealed that the activation domain of a transcription factor, UKTF1 interacted with AtMed15a KIX domain. Thus, our results suggest that in Arabidopsis, activation domain of transcription factors target KIX domain of AtMed15a for their transcriptional responses.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Fatores de Transcrição/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Ligação Proteica , Fatores de Transcrição/genéticaRESUMO
Alzheimer's disease (AD) is a progressive neurodegenerative disorder and the most common type of dementia in elderly ( >65years of age). Excessive extra cellular deposits of amyloid beta (Aß) are a pathological feature of AD. Aß can cause cell death through oxidative damage; recent studies have implicated opening of mPTP as a detrimental event in AD-related mitochondrial dysfunctions. Over the past few years, natural compounds with antioxidant properties have shown promise for intervention in AD.