RESUMO
Interferon-gamma-inducible large GTPases, hGBPs, possess antipathogenic and antitumor activities in human cells. Like hGBP1, its closest homolog, hGBP3 has two domains; an N-terminal catalytic domain and a C-terminal helical domain, connected by an intermediate region. The biochemical function of this protein and the role of its domains in substrate hydrolysis have not yet been investigated. Here, we report that while hGBP3 can produce both GDP and GMP, GMP is the minor product, 30% (unlike 85% in hGBP1), indicating that hGBP3 is unable to produce enhanced GMP. To understand which domain(s) are responsible for this deficiency, we created hGBP3 truncated variants. Surprisingly, GMP production was similar upon deletion of the helical domain, suggesting that in contrast to hGBP1, the helical domain of hGBP3 cannot stimulate the second phosphate cleavage of GTP. We conducted computational and solution studies to understand the underlying basis. We found that the regulatory residue W79, present in the catalytic domain, forms an H-bond with the backbone carbonyl of K76 (located in the catalytic loop) of the substrate-bound hGBP3. However, after gamma-phosphate cleavage of GTP, the W79-containing region does not undergo a conformational change, failing to redirect the catalytic loop toward the beta-phosphate. This is necessary for efficient GMP formation because hGBP homologs utilize the same catalytic residue for both phosphate cleavages. We suggest that the lack of specific interdomain contacts mediated by the helical domain prevents the catalytic loop movement, resulting in reduced GMP formation. These findings may provide insight into how hGBP3 contributes to immunity.
Assuntos
Domínio Catalítico , Proteínas de Ligação ao GTP , Guanosina Trifosfato , Fosfatos , Humanos , Domínio Catalítico/genética , GTP Fosfo-Hidrolases/metabolismo , Guanosina Trifosfato/metabolismo , Fosfatos/metabolismo , Proteínas de Ligação ao GTP/genética , Proteínas de Ligação ao GTP/metabolismoRESUMO
Interferon-gamma-inducible human large GTPases, hGBP1 and hGBP2, have a distinctive feature of hydrolyzing GTP to GDP and GMP through successive phosphate cleavages. In hGBP1, GMP is the major product, which is essential for its anti-pathogenic activities. However, its close homologue hGBP2 produces significantly less GMP, despite having a similar active site architecture. The molecular basis for less GMP formation and catalytic residue(s) in hGBP2 are not fully explored. To address these issues, we performed systematic biochemical, biophysical, and microsecond simulation studies. Our data suggest that the less GMP formation in hGBP2 is due to the lack of H-bond formation between the W79 side-chain (located near the active site) and main-chain carbonyl of K76 (present in the catalytic loop) in the substrate-bound hGBP2. The absence of this H-bond could not redirect the catalytic loop toward the beta phosphate after the cleavage of gamma-phosphate, a step essential for enhanced GMP formation. Furthermore, based on the mutational and structural analyses, this study for the first time indicates that the same residue, T75, mediates both phosphate cleavages in hGBP2 and hGBP1. This suggests the conservation of the catalytic residue in hGBP homologues. These findings emphasize the indispensable role of correct catalytic loop repositioning for efficient beta phosphate cleavage. This led us to propose a new substrate hydrolysis mechanism by hGBP1 and hGBP2, which may also be helpful to understand the GTP hydrolysis in other hGBP homologues. Overall, the study could provide insight into how these two close homologues play crucial roles in host-mediated immunity through different mechanisms.
Assuntos
GTP Fosfo-Hidrolases , Proteínas de Ligação ao GTP , Humanos , Proteínas de Ligação ao GTP/metabolismo , Guanosina Trifosfato/química , GTP Fosfo-Hidrolases/metabolismo , Hidrólise , FosfatosRESUMO
OBJECTIVE: Neuroprotection is a precise target for the treatment of neurodegenerative diseases, ischemic stroke, and traumatic brain injury. Pyrimidine and its derivatives have been proven to use antiviral, anticancer, antioxidant, and antimicrobial activity prompting us to study the neuroprotection and anti-inflammatory activity of the triazole-pyrimidine hybrid on human microglia and neuronal cell model. METHODS: A series of novel triazole-pyrimidine-based compounds were designed, synthesized and characterized by mass spectra, 1HNMR, 13CNMR, and a single X-Ray diffraction analysis. Further, the neuroprotective, anti-neuroinflammatory activity was evaluated by cell viability assay (MTT), Elisa, qRT-PCR, western blotting, and molecular docking. RESULTS: The molecular results revealed that triazole-pyrimidine hybrid compounds have promising neuroprotective and anti-inflammatory properties. Among the 14 synthesized compounds, ZA3-ZA5, ZB2-ZB6, and intermediate S5 showed significant anti-neuroinflammatory properties through inhibition of nitric oxide (NO) and tumor necrosis factor-α (TNF-α) production in LPS-stimulated human microglia cells. From 14 compounds, six (ZA2 to ZA6 and intermediate S5) exhibited promising neuroprotective activity by reduced expression of the endoplasmic reticulum (ER) chaperone, BIP, and apoptosis marker cleaved caspase-3 in human neuronal cells. Also, a molecular docking study showed that lead compounds have favorable interaction with active residues of ATF4 and NF-kB proteins. CONCLUSION: The possible mechanism of action was observed through the inhibition of ER stress, apoptosis, and the NF-kB inflammatory pathway. Thus, our study strongly indicates that the novel scaffolds of triazole-pyrimidine-based compounds can potentially be developed as neuroprotective and anti-neuroinflammatory agents.
Assuntos
Neuroproteção , Fármacos Neuroprotetores , Humanos , NF-kappa B/metabolismo , Triazóis/farmacologia , Triazóis/metabolismo , Simulação de Acoplamento Molecular , Anti-Inflamatórios/farmacologia , Microglia/patologia , Pirimidinas/farmacologia , Pirimidinas/metabolismo , Fármacos Neuroprotetores/farmacologia , Fármacos Neuroprotetores/metabolismo , Lipopolissacarídeos/farmacologiaRESUMO
Glioma is a major brain tumor, and the associated mortality rate is very high. Contemporary therapies provide a chance of survival for 9-12 months. Therefore, a novel approach is essential to improve the survival rate. Sonic hedgehog (Shh) cell signaling is critical for early development in various tumors. This investigation attempted to explore the potential interaction and regulation of Shh-Gli1 cell signaling in association with paired box 6 (Pax6) and isocitrate dehydrogenase 2 (IDH2). The expression pattern of Shh, Gli1, Pax6, and IDH2 was examined by transcriptome analysis, immunohistochemistry, and confocal images. The results suggest the interaction of Shh-Gli1 cell signaling pathway with Pax6 and IDH2 and potential regulation. Thereafter, we performed protein-protein docking and molecular dynamic simulations (MDS) of Gli1 with Pax6 and IDH2. The results suggest differential dynamic interactions of Gli1-IDH2 and Gli1-Pax6. Gli1 knockdown downregulated the expression of Pax6 and upregulated the expression of IDH2. Moreover, Gli1 knockdown decreased the expression of the drug resistance gene MRP1. The knockdown of Pax6 gene in glioma cells downregulated the expression of Gli1 and IDH2 and promoted cell proliferation. Moreover, the efficacy of the treatment of glioma cells with temozolomide (TMZ) and Gli1 inhibitor GANT61 was higher than that of TMZ alone. MDS results revealed that the interactions of Gli1 with IDH2 were stronger and more stable than those with Pax6. Intriguingly, inhibition of Pax6 promoted glioma growth even in the presence of TMZ. However, the tumor-suppressive nature of Pax6 was altered when Gli1 was inhibited by GANT61, and it showed potential oncogenic character, as observed in other cancers. Therefore, we conclude that Pax6 interacted with IDH2 and Gli1 in glioma. Moreover, the Shh-Gli1-IDH2/Pax6 cell signaling axis provides a new therapeutic approach for inhibiting the progression of the disease and mitigating drug resistance in glioma.
Assuntos
Neoplasias Encefálicas , Glioma , Humanos , Proteína GLI1 em Dedos de Zinco/genética , Proteína GLI1 em Dedos de Zinco/metabolismo , Proteína GLI1 em Dedos de Zinco/uso terapêutico , Resistencia a Medicamentos Antineoplásicos , Proteínas Hedgehog/metabolismo , Glioma/tratamento farmacológico , Glioma/metabolismo , Neoplasias Encefálicas/metabolismo , Temozolomida/farmacologia , Fator de Transcrição PAX6/genéticaRESUMO
The binuclear metalloenzyme Helicobacter pylori arginase is important for pathogenesis of the bacterium in the human stomach. Despite conservation of the catalytic residues, this single Trp enzyme has an insertion sequence (-153ESEEKAWQKLCSL165-) that is extremely crucial to function. This sequence contains the critical residues, which are conserved in the homolog of other Helicobacter gastric pathogens. However, the underlying basis for the role of this motif in catalytic function is not completely understood. Here, we used biochemical, biophysical and molecular dynamics simulations studies to determine that Glu155 of this stretch interacts with both Lys57 and Ser152. These interactions are essential for positioning of the motif through Trp159, which is located near Glu155 (His122-Trp159-Tyr125 contact is essential to tertiary structural integrity). The individual or double mutation of Lys57 and Ser152 to Ala considerably reduces catalytic activity with Lys57 to Ala being more significant, indicating they are crucial to function. Our data suggest that the Lys57-Glu155-Ser152 interaction influences the positioning of the loop containing the catalytic His133 so that this His can participate in catalysis, thereby providing a mechanistic understanding into the role of this motif in catalytic function. Lys57 was also found only in the arginases of other Helicobacter gastric pathogens. Based on the non-conserved motif, we found a new molecule, which specifically inhibits this enzyme. Thus, the present study not only provides a molecular basis into the role of this motif in function, but also offers an opportunity for the design of inhibitors with greater efficacy.
Assuntos
Arginase/química , Proteínas de Bactérias/química , Helicobacter pylori/enzimologia , Motivos de Aminoácidos , Sequência de Aminoácidos , Substituição de Aminoácidos , Aminoácidos/química , Animais , Arginase/antagonistas & inibidores , Arginase/genética , Arginina/metabolismo , Proteínas de Bactérias/antagonistas & inibidores , Proteínas de Bactérias/genética , Catálise , Cobalto/metabolismo , Sequência Conservada , Polarização de Fluorescência , Gastrite/microbiologia , Gastrite/veterinária , Helicobacter/enzimologia , Infecções por Helicobacter/microbiologia , Infecções por Helicobacter/veterinária , Helicobacter pylori/genética , Humanos , Hidrólise , Modelos Moleculares , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Mutação de Sentido Incorreto , Mutação Puntual , Estrutura Secundária de Proteína , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Especificidade da EspécieRESUMO
An ancient saffron-based polyherbal formulation, Dawa-ul-Kurkum (DuK), has been used to treat liver ailments and other diseases and was recently evaluated for its anticancer potential against hepatocellular carcinoma (HCC) by our research team. To gain further insight into the lead molecule of DuK, we selected ten active constituents belonging to its seven herbal constituents (crocin, crocetin, safranal, jatamansone, isovaleric acid, cinnamaldehyde, coumaric acid, citral, guggulsterone and dehydrocostus lactone). We docked them with 32 prominent proteins that play important roles in the development, progression and suppression of HCC and those involved in endoplasmic reticulum (ER) stress to identify the binding interactions between them. Three reference drugs for HCC (sorafenib, regorafenib, and nivolumab) were also examined for comparison. The in silico studies revealed that, out of the ten compounds, three of them-viz., Z-guggulsterone, dehydrocostus lactone and crocin-showed good binding efficiency with the HCC and ER stress proteins. Comparison of binding affinity with standard drugs was followed by preliminary in vitro screening of these selected compounds in human liver cancer cell lines. The results provided the basis for selecting Z-guggulsterone as the best-acting phytoconstituent amongst the 10 studied. Further validation of the binding efficiency of Z-guggulsterone was undertaking using molecular dynamics (MD) simulation studies. The effects of Z-guggulsterone on clone formation and cell cycle progression were also assessed. The anti-oxidant potential of Z-guggulsterone was analyzed through DPPH and FRAP assays. qRTPCR was utilized to check the results at the in vitro level. These results indicate that Z-guggulsterone should be considered as the main constituent of DuK instead of the crocin in saffron, as previously hypothesized.
Assuntos
Carcinoma Hepatocelular , Crocus , Neoplasias Hepáticas , Pregnenodionas , Carcinoma Hepatocelular/metabolismo , Humanos , Neoplasias Hepáticas/patologia , Pregnenodionas/farmacologiaRESUMO
Acetaldehyde is a metabolite of ethanol, an important constituent of tobacco pyrolysis and the aldehydic product of lipid peroxidation. Acetaldehyde induced toxicity is mainly due to its binding to cellular macromolecules resulting in the formation of stable adducts accompanied by oxidative stress. The aim of this study was to characterize structural and immunological alterations in human immunoglobulin G (IgG) modified with acetaldehyde in the presence of sodium borohydride, a reducing agent. The IgG modifications were studied by various physicochemical techniques such as fluorescence and CD spectroscopy, free amino group estimation, 2,2-azobis 2-amidinopropane (AAPH) induced red blood cell hemolysis as well as transmission electron microscopy. Molecular docking was also employed to predict the preferential binding of acetaldehyde to IgG. The immunogenicity of native and acetaldehyde-modified IgG was investigated by immunizing female New Zealand white rabbits using native and modified IgG as antigens. Binding specificity and cross reactivity of rabbit antibodies was screened by competitive inhibition ELISA and band shift assays. The modification of human IgG with acetaldehyde results in quenching of the fluorescence of tyrosine residues, decrease in free amino group content, a change in the antioxidant property as well as formation of cross-linked structures in human IgG. Molecular docking reveals strong binding of IgG to acetaldehyde. Moreover, acetaldehyde modified IgG induced high titer antibodies (>1:12800) in the experimental animals. The antibodies exhibited high specificity in competitive binding assay toward acetaldehyde modified human IgG. The results indicate that acetaldehyde induces alterations in secondary and tertiary structure of IgG molecule that leads to formation of neo-epitopes on IgG that enhances its immunogenicity.
Assuntos
Acetaldeído/química , Epitopos/ultraestrutura , Imunoglobulina G/ultraestrutura , Conformação Proteica , Animais , Sítios de Ligação/imunologia , Ensaio de Imunoadsorção Enzimática , Epitopos/imunologia , Eritrócitos/imunologia , Feminino , Hemólise/imunologia , Humanos , Imunoglobulina G/química , Imunoglobulina G/imunologia , Microscopia Eletrônica de Transmissão , Simulação de Acoplamento Molecular , Estresse Oxidativo/imunologia , Ligação Proteica/imunologia , Coelhos , Tirosina/imunologiaRESUMO
Methylglyoxal (MGO), a reactive dicarbonyl metabolite is a potent arginine directed glycating agent which has implications for diabetes-related complications. Dicarbonyl metabolites are produced endogenously and in a state of misbalance, they contribute to cell and tissue dysfunction through protein and DNA modifications causing dicarbonyl stress. MGO is detoxified by glyoxalase 1 (GLO1) system in the cytoplasm. Reactive oxygen species (ROS) are known to aggravate the glycation process. Both the processes are closely linked, and their combined activity is often referred to as "glycoxidation" process. Glycoxidation of proteins has several consequences such as type 2 diabetes mellitus (T2DM), aging etc. In this study, we have investigated the glycation of low-density lipoprotein (LDL) using different concentrations of MGO for varied incubation time periods. The structural perturbations induced in LDL were analyzed by UV-Vis, fluorescence, circular dichroism spectroscopy, molecular docking studies, polyacrylamide gel electrophoresis, FTIR, thermal denaturation studies, Thioflavin T assay and isothermal titration calorimetry. The ketoamine moieties, carbonyl content and HMF content were quantitated in native and glycated LDL. Simulation studies were also done to see the effect of MGO on the secondary structure of the protein. We report structural perturbations, increased carbonyl content, ketoamine moieties and HMF content in glycated LDL as compared to native analog (native LDL). We report the structural perturbations in LDL upon modification with MGO which could obstruct its normal physiological functions and hence contribute to disease pathogenesis and associated complications.
Assuntos
Lipoproteínas LDL/química , Lipoproteínas LDL/metabolismo , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Produtos Finais de Glicação Avançada , Humanos , Conformação Proteica , Aldeído Pirúvico/metabolismo , Aldeído Pirúvico/farmacologiaRESUMO
OBJECTIVE: To compare the safety and efficacy of 10 units of intravenous syntocinon alone with 10 units intravenous syntocinon and 0.25 mg intramuscular ergometrine in the prevention of atonic uterine haemorrhage during caesarean section. METHOD: The quasi-experimental study was conducted at the Maternal and Child Health Centre, Unit I, Pakistan Institute of Medical Sciences, Islamabad, from November 1, 2010 to February 28, 2011. All women undergoing caesarean section were included in the study. Patients were given intravenous 10 units syntocinon alone intraoperatively from November 1 to December 31,2010, while 0.25 mg ergometrine intramuscular was added to 10 units intravenous syntocinon from January 1 to February 28, 2011. Frequency of postpartum haemorrhage, adverse effects of drugs and maternal morbidity and mortality were assessed by using chi square test. P < 0.05 was taken as statistically significant. RESULTS: Of the total number of 701 subjects, 378 (54%) women were given 10 units syntocinon and 323 (46%) were given 0.25 mg ergometrine in addition to 10 units syntocinon. The mean age in the syntocinon group was 28 +/- 3.5 yrs with gestational age of 37.5 +/- 2 wks, while that in syntocinon-ergometrine group was 29 +/- 3.4 years and 38 +/- 2 weeks respectively. Postpartum haemorrhage in the syntocinon group was found in 38 (10%) women versus 05 (1.5%) women) in the other group (p < 0.001). Adverse effects like nausea, vomiting and raised blood pressure were slightly more with syntocinon-ergometrine than syntocinon alone (n = 56; 15.3% vs n = 35; 9.2%), but it was not statistically significant. Post partum haemorrhage was responsible for 40% of maternal mortality during the study period and that was in the syntocinon group. CONCLUSION: Prophylactic ergometrine in addition to syntocinon is superior to syntocinon alone in decreasing frequency of postpartum haemorrhage in caesarean section and associated maternal morbidity and mortality. Regarding safety profile, the two groups showed no statistically significant change.
Assuntos
Cesárea , Ergonovina/uso terapêutico , Ocitócicos/uso terapêutico , Ocitocina/uso terapêutico , Hemorragia Pós-Parto/prevenção & controle , Adulto , Feminino , Humanos , Mortalidade Materna , Paquistão/epidemiologia , Hemorragia Pós-Parto/mortalidade , Gravidez , Resultado do TratamentoRESUMO
Lipid peroxidation (LPO) is a biological process that frequently occurs under physiological conditions. Undue oxidative stress increases the level of LPO; which may further contribute to the development of cancer. 4-Hydroxy-2-nonenal (HNE), one of the principal by-products of LPO, is present in high concentrations in oxidatively stressed cells. HNE rapidly reacts with various biological components, including DNA and proteins; however, the extent of protein degradation by lipid electrophiles is not well understood. The influence of HNE on protein structures will likely have a considerable therapeutic value. This research elucidates the potential of HNE, one of the most researched phospholipid peroxidation products, in modifying low-density lipoprotein (LDL). In this study, we tracked the structural alterations in LDL by HNE using various physicochemical techniques. To comprehend the stability, binding mechanism and conformational dynamics of the HNE-LDL complex, computational investigations were carried out. LDL was altered in vitro by HNE, and the secondary and tertiary structural alterations were examined using spectroscopic methods, such as UV-visible, fluorescence, circular dichroism and fourier transform infrared spectroscopy. Carbonyl content, thiobarbituric acid-reactive-substance (TBARS) and nitroblue tetrazolium (NBT) reduction assays were used to examine changes in the oxidation status of LDL. Thioflavin T (ThT), 1-anilinonaphthalene-8-sulfonic (ANS) binding assay and electron microscopy were used to investigate aggregates formation. According to our research, LDL modified by HNE results in changes in structural dynamics, oxidative stress and the formation of LDL aggregates. The current investigation must characterize HNE's interactions with LDL and comprehend how it can change their physiological or pathological functions.Communicated by Ramaswamy H. Sarma.
Assuntos
Aldeídos , Lipoproteínas LDL , Humanos , Lipoproteínas LDL/química , Lipoproteínas LDL/metabolismo , Aldeídos/metabolismo , Aldeídos/farmacologia , Oxirredução , Peroxidação de LipídeosRESUMO
OBJECTIVE: To develop a scoring system for the prediction of successful vaginal birth after caesarean section, following induction of labour with intra-vaginal E2 gel (Glandin). METHODS: The cross-sectional study was conducted from January 2010 to August 2011, at the Pakistan Institute of Medical Sciences in Islamabad. Trial of labour in previous one caesarean section, undergoing induction with intra-vaginal E2 gel, was attempted in 100 women. They were scored according to six variables; maternal age; gestation; indications of previous caesarean; history of vaginal birth either before or after the previous caesarean; Bishop score and body mass index. Multivariate and univariate logistic regression analysis was used to develop the scoring system. RESULTS: Of the total, 67 (67%) women delivered vaginally, while 33 (33%) ended in repeat caesarean delivery. Among the subjects, 55 (55%) women had no history of vaginal delivery either before or after previous caesarean section; 15 (15%) had history of vaginal births both before and after the previous caesarean; while 30 (30%) had vaginal delivery only after the previous caesarean section. Rates of successful vaginal birth after caesarean increased from 38% in women having a score of 0-3 to 58% in patients scoring 4-6. Among those having a score of 7-9 and 10-12, the success rates were 71% and 86% respectively. CONCLUSION: Increasing scores correlated with the increasing probability of vaginal birth after caesarean undergoing induction of labour.The admission VBAC scoring system is useful in counselling women with previous caesarean for the option of induction of labour or repeat caesarean delivery.
Assuntos
Nascimento Vaginal Após Cesárea , Adulto , Estudos Transversais , Feminino , Humanos , Trabalho de Parto Induzido , Idade Materna , Paquistão , Gravidez , Resultado da Gravidez , Fatores de RiscoRESUMO
Macromolecular crowding has been known to influence the structure and function of many enzymes through excluded volume effects and/or soft interactions. Here, we employed two synthetic macromolecular crowders, Dextrans and poly(ethylene glycol)s (PEGs) with varying molecular masses, to examine how they affected the structure and function of a therapeutically important enzyme, human arginase-I that catalyzes the conversion of l-arginine to l-ornithine and urea. Except at greater concentrations of Dextran 200, Dextrans were observed to slightly reduce the enzymatic activity, indicating that they exert their influence mainly through the excluded volume effects. Similar outcomes were seen with PEGs, with the exception of PEG 1000, where the activity decreased with increasing PEG concentrations, showing the maximum effect at a 20 g/L concentration. This finding suggests that the enzyme function is reduced by the soft interactions of this macromolecule with the enzyme, supported by the binding measurement. Secondary and local tertiary structures and thermodynamic stability were also affected, suggesting that PEG 1000 has an impact on the protein's structure. Furthermore, molecular dynamics simulation studies suggest that the catalytic pocket is disturbed, presumably by the unwinding of neighboring helix 9. As a result, the positioning of nearby Glu277 is altered, which prevents His141 and Glu277 from making contact. This hampers the proton transfer from the catalytic His141 to the intermediate species to form ornithine, a crucial step for the substrate hydrolysis reaction by this arginase. Overall, the knowledge gained from this study might be helpful for understanding how different enzymes work in a crowded/cellular environment.
Assuntos
Arginase , Dextranos , Humanos , Arginase/química , Arginase/metabolismo , Dextranos/química , Polietilenoglicóis/química , Ornitina/metabolismo , Substâncias Macromoleculares/químicaRESUMO
Methyl methanesulfonate (MMS) is a highly toxic DNA-alkylating agent that has a potential to damage the structural integrity of DNA. This work employed multiple biophysical and computational methods to report the MMS mediated structural alterations in the DNA (MMS-DNA). Spectroscopic techniques and gel electrophoresis studies revealed MMS induced exposure of chromophoric groups of DNA; methylation mediated antiâsyn conformational change, DNA fragmentation and reduced nucleic acid stability. MMS induced single-stranded regions in the DNA were observed in nuclease S1 assay. FT-IR results indicated MMS mediated loss of the assigned peaks for DNA, partial loss of C-O ribose, loss of deoxyribose region, C-O stretching and bending of the C-OH groups of hexose sugar, a progressive shift in the assigned guanine and adenine peaks, loss of thymine peak, base stacking and presence of C-O-H vibrations of glucose and fructose, indicating direct strand breaks in DNA due to backbone loss. Isothermal titration calorimetry showed MMS-DNA interaction as exothermic with moderate affinity. Dynamic light scattering studies pointed towards methylation followed by the generation of single-stranded regions. Electron microscopy pictured the loss of alignment in parallel base pairs and showed the formation of fibrous aggregates in MMS-DNA. Molecular docking found MMS in close contact with the ribose sugar of DNA backbone having non-bonded interactions. Molecular dynamic simulations confirmed that MMS is capable of interacting with DNA at two levels, one at the level of nitrogenous bases and another at the DNA backbone. The study offers insights into the molecular interaction of MMS and DNA.Communicated by Ramaswamy H. Sarma.
Assuntos
DNA , Ribose , Dano ao DNA , Reparo do DNA , Metanossulfonato de Metila/toxicidade , Simulação de Acoplamento Molecular , Espectroscopia de Infravermelho com Transformada de FourierRESUMO
Memantine belongs to the class of cognition enhancers that functions as NMDA receptor antagonist, used to treat Alzheimer's disease. The interaction of memantine with DNA was not investigated. In the present study, the interaction of memantine with ct-DNA, as well as its cytotoxicity on cancer cells, was evaluated. UV-visible spectroscopy, steady-state fluorescence spectroscopic studies revealed the interaction between memantine and ct-DNA. The quenching studies, chemical denaturation, (CD), and DNA melting studies showed the groove binding mode of memantine with ct-DNA. The thermodynamic parameters revealed that the interaction between memantine and ct-DNA is enthalpically driven, and the stabilizing forces involved were hydrogen bonding and van der Waals interaction. The groove-binding was also observed by molecular docking studies, which corroborated the findings of spectroscopic investigations. Density function theory calculations confirmed the existence of electron donor and recipient groups. The stability of memantine and DNA interaction, as well as the critical residues involved in the interaction, was identified by molecular dynamics simulations. Memantine showed cytotoxicity towards the cancer cells as compared to normal cells, as observed by MTT assay. Inverted compound microscopy analysis of memantine treated cancer cell lines further confirmed the results obtained by MTT assay.Communicated by Ramaswamy H. Sarma.
Assuntos
DNA , Memantina , Linhagem Celular , DNA/química , Memantina/farmacologia , Simulação de Acoplamento Molecular , Conformação de Ácido Nucleico , Espectrometria de Fluorescência , TermodinâmicaRESUMO
PURPOSE: We designed a comprehensive multiple myeloma targeted sequencing panel to identify common genomic abnormalities in a single assay and validated it against known standards. EXPERIMENTAL DESIGN: The panel comprised 228 genes/exons for mutations, 6 regions for translocations, and 56 regions for copy number abnormalities (CNA). Toward panel validation, targeted sequencing was conducted on 233 patient samples and further validated using clinical FISH (translocations), multiplex ligation probe analysis (MLPA; CNAs), whole-genome sequencing (WGS; CNAs, mutations, translocations), or droplet digital PCR (ddPCR) of known standards (mutations). RESULTS: Canonical immunoglobulin heavy chain translocations were detected in 43.2% of patients by sequencing, and aligned with FISH except for 1 patient. CNAs determined by sequencing and MLPA for 22 regions were comparable in 103 samples and concordance between platforms was R2 = 0.969. Variant allele frequency (VAF) for 74 mutations were compared between sequencing and ddPCR with concordance of R2 = 0.9849. CONCLUSIONS: In summary, we have developed a targeted sequencing panel that is as robust or superior to FISH and WGS. This molecular panel is cost-effective, comprehensive, clinically actionable, and can be routinely deployed to assist risk stratification at diagnosis or posttreatment to guide sequencing of therapies.
Assuntos
Mieloma Múltiplo , Variações do Número de Cópias de DNA , Genômica , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Mieloma Múltiplo/diagnóstico , Mieloma Múltiplo/genética , Mutação , Translocação Genética , Sequenciamento Completo do GenomaRESUMO
Proteins do not operate as individual units, and components of intracellular canonical pathways often cross talk in tumor genesis. We hypothesized that G-protein-coupled receptor 56 (GPR56), transglutaminase (TG2), and nuclear factor-κB (NF-κB) may collaborate in interconnected pathways and contribute to the aggressive behavior of esophageal squamous cell carcinoma (ESCC). Immunohistochemical analysis of GPR56, TG2, and NF-κB was carried out using ESCC tissue microarrays. Immunostaining of all the three proteins revealed a significant increase in their expression in ESCCs as compared with normal epithelia and correlated with their concomitant expression. A significant correlation between GPR56, TG2, and NF-κB was observed that correlated with nodal metastasis and tumor invasion in ESCCs.
Assuntos
Biomarcadores Tumorais/análise , Carcinoma de Células Escamosas/química , Neoplasias Esofágicas/química , NF-kappa B/análise , Receptores Acoplados a Proteínas G/análise , Transglutaminases/análise , Adulto , Western Blotting , Carcinoma de Células Escamosas/patologia , Distribuição de Qui-Quadrado , Neoplasias Esofágicas/patologia , Feminino , Proteínas de Ligação ao GTP , Humanos , Imuno-Histoquímica , Índia , Metástase Linfática , Masculino , Invasividade Neoplásica , Prognóstico , Proteína 2 Glutamina gama-Glutamiltransferase , Análise Serial de Tecidos , Regulação para CimaRESUMO
Insight into the mechanistic binding of bovine serum albumin (BSA) with doxofylline can layout pivotal enlightenment with relevance to pharmacokinetics and pharmacodynamics properties. Herein, many spectroscopic techniques and computational methods had been employed to interpret the structural and binding dynamics of BSA-doxofylline interaction. Doxofylline quenched the intrinsic fluorescence of BSA by static quenching. The stoichiometry and the binding constant of the BSA-doxofylline complex were 1:1 and in the order of 103 M-1. It was also concluded that the binding process was spontaneous and exothermic, primarily based on the thermodynamic study. Circular dichroism and three-dimensional excitation-emission matrix fluorescence results concluded pronounced conformational and microenvironmental changes in BSA structure on binding with doxofylline. The influence of metal ions and vitamins on the binding affinity of the BSA-doxofylline system were also explored. The in vitro findings were further supported by in silico analysis. With a score value of -6.25 kcal/mol, molecular docking showed strong interactions. Molecular dynamics simulation interpretation also suggested the stable binding with lower deviation in the values of RMSD and RMSF obtained by uninterrupted long simulation run. These studies will propose the optimum potency of distribution of the doxofylline into the bloodstream for asthma treatment.
Assuntos
Soroalbumina Bovina , Sítios de Ligação , Dicroísmo Circular , Simulação de Acoplamento Molecular , Ligação Proteica , Soroalbumina Bovina/metabolismo , Espectrometria de Fluorescência , Espectrofotometria Ultravioleta , Teofilina/análogos & derivados , TermodinâmicaRESUMO
Expression of sperm protein 17 (Sp17) mRNA has been reported in various malignancies. In an earlier study, we reported the upregulation of Sp17 transcripts in primary esophageal squamous cell carcinomas (ESCCs) using differential display and detected Sp17 transcripts in 86% of ESCCs by RT-PCR, whereas no transcripts were detected in the paired normal esophageal tissues. Herein we hypothesized that Sp17 might be used as a marker for detecting the response of anticancer therapies in ESCCs. Our results indicated that Sp17 protein levels in esophageal squamous cancer cell lines decreased in response to treatment with (i) the HSP90 activity inhibitor geldanamycin, (ii) the tyrosine kinase inhibitor erlotinib and (iii) cisplatin (chemotherapeutic agent commonly used in management of ESCC). In contrast, the Sp17 levels did not decrease in response to radiation therapy and treatment with the chemotherapeutic agent, gemcitabine. Further investigations showed that cisplatin induced decrease in Sp17 levels was due to transcriptional inhibition and cisplatin-resistant cell lines did not show this decrease in Sp17 levels in response to cisplatin treatment. In addition, we also carried our mass spectophotometric analysis to identify the binding partners of Sp17 to characterize its possible involvement in esophageal tumorigenesis and chemoresistance.
Assuntos
Antígenos de Superfície/metabolismo , Biomarcadores Tumorais/metabolismo , Proteínas de Transporte/metabolismo , Cisplatino/farmacologia , Antígenos de Superfície/genética , Antineoplásicos/farmacologia , Benzoquinonas/farmacologia , Biomarcadores Tumorais/genética , Western Blotting , Proteínas de Ligação a Calmodulina , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patologia , Proteínas de Transporte/genética , Linhagem Celular Tumoral , Cloridrato de Erlotinib , Neoplasias Esofágicas/genética , Neoplasias Esofágicas/metabolismo , Neoplasias Esofágicas/patologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos da radiação , Humanos , Imuno-Histoquímica , Imunoprecipitação , Lactamas Macrocíclicas/farmacologia , Espectrometria de Massas , Proteínas de Membrana , Ligação Proteica , Mapeamento de Interação de Proteínas , Quinazolinas/farmacologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/efeitos da radiaçãoRESUMO
Oxidative stress and inflammation are considered as therapeutic targets in myocardial injury. The aim of the present study was to investigate the protective effect of syringic acid (SA) and syringaldehyde (SYD) on peripheral blood mononuclear cells (PBMCs) of myocardial infarction (MI) patients. PBMCs from MI patients were cultured in the presence and absence of SA and SYD. The level of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and nitric oxide (NO) was estimated. Reactive oxygen species (ROS) formation, oxidation of lipids, proteins, and activity of antioxidant enzymes were also quantified. To further determine biomolecular changes in treated PBMCs, Fourier transform infrared (FTIR) spectroscopic analysis was done. Molecular docking study was also conducted to evaluate the binding interaction of SA and SYD with various target proteins. SA and SYD treated PBMCs of MI patients showed decreased secretion of TNF-α, IL-6, and NO. Moreover, the content of ROS, level of lipid, and protein oxidation showed diminution by treatment with both the compounds. Enhanced antioxidant defense was also observed in treated PBMCs. The FTIR spectra of treated cells revealed safeguarding effect of SA and SYD on biomolecular structure. The molecular docking analysis displayed significant binding affinity of the two compounds towards TNF-α, IL-6, and antioxidant enzymes. Our findings demonstrated potent antioxidant and anti-inflammatory effects of SA and SYD on PBMCs of MI patients. Thus, SA and SYD supplementation might be beneficial in attenuating oxidative stress and inflammation in MI.
Assuntos
Anti-Inflamatórios/farmacologia , Antioxidantes/farmacologia , Benzaldeídos/farmacologia , Ácido Gálico/análogos & derivados , Leucócitos Mononucleares/efeitos dos fármacos , Infarto do Miocárdio/metabolismo , Adulto , Células Cultivadas , Feminino , Ácido Gálico/farmacologia , Glutationa/metabolismo , Humanos , Interleucina-6/metabolismo , Leucócitos Mononucleares/metabolismo , Masculino , Malondialdeído/metabolismo , Pessoa de Meia-Idade , Simulação de Acoplamento Molecular , Infarto do Miocárdio/imunologia , Óxido Nítrico/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Fator de Necrose Tumoral alfa/metabolismoRESUMO
GDF-5 mediated signal transduction regulating chondrogenesis and skeletogenesis involves three different type-I receptors viz. Act-RI, BMPRIA and BMPRIB. BMPRIA and BMPRIB generally shows temporal and spatial co-expression but some spatially different expression pattern has also been observed. BMPRIA receptor is the key receptor implicated in BMP signalling during osteogenesis and is expressed in osteoblasts during the course of bone formation. However, BMPRIB appears to be primarily expressed in mesenchymal pre-cartilage condensations and also found in differentiated osteoblast and chondrocytes. The extracellular pH affects bone cell function and it is experimentally known that mineralization of bone is affected by shift of pH in cultured osteoblast. Here we report the effect of pH on dynamics of water present at the interface of GDF-5:BMPRIA and GDF-5:BMPRIB and binding interaction energy of these complexes. Water dynamics at different pH was analysed using residence time and hydrogen bond relaxation kinetics. pH influences the interaction energy between GDF-5 and BMPRIA and BMPRIB receptors indicating the electrostatic environment modulating the activity of two receptors. This pH dependence of interaction energy is further supported by similar behaviour of hydrogen bond existence of buried water molecules at the interface. In contrast to this the slow and fast exchanging water molecules do not show similar pH dependence of hydrogen bonding relaxation kinetics. Hence; we conclude that only buried water molecule at the interface influences the protein-protein interaction and the electrostatic environment of the extracellular fluid might decide the specificity of the two receptors.