RESUMO
Most mature B-cell malignancies originate from the malignant transformation of germinal center (GC) B cells. The GC reaction appears to have a role in malignant transformation, in which a major player of the GC reaction is BCL6, a key regulator of this process. We now demonstrate that BCL6 protein levels were dramatically decreased in Epstein-Barr virus (EBV)-positive lymphoblastoid cell lines and Burkitt's lymphoma cell lines. Notably, BCL6 degradation was significantly enhanced in the presence of both EBNA3C and FBXO11. Furthermore, the amino-terminal domain of EBNA3C, which contains residues 50-100, interacts directly with FBXO11. The expression of EBNA3C and FBXO11 resulted in a significant induction of cell proliferation. Furthermore, BCL6 protein expression levels were regulated by EBNA3C via the Skp Cullin Fbox (SCF)FBXO11 complex, which mediated its ubiquitylation, and knockdown of FBXO11 suppressed the transformation of lymphoblastoid cell lines. These data provide new insights into the function of EBNA3C in B-cell transformation during GC reaction and raise the possibility of developing new targeted therapies against EBV-associated cancers. IMPORTANCE: The novel revelation in our study involves the suppression of BCL6 expression by the essential Epstein-Barr virus (EBV) antigen EBNA3C, shedding new light on our current comprehension of how EBV contributes to lymphomagenesis by impeding the germinal center reaction. It is crucial to note that while several EBV latent proteins are expressed in infected cells, the collaborative mechanisms among these proteins in regulating B-cell development or inducing B-cell lymphoma require additional investigation. Nonetheless, our findings carry significance for the development of emerging strategies aimed at addressing EBV-associated cancers.
Assuntos
Antígenos Nucleares do Vírus Epstein-Barr , Proteínas F-Box , Herpesvirus Humano 4 , Proteínas Proto-Oncogênicas c-bcl-6 , Humanos , Antígenos Nucleares do Vírus Epstein-Barr/metabolismo , Antígenos Nucleares do Vírus Epstein-Barr/genética , Proteínas Proto-Oncogênicas c-bcl-6/metabolismo , Proteínas Proto-Oncogênicas c-bcl-6/genética , Proteínas F-Box/metabolismo , Proteínas F-Box/genética , Herpesvirus Humano 4/metabolismo , Herpesvirus Humano 4/genética , Linhagem Celular Tumoral , Linfócitos B/metabolismo , Linfócitos B/virologia , Infecções por Vírus Epstein-Barr/metabolismo , Infecções por Vírus Epstein-Barr/virologia , Proteólise , Proliferação de Células , Ubiquitinação , Linfoma de Burkitt/virologia , Linfoma de Burkitt/metabolismo , Linfoma de Burkitt/genética , Linfoma de Burkitt/patologia , Antígenos Virais/metabolismo , Antígenos Virais/genética , Centro Germinativo/metabolismo , Centro Germinativo/virologia , Proteína-Arginina N-MetiltransferasesRESUMO
Nitrogen (N) limits crop yield, and improvement of N nutrition remains a key goal for crop research; one approach to improve N nutrition is identifying plant-interacting, N2-fixing microbes. Rhodotorula mucilaginosa JGTA-S1 is a basidiomycetous yeast endophyte of narrowleaf cattail (Typha angustifolia). JGTA-S1 could not convert nitrate or nitrite to ammonium but harbors diazotrophic (N2-fixing) endobacteria (Pseudomonas stutzeri) that allow JGTA-S1 to fix N2 and grow in a N-free environment; moreover, P. stutzeri dinitrogen reductase was transcribed in JGTA-S1 even under adequate N. Endobacteria-deficient JGTA-S1 had reduced fitness, which was restored by reintroducing P. stutzeri JGTA-S1 colonizes rice (Oryza sativa), significantly improving its growth, N content, and relative N-use efficiency. Endofungal P. stutzeri plays a significant role in increasing the biomass and ammonium content of rice treated with JGTA-S1; also, JGTA-S1 has better N2-fixing ability than free-living P. stutzeri and provides fixed N to the plant. Genes involved in N metabolism, N transporters, and NODULE INCEPTION-like transcription factors were upregulated in rice roots within 24 h of JGTA-S1 treatment. In association with rice, JGTA-S1 has a filamentous phase and P. stutzeri only penetrated filamentous JGTA-S1. Together, these results demonstrate an interkingdom interaction that improves rice N nutrition.
Assuntos
Bactérias/metabolismo , Basidiomycota/metabolismo , Fixação de Nitrogênio/fisiologia , Nitrogênio/metabolismo , Oryza/metabolismo , Oryza/microbiologia , Rhodotorula/metabolismo , Compostos de Amônio , Basidiomycota/crescimento & desenvolvimento , Endófitos/metabolismo , Regulação da Expressão Gênica de Plantas , Oryza/genética , Oryza/crescimento & desenvolvimento , Raízes de Plantas/genética , Raízes de Plantas/metabolismo , Pseudomonas/metabolismo , Pseudomonas stutzeri/metabolismo , Rhodotorula/crescimento & desenvolvimento , Rhodotorula/isolamento & purificação , Simbiose , TranscriptomaRESUMO
BACKGROUND: Reactivation of Epstein Barr virus (EBV) leads to modulation of the viral and cellular epitranscriptome. N6-methyladenosine (m6A) modification is a type of RNA modification that regulates metabolism of mRNAs. Previous reports demonstrated that m6A modification affects the stability and metabolism of EBV encoded mRNAs. However, the effect of reactivation on reprograming of the cellular mRNAs, and how this contributes to successful induction of lytic reactivation is not known. METHODS: Methylated RNA immunoprecipitation sequencing (MeRIP-seq), transcriptomic RNA sequencing (RNA-seq) and RNA pull-down PCR were used to screen and validate differentially methylated targets. Western blotting, quantitative real-time PCR (RT-qPCR) and immunocytochemistry were used to investigate the expression and localization of different proteins. RNA stability and polysome analysis assays were used to detect the half-lives and translation efficiencies of downstream genes. Insertion of point mutation to disrupt the m6A methylation sites was used to verify the effect of m6A methylation on its stability and expression levels. RESULTS: We report that during EBV reactivation the m6A eraser ALKBH5 is significantly downregulated leading to enhanced methylation of the cellular transcripts DTX4 and TYK2, that results in degradation of TYK2 mRNAs and higher efficiency of translation of DTX4 mRNAs. This resulted in attenuation of IFN signaling that promoted progression of viral lytic replication. Furthermore, inhibition of m6A methylation of these transcripts led to increased production of IFN, and a substantial reduction in viral copy number, which suggests abrogation of lytic viral replication. CONCLUSION: Our findings illuminate the significance of m6A modification in overcoming the innate immune response during EBV reactivation. We now report that during lytic reactivation EBV targets the RNA methylation system of the host to attenuate the innate immune response by suppressing the interferon signaling which facilitates successful lytic replication of the virus.
Assuntos
Infecções por Vírus Epstein-Barr , Herpesvirus Humano 4 , Humanos , Herpesvirus Humano 4/genética , Infecções por Vírus Epstein-Barr/genética , Ativação Viral/genética , Replicação Viral/genética , RNARESUMO
The hypoxic microenvironment and metabolic reprogramming are two major contributors to the phenotype of oncogenic virus-infected cells. Infection by Kaposi's sarcoma-associated herpesvirus (KSHV) stabilizes hypoxia-inducible factor 1α (HIF1α) and reprograms cellular metabolism. We investigated the comparative transcriptional regulation of all major genes involved in fatty acid and amino acid metabolism in KSHV-positive and -negative cells grown under normoxic or hypoxic conditions. We show a distinct regulation of genes involved in both fatty acid and amino acid metabolism in KSHV-positive cells grown in either normoxic or hypoxic conditions, with a particular focus on genes involved in the acetyl coenzyme A (acetyl-CoA) pathway. The fatty acid binding protein (FABP) family of genes, specifically FABP1, FABP4, and FABP7, was also observed to be synergistically upregulated in hypoxia by KSHV. This pattern of FABP gene expression was also seen in naturally infected KSHV BC3 or BCBL1 cells when compared to KSHV-negative DG75 or BL41 cells. Two KSHV-encoded antigens, which positively regulate HIF1α, the viral G-protein coupled receptor (vGPCR), and the latency-associated nuclear antigen (LANA) were shown to drive upregulation of the FABP gene transcripts. Suppression of FABPs by RNA interference resulted in an adverse effect on hypoxia-dependent viral reactivation. Overall, this study provides new evidence, which supports a rationale for the inhibition of FABPs in KSHV-positive cells as potential strategies, for the development of therapeutic approaches targeting KSHV-associated malignancies.IMPORTANCE Hypoxia is a detrimental stress to eukaryotes and inhibits several cellular processes, such as DNA replication, transcription, translation, and metabolism. Interestingly, the genome of Kaposi's sarcoma-associated herpesvirus (KSHV) is known to undergo productive replication in hypoxia. We investigated the comparative transcriptional regulation of all major genes involved in fatty acid and amino acid metabolism in KSHV-positive and -negative cells grown under normoxic or hypoxic conditions. Several metabolic pathways were observed differentially regulated by KSHV in hypoxia, specifically, the fatty acid binding protein (FABP) family genes (FABP1, FABP4, and FABP7). KSHV-encoded antigens, vGPCR and LANA, were shown to drive upregulation of the FABP transcripts. Suppression of FABPs by RNA interference resulted in an adverse effect on hypoxia-dependent viral reactivation. Overall, this study provides new evidence, which supports a rationale for the inhibition of FABPs in KSHV-positive cells as potential strategies, for the development of therapeutic approaches targeting KSHV-associated malignancies.
Assuntos
Hipóxia Celular , Proteína 7 de Ligação a Ácidos Graxos/genética , Proteínas de Ligação a Ácido Graxo/genética , Herpesvirus Humano 8/fisiologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Proteínas Supressoras de Tumor/genética , Aminoácidos/metabolismo , Antígenos Virais/genética , Antígenos Virais/metabolismo , Linhagem Celular Tumoral , Proteína 7 de Ligação a Ácidos Graxos/metabolismo , Proteínas de Ligação a Ácido Graxo/metabolismo , Ácidos Graxos/metabolismo , Regulação da Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Interferência de RNA , Receptores de Quimiocinas/genética , Receptores de Quimiocinas/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Proteínas Virais/genética , Proteínas Virais/metabolismo , Ativação ViralRESUMO
Hemoglobin oxidation due to oxidative stress and disease conditions leads to the generation of ROS (reactive oxygen species) and membrane attachment of hemoglobin in-vivo, where its redox activity leads to peroxidative damage of membrane lipids and proteins. Spectrin, the major component of the red blood cell (RBC) membrane skeleton, is known to interact with hemoglobin and, here this interaction is shown to increase hemoglobin peroxidase activity in the presence of reducing substrate ABTS (2', 2'-Azino-Bis-3-Ethylbenzothiazoline-6-Sulfonic Acid). It is also shown that in the absence of reducing substrate, spectrin forms covalently cross-linked aggregates with hemoglobin which display no peroxidase activity. This may have implications in the clearance of ROS and limiting peroxidative damage. Spectrin is found to modulate the peroxidase activity of different hemoglobin variants like A, E, and S, and of isolated globin chains from each of these variants. This may be of importance in disease states like sickle cell disease and HbE-ß-thalassemia, where increased oxidative damage and free globin subunits are present due to the defects inherent in the hemoglobin variants associated with these diseases. This hypothesis is corroborated by lipid peroxidation experiments. The modulatory role of spectrin is shown to extend to other heme proteins, namely catalase and cytochrome-c. Experiments with free heme and Raman spectroscopy of heme proteins in the presence of spectrin show that structural alterations occur in the heme moiety of the heme proteins on spectrin binding, which may be the structural basis of increased enzyme activity.
Assuntos
Hemeproteínas , Antioxidantes , Catalase/genética , Heme , Hemoglobinas/genética , Hemoglobinas/metabolismo , Peroxidase/genética , Peroxidases/genética , Espécies Reativas de Oxigênio , Espectrina/química , Espectrina/genética , Espectrina/metabolismoRESUMO
Characterization of the nanoparticle protein corona has gained tremendous importance lately. The parameters which quantitatively establish a specific nanoparticle-protein interaction need to be measured accurately since good quality data are necessary for the elucidation of the underlying mechanism and accurate molecular dynamics simulation. Here, we have employed surface sensitive second harmonic light scattering (SHLS) for investigating the adsorption of a tetrameric protein, alcohol dehydrogenase (ADH, Saccharomyces cerevisiae 147 kDa), on 16 nm, 27 nm, 41 nm, and 69 nm citrate capped gold nanoparticles (GNPs) in aqueous phosphate buffer at pH 7. We have extracted the binding constant, number of ADH bound per GNP, Gibbs free energy (ΔG°) from the decay of the second harmonic scattered signal as a function of protein concentration using a modified version of the Langmuir adsorption isotherm. The data obtained were checked with another technique, dynamic light scattering, using the same modified Langmuir model (MLM). While the binding constants measured by the two methods are in agreement, the number of ADH bound to each GNP obtained by the two methods varies a lot. In order to further probe this binding independent of a model fitting, we used an orthogonal fluorescence assay which measures the number of ADH bound to a GNP directly, and no model-fitting is necessary. We then used temperature dependent SHLS to measure the heat of adsorption (ΔH°) and entropy (ΔS°) for ADH-GNP corona formation. We found that the equilibrium binding constant (Kb) obtained from SHLS is of the order of 109 M-1 and the formation of the GNP-ADH corona is spontaneous with ΔG° â¼ -55 kJ mol-1. However, the adsorption is modestly endothermic, accompanied by a large increase in entropy. Stated differently, GNP-ADH corona formation is entropically driven. This is perhaps due to the tremendous disruption of the water structure at the negatively charged interface upon the arrival of the protein within the bonding distance to it. We believe that the SHLS technique is highly sensitive and reliable, at very low concentrations of both nanoparticles and proteins, for the quantitative estimation of the thermodynamic parameters of nanoparticle-protein corona formation, where many other techniques may fall short.
Assuntos
Álcool Desidrogenase/química , Ouro/química , Nanopartículas Metálicas/química , Termodinâmica , Adsorção , Álcool Desidrogenase/metabolismo , Ouro/metabolismo , Modelos Moleculares , Tamanho da Partícula , Saccharomyces cerevisiae/enzimologia , Propriedades de SuperfícieRESUMO
Spectrin is a multifunctional, multi-domain protein most well known in the membrane skeleton of mature human erythrocytes. Here we review the literature on the crosstalk of the chaperone activity of spectrin with its other functionalities. We hypothesize that the chaperone activity is derived from the surface exposed hydrophobic patches present in individual "spectrin-repeat" domains and show a competition between the membrane phospholipid binding functionality and chaperone activity of spectrin. Moreover, we show that post-translational modifications such as glycation which shield these surface exposed hydrophobic patches, reduce the chaperone function. On the other hand, oligomerization which is linked to increase of hydrophobicity is seen to increase it. We note that spectrin seems to prefer haemoglobin as its chaperone client, binding with it preferentially over other denatured proteins. Spectrin is also known to interact with unstable haemoglobin variants with a higher affinity than in the case of normal haemoglobin. We propose that chaperone activity of spectrin could be important in the cellular biochemistry of haemoglobin, particularly in the context of diseases.
Assuntos
Espectrina/metabolismo , Animais , Membrana Eritrocítica/química , Membrana Eritrocítica/metabolismo , Hemoglobinas/química , Hemoglobinas/metabolismo , Humanos , Interações Hidrofóbicas e Hidrofílicas , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Chaperonas Moleculares/química , Chaperonas Moleculares/metabolismo , Fosfolipídeos/química , Ligação Proteica , Processamento de Proteína Pós-Traducional , Espectrina/químicaRESUMO
Spectrin-based proteinaceous membrane skeletal network has been found to be implicated in membrane disorders like hereditary spherocytosis (HS). HS greatly affects eryptosis via loss of membrane asymmetry which is seen to be the case in haemoglobin disorders like thalassemia and sickle cell disease as well. The biological implications of the status of membrane asymmetry are strongly correlated to spectrin interactions with aminophospholipids, e.g. PE and PS. Fluorescence and X-ray reflectivity (XRR) measurements of spectrin interactions with small unilamellar vesicles (SUVs) and cushioned bilayers of phospholipids, respectively, were studied. Both the XRR and fluorescence measurements led to the characterization of spectrin orientation on the surface of lipid bilayer of phosphatidylcholine (PC) and PC/aminophospholipid mixed membrane systems showing formation of a uniform layer of spectrin on top of the mixed phospholipid bilayer. Fluorescence studies show that spectrin interacts with PC and phosphatidylethanolamine (PE)/phosphatidylserine (PS) membranes with binding dissociation constants (Kd) in the nanomolar range indicating the role of spectrin in the maintenance of the overall membrane asymmetry of erythrocytes.
Assuntos
Membrana Celular/química , Eritrócitos/citologia , Espectrina/química , Eriptose , Humanos , Bicamadas Lipídicas/química , Fosfolipídeos/química , Esferocitose HereditáriaRESUMO
Molecular chaperones are one of the key players in protein biology and as such their structure and mechanism of action have been extensively studied. However the substrate specificity of molecular chaperones has not been well investigated. This review aims to summarize what is known about the substrate specificity and substrate recognition motifs of chaperones so as to better understand what substrate specificity means in the context of molecular chaperones. Available literature shows that the majority of chaperones have broad substrate range and recognize non-native conformations of proteins depending on recognition of hydrophobic and/or charged patches. Based on these recognition motifs chaperones can select for early, mid or late folding intermediates. Another major contributor to chaperone specificity are the co-chaperones they interact with as well as the sub-cellular location they are expressed in and the inducability of their expression. Some chaperones which have only one or a few known substrates are reported. In their case the mode of recognition seems to be specific structural complementarity between chaperone and substrate. It can be concluded that the vast majority of chaperones do not show a high degree of specificity but recognize elements that signal non-native protein conformation and their substrate range is modulated by the context they function in. However a few chaperones are known that display exquisite specificity of their substrate e.g. mammalian heat shock protein 47 collagen interaction. © 2017 IUBMB Life, 69(9):647-659, 2017.
Assuntos
Motivos de Aminoácidos/genética , Colágeno/química , Proteínas de Choque Térmico HSP47/química , Chaperonas Moleculares/química , Sequência de Aminoácidos/genética , Colágeno/genética , Proteínas de Choque Térmico HSP47/genética , Proteínas de Choque Térmico HSP47/metabolismo , Humanos , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Conformação Proteica , Dobramento de Proteína , Mapas de Interação de Proteínas/genética , Especificidade por SubstratoRESUMO
Spectrin, a major component of the eukaryotic membrane skeleton, has been shown to have chaperone like activity. Here we investigate the pH induced changes in the structure and stability of erythroid and brain spectrin by spectroscopic methods. We also correlate these changes with modulations of chaperone potential at different pH. We have followed the pH induced structural changes by circular dichroism spectroscopy and intrinsic tryptophan fluorescence. It is seen that lowering the pH from 9 has little effect on structure of the proteins till about pH6. At pH4, there is significant change of the secondary structure of the proteins, along with a 5nm hypsochromic shift of the emission maxima. Below pH4 the proteins undergo acid denaturation. Probing exposed hydrophobic patches on the proteins using protein-bound 8-anilinonaphthalene-1-sulfonate fluorescence demonstrates that there is higher solvent accessibility of hydrophobic surfaces in both forms of spectrin at around pH4. Dynamic light scattering and 90° light scattering studies show that the both forms of spectrin forms oligomers at pH~4. Chemical unfolding data shows that these oligomers are less stable than the tetrameric form. Aggregation studies with BSA show that at pH4, both spectrins exhibit better chaperone activity. This enhancement of chaperone like activity appears to result from an increase in regions of solvent-exposed hydrophobicity and oligomeric state of the spectrins which in turn are induced by moderately acid pH. This may have in-vivo implications in cells facing stress conditions where cytoplasmic pH is lowered.
Assuntos
Concentração de Íons de Hidrogênio , Chaperonas Moleculares/química , Estabilidade Proteica , Espectrina/química , Dicroísmo Circular , Chaperonas Moleculares/metabolismo , Conformação Proteica , Espectrometria de Fluorescência , Espectrofotometria Ultravioleta , Triptofano/química , Ureia/químicaRESUMO
BACKGROUND/AIMS: Cytotoxic effect of attenuated Leishmania on liver cancer cells by inducing ROS generation. METHODS: Spectrophotometric study to analyze cell death and levels of different active caspases. Flow cytometric study was done to analyze apoptosis induction and ROS generation and levels of different protein. Western blot analysis was performed to study the levels of protein. Confocal microscopy was done to ascertain the expression of different apoptotic markers. RESULTS: We have now observed that attenuated Leishmania donovani UR6 also has potentiality towards growth inhibition of HepG2 cells and investigated the mechanism of action. The effect is associated with increased DNA fragmentation, rise in number of annexinV positive cells, and cell cycle arrest at G1 phase. The detection of unregulated levels of active PARP, cleaved caspases 3 and 9, cytosolic cytochrome C, Bax, and Bad, along with the observed downregulation of Bcl-2 and loss of mitochondrial membrane potential suggested the involvement of mitochondrial pathway. Enhanced ROS and p53 levels regulate the apoptosis of HepG2 cells. NAC was found to inhibit p53 production but PFT-α has no effect on ROS generation. In conclusion, Leishmania donovani UR6 efficiently induces apoptosis in HepG2 cells through ROS mediated p53 dependent mitochondrial pathway. CONCLUSION: It has been reported earlier that some parasites show prominent cytotoxic effect and prevent tumor growth. From our study we found that Leishmania donovani UR6 efficiently induced apoptosis in HepG2 cells through ROS mediated p53 dependent mitochondrial pathway. This study has rejuvenated the age old idea of bio-therapy.
Assuntos
Apoptose , Leishmania/patogenicidade , Mitocôndrias/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Acetilcisteína/farmacologia , Cálcio/metabolismo , Caspase 3/análise , Caspase 3/metabolismo , Caspase 9/análise , Caspase 9/metabolismo , Citocromos c/metabolismo , Fragmentação do DNA , Regulação para Baixo , Pontos de Checagem da Fase G1 do Ciclo Celular , Células Hep G2 , Temperatura Alta , Humanos , Potencial da Membrana Mitocondrial , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Regulação para Cima/efeitos dos fármacos , Proteína X Associada a bcl-2/metabolismo , Proteína de Morte Celular Associada a bcl/metabolismoRESUMO
Very often conventional therapy, i.e. chemotherapeutic treatment, develops resistance in cancer cells and fails to be effective against disease states. An alternative strategy or a new entity may resolve the problem. Interestingly, the microbial world has begun to be explored in medicinal research as a potential new source to deliver bio-active molecules such as sphingolipids for efficacious cancer treatment. A sphingolipid of microbial origin, especially from Leishmania donovani (LSPL), is a novel entity which may exert anti-cancer activity by regulating cellular growth. The present study reveals that among a range of cancer cells evaluated, LSPL-1 (a component of LSPL) reduces cell viability, annexin exposures and arrests cell cycle in B16F10 cells in a concentration and time dependent manner. Flowcytometric analysis showed that it alters mitochondrial membrane potential and generates a number of ROS positive melanoma cells. It activates p53 at serine anchor region via up-regulation of p21 subunit along with PUMA and NOXA. It also exerts activity in vivo by reducing tumor micro vessel and mitotic index while simultaneously improving the survival rate. The inflammatory responses including elevated level of cytokine-chemokine and increased expression of PCNA and F480 are subdued by LSPL-1 treatment in tumour bearing mice. Besides, it reduces the metastatic outburst of angiogenic factors like VEGF, Ang-2, and CD34 through the involvement of several growth promoting factors. These findings indicate that LSPL-1 may be explored as a potent entity against melanoma growth and the associated angiogenic promotion.
Assuntos
Antineoplásicos/farmacologia , Proliferação de Células/efeitos dos fármacos , Leishmania donovani/química , Neovascularização Patológica/metabolismo , Esfingolipídeos/farmacologia , Animais , Linhagem Celular Tumoral , Feminino , Inflamação/metabolismo , Camundongos Endogâmicos BALB C , Mitocôndrias/metabolismo , Espécies Reativas de Oxigênio/metabolismoRESUMO
Sphingolipids are membrane and intracellular lipids that typically modulate cellular processes to cause cell death. Exogenous administration of sphingolipids may cause restriction of tumour growth and several alternative strategies are being used to control the cell growth. The microbes, their cellular component(s) or metabolites like DHA, EPA and also FTY720 have been employed as new therapeutic entities to regulate the disease condition. The therapeutic efficacy of lipids from Leishmania donovani in rheumatoid arthritis and also in sepsis condition associated with inflammatory diseases is well established. In this study, we explored the apoptotic effect of LSPL-1 (leishmanial sphingolipid-1) in Sarcoma 180 cells towards the regulation of tumour growth. The study using a panel of cancer cell lines revealed that LSPL-1 induces cell death in Sarcoma 180. The apoptotic changes were assessed by annexin exposure and DNA content analysis using flow cytometry. LSPL-1 appears to activate several pro- and anti-apoptotic molecules through reactive oxygen species (ROS) generation and also caspase activation, as determined by Western blot and ELISA analyses. Simultaneously, it may improve the survival rate of mice bearing tumour induced by Sarcoma 180 cells, with pathological changes. LSPL-1 may also suppress the cancer-associated inflammatory responses with the expression of matrix metalloproteinase having inhibitory role. It may regulate several angiogenic factors including VEGF, Ang-2 and CD34 in angiogenic events generated in Sarcoma 180 cell-induced tumour. These studies underline the significance of anti-neoplastic potential of LSPL-1 through apoptosis induction and abrogation of angiogenic responses in Sarcoma 180 cell-associated tumour.
Assuntos
Proliferação de Células/efeitos dos fármacos , Neovascularização Patológica/tratamento farmacológico , Sarcoma 180/tratamento farmacológico , Esfingolipídeos/administração & dosagem , Animais , Apoptose/efeitos dos fármacos , Caspase 3/biossíntese , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Humanos , Leishmania donovani/química , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Camundongos , Neovascularização Patológica/patologia , Espécies Reativas de Oxigênio/metabolismo , Sarcoma 180/patologia , Transdução de Sinais/efeitos dos fármacos , Esfingolipídeos/química , Esfingolipídeos/isolamento & purificação , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Sepsis is the reflection of systemic immune response that manifests in the sequential inflammatory process in presence of infection. This may occur as a result of gram-negative bacterial sepsis including Escherichia coli infection that gives rise to excessive production of inflammatory mediators and causes severe tissue injuries. We have reported earlier that the lipid of attenuated Leishmania donovani suppresses the inflammatory responses in arthritis patients. Using heat killed E. coli stimulated macrophages, we have now investigated the effect of leishmanial total lipid (LTL) isolated from Leishmania donovani (MHO/IN/1978/UR6) for amelioration of the inflammatory mediators and transcriptional factor with suppression of TLR4-CD14 expression. To evaluate the in vivo effect, E. coli induced murine sepsis model was used focusing on the changes in different parameter(s) of lung injury caused by sepsis, namely, edema, vascular permeability, and pathophysiology, and the status of different cytokine-chemokine(s) and adhesion molecule(s). Due to the effect of LTL, E. coli induced inflammatory cytokine-chemokine(s) levels were significantly reduced in serum and bronchoalveolar lavage fluid simultaneously. LTL also improved the lung injury and suppressed the cell adhesion molecules in lung tissue. These findings indicate that LTL may prove to be a potential anti-inflammatory agent and provide protection against gram-negative bacterial sepsis with pulmonary impairment.
Assuntos
Escherichia coli/patogenicidade , Inflamação/tratamento farmacológico , Leishmania donovani/química , Lipídeos/uso terapêutico , Sepse/tratamento farmacológico , Animais , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Escherichia coli/efeitos dos fármacos , Feminino , Lipídeos/farmacologia , Camundongos , Camundongos Endogâmicos BALB C , Microscopia de FluorescênciaRESUMO
IMPORTANCE: Hypoxia can induce the reactivation of Kaposi sarcoma-associated virus (KSHV), which necessitates the synthesis of critical structural proteins. Despite the unfavorable energetic conditions of hypoxia, KSHV utilizes mechanisms to prevent the degradation of essential cellular machinery required for successful reactivation. Our study provides new insights on strategies employed by KSHV-infected cells to maintain steady-state transcription by overcoming hypoxia-mediated metabolic stress to enable successful reactivation. Our discovery that the interaction of latency-associated nuclear antigen with HIF1α and NEDD4 inhibits its polyubiquitination activity, which blocks the degradation of RNA Pol II during hypoxia, is a significant contribution to our understanding of KSHV biology. This newfound knowledge provides new leads in the development of novel therapies for KSHV-associated diseases.
Assuntos
Herpesvirus Humano 8 , Sarcoma de Kaposi , Humanos , Herpesvirus Humano 8/fisiologia , RNA Polimerase II/genética , RNA Polimerase II/metabolismo , Latência Viral/genética , Antígenos Virais/genética , Hipóxia/metabolismo , Replicação ViralRESUMO
Neurotransmitters are released from synaptic vesicles with remarkable precision in response to presynaptic Ca2+ influx but exhibit significant heterogeneity in exocytosis timing and efficacy based on the recent history of activity. This heterogeneity is critical for information transfer in the brain, yet its molecular basis remains poorly understood. Here, we employ a biochemically-defined fusion assay under physiologically-relevant conditions to delineate the minimal protein machinery sufficient to account for different modes of Ca2+-triggered vesicle fusion and short-term facilitation. We find that Synaptotagmin-1, Synaptotagmin-7, and Complexin, synergistically restrain SNARE complex assembly, thus preserving vesicles in a stably docked state at rest. Upon Ca2+ activation, Synaptotagmin-1 induces rapid vesicle fusion, while Synaptotagmin-7 mediates delayed fusion. Competitive binding of Synaptotagmin-1 and Synaptotagmin-7 to the same SNAREs, coupled with differential rates of Ca2+-triggered fusion clamp reversal, govern the kinetics of vesicular fusion. Under conditions mimicking sustained neuronal activity, the Synaptotagmin-7 fusion clamp is destabilized by the elevated basal Ca2+ concentration, thereby enhancing the synchronous component of fusion. These findings provide a direct demonstration that a small set of proteins is sufficient to account for how nerve terminals adapt and regulate the Ca2+-evoked neurotransmitter exocytosis process to support their specialized functions in the nervous system.
RESUMO
Cytoskeletal drugs having enormous therapeutic potential act on the cytoskeletal components like actin, tubulin either by promoting polymerization or destabilizing the same. Here we present the interaction of the popular cytoskeletal drugs such as taxol, latrunculin and cytochalasin with spectrin, a huge protein with multi domains that forms the cytoskeletal network. Particularly, the actin binding domain of spectrin regulates the dynamics of the actin cytoskeleton. We followed the binding of these drugs to its actin binding domain and intact spectrin as well. These drugs bind with moderate affinity (Kb â¼ 104 M-1) and the interaction with actin binding domain is entropy driven and hydrophobic in nature as determined by Van't Hoff plot. The docking studies and molecular dynamics simulations further corroborate the experimental findings. Particularly the higher binding constants in the case of latrunculin and cytochalasin to the actin binding domain of spectrin suggest the binding sites are presumably located in its actin binding domain.Communicated by Ramaswamy H. Sarma.
RESUMO
Different quinazoline derivatives have showed wide spectrum of pharmacological activities. Some 3-(arylideneamino)-phenylquinazoline-4(3H)-ones have been reported to possess antimicrobial activity. The present study has been undertaken to evaluate the anticancer effect of these quinazolinone derivatives. The quinazolinone derivatives were synthesized as reported earlier. Compounds containing NO(2), OH, OCH(3), or OH and OCH(3) as substituent(s) on the arylideneamino group were named as P(3a), P(3b), P(3c), and P(3d) respectively. Out of these, P(3a) and P(3d) showed better cytotoxic activity than P(3b) and P(3c) on a panel of six cancer cell lines of different origin, namely, B16F10, MiaPaCa-2, HCT116, HeLa, MCF7, and HepG2, though the effect was higher in B16F10, HCT116, and MCF7 cells. P(3a) and P(3d) induced death of B16F10 and HCT116 cells was associated with characteristic apoptotic changes like cell shrinkage, nuclear condensation, DNA fragmentation, and annexin V binding. Also, cell cycle arrest at G1 phase, alteration of caspase-3, caspase-9, Bcl-2 and PARP levels, loss of mitochondrial membrane potential, and enhanced level of cytosolic cytochrome c were observed in treated B16F10 cells. Treatment with multiple doses of P(3a) significantly increased the survival rate of B16F10 tumor bearing BALB/c mice by suppressing the volume of tumor while decreasing microvascular density and mitotic index of the tumor cells.
Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Quinazolinas/farmacologia , Animais , Anexina A5/metabolismo , Antineoplásicos/síntese química , Antineoplásicos/uso terapêutico , Caspase 3/metabolismo , Caspase 9/metabolismo , Linhagem Celular Tumoral , Forma Celular/efeitos dos fármacos , Citocromos c/metabolismo , Fragmentação do DNA/efeitos dos fármacos , Pontos de Checagem da Fase G1 do Ciclo Celular/efeitos dos fármacos , Células HCT116 , Células HeLa , Humanos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Camundongos , Neoplasias/tratamento farmacológico , Poli(ADP-Ribose) Polimerases/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Quinazolinas/química , Quinazolinas/uso terapêutico , Transplante HeterólogoRESUMO
Inflammation is a protective immune response against harmful stimuli whose long time continuation results in host disease. Quinazolinones are nitrogen containing heterocyclic compounds with wide spectrum of biological activities. The anticancer effect of a 3-(arylideneamino)-phenylquinazoline-4(3H)-one derivative was reported earlier. The anti-inflammatory effect of these quinazolinone derivatives has now been examined in endotoxin stimulated macrophages and in different in vivo models of inflammation by measuring the proinflammatory cytokines (TNF-α, IL-1ß and IL-6), mediators NO and NF-κB (by ELISA and western blot), and translocation of the nuclear factor kB (by immunocytochemical analysis). To elucidate the in vivo effect, mice endotoxin model was and the various levels of edema, inflammatory pain and vascular permeability were studied. One of the quinazolinone derivatives showed significant anti-inflammatory activity in stimulated macrophage cells by inhibiting the expression of TNF-α, IL-1ß, IL-6, iNOS, COX-2, p-IκB and NF-κBp65. Significant (P<0.01) improvement was observed in the mortality of endotoxemic mice. The carrageenan and formalin-induced paw edema thicknesses were found to be reduced significantly (P<0.01) along with the reduction of pain, vascular permeability and edema induced by complete Freund's adjuvant (P<0.01). These findings indicate that 3-(arylideneamino)-phenylquinazoline-4(3H)-one derivative as a potential anti-inflammatory agent.
Assuntos
Anti-Inflamatórios não Esteroides , Inflamação/tratamento farmacológico , Quinazolinas/farmacologia , Animais , Western Blotting , Permeabilidade Capilar/efeitos dos fármacos , Carragenina , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Citocinas/sangue , Dinoprostona/metabolismo , Feminino , Formaldeído , Inflamação/induzido quimicamente , Inflamação/patologia , Lipopolissacarídeos/toxicidade , Fígado/patologia , Testes de Função Hepática , Macrófagos/efeitos dos fármacos , Macrófagos/patologia , Camundongos , Camundongos Endogâmicos BALB C , Microscopia de Fluorescência , NF-kappa B/metabolismo , Óxido Nítrico/metabolismo , Quinazolinas/síntese química , Quinazolinas/toxicidade , Choque Séptico/patologia , SobrevidaRESUMO
Spectrin is a cytoskeletal protein ubiquitous in metazoan cells that acts as a liaison between the plasma membrane and the cellular interior and imparts mechanical stability to the plasma membrane. Spectrin is known to be highly dynamic, with an appreciable degree of torsional and segmental mobility. In this context, we have earlier utilized the red edge excitation shift (REES) approach to report the retention of restricted solvation dynamics and local structure in the vicinity of spectrin tryptophans on urea denaturation and loss of spectrin secondary structure. As a natural progression of our earlier work, in this work, we carried out a biophysical dissection of tryptophan solvation and rotational dynamics in spectrin and its constituent domains, in order to trace the origin of local structure retention observed in denatured spectrin. Our results show that the ankyrin binding domain (and, to a lesser extent, the ß-tetramerization domain) is capable of retention of local structure, similar to that observed for intact spectrin. However, all α-chain domains studied exhibit negligible retention of local structure on urea denaturation. Such a stark chain-specific retention of local structure could originate from the fact that the ß-chain domains possess specialized functions, where conservation of local (structural) integrity may be a prerequisite for optimum cellular function. To the best of our knowledge, these observations represent one of the first systematic biophysical dissections of spectrin dynamics in terms of its constituent domains and add to emerging literature on comprehensive domain-based analysis of spectrin organization, dynamics, and function.