RESUMEN
G-protein-coupled receptors (GPCRs) activate heterotrimeric G proteins by stimulating guanine nucleotide exchange in the Gα subunit1. To visualize this mechanism, we developed a time-resolved cryo-EM approach that examines the progression of ensembles of pre-steady-state intermediates of a GPCR-G-protein complex. By monitoring the transitions of the stimulatory Gs protein in complex with the ß2-adrenergic receptor at short sequential time points after GTP addition, we identified the conformational trajectory underlying G-protein activation and functional dissociation from the receptor. Twenty structures generated from sequential overlapping particle subsets along this trajectory, compared to control structures, provide a high-resolution description of the order of main events driving G-protein activation in response to GTP binding. Structural changes propagate from the nucleotide-binding pocket and extend through the GTPase domain, enacting alterations to Gα switch regions and the α5 helix that weaken the G-protein-receptor interface. Molecular dynamics simulations with late structures in the cryo-EM trajectory support that enhanced ordering of GTP on closure of the α-helical domain against the nucleotide-bound Ras-homology domain correlates with α5 helix destabilization and eventual dissociation of the G protein from the GPCR. These findings also highlight the potential of time-resolved cryo-EM as a tool for mechanistic dissection of GPCR signalling events.
Asunto(s)
Microscopía por Crioelectrón , Subunidades alfa de la Proteína de Unión al GTP Gs , Receptores Adrenérgicos beta 2 , Humanos , Sitios de Unión , Subunidades alfa de la Proteína de Unión al GTP Gs/química , Subunidades alfa de la Proteína de Unión al GTP Gs/efectos de los fármacos , Subunidades alfa de la Proteína de Unión al GTP Gs/metabolismo , Subunidades alfa de la Proteína de Unión al GTP Gs/ultraestructura , Guanosina Trifosfato/metabolismo , Guanosina Trifosfato/farmacología , Modelos Moleculares , Simulación de Dinámica Molecular , Unión Proteica , Receptores Adrenérgicos beta 2/metabolismo , Receptores Adrenérgicos beta 2/química , Receptores Adrenérgicos beta 2/ultraestructura , Factores de Tiempo , Activación Enzimática/efectos de los fármacos , Dominios Proteicos , Estructura Secundaria de Proteína , Transducción de Señal/efectos de los fármacosRESUMEN
Tetrabromobisphenol A (TBBPA) is a global pollutant. When TBBPA is absorbed by the body through various routes, it can have a wide range of harmful effects on the body. Green tea polyphenols (GTPs) can act as antioxidants, resisting the toxic effects of TBBPA on animals. The effects and mechanisms of GTP and TBBPA on oxidative stress, inflammation and apoptosis in the mouse lung are unknown. Therefore, we established in vivo and in vitro models of TBBPA exposure and GTP antagonism using C57 mice and A549 cells and examined the expression of factors related to oxidative stress, autophagy, inflammation and apoptosis. The results of the study showed that the increase in reactive oxygen species (ROS) levels after TBBPA exposure decreased the expression of autophagy-related factors Beclin1, LC3-II, ATG3, ATG5, ATG7 and ATG12 and increased the expression of p62; oxidative stress inhibits autophagy levels. The increased expression of the pro-inflammatory factors IL-1ß, IL-6 and TNF-α decreased the expression of the anti-inflammatory factor IL-10 and activation of the NF-κB p65/TNF-α pathway. The increased expression of Bax, caspase-3, caspase-7 and caspase-9 and the decreased expression of Bcl-2 activate apoptosis-related pathways. The addition of GTP attenuated oxidative stress levels, restored autophagy inhibition and reduced the inflammation and apoptosis levels. Our results suggest that GTP can attenuate the toxic effects of TBBPA by modulating ROS, reducing oxidative stress levels, increasing autophagy and attenuating inflammation and apoptosis in mouse lung and A549 cells. These results provide fundamental information for exploring the antioxidant mechanism of GTP and further for studying the toxic effects of TBBPA.
Asunto(s)
Lesión Pulmonar , FN-kappa B , Bifenilos Polibrominados , Ratones , Animales , FN-kappa B/genética , FN-kappa B/metabolismo , Antioxidantes/farmacología , Antioxidantes/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Lesión Pulmonar/inducido químicamente , Lesión Pulmonar/tratamiento farmacológico , Estrés Oxidativo , Apoptosis , Inflamación/tratamiento farmacológico , Inflamación/metabolismo , Polifenoles/farmacología , Té , Guanosina Trifosfato/metabolismo , Guanosina Trifosfato/farmacologíaRESUMEN
INTRODUCTION: Di(2-ethylhexyl) phthalate (DEHP) is a common plasticizer. Studies have revealed that DEHP exposure can cause kidney damage. Green tea is among the most popular beverages in China. Green tea polyphenols (GTPs) have been proven to have therapeutic effects on organ damage induced by heavy metal exposure. However, few studies have reported on GTP-relieving DEHP-induced kidney damage. METHODS: C57BL/6J male mice aged 6-8 weeks were treated with distilled water (control group), 1,500 mg/kg/d DEHP + corn oil (model group), 1,500 mg/kg/d DEHP + corn oil + 70 mg/kg GTP (treatment group), corn oil (oil group), and 70 mg/kg GTP (GTP group) by gavage for 8 weeks, respectively. The renal function of mice and renal tissue histopathology of each group were evaluated. The renal tissues of mice in the model, treatment, and control groups were analyzed using high-throughput sequencing. We calculated the differentially expressed microRNAs (miRNAs) and messenger RNAs (mRNAs) using the limma R package, the CIBERSORT algorithm was used to predict immune infiltration, the starBase database was used to screen the miRNA-mRNA regulatory axis, and immunohistochemical analyses were performed to verify protein expression. RESULTS: GTP alleviated the deterioration of renal function, renal inflammation and fibrosis, and mitochondrial and endoplasmic reticulum lesions induced by DEHP in mice. Differential immune infiltrations of plasma, dendritic, T, and B cells were noted between the model and treatment groups. We found that three differentially expressed miRNAs (mmu-miR-383-5p, mmu-miR-152-3p, and mmu-miR-144-3p), three differentially expressed mRNAs (Ddit4, Dusp1, and Snx18), and three differentially expressed proteins (Ddit4, Dusp1, and Snx18) played crucial roles in the miRNA-mRNA-protein regulatory axes when GTPs mitigate DEHP-induced kidney damage in mice. CONCLUSION: GTP can alleviate DEHP-induced kidney damage and regulate immune cell infiltration. We screened four important miRNA-mRNA-protein regulatory axes of GTP, mitigating DEHP-induced kidney damage in mice.
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Dietilhexil Ftalato , MicroARNs , Ácidos Ftálicos , Animales , Ratones , Masculino , Dietilhexil Ftalato/toxicidad , Aceite de Maíz/farmacología , Ratones Endogámicos C57BL , Antioxidantes , Riñón , MicroARNs/genética , MicroARNs/farmacología , ARN Mensajero , Polifenoles/farmacología , Polifenoles/uso terapéutico , Guanosina Trifosfato/farmacologíaRESUMEN
Though the physiological effects of adenosine and adenine nucleotides on purinergic receptors in cancer cells have been well studied, the influence of extracellular guanosine and guanine nucleotides on breast cancer cells remains unclear. Here, we show that extracellular guanosine and guanine nucleotides decrease the viability and proliferation of human breast cancer SKBR-3 cells. Treatment with guanosine or guanine nucleotides increased mitochondrial production of reactive oxygen species (ROS), and modified the cell cycle. Guanosine-induced cell death was suppressed by treatment with adenosine or the equilibrium nucleoside transporter (ENT) 1/2 inhibitor dipyridamole, but was not affected by adenosine receptor agonists or antagonists. These results suggest that guanosine inhibits adenosine uptake through ENT1/2, but does not antagonize adenosine receptors. In contrast, guanosine triphosphate (GTP)-induced cell death was suppressed not only by adenosine and dipyridamole, but also by the A1 receptor agonist 2-chloro-N6-cyclopentyladenosine (CCPA), suggesting that GTP-induced cell death is mediated in part by an antagonistic effect on adenosine A1 receptor. Thus, both guanosine and GTP induce apoptosis of breast cancer cells, but via at least partially different mechanisms.
Asunto(s)
Neoplasias de la Mama , Nucleótidos de Guanina , Humanos , Femenino , Nucleótidos de Guanina/metabolismo , Nucleótidos de Guanina/farmacología , Guanosina/farmacología , Neoplasias de la Mama/tratamiento farmacológico , Guanosina Trifosfato/farmacología , Adenosina/farmacología , Adenosina/metabolismo , DipiridamolRESUMEN
ETHNOPHARMACOLOGICAL RELEVANCE: Rice (Oryza sativa L.), a staple food for a significant portion of the global population, has been recognized for its traditional medicinal properties for centuries. Rice bran, a by-product of rice milling, contains many bioactive compounds with potential pharmaceutical and therapeutic benefits. In recent years, research has highlighted the anti-inflammatory potential of rice bran, contributed by the bioactive components concentrated in their bran but, unfortunately, entrapped in the bran matrix, with limited bioavailability. Previous studies have reported that the enzymatic treatment of rice bran improves the bran's bioactive compound profile but did not investigate its impact on chronic conditions such as inflammation. AIM OF THE STUDY: This study investigates the anti-inflammatory effects of endo-1,4-ß-xylanase (ERB) and Viscozyme (VRB) treated red rice bran extracts against lipopolysaccharide-induced inflammation in RAW264.7 macrophages in comparison with non-enzyme-treated bran (CRB). Further established their efficacy with known anti-inflammatory compounds-ferulic acid (FA), catechin (CAT), γ-tocopherol (GTP), and γ-oryzanol (ORZ). MATERIALS AND METHODS: The RAW 264.7 macrophage cells were pre-treated with non-toxic concentrations (10-200 µg/mL) of FA, CAT, GTP, ORZ, CRB, ERB, and VRB, followed by inflammatory stimulation with LPS for 24 h. Further, the cell supernatant and pellets were harvested to study the anti-inflammatory effects by evaluating and measuring their efficacy in inhibiting pro-inflammatory cytokines (TNF-α, IL-6, IL-10, IL-1ß) and mediators (ROS, NO, PGE2, COX2, iNOS) through biochemical, ELISA, and mRNA expression studies. RESULTS: The findings showed that both ERB and VRB effectively inhibited the production of pro-inflammatory markers (TNF-α, IL-6) and mediators (ROS, NO, PGE2) by downregulating mRNA expressions of inflammatory genes (TNF-α, IL-1ß, IL-6, IL-10, COX2, iNOS) and demonstrated anti-inflammatory efficacy higher than CRB. On comparison, ERB demonstrated exceptional efficacy by causing a reduction of 48% in ROS, 20% in TNF-α, and 23% in PGE2 at 10 µg/mL, surpassing the anti-inflammatory capabilities of all the bioactive compounds, FA and ORZ, respectively. At the same time, VRB exhibited remarkable efficacy by reducing NO production by 52% at 200 µg/mL and IL-6 by 66% at 10 µg/mL, surpassing FA, CAT, ORZ, and GTP. Further, ERB downregulated the mRNA expression of IL-10 and iNOS, while VRB downregulated TNF-α, IL-1ß, and COX2 expression. Both extracts equally downregulated IL-6 expression at 10 µg/mL, demonstrating the efficacy more remarkable/on par with established anti-inflammatory compounds. CONCLUSIONS: Overall, enzyme-treated rice bran/extract, particularly ERB, possesses excellent anti-inflammatory properties, making them promising agents for alternatives to contemporary nutraceuticals/functional food against inflammatory diseases.
Asunto(s)
Catequina , Ácidos Cumáricos , Oryza , Fenilpropionatos , Oryza/química , gamma-Tocoferol/metabolismo , gamma-Tocoferol/farmacología , gamma-Tocoferol/uso terapéutico , Interleucina-10/metabolismo , Catequina/farmacología , Factor de Necrosis Tumoral alfa/metabolismo , Interleucina-6/metabolismo , Dinoprostona/metabolismo , Ciclooxigenasa 2/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Antiinflamatorios/uso terapéutico , Extractos Vegetales/uso terapéutico , Inflamación/tratamiento farmacológico , Inflamación/metabolismo , Macrófagos , ARN Mensajero/metabolismo , Guanosina Trifosfato/metabolismo , Guanosina Trifosfato/farmacología , Guanosina Trifosfato/uso terapéutico , Lipopolisacáridos/farmacologíaRESUMEN
BACKGROUND: Neutrophils depend heavily on glycolysis for energy production under normal conditions. In contrast, neutrophils require energy supplied by mitochondrial oxidative phosphorylation (OXPHOS) during chemotaxis. However, the mechanism by which the energy supply changes from glycolysis to OXPHOS remains unknown. Leucine-rich repeat kinase 2 (LRRK2) is partially present in the outer mitochondrial membrane fraction. Lrrk2-deficient cells show mitochondrial fragmentation and reduced OXPHOS activity. We have previously reported that mitofusin (MFN) 2 is involved in chemotaxis and OXPHOS activation upon chemoattractant N-formyl-Met-Leu-Phe (fMLP) stimulation in differentiated HL-60 (dHL-60) cells. It has been previously reported that LRRK2 binds to MFN2 and partially colocalizes with MFN2 at the mitochondrial membranes. This study investigated the involvement of LRRK2 in chemotaxis and MFN2 activation in neutrophils and dHL-60 cells. METHODS: Lrrk2 knockout neutrophils and Lrrk2 knockdown dHL-60 cells were used to examine the possible involvement of LRRK2 in chemotaxis. Lrrk2 knockdown dHL-60 cells were used a tetracycline-inducible small hairpin RNA (shRNA) system to minimize the effects of LRRK2 knockdown during cell culture. The relationship between LRRK2 and MFN2 was investigated by measuring the GTP-binding activity of MFN2 in Lrrk2 knockdown dHL-60 cells. The effects of LRRK2 kinase activity on chemotaxis were examined using the LRRK2 kinase inhibitor MLi-2. RESULTS: fMLP-induced chemotactic activity was reduced in Lrrk2 knockout neutrophils in vitro and in vivo. Lrrk2 knockdown in dHL-60 cells expressing Lrrk2 shRNA also reduced fMLP-induced chemotactic activity. Lrrk2 knockdown dHL-60 cells showed reduced OXPHOS activity and suppressed mitochondrial morphological change, similar to Mfn2 knockdown dHL-60 cells. The amount of LRRK2 in the mitochondrial fraction and the GTP-binding activity of MFN2 increased upon fMLP stimulation, and the MFN2 GTP-binding activity was suppressed in Lrrk2 knockdown dHL-60 cells. Furthermore, the kinase activity of LRRK2 and Ser935 phosphorylation of LRRK2 were reduced upon fMLP stimulation, and LRRK2 kinase inhibition by MLi-2 increased the migration to fMLP. CONCLUSIONS: LRRK2 is involved in neutrophil chemotaxis and the GTP-binding activity of MFN2 upon fMLP stimulation. On the other hand, the kinase activity of LRRK2 shows a negative regulatory effect on fMLP-induced chemotactic activity in dHL-60 cells. Video Abstract.
Asunto(s)
Quimiotaxis , Neutrófilos , Humanos , Neutrófilos/metabolismo , Células HL-60 , Fosforilación Oxidativa , ARN Interferente Pequeño/metabolismo , Guanosina Trifosfato/metabolismo , Guanosina Trifosfato/farmacología , Proteína 2 Quinasa Serina-Treonina Rica en Repeticiones de Leucina/metabolismo , Proteína 2 Quinasa Serina-Treonina Rica en Repeticiones de Leucina/farmacologíaRESUMEN
This review is based upon evidence from the published effects of green tea polyphenols (GTP) on genotoxic damage induced by metals with carcinogenic potential. First, the relationship between GTP and antioxidant defense system is provided. Subsequently, the processes involved in the oxidative stress generated by metals and their relationship to oxidative DNA damage is examined. The review demonstrated that GTP generally decrease oxidative DNA damage induced by exposure to metals such as arsenic (As), cadmium (Cd), cobalt (Co), copper (Cu), chromium (Cr), iron (Fe), and lead (Pb). The pathways involved in these effects are related to: (1) direct scavenging of free radicals (FR); (2) activation of mechanisms to repair oxidative DNA damage; (3) regulation of the endogenous antioxidant system; and (4) elimination of cells with genetic damage via apoptosis. The results obtained in the studies reviewed demonstrate potential for possible use of GTP to prevent and treat oxidative damage in populations exposed to metals. Further, GTP may be considered as adjuvants to treatments for metal-associated diseases related to oxidative stress and DNA damage.
Asunto(s)
Antioxidantes , Estrés Oxidativo , Antioxidantes/farmacología , Metales/toxicidad , Daño del ADN , Polifenoles/farmacología , Té , Guanosina Trifosfato/metabolismo , Guanosina Trifosfato/farmacologíaRESUMEN
Objective To investigate the effect of Echinococcus granulosus cyst fluid(EgCF) on the cytoskeletal rearrangement and phagocytosis and the migration of macrophages induced by lipopolysaccharide(LPS). Methods Peritoneal macrophages of C57BL/6 mice were isolated and cultured in vitro, and divided into control group and LPS group and LPS combined with EgCF group. After 48 hours of treatment, filamentous actin (F-actin) changes were observed with rhodamine-labelled phalloidin staining and fluorescence microscopy; TranswellTM chamber was used to test cell migration ability and flow cytometry to test cell phagocytosis. After 1 hour of treatment, PI3K and AKT, phosphorylated AKT (p-AKT), Rac1, guanosine triphospho-Rac1 (GTP-Rac1), WASP and Arp2 protein expressions were detected with Western blot analysis. Results Compared with the control group, after LPS stimulation, macrophages were deformed significantly; pseudopodia increased; actin cytoskeleton increased and was more distributed in pseudopodia; the ability of migration and phagocytosis were significantly improved, and the expression of PI3K, p-AKT, GTP-Rac1, WASP and Arp2 proteins significantly increased. EgCF treatment caused cell shrinkage and disappearance of pseudopodia protrusions of LPS-activated cells, and led to the reduced phagocytic and migratory of cells; the protein expression of PI3K, p-AKT, GTP-Rac1, WASP and Arp2 decreased significantly compared with the LPS group. Conclusion LPS induces the migration and enhances phagocytosis of macrophages while EgCF inhibits these effects, which is related to actin cytoskeleton rearrangement.
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Echinococcus granulosus , Lipopolisacáridos , Ratones , Animales , Lipopolisacáridos/farmacología , Echinococcus granulosus/metabolismo , Proteínas Proto-Oncogénicas c-akt , Líquido Quístico/metabolismo , Ratones Endogámicos C57BL , Macrófagos/metabolismo , Fagocitosis , Actinas/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Guanosina Trifosfato/farmacologíaRESUMEN
Mitochondrial ATP-sensitive K+ channels (mitoKATP) have been recently characterized structurally, and possess a protein through which K+ enters mitochondria (MitoKIR), and a regulatory subunit (mitoSUR). The mitoSUR regulatory subunit is an ATP-binding cassette (ABC) protein isoform 8 (ABCB8). Opening these channels is known to be cardioprotective, but the molecular and physiological mechanisms that activate them are not fully known. Here, to better understand the molecular and physiological mechanisms of activators (GTP) and inhibitors (ATP) on the activity of mitoKATP, we exposed isolated mitochondria to both nucleotides. We also used molecular docking directed to the nucleotide-binding domain of human ABCB8/mitoSUR to test a comparative model of ATP and GTP effects. As expected, we find that ATP dose-dependently inhibits mitoKATP activity (IC50 = 21.24 ± 1.4 µM). However, simultaneous exposure of mitochondria to GTP dose-dependently (EC50 = 13.19 ± 1.33 µM) reversed ATP inhibition. Pharmacological and computational studies suggest that GTP reverses ATP activity competitively. Docking directed to the site of crystallized ADP reveals that both nucleotides bind to mitoSUR with high affinity, with their phosphates directed to the Mg2+ ion and the walker A motif of the protein (SGGGKTT). These effects, when combined, result in GTP binding, ATP displacement, mitochondrial ATP-sensitive K+ transport, and lower formation of reactive oxygen species. Overall, our findings demonstrate the basis for ATP and GTP binding in mitoSUR using a combination of biochemical, pharmacological, and computational experiments. Future studies may reveal the extent to which the balance between ATP and GTP actions contributes toward cardioprotection against ischemic events.
Asunto(s)
Adenosina Trifosfato , Canales KATP , Humanos , Canales KATP/metabolismo , Simulación del Acoplamiento Molecular , Adenosina Trifosfato/metabolismo , Mitocondrias , Guanosina Trifosfato/metabolismo , Guanosina Trifosfato/farmacología , Potasio/metabolismoRESUMEN
Background: An increase in the demand for a functional cure has accelerated research on new methods of therapy for chronic hepatitis B, which is mainly focused on restoring antiviral immunity for controlling viral infections. Previously, we had described elongation factor Tu GTP-binding domain containing 2 (EFTUD2) as an innate immune regulator and suggested that it might be an antiviral target. Methods: In this study, we generated the Epro-LUC-HepG2 cell model for screening compounds that target EFTUD2. Plerixafor and resatorvid were screened from 261 immunity and inflammation-related compounds due to their ability to highly upregulate EFTUD2. The effects of plerixafor and resatorvid on hepatitis B virus (HBV) were examined in HepAD38 cells and HBV-infected HepG2-NTCP cells. Results: The dual-luciferase reporter assays showed that the EFTUD2 promoter hEFTUD2pro-0.5 kb had the strongest activity. In Epro-LUC-HepG2 cells, plerixafor and resatorvid significantly upregulated the activity of the EFTUD2 promoter and the expression of the gene and protein. In HepAD38 cells and HBV-infected HepG2-NTCP cells, treatment with plerixafor and resatorvid strongly inhibited HBsAg, HBV DNA, HBV RNAs, and cccDNA in a dose-dependent manner. Furthermore, the anti-HBV effect was enhanced when entecavir was administered along with either of the previous two compounds, and the effect could be blocked by knocking down EFTUD2. Conclusion: We established a convenient model for screening compounds that target EFTUD2 and further identified plerixafor and resatorvid as novel HBV inhibitors in vitro. Our findings provided information on the development of a new class of anti-HBV agents that act on host factors rather than viral enzymes.
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Hepatitis B , Compuestos Heterocíclicos , Humanos , Virus de la Hepatitis B/fisiología , Factor Tu de Elongación Peptídica/farmacología , Movilización de Célula Madre Hematopoyética , Compuestos Heterocíclicos/farmacología , Células Hep G2 , Antivirales/farmacología , Antivirales/uso terapéutico , Guanosina Trifosfato/farmacología , Guanosina Trifosfato/uso terapéutico , Hepatitis B/tratamiento farmacológico , Replicación Viral , ADN Viral , Factores de Elongación de Péptidos/farmacología , Ribonucleoproteína Nuclear Pequeña U5/farmacologíaRESUMEN
Hemostatic powders provide an important treatment approach for time-sensitive hemorrhage control. Conventional hemostatic powders are challenged by the lack of tissue adhesiveness, insufficient hemostatic efficacy, limited infection control, and so forth. This study develops a hemostatic powder from tricomponent GTP coacervates consisting of gelatin, tannic acid (TA), and poly(vinyl alcohol) (PVA). The physical cross-linking by TA results in facile preparation, good storage stability, ease of application to wounds, and removal, which provide good potential for clinical translation. When rehydrated, the coacervate powders rapidly form a cohesive layer with interconnected microporous structure, competent flexibility, switchable wet adhesiveness, and antibacterial properties, which facilitate the hemostatic efficacy for treating irregular, noncompressible, or bacteria-infected wounds. Compared to commercial hemostats, GTP treatment results in significantly accelerated hemostasis in a liver puncture model (â¼19 s, >30% reduction in the hemostatic time) and in a tail amputation model (â¼38 s, >60% reduction in the hemostatic time). In the GTP coacervates, gelatin functioned as the biodegradable scaffold, while PVA introduced the flexible segments to enable shape-adaptability and interfacial interactions. Furthermore, TA contributed to the physical cross-linking, adhesiveness, and antibacterial performance of the coacervates. The study explores the tunability of GTP coacervate powders to enhance their hemostatic and wound healing performances.
Asunto(s)
Gelatina , Hemostáticos , Polvos/farmacología , Gelatina/farmacología , Hemostasis , Hemostáticos/farmacología , Hemostáticos/química , Cicatrización de Heridas , Antibacterianos/farmacología , Antibacterianos/química , Guanosina Trifosfato/farmacologíaRESUMEN
Microtubule (MT) severing enzymes Katanin and Spastin cut the MT into smaller fragments and are being studied extensively usingin-vitroexperiments due to their crucial role in different cancers and neurodevelopmental disorders. It has been reported that the severing enzymes are either involved in increasing or decreasing the tubulin mass. Currently, there are a few analytical and computational models for MT amplification and severing. However, these models do not capture the action of MT severing explicitly, as these are based on partial differential equations in one dimension. On the other hand, a few discrete lattice-based models were used earlier to understand the activity of severing enzymes only on stabilized MTs. Hence, in this study, discrete lattice-based Monte Carlo models that included MT dynamics and severing enzyme activity have been developed to understand the effect of severing enzymes on tubulin mass, MT number, and MT length. It was found that the action of severing enzyme reduces average MT length while increasing their number; however, the total tubulin mass can decrease or increase depending on the concentration of GMPCPP (Guanylyl-(α,ß)-methylene-diphosphonate)-which is a slowly hydrolyzable analogue of GTP (Guanosine triphosphate). Further, relative tubulin mass also depends on the detachment ratio of GTP/GMPCPP and Guanosine diphosphate tubulin dimers and the binding energies of tubulin dimers covered by the severing enzyme.
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Microtúbulos , Tubulina (Proteína) , Tubulina (Proteína)/metabolismo , Tubulina (Proteína)/farmacología , Simulación por Computador , Microtúbulos/metabolismo , Guanosina Trifosfato/metabolismo , Guanosina Trifosfato/farmacología , Guanosina Difosfato/metabolismo , Guanosina Difosfato/farmacologíaRESUMEN
Hyperinsulinism/hyperammonemia (HI/HA) syndrome has been known to be caused by dominant gain-of-function mutations in GLUD1, encoding the mitochondrial enzyme glutamate dehydrogenase. Pathogenic GLUD1 mutations enhance enzymatic activity by reducing its sensitivity to allosteric inhibition by GTP. Two recent independent studies showed that a similar HI/HA phenotype can be caused by biallelic mutations in SLC25A36, encoding pyrimidine nucleotide carrier 2 (PNC2), a mitochondrial nucleotide carrier that transports pyrimidine and guanine nucleotides across the inner mitochondrial membrane: one study reported a single case caused by a homozygous truncating mutation in SLC25A36 resulting in lack of expression of SLC25A36 in patients' fibroblasts. A second study described two siblings with a splice site mutation in SLC25A36, causing reduction of mitochondrial GTP content, putatively leading to hyperactivation of glutamate dehydrogenase. In an independent study, through combined linkage analysis and exome sequencing, we demonstrate in four individuals of two Bedouin Israeli related families the same disease-causing SLC25A36 (NM_018155.3) c.284 + 3A > T homozygous splice-site mutation found in the two siblings. We demonstrate that the mutation, while causing skipping of exon 3, does not abrogate expression of mRNA and protein of the mutant SLC25A36 in patients' blood and fibroblasts. Affected individuals had hyperinsulinism, hyperammonemia, borderline low birth weight, tonic-clonic seizures commencing around 6 months of age, yet normal intellect and no significant other morbidities. Chronic constipation, hypothyroidism, and developmental delay previously described in a single patient were not found. We thus verify that biallelic SLC25A36 mutations indeed cause HI/HA syndrome and clearly delineate the disease phenotype.
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Hiperamonemia , Hiperinsulinismo , Humanos , Glutamato Deshidrogenasa , Guanosina Trifosfato/farmacología , Hiperamonemia/genética , Hiperinsulinismo/genética , Mutación , Síndrome , Proteínas de Transporte de Membrana Mitocondrial/genéticaRESUMEN
Membrane dynamics are important to the integrity and function of mitochondria. Defective mitochondrial fusion underlies the pathogenesis of multiple diseases. The ability to target fusion highlights the potential to fight life-threatening conditions. Here we report a small molecule agonist, S89, that specifically promotes mitochondrial fusion by targeting endogenous MFN1. S89 interacts directly with a loop region in the helix bundle 2 domain of MFN1 to stimulate GTP hydrolysis and vesicle fusion. GTP loading or competition by S89 dislodges the loop from the GTPase domain and unlocks the molecule. S89 restores mitochondrial and cellular defects caused by mitochondrial DNA mutations, oxidative stress inducer paraquat, ferroptosis inducer RSL3 or CMT2A-causing mutations by boosting endogenous MFN1. Strikingly, S89 effectively eliminates ischemia/reperfusion (I/R)-induced mitochondrial damage and protects mouse heart from I/R injury. These results reveal the priming mechanism for MFNs and provide a therapeutic strategy for mitochondrial diseases when additional mitochondrial fusion is beneficial.
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Dinámicas Mitocondriales , Proteínas de Transporte de Membrana Mitocondrial , Ratones , Animales , Proteínas de Transporte de Membrana Mitocondrial/análisis , Proteínas de Transporte de Membrana Mitocondrial/química , Proteínas de Transporte de Membrana Mitocondrial/genética , Mitocondrias , Hidrólisis , Guanosina Trifosfato/análisis , Guanosina Trifosfato/farmacología , Proteínas Mitocondriales/genética , Proteínas Mitocondriales/análisis , Proteínas Mitocondriales/farmacologíaRESUMEN
The rapid healing of impaired intestinal surface plays a role in maintaining intestinal homeostasis. This study investigated the effect of calcium-sensing receptor (CaSR) on the migration and proliferation of intestinal porcine epithelial cells (IPEC-J2). Results showed that cell migration area and width were increased by R568 (CaSR activator) and decreased by NPS2143 (CaSR inhibitor). The protein level of GTP-rac1 and the phosphorylation of phospholipase C gamma 1 (PLCγ1) were increased by 2 µM R568. Furthermore, R568 + 120 µM NSC23766 (Rac1 inhibitor) and R568 + 1 µM U73122 (PLCγ1 inhibitor) decreased the protein level of GTP-rac1 and the phosphorylated PLCγ1, respectively, and both inhibited cell migration compared with R568. In addition, spermine increased the protein expression levels of CaSR and the levels of GTP-rac1 and the phosphorylated PLCγ1 and thereby promoted the migration of IPEC-J2 cells. Moreover, R568 improved the proliferation of the IPEC-J2 cells. Spermine increased cell proliferation, but NPS2143 incubated with spermine decreased cell proliferation compared with the spermine group. This study suggests that CaSR activation increased cell migration by activating Rac1 and PLCγ1 signaling and improved cell proliferation, and both effects were regulated by spermine by activating CaSR.
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Receptores Sensibles al Calcio , Espermina , Porcinos , Animales , Espermina/metabolismo , Espermina/farmacología , Proliferación Celular , Células Epiteliales/metabolismo , Guanosina Trifosfato/metabolismo , Guanosina Trifosfato/farmacologíaRESUMEN
OBJECTIVE: Epidemiology studies indicate that green tea polyphenols (GTP) perform a protective effect on cardiovascular diseases, but the underlying mechanisms are complex. The present study aimed to investigate the effect of GTP on high-fat diets (HFD) induced-early vascular aging. METHODS: Six-week young adult Wistar rats were fed with standard chow or HFD in the presence and absence of GTP (200 mg/kg body weight) for 18 weeks. In vitro experiment, human umbilical vascular endothelial cells (HUVECs) were treated with palmitic acid (PA) and GTP. RESULTS: The results showed that GTP alleviated the disorganized arterial wall and the increased intima-media thickness induced by HFD. In addition, the vascular oxidative injury was suppressed following GTP treatment. Furthermore, GTP elevated the ratio of LC3-II/LC3-I and suppressed expression of p62/SQSTM1, and restored SIRT3 expression in the aorta of HFD rats. Consistently, in cultured HUVECs, GTP inhibited cell senescence indicated by SA-ß-gal and promoted endothelial autophagy compared with the PA treatment group. The activity of SIRT3 was specifically inhibited by 3-TYP, and the protective effect of GTP was consequently abolished. CONCLUSION: The findings indicated that GTP protected against early vascular senescence in young HFD rats via ameliorating oxidative injury and promoting autophagy which was partially regulated by the SIRT3 pathway.
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Dieta Alta en Grasa , Sirtuina 3 , Animales , Ratas , Envejecimiento , Antioxidantes/farmacología , Autofagia , Grosor Intima-Media Carotídeo , Dieta Alta en Grasa/efectos adversos , Células Endoteliales/metabolismo , Guanosina Trifosfato/farmacología , Ácido Palmítico/farmacología , Polifenoles/farmacología , Ratas Wistar , Proteína Sequestosoma-1/metabolismo , Sirtuina 3/metabolismo , Sirtuina 3/farmacología , Té/metabolismoRESUMEN
Infection with human papillomavirus (HPV) is relatively common and certain high-risk HPV strains can induce epithelial dysplasia, increasing the risk of cervical cancer. Green tea polyphenol (GTP) preparations exhibit diverse anti-inflammatory, antioxidative, and antitumor properties In Vitro and In Vivo. Topical GTP application has been recommended as a treatment for genital warts, but the effect of GTP treatment on HPV infection and HPV-associated cancer remains to be established. The present study aimed to explore the mechanism by which GTP affected HPV type 16 (HPV-16)-positive immortalized human cervical epithelial cells. Survival, apoptosis, and autophagocytosis of these cells following GTP treatment was assessed using CCK-8 assay, flow cytometry, and monodansylcadaverine (MDC) staining. These cells were further transfected with an shRNA specific for Nrf2 to generate stable Nrf2-knockdown cells. The levels of Caspase-3, Bcl-2, Bax, P53, Rb, HPV-16 E6, HPV-16 E7, P62, Beclin1 and LC3B were determined via Western blotting. These analyses revealed that GTP treatment induced autophagy and apoptosis in HPV-16-positive cells, while Nrf2 gene knockdown reversed GTP-induced autophagic and apoptotic effects. Together, these results suggested that GTP could alleviate HPV infection and HPV-associated precancerous lesions In Vitro by regulating the Nrf2 pathway, highlighting the therapeutic potential of GTP in treating HPV infection.
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Proteínas Oncogénicas Virales , Infecciones por Papillomavirus , Neoplasias del Cuello Uterino , Apoptosis , Autofagia , Células Epiteliales/metabolismo , Femenino , Guanosina Trifosfato/farmacología , Guanosina Trifosfato/uso terapéutico , Papillomavirus Humano 16/genética , Papillomavirus Humano 16/metabolismo , Humanos , Factor 2 Relacionado con NF-E2/genética , Proteínas Oncogénicas Virales/genética , Proteínas Oncogénicas Virales/metabolismo , Proteínas Oncogénicas Virales/farmacología , Proteínas E7 de Papillomavirus/genética , Proteínas E7 de Papillomavirus/metabolismo , Proteínas E7 de Papillomavirus/farmacología , Infecciones por Papillomavirus/tratamiento farmacológico , Polifenoles/farmacología , Polifenoles/uso terapéutico , Té , Neoplasias del Cuello Uterino/patologíaRESUMEN
Enterococcus faecalis (E. faecalis) belongs to lactic acid bacteria which can be used as a probiotic additive and feed, bringing practical value to the health of humans and animals. The prebiotic function of tea polyphenols lays a foundation for green tea polyphenols (GTP) to repair the adverse changes of E. faecalis under stress conditions. In this study, RNA-sequence analysis was used to explore the protective effect of GTP on E. faecalis under bile salt stress. A total of 50 genes were found to respond to GTP under bile salts stress, containing 18 up-regulated and 32 down-regulated genes. The results showed that multiple genes associated with cell wall and membrane, transmembrane transport, nucleotide transport and metabolism were significantly differentially expressed (P < 0.05), while GTP intervention can partly alleviate the detrimental effects of bile salt on amino acid metabolism and transport. The present study provides the whole genome transcriptomics of E. faecalis under bile salt stress and GTP intervention which help us understand the growth and mechanism of continuous adaptation of E. faecalis under stress conditions.
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Enterococcus faecalis , Polifenoles , Animales , Antioxidantes/farmacología , Bilis , Ácidos y Sales Biliares/metabolismo , Ácidos y Sales Biliares/farmacología , Enterococcus faecalis/genética , Guanosina Trifosfato/metabolismo , Guanosina Trifosfato/farmacología , Polifenoles/farmacología , RNA-Seq , Estrés Salino , Té/química , TranscriptomaRESUMEN
RUN and FYVE domain-containing 3 (Rufy3) is a well-known adapter protein of a small GTPase protein family and is bound to the activated Ras family protein to maintain neuronal polarity. However, in experimental subarachnoid hemorrhage (SAH), the role of Rufy3 has not been investigated. Consequently, we aimed to investigate the potential role of Rufy3 in an in vivo model of SAH-induced early brain injury (EBI). In addition, we investigated the relevant brain-protective mechanisms. Oxyhemoglobin (OxyHb) stimulation of cultured primary neurons simulated vitro SAH condition. The SAH rat model was induced by infusing autologous blood into the optic chiasma pool and treating the rats with lentivirus-negative control 1 (LV-NC1), lentivirus-Rufy3 shRNA (LV-shRNA), lentivirus-negative control 2 (LV-NC2), lentivirus-Rufy3 (LV-Rufy3), or 8-pCPT-2'-O-Me-cAMP (8p-CPT) (Rap1 agonist). In experiment one, we found that the protein level of Rufy3 decreased and neuronal axon injury in the injured neurons but was rectified by LV-Rufy3 treatment. In experiment two, mRNA and protein levels of Rufy3 were downregulated in brain tissue and reached the lowest level at 24 h after SAH. In addition, the expression of Myelin Basic Protein was downregulated and that of anti-hypophosphorylated neurofilament H (N52) was upregulated after SAH. In experiments three and four, Rufy3 overexpression (LV-Rufy3) increased the interactions between Rufy3 and Rap1, the level of Rap1-GTP, and the ratio of Rap1-GTP/total GTP. In addition, LV-Rufy3 treatment inhibited axon injury and accelerated axon repair by activating the Rap1/Arap3/Rho/Fascin signaling pathway accompanied by upregulated protein expression levels of ARAP3, Rho, Fascin, and Facin. LV-Rufy3 also enhanced synaptic plasticity by activating the Rap1/MEK/ERK/synapsin I signaling pathway accompanied by upregulated protein expression levels of ERK1, p-ERK1, MEK1, p-MEK1, synaspin I, and p-synaspin I. Moreover, LV-Rufy3 also alleviated brain damage indicators, including cortical neuronal cell apoptosis and degeneration, brain edema, and cognitive impairment after SAH. However, the downregulation of Rufy3 had the opposite effect and aggravated EBI induced by SAH. Notably, the combined application of LV-Rufy3 and 8p-CPT showed a significant synergistic effect on the aforementioned parameters. Our findings suggest that enhanced Rufy3 expression may reduce EBI by inhibiting axon injury and promoting neuronal axon repair and synaptic plasticity after SAH.
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Lesiones Encefálicas , Hemorragia Subaracnoidea , Animales , Apoptosis , Axones/metabolismo , Lesiones Encefálicas/complicaciones , Lesiones Encefálicas/metabolismo , Guanosina Trifosfato/metabolismo , Guanosina Trifosfato/farmacología , Plasticidad Neuronal , Neuronas/metabolismo , ARN Interferente Pequeño/metabolismo , Ratas , Hemorragia Subaracnoidea/complicaciones , Hemorragia Subaracnoidea/genéticaRESUMEN
Tuberculosis claims significantly more than one million lives each year. A feasible way to face the issue of drug resistance is the development of new antibiotics. Bacterial uridine 5'-monophosphate (UMP) kinase is a promising target for novel antibiotic discovery as it is essential for bacterial survival and has no counterpart in human cells. The UMP kinase from M. tuberculosis is also a model of particular interest for allosteric regulation with two effectors, GTP (positive) and UTP (negative). In this study, using X-ray crystallography and cryo-electron microscopy, we report for the first time a detailed description of the negative effector UTP-binding site of a typical Gram-positive behaving UMP kinase. Comparison between this snapshot of low affinity for Mg-ATP with our previous 3D-structure of the GTP-bound complex of high affinity for Mg-ATP led to a better understanding of the cooperative mechanism and the allosteric regulation of UMP kinase. Thermal shift assay and circular dichroism experiments corroborate our model of an inhibition by UTP linked to higher flexibility of the Mg-ATP-binding domain. These new structural insights provide valuable knowledge for future drug discovery strategies targeting bacterial UMP kinases.