RESUMEN
G protein-coupled receptor (GPCR) signaling, mediated by hetero-trimeric G proteins, can be differentially controlled by agonists. At a molecular level, this is thought to occur principally via stabilization of distinct receptor conformations by individual ligands. These distinct conformations control subsequent recruitment of transducer and effector proteins. Here, we report that ligand efficacy at the calcitonin GPCR (CTR) is also correlated with ligand-dependent alterations to G protein conformation. We observe ligand-dependent differences in the sensitivity of the G protein ternary complex to disruption by GTP, due to conformational differences in the receptor-bound G protein hetero-trimer. This results in divergent agonist-dependent receptor-residency times for the hetero-trimeric G protein and different accumulation rates for downstream second messengers. This study demonstrates that factors influencing efficacy extend beyond receptor conformation(s) and expands understanding of the molecular basis for how G proteins control/influence efficacy. This has important implications for the mechanisms that underlie ligand-mediated biased agonism. VIDEO ABSTRACT.
Asunto(s)
Proteínas de Unión al GTP/química , Guanosina Trifosfato/farmacología , Receptores de Calcitonina/agonistas , Receptores de Calcitonina/química , Adenosina Difosfato/biosíntesis , Animales , Células COS , Chlorocebus aethiops , Proteínas de Unión al GTP/metabolismo , Guanosina Trifosfato/metabolismo , Humanos , Ligandos , Conformación Proteica , Multimerización de Proteína , Receptores de Calcitonina/metabolismoRESUMEN
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
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.
Asunto(s)
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
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.
Asunto(s)
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
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.
Asunto(s)
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
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
During host colonization, bacteria use the alarmones (p)ppGpp to reshape their proteome by acting pleiotropically on DNA, RNA, and protein synthesis. Here, we elucidate how the initiating ribosome senses the cellular pool of guanosine nucleotides and regulates the progression towards protein synthesis. Our results show that the affinity of guanosine triphosphate (GTP) and the inhibitory concentration of ppGpp for the 30S-bound initiation factor IF2 vary depending on the programmed mRNA. The TufA mRNA enhanced GTP affinity for 30S complexes, resulting in improved ppGpp tolerance and allowing efficient protein synthesis. Conversely, the InfA mRNA allowed ppGpp to compete with GTP for IF2, thus stalling 30S complexes. Structural modeling and biochemical analysis of the TufA mRNA unveiled a structured enhancer of translation initiation (SETI) composed of two consecutive hairpins proximal to the translation initiation region (TIR) that largely account for ppGpp tolerance under physiological concentrations of guanosine nucleotides. Furthermore, our results show that the mechanism enhancing ppGpp tolerance is not restricted to the TufA mRNA, as similar ppGpp tolerance was found for the SETI-containing Rnr mRNA. Finally, we show that IF2 can use pppGpp to promote the formation of 30S initiation complexes (ICs), albeit requiring higher factor concentration and resulting in slower transitions to translation elongation. Altogether, our data unveil a novel regulatory mechanism at the onset of protein synthesis that tolerates physiological concentrations of ppGpp and that bacteria can exploit to modulate their proteome as a function of the nutritional shift happening during stringent response and infection.
Asunto(s)
Guanosina Tetrafosfato/farmacología , Iniciación de la Cadena Peptídica Traduccional , ARN Mensajero/metabolismo , Ribosomas/efectos de los fármacos , Ribosomas/metabolismo , Unión Competitiva , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Guanosina Tetrafosfato/metabolismo , Guanosina Trifosfato/metabolismo , Guanosina Trifosfato/farmacología , Interacciones Huésped-Patógeno/fisiología , Cinética , Conformación de Ácido Nucleico , Iniciación de la Cadena Peptídica Traduccional/efectos de los fármacos , Iniciación de la Cadena Peptídica Traduccional/fisiología , Factor Tu de Elongación Peptídica/metabolismo , Biosíntesis de Proteínas/efectos de los fármacos , ARN Mensajero/química , ARN Mensajero/efectos de los fármacos , ARN Mensajero/genéticaRESUMEN
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
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.
Asunto(s)
Hiperamonemia , Hiperinsulinismo , Humanos , Glutamato Deshidrogenasa , Guanosina Trifosfato/farmacología , Hiperamonemia/genética , Hiperinsulinismo/genética , Mutación , Síndrome , Proteínas de Transporte de Membrana Mitocondrial/genéticaRESUMEN
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
Organelles display characteristic morphologies that are intimately tied to their cellular function, but how organelles are shaped is poorly understood. The endoplasmic reticulum is particularly intriguing, as it comprises morphologically distinct domains, including a dynamic network of interconnected membrane tubules. Several membrane proteins have been implicated in network formation, but how exactly they mediate network formation and whether they are all required are unclear. Here we reconstitute a dynamic tubular membrane network with purified endoplasmic reticulum proteins. Proteoliposomes containing the membrane-fusing GTPase Sey1p (refs 6, 7) and the curvature-stabilizing protein Yop1p (refs 8, 9) from Saccharomyces cerevisiae form a tubular network upon addition of GTP. The tubules rapidly fragment when GTP hydrolysis of Sey1p is inhibited, indicating that network maintenance requires continuous membrane fusion and that Yop1p favours the generation of highly curved membrane structures. Sey1p also forms networks with other curvature-stabilizing proteins, including reticulon and receptor expression-enhancing proteins (REEPs) from different species. Atlastin, the vertebrate orthologue of Sey1p, forms a GTP-hydrolysis-dependent network on its own, serving as both a fusion and curvature-stabilizing protein. Our results show that organelle shape can be generated by a surprisingly small set of proteins and represents an energy-dependent steady state between formation and disassembly.
Asunto(s)
Retículo Endoplásmico/química , Retículo Endoplásmico/metabolismo , GTP Fosfohidrolasas/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Retículo Endoplásmico/efectos de los fármacos , Retículo Endoplásmico/ultraestructura , Guanosina Trifosfato/metabolismo , Guanosina Trifosfato/farmacología , Hidrólisis/efectos de los fármacos , Fusión de Membrana , Proteínas de Transporte de Membrana/ultraestructura , Proteolípidos/química , Proteolípidos/metabolismo , Proteolípidos/ultraestructura , Saccharomyces cerevisiae/efectos de los fármacos , Saccharomyces cerevisiae/ultraestructura , Proteínas de Saccharomyces cerevisiae/ultraestructura , Proteínas de Transporte Vesicular/ultraestructuraRESUMEN
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.
Asunto(s)
Receptores Sensibles al Calcio , Espermina , Porcinos , Animales , Espermina/metabolismo , Espermina/farmacología , Proliferación Celular , Células Epiteliales/metabolismo , Guanosina Trifosfato/metabolismo , Guanosina Trifosfato/farmacologíaRESUMEN
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.
Asunto(s)
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
BACKGROUND: The plant growth is influenced by multiple interactions with biotic (microbial) and abiotic components in their surroundings. These microbial interactions have both positive and negative effects on plant. Plant growth promoting bacterial (PGPR) interaction could result in positive growth under normal as well as in stress conditions. METHODS: Here, we have screened two PGPR's and determined their potential in induction of specific gene in host plant to overcome the adverse effect of biotic stress caused by Magnaporthe grisea, a fungal pathogen that cause blast in rice. We demonstrated the glucanase protein mode of action by performing comparative modeling and molecular docking of guanosine triphosphate (GTP) ligand with the protein. Besides, molecular dynamic simulations have been performed to understand the behavior of the glucanase-GTP complex. RESULTS: The results clearly showed that selected PGPR was better able to induce modification in host plant at morphological, biochemical, physiological and molecular level by activating the expression of ß-1,3-glucanases gene in infected host plant. The docking results indicated that Tyr75, Arg256, Gly258, and Ser223 of glucanase formed four crucial hydrogen bonds with the GTP, while, only Val220 found to form hydrophobic contact with ligand. CONCLUSIONS: The PGPR able to induce ß-1,3-glucanases gene in host plant upon pathogenic interaction and ß-1,3-glucanases form complex with GTP by hydrophilic interaction for induction of defense cascade for acquiring resistance against Magnaporthe grisea.
Asunto(s)
Magnaporthe , Oryza , Antifúngicos/metabolismo , Antifúngicos/farmacología , Bacterias , Guanosina Trifosfato/metabolismo , Guanosina Trifosfato/farmacología , Ligandos , Magnaporthe/metabolismo , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Oryza/genética , Enfermedades de las Plantas/microbiologíaRESUMEN
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.
Asunto(s)
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
Although naturally occurring low-molecular-weight compounds have many known roles within the cell, these do not usually involve the direct inhibition of protein-protein interactions. Based on the results of high-throughput screening of a library of bioactive compounds and neurotransmitters, we report here that the four nucleoside triphosphates ATP, GTP, CTP and UTP inhibit the SH2 domain of the tumor-related transcription factor STAT5b. ATP and GTP are the most active nucleoside triphosphates and show specificity for STAT5b over STAT5a, STAT3, STAT6 and the p53-binding protein HDM2. As the inhibition constant of ATP against STAT5b is significantly lower than published values for the intracellular ATP concentration, our data suggest that ATP might inhibit the protein-protein interactions of STAT5b in living cells.
Asunto(s)
Adenosina Trifosfato/farmacología , Factor de Transcripción STAT5/antagonistas & inhibidores , Guanosina Trifosfato/farmacología , Humanos , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Bibliotecas de Moléculas Pequeñas , Dominios Homologos srcRESUMEN
Chloroplasts evolved from cyanobacterial endosymbiotic ancestors and their division is a complex process initiated by the assembly of cytoskeletal FtsZ (Filamentous temperature sensitive Z) proteins into a ring structure at the division site (Z-ring). The cyanobacterial Z-ring positioning system (MinCDE proteins) is also conserved in chloroplasts, except that MinC was lost and replaced by the eukaryotic ARC3 (accumulation and replication of chloroplasts). Both MinC and ARC3 act as negative regulators of FtsZ assembly, but ARC3 bears little sequence similarity with MinC. Here, light scattering assays, co-sedimentation, GTPase assay and transmission electron microscopy in conjunction with single-particle analysis have been used to elucidate the structure of ARC3 and its effect on its main target in chloroplast division, FtsZ2. Analysis of FtsZ2 in vitro assembly reactions in the presence and absence of GMPCPP showed that ARC3 promotes FtsZ2 debundling and disassembly of existing filaments in a concentration-dependent manner and requires GTP hydrolysis. Three-dimensional reconstruction of ARC3 revealed an almost circular molecule in which the FtsZ-binding N-terminus and the C-terminal PARC6 (paralog of ARC6)-binding MORN (Membrane Occupation and Recognition Nexus) domain are in close proximity and suggest a model for PARC6-enabled binding of ARC3 to FtsZ2. The latter is corroborated by in vivo data.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Cloroplastos/metabolismo , Regulación de la Expresión Génica de las Plantas , Guanosina Trifosfato/metabolismo , Arabidopsis/efectos de los fármacos , Arabidopsis/metabolismo , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , División Celular , Cloroplastos/efectos de los fármacos , Cloroplastos/genética , Cloroplastos/ultraestructura , Citoesqueleto/metabolismo , Citoesqueleto/ultraestructura , Células Eucariotas/metabolismo , Células Eucariotas/ultraestructura , Guanosina Trifosfato/análogos & derivados , Guanosina Trifosfato/farmacología , Cinética , Proteínas Nucleares/química , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Pichia/genética , Pichia/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismoRESUMEN
Nitric oxide sensitive guanylyl cyclase (NOsGC) is a heterodimeric enzyme consisting of one α and one ß subunit. Each subunit consists of four domains: the N-terminal heme-nitric oxide oxygen binding (HNOX) domain, a PAS domain, a coiled-coil domain and the C-terminal catalytic domain. Upon activation by the endogenous ligand NO or activating drugs, NOsGC catalyses the conversion of GTP to cGMP. Although several crystal structures of the isolated domains are known, the structure of the full-length enzyme and the interdomain conformational changes during activation remain unsolved to date. In the current study, we performed protein thermal shift assays of purified NOsGC to identify discrete conformational states amenable to further analysis e.g. by crystallisation. A non-hydrolysable substrate analogue binding to the catalytic domain led to a subtle change in melting temperature. An activator drug binding to the HNOX domain led to a small increase. However, the combination of substrate analogue and activator drug led to a marked synergistic increase from 51⯰C to 60⯰C. This suggests reciprocal communication between HNOX domain and catalytic domain and formation of a stable activated conformation amenable to further biophysical characterization.
Asunto(s)
Benzoatos/farmacología , Activadores de Enzimas/farmacología , Guanosina Trifosfato/farmacología , Hemo/metabolismo , Guanilil Ciclasa Soluble/metabolismo , Sitios de Unión , Dominio Catalítico , Cloruros/farmacología , GMP Cíclico/metabolismo , Relación Dosis-Respuesta a Droga , Sinergismo Farmacológico , Activación Enzimática , Estabilidad de Enzimas , Guanosina Trifosfato/análogos & derivados , Guanosina Trifosfato/metabolismo , Hemo/química , Humanos , Compuestos de Manganeso/farmacología , Unión Proteica , Conformación Proteica , Desnaturalización Proteica , Guanilil Ciclasa Soluble/química , Guanilil Ciclasa Soluble/genética , Relación Estructura-Actividad , Temperatura de TransiciónRESUMEN
The release of nucleic acids and derivatives after tissue-injury may affect cellular immune-response. We studied the impact of extracellular ribo-, desoxyribonucleotides and nucleosides on T-cell immunity. Peripheral-blood-mononuclear-cells (PBMCs) or isolated CD3+T-cells obtained from 6 healthy donors were stimulated via CD3/CD28 Dynabeads or dendritic cells (DCs) in the presence or absence of pyrimidine-, purine-nucleotides and -nucleosides (range 2-200µM). Addition of deoxy-, guanosine-triphosphate (dGTP, GTP) and guanosine resulted concentration dependent in a complete, adenosine-triphosphate (ATP) in a partial inhibition of the induced T-cell-proliferation. Deoxyadenosine-triphosphate (dATP), adenosine and the pyrimidine-ribo- and -deoxyribonucleotides displayed no inhibitory capacity. Inhibitory effects of dGTP and GTP, but not of guanosine and ATP were culture-media-dependent and could be almost abrogated by use of the serum-free lymphocyte-culture-media X-Vivo15 instead of RPMI1640 with standard-supplementation. In contrast to RPMI1640, X-Vivo15 resulted in a significant down-regulation of the cell-surface-located ectonucleotidases CD39 (Ecto-Apyrase) and CD73 (Ecto-5'-Nucleotidase), critical for the extracellular nucleotides-hydrolysis to nucleosides, explaining the loss of inhibition mediated by dGTP and GTP, but not Guanosine. In line with previous findings ATP was found to exert immunosuppressive effects on T-cell-proliferation. Purine-nucleotides, dGTP and GTP displayed a higher inhibitory capacity, but seem to be strictly dependent on the microenvironmental conditions modulating the responsiveness of the respective T-lymphocytes. Further evaluation of experimental and respective clinical settings should anticipate these findings.