RESUMO
Chromosome conformation capture technologies have revealed important insights into genome folding. Yet, how spatial genome architecture is related to gene expression and cell fate remains unclear. We comprehensively mapped 3D chromatin organization during mouse neural differentiation in vitro and in vivo, generating the highest-resolution Hi-C maps available to date. We found that transcription is correlated with chromatin insulation and long-range interactions, but dCas9-mediated activation is insufficient for creating TAD boundaries de novo. Additionally, we discovered long-range contacts between gene bodies of exon-rich, active genes in all cell types. During neural differentiation, contacts between active TADs become less pronounced while inactive TADs interact more strongly. An extensive Polycomb network in stem cells is disrupted, while dynamic interactions between neural transcription factors appear in vivo. Finally, cell type-specific enhancer-promoter contacts are established concomitant to gene expression. This work shows that multiple factors influence the dynamics of chromatin interactions in development.
Assuntos
Cromatina/metabolismo , Genoma , Neurogênese , Animais , Fator de Ligação a CCCTC , Células-Tronco Embrionárias/metabolismo , Elementos Facilitadores Genéticos , Éxons , Expressão Gênica , Redes Reguladoras de Genes , Camundongos , Regiões Promotoras Genéticas , Proteínas Repressoras/metabolismo , Fatores de Transcrição/metabolismoRESUMO
Papain-like protease (PLpro), a non-structural protein encoded by SARS-CoV-2, is an important therapeutic target. Regions 1 and 5 of an existing drug, GRL0617, can be optimized to produce cooperativity with PLpro binding, resulting in stronger binding affinity. This work investigated the origin of the cooperativity using molecular dynamics simulations combined with the interaction entropy (IE) method. The regions' improvement exhibits cooperativity by calculating the binding free energies between the complex of PLpro-inhibitor. The thermodynamic integration method further verified the cooperativity generated in the drug improvement. To further determine the specific source of cooperativity, enthalpy and entropy in the complexes were calculated using molecular mechanics/generalized Born surface area and IE. The results show that the entropic change is an important contributor to the cooperativity. Our study also identified residues P248, Q269, and T301 that play a significant role in cooperativity. The optimization of the inhibitor stabilizes these residues and minimizes the entropic loss, and the cooperativity observed in the binding free energy can be attributed to the change in the entropic contribution of these residues. Based on our research, the application of cooperativity can facilitate drug optimization, and provide theoretical ideas for drug development that leverage cooperativity by reducing the contribution of entropy through multi-locus binding.
Assuntos
COVID-19 , SARS-CoV-2 , Humanos , Entropia , Simulação de Dinâmica MolecularRESUMO
Recent studies have shown that DNA methylation is an important epigenetic marker. Two prominent forms are methylation of the C5 position of cytosine and methylation of the C6 position of adenine. Given the vital significance of DNA methylation, investigating the mechanisms that influence protein binding remains a compelling pursuit. This study used molecular dynamics simulations to investigate the binding patterns of R2R3 protein and four differentially methylated DNAs. The alanine scanning combined with interaction entropy method was used to identify key residues that respond to different methylation patterns. The order of protein binding ability to DNA is as follows: unmethylated DNA > A11 methylation (5'-A6mAC-3') (6m2A system) > A10 methylation (5'-6mAAC-3') (6m1A system) > both A10 and A11 methylation (5'-6mA6mAC-3') (6mAA system) > C12 methylation (5'-AA5mC-3') (5mC system). All methylation systems lead to the sixth α helix (H6) (residues D105 to L116) moving away from the binding interface, and in the 5mC and 6m1A systems, the third α helix (H3) (residues G54 to L65) exhibits a similar trend. When the positively charged amino acids in H3 and H6 move away from the binding interface, their electrostatic and van der Waals interactions with the negatively charged DNA are weakened. Structural changes induced by methylation contributed to the destabilization of the hydrogen bond network near the original binding site, except for the 6m2A system. Moreover, there is a positive correlation between the number of methylated sites and the probability of distorting the DNA structure. Our study explores how different methylation patterns affect binding and structural adaptability, and have implications for drug discovery and understanding diseases related to abnormal methylation.
Assuntos
5-Metilcitosina , DNA , Cinética , AdeninaRESUMO
Human trophoblast cell apoptosis may induce miscarriage. Trophoblast cells are sensitive to environmental BaP-7,8-dihydrodiol-9,10-epoxide (BPDE). However, how BPDE induces human trophoblast cell apoptosis is still largely elusive. In this work, we used BPDE-treated human trophoblast cells and villous tissues collected from recurrent miscarriage and health control groups to explore the underlying mechanism of BPDE-induced human trophoblast cell apoptosis. Continued with our recent work, we found that lncRNA HZ01 (lnc-HZ01) could induce human trophoblast cell apoptosis. In mechanism, lnc-HZ01 up-regulated p53 expression level by suppressing its MDM2-mediated proteasomal degradation. Meanwhile, we found that p53 acted as lnc-HZ01 transcription factor and promoted lnc-HZ01 transcription. Thus, lnc-HZ01 and p53 composed a positive feedback loop in human trophoblast cells. In normal trophoblast cells, relatively low levels of lnc-HZ01 and p53 suppressed p53/caspase-3 apoptosis pathway, giving normal pregnancy. Upon BPDE exposure, BPDE up-regulated the expression levels of lnc-HZ01 and p53, triggered this positive feedback loop, activated the p53/caspase-3 apoptosis pathway, and then induced miscarriage. Collectively, we discovered new mechanism by which lnc-HZ01 regulated BPDE-induced human trophoblast cell apoptosis, providing scientific basis for the diagnosis and treatment of unexplained recurrent miscarriage.
Assuntos
Aborto Habitual , RNA Longo não Codificante , 7,8-Di-Hidro-7,8-Di-Hidroxibenzo(a)pireno 9,10-óxido/metabolismo , 7,8-Di-Hidro-7,8-Di-Hidroxibenzo(a)pireno 9,10-óxido/toxicidade , Aborto Habitual/induzido quimicamente , Aborto Habitual/metabolismo , Apoptose , Caspase 3/metabolismo , Retroalimentação , Feminino , Humanos , Gravidez , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Trofoblastos/metabolismo , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismoRESUMO
A novel function of retinoid X receptor beta (RXRß) in endothelial cells has been reported by us during the formation of atherosclerosis. Here, we extended the study to explore the cellular mechanisms of RXRß protein stability regulation. In this study, we discovered that murine double minute-2 (MDM2) acts as an E3 ubiquitin ligase to target RXRß for degradation. The result showed that MDM2 directly interacted with and regulated RXRß protein stability. MDM2 promoted RXRß poly-ubiquitination and degradation by proteasomes. Moreover, mutated MDM2 RING domain (C464A) or treatment with an MDM2 inhibitor targeting the RING domain of MDM2 lost the ability of MDM2 to regulate RXRß protein expression and ubiquitination. Furthermore, treatment with MDM2 inhibitor alleviated oxidized low-density lipoprotein-induced mitochondrial damage, activation of TLR9/NF-κB and NLRP3/caspase-1 pathway and production of pro-inflammatory cytokines in endothelial cells. However, all these beneficial effects were reduced by the transfection of RXRß siRNA. Moreover, pharmacological inhibition of MDM2 attenuated the development of atherosclerosis and reversed mitochondrial damage and related inflammation in the atherosclerotic process in LDLr-/- mice, along with the increased RXRß protein expression in the aorta. Therefore, our study uncovers a previously unknown ubiquitination pathway and suggests MDM2-mediated RXRß ubiquitination as a new therapeutic target in atherosclerosis.
Assuntos
Aterosclerose , Proteínas Proto-Oncogênicas c-mdm2 , Animais , Aterosclerose/genética , Células Endoteliais/metabolismo , Inflamação/genética , Camundongos , Proteínas Proto-Oncogênicas c-mdm2/genética , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Proteína Supressora de Tumor p53/metabolismo , UbiquitinaçãoRESUMO
The compound, 2,3,5,4'-tetrahydroxystilbene-2-O-ß-d-glucoside (TSG), a primary bioactive polyphenolic component of Polygonum multiflorum exerts numerous pharmacological activities. However, its protective effect against non-alcoholic steatohepatitis (NASH), in the context of metabolic syndrome, remains poorly understood. The aim of the present study is to evaluate the effects of TSG treatment on middle-aged (12-mo-old) male LDLr-/- mice, which were fed a high fat diet for 12 weeks to induce metabolic syndrome and NASH. At the end of the experiment, the blood samples of mice were collected for determination of metabolic parameters. Liver and aorta tissues were collected for analysis, such as histology, immunofluorescence, hepatic lipid content, real-time PCR, and western blot. Our data show that TSG treatment improved the different aspects of NASH (steatosis, inflammation, and fibrosis) and atherosclerosis, as well as some of the metabolic basal characteristics. These modulatory effects of TSG are mediated, at least in part, through regulating key regulators of lipid metabolism (SREBP1c, PPARα and their target genes, ABCG5 and CYP7A1), inflammation (CD68, TNF-α, IL-6 and ICAM), fibrosis (α-SMA and TNFß) and oxidative stress (NADPH-oxidase 2/4, CYP2E1 and antioxidant enzymes). These results suggest that TSG may be a promising candidate for preventing and treating the progression of NASH.
Assuntos
Envelhecimento/patologia , Aterosclerose/tratamento farmacológico , Glucosídeos/uso terapêutico , Hepatopatia Gordurosa não Alcoólica/tratamento farmacológico , Estilbenos/uso terapêutico , Animais , Aorta/efeitos dos fármacos , Aorta/metabolismo , Aterosclerose/etiologia , Dieta Hiperlipídica/efeitos adversos , Glucosídeos/farmacologia , Metabolismo dos Lipídeos , Fígado/efeitos dos fármacos , Fígado/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Hepatopatia Gordurosa não Alcoólica/etiologia , Receptores de LDL/genética , Estilbenos/farmacologiaRESUMO
KEY POINTS: Abnormal mitochondrial morphology and function in cardiomyocytes are frequently observed under persistent Gq protein-coupled receptor (Gq PCR) stimulation. Cardiac signalling mechanisms for regulating mitochondrial morphology and function under pathophysiological conditions in the heart are still poorly understood. We demonstrate that a downstream kinase of Gq PCR, protein kinase D (PKD) induces mitochondrial fragmentation via phosphorylation of dynamin-like protein 1 (DLP1), a mitochondrial fission protein. The fragmented mitochondria enhance reactive oxygen species generation and permeability transition pore opening in mitochondria, which initiate apoptotic signalling activation. This study identifies a novel PKD-specific substrate in cardiac mitochondria and uncovers the role of PKD on cardiac mitochondria, with special emphasis on the molecular mechanism(s) underlying mitochondrial injury with abnormal mitochondrial morphology under persistent Gq PCR stimulation. These findings provide new insights into the molecular basis of cardiac mitochondrial physiology and pathophysiology, linking Gq PCR signalling with the regulation of mitochondrial morphology and function. ABSTRACT: Regulation of mitochondrial morphology is crucial for the maintenance of physiological functions in many cell types including cardiomyocytes. Small and fragmented mitochondria are frequently observed in pathological conditions, but it is still unclear which cardiac signalling pathway is responsible for regulating the abnormal mitochondrial morphology in cardiomyocytes. Here we demonstrate that a downstream kinase of Gq protein-coupled receptor (Gq PCR) signalling, protein kinase D (PKD), mediates pathophysiological modifications in mitochondrial morphology and function, which consequently contribute to the activation of apoptotic signalling. We show that Gq PCR stimulation induced by α1 -adrenergic stimulation mediates mitochondrial fragmentation in a fission- and PKD-dependent manner in H9c2 cardiac myoblasts and rat neonatal cardiomyocytes. Upon Gq PCR stimulation, PKD translocates from the cytoplasm to the outer mitochondrial membrane (OMM) and phosphorylates a mitochondrial fission protein, dynamin-like protein 1 (DLP1), at S637. PKD-dependent phosphorylation of DLP1 initiates DLP1 association with the OMM, which then enhances mitochondrial fragmentation, mitochondrial superoxide generation, mitochondrial permeability transition pore opening and apoptotic signalling. Finally, we demonstrate that DLP1 phosphorylation at S637 by PKD occurs in vivo using ventricular tissues from transgenic mice with cardiac-specific overexpression of constitutively active Gαq protein. In conclusion, Gq PCR-PKD signalling induces mitochondrial fragmentation and dysfunction via PKD-dependent DLP1 phosphorylation in cardiomyocytes. This study is the first to identify a novel PKD-specific substrate, DLP1 in mitochondria, as well as the functional role of PKD in cardiac mitochondria. Elucidation of these molecular mechanisms by which PKD-dependent enhanced fission mediates cardiac mitochondrial injury will provide novel insight into the relationship among mitochondrial form, function and Gq PCR signalling.
Assuntos
Dinaminas/metabolismo , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/metabolismo , Mitocôndrias/patologia , Dinâmica Mitocondrial , Miócitos Cardíacos/patologia , Proteína Quinase C/metabolismo , Animais , Camundongos , Camundongos Transgênicos , Mitocôndrias/metabolismo , Proteínas de Transporte da Membrana Mitocondrial , Poro de Transição de Permeabilidade Mitocondrial , Miócitos Cardíacos/metabolismo , Fosforilação , Ratos , Ratos Sprague-Dawley , Transdução de SinaisRESUMO
Despite a general view that asparagine synthetase generates asparagine as an amino acid for long-distance transport of nitrogen to sink organs, its role in nitrogen metabolic pathways in floral organs during seed nitrogen filling has remained undefined. We demonstrate that the onset of pollination in Arabidopsis induces selected genes for asparagine metabolism, namely ASN1 (At3g47340), GLN2 (At5g35630), GLU1 (At5g04140), AapAT2 (At5g19950), ASPGA1 (At5g08100) and ASPGB1 (At3g16150), particularly at the ovule stage (stage 0), accompanied by enhanced asparagine synthetase protein, asparagine and total amino acids. Immunolocalization confined asparagine synthetase to the vascular cells of the silique cell wall and septum, but also to the outer and inner seed integuments, demonstrating the post-phloem transport of asparagine in these cells to developing embryos. In the asn1 mutant, aberrant embryo cell divisions in upper suspensor cell layers from globular to heart stages assign a role for nitrogen in differentiating embryos within the ovary. Induction of asparagine metabolic genes by light/dark and nitrate supports fine shifts of nitrogen metabolic pathways. In transgenic Arabidopsis expressing promoterCaMV35S ::ASN1 fusion, marked metabolomics changes at stage 0, including a several-fold increase in free asparagine, are correlated to enhanced seed nitrogen. However, specific promoterNapin2S ::ASN1 expression during seed formation and a six-fold increase in asparagine toward the desiccation stage result in wild-type seed nitrogen, underlining that delayed accumulation of asparagine impairs the timing of its use by releasing amide and amino nitrogen. Transcript and metabolite profiles in floral organs match the carbon and nitrogen partitioning to generate energy via the tricarboxylic acid cycle, GABA shunt and phosphorylated serine synthetic pathway.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimologia , Arabidopsis/metabolismo , Aspartato-Amônia Ligase/metabolismo , Nitrogênio/metabolismo , Sementes/enzimologia , Sementes/metabolismo , Aminoácidos/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Aspartato-Amônia Ligase/genética , Regulação da Expressão Gênica de Plantas/genética , Regulação da Expressão Gênica de Plantas/fisiologia , Floema/enzimologia , Floema/genética , Floema/metabolismo , Plantas Geneticamente Modificadas/enzimologia , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/metabolismo , Sementes/genéticaRESUMO
The aim of this study was to evaluate the potential effects of 2,3,4',5-tetrahydroxystilbene-2-O-ß-d-glucoside (TSG) on the development of atherosclerotic plaque in ApoE-/- mice, and explore the mechanisms involved. Our data showed that after 8 weeks of treatment, TSG ameliorated serum levels of total cholesterol, triglyceride, and low density lipoprotein cholesterol, and increased serum levels of high density lipoprotein cholesterol in ApoE-/- mice. TSG suppressed hepatic steatosis, the formation of atherosclerotic lesions, and the formation of macrophage foam cells in ApoE-/- mice. Moreover, TSG improved the expressions of hepatic SR-BI, ABCG5, and CYP7A1, and up-regulated the protein expressions of aortic ABCA1 and ABCG1. An in-vitro study showed that TSG promoted macrophage cholesterol efflux and increased the protein expressions of ABCA1 and ABCG1. Our findings provide evidence for a positive role of TSG in preventing atherosclerosis by promoting reverse cholesterol transport. These effects may be achieved by stimulating cholesterol efflux through ABCA1 and ABCG1, promoting SR-BI-mediated cholesterol uptake in the liver, increasing secretion of cholesterol into bile by ABCG5, and improving cholesterol metabolism by the CYP7A1 pathway. In addition, antioxidative and anti-inflammatory effects of TSG may also contribute to its inhibitory effects on atherosclerosis. Further study is needed to investigate whether other potential mechanisms are involved in TSG-mediated atheroprotection.
Assuntos
Apolipoproteínas E/deficiência , Aterosclerose/tratamento farmacológico , Aterosclerose/metabolismo , Colesterol/metabolismo , Glucosídeos/uso terapêutico , Estilbenos/uso terapêutico , Animais , Aterosclerose/complicações , Aterosclerose/patologia , Transporte Biológico/efeitos dos fármacos , Fígado Gorduroso/complicações , Fígado Gorduroso/tratamento farmacológico , Células Espumosas/efeitos dos fármacos , Células Espumosas/metabolismo , Glucosídeos/farmacologia , Hiperlipidemias/complicações , Hiperlipidemias/tratamento farmacológico , Hiperlipidemias/metabolismo , Inflamação/complicações , Inflamação/patologia , Metabolismo dos Lipídeos/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Estresse Oxidativo/efeitos dos fármacos , Células RAW 264.7 , Estilbenos/farmacologiaRESUMO
Background Two pivotal Phase III trials compared the efficacy of palonosetron, ondansetron and granisetron, combined with dexamethasone, for the prevention of nausea and vomiting following highly emetogenic chemotherapy. However, an economic evaluation of these three regimens in the real-world setting of Chinese adult patients has not been determined. Objectives To estimate, from the perspective of the Chinese healthcare system, which of these frequently used strategies consisting of 0.25 mg palonosetron (0.25P), 16 mg ondansetron (Onda), and 3 mg granisetron (Gran), is the most cost-effective option in patients following highly emetogenic chemotherapy. Methods A Markov decision-analytic model was developed. The health and economic outcomes of the three strategies; 0.25P, Onda, and Gran were investigated. The clinical and utility data were taken from published studies. The cost data were calculated according to current local Chinese practices. Sensitivity analyses were performed to determine the impact of uncertainty regarding the results. Results The base-case analysis showed that the 0.25P strategy yielded maximum health benefits compared with the other two strategies. However, the probabilistic sensitivity analysis demonstrated that the Gran strategy was the most cost-effective approach when the willingness-to-pay threshold was not more than US$22,515/quality-adjusted life year. Moreover, palonosetron is not cost-effective in preventing 'overall' nausea and vomiting following highly emetogenic chemotherapy in Chinese patients. Conclusions Our analysis suggests that, compared with palonosetron and ondansetron, 3 mg granisetron may be a cost-effective treatment option in the current Chinese healthcare setting.
Assuntos
Antieméticos/administração & dosagem , Dexametasona/administração & dosagem , Náusea/prevenção & controle , Vômito/prevenção & controle , Adulto , Antieméticos/economia , Antieméticos/uso terapêutico , Antineoplásicos/efeitos adversos , Povo Asiático , Ensaios Clínicos Fase III como Assunto , Análise Custo-Benefício , Granisetron/administração & dosagem , Humanos , Isoquinolinas/administração & dosagem , Náusea/induzido quimicamente , Ondansetron/efeitos adversos , Palonossetrom , Quinuclidinas/administração & dosagem , Ensaios Clínicos Controlados Aleatórios como Assunto , Receptores 5-HT3 de Serotonina/efeitos dos fármacos , Vômito/induzido quimicamenteRESUMO
Maspin, a non-inhibitory member of serine protease family, acts as an effective tumor suppressor by inhibiting cell inhesion and mobility. We found that exogenous wild-type rMaspin had a low effect on tumor growth in vivo. However, when the peptide Arg-Gly-Asp-hexahistidine (RGD-6His) was introduced into rMaspin, the modified rMaspin showed significant inhibitory activity in angiogenic assays and tumor-bearing animal models. Overall, our data suggested that both the RGD and hexahistidine fragments contributed to improve the fusion protein activity and polyhistidine peptide could be considered as flexible linker to separate RGD and Maspin moieties to avoid function interference. Besides, it is an efficient tag to achieve purified recombinant proteins. Furthermore, rMaspin fusing with RGD and hexahistidine could be a viable anticancer candidate.
Assuntos
Antineoplásicos/farmacologia , Histidina/farmacologia , Oligopeptídeos/farmacologia , Serpinas/farmacologia , Inibidores da Angiogênese/química , Inibidores da Angiogênese/farmacologia , Animais , Antineoplásicos/química , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Escherichia coli/genética , Escherichia coli/metabolismo , Feminino , Regulação da Expressão Gênica , Genes Supressores de Tumor , Histidina/química , Células Endoteliais da Veia Umbilical Humana , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Neoplasias/tratamento farmacológico , Oligopeptídeos/química , Proteínas Recombinantes/química , Proteínas Recombinantes/farmacologia , Serpinas/química , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
BACKGROUND/AIMS: salusin-ß is considered to be a potential pro-atherosclerotic factor. Regulation and function of vascular smooth muscle cells (VSMCs) are important in the progression of atherosclerosis. Peroxisome proliferator-activated receptor gamma (PPARγ) exerts a vascular protective role beyond its metabolic effects. Salusin-ß has direct effects on VSMCs. The aim of the present study was to assess the effect of salusin-ß on PPARγ gene expression in primary cultured rat VSMCs. METHODS: Western blotting analysis, real-time PCR and transient transfection approach were used to determine expression of target proteins. Specific protein knockdown was performed with siRNA transfection. Cell proliferation was determined by 5-bromo-2'-deoxyuridine incorporation. The levels of inflammation indicators interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) were determined using enzyme-linked immunosorbent assay. RESULTS: Salusin-ß negatively regulated PPARγ gene expression at protein, mRNA and gene promoter level in VSMCs. The inhibitory effect of salusin-ß on PPARγ gene expression contributed to salusin-ß-induced VSMCs proliferation and inflammation in vitro. IκBα-NF-κB activation, but not NF-κB p50 or p65, mediated the salusin-ß-induced inhibition of PPARγ gene expression. Salusin-ß induced nuclear translocation of histone deacetylase 3 (HDAC3). HDAC3 siRNA prevented salusin-ß-induced PPARγ reduction. Nuclear translocation of HDAC3 in response to salusin-ß was significantly reversed by an IκBα inhibitor BAY 11-7085. Furthermore, IκBα-HDAC3 complex was present in the cytosol of VSMCs but interrupted after salusin-ß treatment. CONCLUSION: IκBα-HDAC3 pathway may contribute to salusin-ß-induced inhibition of PPARγ gene expression in VSMCs.
Assuntos
Regulação da Expressão Gênica/efeitos dos fármacos , Peptídeos e Proteínas de Sinalização Intercelular/farmacologia , Músculo Liso Vascular/citologia , Miócitos de Músculo Liso/metabolismo , PPAR gama/genética , Animais , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Proliferação de Células/efeitos dos fármacos , Regulação para Baixo/efeitos dos fármacos , Inibidores de Histona Desacetilases/farmacologia , Histona Desacetilases/metabolismo , Proteínas I-kappa B/metabolismo , Inflamação/genética , Inflamação/patologia , Ligantes , Masculino , Miócitos de Músculo Liso/efeitos dos fármacos , Inibidor de NF-kappaB alfa , NF-kappa B/metabolismo , PPAR gama/metabolismo , Prostaglandina D2/análogos & derivados , Prostaglandina D2/farmacologia , Transporte Proteico/efeitos dos fármacos , RNA Interferente Pequeno/metabolismo , Ratos Sprague-DawleyRESUMO
Despite the clear mitogenic effect of salusin-ß on vascular smooth muscle cells (VSMCs), which contributes to its proatherosclerotic effects, additional studies are needed to explore its underlying mechanisms. The aim of this study was to investigate the mechanism of salusin-ß's effects on VSMCs cell cycle regulation and the possible signal pathways. Salusin-ß accelerated the G1/S phase transition in VSMCs and increased the expression levels of cyclins D1 and E. Silencing either cyclin D1 or cyclin E gene inhibited salusin-ß-induced VSMCs proliferation, cell cycle progression, phosphorylation of the Rb protein, and dissociation of the E2F-Rb complex. Importantly, expression of cyclin E was also induced by cyclin D1. Next, we found that salusin-ß increased the protein expressions of activator protein-1 (AP-1) subunits c-Jun and c-Fos, and enhanced binding of AP-1 to the promoter region of cyclin D1. In addition, inhibition of AP-1 activity could lead to significant suppression of salusin-ß-induced cyclin D1 expression. Furthermore, MPAKs pathways were found to mediate salusin-ß-induced VSMCs proliferation, cyclin D1, cyclin E, c-Jun, and c-Fos expression. These results suggest that salusin-ß promotes cell cycle progression of VSMCs by upregulating the cyclin D1 and cyclin E, in an AP-1-dependent manner through mitogen-activated protein kinases signaling pathways.
Assuntos
Proliferação de Células/fisiologia , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Músculo Liso Vascular/fisiologia , Miócitos de Músculo Liso/fisiologia , Animais , Ciclo Celular/fisiologia , Células Cultivadas , Ciclina D1/metabolismo , Ciclina E/metabolismo , Fator Promotor de Maturação/metabolismo , Ratos , Transdução de SinaisRESUMO
This paper investigates the finite-time consensus problem of leader-following multiagent systems. The dynamical models for all following agents and the leader are assumed the same general form of linear system, and the interconnection topology among the agents is assumed to be switching and undirected. We mostly consider the continuous-time case. By assuming that the states of neighbouring agents are known to each agent, a sufficient condition is established for finite-time consensus via a neighbor-based state feedback protocol. While the states of neighbouring agents cannot be available and only the outputs of neighbouring agents can be accessed, the distributed observer-based consensus protocol is proposed for each following agent. A sufficient condition is provided in terms of linear matrix inequalities to design the observer-based consensus protocol, which makes the multiagent systems achieve finite-time consensus under switching topologies. Then, we discuss the counterparts for discrete-time case. Finally, we provide an illustrative example to show the effectiveness of the design approach.
Assuntos
Técnicas de Apoio para a Decisão , Reconhecimento Automatizado de Padrão/métodos , Inteligência Artificial , Retroalimentação , Modelos Teóricos , Processamento de Sinais Assistido por ComputadorRESUMO
The design of small molecule inhibitors that target the programmed death ligand-1 (PD-L1) is a forefront issue in immune checkpoint blocking therapy. Small-molecule inhibitors have been shown to exert therapeutic effects by inducing dimerization of the PD-L1 protein, however, the specific mechanisms underlying this dimerization process remain largely unexplored. Furthermore, there is a notable lack of comparative studies examining the binding modes of structurally diverse inhibitors. In view of the research gaps, this work employed molecular dynamics simulations to meticulously examine the interactions between two distinct types of inhibitors and PD-L1 in both monomeric and dimeric forms, and predicted the dimerization mechanism. The results revealed that inhibitors initially bind to a PD-L1 monomer, subsequently attracting another monomer to form a dimer. Notably, symmetric inhibitors observed superior binding efficiency compared to other inhibitors. Key residues, including Ile54, Tyr56, Met115 and Tyr123 played a leading role in binding. Structurally, symmetric inhibitors were capable of thoroughly engaging the binding pocket, promoting a more symmetrical formation of PD-L1 dimers. Furthermore, symmetric inhibitors formed more extensive hydrophobic interactions with protein residues. The insights garnered from this research are expected to significantly contribute to the rational design and optimization of small molecule inhibitors targeting PD-L1.
Assuntos
Antígeno B7-H1 , Receptor de Morte Celular Programada 1 , Dimerização , Antígeno B7-H1/metabolismo , Receptor de Morte Celular Programada 1/metabolismo , Bibliotecas de Moléculas Pequenas/química , Simulação de Dinâmica MolecularRESUMO
DNA methylation as an important epigenetic marker, has gained attention for the significance of three oxidative modifications (hydroxymethyl-C (hmC), formyl-C (fC), and carboxyl-C (caC)). Mutations occurring in the methyl-CpG-binding domain (MBD) of MeCP2 result in Rett. However, uncertainties persist regarding DNA modification and MBD mutation-induced interaction changes. Here, molecular dynamics simulations were used to investigate the underlying mechanisms behind changes due to different modifications of DNA and MBD mutations. Alanine scanning combined with the interaction entropy method was employed to accurately evaluate the binding free energy. The results show that MBD has the strongest binding ability for mCDNA, followed by caC, hmC, and fCDNA, with the weakest binding ability observed for CDNA. Further analysis revealed that mC modification induces DNA bending, causing residues R91 and R162 closer to the DNA. This proximity enhances van der Waals and electrostatic interactions. Conversely, the caC/hmC and fC modifications lead to two loop regions (near K112 and K130) closer to DNA, respectively. Furthermore, DNA modifications promote the formation of stable hydrogen bond networks, however mutations in the MBD significantly reduce the binding free energy. This study provides detailed insight into the effects of DNA modifications and MBD mutations on binding ability. It emphasizes the necessity for research and development of targeted Rett compounds that induce conformational compatibility between MBD and DNA, enhancing the stability and strength of their interactions.
Assuntos
Síndrome de Rett , Humanos , Síndrome de Rett/genética , Síndrome de Rett/metabolismo , Proteína 2 de Ligação a Metil-CpG/química , DNA/química , Mutação , Metilação de DNA , Ligação ProteicaRESUMO
OBJECTIVES: This study was to explore the potential effects and mechanism of naringenin against vascular senescence in atherosclerosis focusing on the SIRT1-mediated signalling pathway. METHODS: Aged apoE-/- mice were administrated with naringenin continuously for three months. Lipid parameters in serum and pathological changes and associated protein expression in aorta were examined. In vitro, endothelial cells were treated with H2O2 to induce senescence. KEY FINDINGS: Dyslipidemia, atherosclerotic lesion formation and vascular senescence were found in ApoE-/- mice, which were significantly ameliorated by naringenin treatment. Naringenin decreased reactive oxygen species overproduction and enhanced the activities of antioxidant enzymes in aorta. It also decreased mitoROS production and increased protein expressions of mitochondrial biogenesis-related genes in aorta. Moreover, naringenin treatment enhanced aortic protein expression and activity of SIRT1. Meanwhile, naringenin increased deacetylation and protein expression of SIRT1's target genes FOXO3a and PGC1α. In vitro study, the benefits of naringenin on endothelial senescence, oxidative stress and mitochondrial injury as well as protein expressions and acetylated levels of FOXO3a and PGC1α were diminished in cells transfected with SIRT1 siRNA. CONCLUSIONS: Naringenin could ameliorate vascular senescence and atherosclerosis and the activation of SIRT1, with subsequent deacetylation and regulation of FOXO3a and PGC1α, is involved in this process.
Assuntos
Aterosclerose , Células Endoteliais , Camundongos , Animais , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/metabolismo , Células Endoteliais/metabolismo , Sirtuína 1/metabolismo , Peróxido de Hidrogênio , Camundongos Knockout para ApoE , Aterosclerose/metabolismo , Estresse Oxidativo , Apolipoproteínas E/metabolismo , Senescência CelularRESUMO
Environmental benzo(a)pyrene (BaP) and its ultimate metabolite BPDE (benzo(a)pyrene-7,8-dihydrodiol-9,10-epoxide) are universal and inevitable persistent organic pollutants and endocrine disrupting chemicals. Angiogenesis in placental decidua plays a pivotal role in healthy pregnancy. Ferroptosis is a newly identified and iron-dependent cell death mode. However, till now, BaP/BPDE exposure, ferroptosis, defective angiogenesis, and miscarriage have never been correlated; and their regulatory mechanisms have been rarely explored. In this study, we used assays with BPDE-exposed HUVECs (human umbilical vein endothelial cells), decidual tissues and serum samples collected from unexplained recurrent miscarriage and their matched healthy control groups, and placental tissues of BaP-exposed mouse miscarriage model. We found that BaP/BPDE exposure caused ferroptosis and then directly suppressed angiogenesis and eventually induced miscarriage. In mechanism, BaP/BPDE exposure up-regulated free Fe2+ level and promoted lipid peroxidation and also up-regulated MARCHF1 (a novel E3 ligase of GPX4) level to promote the ubiquitination degradation of GPX4, both of which resulted in HUVEC ferroptosis. Furthermore, we also found that GPX4 protein down-regulated the protein levels of VEGFA and ANG-1, two key proteins function for angiogenesis, and thus suppressed HUVEC angiogenesis. In turn, supplement with GPX4 could suppress ferroptosis, recover angiogenesis, and alleviate miscarriage. Moreover, the levels of free Fe2+ and VEGFA in serum might predict the risk of miscarriage. Overall, this study uncovered the crosstalk among BaP/BPDE exposure, ferroptosis, angiogenesis, and miscarriage, discovering novel toxicological effects of BaP/BPDE on human reproductive health. This study also warned the public to avoid exposure to polycyclic aromatic hydrocarbons during pregnancy to effectively prevent adverse pregnancy outcomes.
Assuntos
Aborto Espontâneo , Ferroptose , Camundongos , Animais , Gravidez , Humanos , Feminino , 7,8-Di-Hidro-7,8-Di-Hidroxibenzo(a)pireno 9,10-óxido/metabolismo , 7,8-Di-Hidro-7,8-Di-Hidroxibenzo(a)pireno 9,10-óxido/farmacologia , Benzo(a)pireno , Células Endoteliais/metabolismo , Placenta/metabolismo , ProteínasRESUMO
Environmental Benzo(a)pyrene (BaP) and its ultimate metabolite BPDE (benzo(a)pyrene-7,8-dihydrodiol-9,10-epoxide) are typical persistent organic pollutants and endocrine disrupting chemicals. BaP/BPDE exposure might cause human trophoblast cell dysfunctions and induce miscarriage. However, the underlying mechanisms remain largely elusive. In this study, we found that BPDE exposure induced human trophoblast cell pyroptosis by up-regulating NLRP3/Caspase1/GSDMD pathway. We also identified that lnc-HZ14 was highly expressed in BPDE-exposed trophoblast cells and in recurrent miscarriage (RM) vs healthy control (HC) villous tissues. Lnc-HZ14 promoted trophoblast cell pyroptosis by promoting IRF1-mediated ZBP1 transcription, increasing METTL3-mediated m6A methylation on NLRP3 mRNA and its stability, and also enhancing ZBP1/NLRP3 protein interactions. Knockdown of lnc-HZ14/ZBP1/NLRP3 axis could efficiently alleviate BPDE-induced trophoblast cell pyroptosis. Higher level of pyroptosis, as indicated by the up-regulation of lnc-HZ14/ZBP1/NLRP3 axis, was found in RM vs HC villous tissues. In BaP-exposed mouse model, BaP exposure induced placental tissue pyroptosis and miscarriage by up-regulating murine Zbp1/Nlrp3 axis, and knockdown of Nlrp3 could efficiently reduce placenta pyroptosis and alleviate BaP-induced mouse miscarriage. Serum IL-1ß protein level might act as a promising indicator to predict the risk of miscarriage. These findings provided new insights into BaP/BPDE-induced trophoblast cell pyroptosis and miscarriage and might be helpful for further assessment of the toxicological effects of BaP/BPDE on the female reproduction.
Assuntos
7,8-Di-Hidro-7,8-Di-Hidroxibenzo(a)pireno 9,10-óxido , Aborto Espontâneo , Gravidez , Humanos , Feminino , Camundongos , Animais , Trofoblastos , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Piroptose , Aborto Espontâneo/induzido quimicamente , Aborto Espontâneo/metabolismo , Benzo(a)pireno/metabolismo , Placenta/metabolismo , Metiltransferases/metabolismo , Proteínas de Ligação a RNA/metabolismo , Proteínas de Ligação a RNA/farmacologiaRESUMO
The proliferation of vascular smooth muscle cells (VSMCs) induced by injury to the intima of arteries is an important etiologic factor in vascular proliferative disorders such as atherosclerosis and restenosis. 2,3,4',5-Tetrahydroxystilbene-2-O-ß-D-glucoside (TSG), an active component extracted from Polygonum multiflorum, has been found to have an antiatherosclerotic effect. The aim of this study was to investigate the effects of TSG on platelet derived growth factor (PDGF)-BB induced VSMCs proliferation and to explore the possible mechanisms of such effects. Pretreatment of VSMCs with TSG significantly inhibited PDGF-BB-induced cell proliferation in a concentration-dependent but not time-dependent manner. In addition, flow cytometry analysis of the DNA content revealed blocking of the PDGF-BB-inducible cell cycle progression by TSG. On the contrary, an inhibitory effect of TSG on VSMCs proliferation and expression of cell cycle regulators were markedly attenuated by addition of an nitric oxide (NO) synthase inhibitor, a soluble guanylate cyclase inhibitor and a cyclic GMP (cGMP)-dependent protein kinase (PKG) inhibitor: N(G)-nitro-L-arginine methyl ester (L-NAME), 1H-[1,2,4] oxadiazolo [4,3-α] quinoxalin-1-one (ODQ) and KT5823, respectively. It was also demonstrated that TSG enhanced NO and cGMP formation through up-regulating endothelial NO synthase expression in VSMCs. The findings indicate that TSG inhibited VSMCs proliferation induced by PDGF-BB may involve the NO/cGMP/PKG signal pathway.