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Microfibril-associated glycoprotein-1 (MAGP1), a crucial extracellular matrix protein, contributes to the initiation and progression of different cancers. However, the role of MAGP1 in laryngeal cancer is not clear. The purpose of this study was to investigate the clinical significance and biological function of MAGP1 in laryngeal cancer. MAGP1 was upregulated in public databases and laryngeal cancer tissues, and high MAGP1 expression led to a poor prognosis and was identified as an independent prognostic marker. Knocking-down MAGP1 inhibited laryngeal cancer cell growth and metastasis. According to gene set enrichment analysis, high MAGP1 expression revealed enrichment in Wnt/ß-catenin signaling and knocking-down MAGP1 in laryngeal cancer cells also caused degradation, de-activation, re-location and loss of stability of ß-catenin. Additionally, we observed MAGP1 in laryngeal cancer cells inhibits angiogenesis in an MMP7-dependent way. In conclusion, our study suggests a clinical role of MAGP1 in laryngeal cancer, signifying its potential as a therapeutic target in the future.
Assuntos
Neoplasias Laríngeas , beta Catenina , Humanos , Angiogênese/metabolismo , beta Catenina/genética , beta Catenina/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Regulação Neoplásica da Expressão Gênica , Glicoproteínas/genética , Neoplasias Laríngeas/genética , Neoplasias Laríngeas/metabolismo , Metaloproteinase 7 da Matriz/genética , Metaloproteinase 7 da Matriz/metabolismo , Via de Sinalização WntRESUMO
Dilated cardiomyopathy (DCM) is a primary cause of heart failure (HF), with the incidence of HF increasing consistently in recent years. DCM pathogenesis involves a combination of inherited predisposition and environmental factors. Endocrine-disrupting chemicals (EDCs) are exogenous chemicals that interfere with endogenous hormone action and are capable of targeting various organs, including the heart. However, the impact of these disruptors on heart disease through their effects on genes remains underexplored. In this study, we aimed to explore key DCM-related genes using machine learning (ML) and the construction of a predictive model. Using the Gene Expression Omnibus (GEO) database, we screened differentially expressed genes (DEGs) and performed enrichment analyses of Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways related to DCM. Through ML techniques combining maximum relevance minimum redundancy (mRMR) and least absolute shrinkage and selection operator (LASSO) logistic regression, we identified key genes for predicting DCM (IL1RL1, SEZ6L, SFRP4, COL22A1, RNASE2, HB). Based on these key genes, 79 EDCs with the potential to affect DCM were identified, among which 4 (3,4-dichloroaniline, fenitrothion, pyrene, and isoproturon) have not been previously associated with DCM. These findings establish a novel relationship between the EDCs mediated by key genes and the development of DCM.
Assuntos
Cardiomiopatia Dilatada , Disruptores Endócrinos , Cardiopatias , Humanos , Coração , Biologia Computacional , Disruptores Endócrinos/toxicidade , Aprendizado de MáquinaRESUMO
BACKGROUND: Yeast is often used to build cell factories to produce various chemicals or nutrient substances, which means the yeast has to encounter stressful environments. Previous research reported that unsaturated fatty acids were closely related to yeast stress resistance. Engineering unsaturated fatty acids may be a viable strategy for enhancing the stress resistance of cells. RESULTS: In this study, two desaturase genes, OLE1 and FAD2 from Z. rouxii, were overexpressed in S. cerevisiae to determine how unsaturated fatty acids affect cellular stress tolerance of cells. After cloning and plasmid recombination, the recombinant S. cerevisiae cells were constructed. Analysis of membrane fatty acid contents revealed that the recombinant S. cerevisiae with overexpression of OLE1 and FAD2 genes contained higher levels of fatty acids C16:1 (2.77 times), C18:1 (1.51 times) and C18:2 (4.15 times) than the wild-type S. cerevisiae pY15TEF1. In addition, recombinant S. cerevisiae cells were more resistant to multiple stresses, and exhibited improved membrane functionality, including membrane fluidity and integrity. CONCLUSION: These findings demonstrated that strengthening the expression of desaturases was beneficial to stress tolerance. Overall, this study may provide a suitable means to build a cell factory of industrial yeast cells with high tolerance during biological manufacturing. © 2023 Society of Chemical Industry.
Assuntos
Ácidos Graxos Dessaturases , Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Ácidos Graxos Dessaturases/genética , Ácidos Graxos Dessaturases/metabolismo , Estearoil-CoA Dessaturase/genética , Estearoil-CoA Dessaturase/metabolismo , Ácidos Graxos Insaturados/metabolismo , Ácidos Graxos/metabolismoRESUMO
Alzheimer's disease (AD) is a common neurodegenerative disease with the main manifestations of progressive cognitive dysfunction,behavioral disorders,and gradual decline of living ability.The etiology of AD is complex,and the pathogenesis of this disease remains controversial.Calcium signaling plays an important role in regulating neuronal activities,including neurotransmitter release,synaptic plasticity,memory storage,and neuronal apoptosis.Increasing studies have shown that neuronal calcium dyshomeostasis is a major pathological factor in the occurrence and development of AD.This article reviews the role and research progress in intracellular calcium dyshomeostasis in AD,including the relationship between calcium homeostasis and amyloid ß,the role of calcium/calmodulin-dependent protein kinases in tau phosphorylation,calcium signaling pathways,the relationship between calcium homeostasis and mitochondrial function,autophagy,and neuroinflammation.
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Doença de Alzheimer , Cálcio , Homeostase , Doença de Alzheimer/metabolismo , Humanos , Cálcio/metabolismo , Peptídeos beta-Amiloides/metabolismo , Sinalização do Cálcio/fisiologia , Mitocôndrias/metabolismo , Proteínas tau/metabolismo , Autofagia/fisiologia , Animais , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Neurônios/metabolismo , FosforilaçãoRESUMO
Two zero-dimensional inorganic-organic hybrids, namely, [C4mim][Cd(TCDPPA)3] (1) and [C4mpy][Cd(TCDPPA)3] (2), where (TCDPPA)- = 2,2,2-trichloro-N-(di(pyrrolidin-1-yl)phosphoryl)acetamide, (C4mim)+ = 1-butyl-3-methylimidazolium, and (C4mpy)+ = 1-butyl-4-methylpyridinium, have been synthesized via metathesis reactions and characterized systematically. These ionic cadmium-containing inorganic-organic hybrid compounds are assembled from a bulky organic cation and a complex anion constructed from the chelation of three TCDPPA ligands to one cadmium ion. These compounds possess wide band gaps and emit in the deep-blue region intensely with a quantum yield as high as 34.04%. The success of this work provides a new method for the design and fabrication of high-efficiency blue-emitting materials.
RESUMO
Zygosaccharomyces rouxii has excellent fermentation performance and good tolerance to osmotic stress. Acetyl-CoA is a crucial intermediate precursor in the central carbon metabolic pathway of yeast. This study investigated the effect of engineering acetyl-CoA metabolism on the membrane functionality and stress tolerance of yeast. Firstly, exogenous supplementation of acetyl-CoA improved the biomass and the ability of unsaturated fatty acid synthesis of Z. rouxii under salt stress. Q-PCR results suggested that the gene ACSS (coding acetyl-CoA synthetase) was significantly up-expressed. Subsequently, the gene ACSS from Z. rouxii was transformed and heterologously expressed in S. cerevisiae. The recombinant cells exhibited better multiple stress (salt, acid, heat, and cold) tolerance, higher fatty acid contents, membrane integrity, and fluidity. Our findings may provide a suitable means to enhance the stress tolerance and fermentation efficiency of yeast under harsh fermentation environments.
Assuntos
Saccharomyces cerevisiae , Zygosaccharomyces , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Acetilcoenzima A/metabolismo , Acetilcoenzima A/farmacologia , Zygosaccharomyces/genética , FermentaçãoRESUMO
Physical injury carried by dried process was an inevitable and hostile problem which could seriously affect the quality and viability of microbial agents. In this study, heat preadaptation was successfully applied as a pretreatment to fight against the physical stresses encountered during freeze-dried and spray-dried process and develop a high activity Tetragenococcus halophilus powder. The results indicated T. halophilus cells maintained a higher viability in dried powder when cells were treated with heat preadaptation before dried process. Flow cytometry analysis illustrated that heat preadaptation contributed to maintain a high membrane integrity during dried process. Besides, glass transition temperatures of dried powder increased when cells were preheated, which further verified that higher stability was obtained in group preadaptation during shelf life. Additionally, dried powder prepared by heat shock presented a better fermentation performance, suggesting heat preadaptation may be a promising strategy to prepare bacterial powder by freeze drying or spray drying.
Assuntos
Lactobacillales , Secagem por Atomização , Pós , Liofilização/métodos , TemperaturaRESUMO
Cav1.2 Ca2+ channels, a type of voltage-gated L-type Ca2+ channel, are ubiquitously expressed, and the predominant Ca2+ channel type, in working cardiac myocytes. Cav1.2 channels are regulated by the direct interactions with calmodulin (CaM), a Ca2+-binding protein that causes Ca2+-dependent facilitation (CDF) and inactivation (CDI). Ca2+-free CaM (apoCaM) also contributes to the regulation of Cav1.2 channels. Furthermore, CaM indirectly affects channel activity by activating CaM-dependent enzymes, such as CaM-dependent protein kinase II and calcineurin (a CaM-dependent protein phosphatase). In this article, we review the recent progress in identifying the role of apoCaM in the channel 'rundown' phenomena and related repriming of channels, and CDF, as well as the role of Ca2+/CaM in CDI. In addition, the role of CaM in channel clustering is reviewed.
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Canais de Cálcio Tipo L , Calmodulina , Calmodulina/metabolismo , Canais de Cálcio Tipo L/genética , Canais de Cálcio Tipo L/metabolismo , Miócitos Cardíacos/metabolismo , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Cálcio/metabolismoRESUMO
Reperfusion after ischemia would cause massive myocardial injury, which leads to oxidative stress (OS). Calcium homeostasis imbalance plays an essential role in myocardial OS injury. CaV1.2 calcium channel mediates calcium influx into cardiomyocytes, and its activity is modulated by a region of calpastatin (CAST) domain L, CSL54-64. In this study, the effect of Ahf-caltide, derived from CSL54-64, on myocardial OS injury was investigated. Ahf-caltide decreased the levels of LDH, MDA and ROS and increased heart rate, coronary flow, cell survival and SOD activity during OS. In addition, Ahf-caltide permeated into H9c2 cells and increased CaV1.2, CaVß2 and CAST levels by inhibiting protein degradation. At different Ca2+ concentrations (25 nM, 10 µM, 1 mM), the binding of CSL to the IQ motif in the C terminus of the CaV1.2 channel was increased in a H2O2 concentration-dependent manner. CSL54-64 was predicted to be responsible for the binding of CSL to CaV1.2. In conclusion, Ahf-caltide exerted a cardioprotective effect on myocardial OS injury by stabilizing CaV1.2 protein expression. Our study, for the first time, proposed that restoring calcium homeostasis by targeting the CaV1.2 calcium channel and its regulating factor CAST could be a novel treatment for myocardial OS injury.
Assuntos
Cálcio , Peróxido de Hidrogênio , Cálcio/metabolismo , Peróxido de Hidrogênio/farmacologia , Peróxido de Hidrogênio/metabolismo , Canais de Cálcio Tipo L/metabolismo , Miócitos Cardíacos/metabolismo , Peptídeos/farmacologia , Estresse OxidativoRESUMO
Numerous studies have shown that severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can infect host cells through binding to angiotensin I converting enzyme 2 (ACE2) expressing in various tissues and organs. In this study, we deeply analyzed the single-cell expression profiles of ACE2 in fetal and adult human hearts to explore the potential mechanism of SARS-CoV-2 harming the heart. The molecular docking software was used to simulate the binding of SARS-CoV-2 and its variant spike protein with ACE2. The genes closely related to ACE2 in renin-angiotensin system (RAS) were identified by constructing a protein-protein interaction network. Through the analysis of single-cell transcription profiles at different stages of human embryos, we found that the expression level of ACE2 in ventricular myocytes was increased with embryonic development. The results of single-cell sequencing analysis showed that the expression of ACE2 in ventricular myocytes was upregulated in heart failure induced by dilated cardiomyopathy compared with normal hearts. The upregulation of ACE2 increases the risk of infection with SARS-CoV-2 in fetal and adult human hearts. We also further confirmed the expression of ACE2 and ACE2-related genes in normal and SARS-CoV-2-infected human pluripotent stem cell-derived cardiomyocytes. In addition, the pathway analysis revealed that ACE2 may regulate the differently expressed genes in heart failure through calcium signaling pathway and Wnt signaling pathway.
Assuntos
Enzima de Conversão de Angiotensina 2/metabolismo , COVID-19 , SARS-CoV-2 , Adulto , Enzima de Conversão de Angiotensina 2/genética , Feminino , Humanos , Simulação de Acoplamento Molecular , Peptidil Dipeptidase A/genética , Peptidil Dipeptidase A/metabolismo , Gravidez , Sistema Renina-AngiotensinaRESUMO
NaV1.5 channel is an integral membrane protein involved in the initiation and conduction of action potentials. IQ motif is located in the C-terminal domain of NaV1.5 sodium channel, which is highly conserved in human sodium channel subtypes. IQ motif is involved in the Ca2+-dependent regulation through interaction with the regulatory proteins such as calpastatin domain L (CSL). Mutations in SCN5A, the gene encoding NaV1.5 channel, have been linked to many cardiac arrhythmias, such as Long QT syndrome type 3 (LQT3) and Brugada syndrome (BRS). LQT3-associated mutations in NaV1.5 IQ motif, IQQ1909R and IQR1913H, have been reported to affect the late INa. A BRS-associated mutation in NaV1.5 IQ motif, IQA1924T, has been reported to affect the peak INa. But the detailed pathogenic mechanisms of LQT3 and BRS remains unclear. To explore the binding properties of CSL to IQ motif and its muants associated with LQT3/BRS, molecular docking and GST pull down assay were performed in this study. As a result, S58 and E59 in CSL activating channel effect region L54-64 were involved in the conformation of the CSL/IQWT complex by protein-protein docking. IQ motif could bind to CSL in a [CSL]-dependent and [Ca2+]-dependent manner by pull down assay. However, the binding affinities of IQQ1909R and IQR1913H to CSL were decreased and its reaction rates with CSL were slower. The binding characteristics of IQA1924T to CSL was opposite in a [Ca2+]-dependent manner and its binding efficacy became smaller. The changes of the binding characteristics of IQmutants to CSL would affect the regulation of NaV1.5 channel, which may be related to LQT3 and BRS.
Assuntos
Síndrome de Brugada , Síndrome do QT Longo , Síndrome de Brugada/genética , Proteínas de Ligação ao Cálcio/genética , Humanos , Síndrome do QT Longo/genética , Simulação de Acoplamento Molecular , Mutação , Canal de Sódio Disparado por Voltagem NAV1.5/metabolismo , Canais de Sódio/genéticaRESUMO
Biofilm on dental restorative materials is an important determinant in the etiology of secondary caries development. Formation of biofilm involves adhesion of bacteria onto substrate, bacterial cell, and biofilm surfaces. Glucosyltransferase B and C (GtfB and GtfC) are essential factors for regulation of Streptococcus mutans biofilm formation, but the mechanisms involving different kinds of bacterial adhesion still lack detailed description. In this study, nanoscale adhesion force measurement was performed using atomic force microscopy. Bacteria-coated cantilevers were used to probe S. mutans adhesion to substrates, bacterial cells, and early biofilms. Two representative dental materials, glass ionomer cement (GIC) and composite resin, served as substrates. It was found that deletion of gtfB and gtfC genes both reduced adhesion forces of S. mutans toward substrate and bacterial cell surfaces (P < 0.05). Notably, reduction of the gtfB gene remarkably decreased bacterial adhesion to biofilm surfaces (P < 0.05), while gtfC showed no obvious effect during this stage. Biofilms cultured on GIG further decreased cell-biofilm adhesion, compared with those on resin (P < 0.05). Confocal fluorescence images and scanning electron microscopy images showed that deletion of gtfB lead to reduced microcolony formation and less production of exopolysaccharides (EPSs) in the biofilm, and after bacterial culturing on GIC, the EPS content was further decreased. Our findings suggest that EPSs mainly mediate bacterial adhesion to early biofilm surface. Deletion of gtfB and coculture with GIC could significantly reduce the cell-biofilm adhesion, which is probably through decreasing of EPS production. gtfB exerts a critical role in the bacterial adhesion for the whole process of biofilm development, while gtfC possibly works only in the early stages.
Assuntos
Glucosiltransferases , Streptococcus mutans , Streptococcus mutans/metabolismo , Microscopia de Força Atômica , Glucosiltransferases/genética , Glucosiltransferases/metabolismo , Biofilmes , Aderência BacterianaRESUMO
PURPOSE: To aim of the study was to explore the possible mechanisms for the decreased contraction capacity of the palatopharyngeal muscle in cases with obstructive sleep apnea hypopnea syndrome (OSAHS). METHODS: Palatopharyngeal muscle specimens from patients with OSAHS were taken as the case group. Palatopharyngeal muscle tissue by surgical removal of oropharyngeal malignant tumors was used as a control cohort. The palatopharyngeal muscle contraction capacity was measured by assessing diaphragm peak-twitching force / cross-sectional area (Pt/CSA), fatigue index (FI) twitch tension, and force per cross-sectional area (Force/CSA). Myofibril and sarcoplasmic reticulum (SR) ultra-structures were observed by electron microscopy. The intra-cellular calcium concentration was measured by fluorescence spectrophotometry. DHPRα1s and RyR1 expression profiles were probed through RT-qPCR and Western blot, and the colocalization of them was determined by immunofluorescence. RESULTS: In comparison with the control cohort, the OSAHS cohort demonstrated decreased Pt/CSA (P < 0.01), FI twitch tension (P < 0.01), together with contraction capacity (P < 0.01). This cohort also had lower intra-cellular [Ca2+] of palatopharyngeal muscle cells with abnormal ultrastructure of sarcoplasmic reticulum (SR) (P < 0.01). In addition, transcriptomic (P < 0.01) and proteomic expression (P < 0.01) for RyR1 and DHPRα1s were markedly reduced within OSAHS cohort, although the degree of colocalization of them was not altered. CONCLUSION: RyR1 and DHPRα1s downregulation may disrupt intra-cellular [Ca2+] homeostasis and subsequently decrease the palatopharyngeal muscle contraction capacity in patients with OSAHS, thus providing a novel insight into the pathogenesis of OSAHS.
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Canal de Liberação de Cálcio do Receptor de Rianodina , Apneia Obstrutiva do Sono , Humanos , Canal de Liberação de Cálcio do Receptor de Rianodina/genética , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Proteômica , Músculos Faríngeos , Diafragma , Contração Muscular/fisiologiaRESUMO
Food fermentation is driven by microorganisms, which usually coexist as multispecies biofilms. The activities and interactions of functional microorganisms and pathogenic bacteria in biofilms have important implications for the quality and safety of fermented foods. It was verified that the biofilm lifestyle benefited the fitness of microorganisms in harsh environments and intensified the cooperation and competition between biofilm members. This review focuses on multispecies biofilm formation, microbial interactions and communication in biofilms, and the application of multispecies biofilms in food fermentation. Microbial aggregation and adhesion are important steps in the early stage of multispecies biofilm formation. Different biofilm-forming abilities and strategies among microorganisms lead to several types of multispecies biofilm formation. The spatial distribution of multispecies biofilms reflects microbial interactions and biofilm function. Then, we discuss the intrinsic factors and external manifestations of multispecies biofilm system succession. Several typical interspecies cooperation and competition modes and mechanisms of microbial communication were reviewed in this review. The main limitations of the studies included in this review are the relatively small number of studies of biofilms formed by functional microorganisms during fermentation and the lack of direct evidence for the formation process of multispecies biofilms and microbial interactions and communication within biofilms. This review aims to provide the food industry with a sufficient understanding of multispecies biofilms in food fermentation. Practical Application: Meanwhile, it offers a reference value for better controlling and utilizing biofilms during food fermentation process, and the improvement of the yield, quality, and safety of fermented products including Chinese Baijiu, cheeese,kefir, soy sauce, kombucha, and fermented olive.
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Biofilmes , Interações Microbianas , Bactérias , Comunicação , FermentaçãoRESUMO
The accumulation of ethanol has a negative effect on the viability and fermentation performance of microorganisms during the production of fermented foods because of its toxicity. In this study, we investigated the effect of co-culture with Tetragenococcus halophilus on ethanol stress resistance of Zygosaccharomyces rouxii. The result showed that co-culture with T. halophilus promoted cell survival of Z. rouxii under ethanol stress, and the tolerance improved with increasing co-culture time when ethanol content was 8%. Physiological analysis showed that the co-cultured Z. rouxii cells maintained higher intracellular content of trehalose and amino acids including tyrosine, tryptophan, arginine and proline after 8% ethanol stress for 90 min. The membrane integrity analysis and biophysical analysis of the cell surface indicated that the presence of ethanol resulted in cell membrane damage and changes of Young's modulus value and roughness of cell surface. While the co-cultured Z. rouxii cells exhibited better membrane integrity, stiffer and smoother cell surface than single-cultured cells under ethanol stress. As for transcriptomic analyses, the genes involved in unsaturated fatty acid biosynthesis, trehalose biosynthesis, various types of N-glycan biosynthesis, inositol phosphate metabolism, MAPK signaling pathway and tight junction had higher expression in co-cultured Z. rouxii cells with down-regulation of majority of gene expression after stress. And these genes may function in the improvement of ethanol tolerance of Z. rouxii in co-culture.
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Enterococcaceae/crescimento & desenvolvimento , Etanol/metabolismo , Saccharomycetales/crescimento & desenvolvimento , Saccharomycetales/metabolismo , Aminoácidos/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Técnicas de Cocultura , Enterococcaceae/química , Enterococcaceae/genética , Enterococcaceae/metabolismo , Fermentação , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Saccharomycetales/química , Saccharomycetales/genética , Propriedades de SuperfícieRESUMO
Calmodulin (CaM) mutations are associated with congenital long QT (LQT) syndrome (LQTS), which may be related to the dysregulation of the cardiac-predominant Ca2+ channel isoform CaV1.2. Among various mutants, CaM-E141G was identified as a critical missense variant. However, the interaction of this CaM mutant with the CaV1.2 channel has not been determined. In this study, by utilizing a semiquantitative pull-down assay, we explored the interaction of CaM-E141G with CaM-binding peptide fragments of the CaV1.2 channel. Using the patch-clamp technique, we also investigated the electrophysiological effects of the mutant on CaV1.2 channel activity. We found that the maximum binding (Bmax) of CaM-E141G to the proximal COOH-terminal region, PreIQ-IQ, PreIQ, IQ, and NT (an NH2-terminal peptide) was decreased (by 17.71-59.26%) compared with that of wild-type CaM (CaM-WT). In particular, the Ca2+-dependent increase in Bmax became slower with the combination of CaM-E141G + PreIQ and IQ but faster in the case of NT. Functionally, CaM-WT and CaM-E141G at 500 nM Ca2+ decreased CaV1.2 channel activity to 24.88% and 55.99%, respectively, compared with 100 nM Ca2+, showing that the inhibitory effect was attenuated in CaM-E141G. The mean open time of the CaV1.2 channel was increased, and the number of blank traces with no channel opening was significantly decreased. Overall, CaM-E141G exhibits disrupted binding with the CaV1.2 channel and induces a flickering gating mode, which may result in the dysfunction of the CaV1.2 channel and, thus, the development of LQTS. The present study is the first to investigate the detailed binding properties and single-channel gating mode induced by the interaction of CaM-E141G with the CaV1.2 channel.
Assuntos
Canais de Cálcio Tipo L/genética , Calmodulina/genética , Síndrome do QT Longo/genética , Animais , Cálcio/metabolismo , Sinalização do Cálcio/genética , Cobaias , Humanos , Ativação do Canal Iônico/genética , Cinética , Síndrome do QT Longo/metabolismo , Síndrome do QT Longo/fisiopatologia , Mutação de Sentido Incorreto/genética , Técnicas de Patch-Clamp , Peptídeos/genética , Ligação Proteica/genética , Isoformas de Proteínas/genéticaRESUMO
Calmodulin (CaM) was reported to interact with PreIQ and IQ of CaV1.2 channels, but to date, no explicit binding sites of CaM were illustrated. Therefore, in the present study, we firstly used MOE (Molecular Operating Environment) for protein-protein docking and we found that the most likely residues of CaM that play an important role in the interface are concentrated in central linker region. Next we examined the binding properties of CaM and its mutants to PreIQ and IQ by GST pull-down assays. Here we confirmed that CaM binds to PreIQ and IQ in a concentration-dependent and [Ca2+]-dependent manner. However, silencing the effect of N-lobe and C-lobe by mutating two Ca2+ binding sites of each lobe abolished [Ca2+]-dependence of CaM binding, but could not influence the combination. And the mutant in central linker reduced the binding of CaM/PreIQ and CaM/IQ especially at low [Ca2+]. We confirmed that N-lobe and C-lobe play vital role in sensing the change of Ca2+, and found that the central linker of CaM is involved in the binding of CaM to CaV1.2 channels in particular at low [Ca2+], not only participates in the combination with PreIQ, but also with IQ.
Assuntos
Canais de Cálcio Tipo L/metabolismo , Calmodulina/química , Calmodulina/genética , Mutação/genética , Sequência de Aminoácidos , Animais , Sítios de Ligação , Epitopos/metabolismo , Cobaias , Ligantes , Simulação de Acoplamento Molecular , Proteínas Mutantes/metabolismo , Ligação Proteica , Domínios ProteicosRESUMO
Intracellular calcium is related to cardiac hypertrophy. The CaV1.2 channel and Ca2+/calmodulin-dependent protein kinase II (CaMKII) and CaM regulate the intracellular calcium content. However, the differences in CaMKII and CaM in cardiac hypertrophy are still conflicting and are worthy of studying as drug targets. Therefore, in this study, we aim to investigate the roles and mechanism of CaM and CaMKII on CaV1.2 in pathological myocardial hypertrophy. The results showed that ISO stimulation caused SD rat heart and cardiomyocyte hypertrophy. In vivo, the HW/BW, LVW/BW, cross-sectional area, fibrosis ratio and ANP expression were all increased. There were no differences in CaV1.2 channel expression in the in vivo model or the in vitro model, but the ISO stimulation induced channel activity, and the [Ca2+]i increased. The protein expression levels of CaMKII and p-CaMKII were all increased in the ISO group, but the CaM expression level decreased. AIP inhibited ANP, CaMKII and p-CaMKII expression, and ISO-induced [Ca2+]i increased. AIP also reduced HDAC4, p-HDAC and MEF2C expression. However, CMZ did not play a cardiac hypertrophy reversal role in vitro. In conclusion, we considered that compared with CaM, CaMKII may be a much more important drug target in cardiac hypertrophy reversal.
Assuntos
Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Calmodulina/metabolismo , Cardiomegalia/induzido quimicamente , Cardiomegalia/metabolismo , Animais , Canais de Cálcio Tipo L/metabolismo , Cardiomegalia/patologia , Modelos Animais de Doenças , Histona Desacetilases/metabolismo , Isoproterenol , Fatores de Transcrição MEF2/metabolismo , Masculino , Fosforilação , Ratos Sprague-DawleyRESUMO
N-methyl berbamine (N-MB) is a berberine derivative. Its analogue berbamine has been reported to have remarkable antiarrhythmic and ischemic protective effects. However, the pharmacological effects of N-MB are ill-defined. In this study, molecular docking was used to evaluate the binding of N-MB to CaV1.2 Ca2+ and KV11.1 K+ channels, and the effects of N-MB on action potential and ionic currents were observed in the ventricular myocytes of rabbits, HEK293 cells stably transfected with the hCaV1.2 gene and CHO cells stably transfected with hERG (human ether-a-go-go related gene). The results showed that N-MB was able to bind to both CaV1.2 and KV11.1 channels. Following a perfusion with N-MB, the durations of action potentials (APD20, APD50 and APD90) were extended, and the outward tail current, Itail, as well as the hERG current, IhERG, were inhibited, while the amplitude of action potential (APA) was only slightly reduced. N-MB also decreased the peak amplitude of the L-type Ca2+ channel current, ICaL, as well as the CaV1.2 current, ICaV1.2; this may limit the prolongation of APD. In conclusion, N-MB is a potent and natural antiarrhythmic multitarget drug that may elicit its antiarrhythmic effect through blocking both Ca2+ and K+ channel currents.
Assuntos
Antiarrítmicos , Benzilisoquinolinas/farmacologia , Bloqueadores dos Canais de Cálcio , Bloqueadores dos Canais de Potássio , Potenciais de Ação/efeitos dos fármacos , Benzilisoquinolinas/químicaRESUMO
To understand the mechanism underlying the regression of cardiac hypertrophy, we investigated the pathological changes after isoproterenol (ISO) withdrawal in ISO-induced cardiomyopathy models in rats and neonatal cardiomyocytes. Cardiac hypertrophy was induced in rats by two weeks of ISO administration; however, the hypertrophy did not regress after three weeks of natural maintenance after ISO administration was withdrawn (ISO-wdr group). The remaining hypertrophy in the ISO-wdr group was accompanied by a sustained increase in the level of phosphorylated Ca2+/calmodulin-dependent protein kinase II (p-CaMKII). Additionally, the increased expression levels of histone deacetylase 4 (HDAC4) and the CaV1.2 channel and amounts of CaMKII bound with HDAC4 and CaV1.2 were not recovered in the ISO-wdr group. The results in cardiomyocyte models were similar to those seen in rat models. Losartan, metoprolol or amlodipine neither ameliorated the increase in atrial natriuretic peptide nor inhibited the increase in p-CaMKII and bound CaMKII. In contrast, autocamtide-2-related inhibitor peptide, a CaMKII inhibitor, reduced these increases. This study investigated the phosphorylation status of CaMKII after hypertrophic stimulus was withdrawn for the first time and proposed that CaMKII as well as its complexes with CaV1.2 could be potential targets to achieve effective regression of cardiac hypertrophy.