RESUMO
Recent studies implicate a strong association between elevated plasma branched-chain amino acids (BCAAs) and insulin resistance (IR). However, a causal relationship and whether interrupted BCAA homeostasis can serve as a therapeutic target for diabetes remain to be established experimentally. In this study, unbiased integrative pathway analyses identified a unique genetic link between obesity-associated IR and BCAA catabolic gene expression at the pathway level in human and mouse populations. In genetically obese (ob/ob) mice, rate-limiting branched-chain α-keto acid (BCKA) dehydrogenase deficiency (i.e., BCAA and BCKA accumulation), a metabolic feature, accompanied the systemic suppression of BCAA catabolic genes. Restoring BCAA catabolic flux with a pharmacological inhibitor of BCKA dehydrogenase kinase (BCKDK) ( a suppressor of BCKA dehydrogenase) reduced the abundance of BCAA and BCKA and markedly attenuated IR in ob/ob mice. Similar outcomes were achieved by reducing protein (and thus BCAA) intake, whereas increasing BCAA intake did the opposite; this corroborates the pathogenic roles of BCAAs and BCKAs in IR in ob/ob mice. Like BCAAs, BCKAs also suppressed insulin signaling via activation of mammalian target of rapamycin complex 1. Finally, the small-molecule BCKDK inhibitor significantly attenuated IR in high-fat diet-induced obese mice. Collectively, these data demonstrate a pivotal causal role of a BCAA catabolic defect and elevated abundance of BCAAs and BCKAs in obesity-associated IR and provide proof-of-concept evidence for the therapeutic validity of manipulating BCAA metabolism for treating diabetes.
Assuntos
Aminoácidos de Cadeia Ramificada/metabolismo , Resistência à Insulina/fisiologia , Insulina/metabolismo , Obesidade/metabolismo , Animais , Dieta Hiperlipídica , Expressão Gênica , Estudo de Associação Genômica Ampla , Teste de Tolerância a Glucose , Humanos , Masculino , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Metaboloma , Camundongos , Obesidade/genética , Proteínas Quinases/metabolismo , Transdução de Sinais/fisiologiaRESUMO
Evidence has demonstrated that sleep-related memory consolidation declines in ageing. However, little is known about age-related changes to sleep-related emotional memory consolidation, especially when considering the positivity effect observed in older adults. In the present study, we sought to explore whether there is a positive emotional bias in sleep-related memory consolidation among healthy older adults. Young and older adults were randomly assigned either into a sleep or wake condition. All participants encoded positive, negative, and neutral stimuli and underwent recognition tests immediately (test 1), after a 12-hour sleep/wake interval (test 2), and 3 days after test 2 (test 3). Results showed that age-related differences of sleep beneficial effect were modulated by emotion valence. In particular, sleep selectively enhanced positive memory in older adults, while in young adults sleep beneficial effect was manifested in neutral memory. Moreover, the sleep beneficial effect can be maintained at least 3 days in both young and older adults. These findings suggest that older adults had preserved but positive bias of sleep-related memory consolidation, which could be one of the underlying mechanisms for their generally better emotional well-being in daily life. These findings highlight the dynamic interplay among sleep and emotional memory in older adults.
Assuntos
Envelhecimento , Emoções , Consolidação da Memória/fisiologia , Sono/fisiologia , Adolescente , Adulto , Idoso , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Reconhecimento Psicológico , Adulto JovemRESUMO
OBJECTIVE: Mitochondrial pyruvate dehydrogenase kinases 1-4 (PDKs1-4) negatively regulate activity of the pyruvate dehydrogenase complex (PDC) by reversible phosphorylation. PDKs play a pivotal role in maintaining energy homeostasis and contribute to metabolic flexibility by attenuating PDC activity in various mammalian tissues. Cumulative evidence has shown that the up-regulation of PDK4 expression is tightly associated with obesity and diabetes. In this investigation, we test the central hypothesis that PDKs1-4 are a pharmacological target for lowering glucose levels and restoring insulin sensitivity in obesity and type 2 diabetes (T2D). METHODS: Diet-induced obese (DIO) mice were treated with a liver-specific pan-PDK inhibitor 2-[(2,4-dihydroxyphenyl) sulfonyl]isoindoline-4,6-diol (PS10) for four weeks, and results compared with PDK2/PDK4 double knockout (DKO) mice on the same high fat diet (HFD). RESULTS: Both PS10-treated DIO mice and HFD-fed DKO mice showed significantly improved glucose, insulin and pyruvate tolerance, compared to DIO controls, with lower plasma insulin levels and increased insulin signaling in liver. In response to lower glucose levels, phosphorylated AMPK in PS10-treated DIO and HFD-fed DKO mice is upregulated, accompanied by decreased nuclear carbohydrate-responsive element binding protein (ChREBP). The reduced ChREBP signaling correlates with down-regulation of hepatic lipogenic enzymes (ACC1, FAS, and SCD1), leading to markedly diminished hepatic steatosis in both study groups, with lower circulating cholesterol and triacylglyceride levels as well as reduced fat mass. PS10-treated DIO as well as DKO mice showed predominant fatty acid over glucose oxidation. However, unlike systemic DKO mice, increased hepatic PDC activity alone in PS10-treated DIO mice does not raise the plasma total ketone body level. CONCLUSION: Our findings establish that specific targeting of hepatic PDKs with the PDK inhibitor PS10 is an effective therapeutic approach to maintaining glucose and lipid homeostasis in obesity and T2D, without the harmful ketoacidosis associated with systemic inhibition of PDKs.
Assuntos
Insulina/metabolismo , Lipogênese , Proteínas Nucleares/metabolismo , Obesidade/metabolismo , Complexo Piruvato Desidrogenase/metabolismo , Transdução de Sinais , Fatores de Transcrição/metabolismo , Animais , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos , Dieta Hiperlipídica/efeitos adversos , Glucose/metabolismo , Isoindóis/farmacologia , Fígado/efeitos dos fármacos , Fígado/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Obesidade/etiologia , Complexo Piruvato Desidrogenase/antagonistas & inibidores , Complexo Piruvato Desidrogenase/genética , Sulfonas/farmacologiaRESUMO
Aging associates with sleep dysfunction as well as brain alterations. However, the association between age-related brain alterations and their subjective sleep changes is less understood. To address this issue, we recorded T1 weighted structural and resting-state functional magnetic resonance imaging from both young (nâ¯=â¯62) and older adults (nâ¯=â¯108). In addition, all participants completed a battery of psychometric tests, including the Pittsburg Sleep Quality Index. We found that the age-related atrophy of cerebral gray matter, hippocampal and thalamic volume were associated with subjective sleep decline, and the atrophy of cerebral gray matter mediated the age effect on sleep. In addition, older adults exhibited decreased functional connectivity within the medial temporal lobe subsystem than their young counterparts. Moreover, there is a significant positive association between sleep and functional connectivity in young but not in older adults. In light of our findings, we suggest a neuropathological model in which age-related brain alterations may partially explain the well-documented declines in sleep with aging.
Assuntos
Envelhecimento/patologia , Envelhecimento/fisiologia , Encéfalo/diagnóstico por imagem , Encéfalo/fisiopatologia , Transtornos do Sono-Vigília/diagnóstico por imagem , Transtornos do Sono-Vigília/fisiopatologia , Adolescente , Adulto , Idoso , Atrofia , Encéfalo/patologia , Feminino , Substância Cinzenta/diagnóstico por imagem , Substância Cinzenta/patologia , Substância Cinzenta/fisiopatologia , Humanos , Masculino , Pessoa de Meia-Idade , Modelos Neurológicos , Vias Neurais/diagnóstico por imagem , Vias Neurais/patologia , Vias Neurais/fisiopatologia , Tamanho do Órgão , Sono/fisiologia , Transtornos do Sono-Vigília/patologia , Adulto JovemRESUMO
The health risk of triadimefon (TF) to cardiovascular system of human is still unclear, especially to pesticide suicides population, occupational population (farmers, retailers and pharmaceutical workers), and special population (young children and infants, pregnant women, older people, and those with compromised immune systems) who are at a greater risk. Therefore, firstly we explored the toxic effects and possible mechanism of cardiovascular toxicity induced by TF using zebrafish model. Zebrafish at stage of 48 h post fertilization (hpf) exposed to TF for 24 h exhibited morphological malformations which were further confirmed by histopathologic examination, including pericardial edema, circulation abnormalities, serious venous thrombosis and increased distance between the sinus venosus (SV) and bulbus arteriosus (BA) regions of the heart. In addition to morphological changes, TF induced functional deficits in the heart of zebrafish, including bradycardia and a significant reduced cardiac output that became more serious at higher concentrations. To better understand the possible molecular mechanisms underlying cardiovascular toxicity in zebrafish, we investigated the transcriptional level of genes related to calcium signaling pathway and cardiac muscle contraction. Q-PCR (quantitative real-time polymerase chain reaction) results demonstrated that the expression level of genes related to ATPase (atp2a1l, atp1b2b, atp1a3b), calcium channel (cacna1ab, cacna1da) and cardiac troponin C (tnnc1a) were significantly decreased after TF exposure. For the first time, the present study revealed that TF exposure had observable morphological and functional negative impacts on cardiovascular system of zebrafish. Mechanistically, this toxicity might result from the pressure of down-regulation of genes associated with calcium signaling pathway and cardiac muscle contraction following TF exposure. These findings generated here can provide information for better pesticide poisoning treatments, occupational disease prevention, and providing theoretical foundation for risk management measures.
Assuntos
Sistema Cardiovascular/efeitos dos fármacos , Triazóis/toxicidade , Poluentes Químicos da Água/toxicidade , Animais , Regulação para Baixo , Embrião não Mamífero/efeitos dos fármacos , Coração/efeitos dos fármacos , Frequência Cardíaca/efeitos dos fármacos , Humanos , Reação em Cadeia da Polimerase em Tempo Real , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/genéticaRESUMO
A period of post-learning sleep benefits memory consolidation compared with an equal-length wake interval. However, whether this sleep-based memory consolidation changes as a function of age remains controversial. Here we report a meta-analysis that investigates the age differences in the sleep-based memory consolidation in two types of memory: declarative memory and procedural memory. The meta-analysis included 22 comparisons of the performance between young adults (N =640) and older adults (N =529) on behavioral tasks measuring sleep-based memory consolidation. Our results showed a significant overall sleep-based beneficial effect in young adults but not in older adults. However, further analyses suggested that the age differences were mainly manifested in sleep-based declarative memory consolidation but not in procedural memory consolidation. We discussed the possible underlying mechanisms for the age-related degradation in sleep-based memory consolidation. Further research is needed to determine the crucial components for sleep-related memory consolidation in older adults such as age-related changes in neurobiological and cardiovascular functions, which may play an important role in this context and have the potential to delineate the interrelationships between age-related changes in sleep and memory.
Assuntos
Envelhecimento/fisiologia , Consolidação da Memória/fisiologia , Sono/fisiologia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Humanos , Pessoa de Meia-Idade , Adulto JovemRESUMO
Pyruvate dehydrogenase kinases 1-4 (PDK1-4) negatively control activity of the pyruvate dehydrogenase complex (PDC) and are up-regulated in obesity, diabetes, heart failure, and cancer. We reported earlier two novel pan-PDK inhibitors PS8 [4-((5-hydroxyisoindolin-2-yl)sulfonyl)benzene-1,3-diol] (1) and PS10 [2-((2,4-dihydroxyphenyl)sulfonyl)isoindoline-4,6-diol] (2) that targeted the ATP-binding pocket in PDKs. Here, we developed a new generation of PDK inhibitors by extending the dihydroxyphenyl sulfonylisoindoline scaffold in 1 and 2 to the entrance region of the ATP-binding pocket in PDK2. The lead inhibitor (S)-3-amino-4-(4-((2-((2,4-dihydroxyphenyl)sulfonyl)isoindolin-5-yl)amino)piperidin-1-yl)-4-oxobutanamide (17) shows a â¼8-fold lower IC50 (58 nM) than 2 (456 nM). In the crystal structure, the asparagine moiety in 17 provides additional interactions with Glu-262 from PDK2. Treatment of diet-induced obese mice with 17 resulted in significant liver-specific augmentation of PDC activity, accompanied by improved glucose tolerance and drastically reduced hepatic steatosis. These findings support 17 as a potential glucose-lowering therapeutic targeting liver for obesity and type 2 diabetes.
Assuntos
Inibidores Enzimáticos/farmacologia , Indóis/farmacologia , Isoenzimas/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Animais , Cristalografia por Raios X , Feminino , Indóis/química , Concentração Inibidora 50 , Camundongos , Piruvato Desidrogenase Quinase de Transferência de AcetilRESUMO
The widely used organotins have the potential to disrupt the endocrine system, but little is known of underlying mechanisms of azocyclotin toxicity in fish. The objective of the present study was to investigate the impact of azocyclotin on reproduction in zebrafish. Adult zebrafish were exposed to 0.09 and 0.45µg/L azocyclotin for 21days, and effects on steroid hormones and mRNA expression of the genes belonging to the hypothalamic-pituitary-gonad (HPG) axis were investigated. Mass spectrometry methodology was developed to profile steroids within the metabolome of the gonads. They were disrupted as a result of azocyclotin exposure. Alterations in the expression of key genes associated with reproductive endocrine pathways in the pituitary (lhß), gonad (cyp19a1a, cyp17a1 and 17ß-hsd3), and liver (vtg1, vtg2, cyp1a1, comt, ugt1a and gstp1) were correlated with significant reductions in estrogen in both sexes and increased testosterone in females. Azocyclotin-induced down-regulation of cyp19a1a in males suggested a reduction in the rate of estrogen biosynthesis, while up-regulation of hepatic cyp1a1 and comt in both sexes suggested an increase in estrogen biotransformation and clearance. Azocyclotin also induced change in the expression of 17ß-hsd3, suggesting increased bioavailability of 11-ketotestosterone (11-KT) in the blood. Furthermore, the down-regulation of lhß expression in the brains of azocyclotin-exposed fish was associated with inhibition of oocyte maturation in females and retarded spermatogenesis in males. As a histological finding, retarded development of the ovaries was found to be an important cause for decreased fecundity, with down-regulation of vtg suspected to be a likely underlying mechanism. Additionally, relatively high concentrations of azocyclotin in the gonads may have directly caused toxicity, thereby impairing gametogenesis and reproduction. Embryonic or larval abnormalities occurred in the F1 generation along with accumulated burdens of azocyclotin in F1 eggs, following parental exposure. Overall, our results indicate that exposure to azocyclotin can impair reproduction in fish, and induce toxicity related abnormalities in non-exposed offspring.
Assuntos
Disruptores Endócrinos/toxicidade , Metaboloma/efeitos dos fármacos , Compostos Orgânicos de Estanho/toxicidade , Reprodução/efeitos dos fármacos , Transcrição Gênica/efeitos dos fármacos , Poluentes Químicos da Água/toxicidade , Peixe-Zebra/fisiologia , Animais , Aromatase/genética , Aromatase/metabolismo , Regulação para Baixo/efeitos dos fármacos , Disruptores Endócrinos/química , Feminino , Gônadas/efeitos dos fármacos , Gônadas/metabolismo , Hipotálamo/efeitos dos fármacos , Hipotálamo/metabolismo , Fígado/efeitos dos fármacos , Fígado/metabolismo , Masculino , Compostos Orgânicos de Estanho/química , Hipófise/efeitos dos fármacos , Hipófise/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Esteroides/metabolismo , Testosterona/análogos & derivados , Testosterona/sangue , Regulação para Cima/efeitos dos fármacos , Poluentes Químicos da Água/química , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismoRESUMO
BACKGROUND: Although metabolic reprogramming is critical in the pathogenesis of heart failure, studies to date have focused principally on fatty acid and glucose metabolism. Contribution of amino acid metabolic regulation in the disease remains understudied. METHODS AND RESULTS: Transcriptomic and metabolomic analyses were performed in mouse failing heart induced by pressure overload. Suppression of branched-chain amino acid (BCAA) catabolic gene expression along with concomitant tissue accumulation of branched-chain α-keto acids was identified as a significant signature of metabolic reprogramming in mouse failing hearts and validated to be shared in human cardiomyopathy hearts. Molecular and genetic evidence identified the transcription factor Krüppel-like factor 15 as a key upstream regulator of the BCAA catabolic regulation in the heart. Studies using a genetic mouse model revealed that BCAA catabolic defect promoted heart failure associated with induced oxidative stress and metabolic disturbance in response to mechanical overload. Mechanistically, elevated branched-chain α-keto acids directly suppressed respiration and induced superoxide production in isolated mitochondria. Finally, pharmacological enhancement of branched-chain α-keto acid dehydrogenase activity significantly blunted cardiac dysfunction after pressure overload. CONCLUSIONS: BCAA catabolic defect is a metabolic hallmark of failing heart resulting from Krüppel-like factor 15-mediated transcriptional reprogramming. BCAA catabolic defect imposes a previously unappreciated significant contribution to heart failure.
Assuntos
Aminoácidos de Cadeia Ramificada/genética , Aminoácidos de Cadeia Ramificada/metabolismo , Insuficiência Cardíaca/genética , Insuficiência Cardíaca/metabolismo , Animais , Insuficiência Cardíaca/patologia , Humanos , Masculino , Metabolismo/fisiologia , Metabolômica , Camundongos , Camundongos Knockout , TranscriptomaRESUMO
A reliable and rapid method has been optimized to determine the residue of amicarthiazol in soil and environmental water samples. After extraction and evaporation, the extraction was carried out with solid phase extraction (SPE) cleanup using HLB cartridge (only soil samples) and for the quantitative determination by ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). The resulting residues of amicarthiazol were analyzed by a gradient separation performed on a UPLC system with a C18 column, methanol and water containing 0.1% (v v(-1)) formic acid as the mobile phase in the mode of electrospray positive ionization (ESI(+)) and multiple reaction monitoring (MRM). Results showed that the recoveries for spiked samples were 74.4-97.1% and 72.1-109.9% for soil and water, respectively, with the relative standard deviation (RSD) less than 10.2% when fortified at 10, 100 and 1000µgL(-1). The limits of detection (LODs) and the limits of quantification (LOQs) for matrix matched standards ranged from 0.073-0.425µgL(-1) and 0.243-1.42µgL(-1). The intra-day precision (n=5) and the inter-day precision over 10 days (n=10) for the amicarthiazol in soils and water samples spiked at 100µgL(-1) was 7.9% and 15.9%, respectively. Results indicated that the developed method could be a helpful tool for the controlling and monitoring of the risks posed by amicarthiazol to human health and environment safety.
Assuntos
Anilidas/análise , Cromatografia Líquida/métodos , Poluentes do Solo/análise , Espectrometria de Massas em Tandem/métodos , Tiazóis/análise , Poluentes Químicos da Água/análise , Resíduos de Drogas/análise , Fungicidas Industriais/análise , Extração em Fase SólidaRESUMO
The mitochondrial branched-chain α-ketoacid dehydrogenase complex (BCKDC) is negatively regulated by reversible phosphorylation.BCKDC kinase (BDK) inhibitors that augment BCKDC flux have been shown to reduce branched-chain amino acid (BCAA) concentrations in vivo. In the present study, we employed high-throughput screens to identify compound 3,6- dichlorobenzo[b]thiophene-2-carboxylic acid (BT2) as a novel BDK inhibitor (IC(50) = 3.19 µM). BT2 binds to the same site in BDK as other known allosteric BDK inhibitors, including (S)-α-cholorophenylproprionate ((S)-CPP). BT2 binding to BDK triggers helix movements in the N-terminal domain, resulting in the dissociation of BDK from the BCKDC accompanied by accelerated degradation of the released kinase in vivo. BT2 shows excellent pharmacokinetics (terminal T(1/2) = 730 min) and metabolic stability (no degradation in 240 min), which are significantly better than those of (S)-CPP. BT2, its analog 3-chloro-6-fluorobenzo[ b]thiophene-2-carboxylic acid (BT2F), and a prodrug of BT2 (i.e. N-(4-acetamido-1,2,5-oxadiazol-3-yl)-3,6-dichlorobenzo[ b]thiophene-2-carboxamide (BT3)) significantly increase residual BCKDC activity in cultured cells and primary hepatocytes from patients and a mouse model of maple syrup urine disease. Administration of BT2 at 20 mg/kg/day to wild-type mice for 1 week leads to nearly complete dephosphorylation and maximal activation of BCKDC in heart, muscle, kidneys, and liver with reduction in plasma BCAA concentrations. The availability of benzothiophene carboxylate derivatives as stable BDK inhibitors may prove useful for the treatment of metabolic disease caused by elevated BCAA concentrations.
Assuntos
Inibidores Enzimáticos/farmacologia , Hepatócitos/enzimologia , Complexo Cetoglutarato Desidrogenase/antagonistas & inibidores , Complexo Cetoglutarato Desidrogenase/metabolismo , Proteólise/efeitos dos fármacos , Tiofenos/farmacologia , Regulação Alostérica/efeitos dos fármacos , Animais , Linhagem Celular , Relação Dose-Resposta a Droga , Estabilidade Enzimática/efeitos dos fármacos , Estabilidade Enzimática/genética , Hepatócitos/patologia , Humanos , Complexo Cetoglutarato Desidrogenase/genética , Camundongos , Camundongos Knockout , Tiofenos/farmacocinéticaRESUMO
Pyruvate dehydrogenase kinase isoforms (PDKs 1-4) negatively regulate activity of the mitochondrial pyruvate dehydrogenase complex by reversible phosphorylation. PDK isoforms are up-regulated in obesity, diabetes, heart failure, and cancer and are potential therapeutic targets for these important human diseases. Here, we employed a structure-guided design to convert a known Hsp90 inhibitor to a series of highly specific PDK inhibitors, based on structural conservation in the ATP-binding pocket. The key step involved the substitution of a carbonyl group in the parent compound with a sulfonyl in the PDK inhibitors. The final compound of this series, 2-[(2,4-dihydroxyphenyl)sulfonyl]isoindoline-4,6-diol, designated PS10, inhibits all four PDK isoforms with IC50 = 0.8 µM for PDK2. The administration of PS10 (70 mg/kg) to diet-induced obese mice significantly augments pyruvate dehydrogenase complex activity with reduced phosphorylation in different tissues. Prolonged PS10 treatments result in improved glucose tolerance and notably lessened hepatic steatosis in the mouse model. The results support the pharmacological approach of targeting PDK to control both glucose and fat levels in obesity and type 2 diabetes.
Assuntos
Diabetes Mellitus Tipo 2/tratamento farmacológico , Inibidores Enzimáticos , Fígado Gorduroso/tratamento farmacológico , Isoindóis/química , Isoindóis/farmacologia , Obesidade/tratamento farmacológico , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Sulfonas/química , Sulfonas/farmacologia , Animais , Diabetes Mellitus Tipo 2/enzimologia , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/patologia , Sistemas de Liberação de Medicamentos , Desenho de Fármacos , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Fígado Gorduroso/enzimologia , Fígado Gorduroso/genética , Fígado Gorduroso/patologia , Proteínas de Choque Térmico HSP90 , Humanos , Isoenzimas/antagonistas & inibidores , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/metabolismo , Masculino , Camundongos , Camundongos Obesos , Obesidade/enzimologia , Obesidade/genética , Obesidade/patologia , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Piruvato Desidrogenase Quinase de Transferência de AcetilRESUMO
Chronic hepatitis B virus (HBV) infection is a major cause of hepatocellular carcinoma (HCC) worldwide. The pre-S2 mutant large HBV surface protein (Δ2 LHBS), which contains an in-frame deletion of approximately 17 amino acids in LHBS, is highly associated with risks and prognoses of HBV-induced HCC. It was previously reported that Δ2 LHBS interacts with the Jun activation domain-binding protein 1 (JAB1), a zinc metalloprotease. This promotes the degradation of the cell cycle regulator p27(Kip1) and is believed to be the major mechanism for Δ2 LHBS-induced HCC. In this study, it was found that the interaction between JAB1 and Δ2 LHBS is facilitated by divalent metal Zn(2+) ions. The binding of JAB1 to Δ2 LHBS requires the JAB1/CSN5 MPN metalloenzyme (JAMM) motif and residue H138 that binds to Zn(2+) ions in JAB1. Isothermal titration calorimetry showed that Δ2 LHBS binds directly to Zn(2+) ions in a two-site binding mode. Residues H71 and H116 in Δ2 LHBS, which also contact Zn(2+) ions, are also indispensable for Δ2 LHBS-mediated p27(Kip1) degradation in human HuH7 cells. These results suggest that developing drugs that interrupt interactions between Δ2 LHBS and JAB1 can be used to mitigate Δ2 LHBS-associated risks for HCC.
Assuntos
Carcinoma Hepatocelular/enzimologia , Antígenos de Superfície da Hepatite B/metabolismo , Vírus da Hepatite B/metabolismo , Hepatite B Crônica/enzimologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Neoplasias Hepáticas/enzimologia , Peptídeo Hidrolases/metabolismo , Precursores de Proteínas/metabolismo , Zinco/metabolismo , Motivos de Aminoácidos , Complexo do Signalossomo COP9 , Carcinoma Hepatocelular/virologia , Linhagem Celular Tumoral , Antígenos de Superfície da Hepatite B/genética , Vírus da Hepatite B/genética , Hepatite B Crônica/virologia , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/química , Peptídeos e Proteínas de Sinalização Intracelular/genética , Neoplasias Hepáticas/virologia , Peptídeo Hidrolases/química , Peptídeo Hidrolases/genética , Ligação Proteica , Precursores de Proteínas/genéticaRESUMO
The branched-chain amino acids (BCAAs) leucine, isoleucine, and valine are elevated in maple syrup urine disease, heart failure, obesity, and type 2 diabetes. BCAA homeostasis is controlled by the mitochondrial branched-chain α-ketoacid dehydrogenase complex (BCKDC), which is negatively regulated by the specific BCKD kinase (BDK). Here, we used structure-based design to develop a BDK inhibitor, (S)-α-chloro-phenylpropionic acid [(S)-CPP]. Crystal structures of the BDK-(S)-CPP complex show that (S)-CPP binds to a unique allosteric site in the N-terminal domain, triggering helix movements in BDK. These conformational changes are communicated to the lipoyl-binding pocket, which nullifies BDK activity by blocking its binding to the BCKDC core. Administration of (S)-CPP to mice leads to the full activation and dephosphorylation of BCKDC with significant reduction in plasma BCAA concentrations. The results buttress the concept of targeting mitochondrial BDK as a pharmacological approach to mitigate BCAA accumulation in metabolic diseases and heart failure.
Assuntos
Proteínas Mitocondriais/química , Inibidores de Proteínas Quinases/química , Proteínas Quinases/química , Estrutura Terciária de Proteína , Regulação Alostérica , Animais , Sítios de Ligação/genética , Cromatografia Líquida , Cristalografia por Raios X , Isoleucina/sangue , Isoleucina/metabolismo , Cinética , Leucina/sangue , Leucina/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos ICR , Proteínas Mitocondriais/antagonistas & inibidores , Proteínas Mitocondriais/metabolismo , Modelos Moleculares , Estrutura Molecular , Mutação , Fenilpropionatos/química , Fenilpropionatos/metabolismo , Fenilpropionatos/farmacologia , Fosforilação , Ligação Proteica , Inibidores de Proteínas Quinases/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Proteínas Quinases/metabolismo , Espectrometria de Massas em Tandem , Valina/sangue , Valina/metabolismoRESUMO
Sequence homologs of the small MutS-related (Smr) domain, the C-terminal endonuclease domain of MutS2, also exist as stand-alone proteins. In this study, we report the crystal structure of a proteolyzed fragment of YdaL (YdaL39-175), a stand-alone Smr protein from Escherichia coli. In this structure, residues 86-170 assemble into a classical Smr core domain and are embraced by an N-terminal extension (residues 40-85) with an α/ß/α fold. Sequence alignment indicates that the N-terminal extension is conserved among a number of stand-alone Smr proteins, suggesting structural diversity among Smr domains. We also discovered that the DNA binding affinity and endonuclease activity of the truncated YdaL39-175 protein were slightly lower than those of full-length YdaL1-187, suggesting that residues 1-38 may be involved in DNA binding.
Assuntos
Endonucleases/química , Proteínas de Escherichia coli/química , Fragmentos de Peptídeos/química , Motivos de Aminoácidos , Sequência de Aminoácidos , Sequência Conservada , Cristalografia por Raios X , Ligação de Hidrogênio , Dados de Sequência Molecular , Proteína MutS de Ligação de DNA com Erro de Pareamento/química , Polinucleotídeos/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , Alinhamento de Sequência , Homologia Estrutural de ProteínaRESUMO
The sliding clamp is a key component of DNA polymerase III (Pol III) required for genome replication. It is known to function with diverse DNA repair proteins and cell cycle-control proteins, making it a potential drug target. To extend our understanding of the structure/function relationship of the sliding clamp, we solved the crystal structure of the sliding clamp from Mycobacterium tuberculosis (M. tuberculosis), a human pathogen that causes most cases of tuberculosis (TB). The sliding clamp from M. tuberculosis forms a ring-shaped head-to-tail dimer with three domains per subunit. Each domain contains two α helices in the inner ring that lie against two ß sheets in the outer ring. Previous studies have indicated that many Escherichia coli clamp-binding proteins have a conserved LF sequence, which is critical for binding to the hydrophobic region of the sliding clamp. Here, we analyzed the binding affinities of the M. tuberculosis sliding clamp and peptides derived from the α and δ subunits of Pol III, which indicated that the LF motif also plays an important role in the binding of the α and δ subunits to the sliding clamp of M. tuberculosis.
Assuntos
Proteínas de Bactérias/química , DNA Polimerase III/química , DNA Polimerase Dirigida por DNA/química , Mycobacterium tuberculosis/enzimologia , Sequência de Aminoácidos , Cristalografia por Raios X , Dados de Sequência Molecular , Peptídeos/química , Estrutura Secundária de Proteína , Estrutura Terciária de ProteínaRESUMO
A heterologous direct competitive enzyme-linked immunosorbent assay (ELISA) for parathion residue determination is described based on a monoclonal antibody and a new competitor. The effects of several physicochemical factors, such as methanol concentration, ionic strength, pH value, and sample matrix, on the performance of the ELISA were optimized for the sake of obtaining a satisfactory assay sensitivity. Results showed that when the assay medium was in the optimized condition (phosphate buffer solution [PBS] containing 10% [v/v] methanol and 0.2 mol/L NaCl at a pH value of 5.0), the sensitivity (estimated as the IC(50) value) and the limit of detection (LOD, estimated as the IC(10) value) were 1.19 and 0.08 ng/ml, respectively. The precision investigation indicated that the intraassay precision values all were below 10% and that the interassay precision values ranged from 4.89 to 19.12%. In addition, the developed ELISA showed a good linear correlation (r(2)=0.9962) to gas chromatography within the analyte's concentration range of 0.1 to 16 ng/ml. When applied to the fortified samples (parathion adding level: 5-15 microg/kg), the developed ELISA presented mean recoveries of 127.46, 122.52, 91.92, 124.01, 129.72, 99.37, and 87.17% for tomato, cucumber, banana, apple, orange, pear, and sugarcane, respectively. Results indicated that the established ELISA is a potential tool for parathion residue determination.
Assuntos
Ensaio de Imunoadsorção Enzimática/métodos , Análise de Alimentos/métodos , Inseticidas/análise , Paration/análise , Ensaio de Imunoadsorção Enzimática/instrumentação , Análise de Alimentos/instrumentação , Frutas/química , Concentração de Íons de Hidrogênio , Inseticidas/química , Metanol , Estrutura Molecular , Concentração Osmolar , Paration/química , Verduras/químicaRESUMO
Using a simple test for rapid identification and quantification of pesticide multiresidues in food and environmental samples is a long-cherished approach for practical monitoring purposes. Here two gold-based lateral-flow strips (strip A and strip B) were investigated for simultaneous detection of carbofuran and triazophos. For the strip A format, a bispecific monoclonal antibody (BsMcAb) against both carbofuran and triazophos was employed to prepare the immunogold probe. For the strip B format, anti-carbofuran monoclonal antibody (McAb) and anti-triazophos McAb separately labeled with colloidal gold were combined as detector reagents. By comparison of visual results from pesticide standard tests between the two formats, the strip B assay manifested higher sensitivities for both pesticides. Analysis of spiked water samples by the preferable strip indicated that the detection limits for carbofuran and triazophos were 32 and 4 microg/L, respectively. The strength of the portable one-step strip assay was in the simultaneous screening for two pesticides within a short time (8-10 min) without any equipment.
Assuntos
Anticorpos Monoclonais/imunologia , Carbofurano/análise , Imuno-Histoquímica/métodos , Organotiofosfatos/análise , Praguicidas/análise , Triazóis/análise , Poluentes Químicos da Água/análise , Água/análise , Carbofurano/imunologia , Organotiofosfatos/imunologia , Praguicidas/imunologia , Triazóis/imunologia , Poluentes Químicos da Água/imunologiaRESUMO
Environmental and food safety issues now are recognized internationally, and pesticide residues play key roles as environment and food pollutants. It is crucial to develop methods for rapid determination of pesticide residues in environments and foods. A one-step strip based on nanocolloidal-gold-labeled monoclonal antibodies for detection of triazophos residue was developed. The nanocolloidal gold, with an average particle diameter of 25 nm (G25), was labeled to an antitriazophos monoclonal antibody. This conjugate was dispensed on the conjugate pad of a porous glass fiber. Ovalbumin hapten and goat anti-mouse IgG were dispensed on the nitrocellulose membrane and served as the test line (T-line) and control line (C-line), respectively. After conditions optimization, the one-step strip was finally developed for the residue determination of triazophos. The limit of detection (LOD) of the strip was 4 ng/mL for standard. The detection was not affected by the pH of the liquid sample but low total ion concentration will induce illegible C-line and T-line. The LOD for spiked samples of soil and water was 5ng/mL, with run time of no more than 10min.