RESUMO
Cigarette smoke contains over 4800 compounds, including at least 200 toxicants or endocrine disruptors. Currently, effects of cigarette smoke on thyroid hormone (TH) levels remains to be clarified. Here, we demonstrate that cigarette smoke extract (CSE) possesses thyroid hormone properties and acts synergistically as a partial agonist for thyroid hormone receptors (TRs) in the presence of TH. In transient gene expression experiments, CSE stimulated transcriptional activity with TH in a dose-dependent manner. Stimulatory effects were observed with physiological TH concentrations, although CSE did not activate TRs without TH. CSE (5%) dissolved in phosphate-buffered saline (PBS) supplemented with 1 nM TH was approximately comparable to 3.2±0.1 and 2.3±0.2 nM of TRα1 and TRß1, respectively. To illustrate probable mechanisms of the CSE agonistic activity, effects on TR mediated transcriptional functions with cofactors were investigated. With a mammalian two-hybrid assay, CSE recruited the nuclear coactivators glucocorticoid receptor interacting protein 1 (GRIP1) and steroid receptor coactivator 1 (SRC1) to the TR. Unsaturated carbonyl compounds, acrolein, crotonaldehyde, and methyl vinyl ketone, representative constituents of CSE, retained such agonistic properties and possibly contributed to stimulatory effects. The results suggest that CSE recruits a transcriptional activator and may reinforce TH binding to the TR additively, resulting in gene expression. CSE partially agonizes TH action and may disturb the function of various nuclear hormone receptor types and their cofactors to disrupt the physiological processes.
Assuntos
Nicotiana/efeitos adversos , Receptores dos Hormônios Tireóideos/efeitos dos fármacos , Fumaça/efeitos adversos , Hormônios Tireóideos/farmacologia , Transcrição Gênica/efeitos dos fármacos , Proteínas de Transporte/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Células HEK293 , Humanos , Malato Desidrogenase/biossíntese , Proteínas do Tecido Nervoso/efeitos dos fármacos , Coativador 1 de Receptor Nuclear/genética , Receptores dos Hormônios Tireóideos/genética , Fumaça/análise , Receptores alfa dos Hormônios Tireóideos/efeitos dos fármacos , Receptores alfa dos Hormônios Tireóideos/genética , Receptores beta dos Hormônios Tireóideos/efeitos dos fármacos , Receptores beta dos Hormônios Tireóideos/genética , Nicotiana/químicaRESUMO
Malic enzyme 1 (ME1) is a multifunctional protein involved in glycolysis, the citric acid cycle, NADPH production, glutamine metabolism, and lipogenesis. It is overexpressed in various cancers. We examined the expression of ME1 in 119 oral squamous cell carcinomas (OSCCs) using immunohistochemistry. Malic enzyme 1 expression was moderate to strong in 57 (48%) OSCCs and correlated with pT, pN, clinical stage, and histological grade. In 37 cases with prognostic evaluation, moderate to strong ME1 expression indicated a worse prognosis than did weak ME1 expression. Malic enzyme 1 knockdown or inactivation by lanthanide inhibited cell proliferation and motility and suppressed the epithelial-mesenchymal transition in HSC3 human OSCC cells. Knockdown of ME1 also shifted energy metabolism from aerobic glycolysis and lactate fermentation to mitochondrial oxidative phosphorylation, and the redox status from reductive to oxidative. In a mouse tumor model, lanthanide suppressed tumor growth and increased survival time. These findings reveal that ME1 is a valid target for molecular therapy in OSCC.
Assuntos
Carcinoma de Células Escamosas/enzimologia , Citosol/enzimologia , Malato Desidrogenase/biossíntese , Neoplasias Bucais/enzimologia , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/patologia , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Movimento Celular/genética , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Progressão da Doença , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Transição Epitelial-Mesenquimal/genética , Regulação Neoplásica da Expressão Gênica , Humanos , Imuno-Histoquímica , Elementos da Série dos Lantanídeos/farmacologia , Malato Desidrogenase/antagonistas & inibidores , Malato Desidrogenase/genética , Neoplasias Bucais/genética , Neoplasias Bucais/patologia , Oligonucleotídeos Antissenso/genética , Oxirredução/efeitos dos fármacos , Transplante HeterólogoRESUMO
BACKGROUND: Advanced non-small cell lung cancer (NSCLC) is an aggressive tumor that is treated with a combination of chemotherapy and radiation if the patient is not a candidate for surgery. Predictive biomarkers for response to radiotherapy are lacking in this patient population, making it a non-tailored therapy regimen with unknown outcome. Twenty to 30 % of NSCLC harbor an activating mutation in KRAS that may confer radioresistance. We hypothesized that mutant KRAS can regulate glutamine metabolism genes in NSCLC and maintain tumor redox balance through transamination reactions that generate cytosolic NADPH via malic enzyme 1 (ME1), which may contribute to radioresistance. FINDINGS: A doxycycline-inducible mouse model of KRAS (G12D) driven NSCLC and patient data was analyzed from multiple publicly accessible databases including TCGA, CCLE, NCBI GEO and Project Achilles. ME1 expression was found to be mutant KRAS associated in both a NSCLC mouse model and human NSCLC cancer cell lines. Perturbing glutamine metabolism sensitized mutant KRAS, but not wild-type KRAS NSCLC cell lines to radiation treatment. NSCLC survival analysis revealed that patients with elevated ME1 and GOT1 expression had significantly worse outcomes after radiotherapy, but this was not seen after chemotherapy alone. CONCLUSIONS: KRAS driven glutamine metabolism genes, specifically ME1 and GOT1 reactions, may be a predictive marker and potential therapeutic target for radiotherapy in NSCLC.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/radioterapia , Genes ras , Neoplasias Pulmonares/radioterapia , Malato Desidrogenase/biossíntese , Proteínas de Neoplasias/biossíntese , Animais , Aspartato Aminotransferase Citoplasmática/biossíntese , Aspartato Aminotransferase Citoplasmática/genética , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/mortalidade , Carcinoma Pulmonar de Células não Pequenas/patologia , Linhagem Celular Tumoral , Bases de Dados Factuais , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica/genética , Glutamina/metabolismo , Humanos , Estimativa de Kaplan-Meier , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/mortalidade , Neoplasias Pulmonares/patologia , Malato Desidrogenase/análise , Malato Desidrogenase/genética , Camundongos , Camundongos Transgênicos , Mutação de Sentido Incorreto , NADP/metabolismo , Proteínas de Neoplasias/análise , Proteínas de Neoplasias/genética , Oxirredução , Mutação Puntual , Prognóstico , Interferência de RNA , RNA Mensageiro/biossíntese , RNA Neoplásico/biossíntese , RNA Interferente Pequeno/genética , Tolerância a Radiação/genética , Proteínas Recombinantes de Fusão/biossíntese , Ensaio Tumoral de Célula-Tronco , Regulação para CimaRESUMO
Malic enzyme 1 (ME1) links the glycolytic and citric acid cycles and is important for NADPH production, glutamine metabolism, and lipogenesis. Recently, its deregulation has been implicated in the progression of various cancers. However, the role of ME1 in the progression of hepatocellular carcinoma (HCC) remains unclear. In this study, we utilized short hairpin RNA-mediated gene silencing to investigate the biological effects of ME1 depletion in HCC and determined its prognostic significance in HCC. ME1 expression was examined by real-time (RT)-PCR and Western blot using five HCC cell lines and one normal liver cell line. We used polyethylenimine nanoparticles to deliver a short hairpin RNA to induce cessation of ME1 expression in HCC cells. Changes in NADPH production and reactive oxygen species (ROS) production were studied. Metastatic potentials of HCC cells were evaluated in vitro. Furthermore, we evaluated the protein level of ME1 in para-tumor and cancerous tissues of 65 HCC patients with detailed clinical, pathological, and clinical follow-up data. Patients' survivals were further assessed as well. Upregulated ME1 expression was observed in HCC cell lines. Downregulation of ME1 attenuated NADPH production and stimulated ROS production. Silencing ME1 was noted to inhibit migratory and invasive properties of HCC cells by inducing the E-cadherin expression and decreasing of N-cadherin and vimentin expression in a ROS-dependent pathway. Overexpression of ME1 was observed in a major fraction of HCC samples. Higher level of ME1 in tumors was significantly associated with reduced overall survival (Kaplan-Meier analysis, P = 0.024) and reduced progression-free survival (Kaplan-Meier analysis, P = 0.011). Inhibition of ME1 expression decreases HCC metastasis via suppression of epithelial-mesenchymal transition (EMT) processes in ROS-induced pathways. ME1 overexpression associates with unfavorable prognoses in patients with HCC, suggesting that ME1 is a poor prognostic predictor of hepatocellular carcinoma.
Assuntos
Biomarcadores Tumorais/biossíntese , Carcinoma Hepatocelular/genética , Transição Epitelial-Mesenquimal/genética , Neoplasias Hepáticas/genética , Malato Desidrogenase/biossíntese , Idoso , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Intervalo Livre de Doença , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Estimativa de Kaplan-Meier , Neoplasias Hepáticas/patologia , Malato Desidrogenase/genética , Masculino , Pessoa de Meia-Idade , Prognóstico , RNA Interferente Pequeno , Espécies Reativas de Oxigênio/metabolismoRESUMO
Metabolic modifications during the developmental period can extend longevity. We found that malic enzyme (Men) overexpression during the larval period lengthened the lifespan of Drosophila. Men overexpression by S106-GeneSwitch-Gal4 driver increased pyruvate content and NADPH/NADP(+) ratio but reduced triglyceride, glycogen, and ATP levels in the larvae. ROS levels increased unexpectedly in Men-overexpressing larvae. Interestingly, adults exposed to larval Men-overexpression maintained ROS tolerance with enhanced expression levels of glutathione-S-transferase D2 and thioredoxin-2. Our results suggest that metabolic changes mediated by Men during development might be related to the control of ROS tolerance and the longevity of Drosophila.
Assuntos
Proteínas de Drosophila/biossíntese , Drosophila melanogaster/crescimento & desenvolvimento , Longevidade/fisiologia , Malato Desidrogenase/biossíntese , Trifosfato de Adenosina/metabolismo , Animais , Proteínas de Drosophila/genética , Drosophila melanogaster/enzimologia , Drosophila melanogaster/genética , Glutationa Transferase/metabolismo , Glicogênio/metabolismo , Larva/enzimologia , Larva/genética , Larva/crescimento & desenvolvimento , Longevidade/genética , Malato Desidrogenase/genética , NADP/metabolismo , Ácido Pirúvico/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Tiorredoxinas/metabolismo , Triglicerídeos/metabolismoRESUMO
The tricarboxylic acid (TCA) cycle is a central route for oxidative metabolism. Besides being responsible for the production of NADH and FADH2, which fuel the mitochondrial electron transport chain to generate ATP, the TCA cycle is also a robust source of metabolic intermediates required for anabolic reactions. This is particularly important for highly proliferating cells, like tumour cells, which require a continuous supply of precursors for the synthesis of lipids, proteins and nucleic acids. A number of mutations among the TCA cycle enzymes have been discovered and their association with some tumour types has been established. In this review we summarise the current knowledge regarding alterations of the TCA cycle in tumours, with particular attention to the three germline mutations of the enzymes succinate dehydrogenase, fumarate hydratase and isocitrate dehydrogenase, which are involved in the pathogenesis of tumours, and to the aberrant regulation of TCA cycle components that are under the control of oncogenes and tumour suppressors.
Assuntos
Ciclo do Ácido Cítrico/genética , Metabolismo Energético/genética , Mitocôndrias/patologia , Neoplasias/patologia , Aconitato Hidratase/biossíntese , Proliferação de Células/fisiologia , Ciclo do Ácido Cítrico/fisiologia , Fumarato Hidratase/genética , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/fisiologia , Isocitrato Desidrogenase/genética , Malato Desidrogenase/biossíntese , Mitocôndrias/genética , Mitocôndrias/metabolismo , Mutação , Neoplasias/genética , Neoplasias/metabolismo , Fosforilação Oxidativa , Prolil Hidroxilases/metabolismo , Proteínas Serina-Treonina Quinases/biossíntese , Piruvato Desidrogenase Quinase de Transferência de Acetil , Transdução de Sinais/genética , Succinato Desidrogenase/genéticaRESUMO
Translation of mRNA into a polypeptide chain is a highly accurate process. Many prokaryotic and eukaryotic viruses, however, use leaky termination of translation to optimize their coding capacity. Although growing evidence indicates the occurrence of ribosomal readthrough also in higher organisms, a biological function for the resulting extended proteins has been elucidated only in very few cases. Here, we report that in human cells programmed stop codon readthrough is used to generate peroxisomal isoforms of cytosolic enzymes. We could show for NAD-dependent lactate dehydrogenase B (LDHB) and NAD-dependent malate dehydrogenase 1 (MDH1) that translational readthrough results in C-terminally extended protein variants containing a peroxisomal targeting signal 1 (PTS1). Efficient readthrough occurs at a short sequence motif consisting of a UGA termination codon followed by the dinucleotide CU. Leaky termination at this stop codon context was observed in fungi and mammals. Comparative genome analysis allowed us to identify further readthrough-derived peroxisomal isoforms of metabolic enzymes in diverse model organisms. Overall, our study highlights that a defined stop codon context can trigger efficient ribosomal readthrough to generate dually targeted protein isoforms. We speculate that beyond peroxisomal targeting stop codon readthrough may have also other important biological functions, which remain to be elucidated.
Assuntos
Códon de Terminação/genética , L-Lactato Desidrogenase/genética , Malato Desidrogenase/genética , Biossíntese de Proteínas , Receptores Citoplasmáticos e Nucleares/genética , Ribossomos/genética , Fungos/genética , Células HeLa , Humanos , Isoenzimas/biossíntese , Isoenzimas/genética , L-Lactato Desidrogenase/biossíntese , Malato Desidrogenase/biossíntese , Motivos de Nucleotídeos/genética , Receptor 1 de Sinal de Orientação para Peroxissomos , Peroxissomos/genética , RNA Mensageiro/genética , Ustilago/genéticaRESUMO
Hop (Humulus lupulus L.) pomace contains procyanidin-rich polyphenols, which are large oligomeric compounds of catechin. We studied the effect of high dose (1%) of dietary hop pomace polyphenols (HPs) in Otsuka Long-EvansTokushima Fatty (OLETF) rats, an animal model of type 2 diabetes. By 70 days, the rats fed HPs tended to have a lower body weight and reduced mesenteric white adipose tissue weight than the rats fed a control diet. Triglyceride levels in both plasma and liver tended to be lower in the HPs-fed group than in the control group. Dietary HPs substantially suppressed the activities of hepatic fatty acid synthetase, glucose-6-phosphate dehydrogenase, and malic enzyme, through the suppression of SREBP1c mRNA expression in OLETF rats. Moreover, in the HPs-fed group, monocyte chemotactic protein-1 (MCP-1) expression and fasting blood glucose levels at 40 days, and hemoglobin A1c (HbA1c) levels at 70 days were significantly lower than those in the control group. Thus, dietary HPs may exert an ameliorative function on hepatic fatty acid metabolism, glucose metabolism, and inflammatory response accompanying the increase of the adipose tissue mass in OLETF rats.
Assuntos
Tecido Adiposo/metabolismo , Catequina/farmacologia , Diabetes Mellitus Tipo 2/sangue , Suplementos Nutricionais , Jejum/sangue , Humulus/química , Tecido Adiposo/patologia , Animais , Glicemia/metabolismo , Catequina/química , Diabetes Mellitus Tipo 2/tratamento farmacológico , Diabetes Mellitus Tipo 2/patologia , Modelos Animais de Doenças , Glucosefosfato Desidrogenase/biossíntese , Malato Desidrogenase/biossíntese , Masculino , Ratos , Proteína de Ligação a Elemento Regulador de Esterol 1/biossíntese , Triglicerídeos/sangueRESUMO
The expression of the enzyme-coding genes, controlling glucose-phosphate isomerase (GPI), malate dehydrogenase (MDH), and alcohol dehydrogenase (ADH), was examined in nongerminating seeds of sugarbeet after Triton X-100 (TX-100) and colchicine treatment. Two types of changes revealed included modification of the enzymatic loci expression (change of the isozyme electrophoretic mobility) and inactivation of standard profiles. In the MDH and GPI systems, these processes were found to be associated. Complete isozyme modification was accompanied with the disappearance of standard profiles. In the ADH system, the treatment with TX-100 and colchicine gave rise to two independent processes, including silencing of the Adh1 locus and the appearance of the ADH isozymes with abnormal electrophoretic mobility, which were probably the products of the Adh2 locus. It was suggested that the effect of TX-100 and colchicine on the expression of the enzyme-encoding genes examined depended on the intracellular localization of the encoded enzymes.
Assuntos
Beta vulgaris/enzimologia , Colchicina/farmacologia , Genes de Plantas , Octoxinol/farmacologia , Sementes/enzimologia , Álcool Desidrogenase/biossíntese , Álcool Desidrogenase/genética , Beta vulgaris/efeitos dos fármacos , Beta vulgaris/genética , Germinação , Glucose-6-Fosfato Isomerase/biossíntese , Glucose-6-Fosfato Isomerase/genética , Isoenzimas/biossíntese , Isoenzimas/genética , Malato Desidrogenase/biossíntese , Malato Desidrogenase/genética , Sementes/efeitos dos fármacos , Sementes/genéticaRESUMO
Foram avaliadas as alterações no metabolismo materno durante a prenhez em ratas Wistar, prenhes e não-prenhes, submetidas à restrição protéica, que receberam dietas isocalóricas (15,74 kJ/g), controle ou hipoprotéica (17 por cento versus 6 por cento), distribuídas em quatro grupos (n = 7), quais sejam: controle não-prenhe (CNP) e prenhe (CP) e hipoprotéico não-prenhe (HNP) e prenhe (HP), do 1º ao 18º dia de prenhez. Parâmetros bioquímicos, hormonais e relacionados à síntese de lipídios foram considerados. Utilizou-se ANOVA a duas vias seguido de teste Tukey-HSD e teste t de Student, significância de p < 0,05. A restrição protéica elevou a síntese de lipídios e a atividade da enzima málica (EM) no fígado (FIG) e reduziu a massa ( por cento) e a razão lipí+dio/glicogênio nesse tecido, bem como reduziu a ingestão protéica (total e por cento), o conteúdo ( por cento) de lipídios na glândula mamária (GMA), as proteínas e a albumina séricas, com consequente redução nas massas da placenta e fetos. A prenhez reduziu a proteinemia, a albuminemia, a síntese de lipídios, a atividade da EM, os lipídios e o glicogênio no FIG. Mas elevou a massa corporal final, a massa ( por cento) do tecido adiposo gonadal (GON), do FIG e da GMA, e reduziu a massa ( por cento) da carcaça (CARC), a síntese e o conteúdo de lipídios no GON e, na GMA, o conteúdo de lipídios. A insulinemia elevou-se na prenhez, com glicemia reduzida, caracterizando resistência hormonal. A leptina e a prolactina também se elevaram na prenhez, sendo o aumento maior no HP. A restrição protéica na prenhez modificou o metabolismo materno, alterando a síntese de lipídios no FIG e o perfil hormonal, além de reduzir a massa da placenta e dos fetos.
Metabolism alterations were evaluated in female Wistar rats (dams) during pregnancy. Pregnant and non-pregnant dams submitted to protein restriction, were fed isocaloric (15.74 kJ/g), control or hypoproteic (17 percent vs. 6 percent) diets, and distributed in four Groups (n=7) as follows: non-pregnant control (NPC), pregnant control (PC), non-pregnant hypoproteic (NPH), and pregnant hypoproteic (PH); from Day 1 to Day 18 of pregnancy. Biochemical, hormonal and metabolic parameters related to lipid synthesis were assessed. The two-way ANOVA, followed by Tukey-HSD and Student-t tests were used, with a significance of p< 0.05. Protein restriction elevated lipid synthesis and malic enzyme (ME) activity in the liver, and reduced mass and the lipid/glycogen ratio in this tissue; it also lowered protein ingestion (total and percent), lipid content ( percent) in the mammary gland (MAG), serum proteins and albumin, with consequent reduction of placenta and fetal masses. Pregnancy reduced serum protein and albumin concentrations, lipid synthesis, ME activity, hepatic lipid and glycogen content. However, it increased final body mass; increased relative masses of gonad (GON), liver and MAG; but reduced lipid synthesis and content of GON, lipid content of MAG and the relative mass of carcass. Pregnancy Insulinemia increased during pregnancy with reduced glycemia, characterizing hormonal resistance. Leptin and prolactin were also increased during pregnancy, being the highest increase in observed in HP rats. Protein restriction in pregnancy modified maternal metabolism, altering lipid synthesis in the liver and hormonal profile and decreasing the placenta and fetus masses.
Assuntos
Animais , Feminino , Gravidez , Ratos , Dieta com Restrição de Proteínas/efeitos adversos , Fenômenos Fisiológicos da Nutrição Materna/fisiologia , Análise de Variância , Tecido Adiposo/metabolismo , Feto/metabolismo , Gônadas/metabolismo , Hormônios/biossíntese , Lipídeos/biossíntese , Glicogênio Hepático/biossíntese , Fígado/química , Fígado/enzimologia , Modelos Animais , Malato Desidrogenase/biossíntese , Glândulas Mamárias Animais/metabolismo , Placenta/metabolismo , Ratos WistarRESUMO
The response to initial glucocorticoid therapy in childhood acute lymphoblastic leukaemia (ALL) reliably predicts the response to multiagent chemotherapy. Patients resistant to glucocorticoids (prednisone poor responders (PPR)) have a poorer event-free survival compared to glucocorticoid-sensitive patients (prednisone good responders (PGR)). A case-control study was performed to investigate differential protein expression in leukaemic blasts from PGR and PPR childhood ALL patients. Two-dimensional gel electrophoresis (2-DE) was used for an unsupervised screening and surface enhanced laser desorption/ionisation-time of flight mass spectrometry (SELDI-TOF MS) for the characterisation of protein spots. In difference maps of average gels for the proteomes of each responder group, differentially expressed proteins were identified after tryptic digestion and spotting onto H4-SELDI-TOF-MS chips. Proteins overexpressed in PPR were Catalase, RING finger protein 22 alpha, Valosin-containing protein (VCP) and a G-protein-coupled receptor. Proteins overexpressed in PGR were protein kinase C and malate dehydrogenase. Valosin-containing protein was chosen for validation and quantification by Western blot analysis in a second case-control group of ALL patients. In this second independent cohort, median VCP expression (P25-P75) was 0.15 (0.11-0.28) in PGR and 0.34 (0.14-0.99) in PPR patients (P = 0.04). We conclude that high VCP expression is associated with poor prednisone response in childhood ALL patients.
Assuntos
Biomarcadores Tumorais/análise , Proteínas de Ciclo Celular/análise , Resistencia a Medicamentos Antineoplásicos , Glucocorticoides/farmacologia , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , Prednisona/uso terapêutico , Proteoma/análise , Adenosina Trifosfatases , Western Blotting , Estudos de Casos e Controles , Catalase/biossíntese , Proteínas de Ciclo Celular/biossíntese , Proteínas de Ciclo Celular/efeitos dos fármacos , Criança , Pré-Escolar , Eletroforese em Gel Bidimensional , Feminino , Glucocorticoides/uso terapêutico , Humanos , Lactente , Recém-Nascido , Malato Desidrogenase/biossíntese , Masculino , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamento farmacológico , Prednisona/farmacologia , Proteína Quinase C/biossíntese , Receptores Acoplados a Proteínas G/biossíntese , Sensibilidade e Especificidade , Proteína com ValosinaRESUMO
The Candida utilis malic enzyme gene, CME1, was isolated from a cDNA library and characterised on a molecular and biochemical level. Sequence analysis revealed an open reading frame of 1,926 bp, encoding a 641 amino acid polypeptide with a predicted molecular weight of approximately 70.2 kDa. The inferred amino acid sequence suggested a cytosolic localisation for the malic enzyme, as well as 37 and 68% homologies with the malic enzymes of Schizosaccharomyces pombe and Saccharomyces cerevisiae, respectively. Expression of the CME1 gene was subject to carbon catabolite repression and substrate induction, similar to the regulatory mechanisms observed for the C. utilis dicarboxylic acid permease. The CME1 gene was successfully expressed in S. cerevisiae under control of the S. cerevisiae PGK1 promoter and terminator. When coexpressed with the S. pombe malate permease gene (mae1), it resulted in a recombinant S. cerevisiae strain able to completely degrade 90% of the extracellular L-malate within 24 h.
Assuntos
Candida/enzimologia , Proteínas Fúngicas/biossíntese , Expressão Gênica , Malato Desidrogenase/biossíntese , Proteínas Recombinantes/biossíntese , Saccharomyces cerevisiae , Sequência de Aminoácidos , Candida/genética , Clonagem Molecular , Proteínas Fúngicas/genética , Malato Desidrogenase/genética , Dados de Sequência Molecular , Proteínas Recombinantes/genética , Schizosaccharomyces , Homologia de Sequência de AminoácidosRESUMO
Protein profile alterations following exposure to cadmium were examined in marine alga Nannochloropsis oculata through proteomic analysis. Alterations of the protein expression patterns following 10 muM cadmium treatment were analyzed on 2-dimensional gels. Out of 380 protein spots detected on 2-D gel using Coomassie staining, 11 spots were changed significantly following cadmium treatment. Because of the non-availability of molecular background information on this non-sequenced algal species, cross-species protein identification through ESI-Q-TOF MS/MS was used to identify altered proteins. Two newly induced proteins were identified as malate dehydrogenase orthologue and NADH dehydrogenase orthologue. One suppressed protein was identified to be glyceraldehydes 3-phosphate dehydrogenase A. Protein spot showing a 3-fold increase was identified as mitochondrial NADH: ubiquinone oxidoreductase orthologue. However, we could not find any matches in the database from ESI-Q-TOF MS/MS for the remaining seven proteins, thus only partial peptide sequences of these proteins were found.
Assuntos
Cádmio/farmacologia , Clorófitas/metabolismo , Biossíntese de Proteínas/efeitos dos fármacos , Sequência de Aminoácidos , Complexo I de Transporte de Elétrons/biossíntese , Gliceraldeído-3-Fosfato Desidrogenases/biossíntese , Malato Desidrogenase/biossíntese , Dados de Sequência Molecular , NADH Desidrogenase/biossíntese , ProteômicaRESUMO
The glyoxysomal malate dehydrogenase (gMDH) catalyses the formation of oxaloacetate from malate during beta-oxidation of fatty acids in the glyoxysome. A partial Pisum sativum L. (cv. Greenfeast) cDNA was first isolated from a suppression subtractive hybridisation cDNA library obtained from heavy metal stressed plants. The full length cDNA was then isolated by rapid amplification of cDNA ends. The translated sequence showed strong similarity to Cucumis sativus and Citrullus lanatus gMDH including a typical glyoxysome-targeting presequence comprising the PTS2 motif and a cleavage site for a cystein-directed protease. Exposure of pea plants to Cd2+ induced expression of the gMDH gene in mature pea leaves indicating that the enzyme is under environmental control in addition to the normal developmental regulation pattern.
Assuntos
Cádmio/toxicidade , Glioxissomos/enzimologia , Malato Desidrogenase/biossíntese , Malato Desidrogenase/genética , Pisum sativum/genética , Sequência de Aminoácidos , Sequência de Bases , Northern Blotting , Primers do DNA , DNA Complementar/genética , Indução Enzimática/efeitos dos fármacos , Dados de Sequência Molecular , Técnicas de Amplificação de Ácido Nucleico , Pisum sativum/enzimologia , Análise de Sequência de DNA , Homologia de SequênciaRESUMO
In newborns and small mammals, cold-induced adaptive (or nonshivering) thermogenesis is produced primarily in brown adipose tissue (BAT). Heat production is stimulated by the sympathetic nervous system, but it has an absolute requirement for thyroid hormone. We used the thyroid hormone receptor-beta--selective (TR-beta--selective) ligand, GC-1, to determine by a pharmacological approach whether adaptive thermogenesis was TR isoform--specific. Hypothyroid mice were treated for 10 days with varying doses of T3 or GC-1. The level of uncoupling protein 1 (UCP1), the key thermogenic protein in BAT, was restored by either T3 or GC-1 treatment. However, whereas interscapular BAT in T3-treated mice showed a 3.0 degrees C elevation upon infusion of norepinephrine, indicating normal thermogenesis, the temperature did not increase (<0.5 degrees C) in GC-1--treated mice. When exposed to cold (4 degrees C), GC-1--treated mice also failed to maintain core body temperature and had reduced stimulation of BAT UCP1 mRNA, indicating impaired adrenergic responsiveness. Brown adipocytes isolated from hypothyroid mice replaced with T3, but not from those replaced with GC-1, had normal cAMP production in response to adrenergic stimulation in vitro. We conclude that two distinct thyroid-dependent pathways, stimulation of UCP1 and augmentation of adrenergic responsiveness, are mediated by different TR isoforms in the same tissue.
Assuntos
Acetatos/farmacologia , Tecido Adiposo Marrom/fisiologia , Fenóis/farmacologia , Isoformas de Proteínas/efeitos dos fármacos , Receptores dos Hormônios Tireóideos/efeitos dos fármacos , Sistema Nervoso Simpático/fisiologia , Termogênese/fisiologia , Hormônios Tireóideos/fisiologia , Adaptação Fisiológica/efeitos dos fármacos , Adipócitos/efeitos dos fármacos , Adipócitos/metabolismo , Tecido Adiposo Marrom/efeitos dos fármacos , Animais , Proteínas de Transporte/biossíntese , Proteínas de Transporte/genética , Temperatura Baixa , AMP Cíclico/biossíntese , Regulação da Expressão Gênica/efeitos dos fármacos , Glicerolfosfato Desidrogenase/biossíntese , Glicerolfosfato Desidrogenase/genética , Frequência Cardíaca/efeitos dos fármacos , Humanos , Hipotireoidismo/complicações , Hipotireoidismo/tratamento farmacológico , Hipotireoidismo/fisiopatologia , Canais Iônicos , Fígado/efeitos dos fármacos , Fígado/enzimologia , Malato Desidrogenase/biossíntese , Malato Desidrogenase/genética , Masculino , Proteínas de Membrana/biossíntese , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Mitocondriais , Norepinefrina/farmacologia , Isoformas de Proteínas/genética , Isoformas de Proteínas/fisiologia , Ratos , Receptores dos Hormônios Tireóideos/genética , Receptores dos Hormônios Tireóideos/fisiologia , Termogênese/efeitos dos fármacos , Tri-Iodotironina/farmacologia , Tri-Iodotironina/uso terapêutico , Proteína Desacopladora 1RESUMO
In liver, insulin stimulates the transcription of the gene encoding the cytosolic form of malic enzyme (ME) and modulates protein binding to two putative insulin response sequences (IRSs) in the ME promoter. One of these IRSs resembles that identified in the phosphoenolpyruvate carboxykinase (PEPCK) gene, whereas the other resembles that defined in the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene. To assess the functional significance of these changes in protein binding, a series of truncated ME-chloramphenicol acetyl-transferase (CAT) fusion genes were transiently transfected into rat H4IIE hepatoma cells. Deletion of the PEPCK-like IRS motif had no effect on the stimulation of CAT expression by insulin. Instead, the stimulatory effect of insulin was mediated through an AP-1 motif and an Egr-1 binding site that overlaps the GAPDH-like IRS motif. Both the ME AP-1 motif and the AP-1 motif identified in the collagenase-1 gene promoter were able to confer a stimulatory effect of insulin on the expression of a heterologous fusion gene, but surprisingly only the latter was able to confer a stimulatory effect of phorbol esters. Instead, the data suggest that AP-1 binds the ME AP-1 motif in an activated state such that phorbol ester treatment has no additional effect. The collagenase and ME AP-1 motifs were both shown to bind mainly Jun D and Fra-2, with similar affinities. However, the results of a proteolytic clipping bandshift assay suggest that these proteins bind the collagenase and ME AP-1 motifs in distinct conformations, which potentially explain the differences in phorbol ester signaling through these elements.
Assuntos
Insulina/farmacologia , Isoenzimas/biossíntese , Fígado/efeitos dos fármacos , Malato Desidrogenase/biossíntese , Regiões Promotoras Genéticas , Transcrição Gênica/genética , Animais , Colagenases/genética , Citosol/enzimologia , Isoenzimas/genética , Fígado/metabolismo , Neoplasias Hepáticas Experimentais/patologia , Malato Desidrogenase/genética , Ligação Proteica , Ratos , Proteínas Recombinantes de Fusão/biossíntese , Alinhamento de Sequência , Homologia de Sequência do Ácido Nucleico , Transfecção , Células Tumorais CultivadasRESUMO
The ontogeny of the cerebral pyruvate recycling pathway and the cellular localization of associated enzymes, malic enzyme (ME) and phosphoenolpyruvate carboxykinase (PEPCK), have been investigated using a combination of 13C NMR spectroscopy, enzymatic analysis, and molecular biology approaches. Activity of the pathway, using [1,2-(13)C2]acetate as a substrate, was detected by 13C NMR in brain extracts 3 weeks after birth, increasing progressively up to the third month of age. In whole-brain homogenates, ME activity increased to adult levels with the same time course as the recycling pathway. PEPCK activity was low during the first 2 weeks of life and decreased further toward adulthood. ME and PEPCK activity were found in primary cultures of astrocytes and in synaptosomal fractions of adult brain. Primary cultures of cortical neurons showed PEPCK activity but no detectable ME activity. The cytosolic ME gene was expressed in primary cultures of neurons and in astrocytes as well as in the neonatal and adult brain. The PEPCK gene was expressed both in primary cultures of cortical neurons and in astrocytes, but the level of its expression in the neonatal and adult brain was undetectable.
Assuntos
Encéfalo/metabolismo , Malato Desidrogenase/metabolismo , Fosfoenolpiruvato Carboxiquinase (GTP)/metabolismo , Ácido Pirúvico/metabolismo , Animais , Animais Recém-Nascidos , Astrócitos/enzimologia , Encéfalo/enzimologia , Encéfalo/crescimento & desenvolvimento , Encéfalo/ultraestrutura , Células Cultivadas , Espectroscopia de Ressonância Magnética , Malato Desidrogenase/biossíntese , Malato Desidrogenase/ultraestrutura , Neurônios/enzimologia , Especificidade de Órgãos , Fosfoenolpiruvato Carboxiquinase (GTP)/biossíntese , Fosfoenolpiruvato Carboxiquinase (GTP)/ultraestrutura , Ratos , Ratos Wistar , Sinaptossomos/enzimologia , Células Tumorais CultivadasRESUMO
Glyoxysomal malate dehydrogenase (gMDH) is an enzyme of the glyoxylate cycle that participates in degradation of storage oil. We have cloned a cDNA for gMDH from etiolated pumpkin cotyledons that encodes a polypeptide consisting of 356 amino acid residues. The nucleotide and N-terminal amino acid sequences revealed that gMDH is synthesized as a precursor with an N-terminal extrapeptide. The N-terminal presequence of 36 amino acid residues contains two regions homologous to those of other microbody proteins, which are also synthesized as large precursors. To investigate the functions of the N-terminal presequence of gMDH, we generated transgenic Arabidopsis that expressed a chimeric protein consisting of beta-glucuronidase and the N-terminal region of gMDH. Immunological and immunocytochemical studies revealed that the chimeric protein was imported into microbodies such as glyoxysomes and leaf peroxisomes and was then subsequently processed. Site-directed mutagenesis studies showed that the conserved amino acids in the N-terminal presequence, Arg-10 and His-17, function as recognition sites for the targeting to plant microbodies, and Cys-36 in the presequence is responsible for its processing. These results correspond to those from the analyses of glyoxysomal citrate synthase (gCS), which was also synthesized as a large precursor, suggesting that common mechanisms that can recognize the targeting or the processing of gMDH and gCS function in higher plant cells.
Assuntos
Cucurbitaceae/enzimologia , Precursores Enzimáticos/biossíntese , Malato Desidrogenase/biossíntese , Organelas/enzimologia , Sequência de Aminoácidos , Arabidopsis , Clonagem Molecular , Cucurbitaceae/ultraestrutura , DNA Complementar , Precursores Enzimáticos/química , Precursores Enzimáticos/genética , Malato Desidrogenase/química , Malato Desidrogenase/genética , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Organelas/ultraestrutura , Plantas Geneticamente Modificadas , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Sementes/enzimologia , Alinhamento de Sequência , Homologia de Sequência de AminoácidosRESUMO
Recent epidemiological evidence suggests that adult cardiovascular risk is determined by birth weight and factors that influence birth weight, such as maternal nutrition. Data from animal models suggest that an interaction between nutrition and glucocorticoid hormones "programs" increased risk of adult hypertension. Increased fetal exposure to maternal glucocorticoids that is proposed to occur from a reduction in the placental barrier to maternal glucocorticoid, 11beta-hydroxysteroid dehydrogenase, is suggested to program hypertension in the resultant offspring from both glucocorticoid-treated and maternally protein-restricted rats. The extent to which postnatal glucocorticoid stimulation may influence the progression of hypertension in the offspring from protein-restricted rat dams was assessed in 6-week-old male Wistar rats, prenatally exposed to either an 18% casein (control) or 9% casein (low protein) diet. Rats from each dietary group were sham operated, adrenalectomized or adrenalectomized, and treated with 20 mg corticosterone/kg body weight per day. Before surgery, systolic blood pressure was significantly higher in the low protein-exposed rats compared with controls (165+/-3.8 versus 142+/-3.3 mm Hg, P<.0001). Adrenalectomy of the low protein-exposed animals significantly reduced the blood pressure to control levels, while corticosterone replacement restored the hypertensive state. No effect of adrenalectomy on blood pressure was observed in 18% casein controls. In both dietary groups adrenalectomy decreased brain, but not hepatic, glucocorticoid-sensitive enzyme activities and corticosterone treatment elevated activities of all enzymes. The data suggest that maternal diet-induced hypertension is dependent on an intact adrenal gland postnatally and that glucocorticoids are key trophic agents in maintaining the high blood pressure.
Assuntos
Adrenalectomia , Pressão Sanguínea/fisiologia , Corticosterona/farmacologia , Dieta com Restrição de Proteínas , Hipertensão/fisiopatologia , Efeitos Tardios da Exposição Pré-Natal , 11-beta-Hidroxiesteroide Desidrogenases , Análise de Variância , Animais , Pressão Sanguínea/efeitos dos fármacos , Encéfalo/enzimologia , Caseínas , Indução Enzimática , Feminino , Glutamato-Amônia Ligase/biossíntese , Glicerolfosfato Desidrogenase/biossíntese , Hidroxiesteroide Desidrogenases/metabolismo , Hipertensão/etiologia , Fígado/enzimologia , Malato Desidrogenase/biossíntese , Masculino , Gravidez , Ratos , Ratos Wistar , Tirosina Transaminase/biossínteseRESUMO
Leptin is thought to exert its actions on energy homeostasis through the long form of the leptin receptor (OB-Rb), which is present in the hypothalamus and in certain peripheral organs, including adipose tissue. In this study, we examined whether leptin has direct effects on the function of brown and white adipose tissue (BAT and WAT, respectively) at the metabolic and molecular levels. The chronic peripheral intravenous administration of leptin in vivo for 4 d resulted in a 1.6-fold increase in the in vivo glucose utilization index of BAT, whereas no significant change was found after intracerebroventricular administration compared with pair-fed control rats, compatible with a direct effect of leptin on BAT. The effect of leptin on WAT fat pads from lean Zucker Fa/ fa rats was assessed ex vivo, where a 9- and 16-fold increase in the rate of lipolysis was observed after 2 h of exposure to 0.1 and 10 nM leptin, respectively. In contrast, no increase in lipolysis was observed in the fat pads from obese fa/fa rats, which harbor an inactivating mutation in the OB-Rb. At the level of gene expression, leptin treatment for 24 h increased malic enzyme and lipoprotein lipase RNA 1.8+/-0.17 and 1.9+/-0.14-fold, respectively, while aP2 mRNA levels were unaltered in primary cultures of brown adipocytes from lean Fa/fa rats. Importantly, however, no significant effect of leptin was observed on these genes in brown adipocytes from obese fa/fa animals. The presence of OB-Rb receptors in adipose tissue was substantiated by the detection of its transcripts by RT-PCR, and leptin treatment in vivo and in vitro activated the specific STATs implicated in the signaling pathway of the OB-Rb. Taken together, our data strongly suggest that leptin has direct effects on BAT and WAT, resulting in the activation of the Jak/STAT pathway and the increased expression of certain target genes, which may partially account for the observed increase in glucose utilization and lipolysis in leptin-treated adipose tissue.