RESUMEN
Utilization of abundant and cheap carbon sources can effectively reduce the production cost and enhance the economic feasibility. Acetate is a promising carbon source to achieve cost-effective microbial processes. In this study, we engineered an Escherichia coli strain to produce itaconic acid from acetate. As acetate is known to inhibit cell growth, we initially screened for a strain with a high tolerance to 10 g/L of acetate in the medium, and the W strain was selected as the host. Subsequently, the WC strain was obtained by overexpression of cad (encoding cis-aconitate decarboxylase) using a synthetic promoter and 5' UTR. However, the WC strain produced only 0.13 g/L itaconic acid because of low acetate uptake. To improve the production, the acetate assimilating pathway and glyoxylate shunt pathway were amplified by overexpression of pathway genes as well as its deregulation. The resulting strain, WCIAG4 produced 3.57 g/L itaconic acid (16.1% of theoretical maximum yield) after 88 hr of fermentation with rapid acetate assimilation. These efforts support that acetate can be a potential feedstock for biochemical production with engineered E. coli.
Asunto(s)
Ácido Acético/metabolismo , Aconitato Hidratasa , Proteínas de Escherichia coli , Escherichia coli , Ingeniería Metabólica , Succinatos/metabolismo , Aconitato Hidratasa/biosíntesis , Aconitato Hidratasa/genética , Escherichia coli/genética , Escherichia coli/crecimiento & desarrollo , Proteínas de Escherichia coli/biosíntesis , Proteínas de Escherichia coli/genéticaRESUMEN
Ginsenoside Re is an active component in ginseng that has attracted much attention because of its evident therapeutic effects on the cardiovascular system. However, little basic information is available on the mechanisms and pharmacological effects of ginsenoside Re. The potential mechanisms and protective effects of Re on H2O2-induced oxidative injury in human umbilical vein endothelial cells (HUVECs) were investigated in this study. An oxidative injury model was established using H2O2. The anti-oxidative effects of Re were determined using a series of experiments, such as MTT and anti-oxidative indicator assays. The potential protective mechanisms of Re were explored at the proteomic level, and differentially expressed proteins were validated by quantitative real-time polymerase chain reaction and western blotting. Results indicated that Re could be a potential anti-oxidant to protect HUVECs against oxidative stress damage. Proteomic analysis showed that the expression of 23 protein spots was upregulated in Re and H2O2 groups to resist oxidative stress, 15 of which were identified by their mass spectrum. These upregulated proteins were involved in stress response, anti-oxidative systems, protein synthesis, regulation of transcription and post-translational modifications, and repair of mitochondrial functions. This study may provide new insights into the mechanisms of ginsenoside Re in protecting the cardiovascular system.
Asunto(s)
Ginsenósidos/farmacología , Células Endoteliales de la Vena Umbilical Humana/efectos de los fármacos , Peróxido de Hidrógeno/metabolismo , Estrés Oxidativo/efectos de los fármacos , Proteómica , Aconitato Hidratasa/biosíntesis , Aconitato Hidratasa/genética , Anexina A3/biosíntesis , Anexina A3/genética , Sistema Cardiovascular , Proliferación Celular/efectos de los fármacos , Glutatión Peroxidasa/efectos de los fármacos , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Humanos , Peróxido de Hidrógeno/toxicidad , L-Lactato Deshidrogenasa/efectos de los fármacos , Malondialdehído/metabolismo , Potencial de la Membrana Mitocondrial , Mitocondrias/fisiología , Óxido Nítrico/metabolismo , Peroxirredoxinas/biosíntesis , Peroxirredoxinas/genética , Biosíntesis de Proteínas/efectos de los fármacos , Procesamiento Proteico-Postraduccional , Proteínas/efectos de los fármacos , Proteínas/genética , Superóxido Dismutasa/efectos de los fármacosRESUMEN
Folding of aggregation prone recombinant proteins through co-expression of chaperonin GroEL and GroES has been a popular practice in the effort to optimize preparation of functional protein in Escherichia coli. Considering the demand for functional recombinant protein products, it is desirable to apply the chaperone assisted protein folding strategy for enhancing the yield of properly folded protein. Toward the same direction, it is also worth attempting folding of multiple recombinant proteins simultaneously over-expressed in E. coli through the assistance of co-expressed GroEL-ES. The genesis of this thinking was originated from the fact that cellular GroEL and GroES assist in the folding of several endogenous proteins expressed in the bacterial cell. Here we present the experimental findings from our study on co-expressed GroEL-GroES assisted folding of simultaneously over-expressed proteins maltodextrin glucosidase (MalZ) and yeast mitochondrial aconitase (mAco). Both proteins mentioned here are relatively larger and aggregation prone, mostly form inclusion bodies, and undergo GroEL-ES assisted folding in E. coli cells during over-expression. It has been reported that the relative yield of properly folded functional forms of MalZ and mAco with the exogenous GroEL-ES assistance were comparable with the results when these proteins were overexpressed alone. This observation is quite promising and highlights the fact that GroEL and GroES can assist in the folding of multiple substrate proteins simultaneously when over-expressed in E. coli. This method might be a potential tool for enhanced production of multiple functional recombinant proteins simultaneously in E. coli.
Asunto(s)
Chaperonina 10/fisiología , Chaperonina 60/fisiología , Escherichia coli/metabolismo , Aconitato Hidratasa/biosíntesis , Aconitato Hidratasa/genética , Escherichia coli/crecimiento & desarrollo , Proteínas de Escherichia coli/biosíntesis , Proteínas de Escherichia coli/genética , Glicósido Hidrolasas/biosíntesis , Glicósido Hidrolasas/genética , Pliegue de Proteína , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genética , Activación TranscripcionalRESUMEN
High intensity training induces muscle damage in dystrophin-deficient mdx mice, an animal model for Duchenne muscular dystrophy. However, low intensity training (LIT) rescues the mdx phenotype and even reduces the level of protein carbonylation, a marker of oxidative damage. Until now, beneficial effects of LIT were mainly assessed at the physiological level. We investigated the effects of LIT at the molecular level on 8-week-old wild-type and mdx muscle using 2D Western blot and protein-protein interaction analysis. We found that the fast isoforms of troponin T and myosin binding protein C as well as glycogen phosphorylase were overcarbonylated and downregulated in mdx muscle. Some of the mitochondrial enzymes of the citric acid cycle were overcarbonylated, whereas some proteins of the respiratory chain were downregulated. Of functional importance, ATP synthase was only partially assembled, as revealed by Blue Native PAGE analysis. LIT decreased the carbonylation level and increased the expression of fast isoforms of troponin T and of myosin binding protein C, and glycogen phosphorylase. In addition, it increased the expression of aconitate hydratase and NADH dehydrogenase, and fully restored the ATP synthase complex. Our study demonstrates that the benefits of LIT are associated with lowered oxidative damage as revealed by carbonylation and higher expression of proteins involved in energy metabolism and muscle contraction. Potentially, these results will help to design therapies for DMD based on exercise mimicking drugs.
Asunto(s)
Metabolismo Energético/fisiología , Contracción Muscular/fisiología , Músculo Esquelético/metabolismo , Condicionamiento Físico Animal/métodos , Carbonilación Proteica/fisiología , Aconitato Hidratasa/biosíntesis , Animales , Proteínas Portadoras/biosíntesis , Proteínas Portadoras/genética , Ciclo del Ácido Cítrico/fisiología , Modelos Animales de Enfermedad , Regulación hacia Abajo , Distrofina/genética , Glucógeno Fosforilasa/biosíntesis , Glucógeno Fosforilasa/genética , Masculino , Ratones , Ratones Endogámicos mdx , Ratones Transgénicos , Mitocondrias/enzimología , Mitocondrias/metabolismo , ATPasas de Translocación de Protón Mitocondriales/metabolismo , Distrofia Muscular de Duchenne , NADH Deshidrogenasa/biosíntesis , Estrés Oxidativo , Isoformas de Proteínas/genética , Troponina T/biosíntesis , Troponina T/genéticaRESUMEN
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.
Asunto(s)
Ciclo del Ácido Cítrico/genética , Metabolismo Energético/genética , Mitocondrias/patología , Neoplasias/patología , Aconitato Hidratasa/biosíntesis , Proliferación Celular/fisiología , Ciclo del Ácido Cítrico/fisiología , Fumarato Hidratasa/genética , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/fisiología , Isocitrato Deshidrogenasa/genética , Malato Deshidrogenasa/biosíntesis , Mitocondrias/genética , Mitocondrias/metabolismo , Mutación , Neoplasias/genética , Neoplasias/metabolismo , Fosforilación Oxidativa , Prolil Hidroxilasas/metabolismo , Proteínas Serina-Treonina Quinasas/biosíntesis , Piruvato Deshidrogenasa Quinasa Acetil-Transferidora , Transducción de Señal/genética , Succinato Deshidrogenasa/genéticaRESUMEN
Transglutaminase 2 (TGase 2) expression and glycolysis are increased in most renal cell carcinoma (RCC) cell lines compared to the HEK293 kidney cell line. Although increased glycolysis and altered tricarboxylic acid cycle are common in RCC, the detailed mechanism by which this phenomenon occurs remains to be elucidated. In the present study, TGase 2 siRNA treatment lowered glucose consumption and lactate levels by about 20-30 % in RCC cells; conversely, high expression of TGase 2 increased glucose consumption and lactate production together with decreased mitochondrial aconitase (Aco 2) levels. In addition, TGase 2 siRNA increased mitochondrial membrane potential and ATP levels by about 20-30 % and restored Aco 2 levels in RCC cells. Similarly, Aco 2 levels and ATP production decreased significantly upon TGase 2 overexpression in HEK293 cells. Therefore, TGase 2 leads to depletion of Aco 2, which promotes glycolytic metabolism in RCC cells.
Asunto(s)
Carcinoma de Células Renales/enzimología , Proteínas de Unión al GTP/biosíntesis , Neoplasias Renales/enzimología , Transglutaminasas/biosíntesis , Aconitato Hidratasa/biosíntesis , Regulación hacia Abajo , Regulación Enzimológica de la Expresión Génica , Glucosa/metabolismo , Glucólisis/efectos de los fármacos , Células HEK293 , Humanos , Potencial de la Membrana Mitocondrial , Proteína Glutamina Gamma Glutamiltransferasa 2 , Células Tumorales Cultivadas , Proteína Supresora de Tumores del Síndrome de Von Hippel-Lindau/metabolismoRESUMEN
The Fe-S cluster of mitochondrial aconitase is rapidly and selectively inactivated by oxidants, yielding an inactive enzyme that can be reactivated by reductants and iron in vivo. In order to elucidate the metabolic impact of oxidant-dependent aconitase inhibition over the citric acid cycle, the respiratory chain reactions, and reactive species formation, we performed a metabolic analysis using isolated mitochondria from different rat tissues. Titrations with fluorocitrate showed IC50 for aconitase inhibition ranging from 7 to 24 µM. The aconitase inhibition threshold in mitochondrial oxygen consumption was determined to range from 63 to 98%. Of the tissues examined, brain and heart exhibited the highest values in the flux control coefficient (> 0.95). Aconitase-specific activity varied widely among tissues examined from ~60 mU/mg in liver to 321 mU/mg in kidney at 21% O2. In brain and heart, aconitase-specific activity increased by 42 and 12%, respectively, at 2% O2 reflecting aconitase inactivation by oxygen-derived oxidants at 21% O2. Both mitochondrial membrane potential and hydrogen peroxide production significantly decreased upon aconitase inhibition in heart and brain mitochondria. These results indicate that aconitase can exert control over respiration (with tissue specificity) and support the hypothesis that inactivation of aconitase may provide a control mechanism to prevent O2(â-) and H2O2 formation by the respiratory chain.
Asunto(s)
Aconitato Hidratasa/biosíntesis , Peróxido de Hidrógeno/metabolismo , Mitocondrias/enzimología , Consumo de Oxígeno/fisiología , Superóxidos/metabolismo , Aconitato Hidratasa/antagonistas & inhibidores , Animales , Encéfalo/metabolismo , Ciclo del Ácido Cítrico/fisiología , Transporte de Electrón/fisiología , Potencial de la Membrana Mitocondrial/fisiología , Mitocondrias/metabolismo , Miocardio/metabolismo , Estrés Oxidativo , Ratas , Ratas WistarRESUMEN
Itaconic acid is an unsaturated dicarboxylic acid which has a high potential as a biochemical building block. It can be microbially produced from some Aspergillus species, such as Aspergillus itaconicus and Aspergillus terreus. However, the achieved titers are significantly lower as compared to the citric acid production by A. niger. Heterologous expression of cis-aconitate decarboxylase in A. niger leads to the accumulation of small amounts of itaconic acid. Additional expression of aconitase, the second enzyme metabolically linking citric acid and itaconic acid improves productivity. However, proper organelle targeting of the enzymes appears to be an important point to consider. Here we compare the mitochondrial expression with the cytosolic expression of cis-aconitate decarboxylase or aconitase in A. niger. Heterologous expression of both enzymes in the mitochondria doubles the productivity compared to strains which express the enzymes in the cytosol. It is essential to target enzymes to the correct compartment in order to establish a proper flux through a compartmentalized pathway.
Asunto(s)
Aspergillus niger/metabolismo , Succinatos/metabolismo , Aconitato Hidratasa/biosíntesis , Aconitato Hidratasa/genética , Aspergillus niger/genética , Carboxiliasas/biosíntesis , Carboxiliasas/genética , Ácido Cítrico/metabolismo , Citosol/metabolismo , Proteínas Fúngicas/biosíntesis , Proteínas Fúngicas/genética , Ingeniería Metabólica/métodos , Mitocondrias/enzimología , Mitocondrias/genéticaRESUMEN
Alterations in energy metabolism play a major role in cancer development. Aconitase (ACO2) is an essential enzyme located in the mitochondria and catalyzes the interconversion of citrate and isocitrate in the tricarboxylic acid cycle. Recent studies suggest that the expression of ACO2 may be altered in certain types of cancer. The purpose of this study was to examine ACO2 expression in clinical tumor specimens from patients with gastric cancer and to evaluate the clinical relevance of ACO2 expression in gastric cancer. A total of 456 paraffin-embedded gastric cancer tissues and 30 pairs of freshly frozen tissues were used in this study. Real-time quantitative reverse transcription polymerase chain reaction, western blotting, and immunohistochemical staining were performed to measure ACO2 expression in tumor tissues and matched adjacent non-tumorous tissues. The results showed that the expression of ACO2 was significantly down-regulated in gastric cancer tissues compared with matched adjacent nontumorous tissues and was associated with clinical stage (p = 0.001), T classification (p = 0.027), N classification (p = 0.012), M classification (p = 0.002), and pathological differentiation states (p = 0.036). Patients with lower ACO2 expression had a shorter survival time than those with higher ACO2 expression. Univariate and multivariate analyses indicated that ACO2 expression functions as an independent prognostic factor (p < 0.001). Our data suggested that ACO2 could play an important role in gastric cancer and may potentially serve as a prognostic biomarker.
Asunto(s)
Aconitato Hidratasa/antagonistas & inhibidores , Biomarcadores de Tumor/antagonistas & inhibidores , Regulación Neoplásica de la Expresión Génica , Proteínas Mitocondriales/antagonistas & inhibidores , Neoplasias Gástricas/diagnóstico , Neoplasias Gástricas/enzimología , Aconitato Hidratasa/biosíntesis , Aconitato Hidratasa/genética , Anciano , Biomarcadores de Tumor/biosíntesis , Biomarcadores de Tumor/genética , Femenino , Estudios de Seguimiento , Humanos , Masculino , Persona de Mediana Edad , Proteínas Mitocondriales/biosíntesis , Proteínas Mitocondriales/genética , Pronóstico , ARN Mensajero/antagonistas & inhibidores , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Neoplasias Gástricas/genética , Tasa de Supervivencia/tendenciasRESUMEN
The role of the CcpC regulatory protein as a repressor of the genes encoding the tricarboxylic acid branch enzymes of the Krebs cycle (citrate synthase, citZ; aconitase, citB; and isocitrate dehydrogenase, citC) has been established for both Bacillus subtilis and Listeria monocytogenes. In addition, hyperexpression of citB-lacZ reporter constructs in an aconitase null mutant strain has been reported for B. subtilis. We show here that such hyperexpression of citB occurs in L. monocytogenes as well as in B. subtilis and that in both species the hyperexpression is unexpectedly dependent on CcpC. We propose a revision of the existing CcpC-citB regulatory scheme and suggest a mechanism of regulation in which CcpC represses citB expression at low citrate levels and activates citB expression when citrate levels are high.
Asunto(s)
Aconitato Hidratasa/biosíntesis , Bacillus subtilis/enzimología , Bacillus subtilis/genética , Regulación Bacteriana de la Expresión Génica , Listeria monocytogenes/enzimología , Listeria monocytogenes/genética , Proteínas Represoras/metabolismo , Fusión Artificial Génica , Eliminación de Gen , Genes Reporteros , beta-Galactosidasa/biosíntesis , beta-Galactosidasa/genéticaRESUMEN
Metabolic syndrome (MetS) is commonly associated with elevated renin-angiotensin system, oxidative stress, and steatohepatitis with down-regulation of uncoupling proteins (UCPs). However, the mechanisms linking renin-angiotensin system, steatosis, and UCP2 to hepatic oxidative damage during insulin resistance are not described. To test the hypothesis that angiotensin receptor activation contributes to decreased hepatic UCP2 expression and aconitase activity and to increased oxidative damage after increased glucose intake in a model of MetS, lean and obese Long Evans rats (n = 10/group) were randomly assigned to the following groups: 1) untreated Long Evans Tokushima Otsuka (lean, strain control), 2) untreated Otsuka Long Evans Tokushima Fatty (OLETF) (MetS model), 3) OLETF + angiotensin receptor blocker (ARB) (10 mg olmesartan/kg·d × 6 wk), 4) OLETF + high glucose (HG) (5% in drinking water × 6 wk), and 5) OLETF + ARB + HG (ARB/HG × 6 wk). HG increased body mass (37%), plasma triglycerides (TGs) (35%), plasma glycerol (87%), plasma free fatty acids (28%), and hepatic nitrotyrosine (74%). ARB treatment in HG decreased body mass (12%), plasma TG (15%), plasma glycerol (23%), plasma free fatty acids (14%), and hepatic TG content (42%), suggesting that angiotensin receptor type 1 (AT1) activation and increased adiposity contribute to the development of obesity-related dyslipidemia. ARB in HG also decreased hepatic nitrotyrosine and increased hepatic UCP2 expression (59%) and aconitase activity (40%), as well as antioxidant enzyme activities (50-120%), suggesting that AT1 activation also contributes to protein oxidation, impaired lipid metabolism, and antioxidant metabolism in the liver. Thus, in addition to promoting obesity-related hypertension, AT1 activation may also impair lipid metabolism and antioxidant capacity, resulting in steatosis via decreased UCP2 and tricarboxylic acid cycle activity.
Asunto(s)
Aconitato Hidratasa/biosíntesis , Antagonistas de Receptores de Angiotensina/farmacología , Regulación Enzimológica de la Expresión Génica , Resistencia a la Insulina , Canales Iónicos/biosíntesis , Hígado/metabolismo , Proteínas Mitocondriales/biosíntesis , Succinato Deshidrogenasa/biosíntesis , Animales , Antioxidantes/metabolismo , Ácidos Grasos/metabolismo , Hígado Graso , Hígado/enzimología , Masculino , Obesidad/metabolismo , Estrés Oxidativo , Ratas , Ratas Long-Evans , Proteína Desacopladora 2RESUMEN
IscA is a key member of the iron-sulfur cluster assembly machinery in prokaryotic and eukaryotic organisms; however, the physiological function of IscA still remains elusive. In the present paper we report the in vivo evidence demonstrating the iron-binding activity of IscA in Escherichia coli cells. Supplement of exogenous iron (1 µM) in M9 minimal medium is sufficient to maximize the iron binding in IscA expressed in E. coli cells under aerobic growth conditions. In contrast, IscU, an iron-sulfur cluster assembly scaffold protein, or CyaY, a bacterial frataxin homologue, fails to bind any iron in E. coli cells under the same experimental conditions. Interestingly, the strong iron-binding activity of IscA is greatly diminished in E. coli cells under anaerobic growth conditions. Additional studies reveal that oxygen in medium promotes the iron binding in IscA, and that the iron binding in IscA in turn prevents formation of biologically inaccessible ferric hydroxide under aerobic conditions. Consistent with the differential iron-binding activity of IscA under aerobic and anaerobic conditions, we find that IscA and its paralogue SufA are essential for the iron-sulfur cluster assembly in E. coli cells under aerobic growth conditions, but not under anaerobic growth conditions. The results provide in vivo evidence that IscA may act as an iron chaperone for the biogenesis of iron-sulfur clusters in E. coli cells under aerobic conditions.
Asunto(s)
Proteínas Portadoras/química , Proteínas Portadoras/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Proteínas de Unión a Hierro/química , Proteínas de Unión a Hierro/metabolismo , Proteínas Hierro-Azufre/metabolismo , Aconitato Hidratasa/biosíntesis , Proteínas Bacterianas/metabolismo , Proteínas Portadoras/genética , Espectroscopía de Resonancia por Spin del Electrón , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Proteínas de Unión a Hierro/genética , Oxígeno , EspectrofotometríaRESUMEN
Iron-sulfur proteins play an essential role in many biologic processes. Hence, understanding their assembly is an important goal. In Escherichia coli, the protein IscA is a product of the isc (iron-sulfur cluster) operon and functions in the iron-sulfur cluster assembly pathway in this organism. IscA is conserved in evolution, but its function in mammalian cells is not known. Here, we provide evidence for a role for a human homologue of IscA, named IscA1, in iron-sulfur protein biogenesis. We observe that small interfering RNA knockdown of IscA1 in HeLa cells leads to decreased activity of two mitochondrial iron-sulfur enzymes, succinate dehydrogenase and mitochondrial aconitase, as well as a cytosolic iron-sulfur enzyme, cytosolic aconitase. IscA1 is observed both in cytosolic and mitochondrial fractions. We find that IscA1 interacts with IOP1 (iron-only hydrogenase-like protein 1)/NARFL (nuclear prelamin A recognition factor-like), a cytosolic protein that plays a role in the cytosolic iron-sulfur protein assembly pathway. We therefore propose that human IscA1 plays an important role in both mitochondrial and cytosolic iron-sulfur cluster biogenesis, and a notable component of the latter is the interaction between IscA1 and IOP1.
Asunto(s)
Aconitato Hidratasa/biosíntesis , Citosol/metabolismo , Hidrogenasas/metabolismo , Proteínas Hierro-Azufre/metabolismo , Mitocondrias/metabolismo , Proteínas Mitocondriales/biosíntesis , Succinato Deshidrogenasa/biosíntesis , Aconitato Hidratasa/genética , Animales , Células COS , Chlorocebus aethiops , Técnicas de Silenciamiento del Gen , Células HeLa , Humanos , Hidrogenasas/genética , Proteínas Hierro-Azufre/antagonistas & inhibidores , Proteínas Hierro-Azufre/genética , Proteínas Mitocondriales/antagonistas & inhibidores , Proteínas Mitocondriales/genética , Proteínas Mitocondriales/metabolismo , Unión Proteica/fisiología , ARN Interferente Pequeño , Succinato Deshidrogenasa/genéticaRESUMEN
A major portion of the over expressed yeast mitochondrial aconitase, a large 82 kDa monomeric TCA cycle enzyme, in Escherichia coli led to the formation of inclusion bodies. Bacterial chaperonin GroEL mediated the correct folding of aconitase with the assistance of its co-chaperonin GroES in an ATP dependent manner. Till date the chaperonin assisted folding of aconitase was limited to the shake flask studies with relatively low yields of folded aconitase. No attempt had yet been made to enhance the yield of chaperone mediated folding of aconitase using a bioreactor. The current report deals with the effect of co-expression of GroEL/GroES in the production of soluble, biologically active recombinant aconitase in E. coli by cultivation in a bioreactor at different temperatures under optimized conditions. It revealed that the yield of functional aconitase was enhanced, either in presence of co-expressed GroEL/ES or at low temperature cultivation. However, the outcome from the chaperone assisted folding of aconitase was more pronounced at lower temperature. A 3-fold enhancement in the yield of functional aconitase from the bioreactor based chaperone assisted folding was obtained as compared to the shake flask study. Hence, the present study provides optimized conditions for increasing the yield of functional aconitase by batch cultivation in a bioreactor.
Asunto(s)
Aconitato Hidratasa/biosíntesis , Reactores Biológicos , Escherichia coli/genética , Chaperonas Moleculares/metabolismo , Aconitato Hidratasa/genética , Aconitato Hidratasa/aislamiento & purificación , Aconitato Hidratasa/metabolismo , Biomasa , Cromatografía en Gel , Electroforesis en Gel de Poliacrilamida , Pliegue de Proteína , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismoRESUMEN
The biogenesis of cytosolic iron-sulfur (Fe-S) proteins in mammalian cells is poorly understood. In Saccharomyces cerevisiae, there is a pathway dedicated to cytosolic Fe-S protein maturation that involves several essential proteins. One of these is Nar1, which intriguingly is homologous to iron-only hydrogenases, ancient enzymes that catalyze the formation of hydrogen gas in anaerobic bacteria. There are two orthologues of Nar1 in mammalian cells, iron-only hydrogenase-like protein 1 (IOP1) and IOP2 (also known as nuclear prelamin A recognition factor). We examined IOP1 for a potential role in mammalian cytosolic Fe-S protein biogenesis. We found that knockdown of IOP1 in both HeLa and Hep3B cells decreases the activity of cytosolic aconitase, an Fe-S protein, but not that of mitochondrial aconitase. Knockdown of IOP2, in contrast, had no effect on either. The decrease in aconitase activity upon IOP1 knockdown is rescued by expression of a small interference RNA-resistant version of IOP1. Upon loss of its Fe-S cluster, cytosolic aconitase is known to be converted to iron regulatory protein 1, and consistent with this, we found that IOP1 knockdown increases transferrin receptor 1 mRNA levels and decreases ferritin heavy chain protein levels. IOP1 knockdown also leads to a decrease in activity of xanthine oxidase, a distinct cytosolic Fe-S protein. Taken together, these results provide evidence that IOP1 is involved in mammalian cytosolic Fe-S protein maturation.
Asunto(s)
Citosol/metabolismo , Hidrogenasas/metabolismo , Metaloproteínas/biosíntesis , Biosíntesis de Proteínas/fisiología , Aconitato Hidratasa/biosíntesis , Aconitato Hidratasa/genética , Apoferritinas/biosíntesis , Apoferritinas/genética , Bacterias Anaerobias/genética , Bacterias Anaerobias/metabolismo , Células HeLa , Humanos , Hidrogenasas/genética , Hierro/metabolismo , Proteína 1 Reguladora de Hierro/biosíntesis , Proteína 1 Reguladora de Hierro/genética , Proteínas Hierro-Azufre/genética , Proteínas Hierro-Azufre/metabolismo , Metaloproteínas/genética , Mitocondrias/genética , Mitocondrias/metabolismo , Interferencia de ARN , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Receptores de Transferrina/biosíntesis , Receptores de Transferrina/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Homología de Secuencia de Aminoácido , Azufre/metabolismo , Xantina Oxidasa/biosíntesis , Xantina Oxidasa/genéticaRESUMEN
The activity of mitochondria induces, as a byproduct, a variety of post-translational modifications in associated proteins, which have functional downstream consequences for processes such as apoptosis, autophagy, and plasticity; e.g., reactive oxygen species (ROS), which induce N-formyl-kynurenine from oxidized tryptophans in certain mitochondrial proteins which are localized in close spatial proximity to their source. This type of fast molecular changes has profound influence on cell death and survival with implications in a number of pathologies. The quantitative and differential analysis of bovine heart mitochondria by four 2D-PAGE methods, including 2D-PAGE with high-resolution IEF as first dimension, revealed that due to limited resolution, those methods employing blue native-, tricine-urea-, and 16-BAC-PAGE as the first dimension are less applicable for the differential quantitative analysis of redundant protein spots which might give insight into post-translational modifications that are relevant in age- and stress-related changes. Moreover, 2D-PAGE with high resolution IEF was able to resolve a surprisingly large number of membrane proteins from mitochondrial preparations. For aconitase-2, an enzyme playing an important role in mitochondrial aging, a more thorough molecular analysis of all separable isoforms was performed, leading to the identification of two particular N-formylkynurenine modifications. Next to protein redundancy, native protein-protein interactions, with the potential of relating certain post-translational modification patterns to distinct oligomeric states, e.g., oxidative phosphorylation super complexes, might provide novel and (patho-) physiologically relevant information. Among proteins identified, 14 new proteins (GenBank entries), previously not associated with mitochondria, were found.
Asunto(s)
Aconitato Hidratasa/biosíntesis , Aconitato Hidratasa/química , Quinurenina/análogos & derivados , Mitocondrias Cardíacas/enzimología , Especies Reactivas de Oxígeno/metabolismo , Aconitato Hidratasa/genética , Animales , Biomarcadores/metabolismo , Bovinos , Perfilación de la Expresión Génica , Humanos , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/metabolismo , Quinurenina/químicaRESUMEN
The ability of the fungal pathogen Cryptococcus neoformans to evade the mammalian innate immune response and cause disease is partially due to its ability to respond to and survive nitrosative stress. In this study, we use proteomic and genomic approaches to elucidate the response of C. neoformans to nitric oxide stress. This nitrosative stress response involves both transcriptional, translational, and posttranslational regulation. Proteomic and genomic analyses reveal changes in expression of stress response genes. In addition, genes involved in cell wall organization, respiration, signal transduction, transport, transcriptional control, and metabolism show altered expression under nitrosative conditions. Posttranslational modifications of transaldolase (Tal1), aconitase (Aco1), and the thiol peroxidase, Tsa1, are regulated during nitrosative stress. One stress-related protein up-regulated in the presence of nitric oxide stress is glutathione reductase (Glr1). To further investigate its functional role during nitrosative stress, a deletion mutant was generated. We show that this glr1Delta mutant is sensitive to nitrosative stress and macrophage killing in addition to being avirulent in mice. These studies define the response to nitrosative stress in this important fungal pathogen.
Asunto(s)
Cryptococcus neoformans/efectos de los fármacos , Cryptococcus neoformans/genética , Cryptococcus neoformans/patogenicidad , Regulación Fúngica de la Expresión Génica , Óxido Nítrico/farmacología , Estrés Oxidativo/efectos de los fármacos , Procesamiento Proteico-Postraduccional , Transcripción Genética , Aconitato Hidratasa/biosíntesis , Aconitato Hidratasa/genética , Animales , Cryptococcus neoformans/enzimología , Cryptococcus neoformans/crecimiento & desarrollo , Relación Dosis-Respuesta a Droga , Femenino , Genes Fúngicos , Genómica , Ratones , Ratones Endogámicos CBA , Óxido Nítrico/toxicidad , Peroxidasas/biosíntesis , Peroxidasas/genética , Proteómica , Transaldolasa/biosíntesis , Transaldolasa/genética , VirulenciaRESUMEN
Deletion of gene Rv3676 in Mycobacterium tuberculosis coding for a transcription factor belonging to the cAMP receptor protein (CRP) family caused growth defects in laboratory medium, in bone marrow-derived macrophages and in a mouse model of tuberculosis. Transcript profiling of M. tuberculosis grown in vitro identified 16 genes with significantly altered expression in the mutant compared with the wild type. Analysis of the DNA sequences upstream of the corresponding open reading frames revealed that 12 possessed sequences related to a consensus CRP binding site that could represent the sites of action of Rv3676. These included rpfA, lprQ, whiB1 and ahpC among genes with enhanced expression in the wild type, and Rv3616c-Rv3613c, Rv0188 and lipQ among genes exhibiting enhanced expression in the mutant. The activity of an rpfA::lacZ promoter fusion was lowered in the Rv3676 mutant and by mutation of the predicted Rv3676 binding site. Moreover, the product of Rv3676 (isolated as a TrxA fusion protein) interacted specifically with the rpfA promoter, and binding was inhibited by mutation of the Rv3676 site. Although Rv3676 retains four of the six amino acid residues that bind cAMP in Escherichia coli CRP addition of cAMP did not enhance Rv3676 binding at the rpfA promoter in vitro. In summary, it has been shown that Rv3676 is a direct regulator of rpfA expression, and because rpfA codes for a resuscitation promoting factor this may implicate Rv3676 in reactivation of dormant M. tuberculosis infections.
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Aconitato Hidratasa/biosíntesis , Proteínas Bacterianas/biosíntesis , Proteína Receptora de AMP Cíclico/fisiología , Regulación Bacteriana de la Expresión Génica , Mycobacterium tuberculosis/patogenicidad , Transcripción Genética , Aconitato Hidratasa/genética , Secuencia de Aminoácidos , Animales , Fusión Artificial Génica , Proteínas Bacterianas/genética , Recuento de Colonia Microbiana , Proteína Receptora de AMP Cíclico/genética , Modelos Animales de Enfermedad , Femenino , Eliminación de Gen , Genes Bacterianos/fisiología , Genes Reporteros/fisiología , Operón Lac/fisiología , Pulmón/microbiología , Macrófagos/microbiología , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/crecimiento & desarrollo , Análisis de Secuencia por Matrices de Oligonucleótidos , Secuencias Reguladoras de Ácidos Nucleicos , Bazo/microbiología , Tuberculosis/microbiología , Virulencia/genética , beta-Galactosidasa/genética , beta-Galactosidasa/metabolismoRESUMEN
The mitochondrial aconitase (mACON) containing a [4Fe-4S] cluster is regarded as the key enzyme for citrate oxidation in the epithelial cells of human prostate. In vitro studies using the human prostatic carcinoma cells, PC-3 cells, found that both hemin and ferric ammonium citrate (FAC) significantly increased mACON enzymatic activity and gene expression. The effect of FAC on mACON was enhanced 2-fold by co-treating with ascorbic acid but blocked by co-treating with iron chelator, deferoxamine mesylate. Hemin treatments blocked 30% of citrate secretion from PC-3 cells but upregualted 2-fold of intracellular ATP biosynthesis. Results from reporter assay by using a cytomegalovirus enhance/promoter driven luciferase mRNA ligated to the iron response element (IRE) of mACON as a reporter construct demonstrated that modulation of FAC on gene translation of mACON gene is dependent on the IRE. Transient gene expression assays indicated that upregulation of mACON gene transcription by FAC may through the putative antioxidant response element (ARE) signal pathway. This study provides the first evidence of the biologic mechanism of human mACON gene translation/transcription and suggests a regulatory link between the energy utilization and the iron metabolism in human prostatic carcinoma cells.
Asunto(s)
Aconitato Hidratasa/biosíntesis , Hierro/metabolismo , Mitocondrias/enzimología , Neoplasias de la Próstata/enzimología , Adenosina Trifosfato/metabolismo , Ácido Ascórbico/farmacología , Secuencia de Bases , Línea Celular Tumoral , Proliferación Celular , Quelantes/farmacología , Citratos/metabolismo , Deferoxamina/farmacología , Relación Dosis-Respuesta a Droga , Compuestos Férricos/farmacología , Regulación Neoplásica de la Expresión Génica , Genes Reporteros , Vectores Genéticos , Hemina/química , Hemina/farmacología , Humanos , Immunoblotting , Masculino , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Biosíntesis de Proteínas , Compuestos de Amonio Cuaternario/farmacología , Transducción de Señal , Transcripción Genética , Regulación hacia ArribaRESUMEN
BACKGROUND: Mitochondrial aconitase (mACON), an iron-requiring enzyme, is a major target of nitric oxide (NO) in cells, which causes the oxidant-mediated disruption of the [4Fe-4S] prosthetic group of the enzyme. In this study, the effect of NO on mACON enzymatic activity and gene expression were investigated. METHODS: Three NO generators, sodium nitroprusside (SNP), S-nitoso-N-acetylpenicillamine (SNAP), and 3-morpholinosydnonimine (SIN) were used to determine the regulation of mACON enzymatic activity by NO. The effect of SNP on mACON, which modulates citrate secretion and cellular bioenergetics in PC-3 cells, was investigated by determining the effect of SNP on mACON gene expression using Western blot and transient gene expression assays. RESULTS: SNP upregulated mACON enzymatic activity and gene expression in PC-3 cells. However, treating cells with other NO generators, SNAP and SIN, resulted in decreased mACON enzymatic activity. The addition of ascorbic acid to the SNP treatment resulted in a decrease in mACON enzymatic activity and gene expression. Our results showed that both SNP and dibutyryl-cAMP increased the mACON promoter activity 2-fold while the effect was blocked by adding H-89. Mutation of the cAMP response element (CRE) to the AGAGCT abolished the activating effects of SNP and dibutyryl-cAMP on mACON promoter activity. CONCLUSIONS: These results establish the function of nitroprusside as a signaling molecule for mACON gene expression through the cAMP signal transduction pathway in human prostatic carcinoma cells.