Aconitase Research Articles

Physiological and proteomic analyses of coix seed aging during storage

Although a series of physio-biochemical changes of coix seed occur during the storage, the underlying mechanism remains unknown. The present study aimed to investigate the aging mechanism of coix seed during storage. Proteome patterns of coix seed stored for 1-month, 5-month and 10-month at room temperature were compared using 2-dimensional gel electrophoresis (2-DE) and mass spectra. Thirty-one differentially expressed proteins (DEPs) were detected, which involved seven pathways including starch and sucrose metabolism, carbon metabolism, RNA transport, proteasome, protein processing in endoplasmic reticulum, ribosome, and RNA degradation. Sucrose synthase 1 was associated with sucrose metabolism and affected the sucrose content during the storage. Increased ambient temperature enhanced respiration of coix after 5-month storage, and overexpression of aconitate hydratase 2 promoted the generation of energy. In addition, the proteins involved in the antioxidant system and resistance stimulus of coix seed were mostly up-regulated during storage.

ACO2 mutations: A novel phenotype associating severe optic atrophy and spastic paraplegia.

Aconitase 2 ( ACO2 ) encodes the mitochondrial aconitase (ACO2), an enzyme catalyzing interconversion of citrate into isocitrate in the Krebs cycle. ACO2 mutations have been initially associated with infantile cerebellar-retinal degeneration combining optic atrophy, retinal degeneration, severe encephalopathy, epilepsy, and cerebellar ataxia1–3; subsequently, ACO2 mutations have also been associated with milder presentations including isolated optic atrophy2 or cerebellar ataxia without optic atrophy.4 We report here a patient presenting with a novel ACO2 phenotype associating optic atrophy with spastic paraplegia. This article is dedicated to the memory of Pr Christian Hamel, who died prematurely on August 15, 2017.

α-Lactalbumin-oleic acid complex kills tumor cells by inducing excess energy metabolism but inhibiting mRNA expression of the related enzymes

Previous studies have demonstrated that the anti-tumor α-lactalbumin-oleic acid complex (α-LA-OA) may target the glycolysis of tumor cells. However, few data are available regarding the effects of α-LA-OA on energy metabolism. In this study, we measured glycolysis and mitochondrial functions in HeLa cells in response to α-LA-OA using the XF flux analyzer (Seahorse Bioscience, North Billerica, MA). The gene expression of enzymes involved in glycolysis, tricarboxylic acid cycle, electron transfer chain, and ATP synthesis were also evaluated. Our results show that α-LA-OA significantly enhanced the basal glycolysis and glycolytic capacity. Mitochondrial oxidative phosphorylation, including the basal respiration, maximal respiration, spare respiratory capacity and ATP production were also improved in response to α-LA-OA. The enhanced mitochondrial functions maybe partly due to the increased capacity of utilizing fatty acids and glutamine as the substrate. However, the gene expressions of pyruvate kinase M2, lactate dehydrogenase A, aconitate hydratase, and isocitrate dehydrogenase 1 were inhibited, suggesting an insufficient ability for the glycolysis process and the tricarboxylic acid cycle. The increased expression of acetyl-coenzyme A acyltransferase 2, a central enzyme involved in the β-oxidation of fatty acids, would enhance the unbalance due to the decreased expression of electron transfer flavoprotein β subunit, which acts as the electron acceptor. These results indicated that α-LA-OA may induce oxidative stress due to conditions in which the ATP production is exceeding the energy demand. Our results may help clarify the mechanism of apoptosis induced by reactive oxygen species and mitochondrial destruction.

Effect of CO2 on NADH production of denitrifying microbes via inhibiting carbon source transport and its metabolism

The potential effect of CO2 on environmental microbes has drawn much attention recently. As an important section of the nitrogen cycle, biological denitrification requires electron donor to reduce nitrogen oxide. Nicotinamide adenine dinucleotide (NADH), which is formed during carbon source metabolism, is a widely reported electron donor for denitrification. Here we studied the effect of CO2 on NADH production and carbon source utilization in the denitrifying microbe Paracoccus denitrificans. We observed that NADH level was decreased by 45.5% with the increase of CO2 concentration from 0 to 30,000ppm, which was attributed to the significantly decreased utilization of carbon source (i.e., acetate). Further study showed that CO2 inhibited carbon source utilization because of multiple negative influences: (1) suppressing the growth and viability of denitrifier cells, (2) weakening the driving force for carbon source transport by decreasing bacterial membrane potential, and (3) downregulating the expression of genes encoding key enzymes involved in intracellular carbon metabolism, such as citrate synthase, aconitate hydratase, isocitrate dehydrogenase, succinate dehydrogenase, and fumarate reductase. This study suggests that the inhibitory effect of CO2 on NADH production in denitrifiers might deteriorate the denitrification performance in an elevated CO2 climate scenario.

Thermodynamic and Kinetic Analyses of Iron Response Element (IRE)-mRNA Binding to Iron Regulatory Protein, IRP1

Comparison of kinetic and thermodynamic properties of IRP1 (iron regulatory protein1) binding to FRT (ferritin) and ACO2 (aconitase2) IRE-RNAs, with or without Mn2+, revealed differences specific to each IRE-RNA. Conserved among animal mRNAs, IRE-RNA structures are noncoding and bind Fe2+ to regulate biosynthesis rates of the encoded, iron homeostatic proteins. IRP1 protein binds IRE-RNA, inhibiting mRNA activity; Fe2+ decreases IRE-mRNA/IRP1 binding, increasing encoded protein synthesis. Here, we observed heat, 5 °C to 30 °C, increased IRP1 binding to IRE-RNA 4-fold (FRT IRE-RNA) or 3-fold (ACO2 IRE-RNA), which was enthalpy driven and entropy favorable. Mn2+ (50 µM, 25 °C) increased IRE-RNA/IRP1 binding (Kd) 12-fold (FRT IRE-RNA) or 6-fold (ACO2 IRE-RNA); enthalpic contributions decreased ~61% (FRT) or ~32% (ACO2), and entropic contributions increased ~39% (FRT) or ~68% (ACO2). IRE-RNA/IRP1 binding changed activation energies: FRT IRE-RNA 47.0 ± 2.5 kJ/mol, ACO2 IRE-RNA 35.0 ± 2.0 kJ/mol. Mn2+ (50 µM) decreased the activation energy of RNA-IRP1 binding for both IRE-RNAs. The observations suggest decreased RNA hydrogen bonding and changed RNA conformation upon IRP1 binding and illustrate how small, conserved, sequence differences among IRE-mRNAs selectively influence thermodynamic and kinetic selectivity of the protein/RNA interactions.

Triphenyltin recognition by primary structures of effector proteins and the protein network of Bacillus thuringiensis during the triphenyltin degradation process

Herein, triphenyltin (TPT) biodegradation efficiency and its transformation pathway have been elucidated. To better understand the molecular mechanism of TPT degradation, the interactions between amino acids, primary structures, and quaternary conformations of effector proteins and TPT were studied. The results verified that TPT recognition and binding depended on amino acid sequences but not on secondary, tertiary or quaternary protein structure. During this process, TPT could change the molecular weight and isoelectric point of effector proteins, induce their methylation or demethylation, and alter their conformation. The effector proteins, alkyl hydroperoxide reductase and acetyl-CoA acetyltransferase, recognizing TPT were crucial to TPT degradation. Electron transfer flavoprotein subunit alpha, phosphoenolpyruvate carboxykinase, aconitate hydratase, branched-chain alpha-keto acid dehydrogenase E1 component, biotin carboxylase and superoxide dismutase were related to energy and carbon metabolism, which was consistent with the results in vivo. The current findings develop a new approach for investigating the interactions between proteins and target compounds.

Proteomics analysis of pleomorphic adenoma of the human parotid gland.

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Serological Reactivity and Identification of Immunoglobulin E-Binding Polypeptides of Ganoderma applanatum Crude Spore Cytoplasmic Extract in Puerto Rican Subjects

Background: The allergenic potential of Ganoderma applanatum basidiospores has been demonstrated previously in Puerto Rico. However, basidiomycete allergens are not available for inclusion in allergy diagnostic panels. Therefore, we sought to confirm allergic sensitization to G. applanatum crude spore cytoplasmic extract through reactivity in serological assays and detection of immunoglobulin E (IgE)-binding polypeptides. Methods: Via an indirect ELISA, serological reactivity was compared between groups of individuals with different allergic profiles. Group 1 (n = 51) consisted of individuals with sIgE to the allergens included in the diagnostic panels; group 2 (n = 14) comprised individuals with no sIgE to the allergens tested; and group 3 (n = 22) included individuals with no allergic history. To visualize IgE-binding polypeptides, group 1 sera were examined via Western blotting (WB). Polypeptide bands with the highest reactivity were analyzed by mass spectrometry (MS) for putative identification. Results: The serological reactivity of group 1 was significantly higher than that of group 3 in an indirect ELISA (p = 0.03). Sixty-five percent of group 1 individuals showed reactivity to polypeptide bands in WB. Bands of 81 and 56 kDa had the highest reactivity proportions among the reactive sera, followed by a 45-kDa band. MS analysis of these 3 polypeptides suggests that they are basidiomycete-derived enzymes with aconitate hydratase, catalase, and enolase functions. Conclusions:G. applanatum spores have allergenic components recognized by Puerto Rican individuals, which could eventually be considered as markers in cases of fungal allergy and be included in diagnostic allergen panels in Puerto Rico and tropical regions.

Mitochondrial O-GlcNAc Transferase (mOGT) Regulates Mitochondrial Structure, Function, and Survival in HeLa Cells

O-Linked N-acetylglucosamine transferase (OGT) catalyzes O-GlcNAcylation of target proteins and regulates numerous biological processes. OGT is encoded by a single gene that yields nucleocytosolic and mitochondrial isoforms. To date, the role of the mitochondrial isoform of OGT (mOGT) remains largely unknown. Using high throughput proteomics, we identified 84 candidate mitochondrial glycoproteins, of which 44 are novel. Notably, two of the candidate glycoproteins identified (cytochrome oxidase 2 (COX2) and NADH:ubiquinone oxidoreductase core subunit 4 (MT-ND4)) are encoded by mitochondrial DNA. Using siRNA in HeLa cells, we found that reducing endogenous mOGT expression leads to alterations in mitochondrial structure and function, including Drp1-dependent mitochondrial fragmentation, reduction in mitochondrial membrane potential, and a significant loss of mitochondrial content in the absence of mitochondrial ROS. These defects are associated with a compensatory increase in oxidative phosphorylation per mitochondrion. mOGT is also critical for cell survival; siRNA-mediated knockdown of endogenous mOGT protected cells against toxicity mediated by rotenone, a complex I inhibitor. Conversely, reduced expression of both nucleocytoplasmic (ncOGT) and mitochondrial (mOGT) OGT isoforms is associated with increased mitochondrial respiration and elevated glycolysis, suggesting that ncOGT is a negative regulator of cellular bioenergetics. Last, we determined that mOGT is probably involved in the glycosylation of a restricted set of mitochondrial targets. We identified four proteins implicated in mitochondrial biogenesis and metabolism regulation as candidate substrates of mOGT, including leucine-rich PPR-containing protein and mitochondrial aconitate hydratase. Our findings suggest that mOGT is catalytically active in vivo and supports mitochondrial structure, health, and survival, whereas ncOGT predominantly regulates cellular bioenergetics.

Isolation and characterization of Aconitate hydratase 4 (Aco4) from soybean

Aconitase catalyzes the reversible isomerization of two tricarboxylic acids, citrate and isocitrate, during the Krebs cycle. Five aconitase genes, namely, Aco1, Aco2, Aco3, Aco4, and Aco5, have bee...

Genome-Wide SNPs Identification and Determination of Proteins Associated with Stress Response in Sorghum (<i>Sorghum bicolor</i> L. Monech) Accessions

Current efforts in sorghum breeding programs are exploiting genotyping-by-sequencing (GBS) data to provide full-genome scans for desired traits. The aim of this study was to utilize GBS approach for the identification of genomic regions associated with stress response in sorghum (Sorghum bicolor L. Monech) accessions. DNA samples of twenty sorghum accessions, having different response to drought, were used to prepare GBS libraries for sequencing. SNPs were called using the TASSELGBS pipeline and the tags that present at least 10 times in the dataset were considered and aligned to the reference genome of Sorghum bicolor. The identified SNPs were all compared with the published sorghum transcript related to stress response gene activity. Overall; 94.40% tags were aligned and 69,736 putative SNPs positions were identified. Blast search revealed homology to annotated heat and drought–tolerance associated genes which code for ATPases, Peroxidase, Hydrophobic protein LTI6A, Aquaporin SIP2-1, Aconitate hydratase and phosphatidylinositol-4-phosphate-5-kinase. The phylogeny of the 20 accessions was constructed using the generated SNPs data. Phylogenetic analysis data showed that the phenotypically tolerant line (El9) makes a separate cluster and the same for the accessions HSD8653 and HSD5612 near to the cluster that includes most accessions with known post-flowering drought tolerance (HSD7410, HDS10033, HSD8552, GESHEISH and HSD8849). Post-flowering drought sensitive accessions (Tabat, Wadahmed, HSD6468 and HSD6478) formed a separate cluster while the sensitive accession HSD9959 and the tolerant accessions HSD8511 and HSD9566 were distributed between the two clusters. Thus, cluster analysis confirmed the variation among accessions in post-flowering drought tolerance. With further validation, these markers may be used for marker assisted selection for breeding new sorghum genotypes with stress adaptation.

Molecular Evolution and Expression Divergence of the Aconitase (ACO) Gene Family in Land Plants.

Aconitase (ACO) is a key enzyme that catalyzes the isomerization of citrate to isocitrate in the tricarboxylic acid (TCA) and glyoxylate cycles. The function of ACOs has been well studied in model plants, such as Arabidopsis. In contrast, the evolutionary patterns of the ACO family in land plants are poorly understood. In this study, we systematically examined the molecular evolution and expression divergence of the ACO gene family in 12 land plant species. Thirty-six ACO genes were identified from the 12 land plant species representing the four major land plant lineages: Bryophytes, lycophytes, gymnosperms, and angiosperms. All of these ACOs belong to the cytosolic isoform. Three gene duplication events contributed to the expansion of the ACO family in angiosperms. The ancestor of angiosperms may have contained only one ACO gene. One gene duplication event split angiosperm ACOs into two distinct clades. Two clades showed a divergence in selective pressure and gene expression patterns. The cis-acting elements that function in light responsiveness were most abundant in the promoter region of the ACO genes, indicating that plant ACO genes might participate in light regulatory pathways. Our findings provide comprehensive insights into the ACO gene family in land plants.

Allozymic Variation and Relationships within Viola sect. Viola (Violaceae) in Iran

AbstractGenetic variation and differentiation in taxa of the genus Viola L. sect. Viola, subsections Viola and Rostratae (Kupffer) W. Becker, were studied from natural populations occurring in Iran. Two isoenzyme systems, Phosphoglucoisomerase (PGI) and Aconitase (ACO), encoding four putative loci, were employed to detect interspecific and intraspecific genetic variation. Considering the patterns of isoenzyme variation in the studied taxa, it is evident that V. caspia (Rupr.) Freyn subsp. caspia and V. capia subsp. sylvestroides Marcussen are closely related. The species V. alba Bess. subsp. alba and V. sintenisii W. Becker are isoenzymatically well characterized as distinct genetic entities.

New recombinant strains of the yeast Yarrowia lipolytica with overexpression of the aconitate hydratase gene for the obtainment of isocitric acid from rapeseed oil

The yeast Yarrowia lipolytica is capable of high-intensity synthesis (overproduction) of citric (CA) and isocitric (ICA) acids under nitrogen limitation. The ratio of the synthesized acids depends on the producing strains used and the expression level of the aconitate hydratase gene (ACO1). Recombinant variants with overexpression of the multicopy ACO1 gene have been obtained based on the natural ICA-producing strain Y. lipolytica 672. A recombinant strain Y. lipolytica 20, which has an isocitrate-citrate ratio shifted towards ICA (2.3: 1) as compared to the parental strain (1.1: 1), has been selected. Culturing of the 20 variant in a 10 L reactor has resulted in the production of 72.6 g/L of ICA and 29.0 g/L of CA with a ratio of 2.5: 1. This makes it possible to regard Y. lipolytica 20 as a promising producer for the development of an industrial process for isocitrate production.

Enhanced itaconic acid production by self-assembly of two biosynthetic enzymes in Escherichia coli.

Here, we described a novel strategy for the production of itaconic acid in Escherichia coli by self-assembly of aconitase (ACO) and cis-aconitate decarboxylase (CAD) existing in the metabolic pathway of itaconic acid via the protein-peptide interactions of PDZ domain and PDZ ligand. Co-expression of ACO and CAD in E. coli (uCA) resulted in low levels of itaconate (117.25 mg/L) after 48 h fermentation while the itaconate titre was significantly improved up to 222.15 mg/L by self-assembly of ACO-PDZ (APd) and CAD-PDZlig (CPl) in E. coli (sPP) under the same conditions. To further confirm the effect of self-assembly, the itaconate catalyzed from sodium citrate was determined. The sPP was extra efficacious in the early catalytic period, showing approximately threefold itaconate yields increased after 2 h catalysis, when compared to uCA. Furthermore, the itaconate production of sPP was increased from 5 to 8.7 g/L after 30 h of reaction compared to uCA. This self-assembly strategy showed remarkable potential for the further improvement of itaconate production. Biotechnol. Bioeng. 2017;114: 457-462. © 2016 Wiley Periodicals, Inc.

Effects of Melaxen and Valdoxan on Free-Radical Oxidation in Rat Heart with Experimental Hyperthyroidism

The rate of free-radical oxidation in rat heart with experimental hyperthyroidism (EH) was estimated after Melaxen and Valdoxan administration. It was found that the content of protein carbonyl groups increased if the pathology developed in cardiomyocytes. This indicated that they were oxidized. Increased concentrations of primary lipid peroxidation products (diene conjugates), inhibition of aconitate hydratase activity, and accumulation of citrate were noted. Injection of Melaxen and Valdoxan to rats with EH normalized these parameters. The melatonin-correcting capability of the used drugs and the antioxidant activity of melatonin probably facilitated normalization of free-radical homeostasis in rat cardiomyocytes with the pathology.

Metagenomic Insights into Effects of Chemical Pollutants on Microbial Community Composition and Function in Estuarine Sediments Receiving Polluted River Water.

Pyrosequencing and metagenomic profiling were used to assess the phylogenetic and functional characteristics of microbial communities residing in sediments collected from the estuaries of Rivers Oujiang (OS) and Jiaojiang (JS) in the western region of the East China Sea. Another sediment sample was obtained from near the shore far from estuaries, used for contrast (CS). Characterization of estuary sediment bacterial communities showed that toxic chemicals potentially reduced the natural variability in microbial communities, while they increased the microbial metabolic enzymes and pathways. Polycyclic aromatic hydrocarbons (PAHs) and nitrobenzene were negatively correlated with the bacterial community variation. The dominant class in the sediments was Gammaproteobacteria. According to Kyoto Encyclopedia of Genes and Genomes (KEGG) enzyme profiles, dominant enzymes were found in estuarine sediments, which increased greatly, such as 2-oxoglutarate synthase, acetolactate synthase, inorganic diphosphatase, and aconitate hydratase. In KEGG pathway profiles, most of the pathways were also dominated by specific metabolism in these sediments and showed a marked increase, for instance alanine, aspartate, and glutamate metabolism, carbon fixation pathways in prokaryotes, and aminoacyl-tRNA biosynthesis. The estuarine sediment bacterial diversity varied with the polluted river water inputs. In the estuary receiving river water from the more seriously polluted River Oujiang, the sediment bacterial community function was more severely affected.

Differential protein expression during sperm maturation and capacitation in an hermaphroditic bivalve,Pecten maximus(Linnaeus, 1758)

In order to investigate the mechanisms of final maturation and capacitation of spermatozoa in Pecten maximus, we used a 2D proteomic approach coupled with MALDI-TOF/TOF mass spectrometry (MS) and bioinformatics search against the Pecten database, to set up a reference map of the proteome of spawned spermatozoa, and identified 133 proteins on the basis of the EST database. These proteins are mainly involved in energy production, ion and electron transport (44%), cell movement (22%) and developmental processes (10%). Comparison between proteomes of spermatozoa collected before and after transit through the genital ducts of P. maximus led to the identification of differentially expressed proteins. Most of them are associated with energy metabolism (aconitate hydratase, malate dehydrogenase, glyceraldehyde-3-phosphate dehydrogenase), indicating important modifications of energy production during transit in gonoducts, potentially linked with acquisition of sperm motility. Three proteins involved in cell movement (Tektin-2, tubulin and microtubule-associated protein RP/EB family member 3) were down-regulated in spermatozoa stripped from the gonad. 40S ribosomal protein SA, involved in maturation of 40S ribosomal subunits, was also found to be down-regulated in spermatozoa obtained by induced spawning, suggesting reduction of the efficiency of RNA translation, a characteristic of late spermatozoon differentiation. These results confirm that maturation processes of P. maximus spermatozoa during transit through the gonoduct involve RNA translation, energy metabolism and structural proteins implicated in cell movement. Spermatozoa maturation processes clearly differ between P. maximus and gonochoric or alternately hermaphroditic bivalves, potentially in relation to reproductive strategies: the final maturation of the spermatozoon along the genital tract probably contributes to reduction of autofertilization in this simultaneously hermaphroditic species.

Bottom-up proteomics suggests an association between differential expression of mitochondrial proteins and chronic fatigue syndrome.

Chronic fatigue syndrome (CFS) is a debilitating and complex disorder characterized by unexplained fatigue not improved by rest. An area of investigation is the likely connection of CFS with defective mitochondrial function. In a previous work, we investigated the proteomic salivary profile in a couple of monozygotic twins discordant for CFS. Following this work, we analyzed mitochondrial proteins in the same couple of twins. Nano-liquid chromatography electrospray ionization mass spectrometry (nano-LC-MS) was used to study the mitochondria extracted from platelets of the twins. Subsequently, we selected three proteins that were validated using western blot analysis in a big cohort of subjects (n=45 CFS; n=45 healthy), using whole saliva (WS). The selected proteins were as follows: aconitate hydratase (ACON), ATP synthase subunit beta (ATPB) and malate dehydrogenase (MDHM). Results for ATPB and ACON confirmed their upregulation in CFS. However, the MDHM alteration was not confirmed. Thereafter, seeing the great variability of clinical features of CFS patients, we decided to analyze the expression of our proteins after splitting patients according to clinical parameters. For each marker, the values were actually higher in the group of patients who had clinical features similar to the ill twin. In conclusion, these results suggest that our potential markers could be one of the criteria to be taken into account for helping in diagnosis. Furthermore, the identification of biomarkers present in particular subgroups of CFS patients may help in shedding light upon the complex entity of CFS. Moreover, it could help in developing tailored treatments.

Влияние мелаксена и вальдоксана на свободнорадикальное окисление в сердце крыс при экспериментальном гипертиреозе

Проведена оценка интенсивности свободнорадикального окисления в сердце крыс при введении мелаксена и вальдоксана при экспериментальном гипертиреозе. Установлено, что при развитии патологии в кардиомиоцитах происходило увеличение содержания карбонильных групп белков, что свидетельствует об их окислительной модификации. При этом отмечено возрастание концентрации первичных продуктов перекисного окисления липидов (диеновых конъюгатов), ингибирование активности аконитатгидратазы и накопление цитрата. Введение мелаксена и вальдоксана крысам при экспериментальном гипертиреозе приводило к изменению исследуемых параметров в сторону нормы. Вероятно, мелатонин-корригирующая способность используемых препаратов и антиоксидантная активность мелатонина способствовали нормализации свободнорадикального гомеостаза в кардиомиоцитах крыс с патологией.