RESUMEN
Pathogenic bacteria employ complex systems to cope with metal ion shortage conditions and propagate in the host. IsrR is a regulatory RNA (sRNA) whose activity is decisive for optimum Staphylococcus aureus fitness upon iron starvation and for full virulence. IsrR down-regulates several genes encoding iron-containing enzymes to spare iron for essential processes. Here, we report that IsrR regulates the tricarboxylic acid (TCA) cycle by controlling aconitase (CitB), an iron-sulfur cluster-containing enzyme, and its transcriptional regulator, CcpE. This IsrR-dependent dual-regulatory mechanism provides an RNA-driven feedforward loop, underscoring the tight control required to prevent aconitase expression. Beyond its canonical enzymatic role, aconitase becomes an RNA-binding protein with regulatory activity in iron-deprived conditions, a feature that is conserved in S. aureus. Aconitase not only negatively regulates its own expression, but also impacts the enzymes involved in both its substrate supply and product utilization. This moonlighting activity concurrently upregulates pyruvate carboxylase expression, allowing it to compensate for the TCA cycle deficiency associated with iron scarcity. These results highlight the cascade of complex posttranscriptional regulations controlling S. aureus central metabolism in response to iron deficiency.
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Aconitato Hidratasa , Proteínas Bacterianas , Ciclo del Ácido Cítrico , Regulación Bacteriana de la Expresión Génica , Staphylococcus aureus , Aconitato Hidratasa/metabolismo , Aconitato Hidratasa/genética , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genética , Hierro/metabolismo , Deficiencias de Hierro , ARN Bacteriano/metabolismo , ARN Bacteriano/genética , ARN Pequeño no Traducido/metabolismo , ARN Pequeño no Traducido/genética , Staphylococcus aureus/genética , Staphylococcus aureus/metabolismo , Staphylococcus aureus/enzimologíaRESUMEN
AIMS: Ferroptosis is a form of iron-regulated cell death implicated in ischemic heart disease. Our previous study revealed that Sirtuin 3 (SIRT3) is associated with ferroptosis and cardiac fibrosis. In this study, we tested whether the knockout of SIRT3 in cardiomyocytes (SIRT3cKO) promotes mitochondrial ferroptosis and whether the blockade of ferroptosis would ameliorate mitochondrial dysfunction. METHODS AND RESULTS: Mitochondrial and cytosolic fractions were isolated from the ventricles of mice. Cytosolic and mitochondrial ferroptosis were analyzed by comparison to SIRT3loxp mice. An echocardiography study showed that SIRT3cKO mice developed heart failure as evidenced by a reduction of EF% and FS% compared to SIRT3loxp mice. Comparison of mitochondrial and cytosolic fractions of SIRT3cKO and SIRT3loxp mice revealed that, upon loss of SIRT3, mitochondrial, but not cytosolic, total lysine acetylation was significantly increased. Similarly, acetylated p53 was significantly upregulated only in the mitochondria. These data demonstrate that SIRT3 is the primary mitochondrial deacetylase. Most importantly, loss of SIRT3 resulted in significant reductions of frataxin, aconitase, and glutathione peroxidase 4 (GPX4) in the mitochondria. This was accompanied by a significant increase in levels of mitochondrial 4-hydroxynonenal. Treatment of SIRT3cKO mice with the ferroptosis inhibitor ferrostatin-1 (Fer-1) for 14 days significantly improved preexisting heart failure. Mechanistically, Fer-1 treatment significantly increased GPX4 and aconitase expression/activity, increased mitochondrial ironsulfur clusters, and improved mitochondrial membrane potential and Complex IV activity. CONCLUSIONS: Inhibition of ferroptosis ameliorated cardiac dysfunction by specifically targeting mitochondrial aconitase and ironsulfur clusters. Blockade of mitochondrial ferroptosis may be a novel therapeutic target for mitochondrial cardiomyopathies.
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Aconitato Hidratasa , Ferroptosis , Ratones Noqueados , Miocitos Cardíacos , Fenilendiaminas , Sirtuina 3 , Animales , Sirtuina 3/metabolismo , Sirtuina 3/genética , Miocitos Cardíacos/metabolismo , Miocitos Cardíacos/efectos de los fármacos , Aconitato Hidratasa/metabolismo , Ferroptosis/efectos de los fármacos , Ratones , Acetilación , Fenilendiaminas/farmacología , Mitocondrias/metabolismo , Mitocondrias/efectos de los fármacos , Proteínas Hierro-Azufre/metabolismo , Proteínas Hierro-Azufre/genética , Hierro/metabolismo , Frataxina , Fosfolípido Hidroperóxido Glutatión Peroxidasa/metabolismo , Fosfolípido Hidroperóxido Glutatión Peroxidasa/genética , Mitocondrias Cardíacas/metabolismo , Mitocondrias Cardíacas/efectos de los fármacos , Proteínas de Unión a Hierro/metabolismo , Proteínas de Unión a Hierro/genética , Insuficiencia Cardíaca/metabolismo , Insuficiencia Cardíaca/genética , Citosol/metabolismo , CiclohexilaminasRESUMEN
We discuss the role of epigenetic changes at the level of promoter methylation of the key enzymes of carbon metabolism in the regulation of respiration by light. While the direct regulation of enzymes via modulation of their activity and post-translational modifications is fast and readily reversible, the role of cytosine methylation is important for providing a prolonged response to environmental changes. In addition, adenine methylation can play a role in the regulation of transcription of genes. The mitochondrial and extramitochondrial forms of several enzymes participating in the tricarboxylic acid cycle and associated reactions are regulated via promoter methylation in opposite ways. The mitochondrial forms of citrate synthase, aconitase, fumarase, NAD-malate dehydrogenase are inhibited while the cytosolic forms of aconitase, fumarase, NAD-malate dehydrogenase, and the peroxisomal form of citrate synthase are activated. It is concluded that promoter methylation represents a universal mechanism of the regulation of activity of respiratory enzymes in plant cells by light. The role of the regulation of the mitochondrial and cytosolic forms of respiratory enzymes in the operation of malate and citrate valves and in controlling the redox state and balancing the energy level of photosynthesizing plant cells is discussed.
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Fumarato Hidratasa , Malato Deshidrogenasa , Malato Deshidrogenasa/genética , Malato Deshidrogenasa/metabolismo , Citrato (si)-Sintasa/genética , Citrato (si)-Sintasa/metabolismo , Fumarato Hidratasa/genética , Ácidos Tricarboxílicos/metabolismo , Ciclo del Ácido Cítrico , Plantas/genética , Plantas/metabolismo , Aconitato Hidratasa/genética , Aconitato Hidratasa/metabolismo , Metilación de ADN/genética , RespiraciónRESUMEN
PURPOSE: Heterozygous mutations in the AFG3L2 gene (encoding a mitochondrial protease indirectly reflecting on OPA1 cleavage) and ACO2 gene (encoding the mitochondrial enzyme aconitase) are associated with isolated forms of Dominant Optic Atrophy (DOA). We aimed at describing their neuro-ophthalmological phenotype as compared with classic OPA1-related DOA. DESIGN: Cross-sectional study. METHODS: The following neuro-ophthalmological parameters were collected: logMAR visual acuity (VA), color vision, mean deviation and foveal threshold at visual fields, average and sectorial retinal nerve fiber layer (RNFL), and ganglion cell layer (GCL) thickness on optical coherence tomography. ACO2 and AFG3L2 patients were compared with an age- and sex-matched group of OPA1 patients with a 1:2 ratio. All eyes were analyzed using a clustered Wilcoxon rank sum test with the Rosner-Glynn-Lee method. RESULTS: A total of 44 eyes from 23 ACO2 patients and 26 eyes from 13 AFG3L2 patients were compared with 143 eyes from 72 OPA1 patients. All cases presented with bilateral temporal-predominant optic atrophy with various degree of visual impairment. Comparison between AFG3L2 and OPA1 failed to reveal any significant difference. ACO2 patients compared to both AFG3L2 and OPA1 presented overall higher values of nasal RNFL thickness (P = .029, P = .023), average thickness (P = .012, P = .0007), and sectorial GCL thickness. These results were confirmed also comparing separately affected and subclinical patients. CONCLUSIONS: Clinically, DOA remains a fairly homogeneous entity despite the growing genetic heterogeneity. ACO2 seems to be associated with an overall better preservation of retinal ganglion cells, probably depending on the different pathogenic mechanism involving mtDNA maintenance, as opposed to AFG3L2, which is involved in OPA1 processing and is virtually indistinguishable from classic OPA1-DOA.
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ATPasas Asociadas con Actividades Celulares Diversas , Aconitato Hidratasa , GTP Fosfohidrolasas , Atrofia Óptica Autosómica Dominante , Células Ganglionares de la Retina , Tomografía de Coherencia Óptica , Agudeza Visual , Campos Visuales , Adolescente , Adulto , Anciano , Femenino , Humanos , Masculino , Persona de Mediana Edad , Adulto Joven , Aconitato Hidratasa/genética , Proteasas ATP-Dependientes/genética , Proteasas ATP-Dependientes/metabolismo , ATPasas Asociadas con Actividades Celulares Diversas/genética , Estudios Transversales , Estudios de Asociación Genética , GTP Fosfohidrolasas/genética , Proteínas Mitocondriales/genética , Mutación , Fibras Nerviosas/patología , Atrofia Óptica Autosómica Dominante/genética , Atrofia Óptica Autosómica Dominante/fisiopatología , Atrofia Óptica Autosómica Dominante/diagnóstico , Fenotipo , Células Ganglionares de la Retina/patología , Agudeza Visual/fisiología , Campos Visuales/fisiologíaRESUMEN
Parkinson's disease (PD) is characterized by α-synuclein aggregation in dopaminergic (DA) neurons, which are sensitive to oxidative stress. Mitochondria aconitase 2 (ACO2) is an essential enzyme in the tricarboxylic acid cycle that orchestrates mitochondrial and autophagic functions to energy metabolism. Though widely linked to diseases, its relation to PD has not been fully clarified. Here we revealed that the peripheral ACO2 activity was significantly decreased in PD patients and associated with their onset age and disease durations. The knock-in mouse and Drosophila models with the A252T variant displayed aggravated motor deficits and DA neuron degeneration after 6-OHDA and rotenone-induction, and the ACO2 knockdown or blockade cells showed features of mitochondrial and autophagic dysfunction. Moreover, the transcription of autophagy-related genes LC3 and Atg5 was significantly downregulated via inhibited histone acetylation at the H3K9 and H4K5 sites. These data provided multi-dimensional evidences supporting the essential roles of ACO2, and as a potential early biomarker to be used in clinical trials for assessing the effects of antioxidants in PD. Moreover, ameliorating energy metabolism by targeting ACO2 could be considered as a potential therapeutic strategy for PD and other neurodegenerative disorders.
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Enfermedad de Parkinson , Humanos , Ratones , Animales , Enfermedad de Parkinson/metabolismo , Histonas/metabolismo , Acetilación , Mitocondrias/metabolismo , Autofagia , Aconitato Hidratasa/genéticaRESUMEN
Bombyx mori nucleopolyhedrovirus (BmNPV) is a specific pathogen of Bombyx mori that can significantly impede agricultural development. Accumulating evidence indicates that the viral proliferation in the host requires an ample supply of energy. However, the correlative reports of baculovirus are deficient, especially on the acetylation modification of tricarboxylic acid cycle (TCA cycle) metabolic enzymes. Our recent quantitative analysis of protein acetylome revealed that mitochondrial aconitase (ACO2) could be modified by (de)acetylation at lysine 56 (K56) during the BmNPV infection; however, the underlying mechanism is yet unknown. In order to understand this regulatory mechanism, the modification site K56 was mutated to arginine (Lys56Arg; K56R) to mimic deacetylated lysine. The results showed that mimic deacetylated mitochondrial ACO2 restricted enzymatic activity. Although the ATP production was enhanced after viral infection, K56 deacetylation of ACO2 suppressed BmN cellular ATP levels and mitochondrial membrane potential by affecting citrate synthase and isocitrate dehydrogenase activities compared with wild-type ACO2. Furthermore, the deacetylation of exogenous ACO2 lowered BmNPV replication and generation of progeny viruses. In summary, our study on ACO2 revealed the potential mechanism underlying WT ACO2 promotes the proliferation of BmNPV and K56 deacetylation of ACO2 eliminates this promotional effect, which might provide novel insights for developing antiviral strategies.
Asunto(s)
Aconitato Hidratasa , Bombyx , Animales , Aconitato Hidratasa/metabolismo , Lisina/metabolismo , Línea Celular , Adenosina Trifosfato/metabolismoRESUMEN
BACKGROUND: Ketamine, an N-methyl-D-aspartate (NMDA) receptor antagonist, is widely used as a general anaesthetic. However, the mechanisms of analgesic/anaesthetic effects induced by ketamine are only partially understood. Previously, studies have demonstrated that various general anaesthetics affect the primary somatosensory cortex (S1), a potential target of general anaesthetics in the central nervous system. However, it is unknown if astrocyte activities affect ketamine's effects on information transmission in S1 pyramidal neurons. METHODS: The whole-cell patch-clamp technique was employed to study the role of astrocytes in ketamine-induced anaesthetic actions. The whole-cell patch-clamp method was used to record the spontaneous postsynaptic currents (SPSCs) of rat S1 pyramidal neurons. We used the glia-selective inhibitor of the aconitase enzyme fluorocitrate (FC), to test if astrocyte activities alter the effects of ketamine on S1 pyramidal neurons. RESULTS: Ketamine lowered the SPSCs of rat S1 pyramidal neurons in a concentration-dependent manner at clinically relevant doses. The concentration-effect curve revealed that ketamine had an EC50 value of 462.1 M for suppressing SPSCs. In rat S1 pyramidal neurons, the glia-selective metabolic inhibitor fluorocitrate (FC), which inhibits the aconitase enzyme, lowered the amplitude and frequency of SPSCs. The inhibitory impact of ketamine on the amplitude and frequency of SPSCs was significantly amplified in the presence of FC. CONCLUSIONS: Astrocytes impact the effects of ketamine on pre- and postsynaptic components and play a role in synaptic transmission.
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Anestésicos Generales , Ketamina , Animales , Ratas , Ketamina/farmacología , Astrocitos , Corteza Somatosensorial , Transmisión Sináptica , Aconitato HidratasaRESUMEN
Fe-S clusters are essential cofactors mediating electron transfer in respiratory and metabolic networks. However, obtaining active [4Fe-4S] proteins with heterologous expression is challenging due to (i) the requirements for [4Fe-4S] cluster assembly, (ii) the O2 lability of [4Fe-4S] clusters, and (iii) copurification of undesired proteins (e.g., ferredoxins). Here, we established a facile and efficient protocol to express mature [4Fe-4S] proteins in the PURE system under aerobic conditions. An enzyme aconitase and thermophilic ferredoxin were selected as model [4Fe-4S] proteins for functional verification. We first reconstituted the SUF system in vitro via a stepwise manner using the recombinant SUF subunits (SufABCDSE) individually purified from E. coli. Later, the incorporation of recombinant SUF helper proteins into the PURE system enabled mRNA translation-coupled [4Fe-4S] cluster assembly under the O2-depleted conditions. To overcome the O2 lability of [4Fe-4S] Fe-S clusters, an O2-scavenging enzyme cascade was incorporated, which begins with formate oxidation by formate dehydrogenase for NADH regeneration. Later, NADH is consumed by flavin reductase for FADH2 regeneration. Finally, bifunctional flavin reductase, along with catalase, removes O2 from the reaction while supplying FADH2 to the SufBC2D complex. These amendments enabled a one-pot, two-step synthesis of mature [4Fe-4S] proteins under aerobic conditions, yielding holo-aconitase with a maximum concentration of â¼0.15 mg/mL. This renovated system greatly expands the potential of the PURE system, paving the way for the future reconstruction of redox-active synthetic cells and enhanced cell-free biocatalysis.
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Proteínas Hierro-Azufre , Proteínas Hierro-Azufre/genética , Proteínas Hierro-Azufre/metabolismo , Escherichia coli/metabolismo , NAD/metabolismo , Ferredoxinas/genética , Ferredoxinas/metabolismo , Aconitato Hidratasa/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Oxidorreductasas/genética , Oxidorreductasas/metabolismo , Flavinas/metabolismoRESUMEN
Pathogenic biallelic variants in ACO2, which encodes the enzyme mitochondrial aconitase, are associated with the very rare diagnosis of ACO2-related infantile cerebellar retinal degeneration (OMIM 614559). We describe the diagnostic odyssey of a 4-year-old female patient with profound global developmental delays, microcephaly, severe hypotonia, retinal dystrophy, seizures, and progressive cerebellar atrophy. Whole-exome sequencing revealed 2 variants in ACO2; c.2105_2106delAG (p.Gln702ArgfsX9), a likely pathogenic variant, and c.988C>T (p.Pro330Ser) which was classified as a variant of uncertain significance (VUS). While the VUS was confirmed to be maternally inherited, the phase of the other variant could not be confirmed due to lack of a paternal sample. Functional biochemical studies were performed on a research basis to clarify the interpretation of the VUS, which enabled clinical confirmation of the diagnosis of ACO2-related infantile cerebellar retinal degeneration for our patient.
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Microcefalia , Malformaciones del Sistema Nervioso , Distrofias Retinianas , Femenino , Humanos , Niño , Preescolar , Aconitato Hidratasa , Distrofias Retinianas/diagnóstico , Distrofias Retinianas/genética , AtrofiaRESUMEN
Accumulating evidence indicates that mitochondria play crucial roles in immunity. However, the role of the mitochondrial Krebs cycle in immunity remains largely unknown, in particular at the organism level. Here we show that mitochondrial aconitase, ACO-2, a Krebs cycle enzyme that catalyzes the conversion of citrate to isocitrate, inhibits immunity against pathogenic bacteria in C. elegans. We find that the genetic inhibition of aco-2 decreases the level of oxaloacetate. This increases the mitochondrial unfolded protein response, subsequently upregulating the transcription factor ATFS-1, which contributes to enhanced immunity against pathogenic bacteria. We show that the genetic inhibition of mammalian ACO2 increases immunity against pathogenic bacteria by modulating the mitochondrial unfolded protein response and oxaloacetate levels in cultured cells. Because mitochondrial aconitase is highly conserved across phyla, a therapeutic strategy targeting ACO2 may eventually help properly control immunity in humans.
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Aconitato Hidratasa , Caenorhabditis elegans , Humanos , Animales , Aconitato Hidratasa/genética , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Ácido Oxaloacético , Oxaloacetatos , Respuesta de Proteína Desplegada , Mamíferos/metabolismoRESUMEN
The tricarboxylic acid (TCA) cycle is a central route for generating cellular energy and precursors for biosynthetic pathways. Emerging evidences have shown that the aberrations of metabolic enzymes which affect the integrity of TCA cycle are implicated in various tumour pathological processes. Interestingly, several TCA enzymes exhibit the characteristics of RNA binding properties, and their long non-coding RNA (lncRNA) partners play critical regulatory roles in regulating the function of TCA cycle and tumour progression. In this review, we will discuss the functional roles of RNA binding proteins and their lncRNA partners in TCA cycle, with emphasis placed on the cancer progression. A further understanding of RNA binding proteins and their lncRNA partners in TCA cycle, as well as their molecular mechanisms in oncogenesis, will aid in developing novel layers of metabolic targets for cancer therapy in the near future.Abbreviations: CS: citrate synthase. AH: aconitase, including ACO1, and ACO2. IDH: isocitrate dehydrogenase, including IDH1, IDH2, and IDH3. KGDHC: α-ketoglutarate dehydrogenase complex, including OGDH, DLD, and DLST. SCS: succinyl-CoA synthase, including SUCLG1, SUCLG2, and SUCLA2. SDH: succinate dehydrogenase, including SDHA, SDHB, SDHC, and SDHD. FH: fumarate hydratase. MDH: malate dehydrogenase, including MDH1 and MDH2. PC: pyruvate carboxylase. ACLY: ATP Citrate Lyase. NIT: nitrilase. GAD: glutamate decarboxylase. ABAT: 4-aminobutyrate aminotransferase. ALDH5A1: aldehyde dehydrogenase 5 family member A1. ASS: argininosuccinate synthase. ASL: adenylosuccinate synthase. DDO: D-aspartate oxidase. GOT: glutamic-oxaloacetic transaminase. GLUD: glutamate dehydrogenase. HK: hexokinase. PK: pyruvate kinase. LDH: lactate dehydrogenase. PDK: pyruvate dehydrogenase kinase. PDH: pyruvate dehydrogenase complex. PHD: prolyl hydroxylase domain protein.
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Neoplasias , ARN Largo no Codificante , Humanos , Carcinogénesis , Aconitato Hidratasa , Proteínas de Unión al ARNRESUMEN
Certain metabolic interventions such as caloric restriction, fasting, exercise, and a ketogenic diet extend lifespan and/or health span. However, their benefits are limited and their connections to the underlying mechanisms of aging are not fully clear. Here, these connections are explored in terms of the tricarboxylic acid (TCA) cycle (Krebs cycle, citric acid cycle) to suggest reasons for the loss of effectiveness and ways of overcoming it. Specifically, the metabolic interventions deplete acetate and likely reduce the conversion of oxaloacetate to aspartate, thereby inhibiting the mammalian target of rapamycin (mTOR) and upregulating autophagy. Synthesis of glutathione may provide a high-capacity sink for amine groups, facilitating autophagy, and prevent buildup of alpha-ketoglutarate, supporting stem cell maintenance. Metabolic interventions also prevent the accumulation of succinate, thereby slowing DNA hypermethylation, facilitating the repair of DNA double-strand breaks, reducing inflammatory and hypoxic signaling, and lowering reliance on glycolysis. In part through these mechanisms, metabolic interventions may decelerate aging, extending lifespan. Conversely, with overnutrition or oxidative stress, these processes function in reverse, accelerating aging and impairing longevity. Progressive damage to aconitase, inhibition of succinate dehydrogenase, and downregulation of hypoxia-inducible factor-1α, and phosphoenolpyruvate carboxykinase (PEPCK) emerge as potentially modifiable reasons for the loss of effectiveness of metabolic interventions.
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Aconitato Hidratasa , Ciclo del Ácido Cítrico , Aconitato Hidratasa/metabolismo , Glucólisis , ADN/metabolismoRESUMEN
This paper presents an analysis of the regulation activity of the partially purified preparations of cellular aconitate hydratase (AH) on the yeast Yarrowia lipolytica cultivated at extreme pH. As a result of purification, enzyme preparations were obtained from cells grown on media at pH 4.0, 5.5, and 9.0, purified by 48-, 46-, and 51-fold and having a specific activity of 0.43, 0.55 and 0.36 E/mg protein, respectively. The kinetic parameters of preparations from cells cultured at extreme pH demonstrated: (1) an increase in the affinity for citrate and isocitrate; and (2) a shift in the pH optima to the acidic and alkaline side in accordance with the modulation of the medium pH. The regulatory properties of the enzyme from cells subjected to alkaline stress showed increased sensitivity to Fe2+ ions and high peroxide resistance. Reduced glutathione (GSH) stimulated AH, while oxidized glutathione (GSSG) inhibited AH. A more pronounced effect of both GSH and GSSG was noted for the enzyme obtained from cells grown at pH 5.5. The data obtained provide new approaches to the use of Y. lipolytica as a model of eukaryotic cells demonstrating the development of a stress-induced pathology and to conducting a detailed analysis of enzymatic activity for its correction.
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Aconitato Hidratasa , Yarrowia , Aconitato Hidratasa/metabolismo , Oxidación-Reducción , Concentración de Iones de HidrógenoRESUMEN
BACKGROUND/AIM: Renal cell carcinoma (RCC) continues to pose a challenge due to our limited understanding of its underlying pathophysiology. Aconitase 2 (ACO2) is a mitochondrial Fe-S cluster enzyme that catalyzes the stereospecific isomerization of citrate to isocitrate in the second step of the Krebs cycle. We investigated the relationship between ACO2 protein expression and the clinical course of RCC. MATERIALS AND METHODS: Tumor samples were evaluated in a commercial tissue microarray for ACO2 expression using the H-score. The tissue microarrays contained a total of 96 cores from primary tumors, matched metastases, and matched adjacent tissues derived from 32 patients with RCC. The mean follow-up was 82.74 months. Correlation analysis of clinicopathological data and survival was performed. Expression levels of ACO2 mRNA were compared using publicly available data. RESULTS: All the tissue samples showed cytoplasmic ACO2 expression, with median H-scores of 139.7, 130.3 and 166.7 in primary tumor, metastatic tissue, and matched control tissue, respectively. A significantly higher ACO2 expression was found in normal tissues compared to primary and metastatic RCC. The analysis demonstrated a significantly positive correlation between ACO2 expression in primary tumors and their metastases. The results also showed a significant correlation between the expression of ACO2 and worse overall survival among patients with RCC. CONCLUSION: ACO2 may be used as a prognostic factor in RCC. Significant alterations in ACO2 expression are thought to occur in the early stages of RCC carcinogenesis. Considering the physiological role of ACO2, its dysregulation may constitute an adaptive trait of RCC for escaping the equilibrium phase of immunoediting.
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Carcinoma de Células Renales , Neoplasias Renales , Humanos , Carcinoma de Células Renales/patología , Neoplasias Renales/patología , Pronóstico , Biomarcadores de Tumor/metabolismo , ARN Mensajero , Aconitato HidratasaRESUMEN
Mitochondrial aconitase (ACO2) has been postulated as a redox sensor in the tricarboxylic acid cycle. Its high sensitivity towards reactive oxygen and nitrogen species is due to its particularly labile [4Fe-4S]2+ prosthetic group which yields an inactive [3Fe-4S]+ cluster upon oxidation. Moreover, ACO2 was found as a main oxidant target during aging and in pathologies where mitochondrial dysfunction is implied. Herein, we report the expression and characterization of recombinant human ACO2 and its interaction with frataxin (FXN), a protein that participates in the de novo biosynthesis of Fe-S clusters. A high yield of pure ACO2 (≥99%, 22 ± 2 U/mg) was obtained and kinetic parameters for citrate, isocitrate, and cis-aconitate were determined. Superoxide, carbonate radical, peroxynitrite, and hydrogen peroxide reacted with ACO2 with second-order rate constants of 108, 108, 105, and 102 M-1 s-1, respectively. Temperature-induced unfolding assessed by tryptophan fluorescence of ACO2 resulted in apparent melting temperatures of 51.1 ± 0.5 and 43.6 ± 0.2 °C for [4Fe-4S]2+ and [3Fe-4S]+ states of ACO2, sustaining lower thermal stability upon cluster oxidation. Differences in protein dynamics produced by the Fe-S cluster redox state were addressed by molecular dynamics simulations. Reactivation of [3Fe-4S]+-ACO2 by FXN was verified by activation assays and direct iron-dependent interaction was confirmed by protein-protein interaction ELISA and fluorescence spectroscopic assays. Multimer modeling and protein-protein docking predicted an ACO2-FXN complex where the metal ion binding region of FXN approaches the [3Fe-4S]+ cluster, supporting that FXN is a partner for reactivation of ACO2 upon oxidative cluster inactivation.
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Proteínas de Unión a Hierro , Proteínas Hierro-Azufre , Humanos , Proteínas de Unión a Hierro/genética , Proteínas de Unión a Hierro/metabolismo , Oxidación-Reducción , Superóxidos/metabolismo , Aconitato Hidratasa/metabolismo , Proteínas Hierro-Azufre/genética , Proteínas Hierro-Azufre/metabolismo , Espectroscopía de Resonancia por Spin del Electrón , FrataxinaRESUMEN
Acute kidney injury (AKI) is a serious and frequently observed disease associated with high morbidity and mortality. Weighted gene co-expression network analysis (WGCNA) is a research method that converts the relationship between tens of thousands of genes and phenotypes into the association between several gene sets and phenotypes. We screened potential target genes related to AKI through WGCNA to provide a reference for the diagnosis and treatment of AKI. Key biomolecules of AKI were investigated based on transcriptome analysis. RNA sequencing data from 39 kidney biopsy specimens of AKI patients and 9 normal subjects were downloaded from the GEO database. By WGCNA, the top 20% of mRNAs with the largest variance in the data matrix were used to construct a gene co-expression network with a p-value < 0.01 as a screening condition, showing that the blue module was most closely associated with AKI. Thirty-two candidate biomarker genes were screened according to the threshold values of |MM|≥0.86 and |GS|≥0.4, and PPI and enrichment analyses were performed. The top three genes with the most connected nodes, alanine-glyoxylate aminotransferase 2(AGXT2), serine hydroxymethyltransferase 1(SHMT1) and aconitase 2(ACO2), were selected as the central genes based on the PPI network. A rat AKI model was constructed, and the mRNA and protein expression levels of the central genes in the model and control groups were verified by PCR and immunohistochemistry experiments. The results showed that the relative mRNA expression and protein levels of AGXT2, SHMT1 and ACO2 showed a decrease in the model group. In conclusion, we inferred that there is a close association between AGXT2, SHMT1 and ACO2 genes and the development of AKI, and the down-regulation of their expression levels may induce AKI.
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Lesión Renal Aguda , Glicina Hidroximetiltransferasa , Animales , Ratas , Lesión Renal Aguda/diagnóstico , Lesión Renal Aguda/genética , Lesión Renal Aguda/metabolismo , Biomarcadores , Perfilación de la Expresión Génica/métodos , Redes Reguladoras de Genes , Glicina Hidroximetiltransferasa/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Aconitato Hidratasa/metabolismoRESUMEN
Iron regulatory proteins (IRPs) control the translation of animal cell mRNAs encoding proteins with diverse roles. This includes the iron storage protein ferritin and the tricarboxylic cycle (TCA) enzyme mitochondrial aconitase (ACO2) through iron-dependent binding of IRP to the iron responsive element (IRE) in the 5' untranslated region (UTR). To further elucidate the mechanisms allowing IRPs to control translation of 5' IRE-containing mRNA differentially, we focused on Aco2 mRNA, which is weakly controlled versus the ferritins. Rat liver contains two classes of Aco2 mRNAs, with and without an IRE, due to alterations in the transcription start site. Structural analysis showed that the Aco2 IRE adopts the canonical IRE structure but lacks the dynamic internal loop/bulge five base pairs 5' of the CAGUG(U/C) terminal loop in the ferritin IREs. Unlike ferritin mRNAs, the Aco2 IRE lacks an extensive base-paired flanking region. Using a full-length Aco2 mRNA expression construct, iron controlled ACO2 expression in an IRE-dependent and IRE-independent manner, the latter of which was eliminated with the ACO23C3S mutant that cannot bind the FeS cluster. Iron regulation of ACO23C3S encoded by the full-length mRNA was completely IRE-dependent. Replacement of the Aco23C3S 5' UTR with the Fth1 IRE with base-paired flanking sequences substantially improved iron responsiveness, as did fusing of the Fth1 base-paired flanking sequences to the native IRE in the Aco3C3S construct. Our studies further define the mechanisms underlying the IRP-dependent translational regulatory hierarchy and reveal that Aco2 mRNA species lacking the IRE contribute to the expression of this TCA cycle enzyme.
Asunto(s)
Hierro , Proteínas de Unión al ARN , Animales , Ratas , Hierro/metabolismo , Proteínas de Unión al ARN/química , Biosíntesis de Proteínas , Ferritinas/metabolismo , Proteínas Reguladoras del Hierro/genética , Proteínas Reguladoras del Hierro/metabolismo , Aconitato Hidratasa/genética , Aconitato Hidratasa/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Conformación de Ácido NucleicoRESUMEN
Developing a microbial chassis with efficient enzymes is key to the synthesis of products by metabolic engineering. The wide distribution of desired pathway enzymes across several species and categories is posing major challenges in screening and selection of the same for pathway reconstruction. One such key enzyme is isopropylmalate isomerase (IPMI) of leucine/isoleucine biosynthetic pathway. The enzymes reported earlier as citraconase and maleate hydratase in Arthrobacter sp. and Pseudomonas sp. respectively, were found to have the characteristics of IPMI. If a systematic study is undertaken to show that these orphan enzymes indeed are part of the aconitase family of enzymes, these reported ones will add to the repertoire of enzymes available for branch-chained amino acid pathway engineering. This work is focused on functional characterisation of the enzymes citraconase and maleate hydratase based on the properties of IPMI. The partially sequenced gene of maleate hydratase reported earlier served as a template to identify the respective genes in these organisms which is found to be that of IPMI with conserved regions in the active site. The native enzymes and the IPMI of A. globiformis and P. pseudoalcaligenes, expressed in E. coli acted upon all the substrates in the forward direction comprising of D-citramalate, citraconate & D-erythro-3-methylmalate. In the reverse direction all the enzymes converted citraconate to D-citramalate with high activity. The estimated equilibrium ratio was same for both the native enzyme and the over-expressed IPMI which is 96:1.5:2.5 for D-citramalate: citraconate: D-erythro-3-methylmalate. The iron requirement for both enzymes which is characteristic of IPMI is ascertained by chelation and reconstitution of the same. Therefore, this work elucidated the broad specificity and the reactions in equilibrium catalysed by these enzymes like that of IPMI, paving way for the integration of these two efficient candidates into aconitase family of enzymes facilitating pathway engineering.