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Functional identity of Drosophila melanogaster Indy as a cation-independent, electroneutral transporter for tricarboxylic acid-cycle intermediates.

ABSTRACT: Indy is a gene in Drosophila melanogaster which, when made dysfunctional, leads to an extension of the average adult life span of the organism. The present study was undertaken to clone the Indy gene-product and to establish its functional identity. We isolated a full-length Indy cDNA from a D. melanogaster cDNA library. The cDNA codes for a protein of 572 amino acids [( Drosophila Indy (drIndy)]. In its amino acid sequence, drIndy exhibits comparable similarity to the two known Na(+)-coupled dicarboxylate transporters in mammals; namely, NaDC1 (35% identity) and NaDC3 (34% identity). We elucidated the functional characteristics of drIndy in two different heterologous expression systems by using mammalian cells and Xenopus laevis oocytes. These studies show that drIndy is a cation-independent electroneutral transporter for a variety of tricarboxylic acid-cycle intermediates, with preference for citrate compared with succinate. These characteristics of drIndy differ markedly from those of NaDC1 and NaDC3, indicating that neither of these latter transporters is the mammalian functional counterpart of drIndy. Since drIndy is a transporter for tricarboxylic acid-cycle intermediates, dysfunction of the Indy gene may lead to decreased production of metabolic energy in cells, analogous to caloric restriction. This might provide the molecular basis for the observation that disruption of the Indy gene function in Drosophila leads to extension of the average adult life span of the organism.

SUBMITTER: Inoue K

PROVIDER: S-EPMC1222911 | biostudies-other | 2002 Oct

REPOSITORIES: biostudies-other

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A non-canonical tricarboxylic acid cycle underlies cellular identity.

Project description:The tricarboxylic acid (TCA) cycle is a central hub of cellular metabolism, oxidizing nutrients to generate reducing equivalents for energy production and critical metabolites for biosynthetic reactions. Despite the importance of the products of the TCA cycle for cell viability and proliferation, mammalian cells display diversity in TCA-cycle activity1,2. How this diversity is achieved, and whether it is critical for establishing cell fate, remains poorly understood. Here we identify a non-canonical TCA cycle that is required for changes in cell state. Genetic co-essentiality mapping revealed a cluster of genes that is sufficient to compose a biochemical alternative to the canonical TCA cycle, wherein mitochondrially derived citrate exported to the cytoplasm is metabolized by ATP citrate lyase, ultimately regenerating mitochondrial oxaloacetate to complete this non-canonical TCA cycle. Manipulating the expression of ATP citrate lyase or the canonical TCA-cycle enzyme aconitase 2 in mouse myoblasts and embryonic stem cells revealed that changes in the configuration of the TCA cycle accompany cell fate transitions. During exit from pluripotency, embryonic stem cells switch from canonical to non-canonical TCA-cycle metabolism. Accordingly, blocking the non-canonical TCA cycle prevents cells from exiting pluripotency. These results establish a context-dependent alternative to the traditional TCA cycle and reveal that appropriate TCA-cycle engagement is required for changes in cell state.

| S-EPMC8934290 | biostudies-literature

Functional features and genomic organization of mouse NaCT, a sodium-coupled transporter for tricarboxylic acid cycle intermediates.

Project description:In the present study, we report on the molecular cloning and functional characterization of mouse NaCT (Na+-coupled citrate transporter), the mouse orthologue of Drosophila Indy. Mouse NaCT consists of 572 amino acids and is highly similar to rat and human NaCTs in primary sequence. The mouse nact gene coding for the transporter is approx. 23 kb long and consists of 12 exons. When expressed in mammalian cells, the cloned transporter mediates the Na+-coupled transport of citrate and succinate. Competition experiments reveal that mouse NaCT also recognizes other tricarboxylic acid cycle intermediates such as malate, fumarate and 2-oxo-glutarate as excellent substrates. The Michaelis-Menten constant for the transport process is 38+/-5 mM for citrate and 37+/-6 mM for succinate at pH 7.5. The transport process is electrogenic and exhibits an obligatory requirement for Na+. Na+-activation kinetics indicates that multiple Na+ ions are involved in the activation process. Extracellular pH has a differential effect on the transport function of mouse NaCT depending on whether the transported substrate is citrate or succinate. The Michaelis-Menten constants for these substrates are also influenced markedly by pH. When examined in the Xenopus laevis oocyte expression system with the two-microelectrode voltage-clamp technique, the transport process mediated by mouse NaCT is electrogenic. The charge-to-substrate ratio is 1 for citrate and 2 for succinate. The most probable transport mechanism predicted by these studies involves the transport of citrate as a tervalent anion and succinate as a bivalent anion with a fixed Na+/substrate stoichiometry of 4:1. The present study provides the first unequivocal evidence for the electrogenic nature of mammalian NaCT.

| S-EPMC1224018 | biostudies-other

Studies on the Interaction of the Histone Demethylase KDM5B with Tricarboxylic Acid Cycle Intermediates.

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2021-12-01 | GSE183434 | GEO

Elimination and replenishment of tricarboxylic acid-cycle intermediates in myocardium.

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| S-EPMC1162823 | biostudies-other

Identification of a chemoreceptor for tricarboxylic acid cycle intermediates: differential chemotactic response towards receptor ligands.

Project description:We report the identification of McpS as the specific chemoreceptor for 6 tricarboxylic acid (TCA) cycle intermediates and butyrate in Pseudomonas putida. The analysis of the bacterial mutant deficient in mcpS and complementation assays demonstrate that McpS is the only chemoreceptor of TCA cycle intermediates in the strain under study. TCA cycle intermediates are abundantly present in root exudates, and taxis toward these compounds is proposed to facilitate the access to carbon sources. McpS has an unusually large ligand-binding domain (LBD) that is un-annotated in InterPro and is predicted to contain 6 helices. The ligand profile of McpS was determined by isothermal titration calorimetry of purified recombinant LBD (McpS-LBD). McpS recognizes TCA cycle intermediates but does not bind very close structural homologues and derivatives like maleate, aspartate, or tricarballylate. This implies that functional similarity of ligands, such as being part of the same pathway, and not structural similarity is the primary element, which has driven the evolution of receptor specificity. The magnitude of chemotactic responses toward these 7 chemoattractants, as determined by qualitative and quantitative chemotaxis assays, differed largely. Ligands that cause a strong chemotactic response (malate, succinate, and fumarate) were found by differential scanning calorimetry to increase significantly the midpoint of protein unfolding (T(m)) and unfolding enthalpy (DeltaH) of McpS-LBD. Equilibrium sedimentation studies show that malate, the chemoattractant that causes the strongest chemotactic response, stabilizes the dimeric state of McpS-LBD. In this respect clear parallels exist to the Tar receptor and other eukaryotic receptors, which are discussed.

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Chronic Hyperglycaemia Inhibits Tricarboxylic Acid Cycle in Rat Cardiomyoblasts Overexpressing Glucose Transporter Type 4.

Project description:An oversupply of nutrients with a loss of metabolic flexibility and subsequent cardiac dysfunction are hallmarks of diabetic cardiomyopathy. Even if excess substrate is offered, the heart suffers energy depletion as metabolic fluxes are diminished. To study the effects of a high glucose supply, a stably glucose transporter type 4 (GLUT4)-overexpressing cell line presenting an onset of diabetic cardiomyopathy-like phenotype was established. Long-term hyperglycaemia effects were analysed. Rat cardiomyoblasts overexpressing GLUT4 (H9C2KE2) were cultured under normo- and hyperglycaemic conditions for long-term. Expression profiles of several proteins were compared to non-transfected H9C2 cells (H9C2) using RT-qPCR, proteomics-based analysis, or Western blotting. GLUT4 surface analysis, glucose uptake, and cell morphology changes as well as apoptosis/necrosis measurements were performed using flow cytometry. Additionally, brain natriuretic peptide (BNP) levels, reactive oxygen species (ROS) formation, glucose consumption, and lactate production were quantified. Long-term hyperglycaemia in H9C2KE2 cells induced increased GLUT4 presence on the cell surface and was associated with exaggerated glucose influx and lactate production. On the metabolic level, hyperglycaemia affected the tricarboxylic acid (TCA) cycle with accumulation of fumarate. This was associated with increased BNP-levels, oxidative stress, and lower antioxidant response, resulting in pronounced apoptosis and necrosis. Chronic glucose overload in cardiomyoblasts induced by GLUT4 overexpression and hyperglycaemia resulted in metabolically stimulated proteome profile changes and metabolic alterations on the TCA level.

| S-EPMC9266806 | biostudies-literature

Ae4 (Slc4a9) is an electroneutral monovalent cation-dependent Cl-/HCO3- exchanger.

Project description:Ae4 (Slc4a9) belongs to the Slc4a family of Cl(-)/HCO3 (-) exchangers and Na(+)-HCO3 (-) cotransporters, but its ion transport cycle is poorly understood. In this study, we find that native Ae4 activity in mouse salivary gland acinar cells supports Na(+)-dependent Cl(-)/HCO3 (-) exchange that is comparable with that obtained upon heterologous expression of mouse Ae4 and human AE4 in CHO-K1 cells. Additionally, whole cell recordings and ion concentration measurements demonstrate that Na(+) is transported by Ae4 in the same direction as HCO3 (-) (and opposite to that of Cl(-)) and that ion transport is not associated with changes in membrane potential. We also find that Ae4 can mediate Na(+)-HCO3 (-) cotransport-like activity under Cl(-)-free conditions. However, whole cell recordings show that this apparent Na(+)-HCO3 (-) cotransport activity is in fact electroneutral HCO3 (-)/Na(+)-HCO3 (-) exchange. Although the Ae4 anion exchanger is thought to regulate intracellular Cl(-) concentration in exocrine gland acinar cells, our thermodynamic calculations predict that the intracellular Na(+), Cl(-), and HCO3 (-) concentrations required for Ae4-mediated Cl(-) influx differ markedly from those reported for acinar secretory cells at rest or under sustained stimulation. Given that K(+) ions share many properties with Na(+) ions and reach intracellular concentrations of 140-150 mM (essentially the same as extracellular [Na(+)]), we hypothesize that Ae4 could mediate K(+)-dependent Cl(-)/HCO3 (-) exchange. Indeed, we find that Ae4 mediates Cl(-)/HCO3 (-) exchange activity in the presence of K(+) as well as Cs(+), Li(+), and Rb(+) In summary, our results strongly suggest that Ae4 is an electroneutral Cl(-)/nonselective cation-HCO3 (-) exchanger. We postulate that the physiological role of Ae4 in secretory cells is to promote Cl(-) influx in exchange for K(+)(Na(+)) and HCO3 (-) ions.

| S-EPMC4845690 | biostudies-literature

The tricarboxylic acid cycle in Shewanella oneidensis is independent of Fur and RyhB control.

Project description:BACKGROUND: It is well established in E. coli and Vibrio cholerae that strains harboring mutations in the ferric uptake regulator gene (fur) are unable to utilize tricarboxylic acid (TCA) compounds, due to the down-regulation of key TCA cycle enzymes, such as AcnA and SdhABCD. This down-regulation is mediated by a Fur-regulated small regulatory RNA named RyhB. It is unclear in the ?-proteobacterium S. oneidensis whether TCA is also regulated by Fur and RyhB. RESULTS: In the present study, we showed that a fur deletion mutant of S. oneidensis could utilize TCA compounds. Consistently, expression of the TCA cycle genes acnA and sdhA was not down-regulated in the mutant. To explore this observation further, we identified a ryhB gene in Shewanella species and experimentally demonstrated the gene expression. Further experiments suggested that RyhB was up-regulated in fur mutant, but that AcnA and SdhA were not controlled by RyhB. CONCLUSIONS: These cumulative results delineate an important difference of the Fur-RyhB regulatory cycle between S. oneidensis and other ?-proteobacteria. This work represents a step forward for understanding the unique regulation in S. oneidensis.

| S-EPMC2964681 | biostudies-literature

Association of the malate dehydrogenase-citrate synthase metabolon is modulated by intermediates of the Krebs tricarboxylic acid cycle.

Project description:Mitochondrial malate dehydrogenase (MDH)-citrate synthase (CS) multi-enzyme complex is a part of the Krebs tricarboxylic acid (TCA) cycle 'metabolon' which is enzyme machinery catalyzing sequential reactions without diffusion of reaction intermediates into a bulk matrix. This complex is assumed to be a dynamic structure involved in the regulation of the cycle by enhancing metabolic flux. Microscale Thermophoresis analysis of the porcine heart MDH-CS complex revealed that substrates of the MDH and CS reactions, NAD+ and acetyl-CoA, enhance complex association while products of the reactions, NADH and citrate, weaken the affinity of the complex. Oxaloacetate enhanced the interaction only when it was present together with acetyl-CoA. Structural modeling using published CS structures suggested that the binding of these substrates can stabilize the closed format of CS which favors the MDH-CS association. Two other TCA cycle intermediates, ATP, and low pH also enhanced the association of the complex. These results suggest that dynamic formation of the MDH-CS multi-enzyme complex is modulated by metabolic factors responding to respiratory metabolism, and it may function in the feedback regulation of the cycle and adjacent metabolic pathways.

| S-EPMC8455617 | biostudies-literature

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