Tricarboxylic Acid Cycle Enzymes Research Articles

Gluconeogenic precursor availability regulates flux through the glyoxylate shunt in Pseudomonas aeruginosa

The glyoxylate shunt bypasses the oxidative decarboxylation steps of the tricarboxylic acid (TCA) cycle, thereby conserving carbon skeletons for gluconeogenesis and biomass production. In Escherichia coli, carbon flux is redirected through the first enzyme of the glyoxylate shunt, isocitrate lyase (ICL), following phosphorylation and inactivation of the TCA cycle enzyme, isocitrate dehydrogenase (ICD), by the kinase/phosphatase, AceK. In contrast, mycobacterial species lack AceK and employ a phosphorylation-insensitive isocitrate dehydrogenase (IDH), which is allosterically activated by the product of ICL activity, glyoxylate. However, Pseudomonas aeruginosa expresses IDH, ICD, ICL, and AceK, raising the question of how these enzymes are regulated to ensure proper flux distribution between the competing pathways. Here, we present the structure, kinetics, and regulation of ICL, IDH, and ICD from P. aeruginosa We found that flux partitioning is coordinated through reciprocal regulation of these enzymes, linking distribution of carbon flux to the availability of the key gluconeogenic precursors, oxaloacetate and pyruvate. Specifically, a greater abundance of these metabolites activated IDH and inhibited ICL, leading to increased TCA cycle flux. Regulation was also exerted through AceK-dependent phosphorylation of ICD; high levels of acetyl-CoA (which would be expected to accumulate when oxaloacetate is limiting) stimulated the kinase activity of AceK, whereas high levels of oxaloacetate stimulated its phosphatase activity. In summary, the TCA cycle-glyoxylate shunt branch point in P. aeruginosa has a complex enzymology that is profoundly different from those in other species characterized to date. Presumably, this reflects its predilection for consuming fatty acids, especially during infection scenarios.

Effects of whey protein and conjugated linoleic acid on acrolein-induced cardiac oxidative stress, mitochondrial dysfunction and dyslipidemia in rats

Acrolein is a ubiquitous environmental pollutant. Whey protein and conjugated linoleic acid are widely used weight-loss supplements. We aimed to evaluate blood lipid profiles, oxidative stress and mitochondrial bioenergetics function in hearts of rats treated with acrolein and/or the weight-loss supplements. The animals were orally gavaged with acrolein, whey protein, conjugated linoleic acid, acrolein + whey protein or acrolein + conjugated linoleic acid for six days per week during 30 days. Acrolein caused dyslipidemia and oxidative stress in red blood cells and haert mitochondria. Moreover, it caused dysfunction in mitochondrial bioenergetics by decreasing levels of oxidative phosphorylation enzymes, tricarboxylic acid cycle enzymes and ATP. Co-treatment with acrolein + whey protein and acrolein + conjugated linoleic acid ameliorated acrolein-induced oxidative stress and dysfunction in mitochondrial bioenergetics. This amelioration effect was more prominent in acrolein + conjugated linoleic acid group. Interestingly, co-treatment with acrolein + whey protein negatively affected some markers of cardiac injury such as creatinine kinase-MB, lactate dehydrogenase and homocysteine. Conjugated linoleic acid may also cause dyslipidemia because it increased the levels of triacylglycerol, low density lipoproteins and very low density lipoproteins. In conclusion, using some weight loss supplements such as whey protein may adversely affect the biochemical parameters related to cardiovascular system.

Fatty acid oxidation: An emerging facet of metabolic transformation in cancer

Cancer cells undergo metabolic reprogramming such as enhanced aerobic glycolysis, mutations in the tricarboxylic acid cycle enzymes, and upregulation of de novo lipid synthesis and glutaminolysis. These alterations are pivotal to the development and maintenance of the malignant phenotype of cancer cells in unfavorable tumor microenvironment or metastatic sites. Although mitochondrial fatty acid β-oxidation (FAO) is a primary bioenergetic source, it has not been generally recognized as part of the metabolic landscape of cancer. The last few years, however, have seen a dramatic change in the view of cancer relevance of the FAO pathway. Many recent studies have provided significant evidence to support a “lipolytic phenotype” of cancer. FAO, like other well-defined metabolic pathways involved in cancer, is dysregulated in diverse human malignancies. Cancer cells rely on FAO for proliferation, survival, stemness, drug resistance, and metastatic progression. FAO is also reprogrammed in cancer-associated immune and other host cells, which may contribute to immune suppression and tumor-promoting microenvironment. This article reviews and puts into context our current understanding of multi-faceted roles of FAO in oncogenesis as well as anti-cancer therapeutic opportunities posed by the FAO pathway.

Identification of Missing Carbon Fixation Enzymes as Potential Drug Targets in Mycobacterium Tuberculosis.

Metabolic adaptation to the host environment has been recognized as an essential mechanism of pathogenicity and the growth of Mycobacterium tuberculosis (Mtb) in the lungs for decades. The Mtb uses CO2 as a source of carbon during the dormant or non-replicative state. However, there is a lack of biochemical knowledge of its metabolic networks. In this study, we investigated the CO2 fixation pathways (such as ko00710 and ko00720) most likely involved in the energy production and conversion of CO2 in Mtb. Extensive pathway evaluation of 23 completely sequenced strains of Mtb confirmed the existence of a complete list of genes encoding the relevant enzymes of the reductive tricarboxylic acid (rTCA) cycle. This provides the evidence that an rTCA cycle may function to fix CO2 in this bacterium. We also proposed that as CO2 is plentiful in the lungs, inhibition of CO2 fixation pathways (by targeting the relevant CO2 fixation enzymes) could be used in the expansion of new drugs against the dormant Mtb. In support of the suggested hypothesis, the CO2 fixation enzymes were confirmed as a potential drug target by analyzing a number of attributes necessary to be a good bacterial target.

Mitoregulin: A lncRNA-Encoded Microprotein that Supports Mitochondrial Supercomplexes and Respiratory Efficiency

Mitochondria are composed of many small proteins that control protein synthesis, complex assembly, metabolism, and ion and reactive oxygen species (ROS) handling. We show that a skeletal muscle- and heart-enriched long non-coding RNA, LINC00116, encodes a highly conserved 56-amino-acid microprotein that we named mitoregulin (Mtln). Mtln localizes to the inner mitochondrial membrane, where it binds cardiolipin and influences protein complex assembly. In cultured cells, Mtln overexpression increases mitochondrial membrane potential, respiration rates, and Ca2+ retention capacity while decreasing mitochondrial ROS and matrix-free Ca2+. Mtln-knockout mice display perturbations in mitochondrial respiratory (super)complex formation and activity, fatty acid oxidation, tricarboxylic acid (TCA) cycle enzymes, and Ca2+ retention capacity. Blue-native gel electrophoresis revealed that Mtln co-migrates alongside several complexes, including the complex I assembly module, complex V, and supercomplexes. Under denaturing conditions, Mtln remains in high-molecular-weight complexes, supporting its role as a sticky molecular tether that enhances respiratory efficiency by bolstering protein complex assembly and/or stability.

The Extra-Pathway Interactome of the TCA Cycle: Expected and Unexpected Metabolic Interactions.

The plant tricarboxylic acid (TCA) cycle provides essential precursors for respiration, amino acid biosynthesis, and general nitrogen metabolism; moreover, it is closely involved in biotic stress responses and cellular redox homeostasis. To further understand the in vivo function of the TCA cycle enzymes, we combined affinity purification with proteomics to generate a comprehensive extra-pathway protein-protein interaction network of the plant TCA cycle. We identified 125 extra-pathway interactions in Arabidopsis (Arabidopsis thaliana) mostly related to the mitochondrial electron transport complex/ATP synthesis and amino acid metabolism but also to proteins associated with redox stress. We chose three high-scoring and two low-scoring interactions for complementary bimolecular fluorescence complementation and yeast two-hybrid assays, which highlighted the reliability of our approach, supported the intimate involvement of TCA cycle enzymes within many biological processes, and reflected metabolic changes reported previously for the corresponding mutant lines. To analyze the function of a subset of these interactions, we selected two mutants of mitochondrial glutaredoxin S15 and Amidase, which have not yet been analyzed with respect to their TCA cycle function, and performed metabolite profiling and flux analysis. Consistent with their interactions identified in this study, TCA cycle metabolites and the relative TCA flux of the two mutants were altered significantly.

Ifi27 is indispensable for mitochondrial function and browning in adipocytes

Brown adipose tissue (BAT) is specialized for energy expenditure, but the signaling pathways that regulate BAT metabolism and activity are incompletely understood. Interferon (IFN) signaling is a sophisticated defense mechanism to counteract viral infection. IFNs and interferon-stimulated genes (ISGs) are reported to exert profound effects on adipocytes. IFN-α inducible protein 27 (Ifi27/ISG12a) is a BAT-enriched gene, yet no any studies on its roles in BAT have been reported. Here, we show that Ifi27 protein localizes to mitochondria and the expression of Ifi27 can be induced by β3-adrenergic activation in adipose tissues. Knockdown of Ifi27 leads to reduced expression of key enzymes of tricarboxylic acid cycle (TCA), the subunits of electron transport chain (ETC) and uncoupling protein 1 (Ucp1) in brown and beige adipocytes. Moreover, the browning of subcutaneous white fat induced by β3-adrenergic agonist is also dramatically blocked. Ectopic expression of Ifi27 in brown adipocytes has the opposite effects. Together, these data indicate that Ifi27 regulates mitochondrial function and browning in adipocytes.

Characterizing Lysine Acetylation of Isocitrate Dehydrogenase in Escherichia coli

The Escherichia coli isocitrate dehydrogenase (ICDH) is one of the tricarboxylic acid cycle enzymes, playing key roles in energy production and carbon flux regulation. E. coli ICDH was the first bacterial enzyme shown to be regulated by reversible phosphorylation. However, the effect of lysine acetylation on E. coli ICDH, which has no sequence similarity with its counterparts in eukaryotes, is still unclear. Based on previous studies of E. coli acetylome and ICDH crystal structures, eight lysine residues were selected for mutational and kinetic analyses. They were replaced with acetyllysine by the genetic code expansion strategy or substituted with glutamine as a classic approach. Although acetylation decreased the overall ICDH activity, its effects were different site by site. Deacetylation tests demonstrated that the CobB deacetylase could deacetylate ICDH both in vivo and in vitro, but CobB was only specific for lysine residues at the protein surface. On the other hand, ICDH could be acetylated by acetyl-phosphate chemically in vitro. And in vivo acetylation tests indicated that the acetylation level of ICDH was correlated with the amounts of intracellular acetyl-phosphate. This study nicely complements previous proteomic studies to provide direct biochemical evidence for ICDH acetylation.

Rad53p Activation Alters Chromatin Structure, Induces Respiration and Elevates Cellular ATP Level

Rad53p is an essential kinase in the DNA damage response in Saccharomyces cerevisiae. Here we show that activation of Rad53p, either via genetic manipulation or by treatment with genotoxic chemicals, induces mitochondrial respiration. Activation of Rad53p results in reduced transcription of histone genes and globally decreased DNA nucleosome occupancy. This globally altered chromatin structure leads to increased expression of genes encoding enzymes of tricarboxylic acid cycle (TCA), electron transport chain (ETC), and oxidative phosphorylation (OXPHOS) and increased mitochondrial DNA (mtDNA) copy number, oxygen consumption, and ATP synthesis. These findings are surprising and counterintuitive, since it is widely believed that DNA damage results in downregulation of oxidative metabolism to protect DNA from the effects of reactive oxygen species, produced during ETC and OXPHOS. However, our results indicate that the elevated ATP synthesis improves cell survival under conditions of DNA damage and Rad53p activation. In summary, our study reveals a novel role for Rad53p activation in the restructuring of global nucleosomal chromatin architecture and concomitant metabolic transition from fermentation to respiration.Support or Funding InformationSupported by NIH GM120710This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

A plant-like mitochondrial carrier family protein facilitates mitochondrial transport of di- and tricarboxylates in Trypanosoma brucei

The procyclic form of the human parasite Trypanosoma brucei harbors one single, large mitochondrion containing all tricarboxylic acid (TCA) cycle enzymes and respiratory chain complexes present also in higher eukaryotes. Metabolite exchange among subcellular compartments such as the cytoplasm, the mitochondrion, and the peroxisomes is crucial for redox homeostasis and for metabolic pathways whose enzymes are dispersed among different organelles. In higher eukaryotes, mitochondrial carrier family (MCF) proteins transport TCA-cycle intermediates across the inner mitochondrial membrane. Previously, we identified several MCF members that are essential for T. brucei survival. Among these, only one MCF protein, TbMCP12, potentially could transport dicarboxylates and tricarboxylates. Here, we conducted phylogenetic and sequence analyses and functionally characterised TbMCP12 in vivo. Our results suggested that similarly to its homologues in plants, TbMCP12 transports both dicarboxylates and tricarboxylates across the mitochondrial inner membrane. Deleting this carrier in T. brucei was not lethal, while its overexpression was deleterious. Our results suggest that the intracellular abundance of TbMCP12 is an important regulatory element for the NADPH balance and mitochondrial ATP-production.

Improved neuroprotective effect of resveratrol nanoparticles as evinced by abrogation of rotenone-induced behavioral deficits and oxidative and mitochondrial dysfunctions in rat model of Parkinson's disease.

The objective of the present study was to evaluate the protective effect of resveratrol nanoparticles (NRSV) against rotenone-induced neurodegeneration in rats. NRSV were prepared by temperature-controlled antisolvent precipitation method and characterized for its particle size, shape, and dissolution properties. Moreover, NRSV effects compared with the free resveratrol (RSV). Animals were divided into four groups: (I) control, (II) rotenone (2mg/kg s.c.), (III) RSV (40mg/kg, p.o.) + rotenone, and (IV) NRSV (40mg/kg, p.o.) + rotenone. Animals received treatments 30min before rotenone administration for a period of 35days. Behavioral quantifications were done using rota rod test and rearing behavior after 24h of last dose. Animals were euthanized, and mid brains were isolated for the estimation of tricarboxylic acid cycle enzymes, oxidative measures (lipid peroxidation (LPO), glutathione (GSH), and catalase), and complex-I activity. In addition, histopathological studies were also performed. Our results showed that chronic rotenone treatment causes motor deficits, decreased rearing behavior, mitochondrial dysfunction, and oxidative stress. Furthermore, histological analysis demonstrated neuronal degeneration in rotenone-treated rats. An important finding of the present study was NRSV showed comparatively better efficacy than the RSV treatment in attenuating the rotenone-induced Parkinson's like behavioral alterations, biochemical and histological changes, oxidative stress, and mitochondrial dysfunction in rats.

Effects of Elevated Atmospheric CO2 on Respiratory Rates in Mature Leaves of Two Rice Cultivars Grown at a Free-Air CO2 Enrichment Site and Analyses of the Underlying Mechanisms.

Respiratory CO2 efflux and O2 uptake rates in leaves change in response to the growth CO2 concentration ([CO2]). The degrees of change vary depending on the responses of cellular processes such as nitrogen (N) assimilation and accumulation of organic acids to growth [CO2]. However, the underlying mechanisms remain unclear. Here, we examined the respiratory characteristics of mature leaves of two rice varieties with different yield capacities at different growth stages under ambient and elevated [CO2] conditions at a free-air CO2 enrichment site. We also examined the effect of increased water temperature on leaf respiration. We measured the rates of CO2 efflux and O2 uptake, and determined N contents, primary metabolite contents and maximal activities of respiratory enzymes. The leaf CO2 efflux rates decreased in plants grown at elevated [CO2] in both varieties, and were higher in high-yielding Takanari than in Koshihikari. The leaf O2 uptake rates showed little change with respect to growth [CO2] and variety. The increased water temperature did not significantly affect the CO2 efflux and O2 uptake rates. The N and amino acid contents were significantly higher in Takanari than in Koshihikari. The enhanced N assimilation in Takanari may have consumed more respiratory NADH, leading to higher CO2 efflux rates. In Koshihikari, the ratio of tricarboxylic acid (TCA) cycle intermediates changed and maximal activities of enzymes in the TCA cycle decreased at elevated [CO2]. Therefore, the decreased rates of CO2 efflux in Koshihikari may be due to the decreased activities of TCA cycle enzymes at elevated [CO2].

Hepatoprotective effects of capsaicin and alpha-tocopherol on mitochondrial function in mice fed a high-fat diet

Capsaicin (CAP) and alpha-tocopherol (TOC) have antioxidant properties. We investigated effects of CAP and TOC on mitochondrial oxidative stress and mitochondrial bioenergetics in liver of mice fed HFD. AST, ALT, glucose, homeostasis model assessment-insulin resistance index ((HOMA-IR)) and mitochondrial oxidative stress parameters increased, whereas oxidative phosphorylation (OXPHOS) enzymes, tricarboxylic acid cycle (TCA) enzymes, ATP level and mitochondrial metabolic function (MTT) decreased in mice fed a HFD compared to the fed a standard diet (NC). Treatment of HFD together with CAP (HFC group), TOC (HFT group) or TOC and CAP (HCT group) can ameliorate the examined parameters. Because co-treatment with CAP and TOC displayed a better ameliorating effect on liver redox status and mitochondrial bioenergetics functions, they can be useful to protect against HFD and oxidative stress-related in liver diseases.

Towards an Understanding of Energy Impairment in Huntington's Disease Brain.

This review systematically examines the evidence for shifts in flux through energy generating biochemical pathways in Huntington’s disease (HD) brains from humans and model systems. Compromise of the electron transport chain (ETC) appears not to be the primary or earliest metabolic change in HD pathogenesis. Rather, compromise of glucose uptake facilitates glucose flux through glycolysis and may possibly decrease flux through the pentose phosphate pathway (PPP), limiting subsequent NADPH and GSH production needed for antioxidant protection. As a result, oxidative damage to key glycolytic and tricarboxylic acid (TCA) cycle enzymes further restricts energy production so that while basal needs may be met through oxidative phosphorylation, those of excessive stimulation cannot. Energy production may also be compromised by deficits in mitochondrial biogenesis, dynamics or trafficking. Restrictions on energy production may be compensated for by glutamate oxidation and/or stimulation of fatty acid oxidation. Transcriptional dysregulation generated by mutant huntingtin also contributes to energetic disruption at specific enzymatic steps. Many of the alterations in metabolic substrates and enzymes may derive from normal regulatory feedback mechanisms and appear oscillatory. Fine temporal sequencing of the shifts in metabolic flux and transcriptional and expression changes associated with mutant huntingtin expression remain largely unexplored and may be model dependent. Differences in disease progression among HD model systems at the time of experimentation and their varying states of metabolic compensation may explain conflicting reports in the literature. Progressive shifts in metabolic flux represent homeostatic compensatory mechanisms that maintain the model organism through presymptomatic and symptomatic stages.

Relationship between Escherichia coli growth rate and bacterial susceptibility to ciprofloxacin.

The effect of Escherichia coli growth rate on its susceptibility to ciprofloxacin was investigated using bacteria grown on different carbon sources and harboring mutations in genes encoding tricarboxylic acid cycle enzymes. A 1-h treatment of the wild type (wt) grown on glucose, succinate, malate, α-ketoglutarate or acetate with 0.3 μg ml-1 ciprofloxacin decreased the number of surviving cells (CFU ml-1), 560, 110, 74, 62 and 5 times, respectively. Among the mutants tested, sucB strain, which grew 1.75 times slower than wt, was 7.4-fold more tolerant to 0.3 μg ml-1 of ciprofloxacin than wt. Strong inverse correlations between log(CFU ml-1) after 1-h exposure to 0.3 and 3.0 μg ml-1 ciprofloxacin and the specific growth rate prior to antibiotic treatment (r = - 0.93 and -0.96, respectively) were observed. Data from the current and previous studies on the inhibitory effect of ciprofloxacin on cultures exhibiting a wide range of growth rates (0.01-1.3 h-1) were collated. Statistical analysis revealed a significant inverse correlation between log(CFU ml-1) after exposure to 3.0 μg ml-1 of ciprofloxacin and the specific bacterial growth rate prior to antibiotic exposure (r = -0.92). These data may be used in a design of antibiotic treatment protocols.

Contribution of Extracellular Matrix and Signal Mechanotransduction to Epithelial Cell Damage in Inflammatory Bowel Disease Patients: A Proteomic Study.

This study utilizes 2D-DIGE (difference gel etrophoresis), isotope-coded protein labeling and biochemical assays to characterize protein alteration in ulcerative colitis (UC) and Crohn's disease (CD) in human epithelial cell and mucosal biopsies in inflammatory bowel disease (IBD)-affected patients. The aim of this study is to identify the key molecular signatures involved in epithelial cell structure of IBDs. In non-inflamed UC (QUC) keratins, vimentin, and focal adhesion kinase (7) increased, whereas vinculin and de-tyrosinated α-tubulin decreased; inflammation (IUC) exacerbated molecular changes, being collagen type VI alpha 1 chain (COL6A1), tenascin-C and vimentin increased. In non-inflamed CD (QCD), tenascin C, de-tyrosinated α-tubulin, vinculin, FAK, and Rho-associated protein kinase 1 (ROCK1) decreased while vimentin increased. In inflamed CD (ICD), COL6A1, vimentin and integrin alpha 4 increased. In QUC, cell metabolism is characterized by a decrease of the tricarboxylic acid cycle enzymes and a decrease of short/branched chain specific acyl-CoA dehydrogenase, fatty acid synthase, proliferator-activated receptors alpha, and proliferator-activated receptors gamma. In QCD a metabolic rewiring occurs, as suggested by glycerol-3-phosphate dehydrogenase (GPD2), pyruvate dehydrogenase E1 component subunit beta, NADH dehydrogenase [ubiquinone] iron-sulfur protein 3, and 4-trimethylaminobutyraldehyde dehydrogenase increment, while dihydrolipoyl dehydrogenase decreased. Macroautophagy is activated in QUC and IUC, with increased levels of p62, HSC70, major vault protein, myosin heavy chain 9, whereas it is blunted in QCD and ICD. The differing pattern of extracellular matrix, cytoskeletal derangements, cellular metabolism, and autophagy in UC and CD may contribute to the pathophysiological understanding of these disorders and serve as diagnostic markers in IBD patients.

Ancient Regulatory Role of Lysine Acetylation in Central Metabolism.

ABSTRACTLysine acetylation is a common protein post-translational modification in bacteria and eukaryotes. Unlike phosphorylation, whose functional role in signaling has been established, it is unclear what regulatory mechanism acetylation plays and whether it is conserved across evolution. By performing a proteomic analysis of 48 phylogenetically distant bacteria, we discovered conserved acetylation sites on catalytically essential lysine residues that are invariant throughout evolution. Lysine acetylation removes the residue’s charge and changes the shape of the pocket required for substrate or cofactor binding. Two-thirds of glycolytic and tricarboxylic acid (TCA) cycle enzymes are acetylated at these critical sites. Our data suggest that acetylation may play a direct role in metabolic regulation by switching off enzyme activity. We propose that protein acetylation is an ancient and widespread mechanism of protein activity regulation.

Connecting salt stress signalling pathways with salinity-induced changes in mitochondrial metabolic processes in C3 plants.

Salinity exerts a severe detrimental effect on crop yields globally. Growth of plants in saline soils results in physiological stress, which disrupts the essential biochemical processes of respiration, photosynthesis, and transpiration. Understanding the molecular responses of plants exposed to salinity stress can inform future strategies to reduce agricultural losses due to salinity; however, it is imperative that signalling and functional response processes are connected to tailor these strategies. Previous research has revealed the important role that plant mitochondria play in the salinity response of plants. Review of this literature shows that 2 biochemical processes required for respiratory function are affected under salinity stress: the tricarboxylic acid cycle and the transport of metabolites across the inner mitochondrial membrane. However, the mechanisms by which components of these processes are affected or react to salinity stress are still far from understood. Here, we examine recent findings on the signal transduction pathways that lead to adaptive responses of plants to salinity and discuss how they can be involved in and be affected by modulation of the machinery of energy metabolism with attention to the role of the tricarboxylic acid cycle enzymes and mitochondrial membrane transporters in this process.

Fumarate Hydratase Loss Causes Combined Respiratory Chain Defects

SummaryFumarate hydratase (FH) is an enzyme of the tricarboxylic acid (TCA) cycle mutated in hereditary and sporadic cancers. Despite recent advances in understanding its role in tumorigenesis, the effects of FH loss on mitochondrial metabolism are still unclear. Here, we used mouse and human cell lines to assess mitochondrial function of FH-deficient cells. We found that human and mouse FH-deficient cells exhibit decreased respiration, accompanied by a varying degree of dysfunction of respiratory chain (RC) complex I and II. Moreover, we show that fumarate induces succination of key components of the iron-sulfur cluster biogenesis family of proteins, leading to defects in the biogenesis of iron-sulfur clusters that affect complex I function. We also demonstrate that suppression of complex II activity is caused by product inhibition due to fumarate accumulation. Overall, our work provides evidence that the loss of a single TCA cycle enzyme is sufficient to cause combined RC activity dysfunction.

IDH3 mediates apoptosis of alveolar epithelial cells type 2 due to mitochondrial Ca2+ uptake during hypocapnia

In adult respiratory distress syndrome (ARDS) pulmonary perfusion failure increases physiologic dead-space (VD/VT) correlating with mortality. High VD/VT results in alveolar hypocapnia, which has been demonstrated to cause edema formation, atelectasis, and surfactant depletion, evoked, at least in part, by apoptosis of alveolar epithelial cells (AEC). However, the mechanism underlying the hypocapnia-induced AEC apoptosis is unknown. Here, using fluorescent live-cell imaging of cultured AEC type 2 we could show that in terms of CO2 sensing the tricarboxylic acid cycle enzyme isocitrate dehydrogenase (IDH) 3 seems to be an important player because hypocapnia resulted independently from pH in an elevation of IDH3 activity and subsequently in an increase of NADH, the substrate of the respiratory chain. As a consequence, the mitochondrial transmembrane potential (ΔΨ) rose causing a Ca2+ shift from cytosol into mitochondria, whereas the IDH3 knockdown inhibited these responses. Furthermore, the hypocapnia-induced mitochondrial Ca2+ uptake resulted in reactive oxygen species (ROS) production, and both the mitochondrial Ca2+ uptake and ROS production induced apoptosis. Accordingly, we provide evidence that in AEC type 2 hypocapnia induces elevation of IDH3 activity leading to apoptosis. This finding might give new insight into the pathogenesis of ARDS and may help to develop novel strategies to reduce tissue injury in ARDS.