Tricarboxylic Acid Cycle Enzymes Research Articles

Effect of calcium levels on structure and function of mitochondria in yeast under high glucose fermentation.

In this study, the effects of calcium levels on structure and function of mitochondria under high glucose environment were studied. In the high glucose environment, yeast growth capacity was inhibited, and intracellular reactive oxygen species (ROS) content was increased from 6 h to 12 h, while ROS content was reduced in group with 1 × 10-1 and 1 g/L CaCl2 level from 24 h to 36 h. Exogenous calcium addition had a significant effect on the elevation of intracellular Ca2+ and cytochrome C content in yeast from 6 h to 12 h; mitochondrial membrane potential decreased with the increase of CaCl2 level under high glucose levels. Mitochondrial swelling of yeast was influenced by high glucose levels and showed a regulatory dynamic change by Ca2+ levels. Isocitrate dehydrogenase activity increased in 1 × 10-3 g/L CaCl2 level from 6 h to 12 h, α-ketoglutarate dehydrogenase activity increased with an increase in CaCl2 level from 6 h to 24 h. Calcium affected the structure and function of mitochondria by regulating the intracellular signal, enzymes in tricarboxylic acid cycle, and cytochrome system of yeast under high glucose stress.

Arbuscular mycorrhiza differentially adjusts central carbon metabolism in two contrasting genotypes of Vigna radiata (L.) Wilczek in response to salt stress

The study aimed at investigating Arbuscular Mycorrhiza (AM) mediated metabolic changes in two genotypes of mungbean (Vigna radiata) differing in their salt tolerance in presence of salt stress (100 mM NaCl). Colonisation by Claroideoglomus etunicatum resulted in higher growth, photosynthetic efficiency, total protein content, and lower levels of stress markers, indicating alleviation of stress in mungbean plants. AM differentially upregulated the components of Tricarboxylic acid (TCA) cycle in salt tolerant (ST) and salt sensitive (SS) genotypes that could be correlated to AM-mediated moderation in nutrient uptake. Under salt stress, while maximum increase in the activity of α-ketoglutarate dehydrogenase (65%) was observed in mycorrhizal (M)-ST; the increase in isocitrate dehydrogenase (79%) and fumarase (133%) activities was maximum in M-SS plants over their non-mycorrhizal (NM) counterparts. Apart from TCA, AM also affected gamma-aminobutyric acid (GABA) and glyoxylate pathways. Activities of enzymes implicated in GABA shunt increased in both the genotypes under stress resulting in increase in GABA concentration (46%). Notably, glyoxylate pathway was induced by AM in SS only, wherein M-SS exhibited significantly higher isocitrate lyase (49%) and malate synthase (104%) activities, reflected in higher malic acid concentration (84%), than NM under stress. The results suggest that AM moderates the central carbon metabolism and strategizes towards boosting the formation of stress-alleviating metabolites such as GABA and malic acid, especially in SS, bypassing the steps catalysed by salt-sensitive enzymes in TCA cycle. The study, therefore, advances the understanding on mechanisms by which AM ameliorates salt stress.

Plasmodium Parasite Malate-Quinone Oxidoreductase Functionally Complements a Yeast Deletion Mutant of Mitochondrial Malate Dehydrogenase.

The emergence of drug-resistant variants of malaria-causing Plasmodium parasites is a life-threatening problem worldwide. Investigation of the physiological function of individual parasite proteins is a prerequisite for a deeper understanding of the metabolic pathways required for parasite survival and therefore a requirement for the development of novel antimalarials. A Plasmodium membrane protein, malate-quinone oxidoreductase (MQO), is thought to contribute to the tricarboxylic acid (TCA) cycle and the electron transport chain (ETC) and is an antimalarial drug target. However, there is little information on its expression and function. Here, we investigated the function of Plasmodium falciparum MQO (PfMQO) in mitochondria using a yeast heterologous expression system. Using a yeast deletion mutant of mitochondrial malate dehydrogenase (MDH1), which is expected to be functionally similar to MQO, as a background strain, we successfully constructed PfMQO-expressing yeast. We confirmed that expression of PfMQO complemented the growth defect of the MDH1 deletion, indicating that PfMQO can adopt the metabolic role of MDH1 in energy transduction for growth in the recombinant yeast. Analysis of cell fractions confirmed that PfMQO was expressed and enriched in yeast mitochondria. By measuring MQO activity, we also confirmed that PfMQO expressed in yeast mitochondria was active. Measurement of oxygen consumption rates showed that mitochondrial respiration was driven by the TCA cycle through PfMQO. In addition, we found that MQO activity was enhanced when intact mitochondria were sonicated, indicating that the malate binding site of PfMQO is located facing the mitochondrial matrix. IMPORTANCE We constructed a model organism to study the physiological role and function of P. falciparum malate-quinone oxidoreductase (PfMQO) in a yeast expression system. PfMQO is actively expressed in yeast mitochondria and functions in place of yeast mitochondrial malate dehydrogenase, which catalyzes the oxidation of malate to oxaloacetate in the TCA cycle. The catalytic site for the oxidation of malate in PfMQO, which is a membrane-bound protein, faces into the mitochondrial matrix, not the mitochondrial inner membrane space. Our findings clearly show that PfMQO is a TCA cycle enzyme and is coupled with the ETC via ubiquinone reduction.

Macrophage fumarate hydratase restrains mtRNA-mediated interferon production.

Metabolic rewiring underlies the effector functions of macrophages1-3, but the mechanisms involved remain incompletely defined. Here, using unbiased metabolomics and stable isotope-assisted tracing, we show that an inflammatory aspartate-argininosuccinate shunt is induced following lipopolysaccharide stimulation. The shunt, supported by increased argininosuccinate synthase (ASS1) expression, also leads to increased cytosolic fumarate levels and fumarate-mediated protein succination. Pharmacological inhibition and genetic ablation of the tricarboxylic acid cycle enzyme fumarate hydratase (FH) further increases intracellular fumarate levels. Mitochondrial respiration is also suppressed and mitochondrial membrane potential increased. RNA sequencing and proteomics analyses demonstrate that there are strong inflammatory effects resulting from FH inhibition. Notably, acute FH inhibition suppresses interleukin-10 expression, which leads to increased tumour necrosis factor secretion, an effect recapitulated by fumarate esters. Moreover, FH inhibition, but not fumarate esters, increases interferon-β production through mechanisms that are driven by mitochondrial RNA (mtRNA) release and activation of the RNA sensors TLR7, RIG-I and MDA5. This effect is recapitulated endogenously when FH is suppressed following prolonged lipopolysaccharide stimulation. Furthermore, cells from patients with systemic lupus erythematosus also exhibit FH suppression, which indicates a potential pathogenic role for this process in human disease. We therefore identify a protective role for FH in maintaining appropriate macrophage cytokine and interferon responses.

A combination of simvastatin and low-protein diet increases renal 2-oxoglutarate dehydrogenase activity in rats

Mitochondrial 2-oxoglutarate dehydrogenase complex (2-OGDH) that consists of multiple copies of 3 catalytic subunits (E1, E2, E3) is a regulatory enzyme of tricarboxylic acid cycle. 2-OGDH together with branched chain α-ketoacid dehydrogenase (BCKDH) and pyruvate dehydrogenase (PDH) belongs to the 2-oxoacid dehydrogenases family. It was shown that in protein-restricted rats simvastatin stimulated liver BCKDH, whereas it exerted no effect on BCKDH in rats fed a standard diet. We hypothesized that combination of simvastatin and low-protein diet could have an impact on renal 2-OGDH. The purpose of the study was to determine the effect of combination of simvastatin and low-protein diet on renal 2-OGDH in rats. Simvastatin (80 mg/kg b.wt/day) or the vehicle (0.3% methylcellulose) were administered orally (for 14 days) to rats fed low-protein (8% protein) or standard (23% protein) diet. 2-OGDH activity, protein levels and mRNA levels for E1 and E2 subunits were determined. In addition, serum creatinine level was measured. Results: The combination of simvastatin and low-protein diet elicited the increase of renal 2-OGDH activity that corresponded to the increase of E1 protein, but not of E1 mRNA level. In contrast, simvastatin treatment did not affect 2-OGDH activity, nor protein and mRNA levels of E1 in rats fed standard diet. Serum creatinine levels were not changed upon simvastatin administration in any group. In conclusion, the results of present study indicate that combination of simvastatin and low-protein diet induces stimulation of renal 2-OGDH complex probably at post-transcriptional level.

Skeletal muscle mitochondrial interactome remodeling is linked to functional decline in aged female mice.

Genomic, transcriptomic and proteomic approaches have been used to gain insight into molecular underpinnings of aging in laboratory animals and in humans. However, protein function in biological systems is under complex regulation and includes factors besides abundance levels, such as modifications, localization, conformation and protein-protein interactions. By making use of quantitative chemical cross-linking technologies, we show that changes in the muscle mitochondrial interactome contribute to mitochondrial functional decline in aging in female mice. Specifically, we identify age-related changes in protein cross-links relating to assembly of electron transport system complexes I and IV, activity of glutamate dehydrogenase, and coenzyme-A binding in fatty acid β-oxidation and tricarboxylic acid cycle enzymes. These changes show a remarkable correlation with complex I respiration differences within the same young-old animal pairs. Each observed cross-link can serve as a protein conformational or protein-protein interaction probe in future studies, which will provide further molecular insights into commonly observed age-related phenotypic differences. Therefore, this data set could become a valuable resource for additional in-depth molecular studies that are needed to better understand complex age-related molecular changes.

Citrate synthase from Cyanidioschyzon merolae exhibits high oxaloacetate and acetyl-CoA catalytic efficiency.

Citrate synthase (CS) catalyzes the reaction that produces citrate and CoA from oxaloacetate and acetyl-CoA in the tricarboxylic acid (TCA) cycle. All TCA cycle enzymes are localized to the mitochondria in the model organism, the red alga Cyanidioschyzon merolae. The biochemical properties of CS have been studied in some eukaryotes, but the biochemical properties of CS in algae, including C. merolae, have not been studied. We then performed the biochemical analysis of CS from C. merolae mitochondria (CmCS4). The results showed that the kcat/Km of CmCS4 for oxaloacetate and acetyl-CoA were higher than those of the cyanobacteria, such as Synechocystis sp. PCC 6803, Microcystis aeruginosa PCC 7806 and Anabaena sp. PCC 7120. Monovalent and divalent cations inhibited CmCS4, and in the presence of KCl, the Km of CmCS4 for oxaloacetate and acetyl-CoA was higher in the presence of MgCl2, the Km of CmCS4 for oxaloacetate and acetyl-CoA was higher and kcat lower. However, in the presence of KCl and MgCl2, the kcat/Km of CmCS4 was higher than those of the three cyanobacteria species. The high catalytic efficiency of CmCS4 for oxaloacetate and acetyl-CoA may be a factor in the increased carbon flow into the TCA cycle in C. merolae.

Succinate dehydrogenase variants in paraganglioma: why are B subunit variants 'bad'?

Mutations that predispose to familial pheochromocytoma and paraganglioma include inherited variants in the four genes (SDHA, SDHB, SDHC and SDHD) encoding subunits of succinate dehydrogenase (SDH), an enzyme of the mitochondrial tricarboxylic acid cycle and complex II of the electron transport chain. In heterozygous variant carriers, somatic loss of heterozygosity is thought to result in tumorigenic accumulation of succinate and reactive oxygen species. Inexplicably, variants affecting the SDHB subunit predict worse clinical outcomes. Why? Here we consider two hypotheses. First, relative to SDH A, C and D subunits, the small SDHB subunit might be more intrinsically 'fragile' to missense mutations because of its relatively large fraction of amino acids contacting prosthetic groups and other SDH subunits. We show evidence that supports this hypothesis. Second, the natural pool of human SDHB variants might, by chance, be biased toward severe truncating variants and missense variants causing more disruptive amino acid substitutions. We tested this hypothesis by creating a database of known SDH variants and predicting their biochemical severities. Our data suggest that natural SDHB variants are more pathogenic. It is unclear if this bias is sufficient to explain clinical data. Other explanations include the possibility that SDH subcomplexes remaining after SDHB loss have unique tumorigenic gain-of-function characteristics, and/or that SDHB may have additional unknown tumor-suppressor functions.

Impact of Coenzyme Q10 Supplementation on Skeletal Muscle Respiration, Antioxidants, and the Muscle Proteome in Thoroughbred Horses.

Coenzyme Q10 (CoQ10) is an essential component of the mitochondrial electron transfer system and a potent antioxidant. The impact of CoQ10 supplementation on mitochondrial capacities and the muscle proteome is largely unknown. This study determined the effect of CoQ10 supplementation on muscle CoQ10 concentrations, antioxidant balance, the proteome, and mitochondrial respiratory capacities. In a randomized cross-over design, six Thoroughbred horses received 1600 mg/d CoQ10 or no supplement (control) for 30-d periods separated by a 60-d washout. Muscle samples were taken at the end of each period. Muscle CoQ10 and glutathione (GSH) concentrations were determined using mass spectrometry, antioxidant activities by fluorometry, mitochondrial enzyme activities and oxidative stress by colorimetry, and mitochondrial respiratory capacities by high-resolution respirometry. Data were analyzed using mixed linear models with period, supplementation, and period × supplementation as fixed effects and horse as a repeated effect. Proteomics was performed by tandem mass tag 11-plex analysis and permutation testing with FDR < 0.05. Concentrations of muscle CoQ10 (p = 0.07), GSH (p = 0.75), and malondialdehyde (p = 0.47), as well as activities of superoxide dismutase (p = 0.16) and catalase (p = 0.66), did not differ, whereas glutathione peroxidase activity (p = 0.003) was lower when horses received CoQ10 compared to no supplement. Intrinsic (relative to citrate synthase activity) electron transfer capacity with complex II (ECII) was greater, and the contribution of complex I to maximal electron transfer capacity (FCRPCI and FCRPCIG) was lower when horses received CoQ10 with no impact of CoQ10 on mitochondrial volume density. Decreased expression of subunits in complexes I, III, and IV, as well as tricarboxylic acid cycle (TCA) enzymes, was noted in proteomics when horses received CoQ10. We conclude that with CoQ10 supplementation, decreased expression of TCA cycle enzymes that produce NADH and complex I subunits, which utilize NADH together with enhanced electron transfer capacity via complex II, supports an enhanced reliance on substrates supplying complex II during mitochondrial respiration.

Abrin inactivating mitochondrial ribosome induced anther abortion in cotton CMS line LD6A by damaging mitochondrial membrane structure

Cytoplasmic male sterile (CMS) system has extensively been exploited for hybrid vigor in plant breeding programs. However, its application in many crops is limited due to poor understanding of molecular mechanism of fertility restoration. Using advanced analytical approaches, we aimed to elucidate the molecular pathways, regulating CMS induction and fertility restoration in cotton. Reproductive structures of a novel CMS (LD6A) and its maintainer (LD6B) lines were analyzed for physiological and proteomic changes during the development process. Transmission electron micrographs of anther tapetum showed a relatively indistinct inner ridge of CMS than of LD6B mitochondria at tetrad stage. Furthermore, at pollen mother cell and tetrad stages, CMS anthers accumulated a significantly higher reactive oxygen species than the maintainer line. At the protein level, CMS anthers induced significantly higher Abrin (XP_016705148.2), lower tricarboxylic acid cycle enzyme (malate dehydrogenase, malic enzyme, isocitrate dehydrogenase), and histone acetyltransferase expression than its maintainer line. Suggesting that abnormal sequence of mitochondrial ribosome gene rps4 and rpl10 and high expression of ribosome-inactivating protein Abrin in CMS line damaged mitochondrial membrane. This inhibited energy supply from the non-degraded tapetum, and consequently induced pollen sterility in LD6A. These data provide new insights into CMS mechanism in cotton crops that can be used for developing new CMS germplasm resources.

Quantification and Proteomic Characterization of β-Hydroxybutyrylation Modification in the Hearts of AMPKα2 Knockout Mice

AMP-activated protein kinase alpha 2 (AMPKα2) regulates energy metabolism, protein synthesis, and glucolipid metabolism myocardial cells. Ketone bodies produced by fatty acid β-oxidation, especially β-hydroxybutyrate, are fatty energy–supplying substances for the heart, brain, and other organs during fasting and long-term exercise. They also regulate metabolic signaling for multiple cellular functions. Lysine β-hydroxybutyrylation (Kbhb) is a β-hydroxybutyrate–mediated protein posttranslational modification. Histone Kbhb has been identified in yeast, mouse, and human cells. However, whether AMPK regulates protein Kbhb is yet unclear. Hence, the present study explored the changes in proteomics and Kbhb modification omics in the hearts of AMPKα2 knockout mice using a comprehensive quantitative proteomic analysis. Based on mass spectrometry (LC-MS/MS) analysis, the number of 1181 Kbhb modified sites in 455 proteins were quantified between AMPKα2 knockout mice and wildtype mice; 244 Kbhb sites in 142 proteins decreased or increased after AMPKα2 knockout (fold change >1.5 or <1/1.5, p < 0.05). The regulation of Kbhb sites in 26 key enzymes of fatty acid degradation and tricarboxylic acid cycle was noted in AMPKα2 knockout mouse cardiomyocytes. These findings, for the first time, identified proteomic features and Kbhb modification of cardiomyocytes after AMPKα2 knockout, suggesting that AMPKα2 regulates energy metabolism by modifying protein Kbhb.

Variants of Fumarate Hydratase Gene in Uterine Disorders: A Clinical Trial

Objective: The dysregulation of metabolism is a hallmark of cancer. Enzymes of tricarboxylic acid (TCA) cycle have a key role in pathogenesis of carcinogenesis as oncometabolits. The variants in fumarate hydratase (FH) gene have been linked to sporadic tumors and familial tumor propensity. To asses the prevalence of variants in the FH gene in endometrial carcinoma (EC) and uterine leiomyomas (UL). Material and Methods: This prospective study included 58 patients with EC and 44 with UL. The FH gene variants were analyzed in the DNA samples collected from peripheral circulation. All exons of the FH gene were replicated by polymerase chain reaction utilizing specific primers including intron-exon boundaries and than was sequenced with Sanger method. Results: Only two variants were detected in two different patients. The c.927G>A (p.Pro309Pro) and c.63C>T (p.Ala21Ala) variants were observed in patients with UL, EC respectively. c.927G>A (p.Pro309=) and c.63C>T (p.Ala21=) are rare variants inside the overall population with allele frequency ranges of 0.02428, 0.000014 respectively. Conclusion: The contribution of variants in genes encoding the TCA cycle enzymes in tumor cells has provided understanding fundamental role on metabolic alterations in carcinogenesis. Further studies are needed to discover the novel tumor mutations and identify appropriate treatment.

Enzyme a Biosynthesis System on Manifestation of Metabolic Stress and Glutathione System in the CNS under Aluminium Neurotoxicosis

Alzheimer’s-like disease was simulated in female adult Wistar CRL(WI) WUBR rats by 6-week intragastric administration of aluminium chloride at a dose of 200 mg/kg body mass. In the presence of developed oxidative stress (OS), we found a decrease in the activities of tricarboxylic acid cycle (TCA cycle) enzymes and an increase in the activities of pentose phosphate pathway (PPP) dehydrogenases as well as a reduction of SH-and SS-groups in proteins (P) along with the increased SH/SS ratio and glutathionylation with simultaneous decreases of glutathione (GSH) and the GSH/GSSG ratio and its redox potential in the brain hemispheres. The glutathione system enzymes were changed multidirectionally, with glutathione reductase remaining stable. Decreased activities of GSH biosynthesis enzymes and cysteine content were noticed. The intragastric administration of the CoA biosynthesis modulators D-panthenol (PL), D-pantethine or D-homopantothenate (HPA) at a dose of 200 mg/kg since the 5th week of the experiment caused either reduction or leveling of OS manifestations in blood plasma, an increase in acetyl cholinesterase, normalization of the activities of TCA cycle and PPP enzymes, P-SH level (not the SH/SS ratio) and a considerable reduction of S-glutathionylation as well as increases in GSH level, the GSH/GSSG ratio and redox potential in the hemispheres. The effect of CoA system modulators was manifested in activation of glutathione transferase, a decrease of glutathione peroxidase and less evident activation of GSH biosynthesis enzymes (PL) although they contributed to the elevation of cysteine content due to the reduced protein S-cysteinylation. The levels and the ratio of CoA/acetyl-CoA (except for PL) were not changed by toxicosis and the OS modulators. The feasibility of non-conenzyme effects was confirmed by the administration of HPA. The phenomenon of redox activity of the CoA biosynthesis modulators with clearly directional effects on the glutathione system and the TCA cycle and PPP enzymes during alleviation of OS and aluminium neurotoxicosis is discussed.

The unbroken Krebs cycle. Hormonal-like regulation and mitochondrial signaling to control mitophagy and prevent cell death.

The tricarboxylic acid (TCA) or Krebs cycle, which takes place in prokaryotic cells and in the mitochondria of eukaryotic cells, is central to life on Earth and participates in key events such as energy production and anabolic processes. Despite its relevance, it is not perceived as tightly regulated compared to other key metabolisms such as glycolysis/gluconeogenesis. A better understanding of the functioning of the TCA cycle is crucial due to mitochondrial function impairment in several diseases, especially those that occur with neurodegeneration. This article revisits what is known about the regulation of the Krebs cycle and hypothesizes the need for large-scale, rapid regulation of TCA cycle enzyme activity. Evidence of mitochondrial enzyme activity regulation by activation/deactivation of protein kinases and phosphatases exists in the literature. Apart from indirect regulation via G protein-coupled receptors (GPCRs) at the cell surface, signaling upon activation of GPCRs in mitochondrial membranes may lead to a direct regulation of the enzymes of the Krebs cycle. Hormonal-like regulation by posttranscriptional events mediated by activable kinases and phosphatases deserve proper assessment using isolated mitochondria. Also see the video abstract here: https://youtu.be/aBpDSWiMQyI.

Biallelic variants in OGDH encoding oxoglutarate dehydrogenase lead to a neurodevelopmental disorder characterized by global developmental delay, movement disorder, and metabolic abnormalities

PurposeThis study aimed to establish the genetic cause of a novel autosomal recessive neurodevelopmental disorder characterized by global developmental delay, movement disorder, and metabolic abnormalities. MethodsWe performed a detailed clinical characterization of 4 unrelated individuals from consanguineous families with a neurodevelopmental disorder. We used exome sequencing or targeted-exome sequencing, cosegregation, in silico protein modeling, and functional analyses of variants in HEK293 cells and Drosophila melanogaster, as well as in proband-derived fibroblast cells. ResultsIn the 4 individuals, we identified 3 novel homozygous variants in oxoglutarate dehydrogenase (OGDH) (NM_002541.3), which encodes a subunit of the tricarboxylic acid cycle enzyme α-ketoglutarate dehydrogenase. In silico homology modeling predicts that c.566C>T:p.(Pro189Leu) and c.890C>A:p.(Ser297Tyr) variants interfere with the structure and function of OGDH. Fibroblasts from individual 1 showed that the p.(Ser297Tyr) variant led to a higher degradation rate of the OGDH protein. OGDH protein with p.(Pro189Leu) or p.(Ser297Tyr) variants in HEK293 cells showed significantly lower levels than the wild-type protein. Furthermore, we showed that expression of Drosophila Ogdh (dOgdh) carrying variants homologous to p.(Pro189Leu) or p.(Ser297Tyr), failed to rescue developmental lethality caused by loss of dOgdh. SpliceAI, a variant splice predictor, predicted that the c.935G>A:p.(Arg312Lys)/p.(Phe264_Arg312del) variant impacts splicing, which was confirmed through a mini-gene assay in HEK293 cells. ConclusionWe established that biallelic variants in OGDH cause a neurodevelopmental disorder with metabolic and movement abnormalities.

Inhibition of hepatic energy metabolizing enzymes in murine model exposed to diisononyl phthalate

Background and objectives: Diisononyl phthalate (DINP) is a class of phthalates and phthalates are known to be metabolism disrupting chemicals (MDCs). Numerous MDCs, to which humans are exposed, have an effect on every aspect of energy transduction. They affect the liver by impairing insulin secretion in pancreatic cells and altering the liver’s insulin-dependent glucose metabolism. Methods: For this study, eighteen male albino rats weighing 200±20g were randomly assigned to three groups (of six rats each) and followed for a 14-days period. The groups were: group A or control which was given Tween-80 orally, group B or DINP1 group which was given 20 mg/kg b.wt. DINP, and Group C or DINP2 group which received 200 mg/kg b.wt. DINP. The rats were then sacrificed, their livers were removed, and the glycolytic and oxidative phosphorylation enzyme activities were evaluated. Results: Activities of the glycolytic, tricarboxylic acid cycle and electron transport chain enzymes under investigation were significantly down-regulated with severity observed in decreased activities of hepatic oxidative phosphorylation enzymes when compared with control (P&lt;0.05). Hepatic tissue sections of 20 and 200mg/kg DiNP group revealed distorted cytoarchitecture of hepatocytes ranging from histocellular disarrangement to vaocular changes suggestive of loss of liver integrity or fibrosis. Conclusions: Finally, DINP exposure impairs hepatic energy transduction enzymes as evident in down-regulation of the various enzymes of energy metabolism under investigation and this may invariably be a good tool for the diagnosis of hepatic energy impairment as seen in some disease conditions.

Fumarate hydratase inhibits non-small cell lung cancer metastasis via inactivation of AMPK and upregulation of DAB2.

Lung cancer is one of the leading causes of cancer mortality worldwide. As it is often first diagnosed only when cancer metastasis has already occurred, the development of effective biomarkers for the risk prediction of cancer metastasis, followed by stringent monitoring and the early treatment of high-risk patients, is essential for improving patient survival. Cancer cells exhibit alterations in metabolic pathways that enable them to maintain rapid growth and proliferation, which are quite different from the metabolic pathways of normal cells. Fumarate hydratase (FH, fumarase) is a well-known tricarboxylic acid cycle enzyme that catalyzes the reversible hydration/dehydration of fumarate to malate. The current study sought to investigate the relationship between FH expression levels and the outcome of patients with lung cancer. FH was knocked down in lung cancer cells using shRNA or overexpressed using a vector, and the effect on migration ability was assessed. Furthermore, the role of AMP-activated protein kinase (AMPK) phosphorylation and disabled homolog 2 in the underlying mechanism was investigated using an AMPK inhibitor approach. The results showed that in lung cancer tissues, low FH expression was associated with lymph node metastasis, tumor histology and recurrence. In addition, patients with low FH expression exhibited a poor overall survival in comparison with patients having high FH expression. When FH was overexpressed in lung cancer cells, cell migration was reduced with no effect on cell proliferation. Furthermore, the level of phosphorylated (p-)AMPK, an energy sensor molecule, was upregulated when FH was knocked down in lung cancer cells, and the inhibition of p-AMPK led to an increase in the expression of disabled homolog 2, a tumor suppressor protein. These findings suggest that FH may serve as an effective biomarker for predicting the prognosis of lung cancer and as a therapeutic mediator.

Nuclear localization of mitochondrial TCA cycle enzymes modulates pluripotency via histone acetylation

Pluripotent stem cells hold great promise in regenerative medicine and developmental biology studies. Mitochondrial metabolites, including tricarboxylic acid (TCA) cycle intermediates, have been reported to play critical roles in pluripotency. Here we show that TCA cycle enzymes including Pdha1, Pcb, Aco2, Cs, Idh3a, Ogdh, Sdha and Mdh2 are translocated to the nucleus during somatic cell reprogramming, primed-to-naive transition and totipotency acquisition. The nuclear-localized TCA cycle enzymes Pdha1, Pcb, Aco2, Cs, Idh3a promote somatic cell reprogramming and primed-to-naive transition. In addition, nuclear-localized TCA cycle enzymes, particularly nuclear-targeted Pdha1, facilitate the 2-cell program in pluripotent stem cells. Mechanistically, nuclear Pdha1 increases the acetyl-CoA and metabolite pool in the nucleus, leading to chromatin remodeling at pluripotency genes by enhancing histone H3 acetylation. Our results reveal an important role of mitochondrial TCA cycle enzymes in the epigenetic regulation of pluripotency that constitutes a mitochondria-to-nucleus retrograde signaling mode in different states of pluripotent acquisition.

Acetylation of citrate synthase inhibits Bombyx mori nucleopolyhedrovirus propagation by affecting energy metabolism.

Many studies have confirmed that virus infection cause changes in the expression level and post-translational modifications of tricarboxylic acid cycle (TCA) enzymes. In a previous study, we found that the acetylation level of lysine 336 of Bombyx mori citrate synthase (BmCS) was remarkably unregulated after Bombyx mori nucleopolyhedrovirus (BmNPV) infection. In the present study, we found that BmN cells infected with BmNPV could up-regulate BmCS transient expression and promote the acetylation modification of BmCS. Transient expression vectors for over-expression of wild-type Bmcs and K336 acetylation mimic mutant (K336Q) were constructed to analyze enzyme activity, revealing that acetylation of K336 significantly reduced its activity. The obtained results indicated that BmCS knock-down or K336 acetylation similarly suppressed BmN cellular ATP production and mitochondrial membrane potential. Furthermore, the acetylation of K336 and the reduction of BmCS expression contributed to weakening the replication lever of the BmNPV proliferation and the generation of progeny viruses. In sum, our study on the single lysine 336 acetylation and knock-down of Bmcs revealed the potential mechanism for inhibiting the proliferation of BmNPV, which may provide novel insights for the development of antiviral strategies.

Nuclear Receptor NR1D1 Regulates Abdominal Aortic Aneurysm Development by Targeting the Mitochondrial Tricarboxylic Acid Cycle Enzyme Aconitase-2.

Metabolic disorder increases the risk of abdominal aortic aneurysm (AAA). NRs (nuclear receptors) have been increasingly recognized as important regulators of cell metabolism. However, the role of NRs in AAA development remains largely unknown. We analyzed the expression profile of the NR superfamily in AAA tissues and identified NR1D1 (NR subfamily 1 group D member 1) as the most highly upregulated NR in AAA tissues. To examine the role of NR1D1 in AAA formation, we used vascular smooth muscle cell (VSMC)-specific, endothelial cell-specific, and myeloid cell-specific conditional Nr1d1 knockout mice in both AngII (angiotensin II)- and CaPO4-induced AAA models. Nr1d1 gene expression exhibited the highest fold change among all 49 NRs in AAA tissues, and NR1D1 protein was upregulated in both human and murine VSMCs from AAA tissues. The knockout of Nr1d1 in VSMCs but not endothelial cells and myeloid cells inhibited AAA formation in both AngII- and CaPO4-induced AAA models. Mechanistic studies identified ACO2 (aconitase-2), a key enzyme of the mitochondrial tricarboxylic acid cycle, as a direct target trans-repressed by NR1D1 that mediated the regulatory effects of NR1D1 on mitochondrial metabolism. NR1D1 deficiency restored the ACO2 dysregulation and mitochondrial dysfunction at the early stage of AngII infusion before AAA formation. Supplementation with αKG (α-ketoglutarate, a downstream metabolite of ACO2) was beneficial in preventing and treating AAA in mice in a manner that required NR1D1 in VSMCs. Our data define a previously unrecognized role of nuclear receptor NR1D1 in AAA pathogenesis and an undescribed NR1D1-ACO2 axis involved in regulating mitochondrial metabolism in VSMCs. It is important that our findings suggest αKG supplementation as an effective therapeutic approach for AAA treatment.