The existence of a nonclassical TCA cycle in the nucleus that wires the metabolic-epigenetic circuitry

The scope and variety of the metabolic intermediates from the mitochondrial tricarboxylic acid (TCA) cycle that are engaged in epigenetic regulation of the chromatin function in the nucleus raise an outstanding question about how timely and precise supply/consumption of these metabolites is achieved in the nucleus. We report here the identification of a nonclassical TCA cycle in the nucleus (nTCA cycle). We found that all the TCA cycle-associated enzymes including citrate synthase (CS), aconitase 2 (ACO2), isocitrate dehydrogenase 3 (IDH3), oxoglutarate dehydrogenase (OGDH), succinyl-CoA synthetase (SCS), fumarate hydratase (FH), and malate dehydrogenase 2 (MDH2), except for succinate dehydrogenase (SDH), a component of electron transport chain for generating ATP, exist in the nucleus. We showed that these nuclear enzymes catalyze an incomplete TCA cycle similar to that found in cyanobacteria. We propose that the nTCA cycle is implemented mainly to generate/consume metabolic intermediates, not for energy production. We demonstrated that the nTCA cycle is intrinsically linked to chromatin dynamics and transcription regulation. Together, our study uncovers the existence of a nonclassical TCA cycle in the nucleus that links the metabolic pathway to epigenetic regulation.

The Ginsenoside Rg1 Rescues Mitochondrial Disorders in Aristolochic Acid-Induced Nephropathic Mice

Chronic exposure to aristolochic acid (AA) leads to renal interstitial fibrosis and nephropathy. In this study, we aimed to investigate the renoprotective effects of Panax ginseng extract (GE) and ginsenoside saponin (GS) on AA-induced nephropathy (AAN) in mice. Eighty female C3H/He mice were randomly divided into eight groups, including normal; AA (3 μg/mL for 56 days); AA with GE (125, 250, or 500 mg/kg/d for 14 days); and AA with important GE ingredients, Rg1, Rb1, or Rd (5 mg/kg/d for 14 days). Compared with the AA group, renal injuries were significantly decreased in the GE (250 mg/kg/d), Rb1, and Rg1 treatment groups. Rg1 exhibited the best renoprotection among all GS-treated groups. There were 24 peaks significantly altered among normal, AA, and AA + Rg1 groups, and four mitochondrial proteins were identified, including acyl-CoA synthetase medium-chain family member 2, upregulated during skeletal muscle growth 5 (Usmg5), mitochondrial aconitase 2 (ACO2), and cytochrome c oxidase subunit Va preprotein (COX5a). We demonstrated for the first time that the AAN mechanism and renoprotective effects of Rg1 are associated with expression of mitochondrial proteins, especially ACO2, Usmg5, and COX5a.

ACO2 clinicobiological dataset with extensive phenotype ontology annotation

Pathogenic variants of the aconitase 2 gene (ACO2) are responsible for a broad clinical spectrum involving optic nerve degeneration, ranging from isolated optic neuropathy with recessive or dominant inheritance, to complex neurodegenerative syndromes with recessive transmission. We created the first public locus-specific database (LSDB) dedicated to ACO2 within the “Global Variome shared LOVD” using exclusively the Human Phenotype Ontology (HPO), a standard vocabulary for describing phenotypic abnormalities. All the variants and clinical cases listed in the literature were incorporated into the database, from which we produced a dataset. We followed a rational and comprehensive approach based on the HPO thesaurus, demonstrating that ACO2 patients should not be classified separately between isolated and syndromic cases. Our data highlight that certain syndromic patients do not have optic neuropathy and provide support for the classification of the recurrent pathogenic variants c.220C>G and c.336C>G as likely pathogenic. Overall, our data records demonstrate that the clinical spectrum of ACO2 should be considered as a continuum of symptoms and refines the classification of some common variants.

Operation of a TCA cycle subnetwork in the mammalian nucleus

Nucleic acid and histone modifications critically depend on central metabolism for substrates and co-factors. Although a few enzymes related to the formation of these required metabolites have been reported in the nucleus, the corresponding metabolic pathways are considered to function elsewhere in the cell. Here we show that a substantial part of the mitochondrial tricarboxylic acid (TCA) cycle, the biosynthetic hub of epigenetic modification factors, is operational also in the nucleus. Using 13C-tracer analysis, we identified activity of glutamine-to-fumarate, citrate-to-succinate, and glutamine-to-aspartate routes in the nuclei of HeLa cells. Proximity labeling mass-spectrometry revealed a spatial vicinity of the involved enzymes with core nuclear proteins, supporting their nuclear location. We further show nuclear localization of aconitase 2 and 2-oxoglutarate dehydrogenase in mouse embryonic stem cells. Together, our results demonstrate operation of an extended metabolic pathway in the nucleus warranting a revision of the canonical view on metabolic compartmentalization and gene expression regulation.

Aconitase 2 inhibits the proliferation of MCF-7 cells promoting mitochondrial oxidative metabolism and ROS/FoxO1-mediated autophagic response

Background Deregulation of the tricarboxylic acid cycle (TCA) due to mutations in specific enzymes or defective aerobic metabolism is associated with tumour growth. Aconitase 2 (ACO2) participates in the TCA cycle by converting citrate to isocitrate, but no evident demonstrations of its involvement in cancer metabolism have been provided so far. Methods Biochemical assays coupled with molecular biology, in silico, and cellular tools were applied to circumstantiate the impact of ACO2 in the breast cancer cell line MCF-7 metabolism. Fluorescence lifetime imaging microscopy (FLIM) of NADH was used to corroborate the changes in bioenergetics. Results We showed that ACO2 levels are decreased in breast cancer cell lines and human tumour biopsies. We generated ACO2- overexpressing MCF-7 cells and employed comparative analyses to identify metabolic adaptations. We found that increased ACO2 expression impairs cell proliferation and commits cells to redirect pyruvate to mitochondria, which weakens Warburg-like bioenergetic features. We also demonstrated that the enhancement of oxidative metabolism was supported by mitochondrial biogenesis and FoxO1-mediated autophagy/mitophagy that sustains the increased ROS burst. Conclusions This work identifies ACO2 as a relevant gene in cancer metabolic rewiring of MCF-7 cells, promoting a different utilisation of pyruvate and revealing the potential metabolic vulnerability of ACO2-associated malignancies.

Minor isozymes tailor yeast metabolism to carbon availability

Isozymes are enzymes that differ in sequence but catalyze the same chemical reactions. Despite their apparent redundancy, isozymes are often retained over evolutionary time for reasons that can be unclear. We find that, in yeast, isozymes are strongly enriched in central carbon metabolism. Using a gene expression compendium, we find that many isozyme pairs show anticorrelated expression during the respirofermentative shift, suggesting roles in adapting to changing carbon availability. Building on this observation, we assign function to two minor central carbon isozymes, aconitase 2 (ACO2) and pyruvate kinase 2 (PYK2).ACO2is expressed during fermentation and proves advantageous when glucose is limiting.PYK2is expressed during respiration and proves advantageous for growth on three-carbon substrates.PYK2’s deletion is rescued by expressing the major pyruvate kinase, but only if that enzyme carries mutations mirroringPYK2’s allosteric regulation. Thus, central carbon isozymes enable more precise tailoring of metabolism to changing nutrient availability.ImportanceGene duplication is one of the main evolutionary drivers of new protein function. However, some gene duplicates have nevertheless persisted long-term without apparent divergence in biochemical function. Further, under standard lab conditions, many isozymes have subtle or no knockout phenotypes. These factors make it hard to assess the unique contributions of individual isozymes to fitness. We therefore developed a method to identify experimental perturbations that could expose such contributions, and applied it to yeast gene expression data, revealing a potential role for a set of yeast isozymes in adapting to changing carbon sources. Our experimental confirmation of distinct roles for two “minor” yeast isozymes, including one with no previously described knockout phenotype, highlight that even apparently redundant paralogs can have important and unique functions, with implications for genome-scale metabolic modeling and systems-level studies of quantitative genetics.