Showing papers on "Oxoglutarate dehydrogenase complex published in 2020"

Wheat mitochondrial respiration shifts from the tricarboxylic acid cycle to the GABA shunt under salt stress

Abstract: Mitochondrial respiration and tricarboxylic acid (TCA) cycle activity are required during salt stress in plants to provide ATP and reductants for adaptive processes such as ion exclusion, compatible solute synthesis and reactive oxygen species (ROS) detoxification. However, there is a poor mechanistic understanding of how salinity affects mitochondrial metabolism, particularly respiratory substrate source. To determine the mechanism of respiratory changes under salt stress in wheat leaves, we conducted an integrated analysis of metabolite content, respiratory rate and targeted protein abundance measurements. Also, we investigated the direct effect of salt on mitochondrial enzyme activities. Salt-treated wheat leaves exhibit higher respiration rate and extensive metabolite changes. The activity of the TCA cycle enzymes pyruvate dehydrogenase complex and the 2-oxoglutarate dehydrogenase complex were shown to be directly salt-sensitive. Multiple lines of evidence showed that the γ-aminobutyric acid (GABA) shunt was activated under salt treatment. During salt exposure, key metabolic enzymes required for the cyclic operation of the TCA cycle are physiochemically inhibited by salt. This inhibition is overcome by increased GABA shunt activity, which provides an alternative carbon source for mitochondria that bypasses salt-sensitive enzymes, to facilitate the increased respiration of wheat leaves.

Lower oxygen consumption and Complex I activity in mitochondria isolated from skeletal muscle of fetal sheep with intrauterine growth restriction

Abstract: Fetal sheep with placental insufficiency-induced intrauterine growth restriction (IUGR) have lower hindlimb oxygen consumption rates (OCRs), indicating depressed mitochondrial oxidative phosphorylation capacity in their skeletal muscle. We hypothesized that OCRs are lower in skeletal muscle mitochondria from IUGR fetuses, due to reduced electron transport chain (ETC) activity and lower abundances of tricarboxylic acid (TCA) cycle enzymes. IUGR sheep fetuses (n = 12) were created with mid-gestation maternal hyperthermia and compared with control fetuses (n = 12). At 132 ± 1 days of gestation, biceps femoris muscles were collected, and the mitochondria were isolated. Mitochondria from IUGR muscle have 47% lower State 3 (Complex I-dependent) OCRs than controls, whereas State 4 (proton leak) OCRs were not different between groups. Furthermore, Complex I, but not Complex II or IV, enzymatic activity was lower in IUGR fetuses compared with controls. Proteomic analysis (n = 6/group) identified 160 differentially expressed proteins between groups, with 107 upregulated and 53 downregulated mitochondria proteins in IUGR fetuses compared with controls. Although no differences were identified in ETC subunit protein abundances, abundances of key TCA cycle enzymes [isocitrate dehydrogenase (NAD+) 3 noncatalytic subunit β (IDH3B), succinate-CoA ligase ADP-forming subunit-β (SUCLA2), and oxoglutarate dehydrogenase (OGDH)] were lower in IUGR mitochondria. IUGR mitochondria had a greater abundance of a hypoxia-inducible protein, NADH dehydrogenase 1α subcomplex 4-like 2, which is known to incorporate into Complex I and lower Complex I-mediated NADH oxidation. Our findings show that mitochondria from IUGR skeletal muscle adapt to hypoxemia and hypoglycemia by lowering Complex I activity and TCA cycle enzyme concentrations, which together, act to lower OCR and NADH production/oxidation in IUGR skeletal muscle.

Selective NADH communication from α-ketoglutarate dehydrogenase to mitochondrial transhydrogenase prevents reactive oxygen species formation under reducing conditions in the heart

Abstract: In heart failure, a functional block of complex I of the respiratory chain provokes superoxide generation, which is transformed to H2O2 by dismutation. The Krebs cycle produces NADH, which delivers electrons to complex I, and NADPH for H2O2 elimination via isocitrate dehydrogenase and nicotinamide nucleotide transhydrogenase (NNT). At high NADH levels, α-ketoglutarate dehydrogenase (α-KGDH) is a major source of superoxide in skeletal muscle mitochondria with low NNT activity. Here, we analyzed how α-KGDH and NNT control H2O2 emission in cardiac mitochondria. In cardiac mitochondria from NNT-competent BL/6N mice, H2O2 emission is equally low with pyruvate/malate (P/M) or α-ketoglutarate (α-KG) as substrates. Complex I inhibition with rotenone increases H2O2 emission from P/M, but not α-KG respiring mitochondria, which is potentiated by depleting H2O2-eliminating capacity. Conversely, in NNT-deficient BL/6J mitochondria, H2O2 emission is higher with α-KG than with P/M as substrate, and further potentiated by complex I blockade. Prior depletion of H2O2-eliminating capacity increases H2O2 emission from P/M, but not α-KG respiring mitochondria. In cardiac myocytes, downregulation of α-KGDH activity impaired dynamic mitochondrial redox adaptation during workload transitions, without increasing H2O2 emission. In conclusion, NADH from α-KGDH selectively shuttles to NNT for NADPH formation rather than to complex I of the respiratory chain for ATP production. Therefore, α-KGDH plays a key role for H2O2 elimination, but is not a relevant source of superoxide in heart. In heart failure, α-KGDH/NNT-dependent NADPH formation ameliorates oxidative stress imposed by complex I blockade. Downregulation of α-KGDH may, therefore, predispose to oxidative stress in heart failure.

DHTKD1 and OGDH display substrate overlap in cultured cells and form a hybrid 2-oxo acid dehydrogenase complex in vivo

Abstract: Glutaric aciduria type 1 (GA1) is an inborn error of lysine degradation characterized by a specific encephalopathy that is caused by toxic accumulation of lysine degradation intermediates. Substrate reduction through inhibition of DHTKD1, an enzyme upstream of the defective glutaryl-CoA dehydrogenase, has been investigated as a potential therapy, but revealed the existence of an alternative enzymatic source of glutaryl-CoA. Here, we show that loss of DHTKD1 in glutaryl-CoA dehydrogenase-deficient HEK-293 cells leads to a 2-fold decrease in the established GA1 clinical biomarker glutarylcarnitine and demonstrate that oxoglutarate dehydrogenase (OGDH) is responsible for this remaining glutarylcarnitine production. We furthermore show that DHTKD1 interacts with OGDH, dihydrolipoyl succinyltransferase and dihydrolipoamide dehydrogenase to form a hybrid 2-oxoglutaric and 2-oxoadipic acid dehydrogenase complex. In summary, 2-oxoadipic acid is a substrate for DHTKD1, but also for OGDH in a cell model system. The classical 2-oxoglutaric dehydrogenase complex can exist as a previously undiscovered hybrid containing DHTKD1 displaying improved kinetics towards 2-oxoadipic acid.

Triheptanoin alters [U-13C6]-glucose incorporation into glycolytic intermediates and increases TCA cycling by normalizing the activities of pyruvate dehydrogenase and oxoglutarate dehydrogenase in a chronic epilepsy mouse model.

Abstract: Triheptanoin is anticonvulsant in several seizure models. Here, we investigated changes in glucose metabolism by triheptanoin interictally in the chronic stage of the pilocarpine mouse epilepsy model. After injection of [U-13C6]-glucose (i.p.), enrichments of 13C in intermediates of glycolysis and the tricarboxylic acid (TCA) cycle were quantified in hippocampal extracts and maximal activities of enzymes in each pathway were measured. The enrichment of 13C glucose in plasma was similar across all groups. Despite this, we observed reductions in incorporation of 13C in several glycolytic intermediates compared to control mice suggesting glucose utilization may be impaired and/or glycogenolysis increased in the untreated interictal hippocampus. Triheptanoin prevented the interictal reductions of 13C incorporation in most glycolytic intermediates, suggesting it increased glucose utilization or - as an additional astrocytic fuel - it decreased glycogen breakdown. In the TCA cycle metabolites, the incorporation of 13C was reduced in the interictal state. Triheptanoin restored the correlation between 13C enrichments of pyruvate relative to most of the TCA cycle intermediates in "epileptic" mice. Triheptanoin also prevented the reductions of hippocampal pyruvate dehydrogenase and 2-oxoglutarate dehydrogenase activities. Decreased glycogen breakdown and increased glucose utilization and metabolism via the TCA cycle in epileptogenic brain areas may contribute to triheptanoin's anticonvulsant effects.

Unstructured regions of large enzymatic complexes control the availability of metabolites with signaling functions

Abstract: Metabolites produced via traditional biochemical processes affect intracellular communication, inflammation, and malignancy. Unexpectedly, acetyl-CoA, α-ketoglutarate and palmitic acid, which are chemical species of reactions catalyzed by highly abundant, gigantic enzymatic complexes, dubbed as “metabolons”, have broad “nonmetabolic” signaling functions. Conserved unstructured regions within metabolons determine the yield of these metabolites. Unstructured regions tether functional protein domains, act as spatial constraints to confine constituent enzyme communication, and, in the case of acetyl-CoA production, tend to be regulated by intricate phosphorylation patterns. This review presents the multifaceted roles of these three significant metabolites and describes how their perturbation leads to altered or transformed cellular function. Their dedicated enzymatic systems are then introduced, namely, the pyruvate dehydrogenase (PDH) and oxoglutarate dehydrogenase (OGDH) complexes, and the fatty acid synthase (FAS), with a particular focus on their structural characterization and the localization of unstructured regions. Finally, upstream metabolite regulation, in which spatial occupancy of unstructured regions within dedicated metabolons may affect metabolite availability and subsequently alter cell functions, is discussed.

Effect of AmyTrap, an amyloid-β binding drug, on Aβ induced mitochondrial dysfunction and tau phosphorylation in cultured neuroblastoma cells.

Abstract: Alzheimer’s Disease (AD) is the most common cause of dementia, affecting 25 million people worldwide. Accumulation of Amyloid-β (Aβ) in the mitochondria has been shown to adversely affect key enzymes including pyruvate dehydrogenase (PDH), succinate dehydrogenase (SDH), oxoglutarate dehydrogenase (OGDH). Accumulation of Aβ is also believed to increase Tau expression and pathology. Tau, in its toxic state, results in synaptic damage causing memory and cognitive dysfunction. We are developing a drug to treat AD namely AmyTrap. The active pharmacological ingredient is a retro inverso, Aβ-binding peptide which sequesters Aβ. We wanted to examine the effect of AmyTrap peptide on Aβ-induced mitochondrial dysfunction and Tau phosphorylation. Therefore, we treated SH-SY5Y neuroblastoma cells with wild-type Aβ, a mutant AβY10A, AmyTrap peptide (RI-peptide), or Aβ and RI-peptide for 72 h. The mutant AβY10A is known to impact the self-aggregating property of Aβ as this Tyr10 is essential for self-aggregation. As expected, AβY10A reversed PDH, OGDH and SDH dysfunction to near normal levels. Further, AβY10A successfully reversed Tau phosphorylation, suggesting that Tyr10 is also associated with Aβ-induced cytotoxicity. RI-peptide was able to significantly reverse SDH dysfunction with limited effect on PDH and Tau phosphorylation. The findings are suggestive that the Tyr10 on Aβ plays a critical role in the self-aggregation. Further studies are warranted to expand these findings.