Α-ketoglutarate Dehydrogenase Complex Research Articles

Proteomics Reveals the Obstruction of Cellular ATP Synthesis in the Ruminal Epithelium of Growth-Retarded Yaks.

Growth-retarded yaks are of a high proportion on the Tibetan plateau and reduce the economic income of farmers. Our previous studies discovered a maldevelopment in the ruminal epithelium of growth-retarded yaks, but the molecular mechanisms are still unclear. This study aimed to reveal how the proteomic profile in the ruminal epithelium contributed to the growth retardation of yaks. The proteome of the ruminal epithelium was detected using a high-resolution mass spectrometer. There were 52 proteins significantly differently expressed between the ruminal epithelium of growth-retarded yaks and growth-normal yaks, with 32 downregulated and 20 upregulated in growth-retarded yaks. Functional analysis showed the differently expressed proteins involved in the synthesis and degradation of ketone bodies (p = 0.012), propanoate metabolism (p = 0.018), pyruvate metabolism (p = 0.020), and mineral absorption (p = 0.024). The protein expressions of SLC26A3 and FTH1, enriched in the mineral absorption, were significantly downregulated in growth-retarded yaks. The key enzymes ACAT2 and HMGCS2 enriched in ketone bodies synthesis and key enzyme PCCA enriched in propanoate metabolism had lower protein expressions in the ruminal epithelium of growth-retarded yaks. The ATP concentration and relative mitochondrial DNA copy number in the ruminal epithelium of growth-normal yaks were dramatically higher than those of growth-retarded yaks (p < 0.05). The activities of citrate synthase (CS), the α-ketoglutarate dehydrogenase complex (α-KGDHC), isocitrate dehydrogenase (ICD) in the tricarboxylic acid cycle (TCA), and the mitochondrial respiratory chain complex (MRCC) were significantly decreased in ruminal epithelium of growth-retarded yaks compared to growth-normal yaks (p < 0.05). The mRNA expressions of COQ9, COX4, and LDHA, which are the encoding genes in MRCC I, IV and anaerobic respiration, were also significantly decreased in the ruminal epithelium of growth-retarded yaks (p < 0.05). Correlation analysis revealed that the average daily gain (ADG) was significantly positively correlated to the relative mitochondrial DNA copy number (p < 0.01, r = 0.772) and ATP concentration (p < 0.01, r = 0.728) in the ruminal epithelium, respectively. The ruminal weight was positively correlated to the relative mitochondrial DNA copy number (p < 0.05, r = 0.631) and ATP concentration in ruminal epithelium (p < 0.01, r = 0.957), respectively. The ruminal papillae had a significant positive correlation with ATP concentration in ruminal epithelium (p < 0.01, r = 0.770). These results suggested that growth-retarded yaks had a lower VFA metabolism, ketone bodies synthesis, ion absorption, and ATP synthesis in the ruminal epithelium; it also indicated that the growth retardation of yaks is related to the obstruction of cellular ATP synthesis in rumen epithelial cells.

Complex I activity in hypoxia: implications for oncometabolism.

Certain cancer cells within solid tumors experience hypoxia, rendering them incapable of oxidative phosphorylation (OXPHOS). Despite this oxygen deficiency, these cells exhibit biochemical pathway activity that relies on NAD+. This mini-review scrutinizes the persistent, residual Complex I activity that oxidizes NADH in the absence of oxygen as the electron acceptor. The resulting NAD+ assumes a pivotal role in fueling the α-ketoglutarate dehydrogenase complex, a critical component in the oxidative decarboxylation branch of glutaminolysis - a hallmark oncometabolic pathway. The proposition is that through glutamine catabolism, high-energy phosphate intermediates are produced via substrate-level phosphorylation in the mitochondrial matrix substantiated by succinyl-CoA ligase, partially compensating for an OXPHOS deficiency. These insights provide a rationale for exploring Complex I inhibitors in cancer treatment, even when OXPHOS functionality is already compromised.

Parapyruvate Induces Neurodegeneration in C57BL/6JNarl Mice via Inhibition of the α-Ketoglutarate Dehydrogenase Complex.

Parapyruvate is a substance commonly found in commercial dietary supplements of calcium pyruvate (DSCP) that inhibits the α-ketoglutarate dehydrogenase complex (KGDHC) and has been shown to induce senescence in human Hs68 cells. However, it is unknown whether parapyruvate can induce neurodegeneration. In this study, the parapyruvate content in DSCP was converted to an equivalent dose for mice and administered to the C57BL/6JNarl mice at doses around the equivalent dose for 69 days, including 5, 50, and 500 mg/kg/day. The Morris water maze (MWM) task and the active avoidance test were conducted to assess the learning and memory ability in mice, and then brain tissues were collected for biochemical analyses. The results demonstrated that parapyruvate significantly impaired the learning and memory ability, decreased the KGDHC activity, and promoted the oxidative stress and acetylcholinesterase (AChE) activity in mice in a dose-dependent manner. Additionally, parapyruvate induced Tau and phosphorylated Tau (p-Tau) aggregation at dosages ≥5 mg/kg/day and increased the myelin basic protein (MBP) expression at a dosage of 500 mg/kg/day. These results suggest that the equivalent dose of parapyruvate can induce neurodegeneration in the C57BL/6JNarl mice.

Mitochondrial Alpha-Keto Acid Dehydrogenase Complexes: Recent Developments on Structure and Function in Health and Disease.

The present work delves into the enigmatic world of mitochondrial alpha-keto acid dehydrogenase complexes discussing their metabolic significance, enzymatic operation, moonlighting activities, and pathological relevance with links to underlying structural features. This ubiquitous family of related but diverse multienzyme complexes is involved in carbohydrate metabolism (pyruvate dehydrogenase complex), the citric acid cycle (α-ketoglutarate dehydrogenase complex), and amino acid catabolism (branched-chain α-keto acid dehydrogenase complex, α-ketoadipate dehydrogenase complex); the complexes all function at strategic points and also participate in regulation in these metabolic pathways. These systems are among the largest multienzyme complexes with at times more than 100 protein chains and weights ranging up to ~10 million Daltons. Our chapter offers a wealth of up-to-date information on these multienzyme complexes for a comprehensive understanding of their significance in health and disease.

A hub for regulation of mitochondrial metabolism: Fatty acid and lipoic acid biosynthesis.

Having evolved from a prokaryotic origin, mitochondria retain pathways required for the catabolism of energy-rich molecules and for the biosynthesis of molecules that aid catabolism and/or participate in other cellular processes essential for life of the cell. Reviewed here are details of the mitochondrial fatty acid biosynthetic pathway (FAS II) and its role in building both the octanoic acid precursor for lipoic acid biosynthesis (LAS) and longer-chain fatty acids functioning in chaperoning the assembly of mitochondrial multisubunit complexes. Also covered are the details of mitochondrial lipoic acid biosynthesis, which is distinct from that of prokaryotes, and the attachment of lipoic acid to subunits of pyruvate dehydrogenase, α-ketoglutarate dehydrogenase, and glycine cleavage system complexes. Special emphasis has been placed on presenting what is currently known about the interconnected paths and loops linking the FAS II-LAS pathway and two other mitochondrial realms, the organellar translation machinery and Fe-S cluster biosynthesis and function.

Evaluating the association of biallelic OGDHL variants with significant phenotypic heterogeneity

BackgroundBiallelic variants in OGDHL, encoding part of the α-ketoglutarate dehydrogenase complex, have been associated with highly heterogeneous neurological and neurodevelopmental disorders. However, the validity of this association remains to be confirmed. A second OGDHL patient cohort was recruited to carefully assess the gene-disease relationship.MethodsUsing an unbiased genotype-first approach, we screened large, multiethnic aggregated sequencing datasets worldwide for biallelic OGDHL variants. We used CRISPR/Cas9 to generate zebrafish knockouts of ogdhl, ogdh paralogs, and dhtkd1 to investigate functional relationships and impact during development. Functional complementation with patient variant transcripts was conducted to systematically assess protein functionality as a readout for pathogenicity.ResultsA cohort of 14 individuals from 12 unrelated families exhibited highly variable clinical phenotypes, with the majority of them presenting at least one additional variant, potentially accounting for a blended phenotype and complicating phenotypic understanding. We also uncovered extreme clinical heterogeneity and high allele frequencies, occasionally incompatible with a fully penetrant recessive disorder. Human cDNA of previously described and new variants were tested in an ogdhl zebrafish knockout model, adding functional evidence for variant reclassification. We disclosed evidence of hypomorphic alleles as well as a loss-of-function variant without deleterious effects in zebrafish variant testing also showing discordant familial segregation, challenging the relationship of OGDHL as a conventional Mendelian gene. Going further, we uncovered evidence for a complex compensatory relationship among OGDH, OGDHL, and DHTKD1 isoenzymes that are associated with neurodevelopmental disorders and exhibit complex transcriptional compensation patterns with partial functional redundancy.ConclusionsBased on the results of genetic, clinical, and functional studies, we formed three hypotheses in which to frame observations: biallelic OGDHL variants lead to a highly variable monogenic disorder, variants in OGDHL are following a complex pattern of inheritance, or they may not be causative at all. Our study further highlights the continuing challenges of assessing the validity of reported disease-gene associations and effects of variants identified in these genes. This is particularly more complicated in making genetic diagnoses based on identification of variants in genes presenting a highly heterogenous phenotype such as “OGDHL-related disorders”.

A reversible state of hypometabolism in a human cellular model of sporadic Parkinson’s disease

Sporadic Parkinson’s Disease (sPD) is a progressive neurodegenerative disorder caused by multiple genetic and environmental factors. Mitochondrial dysfunction is one contributing factor, but its role at different stages of disease progression is not fully understood. Here, we showed that neural precursor cells and dopaminergic neurons derived from induced pluripotent stem cells (hiPSCs) from sPD patients exhibited a hypometabolism. Further analysis based on transcriptomics, proteomics, and metabolomics identified the citric acid cycle, specifically the α-ketoglutarate dehydrogenase complex (OGDHC), as bottleneck in sPD metabolism. A follow-up study of the patients approximately 10 years after initial biopsy demonstrated a correlation between OGDHC activity in our cellular model and the disease progression. In addition, the alterations in cellular metabolism observed in our cellular model were restored by interfering with the enhanced SHH signal transduction in sPD. Thus, inhibiting overactive SHH signaling may have potential as neuroprotective therapy during early stages of sPD.

Engineering of two thiamine diphosphate-dependent enzymes for the regioselective condensation of C1-formaldehyde into C4-erythrulose

A number of carboligases, which catalyze condensation of C1- and/or C2-aldehydes into multi-carbon products, have been reported. However, their catalytic activities and/or regioselectivities remained rather low. Thereby, this study has focused on engineering of C1 and C2 carboligases for the regioselective condensation of C1-formaldehyde into C4-erythrulose via C2-glycolaldehyde. The crystal structure of the glyoxylate carboligase from Escherichia coli (EcGCL) was elucidated in complex with glycolaldehyde. A structure-guided rationale generated several mutants, one of whose catalytic activity reached 15.6 M−1·s−1, almost 10 times greater than the wild-type enzyme. Another variant (i.e., EcGCL_R484M/N283Q/L478M/M488L/R284K) has shown significantly increased stability to the glycolaldehyde toxicity, enabling production of glycolaldehyde to 31 mM from 75 mM formaldehyde (conversion: 83 %). Besides, the E1 subunit of α-ketoglutarate dehydrogenase complex from Vibrio vulnificus (VvSucA) was engineered as a regiospecific C2 carboligase for condensation of glycolaldehyde into erythrulose. The combination of EcGCL_R484M/N283Q/L478M/M488L/R284K and VvSucA_K228L led to the cascade production of erythrulose to 8 mM from 90 mM formaldehyde via glycolaldehyde without byproduct formation. This study will contribute to valorization of C1 gases into industrially relevant multi-carbon products in an environment-friendly way.

Defective function of α-ketoglutarate dehydrogenase exacerbates mitochondrial ATP deficits during complex I deficiency

The NDUFS4 knockout (KO) mouse phenotype resembles the human Complex I deficiency Leigh Syndrome. The irreversible succination of protein thiols by fumarate is increased in select regions of the NDUFS4 KO brain affected by neurodegeneration. We report that dihydrolipoyllysine-residue succinyltransferase (DLST), a component of the α-ketoglutarate dehydrogenase complex (KGDHC) of the tricarboxylic acid (TCA) cycle, is succinated in the affected regions of the NDUFS4 KO brain. Succination of DLST reduced KGDHC activity in the brainstem (BS) and olfactory bulb (OB) of KO mice. The defective production of KGDHC derived succinyl-CoA resulted in decreased mitochondrial substrate level phosphorylation (SLP), further aggravating the existing oxidative phosphorylation (OXPHOS) ATP deficit. Protein succinylation, an acylation modification that requires succinyl-CoA, was reduced in the KO mice. Modeling succination of a cysteine in the spatial vicinity of the DLST active site or introduction of succinomimetic mutations recapitulates these metabolic deficits. Our data demonstrate that the biochemical deficit extends beyond impaired Complex I assembly and OXPHOS deficiency, functionally impairing select components of the TCA cycle to drive metabolic perturbations in affected neurons.

Can Genome Sequencing Coupled to Flux Balance Analyses Offer Precision Guidance for Industrial Strain Development? The Lessons from Carbon Trafficking in Corynebacterium glutamicum ATCC 21573.

Systems biology tools offer new prospects for industrial strain selection. For bacteria that are significant for industrial applications, whole-genome sequencing coupled to flux balance analysis (FBA) can help unpack the complex relationships between genome mutations and carbon trafficking. This work investigates the l-tyrosine (l-Tyr) overproducing model system Corynebacterium glutamicum ATCC 21573 with an eye to more rational and precision strain development. Using genome-wide mutational analysis of C. glutamicum, we identified 27,611 single nucleotide polymorphisms and 479 insertion/deletion mutations. Mutations in the carbon uptake machinery have led to phosphotransferase system-independent routes as corroborated with FBA. Mutations within the central carbon metabolism of C. glutamicum impaired the carbon flux, as evidenced by the lower growth rate. The entry to and flow through the tricarboxylic acid cycle was affected by mutations in pyruvate and α-ketoglutarate dehydrogenase complexes, citrate synthase, and isocitrate dehydrogenase. FBA indicated that the estimated flux through the shikimate pathway became larger as the l-Tyr production rate increased. In addition, protocatechuate export was probabilistically impossible, which could have contributed to the l-Tyr accumulation. Interestingly, aroG and cg0975, which have received previous attention for aromatic amino acid overproduction, were not mutated. From the branch point molecule, prephenate, the change in the promoter region of pheA could be an influential contributor. In summary, we suggest that genome sequencing coupled with FBA is well poised to offer rational guidance for industrial strain development, as evidenced by these findings on carbon trafficking in C. glutamicum ATCC 21573.

Dietary α-ketoglutarate alleviates glycinin and β-conglycinin induced damage in the intestine of mirror carp (Cyprinus carpio).

This study investigated the glycinin and β-conglycinin induced intestinal damage and α-ketoglutarate alleviating the damage of glycinin and β-conglycinin in intestine. Carp were randomly divided into six dietary groups: containing fish meal (FM) as the protein source, soybean meal (SM), glycinin (FMG), β-conglycinin (FMc), glycinin+1.0% α-ketoglutarate (AKG) (FMGA), β-conglycinin+1.0% AKG (FMcA). The intestines were collected on 7th, and the hepatopancreas and intestines were collected on 56th. Fish treated with SM and FMc displayed reduced weight gain, specific growth rate, and protein efficiency. On 56th day, Fish fed on SM, FMG and FMc presented lower superoxide dismutase (SOD) activities. FMGA and FMcA had higher SOD activity than those fed on the FMG and FMc, respectively. In intestine, fish fed on the SM diets collected on 7th presented upregulated the expression of transforming growth factor beta (TGFβ1), AMP-activated protein kinase beta (AMPKβ), AMPKγ, and acetyl-CoA carboxylase (ACC). Fish fed FMG presented upregulated expression of tumor necrosis factor alpha (TNF-α), caspase9, and AMPKγ, while downregulated the expression of claudin7 and AMPKα. FMc group presented upregulated expression of TGFβ1, caspase3, caspase8, and ACC. Fish fed FMGA showed upregulated expression of TGFβ1, claudin3c, claudin7, while downregulating the expression of TNF-α and AMPKγ when compared to fish fed FMG diet. FMcA upregulated the expression of TGFβ1, claudin3c than fed on the FMc. In intestine, the villus height and mucosal thickness of the proximal intestine (PI) and the distal intestine (DI) were decreased and crypt depth of the PI and mid intestine (MI) were increased in SM, FMG and FMc. In addition, fish fed on SM, FMG and FMc presented lower citrate synthase (CS), isocitrate dehydrogenase (ICD), α-ketoglutarate dehydrogenase complex (α-KGDHC) Na+/K+-ATPase activity in DI. FMGA had higher CS, ICD, α-KGDHC, and Na+/K+-ATPase activity in PI and MI than those fed on the FMG. FMcA had higher Na+/K+-ATPase activity in MI. In conclusion, dietary soybean meal destroys the intestine's health, the adverse effects are related to the presence of β-conglycinin and glycinin, especially glycinin. AKG may regulate intestinal energy via tricarboxylic acid cycle, thereby alleviating the damage intestinal morphology caused by the dietary soybean antigen proteins.

The α-Ketoglutarate Dehydrogenase Complex as a Hub of Plasticity in Neurodegeneration and Regeneration.

Abnormal glucose metabolism is central to neurodegeneration, and considerable evidence suggests that abnormalities in key enzymes of the tricarboxylic acid (TCA) cycle underlie the metabolic deficits. Significant recent advances in the role of metabolism in cancer provide new insight that facilitates our understanding of the role of metabolism in neurodegeneration. Research indicates that the rate-limiting step of the TCA cycle, the α-ketoglutarate dehydrogenase complex (KGDHC) and its substrate alpha ketoglutarate (KG), serve as a signaling hub that regulates multiple cellular processes: (1) is the rate-limiting step of the TCA cycle, (2) is sensitive to reactive oxygen species (ROS) and produces ROS, (3) determines whether KG is used for energy or synthesis of compounds to support growth, (4) regulates the cellular responses to hypoxia, (5) controls the post-translational modification of hundreds of cell proteins in the mitochondria, cytosol, and nucleus through succinylation, (6) controls critical aspects of transcription, (7) modulates protein signaling within cells, and (8) modulates cellular calcium. The primary focus of this review is to understand how reductions in KGDHC are translated to pathologically important changes that underlie both neurodegeneration and cancer. An understanding of each role is necessary to develop new therapeutic strategies to treat neurodegenerative disease.

Phosphonate analog of 2-oxoglutarate regulates glutamate-glutamine homeostasis and counteracts amyloid beta induced learning and memory deficits in rats

Metabolic alteration is a mainstream concept underlying the cognitive decline in neurodegenerative disorders including Alzheimer's disease (AD). Mitochondrial enzyme α-ketoglutarate dehydrogenase complex (α-KGDHC) seems to play a dual-edged sword role in cytotoxic insult. Here, using succinyl phosphonate (SP), a specific α-KGDHC inhibitor, we aimed to examine its potential action on AD progression. Male Wistar rats were assigned to two separate experiments. First, they were bilaterally microinjected into the dorsal CA1 area by amyloid-beta (Aβ)25-35 for four consecutive days. Seven days after the last injection, they were trained to acquire Morris Water Maze (MWM) task for three successive days when they were treated with SP after each training session. In the second experiment, SP was administered 30 min after the first Aβ microinjection and behavioral tests were performed one week after the last Aβ administration. The activity of glutamate dehydrogenase (GDH), and glutamine synthetase (GS), as key enzymes involved in glutamate-glutamine homeostasis and histological assays were evaluated in the hippocampi. Our behavioral results indicated that post-training SP treatment enhanced task acquisition but did not change memory performance in Aβ-treated rats. However, administration of SP at the time of Aβ injection precludes the deteriorative effect of Aβ and neuronal injury on both spatial learning and memory performances indicating its preventive action against Aβ pathology at its early stages. Measurement of enzymes activity shows that α-KGDHC activity was reduced in the Aβ treated group, and SP administration restored its activity; also, GDH and GS activities were increased and decreased respectively due to Aβ, and SP reversed the action of Aβ on these enzymes. This study proposes that SP possibly a promising therapeutic approach to improve memory impairment in AD, especially in the early phases of this disease.

Genomics and transcriptomics-guided metabolic engineering Corynebacterium glutamicum for l-arginine production

l-arginine is a semi-essential amino acid that is broadly used as food additives and pharmaceutical intermediates. The synthesis of l-arginine is restricted by complex metabolic mechanisms and suboptimal fermentation conditions. Initially, a mutant strain that accumulated 19.4 g/L l-arginine was generated by random mutagenesis. Subsequently, a mutation of the repressor protein (argRG159D) in the l-arginine operon and glutamate synthase (gltD) with 532-fold upregulation were identified to be vital for l-arginine production by multi-omic analysis. Systematic metabolic engineering was used to modify the strain, which included interfering with α-ketoglutarate dehydrogenase complex (ODHC) activity by knocking out serine/threonine-protein kinase (pknG), enhancing the expression of multiple key enzymes in the l-arginine synthesis pathway, and increasing the availability of intracellular cofactor (NADPH) and energy (ATP). Finally, C. glutamicum ARG12 produced 71.3 g/L l-arginine, with a yield of 0.43 g/g glucose by fermentation optimization. This study provides new ideas to boost l-arginine production.

Different responses to glucose overload between two strains of largemouth bass (Micropterus salmoides).

In order to improve the glucose utilization capacity of largemouth bass (Micropterus salmoides), responses to glucose overload between two strains (Y: breeding strain; W: wild strain) were compared at 0, 6, 12, and 24 h after glucose injection (1.67 g/kg). The data revealed that plasma glucose in the Y strain (<12 h) recovered faster than in the W strain (12 h), with the Y strain secreted more insulin within 6 h post-injection. Triglyceride (TG) and low-density lipoprotein-cholesterol (VLDL-CH) content in the Y strain increased, peaking at 12 h, then decreased, whereas the W strain’s TG content was not affected and VLDL-CH content decreased. The hepatic and muscular fatty acid synthetase, liver x receptor-1, and sterol regulatory element-binding protein expressions were consistent with the TG content change. Both strains’ liver and muscle glycogen contents exhibited similar trends to that of the glycogen synthase gene—increasing, then declining, and peaking at 6 and 12 h. The expression levels of hepatic and muscular phosphofructokinase and pyruvate kinase in the Y strain increased, peaking at 12 h. In the W strain, they were suppressed and reached the minimum at 24 h. The mRNA levels of hepatic and muscular phosphoenolpyruvate carboxykinase and glucose-6-phosphatase were enhanced and peaked at 24 h in both strains, hepatic isocitrate dehydrogenase-1, and α-ketoglutarate dehydrogenase complex expression increased after declining, peaking at 12 and 24 h. Two genes in the W strain’s muscles showed a similar trend. Both strains’ transcriptome results identified seven common functional genes for resistance to hyperglycemia that were involved in the circadian rhythm pathway, which is a suggested key pathway for coping with hyperglycemia. Furthermore, 48 differential genes were identified between the two strains, and these genes were enriched in the TGF-beta and cell cycle signaling pathways, indicating that these pathways may be key factors affecting the differential responses to glucose overload. We conducted a comprehensive comparison of glucose overload molecular responses between two strains of M. salmoides, and the results can provide a promising strategy to improve the glucose utilization capacity of M. salmoides based on advantageous pre-existing traits.

Reverse and Forward Electron Flow-Induced H2O2 Formation Is Decreased in α-Ketoglutarate Dehydrogenase (α-KGDH) Subunit (E2 or E3) Heterozygote Knock Out Animals.

α-ketoglutarate dehydrogenase complex (KGDHc), or 2-oxoglutarate dehydrogenase complex (OGDHc) is a rate-limiting enzyme in the tricarboxylic acid cycle, that has been identified in neurodegenerative diseases such as in Alzheimer’s disease. The aim of the present study was to establish the role of the KGDHc and its subunits in the bioenergetics and reactive oxygen species (ROS) homeostasis of brain mitochondria. To study the bioenergetic profile of KGDHc, genetically modified mouse strains were used having a heterozygous knock out (KO) either in the dihydrolipoyl succinyltransferase (DLST+/−) or in the dihydrolipoyl dehydrogenase (DLD+/−) subunit. Mitochondrial oxygen consumption, hydrogen peroxide (H2O2) production, and expression of antioxidant enzymes were measured in isolated mouse brain mitochondria. Here, we demonstrate that the ADP-stimulated respiration of mitochondria was partially arrested in the transgenic animals when utilizing α-ketoglutarate (α-KG or 2-OG) as a fuel substrate. Succinate and α-glycerophosphate (α-GP), however, did not show this effect. The H2O2 production in mitochondria energized with α-KG was decreased after inhibiting the adenine nucleotide translocase and Complex I (CI) in the transgenic strains compared to the controls. Similarly, the reverse electron transfer (RET)-evoked H2O2 formation supported by succinate or α-GP were inhibited in mitochondria isolated from the transgenic animals. The decrease of RET-evoked ROS production by DLST+/− or DLD+/− KO-s puts the emphasis of the KGDHc in the pathomechanism of ischemia-reperfusion evoked oxidative stress. Supporting this notion, expression of the antioxidant enzyme glutathione peroxidase was also decreased in the KGDHc transgenic animals suggesting the attenuation of ROS-producing characteristics of KGDHc. These findings confirm the contribution of the KGDHc to the mitochondrial ROS production and in the pathomechanism of ischemia-reperfusion injury.

α-Ketoglutarate-Mediated DNA Demethylation Sustains T-Acute Lymphoblastic Leukemia upon TCA Cycle Targeting.

Simple SummaryA promising anti-cancer strategy is to target the tumor’s dependence on particular nutrients. However, resistance to single-agent treatment is common due to the adaptability of cancer cells. This research seeks to understand how T acute lymphoblastic leukemia (T-ALL) escapes disruption of the tricarboxylic acid (TCA) cycle, a biochemical pathway critical for T-ALL survival. We show that leukemic cells modify their DNA to increase the activity of other pathways to compensate for diminished TCA cycle function. Our findings will help guide the rational selection of therapies to overcome drug resistance.Despite the development of metabolism-based therapy for a variety of malignancies, resistance to single-agent treatment is common due to the metabolic plasticity of cancer cells. Improved understanding of how malignant cells rewire metabolic pathways can guide the rational selection of combination therapy to circumvent drug resistance. Here, we show that human T-ALL cells shift their metabolism from oxidative decarboxylation to reductive carboxylation when the TCA cycle is disrupted. The α-ketoglutarate dehydrogenase complex (KGDHC) in the TCA cycle regulates oxidative decarboxylation by converting α-ketoglutarate (α-KG) to succinyl-CoA, while isocitrate dehydrogenase (IDH) 1 and 2 govern reductive carboxylation. Metabolomics flux analysis of T-ALL reveals enhanced reductive carboxylation upon genetic depletion of the E2 subunit of KGDHC, dihydrolipoamide-succinyl transferase (DLST), mimicking pharmacological inhibition of the complex. Mechanistically, KGDHC dysfunction causes increased demethylation of nuclear DNA by α-KG-dependent dioxygenases (e.g., TET demethylases), leading to increased production of both IDH1 and 2. Consequently, dual pharmacologic inhibition of the TCA cycle and TET demethylases demonstrates additive efficacy in reducing the tumor burden in zebrafish xenografts. These findings provide mechanistic insights into how T-ALL develops resistance to drugs targeting the TCA cycle and therapeutic strategies to overcome this resistance.

Sustainable production of 4-hydroxyisoleucine with minimised carbon loss by simultaneously utilising glucose and xylose in engineered Escherichia coli

4-Hydroxyisoleucine is a promising drug for diabetes therapy; however, microbial production of 4-hydroxyisoleucine is not economically efficient because of the carbon loss in the form of CO2. This study aims to achieve de novo synthesis of 4-hydroxyisoleucine with minimised carbon loss in engineered Escherichia coli. Initially, an L-isoleucine-producing strain, ILE-5, was established, and the 4-hydroxyisoleucine synthesis pathway was introduced. The flux toward α-ketoglutarate was enhanced by reinforcing the anaplerotic pathway and disrupting competitive pathways. Subsequently, the metabolic flux for 4-hydroxyisoleucine synthesis was redistributed by dynamically modulating the α-ketoglutarate dehydrogenase complex activity, achieving a 4-hydroxyisoleucine production of 16.53 g/L. Finally, carbon loss was minimised by employing the Weimberg pathway, resulting in a 24.5% decrease in sugar consumption and a 31.6% yield increase. The 4-hydroxyisoleucine production by strain IEOH-11 reached 29.16 g/L in a 5-L fermenter. The 4-hydroxyisoleucine yield (0.29 mol/mol sugar) and productivity (0.91 g/(L⋅h)) were higher than those previously reported.

Using Taguchi Method to Determine the Optimum Conditions for Synthesizing Parapyruvate.

The synthesis of parapyruvate is important for the analysis of the content in the pyruvate supplements and the study of aging-related neurodegenerative diseases. However, the pure parapyruvate crystal is not, as yet, commercially available. In this study, we applied the Taguchi’s L9 orthogonal array to investigate the optimal conditions for the preparation of the pure parapyruvate by the alkaline treatment of the pyruvic acid and then followed it with the solvent crystallization steps. We were also interested in revealing the major factors that affect the yield for the synthesized pure parapyruvate crystals. In addition, the parapyruvate-inhibited enzyme kinetic of α-ketoglutarate dehydrogenase complex (KGDHC) was also investigated. We found that the pure parapyruvate could be obtained in combination with an alkaline treatment and two solvent crystallization steps. The main factors affecting the yield of the pure parapyruvate were the concentration of the pyruvic acid (the reactant), the pH of the alkali treatment, the type of solvent used for the crystallization and the volume ratio of solvent used for crystallization. Finally, the optimal conditions could prepare parapyruvate crystals with a high purity of 99.8% and a high yield of 72.8%. In addition, the results demonstrate that parapyruvate is a reversibly competitive inhibitor for KGDHC.

The influence of 5-fluorouracil on the α-ketoglutarate dehydrogenase complex in rat's cardiac muscle - a preliminary study.

5-fluorouracil (5-FU), which is a commonly used chemotherapy agent exerts undesired cardiac toxicity. Mitochondrial dysfunction is thought to be one of potentially important mechanisms of 5-FU- induced cardiotoxicity. α-ketoglutarate dehydrogenase (α-KGDHC) is the key regulatory enzyme of TCA cycle. The complex consists of multiple copies of three catalytic subunits: α-ketoglutarate dehydrogenase (E1), dihydrolipoamide succinyltransferase (E2) and dihydrolipoamide dehydrogenase (E3). α-KGDHC together with branched chain α-ketoacid dehydrogenase (BCKDH) and pyruvate dehydrogenase (PDH), are the members of 2-oxoacid dehydrogenases family that share some structural and functional similarities. Recently, it has been found that 5-FU stimulates BCKDH in rat's cardiac muscle. Therefore, we hypothesize that 5-FU modifies α-KGDHC activity and affects cardiac muscle metabolism. The aim of this study was to determine the effect of 5-FU on α-KGDHC activity and protein levels of E1 and E2 subunits of the complex in rat's cardiac muscle. Wistar male rats were administered with 4 doses of 5-FU, 150 mg/ kg b.wt. each (study group) or 0.3% methylcellulose (control group). α-KGDHC activity was assayed spectrophotometrically. The E1 and E2 proteins levels were quantified by Western blot. 5-FU administration resulted in stimulation of myocardial α-KGDHC activity in rats. In addition, E2 protein level increased in response to 5-FU treatment, while the E1 protein level remained unchanged. Up-regulation of α-KGDHC appears to result from change in E2 subunit protein level. However, the effect of 5-FU on factors modifying α-KGDHC activity at post-translational level cannot be excluded.