Prediction of key milk biomarkers in dairy cows through milk mid-infrared spectra and international collaborations.

At the individual cow level, suboptimum fertility, mastitis, negative energy balance, and ketosis are major issues in dairy farming. These problems are widespread on dairy farms and have an important economic impact. The objectives of this study were (1) to assess the potential of milk mid-infrared (MIR) spectra to predict key biomarkers of energy deficit (citrate, isocitrate, glucose-6 phosphate [glucose-6P], free glucose), ketosis (ß-hydroxybutyrate [BHB] and acetone), mastitis (N-acetyl-ß-d-glucosaminidase activity [NAGase] and lactate dehydrogenase), and fertility (progesterone); (2) to test alternative methodologies to partial least squares (PLS) regression to better account for the specific asymmetric distribution of the biomarkers; and (3) to create robust models by merging large datasets from 5 international or national projects. Benefiting from this international collaboration, the dataset comprised a total of 9,143 milk samples from 3,758 cows located in 589 herds across 10 countries and represented 7 breeds. The samples were analyzed by reference chemistry for biomarker contents, whereas the MIR analyses were performed on 30 instruments from different models and brands, with spectra harmonized into a common format. Four quantitative methodologies were evaluated to address the strongly skewed distribution of some biomarkers. Partial least squares regression was used as the reference basis, and compared with a random modification of distribution associated with PLS (random-downsampling-PLS), an optimized modification of distribution associated with PLS (KennardStone-downsampling-PLS), and support vector machine (SVM). When the ability of MIR to predict biomarkers was too low for quantification, different qualitative methodologies were tested to discriminate low versus high values of biomarkers. For each biomarker, 20% of the herds were randomly removed within all countries to be used as the validation dataset. The remaining 80% of herds were used as the calibration dataset. In calibration, the 3 alternative methodologies outperform the PLS performances for the majority of biomarkers. However, in the external herd validation, PLS provided the best results for isocitrate, glucose-6P, free glucose, and lactate dehydrogenase (coefficient of determination in external herd validation [R2v] = 0.48, 0.58, 0.28, and 0.24, respectively). For other molecules, PLS-random-downsampling and PLS-KennardStone-downsampling outperformed PLS in the majority of cases, but the best results were provided by SVM for citrate, BHB, acetone, NAGase, and progesterone (R2v = 0.94, 0.58, 0.76, 0.68, and 0.15, respectively). Hence, PLS and SVM based on the entire dataset provided the best results for normal and skewed distributions, respectively. Complementary to the quantitative methods, the qualitative discriminant models enabled the discrimination of high and low values for BHB, acetone, and NAGase with a global accuracy around 90%, and glucose-6P with an accuracy of 83%. In conclusion, MIR spectra of milk can enable quantitative screening of citrate as a biomarker of energy deficit and discrimination of low and high values of BHB, acetone, and NAGase, as biomarkers of ketosis and mastitis. Finally, progesterone could not be predicted with sufficient accuracy from milk MIR spectra to be further considered. Consequently, MIR spectrometry can bring valuable information regarding the occurrence of energy deficit, ketosis, and mastitis in dairy cows, which in turn have major influences on their fertility and survival.

Challenges for the development of mutant isocitrate dehydrogenases 1 inhibitors to treat glioma.

Glioma is one of the most common types of brain tumors, and its high recurrence and mortality rates threaten human health. In 2008, the frequent isocitrate dehydrogenase 1 (IDH1) mutations in glioma were reported, which brought a new strategy in the treatment of this challenging disease. In this perspective, we first discuss the possible gliomagenesis after IDH1 mutations (mIDH1). Subsequently, we systematically investigate the reported mIDH1 inhibitors and present a comparative analysis of the ligand-binding pocket in mIDH1. Additionally, we also discuss the binding features and physicochemical properties of different mIDH1 inhibitors to facilitate the future development of mIDH1 inhibitors. Finally, we discuss the possible selectivity features of mIDH1 inhibitors against WT-IDH1 and IDH2 by combining protein-based and ligand-based information. We hope that this perspective can inspire the development of mIDH1 inhibitors and bring potent mIDH1 inhibitors for the treatment of glioma.

Redox Homeostasis and Beyond: The Role of Wild-Type Isocitrate Dehydrogenases for the Pathogenesis of Glioblastoma.

Significance: Glioblastoma is an aggressive and devastating brain tumor characterized by a dismal prognosis and resistance to therapeutic intervention. To support catabolic processes critical for unabated cellular growth and defend against harmful reactive oxygen species, glioblastoma tumors upregulate the expression of wild-type isocitrate dehydrogenases (IDHs). IDH enzymes catalyze the oxidative decarboxylation of isocitrate to α-ketoglutarate (α-KG), NAD(P)H, and CO2. On molecular levels, IDHs epigenetically control gene expression through effects on α-KG-dependent dioxygenases, maintain redox balance, and promote anaplerosis by providing cells with NADPH and precursor substrates for macromolecular synthesis. Recent Advances: While gain-of-function mutations in IDH1 and IDH2 represent one of the most comprehensively studied mechanisms of IDH pathogenic effects, recent studies identified wild-type IDHs as critical regulators of normal organ physiology and, when transcriptionally induced or down regulated, as contributing to glioblastoma progression. Critical Issues: Here, we will discuss molecular mechanisms of how wild-type IDHs control glioma pathogenesis, including the regulation of oxidative stress and de novo lipid biosynthesis, and provide an overview of current and future research directives that aim to fully characterize wild-type IDH-driven metabolic reprogramming and its contribution to the pathogenesis of glioblastoma. Future Directions: Future studies are required to further dissect mechanisms of metabolic and epigenomic reprogramming in tumors and the tumor microenvironment, and to develop pharmacological approaches to inhibit wild-type IDH function. Antioxid. Redox Signal. 39, 923-941.

Isocitrate-dehydrogenase-mutant lower grade glioma in elderly patients: treatment and outcome in a molecularly characterized contemporary cohort.

PURPOSE: Lower-grade glioma (LGG) is rare among patients above the age of 60 ("elderly"). Previous studies reported poor outcome, likely due to the inclusion of isocitrate dehydrogenase (IDH) wildtype astrocytomas and advocated defensive surgical and adjuvant treatment. This study set out to question this paradigm analyzing a contemporary cohort of patients with IDH mutant astrocytoma and oligodendroglioma WHO grade 2 and 3. METHODS: Elderly patients treated in our department for a supratentorial, hemispheric LGG between 2009 and 2019 were retrospectively analyzed for patient-, tumor- and treatment-related factors and progression-free survival (PFS) and compared to patients aged under 60. Inclusion required the availability of subtype-defining molecular data and pre- and post-operative tumor volumes. RESULTS: 207 patients were included, among those 21 elderlies (10%). PFS was comparable between elderly and younger patients (46 vs. 54 months; p = 0.634). Oligodendroglioma was more common in the elderly (76% vs. 46%; p = 0.011). Most patients underwent tumor resection (elderly: 81% vs. younger: 91%; p = 0.246) yielding comparable residual tumor volumes (elderly: 7.8 cm3; younger: 4.1 cm3; p = 0.137). Adjuvant treatment was administered in 76% of elderly and 61% of younger patients (p = 0.163). Uni- and multi-variate survival analyses identified a tumor crossing the midline, surgical strategy, and pre- and post-operative tumor volumes as prognostic factors. CONCLUSION: Elderly patients constitute a small fraction of molecularly characterized LGGs. In contrast to previous reports, favorable surgical and survival outcomes were achieved in our series comparable to those of younger patients. Thus, intensified treatment including maximal safe resection should be advocated in elderly patients whenever feasible.

NADP(H)-dependent biocatalysis without adding NADP(H).

Isocitrate dehydrogenase 1 (IDH1) naturally copurifies and crystallizes in a resting state with a molecule of its exchangeable cofactor, NADP+/NADPH, bound in each monomer of the homodimer. We report electrochemical studies with IDH1 that exploit this property to reveal the massive advantage of nanoconfinement to increase the efficiency of multistep enzyme-catalyzed cascade reactions. When coloaded with ferredoxin NADP+ reductase in a nanoporous conducting indium tin oxide film, IDH1 carries out the complete electrochemical oxidation of 6 mM isocitrate (in 4mL) to 2-oxoglutarate (2OG), using only the NADP(H) that copurified with IDH1 and was carried into the electrode pores as cargo-the system remains active for days. The entrapped cofactor, now quantifiable by cyclic voltammetry, undergoes ~160,000 turnovers during the process. The results from a variety of electrocatalysis experiments imply that the local concentrations of the two nanoconfined enzymes lie around the millimolar range. The combination of crowding and entrapment results in a 102 to 103-fold increase in the efficiency of NADP(H) redox cycling. The ability of the method to drive cascade catalysis in either direction (oxidation or reduction) and remove and replace substrates was exploited to study redox-state dependent differences in cofactor binding between wild-type IDH1 and the cancer-linked R132H variant that catalyzes the "gain of function" reduction of 2OG to 2-hydroxyglutarate instead of isocitrate oxidation. The combined results demonstrate the power of nanoconfinement for facilitating multistep enzyme catalysis (in this case energized and verified electrochemically) and reveal insights into the dynamic role of nicotinamide cofactors as redox (hydride) carriers.

Pharmacophore-based Identification of Potential Mutant Isocitrate Dehydrogenases I/2 Inhibitors: An Explorative Avenue in Cancer Drug Design.

BACKGROUND: Heterozygous mutations in the cytoplasmic and mitochondrial isoforms of isocitrate dehydrogenase enzymes 1 and 2 subtypes have been extensively exploited as viable druggable targets, as they decrease the affinity of isocitrate and higher affinity of D-2-hydroxyglutarate, an oncometabolite. OBJECTIVE: Vorasidenib (AG-881) has recently been reported as a promising dual inhibitor of mutant isocitrate dehydrogenase 1 and 2 with the ability to penetrate the blood-brain barrier towards the treatment of low-grade glioma. In order to combat drug resistance and toxicity levels, this compelled us to further investigate this substance as a basis for the creation of potential selective inhibitors of mutant isocitrate dehydrogenases 1 and 2. METHODS: By employing a wide range of computational techniques, binding moieties of AG-881 that contributed towards its selective binding to isocitrate dehydrogenase enzymes 1 and 2 were identified and subsequently used to generate pharmacophore models for the screening of potential inhibitor drugs that were further assessed by their pharmacokinetics and physicochemical properties. RESULTS: AG-881 was identified as the most favorable candidate for isocitrate dehydrogenase enzyme 1, exhibiting a binding free energy of -28.69 kcal/mol. ZINC93978407 was the most favorable candidatefor isocitrate dehydrogenase enzyme 2, displaying a strong binding free energy of -27.10 kcal/mol. ZINC9449923 and ZINC93978407 towards isocitrate dehydrogenase enzyme 1 and 2 showed good protein structural stability with a low radius of gyration values relative to AG-881. CONCLUSION: We investigated that ZINC9449923 of isocitrate dehydrogenase enzyme 1 and ZINC 93978407 of isocitrate dehydrogenase enzyme 2 could serve as promising candidates for the treatment of lower-grade glioma as they cross the blood-brain barrier, and present with lower toxicity levels relative to AG-881.

Metabolic adaptation and ATP homeostasis in Pseudomonas fluorescens exposed to phosphate stress.

Phosphate (Pi) is essential for life as it is an integral part of the universal chemical energy adenosine triphosphate (ATP), and macromolecules such as, DNA, RNA proteins and lipids. Despite the core roles and the need of this nutrient in living cells, some bacteria can grow in environments that are poor in Pi. The metabolic mechanisms that enable bacteria to proliferate in a low phosphate environment are not fully understood. In this study, the soil microbe Pseudomonas (P.) fluorescens was cultured in a control and a low Pi (stress) medium in order to delineate how energy homeostasis is maintained. Although there was no significant variation in biomass yield in these cultures, metabolites like isocitrate, oxaloacetate, pyruvate and phosphoenolpyruvate (PEP) were markedly increased in the phosphate-starved condition. Components of the glycolytic, glyoxylate and tricarboxylic acid cycles operated in tandem to generate ATP by substrate level phosphorylation (SLP) as NADH-producing enzymes were impeded. The α-ketoglutarate (KG) produced when glutamine, the sole carbon nutrient was transformed into phosphoenol pyruvate (PEP) and succinyl-CoA (SC), two high energy moieties. The metabolic reprogramming orchestrated by isocitrate lyase (ICL), phosphoenolpyruvate synthase (PEPS), pyruvate phosphate dikinase (PPDK), and succinyl-CoA synthetase fulfilled the ATP budget. Cell free extract experiments confirmed ATP synthesis in the presence of such substrates as PEP, oxaloacetate and isocitrate respectively. Gene expression profiling revealed elevated transcripts associated with numerous enzymes including ICL, PEPS, and succinyl-CoA synthetase (SCS). This microbial adaptation will be critical in promoting biological activity in Pi-poor ecosystems.

Recovery of ΔF508-CFTR Function by Citrate.

Treatment of cystic fibrosis relies so far on expensive and sophisticated drugs. A logical approach to rescuing the defective ΔF508-CFTR protein has not yet been published. Therefore, virtual docking of ATP and CFTR activators to the open conformation of the CFTR protein was performed. A new ATP binding site outside of the two known locations was identified. It was located in the cleft between the nucleotide binding domains NBD1 and NBD2 and comprised six basic amino acids in close proximity. Citrate and isocitrate were also bound to this site. Citrate was evaluated for its action on epithelial cells with intact CFTR and defective ΔF508-CFTR. It activated hyaluronan export from human breast carcinoma cells and iodide efflux, and recovered ΔF508-CFTR from premature intracellular degradation. In conclusion, citrate is an activator for ΔF508-CFTR and increases export by defective ΔF508-CFTR into the extracellular matrix of epithelial cells.

Computational study on novel natural compound inhibitor targeting IDH1_R132H.

Isocitrate dehydrogenases (IDH) catalyze the oxidative decarboxylation of isocitrate to 2-oxoglutarate. IDH1 mutation has been reported in various tumors especially Cholangiocarcinoma, while the IDH1_R132H is reported to be the most common mutation of IDH1. IDH1_R132H inhibitors are effective anti-cancer drugs and have shown significant therapeutic effects in clinical. In this study, two novel natural compounds were identified to combine respectively with IDH1_R132H with a stronger binding force with conductive to interaction energy. They also showed low toxicity potential. Molecular dynamics simulation analysis demonstrated that the candidate ligands-IDH1_R132H complexes is stable in natural circumstances with favorable potential energy. Thus, Styraxlignolide F and Tremulacin were screened as promising IDH1_R132H inhibitors. We provide a solid foundation for the design and development of IDH1_R132H targeted drugs.

Development of simultaneous quantitative analysis of tricarboxylic acid cycle metabolites to identify specific metabolites in cancer cells by targeted metabolomic approach.

The tricarboxylic acid (TCA) cycle is one of the most important pathways of energy metabolism, and the profiles of its components are influenced by factors such as diseases and diets. Therefore, the differences in metabolic profile of TCA cycle between healthy and cancer cells have been the focus of studies to understand pathological conditions. In this study, we developed a quantitative method to measure TCA cycle metabolites using LC-MS/MS to obtain useful metabolic profiles for development of diagnostic and therapeutic methods for cancer. We successfully analyzed 11 TCA cycle metabolites by LC MS/MS with high reproducibility by using a PFP column with 0.5% formic acid as a mobile phase. Next, we analyzed the concentration of TCA cycle metabolites in human cell lines (HaCaT: normal skin keratinocytes; A431: skin squamous carcinoma cells; SW480: colorectal cancer cells). We observed reduced concentration of succinate and increased concentration of citrate, 2-hydroxyglutarate, and glutamine in A431 cells as compared with HaCaT cells. On the other hand, decreased concentration of isocitrate, fumarate, and α-ketoglutarate and increased concentration of malate, glutamine, and glutamate in A431 cells were observed in comparison with SW480 cells. These findings suggested the possibility of identifying disease-specific metabolites and/or organ-specific metabolites by using this targeted metabolomic analysis.

Tricarboxylic acid cycle related-metabolites and risk of atrial fibrillation and heart failure.

BACKGROUND: Tricarboxylic acid (TCA) cycle deregulation may predispose to cardiovascular diseases, but the role of TCA cycle-related metabolites in the development of atrial fibrillation (AF) and heart failure (HF) remains unexplored. This study sought to investigate the association of TCA cycle-related metabolites with risk of AF and HF. METHODS: We used two nested case-control studies within the PREDIMED study. During a mean follow-up for about 10 years, 512 AF and 334 HF incident cases matched by age (±5 years), sex and recruitment center to 616 controls and 433 controls, respectively, were included in this study. Baseline plasma levels of citrate, aconitate, isocitrate, succinate, malate and d/l-2-hydroxyglutarate were measured with liquid chromatography-tandem mass spectrometry. Multivariable conditional logistic regression models were fitted to estimate odds ratios (OR) and 95% confidence intervals (95% CI) for metabolites and the risk of AF or HF. Potential confounders included smoking, family history of premature coronary heart disease, physical activity, alcohol intake, body mass index, intervention groups, dyslipidemia, hypertension, type 2 diabetes and medication use. RESULTS: Comparing extreme quartiles of metabolites, elevated levels of succinate, malate, citrate and d/l-2-hydroxyglutarate were associated with a higher risk of AF [ORQ4 vs. Q1 (95% CI): 1.80 (1.21-2.67), 2.13 (1.45-3.13), 1.87 (1.25-2.81) and 1.95 (1.31-2.90), respectively]. One SD increase in aconitate was directly associated with AF risk [OR (95% CI): 1.16 (1.01-1.34)]. The corresponding ORs (95% CI) for HF comparing extreme quartiles of malate, aconitate, isocitrate and d/l-2-hydroxyglutarate were 2.15 (1.29-3.56), 2.16 (1.25-3.72), 2.63 (1.56-4.44) and 1.82 (1.10-3.04), respectively. These associations were confirmed in an internal validation, except for aconitate and AF. CONCLUSION: These findings underscore the potential role of the TCA cycle in the pathogenesis of cardiac outcomes.

Iron control of erythroid microtubule cytoskeleton as a potential target in treatment of iron-restricted anemia.

Anemias of chronic disease and inflammation (ACDI) result from restricted iron delivery to erythroid progenitors. The current studies reveal an organellar response in erythroid iron restriction consisting of disassembly of the microtubule cytoskeleton and associated Golgi disruption. Isocitrate supplementation, known to abrogate the erythroid iron restriction response, induces reassembly of microtubules and Golgi in iron deprived progenitors. Ferritin, based on proteomic profiles, regulation by iron and isocitrate, and putative interaction with microtubules, is assessed as a candidate mediator. Knockdown of ferritin heavy chain (FTH1) in iron replete progenitors induces microtubule collapse and erythropoietic blockade; conversely, enforced ferritin expression rescues erythroid differentiation under conditions of iron restriction. Fumarate, a known ferritin inducer, synergizes with isocitrate in reversing molecular and cellular defects of iron restriction and in oral remediation of murine anemia. These findings identify a cytoskeletal component of erythroid iron restriction and demonstrate potential for its therapeutic targeting in ACDI.

Species Prioritization Based on Spectral Dissimilarity: A Case Study of Polyporoid Fungal Species.

Biological species collections are critical for natural product drug discovery programs. However, prioritization of target species in massive collections remains difficult. Here, we introduce an untargeted metabolomics-based prioritization workflow that uses MS/MS molecular networking to estimate scaffold-level distribution. As a demonstration, we applied the workflow to 40 polyporoid fungal species. Nine species were prioritized as candidates based on the chemical structural and compositional similarity (CSCS) metric. Most of the selected species showed relatively higher richness and uniqueness of metabolites than those of the others. Cryptoporus volvatus, one of the prioritized species, was investigated further. The chemical profiles of the extracts of C. volvatus culture and fruiting bodies were compared, and it was shown that derivative-level diversity was higher in the fruiting bodies; meanwhile, scaffold-level diversity was similar. This showed that the compounds found from a cultured fungus can also be isolated in wild mushrooms. Targeted isolation of the fruiting body extract yielded three unknown (1-3) and six known (4-9) cryptoporic acid derivatives, which are drimane-type sesquiterpenes with isocitric acid moieties that have been reported in this species. Cryptoporic acid T (1) is a trimeric cryptoporic acid reported for the first time. Compounds 2 and 5 exhibited cytotoxicity against HCT-116 cell lines with IC50 values of 4.3 and 3.6 µM, respectively.

Engineering Yarrowia lipolytica for the selective and high-level production of isocitric acid through manipulation of mitochondrial dicarboxylate-tricarboxylate carriers.

During cultivation under nitrogen starvation, Yarrowia lipolytica produces a mixture of citric acid and isocitric acid whose ratio is mainly determined by the carbon source used. We report that mitochondrial succinate-fumarate carrier YlSfc1 controls isocitric acid efflux from mitochondria. YlSfc1 purified and reconstituted into liposomes transports succinate, fumarate, oxaloacetate, isocitrate and α-ketoglutarate. YlSFC1 overexpression determined the inversion of isocitric acid/citric acid ratio towards isocitric acid, resulting in 33.4 ± 1.9 g/L and 43.3 ± 2.8 g/L of ICA production in test-tube cultivation with glucose and glycerol, respectively. These titers represent a 4.0 and 6.3-fold increase compared to the wild type. YlSFC1 gene expression was repressed in the wild type strain grown in glucose-based medium compared to olive oil medium explaining the reason for the preferred citric acid production during Y. lipolytica growth on carbohydrates. Coexpression of YlSFC1 and adenosine monophosphate deaminase YlAMPD genes together with inactivation of citrate mitochondrial carrier YlYHM2 gene enhanced isocitric acid accumulation up to 41.4 ± 4.1 g/L with an isocitric acid/citric acid ratio of 14.3 in a small-scale cultivation with glucose as a carbon source. During large-scale cultivation with glucose pulse-feeding, the engineered strain produced 136.7 ± 2.5 g/L of ICA with a process selectivity of 88.1%, the highest reported titer and selectivity to date. These results represent the first reported isocitric acid secretion by Y. lipolytica as a main organic acid during cultivation on carbohydrate. Moreover, we demonstrate for the first time that the replacement of one mitochondrial transport system for another can be an efficient tool for switching product accumulation.

Sensitive and Reproducible Mass Spectrometry-Compatible RP-UHPLC Analysis of Tricarboxylic Acid Cycle and Related Metabolites in Biological Fluids: Application to Human Urine.

We describe a method for the analysis of organic acids, including those of the tricarboxylic acid cycle (TCA cycle), by mixed-mode reversed-phase chromatography, on a CSH Phenyl-Hexyl column, to accomplish mixed-mode anion-exchange separations, which results in increased retention for acids without the need for ion-pairing reagents or other mobile phase additives. The developed method exhibited good retention time reproducibility for over 650 injections or more than 5 days of continuous operation. Additionally, it showed excellent resolution of the critical pairs, isocitric acid and citric acid as well as malic acid and fumaric acid, among others. The use of hybrid organic-inorganic surface technology incorporated into the hardware of the column not only improved the mass spectral quality and subsequent database match scoring but also increased the recovery of the analytes, showing particular benefit for low concentrations of phosphorylated species. The method was applied to the comparative metabolomic analysis of urine samples from healthy controls and breast cancer positive subjects. Unsupervised PCA analysis showed distinct grouping of samples from healthy and diseased subjects, with excellent reproducibility of respective injection clusters. Finally, abundance plots of selected analytes from the tricarboxylic acid cycle revealed differences between healthy control and disease groups.

Enasidenib and ivosidenib in AML.

The isocitrate dehydrogenases enzymes, IDH1 and IDH2, catalyze the conversion of isocitrate to α-ketoglutarate (αKG) in the cell cytoplasm and mitochondria, respectively, and contribute to generating the dihydronicotinamide-adenine dinucleotide phosphate (NADPH) as reductive potential in different cellular processes. Mutations in IDH1 and IDH2 genes are found collectively in about 20-25% of acute myeloid leukemia (AML) patients. Mutant IDH enzymes have neomorphic activity and convert αKG to the oncometabolite R-2-hydroxyglutarate (R-2-HG) which accumulates at high levels in the cell and hampers the function of αKG-dependent enzymes, including epigenetic regulators, thus leading to altered gene expression and block of differentiation and contributing to leukemia development. Inhibition of the neomorphic mutants induces marked decrease in R-2-HG levels and restores myeloid differentiation. Enasidenib and ivosidenib are potent and selective inhibitors of mutant IDH2 and IDH1, respectively, act as differentiating agents and showed clinical activity in relapsed/refractory (R/R) AML harboring the specific mutation. As single agents, both drugs have been approved by the Food and Drug Administration (FDA) for the treatment of R/R AML. The relevance of IDH targeting within either single agent approach or, most importantly, combinatorial treatments in AML will be discussed.

An acidic residue buried in the dimer interface of isocitrate dehydrogenase 1 (IDH1) helps regulate catalysis and pH sensitivity.

Isocitrate dehydrogenase 1 (IDH1) catalyzes the reversible NADP+-dependent conversion of isocitrate to α-ketoglutarate (αKG) to provide critical cytosolic substrates and drive NADPH-dependent reactions like lipid biosynthesis and glutathione regeneration. In biochemical studies, the forward reaction is studied at neutral pH, while the reverse reaction is typically characterized in more acidic buffers. This led us to question whether IDH1 catalysis is pH-regulated, which would have functional implications under conditions that alter cellular pH, like apoptosis, hypoxia, cancer, and neurodegenerative diseases. Here, we show evidence of catalytic regulation of IDH1 by pH, identifying a trend of increasing kcat values for αKG production upon increasing pH in the buffers we tested. To understand the molecular determinants of IDH1 pH sensitivity, we used the pHinder algorithm to identify buried ionizable residues predicted to have shifted pKa values. Such residues can serve as pH sensors, with changes in protonation states leading to conformational changes that regulate catalysis. We identified an acidic residue buried at the IDH1 dimer interface, D273, with a predicted pKa value upshifted into the physiological range. D273 point mutations had decreased catalytic efficiency and, importantly, loss of pH-regulated catalysis. Based on these findings, we conclude that IDH1 activity is regulated, at least in part, by pH. We show this regulation is mediated by at least one buried acidic residue ∼12 Å from the IDH1 active site. By establishing mechanisms of regulation of this well-conserved enzyme, we highlight catalytic features that may be susceptible to pH changes caused by cell stress and disease.

Mutation of Isocitrate Dehydrogenase 1 in Cholangiocarcinoma Impairs Tumor Progression by Inhibiting Isocitrate Metabolism.

Aim: Isocitrate dehydrogenase 1 (IDH1) is key enzyme involved in cellular metabolism and DNA repair. Mutations in IDH1 occur in up to 25% of cholangiocarcinomas. The present study aimed to explore the features of cellosaurus REB cells with mutant and wide-type IDH1. Methods: To compare the features of IDH1 knockout and mutation in cholangiocarcinoma, we firstly constructed the IDH1 knockout and IDH1 mutation cell lines. We then evaluated the viability of these cell lines using the cell count assay and MTT assay. Next, we determined cell migration and invasion using the Transwell assay. Additionally, to evaluate the effects of IDH1 on cellular metabolism, the levels of α-ketoglutarate (α-KG) and nicotinamide adenine dinucleotide phosphate (NADPH) were determined using enzyme-linked immunosorbent assay. We then applied ChIPbase dataset to explore the genes that were regulated by IDH1. Results: High frequency of mutated IDH1 was observed in the cholangiocarcinoma and IDH1 R132C was presented in more than 80% of mutations. The results showed that IDH1 knockout decreased cell proliferation, migration and invasion, whereas the overexpression of IDH1 in IDH1 knockout cell line recovered its proliferation, migration and invasion capacities. Additionally, IDH1 mutation reduced the levels of NADPH and α-KG. Furthermore, investigation into the underlying mechanisms revealed that IDH1 overexpression induced the expression of aldehyde dehydrogenase 1 thereby promoting cell proliferation, migration and invasion. Conclusion:IDH1 plays an important role in cholangiocarcinoma and its mutation impairs tumor progression in part by inhibition of isocitrate metabolism.

SIRT3 and GCN5L regulation of NADP+- and NADPH-driven reactions of mitochondrial isocitrate dehydrogenase IDH2.

Wild type mitochondrial isocitrate dehydrogenase (IDH2) was previously reported to produce oncometabolite 2-hydroxyglutarate (2HG). Besides, mitochondrial deacetylase SIRT3 has been shown to regulate the oxidative function of IDH2. However, regulation of 2HG formation by SIRT3-mediated deacetylation was not investigated yet. We aimed to study mitochondrial IDH2 function in response to acetylation and deacetylation, and focus specifically on 2HG production by IDH2. We used acetylation surrogate mutant of IDH2 K413Q and assayed enzyme kinetics of oxidative decarboxylation of isocitrate, 2HG production by the enzyme, and 2HG production in cells. The purified IDH2 K413Q exhibited lower oxidative reaction rates than IDH2 WT. 2HG production by IDH2 K413Q was largely diminished at the enzymatic and cellular level, and knockdown of SIRT3 also inhibited 2HG production by IDH2. Contrary, the expression of putative mitochondrial acetylase GCN5L likely does not target IDH2. Using mass spectroscopy, we further identified lysine residues within IDH2, which are the substrates of SIRT3. In summary, we demonstrate that 2HG levels arise from non-mutant IDH2 reductive function and decrease with increasing acetylation level. The newly identified lysine residues might apply in regulation of IDH2 function in response to metabolic perturbations occurring in cancer cells, such as glucose-free conditions.

The heterocyclic compound Tempol inhibits the growth of cancer cells by interfering with glutamine metabolism.

Tempol (4-hydroxy-2,2,6,6-Tetramethylpiperidine-1-oxyl, TPL), a nitroxide compound, inhibits proliferation and increases the vulnerability of cancer cells to apoptosis induced by cytotoxic agents. However, the molecular mechanism of TPL inhibiting cancer cell proliferation has not been fully understood. In this study, we evaluated the metabolic effect of TPL on cancer cells and explored its cancer therapeutic potential. Extracellular flow assays showed that TPL inhibited cellular basal and maximal oxygen consumption rates of mitochondrial. 13C metabolic flux analysis showed that TPL treatment had minimal effect on glycolysis. However, we found that TPL inhibits glutamine metabolism by interfering with the oxidative tricarboxylic acid cycle (TCA) process and reductive glutamine process. We found that the inhibitory effect of TPL on metabolism occurs mainly on the step from citrate to α-ketoglutarate or vice versa. We also found that activity of isocitrate dehydrogenase IDH1 and IDH2, the key enzymes in TCA, were inhibited by TPL treatment. In xenograft mouse model, TPL treatment reduced tumor growth by inhibiting cellular proliferation of xenograft tumors. Thus, we provided a mechanism of TPL inhibiting cancer cell proliferation by interfering with glutamine utilization that is important for survival and proliferation of cancer cells. The study may help the development of a therapeutic strategy of TPL combined with other anticancer medicines.