Molecular characterization of higher plant NAD-dependent isocitrate dehydrogenase: evidence for a heteromeric structure by the complementation of yeast mutants

NAD-dependent isocitrate dehydrogenase (IDH) is a key enzyme controlling the activity of the citric acid cycle. Despite more than 30 years of work, the plant enzyme remains poorly characterized. In this paper, a molecular characterization of the plant IDH is presented. Starting from probes defined according to sequence comparisons, three full-length cDNAs named Ntidha , Ntidhb and Ntidhc encoding different IDH subunits have been isolated from a Nicotiana tabacum cell suspension library. Sequence comparisons of the tobacco IDH subunits with the E. coli NADP-dependent enzyme, and the yeast IDH1 and IDH2 subunits suggested that only IDHa had the capacity to be catalytic as IDHb and IDHc were lacking certain residues implied in catalysis. The ability of antibodies raised against the recombinant IDHa protein to preferentially cross-react with IDH2 indicated that IDHa was more closely related to IDH2 than to IDH1. Complementation of yeast single IDH mutants showed that IDHb and IDHc could replace the function of the yeast regulatory IDH1 subunit. Although IDHa was unable to complement the IDH2 mutant, its catalytic function was revealed by the ability of two heteromeric enzymes, composed of either IDHa with IDHb or IDHa with IDHc, to replace IDH function in a yeast double mutant lacking both subunits. Expression studies at the protein and mRNA levels show that each subunit is present in both root and leaf tissues and that the three IDH genes respond in the same way to nitrate addition. Taken together, such observations suggest that the physiologically active enzyme is composed of the three different subunits. These results show for the first time that the plant IDH is heteromeric and that IDH subunit composition appears to be conserved between plant and animal kingdoms.     

Locations of the regulatory sites for isocitrate dehydrogenase kinase/phosphatase

Isocitrate dehydrogenase (IDH)(1) of Escherichia coli is regulated by a bifunctional protein, IDH kinase/phosphatase. In this paper, we demonstrate that the effectors controlling these activities belong to two distinct classes that differ in mechanism and in the locations of their binding sites. NADPH and isocitrate are representative members of one of these effector classes. NADPH inhibits both IDH kinase and IDH phosphatase, whereas isocitrate inhibits only IDH kinase. Isocitrate can "activate" IDH phosphatase by reversing product inhibition by dephospho-IDH. Mutations in icd, which encodes IDH, had parallel effects on the binding of these ligands to the IDH active site and on their effects on IDH kinase and phosphatase, indicating that these ligands regulate IDH kinase/phosphatase through the IDH active site. Kinetic analyses suggested that isocitrate and NADPH prevent formation of the complex between IDH kinase/phosphatase and its protein substrate. AMP, 3-phosphoglycerate, and pyruvate represent a class of regulatory ligands that is distinct from that which includes isocitrate and NADPH. These ligands bind directly to IDH kinase/phosphatase, a conclusion which is supported by the observation that they inhibit the IDH-independent ATPase activity of this enzyme. These effector classes can also be distinguished by the observation that mutant derivatives of IDH kinase/phosphatase expressed from aceK3 and aceK4 exhibited dramatic changes in their responses to AMP, 3-phosphoglycerate, and pyruvate but not to NADPH and isocitrate.     

NAD(+)-dependent isocitrate dehydrogenase. Cloning, nucleotide sequence, and disruption of the IDH2 gene from Saccharomyces cerevisiae

NAD(+)-dependent isocitrate dehydrogenase from Saccharomyces cerevisiae is composed of two nonidentical subunits, designated IDH1 (Mr approximately 40,000) and IDH2 (Mr approximately 39,000). We have isolated and characterized a yeast genomic clone containing the IDH2 gene. The amino acid sequence deduced from the gene indicates that IDH2 is synthesized as a precursor of 369 amino acids (Mr 39,694) and is processed upon mitochondrial import to yield a mature protein of 354 amino acids (Mr 37,755). Amino acid sequence comparison between S. cerevisiae IDH2 and S. cerevisiae NADP(+)-dependent isocitrate dehydrogenase shows no significant sequence identity, whereas comparison of IDH2 and Escherichia coli NADP(+)-dependent isocitrate dehydrogenase reveals a 33% sequence identity. To confirm the identity of the IDH2 gene and examine the relationship between IDH1 and IDH2, the IDH2 gene was disrupted by genomic replacement in a haploid yeast strain. The disruption strain expressed no detectable IDH2, as determined by Western blot analysis, and was found to lack NAD(+)-dependent isocitrate dehydrogenase activity, indicating that IDH2 is essential for a functional enzyme. Overexpression of IDH2, however, did not result in increased NAD(+)-dependent isocitrate dehydrogenase activity, suggesting that both IDH1 and IDH2 subunits are required for catalytic activity. The disruption strain was unable to utilize acetate as a carbon source and exhibited a 2-fold slower growth rate than wild type strains on glycerol or lactate. This growth phenotype is consistent with NAD(+)-dependent isocitrate dehydrogenase performing an essential role in the oxidative function of the citric acid cycle.     

Structure of the monomeric isocitrate dehydrogenase: evidence of a protein monomerization by a domain duplication

NADP(+)-dependent isocitrate dehydrogenase is a member of the beta-decarboxylating dehydrogenase family and catalyzes the oxidative decarboxylation reaction from 2R,3S-isocitrate to yield 2-oxoglutarate and CO(2) in the Krebs cycle. Although most prokaryotic NADP(+)-dependent isocitrate dehydrogenases (IDHs) are homodimeric enzymes, the monomeric IDH with a molecular weight of 80-100 kDa has been found in a few species of bacteria. The 1.95 A crystal structure of the monomeric IDH revealed that it consists of two distinct domains, and its folding topology is related to the dimeric IDH. The structure of the large domain repeats a motif observed in the dimeric IDH. Such a fusional structure by domain duplication enables a single polypeptide chain to form a structure at the catalytic site that is homologous to the dimeric IDH, the catalytic site of which is located at the interface of two identical subunits.     

Dimerization and Bifunctionality Confer Robustness to the Isocitrate Dehydrogenase Regulatory System in Escherichia coli

An important goal of systems biology is to develop quantitative models that explain how specific molecular features give rise to systems-level properties. Metabolic and regulatory pathways that contain multifunctional proteins are especially interesting to study from this perspective because they have frequently been observed to exhibit robustness: the ability for a system to perform its proper function even as levels of its components change. In this study, we use extensive biochemical data and algebraic modeling to develop and analyze a model that shows how robust behavior arises in the isocitrate dehydrogenase (IDH) regulatory system of Escherichia coli, which was shown in 1985 to experimentally exhibit robustness. E. coli IDH is regulated by reversible phosphorylation catalyzed by the bifunctional isocitrate dehydrogenase kinase/phosphatase (IDHKP), and the level of IDH activity determines whether carbon flux is directed through the glyoxylate bypass (for growth on two-carbon substrates) or the full tricarboxylic acid cycle. Our model, which incorporates recent structural data on IDHKP, identifies several specific biochemical features of the system (including homodimerization of IDH and bifunctionality of IDHKP) that provide a potential explanation for robustness. Using algebraic techniques, we derive an invariant that summarizes the steady-state relationship between the phospho-forms of IDH. We use the invariant in combination with kinetic data on IDHKP to calculate IDH activity at a range of total IDH levels and find that our model predicts robustness. Our work unifies much of the known biochemistry of the IDH regulatory system into a single quantitative framework and highlights the importance of constructing biochemically realistic models in systems biology.     

Cloning and characterization of the gene encoding the IDH1 subunit of NAD(+)-dependent isocitrate dehydrogenase from Saccharomyces cerevisiae.

NAD(+)-dependent isocitrate dehydrogenase from Saccharomyces cerevisiae is composed of two nonidentical subunits, designated IDH1 and IDH2. The gene encoding IDH2 was previously cloned and sequenced (Cupp, J.R., and McAlister-Henn, L. (1991) J. Biol. Chem. 266, 22199-22205), and in this paper we describe the isolation of a yeast genomic clone containing the IDH1 gene. A fragment of the IDH1 gene was amplified by the polymerase chain reaction method utilizing degenerate oligonucleotides based on tryptic peptide sequences of the purified subunit; this fragment was used to isolate a full length IDH1 clone. The nucleotide sequence of the IDH1 coding region was determined and encodes a 360-residue polypeptide including an 11-residue mitochondrial targeting presequence. Amino acid sequence comparison between IDH1 and IDH2 reveals a 42% sequence identity, and both IDH1 and IDH2 show approximately 32% identity to Escherichia coli NAD(P)(+)-dependent isocitrate dehydrogenase. To examine the function of the IDH1 subunit and to determine the metabolic role of NAD(+)-dependent isocitrate dehydrogenase the IDH1 gene was disrupted in a wild type haploid yeast strain and in a haploid strain lacking IDH2. The IDH1 disruption strains expressed no detectable IDH1 as determined by Western blot analysis, and these strains were found to lack NAD(+)-dependent isocitrate dehydrogenase activity indicating that IDH1 is essential for a functional enzyme. Over-expression of IDH1 in a strain containing IDH2 restored wild type activity but did not result in increased levels of activity, suggesting that both IDH1 and IDH2 are required for a functional enzyme. Growth phenotype analysis of the IDH1 disruption strains revealed that they grew at a reduced rate on the nonfermentable carbon sources examined (glycerol, lactate, and acetate), consistent with NAD(+)-dependent isocitrate dehydrogenase performing a critical role in oxidative function of the citric acid cycle. In addition, the IDH1 disruption strains grew at wild type rates in the absence of glutamate, indicating that these strains are not glutamate auxotrophs.     

Dual-Knockout of Mutant Isocitrate Dehydrogenase 1 and 2 Subtypes Towards Glioma Therapy: Structural Mechanistic Insights on the Role of Vorasidenib

Recently, Vorasidenib (AG-881) has been reported as a therapeutic alternative that exerts potent dual inhibitory activity against mIDH1/2 towards the treatment of low-grade glioma. However, structural and dynamic events associated with its dual inhibition mechanism remain unclear. As such, we employ integrative computer-assisted atomistic techniques to provide thorough structural and dynamic insights. Our analysis proved that the dual-targeting ability of AG-881 is mediated by Val255/Val294 within the binding pockets of both mIDH1 and mIDH2 which are shown to elicit a strong intermolecular interaction, thus favoring binding affinity. The structural orientations of AG-881 within the respective hydrophobic pockets allowed favorable interactions with binding site residues which accounted for its high binding free energy of −28.69 kcal/mol and −19.89 kcal/mol towards mIDH1 and mIDH2, respectively. Interestingly, upon binding, AG-881 was found to trigger systemic alterations of mIDH1 and mIDH2 characterized by restricted residue flexibility and a reduction in exposure of residues to the solvent surface area. As a result of these structural alterations, crucial interactions of the mutant enzymes were inhibited, a phenomenon that results in a suppression of the production of oncogenic stimulator 2-HG. Findings therefore provide thorough structural and dynamic insights associated with the dual inhibitory activity of AG-881 towards glioma therapy.     

Bistability in the isocitrate dehydrogenase reaction: an experimentally based theoretical study.

The enzyme isocitrate dehydrogenase (IDH, EC 1.1.1.42) can exhibit activation by one of its products, NADPH. This activation is competitively inhibited by the substrate NADP+, whereas NADPH competes with NADP+ for the catalytic site. Experimental observations briefly presented here have shown that if IDH is coupled to another enzyme, diaphorase (EC 1.8.1.4), which transforms NADPH into NADP+, the system can attain either one of two stable states, corresponding to a low and a high NADPH concentration. The evolution toward either one of these stable states depends on the time of addition of diaphorase to the medium containing IDH and its substrate NADP+. We present a theoretical and numerical analysis of a model for the IDH-diaphorase bienzymatic system, based on the regulatory properties of IDH. The results confirm the occurrence of bistability for parameter values derived from the experiments. Depending on the total concentration of NADP+ plus NADPH and the concentration of IDH, the system can either admit a single steady state or display bistability. We obtain an expression for the critical time t*, before which diaphorase addition leads to the lower steady state and after which addition of the enzyme leads to the upper steady state of NADPH. The analysis is extended to the case where the second substrate of IDH, isocitrate, is consumed in the course of the reaction without being regenerated. Bistability occurs only as a transient phenomenon in these conditions.     

Mutation of the predicted ATP binding site inactivates both activities of isocitrate dehydrogenase kinase/phosphatase

In Escherichia coli, the reversible phosphorylation of isocitrate dehydrogenase (IDH) is catalyzed by a bifunctional protein: IDH kinase/phosphatase. Although both IDH kinase and IDH phosphatase require ATP, the amino acid sequence of IDH kinase/phosphatase contains a single sequence that matches the consensus for ATP binding sites. A mutation that converted the "invariant" lysine (residue 336) of this consensus sequence to a methionine reduced the activities of both IDH kinase and IDH phosphatase by factors of greater than 500, to levels below the detection limits of the assays. The apparent elimination of both IDH kinase and IDH phosphatase by this mutation is consistent with the proposal that these activities share a common ATP binding site and that these reactions may occur at the same active site. Although conversion of Lys336 to a methionine eliminated detectable IDH kinase activity as measured in vitro, the mutant allele retained the ability to complement an aceK deletion mutation, restoring the ability of these cells to grow on minimal acetate medium. Complementation apparently resulted because the mutant protein retained sufficient activity to phosphorylate IDH in vivo. To determine whether the enzymatic assays performed in vitro had correctly reflected the activity of the mutant protein in vivo, we measured the rates at which mutant and wild-type cultures could incorporate [32P]inorganic phosphate into IDH. The wild-type culture achieved maximal incorporation in less than 3 min. In contrast, 32P incorporation was only barely detectable after 30 min in the mutant culture, indicating that the activity of the mutant protein is, indeed, greatly reduced in vivo. The ability of the mutant allele to complement an aceK null mutation thus suggests that IDH kinase/phosphatase levels in wild-type cells are in great excess over what is required for steady-state growth on acetate medium.     

变铅青链霉菌异柠檬酸脱氢酶的异源表达及序列分析

通过同源性引物成功扩增和克隆了变铅青链霉菌(Streptomyces lividans)TK54的异柠檬酸脱氢酶(isocitrate dehydrogenase, IDH) (简称SlIDH)基因icd (GenBank登录号为EU661252).icd的起始密码子为GTG,GC含量为69.55 %,显示了链霉菌基因的高GC含量特征,实现了SlIDH在E.coli中的异源高效表达.0.5 mmol/L的IPTG为最佳诱导条件.SlIDH的分子量约为80 kD.在Mn~(2+)或Mg~(2+)条件下,SlIDH以NADP~+为辅酶时的活性分别为7.94 U/mg及4.00 U/mg,以NAD~+为辅酶时的活性分别为0.58 U/mg及0.27 U/mg,SlIDH更偏爱以NADP~+为辅酶.与不同种属单体IDH的氨基酸序列比对显示,SlIDH与单体IDH的序列一致性均在60 %以上.因此本工作首次以实验性证据初步鉴定了SlIDH为NADP-依赖型单体IDH.本工作为进一步探索单体IDH的结构与功能以及单体IDH与同源二聚体IDH的进化关系奠定了基础.     

A kinetic model of functioning and regulation of Escherichia coli isocitrate dehydrogenase

A Rapid Equilibrium Random Bi Ter mechanism of formation of two dead-end complexes was proposed to describe the experimental data on the functioning of E. coli isocitrate dehydrogenase (IDH). A kinetic model for the enzyme functioning was constructed, which assumes that it is regulated through reversible phosphorylation by its kinase/phosphatase, which in turn is regulated by IDH substrates and central metabolites such as pyruvate (Pyr), 3-phosphoglycerate (3-PG), and AMP. It was shown using the model that increasing the concentration of these effectors results in an increase of the active part of IDH, thus leading to an increase in the Krebs cycle flux. We predict that the ratio of the phosphorylated and free forms of IDH (IDHP/IDH) is more sensitive to AMP, NADPH, and isocitrate concentrations than to Pyr and 3-PG. The model allows a realistic prediction of changes in the IDHP/IDH ratio, which would occur under changes of biosynthetic and energetic loading of the E. coli cell.     

Mutations in isocitrate dehydrogenase 2 accelerate glioma cell migration via matrix metalloproteinase-2 and 9

The gene encoding isocitrate dehydrogenase (IDH) is somatically mutated predominantly in secondary glioblastoma multiforme. Glioma-specific mutations in IDH1 always produced a single amino acid substitution at R132, but mutations in IDH2 were exclusively at R172 which was the analogous site to R132 in IDH1. Mutations of IDH1 and IDH2 led to simultaneous loss and gain of activities in the production of α-ketoglutarate and 2-hydroxyglutarate, respectively. Matrix metalloproteinases (MMPs) are zinc-dependent endoproteinases involved in the degradation of the extracellular matrix. The exact role of IDH2 mutant on MMPs activity and cell migration has not been fully studied. Here, we show that in response to IDH2 mutations, low levels of α-ketoglutarate increased the stabilization of HIF-1α which can contribute to tumor growth. Moreover, mutant IDH2-induced HIF-1α improved the secretion levels of pro-MMP-2 and pro-MMP-9 as well as the conversion from pro-MMP-2 to its active form, giving C6 glioma cells a higher migration potential. The HIF-1α pathway is probably a critical pathway for release of MMPs in the glioma cancer harboring IDH mutant.     

Crystal structure of porcine mitochondrial NADP+-dependent isocitrate dehydrogenase complexed with Mn2+ and isocitrate. Insights into the enzyme mechanism

The crystal structure of porcine heart mitochondrial NADP+-dependent isocitrate dehydrogenase (IDH) complexed with Mn2+ and isocitrate was solved to a resolution of 1.85 A. The enzyme was expressed in Escherichia coli, purified as a fusion protein with maltose binding protein, and cleaved with thrombin to yield homogeneous enzyme. The structure was determined by multiwavelength anomalous diffraction phasing using selenium substitution in the form of selenomethionine as the anomalous scatterer. The porcine NADP+-IDH enzyme is structurally compared with the previously solved structures of IDH from E. coli and Bacillus subtilis that share 16 and 17% identity, respectively, with the mammalian enzyme. The porcine enzyme has a protein fold similar to the bacterial IDH structures with each monomer folding into two domains. However, considerable differences exist between the bacterial and mammalian forms of IDH in regions connecting core secondary structure. Based on the alignment of sequence and structure among the porcine, E. coli, and B. subtilis IDH, a putative phosphorylation site has been identified for the mammalian enzyme. The active site, including the bound Mn2+-isocitrate complex, is highly ordered and, therefore, mechanistically informative. The consensus IDH mechanism predicts that the Mn2+-bound hydroxyl of isocitrate is deprotonated prior to its NADP+-dependent oxidation. The present crystal structure has an active site water that is well positioned to accept the proton and ultimately transfer the proton to solvent through an additional bound water.     

Isocitrate dehydrogenase 1 R132C mutation occurs exclusively in microsatellite stable colorectal cancers with the CpG island methylator phenotype

The CpG Island Methylator Phenotype (CIMP) is fundamental to an important subset of colorectal cancer; however, its cause is unknown. CIMP is associated with microsatellite instability but is also found in BRAF mutant microsatellite stable cancers that are associated with poor prognosis. The isocitrate dehydrogenase 1 (IDH1) gene causes CIMP in glioma due to an activating mutation that produces the 2-hydroxyglutarate oncometabolite. We therefore examined IDH1 alteration as a potential cause of CIMP in colorectal cancer. The IDH1 mutational hotspot was screened in 86 CIMP-positive and 80 CIMP-negative cancers. The entire coding sequence was examined in 81 CIMP-positive colorectal cancers. Forty-seven cancers varying by CIMP-status and IDH1 mutation status were examined using Illumina 450K DNA methylation microarrays. The R132C IDH1 mutation was detected in 4/166 cancers. All IDH1 mutations were in CIMP cancers that were BRAF mutant and microsatellite stable (4/45, 8.9%). Unsupervised hierarchical cluster analysis identified an IDH1 mutation-like methylation signature in approximately half of the CIMP-positive cancers. IDH1 mutation appears to cause CIMP in a small proportion of BRAF mutant, microsatellite stable colorectal cancers. This study provides a precedent that a single gene mutation may cause CIMP in colorectal cancer, and that this will be associated with a specific epigenetic signature and clinicopathological features.     

Isocitrate dehydrogenase 1 R132C mutation occurs exclusively in microsatellite stable colorectal cancers with the CpG island methylator phenotype

The CpG Island Methylator Phenotype (CIMP) is fundamental to an important subset of colorectal cancer; however, its cause is unknown. CIMP is associated with microsatellite instability but is also found in BRAF mutant microsatellite stable cancers that are associated with poor prognosis. The isocitrate dehydrogenase 1 (IDH1) gene causes CIMP in glioma due to an activating mutation that produces the 2-hydroxyglutarate oncometabolite. We therefore examined IDH1 alteration as a potential cause of CIMP in colorectal cancer. The IDH1 mutational hotspot was screened in 86 CIMP-positive and 80 CIMP-negative cancers. The entire coding sequence was examined in 81 CIMP-positive colorectal cancers. Forty-seven cancers varying by CIMP-status and IDH1 mutation status were examined using Illumina 450K DNA methylation microarrays. The R132C IDH1 mutation was detected in 4/166 cancers. All IDH1 mutations were in CIMP cancers that were BRAF mutant and microsatellite stable (4/45, 8.9%). Unsupervised hierarchical cluster analysis identified an IDH1 mutation-like methylation signature in approximately half of the CIMP-positive cancers. IDH1 mutation appears to cause CIMP in a small proportion of BRAF mutant, microsatellite stable colorectal cancers. This study provides a precedent that a single gene mutation may cause CIMP in colorectal cancer, and that this will be associated with a specific epigenetic signature and clinicopathological features.     

Lithium chloride decreases proliferation and migration of C6 glioma cells harboring isocitrate dehydrogenase 2 mutant via GSK-3β

The gene encoding isocitrate dehydrogenase (IDH) is somatically mutated predominantly in secondary glioblastoma multiforme. Mutations of IDH1 and IDH2 lead to simultaneous loss and gain of activities in the production of α-ketoglutarate and 2-hydroxyglutarate, respectively. Lithium chloride was recently proved efficient in inhibiting glioma cell migration. The mechanism of lithium chloride on C6 glioma cells harboring IDH2 mutation has not been studied. Here, we found lithium chloride induced inhibitive effects on cell proliferation of both C6 glioma cells with and without IDH2 mutation, although IDH2 mutation increased the stability of HIF-1α. GSK-3β could be phosphorylated at Ser9 and its activity was inhibited when C6 glioma cells were treated by lithium chloride. The degree of phosphorylation in IDH2^R172G treatment group was lower than that as compared to the control and IDH2 treatment groups. At the same time, the accumulation of β-catenin in C6 cell nucleus was decreased. Moreover, although the β-catenin and HIF-1α increased the secretion of metalloproteinase-2,-9 in C6 glioma cells harboring IDH2 mutation, the migration potential of lithium chloride-treated C6 glioma cells harboring the IDH2 and its mutant was uniform. These results indicated lithium chloride could decrease the proliferation and migration potential of C6 glioma cells harboring IDH2 mutation.     

Isocitrate dehydrogenase kinase/phosphatase

In Escherichia coli, isocitrate dehydrogenase (IDH) is regulated by phosphorylation. This phosphorylation cycle is catalyzed by an unusual, bifunctional protein:IDH kinase/phosphatase. IDH kinase/phosphatase is expressed from a single gene, aceK, and both activities are catalyzed by the same polypeptide. The amino acid sequence of IDH kinase/phosphatase does not exhibit the characteristics which are typical of other protein kinases, although it does contain a consensus ATP binding site. The available evidence suggests that the IDH kinase and IDH phosphatase reactions occur at the same active site and that the IDH phosphatase reaction results from the back reaction of IDH kinase tightly coupled to ATP hydrolysis. The function of the IDH phosphorylation cycle is to control the flux of isocitrate through the glyoxylate bypass. This pathway is essential for growth on acetate because it prevents the quantitative loss of the acetate carbons as CO2 in the Krebs' cycle. IDH kinase/phosphatase monitors general metabolism by responding to the levels of a wide variety of metabolites, many of which activate IDH phosphatase and inhibit IDH kinase. The ability of IDH kinase/phosphatase to monitor general metabolism allows. the IDH phosphorylation cycle to compensate for substantial perturbations of the system, such as a 15-fold overproduction of IDH. The significance of the cellular level of IDH kinase/phosphatase has also been evaluated. The level of this protein is in great excess of that required for steady-state growth on acetate. In contrast, IDH kinase/phosphatase is, in some cases, rate-limiting for the dephosphorylation of IDH which results when preferred carbon sources are added to cultures growing on acetate.     

A glycolysis-based ten-gene signature correlates with the clinical outcome,molecular subtype and IDH1 mutation in glioblastoma

Reprogrammed metabolism is a hallmark of cancer. Glioblastoma (GBM) tumor cells predominantly utilize aerobic glycolysis for the biogenesis of energy and intermediate nutrients. However, in GBM, the clinical significance of glycolysis and its underlying relations with the molecular features such as IDH1 mutation and subtype have not been elucidated yet. Herein, based on glioma datasets including TCGA (The Cancer Genome Atlas), REMBRANDT (Repository for Molecular Brain Neoplasia Data) and GSE16011, we established a glycolytic gene expression signature score (GGESS) by incorporating ten glycolytic genes. Then we performed survival analyses and investigated the correlations between GGESS and IDH1 mutation as well as the molecular subtypes in GBMs. The results showed that GGESS independently predicted unfavorable prognosis and poor response to chemotherapy of GBM patients. Notably, GGESS was high in GBMs of mesenchymal subtype but low in IDH1-mutant GBMs. Furthermore, we found that the promoter regions of tumor-promoting glycolytic genes were hypermethylated in IDH1-mutant GBMs. Finally, we found that high GGESS also predicted poor prognosis and poor response to chemotherapy when investigating IDH1-wildtype GBM patients only. Collectively, glycolysis represented by GGESS predicts unfavorable clinical outcome of GBM patients and is closely associated with mesenchymal subtype and IDH1 mutation in GBMs.     

The isocitrate dehydrogenase phosphorylation cycle. Identification of the primary rate-limiting step

In Escherichia coli, the branch point between the Krebs cycle and the glyoxylate bypass is regulated by the phosphorylation of isocitrate dehydrogenase (IDH). Phosphorylation inactivates IDH, forcing isocitrate through the bypass. This bypass is essential for growth on acetate but does not serve a useful function when alternative carbon sources, such as glucose or pyruvate, are also present. When pyruvate or glucose is added to a culture growing on acetate, the cells responded by dephosphorylating IDH and thus inhibiting the flow of isocitrate through the glyoxylate bypass. In an effort to identify the primary rate-limiting step in the response of IDH phosphorylation to alternative carbon sources, we have examined the response rates of congenic strains of E. coli which express different levels of IDH kinase/phosphatase, the bifunctional protein which catalyzes this phosphorylation cycle. The rate of the pyruvate-induced dephosphorylation of IDH was proportional to the level of IDH kinase/phosphatase, indicating that IDH kinase/phosphatase was primarily rate-limiting for dephosphorylation. However, the identity of the primary rate-limiting step appears to depend on the stimulus, since the rate of dephosphorylation of IDH in response to glucose was independent of the level of IDH kinase/phosphatase.     

Prognosis of Glioblastoma With Oligodendroglioma Component is Associated With the IDH1 Mutation and MGMT Methylation Status

Glioblastoma (GBM) with oligodendroglioma component (GBMO) is a newly described GBM subtype in the 2007 World Health Organization classification. However, its biological and genetic characteristics are largely unknown. We investigated the clinicopathological and molecular features of 34 GBMOs and compared the survival rate of these patients with those of patients with astrocytoma, oligodendroglioma, anaplastic oligoastrocytoma (AOA), and conventional GBMs in our hospital. GBMO could be divided into two groups based on the presence of an IDH1 mutation. The IDH1 mutation was more frequently found in secondary GBMO, which had lower frequencies of EGFR amplification but higher MGMT methylation than the wild type IDH1 group, and patients with mutant IDH1 GBMO were on average younger than those with wild-type IDH1. Therefore, GBMO is a clinically and molecularly heterogeneous subtype, largely belonging to a proneural and classical subtype of GBM. The survival rate of GBMO patients itself was worse than that of AOA patients but not significantly better than that of conventional GBM patients. GBMO survival was independent of the dominant histopathological subtype i.e., astrocyte-dominant or oligodendroglioma -dominant, but it was significantly associated with the IDH1 mutation and MGMT methylation status. Therefore, GBMO should be regarded as a separate entity from AOA and must be classified as a subtype of GBM. However, further study is needed to determine whether it is a pathologic variant or a pattern of GBM because GBMO has a similar prognosis to conventional GBMs.